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Sample records for sarcoplasmic reticulum-based ryanodine

  1. Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

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    V.A. Vizotto

    2007-01-01

    Full Text Available We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2, phospholamban (PLB, and ryanodine channel (RYR2 mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01. The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively. Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

  2. Effect of Zn2+ ions on ryanodine binding to sarcoplasmic reticulum of striated muscles in the presence of pyrithione

    Institute of Scientific and Technical Information of China (English)

    Hong XIE; Ke-ying CHEN; Pei-hong ZHU

    2004-01-01

    AIM: To explore whether the differential effects of Zn2+ on ryanodine binding to the sarcoplasmic reticulum (SR)of skeletal and cardiac muscles resulted from different permeability of the SR to Zn2+. METHODS: [3H]ryanodine binding assays were performed to examine the effect of Zn2+ on ryanodine binding to the SR in the presence of pyrithione sodium (PyNa), a specific Zn2+ ionophore. RESULTS: As a control, PyNa up to 50 μmol/L did not induce any effect on ryanodine binding to the SR of cardiac muscle. But PyNa 1-100 μmol/L increased ryanodine binding in skeletal muscle with maximum binding (222.2 %+20.9 % of the control) and inhibited ryanodine binding to 50 % of the control at about 500 μrnol/L. In the presence of PyNa 10 and 50 μmol/L the dose-dependence of the effect of Zn2+ in cardiac muscle was still monophasic and not changed by PyNa, while the biphasic effect of Zn2+in skeletal muscle became monophasic. CONCLUSION: Different permeability of the SR to Zn2+ may account for the differential effects of Zn2+on ryanodine binding in skeletal and cardiac muscles. PyNa is not a strictly specific Zn2+ ionophore.

  3. Isolation of rat cardiac sarcoplasmic reticulum with improved Ca2+ uptake and ryanodine binding.

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    Feher, J J; Davis, M D

    1991-03-01

    The instability of the oxalate-supported Ca2+ uptake activity of rat cardiac sarcoplasmic reticulum (CSR) in ventricular homogenates most likely accounts for the low specific activity of the rate of oxalate-supported Ca2+ uptake in previously reported fractions of isolated rat CSR. We have found that CSR vesicles with improved Ca2+ transport capabilities can be isolated if 1 M KCl is used to stabilize the CSR activity and to allow the extraction of the CSR from the cellular debris. The average rate of Ca2+ uptake by the isolated rat CSR in the presence of 10 mM oxalate at 37 degrees C was 0.45 mumols/min-mg in the absence of CSR Ca2+ channel blockers and 0.87 mumols/min-mg in the presence of 10 microM ruthenium red. The Ca(2+)-dependent ATPase activity under the conditions of oxlate-supported uptake was 1.25 mumols/min-mg and 0.84 mumols/min-mg in the absence and presence of 10 microM ruthenium red, respectively. The rat CSR vesicles bound 3H-ryanodine with a Kd of 1.45 nM and a Bmax of 3.7 pmol mg. The level of phosphorylated intermediate was 0.30 nmol/mg. The values Bmax, EP and Ca(2+)-ATPase activity are from one-third to one-half of those previously reported for isolated canine CSR vesicles. These results suggest that the isolated rat CSR may be quite similar to dog CSR.

  4. Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

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    Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Ruas, Jorge L; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T; Skurvydas, Albertas; Westerblad, Håkan

    2015-12-15

    High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

  5. Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum.

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    Loy, Ryan E; Orynbayev, Murat; Xu, Le; Andronache, Zoita; Apostol, Simona; Zvaritch, Elena; MacLennan, David H; Meissner, Gerhard; Melzer, Werner; Dirksen, Robert T

    2011-01-01

    The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.

  6. Ryanodine receptor channelopathies

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    Betzenhauser, Matthew J.

    2010-01-01

    Ryanodine receptors (RyR) are intracellular Ca2+-permeable channels that provide the sarcoplasmic reticulum Ca2+ release required for skeletal and cardiac muscle contractions. RyR1 underlies skeletal muscle contraction, and RyR2 fulfills this role in cardiac muscle. Over the past 20 years, numerous mutations in both RyR isoforms have been identified and linked to skeletal and cardiac diseases. Malignant hyperthermia, central core disease, and catecholaminergic polymorphic ventricular tachycardia have been genetically linked to mutations in either RyR1 or RyR2. Thus, RyR channelopathies are both of interest because they cause significant human diseases and provide model systems that can be studied to elucidate important structure–function relationships of these ion channels. PMID:20179962

  7. Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion.

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    Hurne, Alanna M; O'Brien, Jennifer J; Wingrove, Douglas; Cherednichenko, Gennady; Allen, Paul D; Beam, Kurt G; Pessah, Isaac N

    2005-11-04

    Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

  8. Effects of boldine on mouse diaphragm and sarcoplasmic reticulum vesicles isolated from skeletal muscle.

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    Kang, J J; Cheng, Y W

    1998-02-01

    The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle.

  9. Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Lunde, Per; Levin, Kasper

    2000-01-01

    To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after...... the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P training. The 5-wk sprint training induced a significantly higher (P ...-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P

  10. Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Lunde, Per; Levin, Kasper

    2000-01-01

    To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after...... the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P training. The 5-wk sprint training induced a significantly higher (P ...-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P

  11. Malignant hyperthermia domain in the regulation of ca(2+) release channel (ryanodine receptor).

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    Zorzato, F; Ronjat, M; Treves, S

    1997-11-01

    Malignant hyperthermia (MH) is a potentially lethal condition that is manifested in humans as an acute increase of body temperature in response to stress and exposure to volatile anaesthetics (halothane, enflurane) and muscle relaxants. To date, eight point mutations in the ryanodine receptor gene, the Ca(2+) release channel of the skeletal muscle sarcoplasmic reticulum, segregate with the MH phenotype, yet direct evidence linking altered [Ca(2+)](i) homeostasis to mutation in recombinant RYR has been obtained only for one such mutation. Most of these mutations appear in an "MH domain" that is localized at the NH(2) terminus of the skeletal muscle ryanodine receptor Ca(2+) channel. In this review, we summarize the available data concerning the role of the MH domain in the altered functions of the ryanodine receptor Ca(2+) channel. (Trends Cardiovasc Med 1997;7:312-316). © 1997, Elsevier Science Inc.

  12. Ryanodine receptors: allosteric ion channel giants.

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    Van Petegem, Filip

    2015-01-16

    The endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) form major intracellular Ca(2+) stores. Ryanodine receptors (RyRs) are large tetrameric ion channels in the SR and ER membranes that can release Ca(2+) upon triggering. With molecular masses exceeding 2.2MDa, they represent the pinnacle of ion channel complexity. RyRs have adopted long-range allosteric mechanisms, with pore opening resulting in conformational changes over 200Å away. Together with tens of protein and small molecule modulators, RyRs have adopted rich and complex regulatory mechanisms. Structurally related to inositol-1,4,5-trisphosphate receptors (IP3Rs), RyRs have been studied extensively using cryo-electron microscopy (cryo-EM). Along with more recent X-ray crystallographic analyses of individual domains, these have resulted in pseudo-atomic models. Over 500 mutations in RyRs have been linked to severe genetic disorders, which underscore their role in the contraction of cardiac and skeletal muscles. Most of these have been linked to gain-of-function phenotypes, resulting in premature or prolonged leak of Ca(2+) in the cytosol. This review outlines our current knowledge on the structure of RyRs at high and low resolutions, their relationship to IP3Rs, an overview of the most commonly studied regulatory mechanisms, and models that relate disease-causing mutations to altered channel function.

  13. Use-dependence of ryanodine effects on postrest contraction in ferret cardiac muscle.

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    Malecot, C O; Katzung, B G

    1987-04-01

    During an investigation of the effect of ryanodine on contractions in cardiac muscle, it was found that long rest periods removed all or most of the drug's effect. Therefore, we studied the kinetics of block development and recovery from block produced by low concentrations of ryanodine (1-100 pM) on the postrest contractions of ferret papillary muscle. At 100 pM, ryanodine depressed steady-state contraction amplitude slightly (4.2 +/- 1.1% mean +/- SEM, n = 10) but strongly inhibited (40-80%) the first contraction (postrest contraction) elicited on restimulation of the preparation after rest periods of 1 second to 5 minutes. Under control conditions, the nearly maximal potentiation of the twitch occurring after a standard test rest period (30 seconds of rest) was not affected by a preceding conditioning rest of up to 20 minutes. In the presence of 100 pM ryanodine, a conditioning rest increased the amplitude of the twitch elicited after a test rest, and the test rest contraction recovered toward control (drug-free) amplitude monoexponentially (time constant, 582 +/- 105 seconds). Block of postrest contraction could be reinduced by stimulation and occurred faster when higher rates were used (time constants, 758 seconds at 1 Hz and 107 +/- 26 seconds at 3 Hz). Since rest potentiation of twitch tension is believed to be mostly dependent on extra calcium released from the sarcoplasmic reticulum, the results suggest that the ryanodine-induced blockade of calcium release from the sarcoplasmic reticulum is use-dependent and recovers during diastole.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Dynamic Changes in Sarcoplasmic Reticulum Structure in Ventricular Myocytes

    Directory of Open Access Journals (Sweden)

    Amanda L. Vega

    2011-01-01

    sarcoplasmic reticulum (SR and the sarcolemma where Ca2+ release is activated. Here, we tested the hypothesis that the SR is a structurally inert organelle in ventricular myocytes. Our data suggest that rather than being static, the SR undergoes frequent dynamic structural changes. SR boutons expressing functional ryanodine receptors moved throughout the cell, approaching or moving away from the sarcolemma of ventricular myocytes. These changes in SR structure occurred in the absence of changes in [Ca2+] during EC coupling. Microtubules and the molecular motors dynein and kinesin 1(Kif5b were important regulators of SR motility. These findings support a model in which the SR is a motile organelle capable of molecular motor protein-driven structural changes.

  15. Targeting ryanodine receptors for anti-arrhythmic therapy

    Institute of Scientific and Technical Information of China (English)

    Mark D McCAULEY; Xander H T WEHRENS

    2011-01-01

    Antiarrhythmic drugs are a group of pharmaceuticals that suppress or prevent abnormal heart rhythms, which are often associated with substantial morbidity and mortality. Current antiarrhythmic drugs that typically target plasma membrane ion channels have limited clinical success and in some cases have been described as being pro-arrhythmic. However, recent studies suggest that pathological release of calcium (Ca2+) from the sarcoplasmic reticulum via cardiac ryanodine receptors (RyR2) could represent a promising target for antiarrhythmic therapy. Diastolic SR Ca2+ release has been linked to arrhythmogenesis in both the inherited arrhythmia synSeveral classes of pharmaceuticals have been shown to reduce abnormal RyR2 activity and may confer protection against triggered arrhythmias through reduction of SR Ca2+ leak. In this review, we will evaluate the current pharmacological methods for stabilizing RyR2 and suggest treatment modalities based on current evidence of molecular mechanisms.

  16. Separation and formation of ryanodine from dehydroryanodine. Preparation of tritium-labelled ryanodine

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    Sutko, J.L.; Thompson, L.J.; Schlatterer, R.G. and others

    1986-02-01

    The commercially available preparation of the naturally occurring diterpene ester ryanodine contains several compounds in addition to ryanodine. These compounds were separated and purified using high performance liquid chromatography. The two major components, ryanodine and dehydroryanodine represented 90% of the material present. A method for the efficient reduction of dehydroryanodine to ryanodine was developed and used to produce ryanodine having tritium atoms at positions 19 and 20 and a specific activity of 60.8 Ci/mmole.

  17. Molecular nature of sulfhydryl modification by hydrogen peroxide on type 1 ryanodine receptor

    Institute of Scientific and Technical Information of China (English)

    Hong-mei HAN; Ri-sheng WEI; Anthony F LAI; Chang-cheng YIN

    2006-01-01

    Aim: To elucidate the molecular nature of sulfhydryl modification by hydrogen peroxide on type 1 ryanodine receptor (RyRl). Methods: Rabbit skeletal muscle sarcoplasmic reticulum was treated with hydrogen peroxide, then RyRl complex was isolated. The proteins in the complex were analysed by electrophoresis, Western blot and electron microscopy. Results: (1) Hydrogen peroxide induces inter-subunit cross-linking within the tetrameric RyR1 molecule; (2) in parallel to inter-subunit cross-linking, the RyR1 molecule changes morphology; (3) the chemical and morphological changes are reversible: upon reduction by reducing agents, the RyR1 molecule regains its original state. Conclusion: These findings suggest that the molecular mechanism of RyR1 channe1 activity in sarcoplasmic reticulum regulated by hydrogen peroxide is through inter-subunit cross-linking within the tetrameric RyR1 molecule, which in turn induces structural changes of RyR1.

  18. Cations and anions as modifiers of ryanodine binding to the skeletal muscle calcium release channel.

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    Hasselbach, W; Migala, A

    1998-08-01

    Rate and equilibrium measurements of ryanodine binding to terminal cysternae fractions of heavy sarcoplasmic reticulum vesicles demonstrate that its activation by high concentrations of monovalent salts is based on neither elevated osmolarity nor ionic strength. The effect of the ions specifically depends on their chemical nature following the Hofmeister ion series for cations (Li+ < NH+4 < K- approximately Cs+ ryanodine. Activation by rising salt concentrations exhibits saturation kinetics with different dissociation constants (25-11 m) and different degrees of cooperativity (n = 1.5-4.0) for the respective salts. Maximal second order binding rates between 40,000 and 80,000 (m-1 x sec-1) were obtained for chlorides and nitrates of 1a group alkali ions with the exception of lithium supporting only rates of maximally 10,000 (M-1 x sec-1). The nitrogen bases, NH+4 and Tris+, in combination with chloride or nitrate, behave divergently. High maximal binding rates were achieved only with NH4NO3. The dissociation constants for the ryanodine-protein complexes obtained by measurements at equilibrium proved to depend differently on salt concentration, yet, converging to 1-3 nm for the applied salts at saturating concentrations. The salts do not affect dissociation of the ryanodine protein complex proving that the effect of salts on the protein's affinity for ryanodine is determined by their effect on the on-rate of ryanodine binding. ATP and its analogues modify salt action resulting in elevated maximal binding rates and reduction or abolition of binding cooperativity. Linear relations have been obtained by comparing the rates of ryanodine binding at different salt concentrations with the rates or the initial amplitudes (15 sec) of salt induced calcium release from actively

  19. Type 1 ryanodine receptor in cardiac mitochondria: transducer of excitation-metabolism coupling.

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    Beutner, Gisela; Sharma, Virendra K; Lin, Lin; Ryu, Shin-Young; Dirksen, Robert T; Sheu, Shey-Shing

    2005-11-10

    Mitochondria in a variety of cell types respond to physiological Ca(2+) oscillations in the cytosol dynamically with Ca(2+) uptakes. In heart cells, mitochondrial Ca(2+) uptakes occur by a ruthenium red-sensitive Ca(2+) uniporter (CaUP), a rapid mode of Ca(2+) uptake (RaM) and a ryanodine receptor (RyR) localized in the inner mitochondrial membrane (IMM). Three subtypes of RyRs have been described and cloned, however, the subtype identity of the mitochondrial ryanodine receptor (mRyR) is unknown. Using subtype specific antibodies, we characterized the mRyR in the IMM from rat heart as RyR1. These results are substantiated by the absence of RyR protein in heart mitochondria from RyR1 knockout mice. The bell-shape Ca(2+)-dependent [(3)H]ryanodine binding curve and its modulation by caffeine and adenylylmethylenediphosphonate (AMPPCP) give further evidence that mRyR functions pharmacologically like RyR1. Ryanodine prevents mitochondrial Ca(2+) uptake induced by raising extramitochondrial Ca(2+) to 10 microM. Similarly, ryanodine inhibits oxidative phosphorylation stimulated by 10 microM extramitochondrial Ca(2+). In summary, our results show that the mRyR in cardiac muscle has similar biochemical and pharmacological properties to the RyR1 in the sarcoplasmic reticulum (SR) of skeletal muscle. These results could also suggest an efficient mechanism by which mitochondria sequesters Ca(2+) via mRyR during excitation-contraction coupling to stimulate oxidative phosphorylation for ATP production to meet metabolic demands. Thus, the mRyR functions as a transducer for excitation-metabolism coupling.

  20. Biphasic decay of the Ca transient results from increased sarcoplasmic reticulum Ca leak

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    Sankaranarayanan, Rajiv; Li, Yatong; Greensmith, David J.; Eisner, David A.

    2016-01-01

    Key points Ca leak from the sarcoplasmic reticulum through the ryanodine receptor (RyR) reduces the amplitude of the Ca transient and slows its rate of decay.In the presence of β‐adrenergic stimulation, RyR‐mediated Ca leak produces a biphasic decay of the Ca transient with a fast early phase and a slow late phase.Two forms of Ca leak have been studied, Ca‐sensitising (induced by caffeine) and non‐sensitising (induced by ryanodine) and both induce biphasic decay of the Ca transient.Only Ca‐sensitising leak can be reversed by traditional RyR inhibitors such as tetracaine.Ca leak can also induce Ca waves. At low levels of leak, waves occur. As leak is increased, first biphasic decay and then slowed monophasic decay is seen. The level of leak has major effects on the shape of the Ca transient. Abstract In heart failure, a reduction in Ca transient amplitude and contractile dysfunction can by caused by Ca leak through the sarcoplasmic reticulum (SR) Ca channel (ryanodine receptor, RyR) and/or decreased activity of the SR Ca ATPase (SERCA). We have characterised the effects of two forms of Ca leak (Ca‐sensitising and non‐sensitising) on calcium cycling and compared with those of SERCA inhibition. We measured [Ca2+]i with fluo‐3 in voltage‐clamped rat ventricular myocytes. Increasing SR leak with either caffeine (to sensitise the RyR to Ca activation) or ryanodine (non‐sensitising) had similar effects to SERCA inhibition: decreased systolic [Ca2+]i, increased diastolic [Ca2+]i and slowed decay. However, in the presence of isoproterenol, leak produced a biphasic decay of the Ca transient in the majority of cells while SERCA inhibition produced monophasic decay. Tetracaine reversed the effects of caffeine but not of ryanodine. When caffeine (1 mmol l−1) was added to a cell which displayed Ca waves, the wave frequency initially increased before waves disappeared and biphasic decay developed. Eventually (at higher caffeine concentrations), the

  1. Minor sarcoplasmic reticulum membrane components that modulate excitation–contraction coupling in striated muscles

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    Treves, Susan; Vukcevic, Mirko; Maj, Marcin; Thurnheer, Raphael; Mosca, Barbara; Zorzato, Francesco

    2009-01-01

    In striated muscle, activation of contraction is initiated by membrane depolarisation caused by an action potential, which triggers the release of Ca2+ stored in the sarcoplasmic reticulum by a process called excitation–contraction coupling. Excitation–contraction coupling occurs via a highly sophisticated supramolecular signalling complex at the junction between the sarcoplasmic reticulum and the transverse tubules. It is generally accepted that the core components of the excitation–contraction coupling machinery are the dihydropyridine receptors, ryanodine receptors and calsequestrin, which serve as voltage sensor, Ca2+ release channel, and Ca2+ storage protein, respectively. Nevertheless, a number of additional proteins have been shown to be essential both for the structural formation of the machinery involved in excitation–contraction coupling and for its fine tuning. In this review we discuss the functional role of minor sarcoplasmic reticulum protein components. The definition of their roles in excitation–contraction coupling is important in order to understand how mutations in genes involved in Ca2+ signalling cause neuromuscular disorders. PMID:19403606

  2. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    赵文; 姜志胜; 倪菊华; 陈光慧; 刘乃奎; 汤健; 贾弘褆; 唐朝枢

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sar-coplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  3. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  4. Taurine protects cardiac contractility in killifish, Fundulus heteroclitus, by enhancing sarcoplasmic reticular Ca(2+) cycling.

    Science.gov (United States)

    Henry, Elenor F; MacCormack, Tyson J

    2017-05-23

    Intracellular taurine is abundant in many animals and it influences an array of physiological processes, including osmoregulation, metabolism, and cardiac contractility. Taurine is an important osmolyte in teleost hearts, but its role in stress tolerance, cardiac metabolism, and contractility has not been assessed. The goal of this study was to determine if ventricular taurine concentration changes in response to environmental stress and to characterize its influence on contractility. Cardiac taurine concentrations varied in killifish (Fundulus heteroclitus) but were generally maintained following acute environmental challenges. In isometrically contracting ventricular strips, supplemental taurine (40 mmol L(-1)) protected peak tension development (F max) at high stimulation frequencies, an effect abolished by treatment with ryanodine, a blocker of sarcoplasmic reticulum Ca(2+) release. In the presence of ryanodine, taurine-treated preparations were also better able to maintain F max at supraphysiological extracellular Ca(2+) levels, but a prior anoxia exposure abolished this effect. Taurine had no impact on basal F max during or after anoxia, but it provided additive protection to high-frequency contractility post-anoxia. Tissue oxygen consumption and extracellular glucose utilization were unaffected by taurine in non-contracting preparations, indicating that it does not impact energy metabolism. Overall, the results suggest that cardiac taurine levels are well maintained on acute time scales in this highly stress-tolerant species. Supplemental taurine has no effect on aerobic metabolism in vitro, but it significantly improved cardiac contractility in a manner dependent upon sarcoplasmic reticulum Ca(2+) cycling. The data indicate that taurine likely plays an important role in the regulation of cardiac performance in teleosts.

  5. Niflumic acid differentially modulates two types of skeletal ryanodine-sensitive Ca(2+)-release channels.

    Science.gov (United States)

    Oba, T

    1997-11-01

    The effects of niflumic acid on ryanodine receptors (RyRs) of frog skeletal muscle were studied by incorporating sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. Frog muscle had two distinct types of RyRs in the SR: one showed a bell-shaped channel activation curve against cytoplasmic Ca2+ or niflumic acid, and its mean open probability (Po) was increased by perchlorate at 20-30 mM (termed "alpha-like" RyR); the other showed a sigmoidal activation curve against Ca2+ or niflumic acid, with no effect on perchlorate (termed "beta-like" RyR). The unitary conductance and reversal potential of both channel types were unaffected after exposure to niflumic acid when clamped at 0 mV. When clamped at more positive potentials, the beta-like RyR channel rectified this, increasing the unitary current. Treatment with niflumic acid did not inhibit the response of both channels to Ca2+ release channel modulators such as caffeine, ryanodine, and ruthenium red. The different effects of niflumic acid on Po and the unitary current amplitude in both types of channels may be attributable to the lack or the presence of inactivation sites and/or distinct responses to agonists.

  6. Targeting and retention of type 1 ryanodine receptors to the endoplasmic reticulum.

    Science.gov (United States)

    Meur, Gargi; Parker, Andrew K T; Gergely, Fanni V; Taylor, Colin W

    2007-08-10

    Most ryanodine receptors and their relatives, inositol 1,4,5-trisphosphate receptors, are expressed in the sarcoplasmic or endoplasmic reticulum (ER), where they mediate Ca(2+) release. We expressed fragments of ryanodine receptor type 1 (RyR1) in COS cells alone or fused to intercellular adhesion molecule-1 (ICAM-1), each tagged with yellow fluorescent protein, and used confocal imaging and glycoprotein analysis to identify the determinants of ER targeting and retention. Single transmembrane domains (TMD) of RyR1 taken from the first (TMD1-TMD2) or last (TMD5-TMD6) pair were expressed in the ER membrane. TMD3-TMD4 was expressed in the outer mitochondrial membrane. The TMD outer pairs (TMD1-TMD2 and TMD5-TMD6) retained ICAM-1, a plasma membrane-targeted protein, within the ER membrane. TMD1 alone provided a strong ER retention signal and TMD6 a weaker signal, but the other single TMD were unable to retain ICAM-1 in the ER. We conclude that TMD1 provides the first and sufficient signal for ER targeting of RyR1. The TMD outer pairs include redundant ER retention signals, with TMD1 providing the strongest signal.

  7. The mitochondrial ryanodine receptor in rat heart: a pharmaco-kinetic profile.

    Science.gov (United States)

    Altschafl, Beth A; Beutner, Gisela; Sharma, Virendra K; Sheu, Shey-Shing; Valdivia, Héctor H

    2007-07-01

    A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca(2+) fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs(+)-conducting, Ca(2+)-sensitive, large conductance (500-800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca(2+) increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx(a)), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca(2+) dependence of [(3)H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes.

  8. Activation of cardiac ryanodine receptors by cardiac glycosides.

    Science.gov (United States)

    Sagawa, Toshio; Sagawa, Kazuko; Kelly, James E; Tsushima, Robert G; Wasserstrom, J Andrew

    2002-03-01

    This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.

  9. Dependency of calcium alternans on ryanodine receptor refractoriness.

    Directory of Open Access Journals (Sweden)

    Enric Alvarez-Lacalle

    Full Text Available BACKGROUND: Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR. However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2 remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: To investigate how ryanodine receptor (RyR2 refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s. CONCLUSIONS/SIGNIFICANCE: We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.

  10. Upregulation of the CaV 1.1-ryanodine receptor complex in a rat model of critical illness myopathy.

    Science.gov (United States)

    Kraner, Susan D; Wang, Qingbo; Novak, Kevin R; Cheng, Dongmei; Cool, David R; Peng, Junmin; Rich, Mark M

    2011-06-01

    The processes that trigger severe muscle atrophy and loss of myosin in critical illness myopathy (CIM) are poorly understood. It has been reported that muscle disuse alters Ca(2+) handling by the sarcoplasmic reticulum. Since inactivity is an important contributor to CIM, this finding raises the possibility that elevated levels of the proteins involved in Ca(2+) handling might contribute to development of CIM. CIM was induced in 3- to 5-mo-old rats by sciatic nerve lesion and infusion of dexamethasone for 1 wk. Western blot analysis revealed increased levels of ryanodine receptor (RYR) isoforms-1 and -2 as well as the dihydropyridine receptor/voltage-gated calcium channel type 1.1 (DHPR/Ca(V) 1.1). Immunostaining revealed a subset of fibers with elevation of RYR1 and Ca(V) 1.1 that had severe atrophy and disorganization of sarcomeres. These findings suggest increased Ca(2+) release from the sarcoplasmic reticulum may be an important contributor to development of CIM. To assess the endogenous functional effects of increased intracellular Ca(2+) in CIM, proteolysis of α-fodrin, a well-known target substrate of Ca(2+)-activated proteases, was measured and found to be 50% greater in CIM. There was also selective degradation of myosin heavy chain relative to actin in CIM muscle. Taken together, our findings suggest that increased Ca(2+) release from the sarcoplasmic reticulum may contribute to pathology in CIM.

  11. The I4895T Mutation in the Type 1 Ryanodine Receptor Induces Fiber-Type Specific Alterations in Skeletal Muscle that Mimic Premature Aging

    OpenAIRE

    2010-01-01

    The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a non leaky channel and excitation-contraction uncoupling. We characterized age- and fiber type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca2+ release in heterozygous Ryr1I4895T/WT (IT/+) knock-in mice on a mixed genetic backgrou...

  12. Decavanadate interactions with sarcoplasmic reticulum calcium pump

    OpenAIRE

    2007-01-01

    Although not stable, once formed, decameric vanadate (V10) disintegration is in general slow enough to allow the study of its effects even in the micromolar range. Besides, it may become inaccessible to decomposition due to their specific interaction upon target proteins such as the Ca2+-ATPase from sarcoplasmic reticulum (SR). Characterization of the vanadate solutions and interactions with compounds containing phosphate as well as with the SR Ca2+-ATPase was analysed by 51V NMR spectr...

  13. Dihydropyridine receptors actively control gating of ryanodine receptors in resting mouse skeletal muscle fibres

    Science.gov (United States)

    Robin, Gaëlle; Allard, Bruno

    2012-01-01

    Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) in response to depolarization of the muscle membrane. Depolarization is known to elicit a conformational change of the dihydropyridine receptor (DHPR) in the tubular membrane that controls in a time- and voltage-dependent manner the opening of the ryanodine receptor (RyR), the SR Ca2+ release channel. At rest, it is assumed that RyRs are kept in a closed state imposed by the repressive action of DHPRs; however, a direct control of the RyR gating by the DHPR has up to now never been demonstrated in resting adult muscle. In this study, we monitored slow changes in SR Ca2+ content using the Ca2+ indicator fluo-5N loaded in the SR of voltage-clamped mouse muscle fibres. We first show that external Ca2+ removal induced a reversible SR Ca2+ efflux at −80 mV and prevented SR Ca2+ refilling following depolarization-evoked SR Ca2+ depletion. The dihydropyridine compound nifedipine induced similar effects. The rate of SR Ca2+ efflux was also shown to be controlled in a time- and voltage-dependent manner within a membrane potential range more negative than −50 mV. Finally, intracellular addition of ryanodine produced an irreversible SR Ca2+ efflux and kept the SR in a highly depleted state following depolarization-evoked SR Ca2+ depletion. The fact that resting SR Ca2+ efflux is modulated by conformational changes of DHPRs induced by external Ca2+, nifedipine and voltage demonstrates that DHPRs exert an active control on gating of RyRs in resting skeletal muscle. PMID:23006480

  14. Cardiac ryanodine receptor in metabolic syndrome: is JTV519 (K201 future therapy?

    Directory of Open Access Journals (Sweden)

    Dincer UD

    2012-04-01

    Full Text Available U Deniz DincerDepartment of Pharmacology, Ufuk University School of Medicine. Mevlana Bulvari, Balgat, Ankara, TurkeyAbstract: Metabolic syndrome is characterized by a combination of obesity, hypertension, insulin resistance, dyslipidemia, and impaired glucose tolerance. This multifaceted syndrome is often accompanied by a hyperdynamic circulatory state characterized by increased blood pressure, total blood volume, cardiac output, and metabolic tissue demand. Experimental, epidemiological, and clinical studies have demonstrated that patients with metabolic syndrome have significantly elevated cardiovascular morbidity and mortality rates. One of the main and frequent complications seen in metabolic syndrome is cardiovascular disease. The primary endpoints of cardiometabolic risk are coronary and peripheral arterial disease, myocardial infarction, congestive heart failure, arrhythmia, and stroke. Alterations in expression and/or functioning of several key proteins involved in regulating and maintaining ionic homeostasis can cause cardiac disturbances. One such group of proteins is known as ryanodine receptors (intracellular calcium release channels, which are the major channels through which Ca2+ ions leave the sarcoplasmic reticulum, leading to cardiac muscle contraction. The economic cost of metabolic syndrome and its associated complications has a significant effect on health care budgets. Improvements in body weight, blood lipid profile, and hyperglycemia can reduce cardiometabolic risk. However, constant hyperadrenergic stimulation still contributes to the burden of disease. Normalization of the hyperdynamic circulatory state with conventional therapies is the most reasonable therapeutic strategy to date. JTV519 (K201 is a newly developed 1,4-benzothiazepine drug with antiarrhythmic and cardioprotective properties. It appears to be very effective in not only preventing but also in reversing the characteristic myocardial changes and preventing

  15. Inhibition of CaMKII does not attenuate cardiac hypertrophy in mice with dysfunctional ryanodine receptor.

    Directory of Open Access Journals (Sweden)

    Asima Chakraborty

    Full Text Available In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM and by phosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII. Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2ADA impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca2+ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2ADA/ADA mice.

  16. Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Suárez-Isla, B. A.; Irribarra, V.; Oberhauser, A.; Larralde, L.; Bull, R.; Hidalgo, C.; Jaimovich, E.

    1988-01-01

    Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles. These results demonstrate the presence of calcium channels in the sarcoplasmic reticulum from frog skeletal muscle with a pharmacological profile consistent with a role in excitation contraction coupling and with the hypothesis that inositol ( 1,4,5)-trisphosphate is a physiological agonist in this process. PMID:2852037

  17. Quantitative Measurement of Ca2+ in the Sarcoplasmic Reticulum Lumen of Mammalian Skeletal Muscle

    Science.gov (United States)

    Ziman, Andrew P.; Ward, Christopher W.; Rodney, George G.; Lederer, W. Jonathan; Bloch, Robert J.

    2010-01-01

    Skeletal muscle stores Ca2+ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca2+ in the SR ([Ca2+]SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca2+]SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca2+]SR, free is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca2+]SR, free to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca2+ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca2+ and for future studies of the role of SR Ca2+ in healthy and diseased mammalian muscle. PMID:20959112

  18. Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles.

    Science.gov (United States)

    Kaasik, A; Minajeva, A; Paju, K; Eimre, M; Seppet, E K

    1997-11-01

    The present study was undertaken to compare the effects of hypothyroidism and hyperthyroidism on sarcoplasmic reticulum (SR) Ca(2+)-pump activity, together with assessment of the functional role of SR in providing activator Ca2+ under these altered thyroid states. In response to a shift from hypothyroid to hyperthyroid state, a 10 fold and 2 fold increase in SR Ca(2+)-pump activity in atria and ventricles, respectively, were observed. This was associated with the 8-9 fold increases in atrial contractility (+dT/dt) and relaxation (-dT/dt), but only with a 3-4 fold increase in their ventricular counterparts. Also, the recirculation fraction of activator Ca2+ (RFA) increased to a far greater extent in atria (4 fold) than in papillary muscles, and the relative increment in inhibition of developed tension by ryanodine became 3 times larger in atria than in papillary muscles. A positive force-frequency relationship (FFR) was observed in hypothyroid atria, whereas the hyperthyroid atria, hypothyroid and hyperthyroid papillary muscles showed a negative FFR. These results suggest the greater role of transsarcolemmal (SL) Ca2+ and smaller role of SR Ca2+ in activating contraction in hypothyroid atria compared to other preparations. Thyroid hormones decrease the contribution of SL and increase that of SR in providing activator Ca2+ to the greater extent in atria than in ventricles. This effect of thyroid hormones is based on larger stimulation of SR Ca(2+)-pump in atria compared to ventricles.

  19. Cylindrical Spirals in Skeletal Muscles Originate From the Longitudinal Sarcoplasmic Reticulum.

    Science.gov (United States)

    Xu, Jing-Wen; Liu, Fu-Chen; Li, Wei; Zhao, Yu-Ying; Zhao, Dan-Dan; Luo, Yue-Bei; Lu, Jian-Qiang; Yan, Chuan-Zhu

    2016-02-01

    Cylindrical spirals (CSs) are rare but distinct subsarcolemmal accumulations in skeletal muscle fibers. To date, CSs have been reported in only 16 patients with a variety of neuromuscular conditions. The origin and composition of CSs are unknown, although there are some morphologic similarities between CSs and tubular aggregates (TAs). To clarify the nature of CSs, we characterized the sarcoplasmic reticulum (SR) and other intracellular membrane system proteins in CSs of muscle biopsies from 2 adult Chinese siblings. Immunohistochemical studies revealed subsarcolemmal immunoreactivity for sarco/endoplasmic reticulum Ca2þ-ATPase 1 (SERCA 1) in the longitudinal SR, but no immunoreactivity for calsequestrin in the terminal cisternae or type 1 ryanodine receptor (RYR1) in the junctional SR. Muscles biopsied from 2 patients with TAs showed immunoreactivity not only for SERCA1 but also for other SR proteins, including calsequestrin and RYR1. CSs exhibited no immunoreactivity for the Golgi apparatus marker GM130, the nuclear membrane emerin, desmin, the autophagosome marker LC3, the lysosomal membrane marker LAMP2, dystrophin, or myosin. Our results suggest CSs may originate only from the longitudinal SR, whereas TAs are composed of both the junctional and longitudinal SR. Immunochemical staining with antibodies against calsequestrin and RYR1 help to distinguish these 2 pathological alterations.

  20. Alteration of Sarcoplasmic Reticulum Ca2+ Release in Skeletal Muscle from Calpain 3-Deficient Mice

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    Govindan Dayanithi

    2009-01-01

    Full Text Available Mutations of Ca2+-activated proteases (calpains cause muscular dystrophies. Nevertheless, the specific role of calpains in Ca2+ signalling during the onset of dystrophies remains unclear. We investigated Ca2+ handling in skeletal cells from calpain 3-deficient mice. [Ca2+]i responses to caffeine, a ryanodine receptor (RyR agonist, were decreased in −/− myotubes and absent in −/− myoblasts. The −/− myotubes displayed smaller amplitudes of the Ca2+ transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca2+ channels (LCC suppressed the caffeine-induced [Ca2+]i responses in −/− myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR Ca2+ release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca2+ signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.

  1. Bioactive electrospun fish sarcoplasmic proteins as a drug delivery system

    DEFF Research Database (Denmark)

    Stephansen, Karen; Chronakis, Ioannis S.; Jessen, Flemming

    2014-01-01

    Nano-microfibers were made from cod (Gadus morhua) sarcoplasmic proteins (FSP) (Mwelectrospinning technique. The FSP fibers were studied by scanning electron microscopy, and thefiber morphology was found to be strongly dependent on FSP concentration. Interestingly, the FSP...

  2. Sarcoplasmic phospholamban protein is involved in the mechanisms of postresuscitation myocardial dysfunction and the cardioprotective effect of nitrite during resuscitation.

    Directory of Open Access Journals (Sweden)

    Yu Huang

    Full Text Available OBJECTIVES: Sarcoplasmic reticulum (SR Ca(2+-handling proteins play an important role in myocardial dysfunction after acute ischemia/reperfusion injury. We hypothesized that nitrite would improve postresuscitation myocardial dysfunction by increasing nitric oxide (NO generation and that the mechanism of this protection is related to the modulation of SR Ca(2+-handling proteins. METHODS: We conducted a randomized prospective animal study using male Sprague-Dawley rats. Cardiac arrest was induced by intravenous bolus of potassium chloride (40 µg/g. Nitrite (1.2 nmol/g or placebo was administered when chest compression was started. No cardiac arrest was induced in the sham group. Hemodynamic parameters were monitored invasively for 90 minutes after the return of spontaneous circulation (ROSC. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after ROSC. RESULTS: Myocardial function was significantly impaired in the nitrite and placebo groups after resuscitation, whereas cardiac function (i.e., ejection fraction and fractional shortening was significantly greater in the nitrite group than in the placebo group. Nitrite administration increased the level of nitric oxide in the myocardium 5 min after resuscitation compared to the other two groups. The levels of phosphorylated phospholamban (PLB were decreased after resuscitation, and nitrite increased the phosphorylation of phospholamban compared to the placebo. No significant differences were found in the expression of sarcoplasmic reticulum Ca(2+ ATPase (SERCA2a and ryanodine receptors (RyRs. CONCLUSIONS: postresuscitation myocardial dysfunction is associated with the impairment of PLB phosphorylation. Nitrite administered during resuscitation improves postresuscitation myocardial dysfunction by preserving phosphorylated PLB protein during resuscitation.

  3. CaMKII Regulation of Cardiac Ryanodine Receptors and Inositol Triphosphate Receptors

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    Emmanuel eCamors

    2014-05-01

    Full Text Available Ryanodine receptors (RyRs and inositol triphosphate receptors (InsP3Rs are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca2+ signals, triggering muscle contraction and oscillatory Ca2+ waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR, and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and posttranslational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

  4. Calpains and proteasomes mediate degradation of ryanodine receptors in a model of cardiac ischemic reperfusion.

    Science.gov (United States)

    Pedrozo, Zully; Sánchez, Gina; Torrealba, Natalia; Valenzuela, Rodrigo; Fernández, Carolina; Hidalgo, Cecilia; Lavandero, Sergio; Donoso, Paulina

    2010-03-01

    Type-2 ryanodine receptors (RyR2)--the calcium release channels of cardiac sarcoplasmic reticulum--have a central role in cardiac excitation-contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems--calpains, the proteasome and autophagy--on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8h of ischemia followed by 16h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia.

  5. Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

    Science.gov (United States)

    Schuster, Frank; Müller, Rainer; Hartung, Edmund; Roewer, Norbert; Anetseder, Martin

    2005-01-01

    Background Malignant hyperthermia (MH) is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA) enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle. Methods With informed consent, surplus muscle bundles of 7 MHS (susceptible), 7 MHE (equivocal) and 16 MHN (non-susceptible) classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA) 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM) and halothane (0.11; 0.22; 0.44; 0.66 mM) was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles. Conclusion Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia. PMID:15946384

  6. Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals

    Directory of Open Access Journals (Sweden)

    Roewer Norbert

    2005-06-01

    Full Text Available Abstract Background Malignant hyperthermia (MH is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle. Methods With informed consent, surplus muscle bundles of 7 MHS (susceptible, 7 MHE (equivocal and 16 MHN (non-susceptible classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM and halothane (0.11; 0.22; 0.44; 0.66 mM was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p Results Initial length, weight, maximum twitch height, predrug resting tension and predrug twitch height of muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles. Conclusion Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia.

  7. Functional properties of human embryonic stem cell-derived cardiomyocytes: intracellular Ca2+ handling and the role of sarcoplasmic reticulum in the contraction.

    Science.gov (United States)

    Dolnikov, Katya; Shilkrut, Mark; Zeevi-Levin, Naama; Gerecht-Nir, Sharon; Amit, Michal; Danon, Asaf; Itskovitz-Eldor, Joseph; Binah, Ofer

    2006-02-01

    Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC-CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+-handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC-CMs displayed temporally related [Ca2+]i transients and contractions, negative force-frequency relations, and lack of post-rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage-dependent Ca2+ current is present in hESC-CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC-CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC-CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.

  8. Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres.

    Science.gov (United States)

    Coonan, J R; Lamb, G D

    1998-04-01

    The effect of intracellular Cl- on Ca2+ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg2+] and [ATP] and sarcoplasmic reticulum (SR) Ca2+ loading. Both in rat and toad fibres, the presence of 20 mM Cl- in the myoplasm increased Ca2+ leakage from the SR at pCa (i.e. -log10 [Ca2+]) 6.7, but not at pCa 8. Ca2+ uptake was not significantly affected by the presence of Cl-. This Ca2+-dependent effect of Cl- on Ca2+ leakage was most likely due to a direct action on the ryanodine receptor/Ca2+ release channel, and could influence channel sensitivity and the resting [Ca2+] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl- to the myoplasm caused a 40-50% reduction in Ca2+ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl- induces a potential across the SR (lumen negative) which might reduce Ca2+ release via several different mechanisms.

  9. 3D Mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle cryo-EM.

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    Alex Perálvarez-Marín

    Full Text Available The type 1 skeletal muscle ryanodine receptor (RyR1 is principally responsible for Ca(2+ release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208 in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.

  10. Biophysical adaptation of the theory of photo-induced phase transition: model of cooperative gating of cardiac ryanodine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Moskvin, A S [Ural State University, Ekaterinburg, 620083 (Russian Federation); Philipiev, M P [Ural State University, Ekaterinburg, 620083 (Russian Federation); Solovyova, O E [Ural State University, Ekaterinburg, 620083 (Russian Federation); Markhasin, V S [Institute of Immunology and Physiology, Ekaterinburg, 620219 (Russian Federation)

    2005-01-01

    Theory of photo-induced phase transitions has been adapted to describe the cooperative dynamics of the lattice of ryanodine receptors/channels (RyR) in cardiac muscle which regulate the release of the intracellular activator calcium from calcium stores in the sarcoplasmic reticulum (SR) by a process of Ca{sup 2+}-induced Ca{sup 2+} release (CICR). We introduce two main degrees of freedom for RyR channel, fast electronic and slow conformational ones. The RyR lattice response to the L-type channel triggering evolves due to a nucleation process with a step-by-step domino-like opening of RyR channels. Typical mode of RyR lattice functioning in a CICR process implies the fractional release with a robust termination due to the depletion of SR with a respective change in effective conformational strain. The SR overload leads to an unconventional auto-oscillation regime with a spontaneous calcium release. The model is believed to consistently describe the main features of CICR, that is its gradedness, coupled gating, irreversibility, inactivation/adaptation, and spark termination.

  11. Role of Ryanodine Receptor Subtypes in Initiation and Formation of Calcium Sparks in Arterial Smooth Muscle: Comparison with Striated Muscle

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    Maik Gollasch

    2009-01-01

    Full Text Available Calcium sparks represent local, rapid, and transient calcium release events from a cluster of ryanodine receptors (RyRs in the sarcoplasmic reticulum. In arterial smooth muscle cells (SMCs, calcium sparks activate calcium-dependent potassium channels causing decrease in the global intracellular [Ca2+] and oppose vasoconstriction. This is in contrast to cardiac and skeletal muscle, where spatial and temporal summation of calcium sparks leads to global increases in intracellular [Ca2+] and myocyte contraction. We summarize the present data on local RyR calcium signaling in arterial SMCs in comparison to striated muscle and muscle-specific differences in coupling between L-type calcium channels and RyRs. Accordingly, arterial SMC Cav1.2 L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux though RyRs. Downregulation of RyR2 up to a certain degree is compensated by increased SR calcium content to normalize calcium sparks. This indirect coupling between Cav1.2 and RyR in arterial SMCs is opposite to striated muscle, where triggering of calcium sparks is controlled by rapid and direct cross-talk between Cav1.1/Cav1.2 L-type channels and RyRs. We discuss the role of RyR isoforms in initiation and formation of calcium sparks in SMCs and their possible molecular binding partners and regulators, which differ compared to striated muscle.

  12. Crystal structures of ryanodine receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant.

    Science.gov (United States)

    Yuchi, Zhiguang; Yuen, Siobhan M Wong King; Lau, Kelvin; Underhill, Ainsley Q; Cornea, Razvan L; Fessenden, James D; Van Petegem, Filip

    2015-08-06

    Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca(2+)-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2-1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding.

  13. Modeling CaMKII-mediated regulation of L-type Ca2+ channels and ryanodine receptors in the heart

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    Joseph L Greenstein

    2014-04-01

    Full Text Available Excitation-contraction coupling (ECC in the cardiac myocyte is mediated by a number of highly integrated mechanisms of intracellular Ca2+ transport. Voltage- and Ca2+-dependent L-type Ca2+ channels (LCCs allow for Ca2+ entry into the myocyte, which then binds to nearby ryanodine receptors (RyRs and triggers Ca2+ release from the sarcoplasmic reticulum in a process known as Ca2+-induced Ca2+ release. The highly coordinated Ca2+-mediated interaction between LCCs and RyRs is further regulated by the cardiac isoform of the Ca2+/calmodulin-dependent protein kinase (CaMKII. Because CaMKII targets and modulates the function of many ECC proteins, elucidation of its role in ECC and integrative cellular function is challenging and much insight has been gained through the use of detailed computational models. Multiscale models that can both reconstruct the detailed nature of local signaling events within the cardiac dyad and predict their functional consequences at the level of the whole cell have played an important role in advancing our understanding of CaMKII function in ECC. Here, we review experimentally based models of CaMKII function with a focus on LCC and RyR regulation, and the mechanistic insights that have been gained through their application.

  14. Ryanodine prolongs Ca-currents while suppressing contraction in rat ventricular muscle cells.

    OpenAIRE

    Mitchell, M. R.; Powell, T; Terrar, D. A.; Twist, V. W.

    1984-01-01

    Ryanodine (1 microM) suppressed or abolished contraction in response to step depolarization in voltage-clamped cells isolated from adult rat ventricular myocardium. The step depolarizations evoked the second inward current, which is carried largely by Ca ions under these conditions, and there was little or no change in the amplitude of this current when contraction was reduced or abolished by ryanodine. The effects of ryanodine on contraction were, however, accompanied by a prolongation of th...

  15. Muscle Dysfunction in Androgen Deprivation: Role of Ryanodine Receptor

    Science.gov (United States)

    2015-09-01

    TITLE AND SUBTITLE Muscle Dysfunction in Androgen Deprivation: Role of Ryanodine Receptor 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-13-1...required for muscle contraction . RyR1 is a homotetrameric macromolecular protein complex that includes four RyR1 monomers (565kDa each), the RyR1... muscle physiology experiments). Under a microscope, the tibialis anterior (TA) muscle is cut with micro dissection scissors at the distal insertion

  16. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells.

    Science.gov (United States)

    Stern, Michael D; Maltseva, Larissa A; Juhaszova, Magdalena; Sollott, Steven J; Lakatta, Edward G; Maltsev, Victor A

    2014-05-01

    The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic-mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating

  17. Effects of simvastatin on cardiac performance and expression of sarcoplasmic reticular calcium regulatory proteins in rat heart

    Institute of Scientific and Technical Information of China (English)

    Xia ZHENG; Shen-jiang HU

    2005-01-01

    Aim: To investigate the effect of simvastatin on the cardiac contractile function and the alteration of gene and protein expression of the sarcoplasmic calcium regulatory proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA),phospholamban (PLB), and ryanodine receptor 2 (RyR2) in rat hearts. Methods:Langendorff-perfused rat hearts were subjected to 60-min perfusion with different concentrations of simvastatin (1, 3, 10, 30, or 100 μmol/L), and the parameters of cardiac function such as left ventricular developed pressure (LVDP), +dp/dtmax,and -dp/dtmax were determined. The cultured neonatal rat ventricular cardiomyocytes were incubated with simvastatin (1, 3, 10, 30, and 100 μmol/L) for 1 h or 24 h.The levels of SERCA, PLB, and RyR2 expression were measured by reverse transcription-polymerase chain reaction and Western blot. Cytotoxic effect of simvastatin on ventricular cardiomyocytes was assessed by the MTT colorimetric assay.Results: LVDP, +dp/dtmax, and -dp/dtmax of hearts were increased significantly after treatment with simvastatin 3, 10, and 30 μmol/L. In simvastatin-treated isolated hearts, the levels of mRNA expression of SERCA and RyR2 were elevated compared with the control (P<0.05), while the mRNA expression of PLB did not change. After the cultured neonatal rat ventricular cardiomyocytes were incubated with 3, 10, 30, and 100 μmol/L simvastatin for 1 h, SERCA and RyR2 mRNA expressions of cardiomyocytes rose, but there was no alteration in protein expressions. However, with the elongation of simvastatin treatment to 24 h, the protein expression of SERCA and RyR2 were also elevated. Additionally,simvastatin (1-30 μmol/L) had no influence on cell viability of cultured cardiac myocytes, but simvastatin 100 μmol/L inhibited the cell viability. Conclusion:Simvastatin improved cardiac performance accompanied by the elevation of SERCA and RyR2 gene and protein expression.

  18. Discontinuity of sarcoplasmic reticulum in the mid-sarcomere region in flight muscle of dragonflies.

    Science.gov (United States)

    de Eguileor, M; Valvassori, R; Lanzavecchia, G

    1980-01-01

    The sarcoplasmic reticulum organization of dragonfly flight muscles is analyzed, with particular reference to the doubling existing at H-band level. This doubling could be explained as a consequence of a regular discontinuity in the sarcoplasmic reticulum covering myofibrils. In each sarcomere, two sleeves of the sarcoplasmic reticulum seem to overlap forming a telescopic system which can slide outside each other during the lengthening and shortening movements of the fiber.

  19. Biphasic contractions induced by milrinone at low temperature in ferret ventricular muscle: role of the sarcoplasmic reticulum and transmembrane calcium influx.

    Science.gov (United States)

    Malecot, C O; Bers, D M; Katzung, B G

    1986-08-01

    The effects of milrinone were studied in ferret papillary muscle stimulated at various rates and temperatures from 23 degrees to 36 degrees C. In voltage-clamp experiments, 50 micrograms/ml (0.237 mM) milrinone induced a 2.1-fold increase in calcium current at 28 degrees or 36 degrees C. At 50 micrograms/ml, milrinone transiently increased contractility in all muscles at 28 degrees C, but its steady-state effect was either increased (+50%) or decreased (-24.7%) steady-state twitch amplitude. A negative inotropic effect always occurred below 27 degrees C. Milrinone decreased the total twitch duration and split the twitch into two components (P1 and P2) in the absence of any evidence of aberrant conduction. Increasing milrinone concentration from 50 to 300 micrograms/ml decreased P1 and increased P2. Ryanodine (100 mM) or caffeine (10 mM) suppressed P1. Contractions elicited after 30 seconds of rest were also biphasic in the presence of milrinone, but not in its absence. P2 of post-rest contraction was increased by caffeine or calcium (10 mM) and decreased by cobalt (2 mM) when drugs were applied at the beginning of the rest. Ryanodine and caffeine also suppressed P1 of post-rest contraction. The evidence suggests that P1 may be caused by Ca release from the sarcoplasmic reticulum and P2 by increased Ca influx during the action potential via the calcium channel. It is also suggested that P2 may be present under control conditions, but to a lesser extent, and masked by a large P1.

  20. Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating.

    Science.gov (United States)

    Murayama, Takashi; Kurebayashi, Nagomi; Oba, Toshiharu; Oyamada, Hideto; Oguchi, Katsuji; Sakurai, Takashi; Ogawa, Yasuo

    2011-10-14

    The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

  1. Ryanodine Receptors Selectively Interact with L Type Calcium Channels in Mouse Taste Cells.

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    Michelle R Rebello

    Full Text Available WE REPORTED THAT RYANODINE RECEPTORS ARE EXPRESSED IN TWO DIFFERENT TYPES OF MAMMALIAN PERIPHERAL TASTE RECEPTOR CELLS: Type II and Type III cells. Type II cells lack voltage-gated calcium channels (VGCCs and chemical synapses. In these cells, ryanodine receptors contribute to the taste-evoked calcium signals that are initiated by opening inositol trisphosphate receptors located on internal calcium stores. In Type III cells that do have VGCCs and chemical synapses, ryanodine receptors contribute to the depolarization-dependent calcium influx.The goal of this study was to establish if there was selectivity in the type of VGCC that is associated with the ryanodine receptor in the Type III taste cells or if the ryanodine receptor opens irrespective of the calcium channels involved. We also wished to determine if the ryanodine receptors and VGCCs require a physical linkage to interact or are simply functionally associated with each other. Using calcium imaging and pharmacological inhibitors, we found that ryanodine receptors are selectively associated with L type VGCCs but likely not through a physical linkage.Taste cells are able to undergo calcium induced calcium release through ryanodine receptors to increase the initial calcium influx signal and provide a larger calcium response than would otherwise occur when L type channels are activated in Type III taste cells.

  2. Affi-gel blue treatment simplifies the protein composition of sarcoplasmic reticulum vesicles.

    Science.gov (United States)

    Papp, S; Dux, L; Martonosi, A

    1986-04-01

    Sarcoplasmic reticulum vesicles isolated by conventional techniques usually contain, in addition to the recognized sarcoplasmic reticulum components, several other proteins (phosphorylase, myosin, glyceraldehyde-3-phosphate dehydrogenase, etc.) in variable amounts; these proteins complicate the interpretation of chemical modification data. Incubation of sarcoplasmic reticulum vesicles with Affi-Gel blue particles for 1-4 h at 2 degrees C, followed by sedimentation of the Affi-Gel in a clinical centrifuge, simplifies the protein composition by selective adsorption of the accessory proteins, and improves the consistency of the preparations. The Affi-Gel blue treatment is recommended as part of the standard procedure for the isolation of sarcoplasmic reticulum vesicles.

  3. Reduced junctional Na+/Ca2+-exchanger activity contributes to sarcoplasmic reticulum Ca2+ leak in junctophilin-2-deficient mice

    Science.gov (United States)

    Wang, Wei; Landstrom, Andrew P.; Wang, Qiongling; Munro, Michelle L.; Beavers, David; Ackerman, Michael J.; Soeller, Christian

    2014-01-01

    Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development. PMID:25193470

  4. Simulation of the effect of rogue ryanodine receptors on a calcium wave in ventricular myocytes with heart failure

    Science.gov (United States)

    Lu, Luyao; Xia, Ling; Ye, Xuesong; Cheng, Heping

    2010-06-01

    Calcium homeostasis is considered to be one of the most important factors for the contraction and relaxation of the heart muscle. However, under some pathological conditions, such as heart failure (HF), calcium homeostasis is disordered, and spontaneous waves may occur. In this study, we developed a mathematical model of formation and propagation of a calcium wave based upon a governing system of diffusion-reaction equations presented by Izu et al (2001 Biophys. J. 80 103-20) and integrated non-clustered or 'rogue' ryanodine receptors (rogue RyRs) into a two-dimensional (2D) model of ventricular myocytes isolated from failing hearts in which sarcoplasmic reticulum (SR) Ca2+ pools are partially unloaded. The model was then used to simulate the effect of rogue RyRs on initiation and propagation of the calcium wave in ventricular myocytes with HF. Our simulation results show that rogue RyRs can amplify the diastolic SR Ca2+ leak in the form of Ca2+ quarks, increase the probability of occurrence of spontaneous Ca2+ waves even with smaller SR Ca2+ stores, accelerate Ca2+ wave propagation, and hence lead to delayed afterdepolarizations (DADs) and cardiac arrhythmia in the diseased heart. This investigation suggests that incorporating rogue RyRs in the Ca2+ wave model under HF conditions provides a new view of Ca2+ dynamics that could not be mimicked by adjusting traditional parameters involved in Ca2+ release units and other ion channels, and contributes to understanding the underlying mechanism of HF.

  5. Kinetic characterization of Channa striatus muscle sarcoplasmic and myofibrillar protein hydrolysates.

    Science.gov (United States)

    Ghassem, Masomeh; Fern, See Siau; Said, Mamot; Ali, Zainon Mohd; Ibrahim, Saadiah; Babji, Abdul Salam

    2014-03-01

    This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h with an increase in DH. The major amino acids were glutamic acid (16.6%) in thermolysin- myofibrillar hydrolysate followed by aspartic acid (11.1%) in sarcoplasmic protein fraction with no enzyme treatment and lysine (10%) in thermolysin-myofibrillar hydrolysate. The apparent Michaelis constant of proteinase K was lower than thermolysin for both sarcoplasmic and myofibrillar proteins. However, rate of turnover and enzyme efficiency suggested that sarcoplasmic and myofibrillar proteins are suitable substrates for proteinase K and thermolysin hydrolytic reaction, respectively.

  6. Levamisole and ryanodine receptors (I): A contraction study in Ascaris suum

    Science.gov (United States)

    Robertson, Alan P.; Clark, Cheryl L.; Martin, Richard J.

    2010-01-01

    Cholinergic anthelmintics (like levamisole) are important drugs but resistance with reduced responses by the parasite to these compounds is a concern. There is a need to study and understand mechanisms that affect the amplitude of the responses of parasites to these drugs. In this paper, we study interactions of levamisole and ryanodine receptors on contractions of Ascaris suum body muscle flaps. In our second paper, we extend these observations to examine electrophysiological interactions of levamisole, ryanodine receptors (RyRs) and AF2. We report that the maximum force of contraction, gmax, was dependent on the extracellular concentration of calcium but the levamisole EC50(0.8 μM) was not. The relationship between maximum force of contraction and extracellular calcium was described by the Michaelis-Menten equation with a Km of 1.8 mM. Ryanodine inhibited gmax without effect on EC50; ryanodine inhibited only 44% of the maximum contraction (Ki of 40 nM), revealing a ryanodine-insensitive component in the levamisole excitation-contraction pathway. Dantrolene had the same effect as ryanodine but was less potent. The neuropeptide AF2 (1 μM) decreased the levamisole EC50 to 0.2 μM without effect on gmax; 0.1 μM ryanodine and 100 μM dantrolene, inhibited the gmax of the AF2-potentiated levamisole response. High concentrations of caffeine, 30 mM, produced weak contraction of the body flap preparation. Caffeine behaved like ryanodine in that it inhibited the maximum force of contraction, gmax, without effects on the levamisole EC50. Thus, RyRs play a modulatory role in the levamisole-excitation contraction pathway by affecting the maximum force of contraction without an effect on levamisole EC50. The levamisole-excitation contraction coupling is graded and has at least two pathways: one sensitive to ryanodine and one not. PMID:20064566

  7. Cooked sausage batter cohesiveness as affected by sarcoplasmic proteins.

    Science.gov (United States)

    Farouk, M M; Wieliczko, K; Lim, R; Turnwald, S; Macdonald, G A

    2002-05-01

    In the first trial, m. semitendinosus and m. biceps femoris were held at 0, 10 and 35 °C until they entered rigor, and in the second trial, minced m. semitendinosus was washed in water for 15, 30, 45 or 60 min. The samples from both the trials were then used to make a finely comminuted sausage batter. Soluble sarcoplasmic protein (SSP) levels decreased with increasing rigor temperature (P < 0.05) or washing (P < 0.01). Cooked batter shear stress was not affected by SSP level, but batter shear strain decreased with the decreasing SSP level associated with an increasing rigor temperature (P < 0.05) or washing (P < 0.01). Reducing the SSP content lowered the cook yield (P < 0.05) and emulsion stability (P < 0.01) of the batter from the washed samples compared to that of controls. The results suggest that sarcoplasmic proteins are important in determining the strain values (cohesiveness) of cooked sausage batter.

  8. Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations.

    Science.gov (United States)

    Brini, Marisa; Manni, Sabrina; Pierobon, Nicola; Du, Guo Guang; Sharma, Parveen; MacLennan, David H; Carafoli, Ernesto

    2005-04-15

    Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.

  9. Regulation of the cardiac muscle ryanodine receptor by glutathione transferases.

    Science.gov (United States)

    Dulhunty, Angela F; Hewawasam, Ruwani; Liu, Dan; Casarotto, Marco G; Board, Philip G

    2011-05-01

    Glutathione transferases (GSTs) are generally recognized for their role in phase II detoxification reactions. However, it is becoming increasingly apparent that members of the GST family also have a diverse range of other functions that are, in general, unrelated to detoxification. One such action is a specific inhibition of the cardiac isoform of the ryanodine receptor (RyR2) intracellular Ca(2+) release channel. In this review, we compare functional and physical interactions between members of the GST family, including GSTO1-1, GSTA1-1, and GSTM2-2, with RyR2 and with the skeletal isoform of the ryanodine receptor (RyR1). The active part of the muscle-specific GSTM2-2 is localized to its nonenzymatic C-terminal α-helical bundle, centered around α-helix 6. The GSTM2-2 binding site is in divergent region 3 (DR3 region) of RyR2. The sequence differences between the DR3 regions of RyR1 and RyR2 explain the specificity of the GSTs for one isoform of the protein. GSTM2-2 is one of the few known endogenous inhibitors of the cardiac RyR and is likely to be important in maintaining low RyR2 activity during diastole. We discuss interactions between a nonenzymatic member of the GST structural family, the CLIC-2 (type 2 chloride intracellular channel) protein, which inhibits both RyR1 and RyR2. The possibility that the GST and CLIC2 proteins bind to different sites on the RyR, and that different structures within the GST and CLIC proteins bind to RyR channels, is discussed. We conclude that the C-terminal part of GSTM2-2 may provide the basis of a therapeutic compound for use in cardiac disorders.

  10. Different Involvement of Type 1, 2, and 3 Ryanodine Receptors in Memory Processes

    Science.gov (United States)

    Galeotti, Nicoletta; Quattrone, Alessandro; Vivoli, Elisa; Norcini, Monica; Bartolini, Alessandro; Ghelardini, Carla

    2008-01-01

    The administration of the ryanodine receptor (RyR) agonist 4-Cmc (0.003-9 nmol per mouse intracerebroventricularly [i.c.v.]) ameliorated memory functions, whereas the RyR antagonist ryanodine (0.0001-1 nmol per mouse i.c.v.) induced amnesia in the mouse passive avoidance test. The role of the type 1, 2, and 3 RyR isoforms in memory processes was…

  11. Ryanodine is a positive modulator of acetylcholine receptor gating in cochlear hair cells.

    Science.gov (United States)

    Zorrilla de San Martín, Javier; Ballestero, Jimena; Katz, Eleonora; Elgoyhen, A Belén; Fuchs, Paul A

    2007-12-01

    The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the alpha9alpha10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the alpha9alpha10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 microM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 microM ryanodine. This facilitatory effect on current through the AChR could enhance brief ( approximately 1 s) activation of associated calcium-dependent K(+) (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate alpha9alpha10-mediated responses and for potential inner ear therapeutics based on this interaction.

  12. Interactions of vanadate oligomers with sarcoplasmic reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Aureliano, M; Mdeira, V M

    1994-04-28

    Upon addition of sarcoplasmic reticulum (SR), the line width of tetrameric vanadate signal of 51V-NMR spectra narrowed in the presence of ATP and Ca2+, whereas monomeric vanadate line widths were broadened. Thus, ATP decreases the affinity of the enzyme for tetravanadate whereas it induces the interaction with monomeric vanadate. In the presence of Ca2+ it was observed that tetrameric and decameric vanadate bind to SR ATPase whereas monomeric vanadate only binds to SR when ATP is present. However, decameric vanadate clearly differs from vanadate oligomers present in monovanadate solutions in preventing the accumulation of Ca2+ by sarcoplasmic reticulum (SR) vesicles coupled to ATP hydrolysis. Mg2+ increased the inhibitory effect promoted by decavanadate whereas a slight enhancement of Ca2+ uptake was observed in the presence of monovanadate. For 5 mM Mg2+, a nominal 2 mM vanadium 'decavanadate' solution containing about 190 to 200 microM decameric and less than 100 microM monomeric species depressed the rate of Ca2+ uptake by 50% whereas a nominal 2 mM monovanadate solution containing about 662 microM monomeric, 143 microM dimeric and 252 microM tetrameric species had no effect on the rate of Ca2+ accumulation. However, 2 mM 'decavanadate' inhibits by 75% the SR Ca(2+)-ATPase activity whereas the presence of 2 mM 'monovanadate' produces an inhibitory effect below 50%. Therefore, the Ca:ATP stoichiometry of Ca2+ transport is enhanced by monovanadate. In the presence of oxalate, inhibition of SR Ca(2+)-ATPase activity by these solutions is enhanced to 97% and 86% whereas in the presence of the ionophore lasalocid, the inhibitory values were 87% and 19% for 2 mM decavanadate and 2 mM monovanadate solutions, respectively. Apparently, the increase of vesicular Ca2+ concentration counteracts monovanadate inhibition of SR Ca(2+)-ATPase activity but it does not significantly affect decavanadate inhibition.

  13. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

    Science.gov (United States)

    McGarry, S J; Williams, A J

    1993-04-01

    1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+. Single-channels were characteristically modified to along lasting open, but reduced, conductance state when 100 nM ryanodine was added to the cytosolic side of the channel.5. Activation of the cardiac SR Ca2+-release channel was observed with similar concentrations of digitoxin, however, higher concentrations of ouabain were required to increase PO. In contrast, a steroid which is not positively inotropic, chlormadinone acetate, had no effect on either cardiac or

  14. The reaction of N-(1-pyrene)maleimide with sarcoplasmic reticulum.

    Science.gov (United States)

    Papp, S; Kracke, G; Joshi, N; Martonosi, A

    1986-01-01

    The excimer fluorescence of the adduct of N-(1-pyrene)maleimide (PMI) with the Ca2+-ATPase was proposed as a probe of ATPase-ATPase interactions in sarcoplasmic reticulum (Lüdi and Hasselbach, Eur. J. Biochem., 1983, 130:5-8). We tested this proposition by analyzing the spectral properties and stoichiometry of the adducts of pyrenemaleimide with sarcoplasmic reticulum and with dithiothreitol and by comparing the effects of various detergents on the excimer fluorescence of the two adducts, with their influence on the sedimentation characteristics, ATPase activity, and light scattering of the pyrenemaleimide-labeled sarcoplasmic reticulum. These studies indicate that pyrenemaleimide reacts nearly randomly with several SH groups on the Ca2+-ATPase, and suggest that the observed excimer fluorescence of pyrenemaleimide-labeled sarcoplasmic reticulum may reflect intramolecular phenomena rather than ATPase-ATPase interactions. Further work is required to establish the relative contribution of intra- and intermolecular mechanisms to the excimer fluorescence. PMID:2937461

  15. Kinetic characterization of Channa striatus muscle sarcoplasmic and myofibrillar protein hydrolysates

    OpenAIRE

    Ghassem, Masomeh; Fern, See Siau; Said, Mamot; Ali, Zainon Mohd; Ibrahim, Saadiah; Babji, Abdul Salam

    2011-01-01

    This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h ...

  16. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, K.P.

    1978-01-01

    Light and heavy sarcoplasmic reticulum vesicles isolated from rabbit leg muscle have been used in a study of chloride-induced calcium release. The biochemical and morphological data indicate that light sarcoplasmic reticulum vesicles are derived from the longitudinal reticulum and heavy sarcoplasmic reticulum vesicles are derived from the terminal cisternae of the sarcoplasmic reticulum. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP to amounts greater than 100 nmoles Ca/sup + +/ per mg of protein in less than one minute. Light and heavy sarcoplasmic reticulum vesicles each had a biphasic time course of calcium uptake. The initial uptake was followed by a rapid release after approximately one minute, of 30 to 40% of the accumulated calcium, which was then followed by a slower phase of calcium accumulation. Results indicate that the chloride induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization. The release of calcium from the light SR vesicles is probably due to osmotic swelling and the release of calcium from the heavy SR vesicles is probably due to depolarization.

  17. Ryanodine receptors: physiological function and deregulation in Alzheimer disease.

    Science.gov (United States)

    Del Prete, Dolores; Checler, Frédéric; Chami, Mounia

    2014-06-05

    Perturbed Endoplasmic Reticulum (ER) calcium (Ca2+) homeostasis emerges as a central player in Alzheimer disease (AD). Accordingly, different studies have reported alterations of the expression and the function of Ryanodine Receptors (RyR) in human AD-affected brains, in cells expressing familial AD-linked mutations on the β amyloid precursor protein (βAPP) and presenilins (the catalytic core in γ-secretase complexes cleaving the βAPP, thereby generating amyloid β (Aβ) peptides), as well as in the brain of various transgenic AD mice models. Data converge to suggest that RyR expression and function alteration are associated to AD pathogenesis through the control of: i) βAPP processing and Aβ peptide production, ii) neuronal death; iii) synaptic function; and iv) memory and learning abilities. In this review, we document the network of evidences suggesting that RyR could play a complex dual "compensatory/protective versus pathogenic" role contributing to the setting of histopathological lesions and synaptic deficits that are associated with the disease stages. We also discuss the possible mechanisms underlying RyR expression and function alterations in AD. Finally, we review recent publications showing that drug-targeting blockade of RyR and genetic manipulation of RyR reduces Aβ production, stabilizes synaptic transmission, and prevents learning and memory deficits in various AD mouse models. Chemically-designed RyR "modulators" could therefore be envisioned as new therapeutic compounds able to delay or block the progression of AD.

  18. Fluorescent Probes for Insect Ryanodine Receptors: Candidate Anthranilic Diamides

    Directory of Open Access Journals (Sweden)

    Yi Wang

    2014-04-01

    Full Text Available Diamide insecticides with high efficacy against pests and good environmental safety are broadly applied in crop protection. They act at a poorly-defined site in the very complex ryanodine (Ry receptor (RyR potentially accessible to a fluorescent probe. Two N-propynyl analogs of the major anthranilic diamide insecticides chlorantraniliprole (Chlo and cyantraniliprole (Cyan were accordingly synthesized and converted into two fluorescent ligands by click reaction coupling with 3-azido-7-hydroxy-2H-chromen-2-one. The new diamide analogs and fluorescent ligands were shown to be nearly as potent as Chlo and Cyan in inhibition of [3H]Chlo binding and stimulation of [3H]Ry binding in house fly thoracic muscle RyR. Although the newly synthesized compounds had only moderate activity in insect larvicidal activity assays, their high in vitro potency in a validated insect RyR binding assay encourages further development of fluorescent probes for insect RyRs.

  19. Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca(2+) probes in rat ventricular myocytes.

    Science.gov (United States)

    Pahlavan, Sara; Morad, Marin

    2017-09-01

    The details of cardiac Ca(2+) signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca(2+) probes localized to dyadic junctions. To critically monitor ryanodine receptors' (RyR2) Ca(2+) nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, Kd=150nM, or FKBP-GCaMP6, Kd=240nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80nm). The punctate z-line patterns of FKBP,(2)-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2-3s) to detect Ca(2+) sparks, but the probe was effective in marking where Fluo-4 Ca(2+) sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca(2+) signals that: a) had faster kinetics and activated synchronous with ICa(3) but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca(2+) sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca(2+) in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

    Directory of Open Access Journals (Sweden)

    Jerry Curran

    Full Text Available Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+ leak through ryanodine receptors. Beta-adrenergic (β-AR tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII and the subsequent phosphorylation of the ryanodine receptor. When β-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous Ca(2+ waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+ leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+ leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca(2+ leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis

  1. Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling [v1; ref status: indexed, http://f1000r.es/32q

    Directory of Open Access Journals (Sweden)

    Nicola Fameli

    2014-04-01

    Full Text Available Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca2+ waves. In pulmonary artery smooth muscle cells (PASMCs it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP triggers increases in cytoplasmic Ca2+ via L-SR junctions, in a manner that requires initial Ca2+ release from lysosomes and subsequent Ca2+-induced Ca2+ release (CICR via ryanodine receptor (RyR subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca2+ signal information as input data. Simulations of NAADP-dependent junctional Ca2+ transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca2+ signals and simulated Ca2+ transients within L-SR junctions, we estimate that “trigger zones” comprising 60–100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca2+] such that there is a failure to breach the threshold for CICR via RyR3. L

  2. The sarcoplasmic calcium pump - a most efficient ion translocating system.

    Science.gov (United States)

    Hasselbach, W

    1977-04-21

    In contrast to the sodium-potassium transporting plasma membranes, the sarcoplasmic membranes (SR) are highly specialized structures into which only two major intrinsic proteins, a calcium transporting protein and a calcium binding protein are embedded. The calcium transporting protein is a highly asymmetric molecule. It binds two calcium ions with a very high affinity at its external, and two calcium ions with low affinity at the internal section of the molecule. ATP is bound with high afffinity to an external binding site, inducing a conformational change. When the vesicular membranes are exposed to solutions containing Ca++, Mg++ and ATP, ATP is hydrolyzed and simultaneously calcium ions are translocated from the external medium into the vesicular space. When calcium ions are translocated in the opposite direction, ATP is synthesized. The calcium-ATP ratio for ATP cleavage as well as for ATP synthesis is 2. Thus, the SR membranes can transform reversibly chemical into osmotical energy. Inward and outward movements of calcium ions are relatively slow processes connected with the appearance and disappearance of different phosphorylated intermediates. One phosphorylated intermediate is formed by phosphoryltransfer from ATP when calcium ions are present in the medium. In contrast, when calcium ions are absent from the external medium, two different intermediates can be formed by the incorporation of inorganic phosphate. Only when calcium ions present in the internal space of the vesicles are released, the incorporation of inorganic phosphate gives rise to an intermediate who phosphoryl group can be transferred to ADP.

  3. Effects of ryanodine on cardiac contraction, excitation-contraction coupling and "Treppe" in the conscious dog.

    Science.gov (United States)

    Kalthof, B; Sato, N; Iwase, M; Shen, Y T; Mirsky, I; Patrick, T A; Vatner, S F

    1995-10-01

    The effects of ryanodine on left ventricular (LV) function and hemodynamics were studied in 16 conscious dogs, chronically instrumented for measurements of LV pressures and dimensions. Systemic infusion of ryanodine (0.5-4 micrograms/kg i.v.) resulted in a dose-dependent depression of cardiac contraction. For example, ryanodine, 4 micrograms/kg i.v., decreased LV fractional shortening by 30.5 +/- 4.1%, LV dP/dt by 41.5 +/- 4.0% and Vcfc by 37.8 +/- 4.1%, while increasing the isovolumic relaxation time constant, tau, from 23.1 +/- 1.4 to 34.1 +/- 3.6 ms without a major effect on preload or afterload. Ryanodine also depressed (P Treppe") was significantly enhanced (P Treppe" in the conscious dog under the condition of impaired SR calcium release caused by ryanodine, supports the concept that the classical Bowditch "Treppe" reflects either a state of myocardial depression due to alteration in SR calcium handling, or enhanced availability of trans-sarcolemmal Ca2+ influx. This finding may help to understand the discrepancy in the importance of the "Treppe" between conscious animals and more isolated preparations.

  4. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR,HSR) were isolated from rabbit leg muscle using a combination of differential centrifugation and isopycnic zonal ultracentrifugation. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes whereas the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The sucrose HSR vesicles have an additional morphological feature which appears as membrane projections that resemble the SR feet. The freeze-fracture morphology of either type of SR reveals an asymmetric distribution of intramembraneous particles in the same orientation and distribution as the sarcoplasmic reticulum in vivo. Biochemical studies were made on the content of Ca, Mg, ATPase, and protein of the vesicles and phosphorylation of the vesicles. The biochemical and morphological data indicate that the LSR is derived from the longitudinal sarcoplasmic reticulum and the HSR is derived from the terminal cisternae of the sarcoplasmic reticulum, contains junctional SR membrane and has three unique proteins (calsequestrin, an intrinsic 30,000 dalton protein and a 9000 dalton proteolipid).

  5. Contribution of impaired myofibril and ryanodine receptor function to prolonged low-frequency force depression after in situ stimulation in rat skeletal muscle.

    Science.gov (United States)

    Watanabe, Daiki; Kanzaki, Keita; Kuratani, Mai; Matsunaga, Satoshi; Yanaka, Noriyuki; Wada, Masanobu

    2015-06-01

    The aim of this study was to examine whether prolonged low-frequency force depression (PLFFD) that occurs in situ is the result of decreased myofibrillar Ca(2+) sensitivity and/or reduced sarcoplasmic reticulum (SR) Ca(2+) release. Intact rat gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected 30 min following the cessation of stimulation. Skinned fibre and whole muscle analyses were performed in the superficial region composed exclusively of type IIB fibres. Fatiguing stimulation significantly reduced the ratio of force at low frequency to that at high frequency to 65% in skinned fibres (1 vs. 50 Hz) and 73% in whole muscles (20 vs. 100 Hz). In order to evaluate changes in myofibrillar Ca(2+) sensitivity and ryanodine receptor caffeine sensitivity, skinned fibres were activated in Ca(2+)- and caffeine-containing solutions, respectively. Skinned fibres from fatigued muscles displayed decreased caffeine sensitivity together with increased myofibrillar Ca(2+) sensitivity. Treatment with 2,2'-dithiodipyridine and reduced glutathione induced a smaller increase in myofibrillar Ca(2+)sensitivity in fatigued than in rested fibres. In fatigued muscles, S-glutathionylation of troponin I was increased and submaximal SR Ca(2+) release, induced by 4-chloro-m-cresol, was decreased. These findings suggest that in the early stage of PLFFD that occurs in fast-twitch muscles of exercising animals and humans, S-glutathionylation of troponin I may attenuate PLFFD by increasing myofibrillar Ca(2+) sensitivity and that under such a circumstance, PLFFD may be ascribable to failure of SR Ca(2+) release.

  6. Ryanodine Receptor Phosphorylation by CaMKII Promotes Spontaneous Ca2+ Release Events in a Rodent Model of Early Stage Diabetes: the Arrhythmogenic Substrate

    Science.gov (United States)

    Sommese, Leandro; Valverde, Carlos A; Blanco, Paula; Castro, María Cecilia; Rueda, Omar Velez; Kaetzel, Marcia; Dedman, John; Anderson, Mark E.; Mattiazzi, Alicia; Palomeque, Julieta

    2016-01-01

    Background Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented. Objectives to prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes. Methods-Results Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet -induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Micetreated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenes is in vivo, despite ROS increases. Conclusions RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic

  7. The I4895T mutation in the type 1 ryanodine receptor induces fiber-type specific alterations in skeletal muscle that mimic premature aging.

    Science.gov (United States)

    Boncompagni, Simona; Loy, Ryan E; Dirksen, Robert T; Franzini-Armstrong, Clara

    2010-12-01

    The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a nonleaky channel and excitation-contraction uncoupling. We characterized age-dependent and fiber-type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca(2+) release in heterozygous Ryr1(I4895T/WT) (IT/+) knock-in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial-deficient areas in type I fibers. Electrically evoked Ca(2+) release is significantly reduced in single flexor digitorum brevis (FDB) fibers from young and old IT/+ mice. Structural changes are strongly fiber-type specific, affecting type I and IIB/IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT/+ mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age-dependent changes. The observed functional and structural alterations and their similarity to age-associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT/+ mice on an inbred 129S2/SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties.

  8. Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts.

    Science.gov (United States)

    O-Uchi, Jin; Jhun, Bong Sook; Hurst, Stephen; Bisetto, Sara; Gross, Polina; Chen, Ming; Kettlewell, Sarah; Park, Jongsun; Oyamada, Hideto; Smith, Godfrey L; Murayama, Takashi; Sheu, Shey-Shing

    2013-12-01

    Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca(2+) transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca(2+) elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca(2+)-induced ATP production in cardiac H9c2 myoblasts.

  9. Dissociation of FK506-binding protein 12.6 kD from ryanodine receptor in bronchial smooth muscle cells in airway hyperresponsiveness in asthma.

    Science.gov (United States)

    Du, Ying; Zhao, Jianhong; Li, Xi; Jin, Si; Ma, Wan-Li; Mu, Qing; Xu, Shuxiang; Yang, Jie; Rao, Shanshan; Zhu, Liping; Xin, Jianbao; Cai, Peng-Cheng; Su, Yunchao; Ye, Hong

    2014-02-01

    Airway hyperresponsiveness (AHR) in asthma is predominantly caused by increased sensitivity of bronchial smooth muscle cells (BSMCs) to stimuli. The sarcoplasmic reticulum (SR)-Ca(2+) release channel, known as ryanodine receptor (RyR), mediates the contractive response of BSMCs to stimuli. FK506-binding protein 12.6 kD (FKBP12.6) stabilizes the RyR2 channel in a closed state. However, the interaction of FKBP12.6 with RyR2 in AHR remains unknown. This study examined the interaction of FKBP12.6 with RyR2 in BSMCs in AHR of asthma. The interaction of FKBP12.6 with RyR2 and FKBP12.6 expression was determined in a rat asthma model and in BSMCs treated with inflammatory cytokines. The calcium responses to contractile agonists were determined in BSMCs with overexpression and knockdown of FKBP12.6. Asthmatic serum, IL-5, IL-13, and TNF-α enhance the calcium response of BSMCs to contractile agonists and cause dissociation of FKBP12.6 from RyR2 and a decrease in FKBP12.6 gene expression in BSMCs in culture and in ovalbumin (OVA)-sensitized and -challenged rats. Knockdown of FKBP12.6 in BSMCs causes a decrease in the association of RyR2 with FKBP12.6 and an increase in the calcium response of BSMCs. Overexpression of FKBP12.6 increases the association of FKBP12.6 with RyR2, decreases the calcium response of BSMCs, and normalizes airway responsiveness in OVA-sensitized and -challenged rats. Dissociation of FKBP12.6 from RyR2 in BSMCs is responsible for the increased calcium response contributing to AHR in asthma. Manipulating the interaction of FKBP12.6 with RyR2 might be a novel and useful treatment for asthma.

  10. Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote.

    Science.gov (United States)

    Koulen, P; Ehrlich, B E

    2000-07-01

    Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C(2)C(12) mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.

  11. Levamisole and ryanodine receptors (II): An electrophysiological study in Ascaris suum

    Science.gov (United States)

    Puttachary, Sreekanth; Robertson, Alan P.; Clark, Cheryl L.; Martin, Richard J.

    2010-01-01

    Resistance to antinematodal drugs like levamisole has increased and there is a need to understand what factors affect the responses to these anthelmintics. In our previous study, we examined the role of ryanodine receptors in muscle contraction pathways. Here we have examined interactions of levamisole receptors, ryanodine receptors (RyRs), the excitatory neuropeptide AF2, and coupling to electrophysiological responses. We examined the effects of a brief application of levamisole on Ascaris suum body muscle under current-clamp. The levamisole responses were characterized as an initial primary depolarization, followed by a slow secondary depolarizing response. We examined the effects of AF2 (KHEYLRFamide), 1 μM applied for 2 min. We found that AF2 potentiated the secondary response to levamisole and had no significant effect on the primary depolarization [1]. Further, the reversal potentials observed during the secondary response suggested that more than one ion was involved in producing this potential. AF2 potentiated the secondary response in the presence of 30 μM mecamylamine suggesting the effect was independent of levamisole sensitive acetylcholine receptors. The secondary response, potentiated by AF2, appeared to be dependent on cytoplasmic events triggered by the primary depolarization. Ion-substitution experiments showed that the AF2 potentiated secondary response was dependent on extracellular calcium and chloride suggesting a role for the calcium-activated anion channel. Caffeine mimicked the AF2 secondary response and 0.1 μM ryanodine inhibited it. 1.0 μM ryanodine increased spiking showing that it affected membrane excitability. A model is proposed showing ryanodine receptors mediating effects of AF2 on levamisole responses. PMID:20064567

  12. Reduced sarcoplasmic reticulum content of releasable Ca2+ in rat soleus muscle fibres after eccentric contractions

    DEFF Research Database (Denmark)

    Nielsen, J S; Sahlin, K; Ørtenblad, N

    2007-01-01

    AIM: The purpose was to evaluate the effects of fatiguing eccentric contractions (EC) on calcium (Ca2+) handling properties in mammalian type I muscles. We hypothesized that EC reduces both endogenous sarcoplasmic reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+ sensitivity...

  13. Sub-sarcolemmal swelling of sarcoplasmic reticulum after isometric contractions in rat semimembranosus lateralis muscle

    NARCIS (Netherlands)

    Willems, M.E.T.; Huijing, P.A.J.B.M.; Friden, J.

    1999-01-01

    The decline in isometric force, swelling of sarcoplasmic reticulum and loss of desmin was measured in semimembranosus lateralis muscle of male Wistar rats immediately after a short series of brief (500 ms) maximal isometric contractions. For the active muscle, the series ended below (protocol A) and

  14. Effect of losartan on sarcoplasmic reticulum Ca2+ handing proteins in heart failure rabbit

    Institute of Scientific and Technical Information of China (English)

    姚艳

    2006-01-01

    Objective To investigate the effects of losartan on mRNA expression of myocardial sarcoplasmic reticulum calcium handling proteins (SERCA2, RyR2 and PLB) and the role of which in prevention of chronic heart failure in rabbit. Methods After chronic heart failure was

  15. Expression and Localization of Ryanodine Receptors in the Frog Semicircular Canal

    OpenAIRE

    Paola Perin; Laura Botta; Simona Tritto; Umberto Laforenza

    2012-01-01

    Several experiments suggest an important role for store-released Ca2+ in hair cell organs: drugs targeting IP3 and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flat mounts of the simpler nonsensory regions. RyRs were present ...

  16. Sexual Dimorphism in a Reciprocal Interaction of Ryanodine and IP3 Receptors in the Induction of Hyperalgesic Priming.

    Science.gov (United States)

    Khomula, Eugen V; Ferrari, Luiz F; Araldi, Dionéia; Levine, Jon D

    2017-02-22

    Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP3) receptor inhibitor prevented the induction of priming by ryanodine. For IP3 induced priming, females were also more sensitive. IP3-induced priming was prevented by pretreatment with inhibitors of the sarcoendoplasmic reticulum calcium ATPase and ryanodine receptor. Antisense to EsRα prevented the induction of priming by low-dose IP3 in females. The induction of priming by an EsRα agonist was ryanodine receptor-dependent and prevented by the IP3 antagonist. Thus, an EsRα-dependent bidirectional interaction between endoplasmic reticulum IP3 and ryanodine receptor-mediated calcium signaling is present in the induction of hyperalgesic priming, in females. In cultured male DRG neurons, IP3 (100 μm) potentiated depolarization-induced transients produced by extracellular application of high-potassium solution (20 mm, K20), in nociceptors incubated with β-estradiol. This potentiation of depolarization-induced calcium transients was blocked by the IP3 antagonist, and not observed in the absence of IP3 IP3 potentiation was also blocked by ryanodine receptor antagonist. The application of ryanodine (2 nm), instead of IP3, also potentiated K20-induced calcium transients in the presence of β-estradiol, in an IP3 receptor-dependent manner. Our results point to an EsRα-dependent, reciprocal interaction between IP3 and ryanodine receptors that contributes to sex differences in hyperalgesic priming.SIGNIFICANCE STATEMENT The present study demonstrates a mechanism that plays a role in the marked sexual dimorphism observed in a model of the transition to chronic pain, hyperalgesic priming. This mechanism involves a reciprocal interaction between the endoplasmic

  17. A rapid detection method for the ryanodine receptor 1 (C7360G) mutation in Quarter Horses.

    Science.gov (United States)

    Nieto, J E; Aleman, M

    2009-01-01

    Anesthetic-induced malignant hyperthermia has been documented in Quarter Horses and is caused by a single-point mutation in the ryanodine receptor 1 gene at nucleotide C7360G generating a R2454G amino acid substitution. An accurate, faster molecular test that is less prone to contamination would facilitate screening for the mutation in horses intended for breeding, in those undergoing surgical procedures, and in those with clinical signs compatible with malignant hyperthermia. To report a rapid and accurate method for the detection of the ryanodine receptor 1 C7360G mutation. Eleven diseased, 10 healthy, and 225 randomly selected Quarter Horses. This study included horses with the ryanodine receptor 1 C7360G mutation as detected by gene sequencing. Available genomic and complementary DNA extracted from whole blood, hair or skeletal muscle was used for genetic analysis. Real-time polymerase chain reaction (RT-PCR) melting curve analysis was performed by equine specific primers and 2 hybridization probes (sensor and anchor probes) that contain the site of the mutation. Results from this method were blinded and compared with nucleic acid sequencing for validation. A rapid genotyping assay with fluorescence resonance energy transfer probes and melting curve analysis was accurate (100% agreement, K= 1) for identification of affected horses. The prevalence of the mutation in a random population of Quarter Horses was 1.3%. Malignant hyperthermia in Quarter Horses can be rapidly and accurately detected by RT-PCR melting curve genotyping with hybridization probes.

  18. Functional characterization of the cardiac ryanodine receptor pore-forming region.

    Directory of Open Access Journals (Sweden)

    Joanne Euden

    Full Text Available Ryanodine receptors are homotetrameric intracellular calcium release channels. The efficiency of these channels is underpinned by exceptional rates of cation translocation through the open channel and this is achieved at the expense of the high degree of selectivity characteristic of many other types of channel. Crystallization of prokaryotic potassium channels has provided insights into the structures and mechanisms responsible for ion selection and movement in these channels, however no equivalent structural detail is currently available for ryanodine receptors. Nevertheless both molecular modeling and cryo-electron microscopy have identified the probable pore-forming region (PFR of the ryanodine receptor (RyR and suggest that this region contains structural elements equivalent to those of the PFRs of potassium-selective channels. The aim of the current study was to establish if the isolated putative cardiac RyR (RyR2 PFR could form a functional ion channel. We have expressed and purified the RyR2 PFR and shown that function is retained following reconstitution into planar phospholipid bilayers. Our data provide the first direct experimental evidence to support the proposal that the conduction pathway of RyR2 is formed by structural elements equivalent to those of the potassium channel PFR.

  19. Ryanodine Receptor and Insecticides Targeting at Ryanodine Receptor%鱼尼丁受体及以其为靶标的杀虫剂

    Institute of Scientific and Technical Information of China (English)

    郑雪松; 时立波; 茹李军; 苏建亚

    2012-01-01

    Novel diamide insecticides targeting at ryanodine receptor, which had excellent insecticidal activity and mammal safety, attracted much attention from research and creation institutes of pesticides. The isoforms, structures, gated characteristics and toxicology of the insecticides targeting at ryanodine receptor were reviewed in this paper. The biological profile of chlorantraniliprole and its effects on insect development, reproduction and feeding behaviour were also discussed.%以鱼尼丁受体为靶标的双酰胺类杀虫剂由于其较强的杀虫活性和对哺乳动物安全而成为农药研究创制单位关注的热点.对近些年来在鱼尼丁受体的同工型、高级结构、门控特性及以鱼尼丁受体为靶标药剂毒理学等方面的研究进行了综述,总结了双酰胺类杀虫剂代表药剂氯虫苯甲酰胺对昆虫的致死效应以及对昆虫生长发育、生殖、取食行为等方面的影响.

  20. Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin

    National Research Council Canada - National Science Library

    Giuffrida, M G; Villalta, D; Mistrello, G; Amato, S; Asero, R

    2014-01-01

    .... We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients...

  1. Calcium uptake by sarcoplasmic reticulum in the presence of organophosphorus insecticide methyl-parathion

    Energy Technology Data Exchange (ETDEWEB)

    Blasiak, J. [Lodz Univ. (Poland)

    1995-12-31

    Using an isotope labelling technique it has been shown that an organophosphorus insecticide methyl parathion (0,0-diethyl 0-4-nitrophenyl phosphorothionate) depressed calcium uptake by sarcoplasmic reticulum isolated from rabbit hind leg muscle. The effect was significant for insecticide concentrations of 50 and 100 {mu}M and was dose-dependent. The insecticide exerted a more pronounced effect on calcium uptake in the presence of ATP in the reticulum environment than in the absence of ATP. The inhibitory action of methyl parathion on Ca{sup 2+} accumulation by sarcoplasmic reticulum can cause a rise in myoplasmic free Ca{sup 2+}, the essential prerequisite for contracture activation. Because methyl parathion, as well as other organophosphorus insecticides, is primarily neurotoxic, evidence of non-specific effect could be important for assessing its environmental safety. (author). 20 refs, 2 figs.

  2. Time Course of the Response of Myofibrillar and Sarcoplasmic Protein Metabolism to Unweighting of the Soleus Muscle

    Science.gov (United States)

    Munoz, Kathryn A.; Satarug, Soisungwan; Tischler, Marc E.

    1993-01-01

    Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of H-3-phenylaianine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.

  3. [Calcium transport in sarcoplasmic reticulum in the presence of AR-L 115 BS].

    Science.gov (United States)

    Hasselbach, W

    1981-01-01

    2-[(2-Methoxy-4-methylsulfinyl)phenyl]-1H-imidazo[4,5-b]pyridine (AR-L 115 BS) is a substance with positive inotropic activity which does not influence the activity of the sarcoplasmic calcium pump. It can, therefore, be expected that AR-L 115 BS does not interfere with the distribution and movement of calcium in the resting and active muscle.

  4. Transient kinetics of Ca2+ transport of sarcoplasmic reticulum. A comparison of cardiac and skeletal muscle.

    Science.gov (United States)

    Sumida, M; Wang, T; Mandel, F; Froehlich, J P; Schwartz, A

    1978-12-25

    Current evidence supports similar functions and mechanisms for cardiac sarcoplasmic reticulum (CSR) as for skeletal sarcoplasmic reticulum (SSR). It is thought that the slower relaxation rate of cardiac muscle compared to fast skeletal muscle reflects the lower ATPase activity and calcium transport of CSR. Possible quantitative differences is phosphorylation, dephosphorylation, and calcium transport of the isolated preparations are studied using a quench-flow apparatus. The results show that both CSR and SSR bind calcium tightly in the absence of ATP, and coupling of E approximately P formation and calcium transport occurs in the transient phase of ATP hydrolysis. The rate of phosphorylation (t-1/2 - 10 ms) of sarcoplasmic reticulum (SR) preloaded with calcium is the same for cardiac and skeletal preparations. However, the rates of dissociation of extra vesicular calcium (10 s-1 versus 15 s-1), phosphorylation of calcium-free SR, and dephosphorylation of E approximately P (8 s-1 versus 12 s-1) are lower for CSR than for SSR. By computer simulation, the apparent rate constants associated with the reduced rates of phosphorylation of calcium-free SR were: 12 s-1 for CSR and 63 s-1 for SSR in the presence of high Mg2+. The difference in the rates may be partly responsible for the lower levels of ATPase and calcium transport activity with characterize cardiac muscle preparations.

  5. Molecular transformations in sarcoplasmic reticulum of fast-twitch muscle by electro-stimulation.

    Science.gov (United States)

    Heilmann, C; Pette, D

    1979-02-01

    Chronic electro-stimulation of fast-twitch rabbit muscle with the frequency pattern received by a slow-twitch muscle induces a progressive transformation of the sarcoplasmic reticulum. After 2 days stimulation activities of Ca2+-dependent ATPase and of Ca2+ transport begin to decrease, and are paralleled by a progressive decrease in Ca2+-dependent and Ca2+, Mg2+-dependent phosphoprotein formation, reduced rate of dephosphorylation and a rearrangement of the electrophoretic polypeptide and phosphoprotein patterns. These findings suggest a transformation of the sarcoplasmic reticulum to resemble that of a slow-twitch muscle. This transformation is paralleled by increase in time-to-peak of twitch contraction and half relaxation time and occurs before conversion of the myosin light chain pattern is observed. The parallel time course of changes in contractile properties of stimulated muscle and the molecular and functional properties of the sarcoplasmic reticulum emphasizes the definitive role of the latter in determining the twitch characteristics of fast and slow twitch muscles.

  6. Ryanodine受体间相互作用及其与钙释放功能的关系%Oligomeric interaction between ryanodine receptors: potential role in Ca2+ release

    Institute of Scientific and Technical Information of China (English)

    胡晓芳; 朱培闳; 胡钧

    2006-01-01

    在真核生物和原核生物的生物膜上都存在由同种受体蛋白相互连接在一起形成的紧密二维排列.最近的模型计算表明这种排列方式可能是一种新型信号转导机制的结构基础,相邻受体可通过功能上的耦联优化信号处理性能.Ryanodine受体(ryanodine receptor,RyR)/钙释放通道通常在肌肉的肌浆网膜上形成二维晶格排列,该蛋白成为研究受体二维排列及其生理功能的一个很好的模型.本文综述了近几年在RyR相互作用及其二维排列工作模式和生理功能研究方面的进展,着重介绍了我们实验室利用新方法对RyR相互作用及其调控进行的研究工作.我们研究中发现了RyR功能状态对其相互作用的调控,本文对据此提出的RyR二维排列的"动态耦联模型"及其可能的生理功能进行了详细讨论.%Receptor proteins in both eukaryotic and prokaryotic cells often form regular lattice or array in the membrane. Recent theoretical analyses indicate that such arrays may provide a novel mechanism for receptor signaling regulation in cells. The functional coupling between neighboring receptors could improve the signaling performance. The ryanodine receptors (RyR)/calcium release channels usually form 2-D regular lattice in the endoplasmic/sarcoplasmic reticulum membranes. Thus, RyR is a potentially good model to study the function of receptor 2-D array. In this article, we briefly review recent progresses in this research field, including RyR-RyR interaction, RyR array's function and working mechanisms. The investigations performed by new methods in our laboratory are summarized. We demonstrate that the RyR-RyR interaction is modulated by the functional states of RyRs. Accordingly, the mechanism of "dynamic coupling" of RyR array is proposed. Its possible role in RyR-mediated Ca2+ release is discussed.

  7. Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells.

    Science.gov (United States)

    Ohashi, Ryo; Sakata, Shin-ichi; Naito, Asami; Hirashima, Naohide; Tanaka, Masahiko

    2014-04-01

    Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells.

  8. 精-甘-天冬-丝氨酸对脓毒性休克大鼠心肌肌浆网钙转运的影响%Effects of Arg-Gly-Asp-Ser on Ca2 + transport of myocardial sarcoplasmic reticulum in rat septic shock

    Institute of Scientific and Technical Information of China (English)

    吉勇; 赵明; 齐鹰; 董林旺; 吴立玲; 彭师奇; 苏静怡

    1996-01-01

    To study the effects of Arg-Gly-Asp-Ser (RGDS), a synthetic short peptide of fibrinogen degradation, on the Ca2 + transport function of cardiac sarcoplasmic reticulum in rat septic shock.4 h and 14 h after cecal ligation and puncture (CLP) operation on rats. Highly purified membrane of sarcoplasmic reticulum (SR) was prepared from rat hearts. Assays were made of ATP-dependent Ca2 + uptake by cardiac SR and [3H] ryanodine binding to SR. RESULTS: The initial rate and the capacity of SR Ca2 + uptake were increased by 104 % (P<0.01) and 12 % (P<0.05), respectively, paralleled by an increase in Ca2 +-ATPase activity and a decrease in calcium accumulation of myo- cardium of septic rats, whereas the Bmax and Kd values of Ca2+ activated [3H]ryanodine binding to SR were unaffected after RGDS administration. CONCLUTIONS: The results indicated that RGDS have cardioprotective effects of maintaining Ca2+ homeostasis of cardiac myocytes by enhancing SR Ca2 + uptake in rat septic shock.%探讨一种人工合成的纤维蛋白原降解肽片段RGDS对脓毒性休克大鼠心肌肌浆网钙转运功能的影响.方法:大鼠盲肠结扎穿孔术后4 h和14 h分两次尾静脉注射RGDS 5 μmol·kg-1.制备大鼠心肌肌浆网(SR)膜;测定SR Ca2+摄取和[3H]ryanodine受体结合功能.结果:RGDS组大鼠心肌SR摄Ca2+率及摄Ca2+量分别较休克组提高104%(P<0.01)和12%(P<0.05),而心肌SR钙释放通道-[3H]ryanodine受体结合Bmax和Ka值没有明显变化.同时RGDS还可以减轻休克大鼠心肌组织钙聚积.结论:RGDS提高休克大鼠心肌SR Ca2+摄取功能,维持心肌细胞钙稳态,具有心肌保护作用.

  9. Selective destruction of nigrostriatal dopaminergic neurons does not alter [3H]-ryanodine binding in rat striatum

    Directory of Open Access Journals (Sweden)

    Noël F.

    2000-01-01

    Full Text Available Dopamine nigrostriatal neurons are important for motor control and may contain a particularly dense population of ryanodine receptors involved in the control of dopamine release. To test this hypothesis, we used a classical model of unilateral selective lesion of these neurons in rats based on 6-hydroxydopamine (6-OHDA injection into the substantia nigra. Binding of [3H]-GBR 12935, used as a presynaptic marker since it labels specifically the dopamine uptake complex, was dramatically decreased by 83-100% in striatum homogenates after 6-OHDA lesion. On the contrary, no reduction of [3H]-ryanodine binding was observed. The present data indicate that [3H]-ryanodine binding sites present in rat striatum are not preferentially localized in dopaminergic terminals.

  10. Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.

    Science.gov (United States)

    Wagner, Larry E; Groom, Linda A; Dirksen, Robert T; Yule, David I

    2014-08-01

    In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

  11. Effects of tetrandrine on calcium transport, protein fluorescences and membrane fluidity of sarcoplasmic reticulum.

    Science.gov (United States)

    Chen, L Y; Chen, X; Tian, X L; Yu, X H

    2000-10-01

    To understand whether the molecular mechanism of Tetrandrine (Tet)'s pharmacological effects is concerned with sarcoplasmic reticulum calcium transport so as to be involved in myocardial contractility, we observed the effects of Tet on calcium transport and membrane structure of rabbit skeletal muscle sarcoplasmic reticulum vesicles (SR) and rat cardiac sarcoplasmic reticulum vesicles (CSR). Calcium uptake was monitored with a dual-wavelength spectrophotometer. Protein conformation and fluorescence polarization were measured by fluospectrophotometric method and membrane lipids labelled with fluorescence probes for SR, respectively. 128 micromol l(-1) Tet reduced the initial rate of calcium uptake to 59% of control 6 min after reaction. Tet un-competitively inhibited SR Ca(2+), Mg(2+)-ATPase activity, causing the stoichiometric ratio of SR Ca(2+)/ATP to decrease to 1.43 from 2.0 of control. Inhibitory rates on SR Ca(2+),Mg(2+)-ATPase by Tet were reduced from 60% in the absence of phosphate to 50% in the presence of phosphate and reduced from 92% in 1 mmol l(-1) ATP to 60% in 5 mmol l(-1) ATP. Tet markedly reduced SR intrinsic protein fluorescence, while it slightly decreased the thiol(SH)-modified protein fluorescence of SR labelled with N-(3-pyrene)-maleimide. Tet slightly increased fluorescence polarization in the middle and deep layers of SR membrane lipids labelled with 7- or 12-(9-anthroyloxy) stearic acid (AS) probes, whereas it did not change that of SR labelled with 1, 6-diphenyl-1,3,5-hexatrine (DPH). These results revealed that prevention of SR calcium uptake by Tet was due to inhibition of the SR calcium pump Ca(2+),Mg(2+)-ATPase, changes in spatial conformation of the pumps protein molecules and a decrease in the extent of motion of membrane lipid molecules, thus altering the regulation of [Ca(2+)](i) and myocardial contractility.

  12. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

    OpenAIRE

    1993-01-01

    1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular...

  13. Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes

    DEFF Research Database (Denmark)

    Boutrup Stephansen, Karen; Mattebjerg, Maria Ahlm; Wattjes, Jasper;

    2015-01-01

    Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged...... fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 +/- 3 nm, and the zeta...

  14. Involvement of ryanodine receptors in pacemaker Ca2+ oscillation in murine gastric ICC.

    Science.gov (United States)

    Liu, Hong-Nian; Ohya, Susumu; Wang, Jing; Imaizumi, Yuji; Nakayama, Shinsuke

    2005-03-11

    Using a cell cluster preparation from the stomach smooth muscle tissue of mice, we measured intracellular Ca(2+) oscillations in interstitial cells of Cajal (ICCs) in the presence of nifedipine. Pacemaker [Ca(2+)](i) activity in ICCs was significantly suppressed by caffeine application and restored after washout. Application of either ryanodine or FK-506 terminated the pacemaker [Ca(2+)](i) activity irreversibly. Immunostaining of smooth muscle tissue showed that c-Kit-immunopositive cells (that form network-like structure cells in the myenteric plexus, equivalent to ICCs) clearly express ryanodine receptors (RyR). RT-PCR revealed that ICCs (identified with c-Kit-immunoreactivity) predominantly express type 3 RyR (RyR3). Furthermore, the FK-binding proteins 12 and 12.6, both of which would interact with RyR3, were detected. In conclusion, we provide first evidence for the essential contribution of RyR to generating pacemaker activity in gastric motility. Similar mechanisms might account for spontaneous rhythmicity seen in smooth muscle tissues distributed in the autonomic nervous system.

  15. A novel late-onset axial myopathy associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene

    NARCIS (Netherlands)

    Loseth, S.; Voermans, N.C.; Torbergsen, T.; Lillis, S.; Jonsrud, C.; Lindal, S.; Kamsteeg, E.J.; Lammens, M.M.Y.; Broman, M.; Dekomien, G.; Maddison, P.; Muntoni, F.; Sewry, C.; Radunovic, A.; Visser, M. de; Straub, V.; Engelen, B.G.M. van; Jungbluth, H.

    2013-01-01

    Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are a common cause of inherited neuromuscular disorders and have been associated with a wide clinical spectrum, ranging from various congenital myopathies to the malignant hyperthermia susceptibility (MHS) trait without any associated w

  16. Familial Evaluation in Catecholaminergic Polymorphic Ventricular Tachycardia Disease Penetrance and Expression in Cardiac Ryanodine Receptor Mutation-Carrying Relatives

    NARCIS (Netherlands)

    van der Werf, Christian; Nederend, Ineke; Hofman, Nynke; van Geloven, Nan; Ebink, Corne; Frohn-Mulder, Ingrid M. E.; Alings, A. Marco W.; Bosker, Hans A.; Bracke, Frank A.; van den Heuvel, Freek; Waalewijn, Reinier A.; Bikker, Hennie; van Tintelen, J. Peter; Bhuiyan, Zahurul A.; van den Berg, Maarten P.; Wilde, Arthur A. M.

    2012-01-01

    Background-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome associated with mutations in the cardiac ryanodine receptor gene (RYR2) in the majority of patients. Previous studies of CPVT patients mainly involved probands, so current insight into disease

  17. Vanadate oligomer inhibition of passive and active Ca2+ translocation by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M

    2000-05-30

    'Monovanadate' containing mainly monomeric, dimeric and tetrameric vanadate species or 'decavanadate', containing mainly decameric vanadate species inhibits the passive and the active efflux of Ca2+ through the sarcoplasmic reticulum calcium pump. When the efflux of Ca2+ by sarcoplasmic reticulum vesicles is not associated with ATP synthesis both vanadate solutions inhibit the passive efflux of Ca2+. However, only 'decavanadate' exerts noticeable effects when the efflux of Ca2+ is associated with ATP synthesis being the active efflux of Ca2+ almost completely inhibited by decameric species concentration as low as 40 microM.

  18. Atomic force microscopy study of the rabbit skeletal muscle ryanodine receptors in different functional states

    Institute of Scientific and Technical Information of China (English)

    魏青青; 程晓阳; 陈克樱; 胡钧; 李民乾; 朱培闳

    2002-01-01

    Atomic force microscope was applied to investigate the effect of extrinsic phospholipid on the structure of rabbit skeletal muscle ryanodine receptor/calcium release channel (RyR1). In addition, in the presence of extrinsic phospholipid, the height and elasticity of the RyR1s in different functional states were also measured. The results indicate: (i) most of the RyR1s showed a normal structure only in the presence of extrinsic phospholipid; (ii) treatment of the RyR1s with AMP and Ca2+ together could increase their Young's Modulus but not change their apparent height; (iii) no detectable change in either height or Young's Modulus of the RyR1s appeared, if the RyR1s were treated with other activators or inhibitors.

  19. A de novo novel cardiac ryanodine mutation (Ser4155Tyr) associated with catecholaminergic polymorphic ventricular tachycardia.

    Science.gov (United States)

    Mantziari, Lilian; Vassilikos, Vassilios; Anastasakis, Aris; Kotsaka, Xanthippi; Paraskevaidis, Stelios; Styliadis, Ioannis H; Luria, David

    2013-11-01

    We describe the case of a 14-year-old girl with a history of syncopal episodes triggered by stress or exercise. Catecholaminergic polymorphic ventricular tachycardia was diagnosed with the aid of an implantable loop recorder. The genetic testing of the patient and her family revealed a de novo novel missense mutation (Ser4155Tyr) in the exon 90 of the ryanodine receptor gene. This mutation affects a highly conserved residue (S4155) and results to replacement of serine (S) with tyrosine (Y) leading to change in physical and chemical properties. The girl was treated with an implantable defibrillator, metoprolol and flecainide. Over 1 year of follow-up she had no recurrence of ventricular tachycardia. ©2013 Wiley Periodicals, Inc.

  20. Alterations in mitochondria and sarcoplasmic reticulum from heart and skeletal muscle of horizontally casted primates

    Science.gov (United States)

    Sordahl, L. A.; Stone, H. L.

    1982-01-01

    Horizontally body-casted rhesus monkeys are used as an animal model in order to study the physiological changes known as cardiovascular deconditioning which occur during weightless conditions. No difference was found between the experimental and control animals in heart mitochondrial oxidative phosphorylation which indicates that no apparent changes occurred in the primary energy-producing system of the heart. A marked increase in cytochrome oxidase activity was observed in the casted primate heart mitochondria compared to controls, while a 25% decrease in respiratory substrate-supported calcium uptake was found in casted primate heart mitochondria compared to controls. Sacroplasmic reticulum isolated from the primate hearts revealed marked changes in calcium transport activities. It is concluded that the marked depression in cardiac sarcoplasmic reticulum functions indicates altered calcium homeostasis in the casted-primate heart which could be a factor in cardiovascular deconditioning.

  1. Chemical and Functional Characterization of Sarcoplasmic Proteins from Giant Squid (Dosidicus gigas Mantle

    Directory of Open Access Journals (Sweden)

    Rosa Linda Lopez-Enriquez

    2015-01-01

    Full Text Available Modification of pH and NaCl concentration changed the physicochemical properties of sarcoplasmic proteins (SP from jumbo squid mantle and consequently their functional properties. Better results of emulsifying activity index (EAI and foam capacity (FC were exhibited at pH 11 in NaCl absence due to higher solubility. But better emulsifying stability index (ESI was obtained at pH 11 in 0.5 M NaCl, while, foaming stability (FS was better at pH near to isoelectric point (pI. These results suggest that SP from jumbo squid may be a promising ingredient, whose functional properties can be manipulated by changing pH and NaCl concentration.

  2. Conformational changes in the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum detected using phosphorescence polarization.

    Science.gov (United States)

    Restall, C J; Coke, M; Murray, E K; Chapman, D

    1985-02-28

    The technique of time-averaged phosphorescence has been used to study the interaction of calcium ions and ATP with the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum vesicles. The presence of excess calcium ions was found to cause a 20% decrease in the phosphorescence emission anisotropy. This is interpreted as being due to a conformational change in the protein and is supported by data from time-resolved phosphorescence measurements which also show a lowering of the anisotropy. This change in the decay of the emission anisotropy is associated with only minor changes in the rotational relaxation time of the protein and is again suggestive of a conformational change in the protein. In some cases ATP was also observed to lower the time-averaged phosphorescence anisotropy possibly via an interaction with the low-affinity regulatory site of the protein.

  3. Biochemical and morphological characterization of light and heavy sarcoplasmic reticulum vesicles. Volume I

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, Kevin Peter [Univ. of Rochester, NY (United States)

    1978-01-01

    Light (30 to 32.5% sucrose) and heavy (38.5 to 42% sucrose) sarcoplasmic reticulum vesicles (LSR, HSR) were isolated from rabbit leg muscle. They were then diluted and washed with sucrose or KCl and referred to as sucrose or KCl washed vesicles. Thin-section electron microscopy of LSR vesicles reveals empty vesicles of various sizes and shapes where as the HSR vesicles appear as rounded vesicles of uniform size filled with electron dense material. The LSR consists of predominantly Ca2+ + Mg2+ ATPase (80 to 90%), a small amount of the high affinity Ca binding protein (5%), and a 5000 dalton proteolipid. The sucrose HSR vesicles contain the Ca2+ + Mg2+ ATPase (50%), Calsequestrin (25%), high affinity Ca binding protein (5%), one extrinsic 34,000 dalton protein (3%), one intrinsic 30,000 dalton protein (3%), a 9000 dalton proteolipid, and a 5000 dalton proteolipid. The sucrose--washed HSR vesicles contain greater than three times the calcium content of the sucrose washed LSR vesicles where as the KCl--washed vesicles contain less than 15 nmoles Ca2+ mg of protein each. The light and heavy sarcoplasmic reticulum vesicles were both able to accumulate calcium in the presence of ATP. Exchange of methanesulfonate for chloride resulted in the release of calcium from both the light and heavy SR vesicles. Sucrose causes a slight inhibition of chloride--induced calcium release from the heavy SR vesicles but it greatly reduces the release of calcium from the light SR vesicles. Sodium dantrolene (20 uM) has no effect on the release of calcium from the light SR vesicles but it inhibits the release of calcium from the heavy SR vesicles. The results indicate that the chloride--induced release of calcium may be acting by two mechanisms, osmotic swelling and depolarization.

  4. H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes.

    Science.gov (United States)

    Yu, X; Carroll, S; Rigaud, J L; Inesi, G

    1993-04-01

    The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.

  5. Clinical features and ryanodine receptor type 1 gene mutation analysis in a Chinese family with central core disease.

    Science.gov (United States)

    Chang, Xingzhi; Jin, Yiwen; Zhao, Haijuan; Huang, Qionghui; Wang, Jingmin; Yuan, Yun; Han, Ying; Qin, Jiong

    2013-03-01

    Central core disease is a rare inherited neuromuscular disorder caused by mutations in ryanodine receptor type 1 gene. The clinical phenotype of the disease is highly variable. We report a Chinese pedigree with central core disease confirmed by the gene sequencing. All 3 patients in the family presented with mild proximal limb weakness. The serum level of creatine kinase was normal, and electromyography suggested myogenic changes. The histologic analysis of muscle biopsy showed identical central core lesions in almost all of the muscle fibers in the index case. Exon 90-106 in the C-terminal domain of the ryanodine receptor type 1 gene was amplified using polymerase chain reaction. One heterozygous missense mutation G14678A (Arg4893Gln) in exon 102 was identified in all 3 patients. This is the first report of a familial case of central core disease confirmed by molecular study in mainland China.

  6. Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia.

    Science.gov (United States)

    Parker, Remai; Schiemann, Anja H; Langton, Elaine; Bulger, Terasa; Pollock, Neil; Bjorksten, Andrew; Gillies, Robyn; Hutchinson, David; Roxburgh, Richard; Stowell, Kathryn M

    2017-01-01

    Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

  7. Effect of ruthenium red, a ryanodine receptor antagonist in experimental diabetes induced vascular endothelial dysfunction and associated dementia in rats.

    Science.gov (United States)

    Jain, Swati; Sharma, Bhupesh

    2016-10-01

    Diabetes mellitus is considered as a main risk factor for vascular dementia. In the past, we have reported the induction of vascular dementia by experimental diabetes. This study investigates the efficacy of a ruthenium red, a ryanodine receptor antagonist and pioglitazone in the pharmacological interdiction of pancreatectomy diabetes (PaD) induced vascular endothelial dysfunction and subsequent vascular dementia in rats. Attentional set shifting and Morris water-maze test were used for assessment of learning and memory. Vascular endothelial function, blood brain barrier permeability, serum glucose, serum nitrite/nitrate, oxidative stress (viz. aortic superoxide anion, brain thiobarbituric acid reactive species and brain glutathione), brain calcium and inflammation (myeloperoxidase) were also estimated. PaD rats have shown impairment of endothelial function, blood brain barrier permeability, learning and memory along with an increase in brain inflammation, oxidative stress and calcium. Administration of ruthenium red and pioglitazone has significantly attenuated PaD induced impairment of learning, memory, blood brain barrier permeability, endothelial function and biochemical parameters. It may be concluded that ruthenium red, a ryanodine receptor antagonist and pioglitazone, a PPAR-γ agonist may be considered as potent pharmacological agent for the management of PaD induced endothelial dysfunction and subsequent vascular dementia. Ryanodine receptor may be explored further for their possible benefits in vascular dementia.

  8. Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle.

    Science.gov (United States)

    Morrissette, J; Xu, L; Nelson, A; Meissner, G; Block, B A

    2000-11-01

    Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types.

  9. Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle

    DEFF Research Database (Denmark)

    Frías, J A; Cadefau, J A; Prats, C;

    2005-01-01

    -migration of terminal cisternae and t-tubules from R3 to R4, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural...... damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may......Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit...

  10. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    OpenAIRE

    Pierce, D H; Scarpa, A.; Trentham, D R; Topp, M. R.; Blasie, J K

    1983-01-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane...

  11. Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

    Energy Technology Data Exchange (ETDEWEB)

    Yuchi, Zhiguang; Lau, Kelvin; Van Petegem, Filip (UBC)

    2015-02-09

    Ryanodine Receptors (RyRs) are huge Ca{sup 2+} release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

  12. Malignant hyperthermia associated with ryanodine receptor 1 (C7360G) mutation in Quarter Horses.

    Science.gov (United States)

    Aleman, M; Nieto, J E; Magdesian, K G

    2009-01-01

    Anesthetic-induced malignant hyperthermia (MH) has been documented in Quarter Horses with a single point mutation in the ryanodine receptor 1 gene (RyR1) at nucleotide C7360G, generating a R2454G amino acid substitution. However, there have been no reports of nonanesthetic manifestations of MH in horses with the C7360G mutation. To describe clinical manifestations of Quarter Horses with the C7360G mutation. Eleven Quarter Horses with the RyR1 C7360G mutation. This prospective study included horses with suspected MH, undetermined etiology of sudden death, death within hours of onset of rhabdomyolysis, muscle rigidity, stiffness, intermittent sweating, and persistent increases in serum muscle enzyme activities. Whole blood in EDTA and skeletal muscle were processed for genetic and histochemical analysis. Medical records and pedigrees were collected when available. Both anesthetic- and non-anesthetic-associated myopathic manifestations of MH occurred in halter Quarter Horses with mutation of RyR1. The disease is inherited as an autosomal dominant trait. Clinical and laboratory abnormalities were similar in both forms. Rhabdomyolysis was a common finding in both groups of horses. Skeletal muscle histochemical findings were nonspecific and compatible with a noninflammatory myopathic process. MH is a potentially fatal disease of Quarter Horses that could be triggered by halogenated anesthetics and other nonanesthetic factors that may include exercise, stress, breeding, illnesses, and concurrent myopathies.

  13. Intermolecular failure of L-type Ca2+ channel and ryanodine receptor signaling in hypertrophy.

    Directory of Open Access Journals (Sweden)

    Ming Xu

    2007-02-01

    Full Text Available Pressure overload-induced hypertrophy is a key step leading to heart failure. The Ca(2+-induced Ca(2+ release (CICR process that governs cardiac contractility is defective in hypertrophy/heart failure, but the molecular mechanisms remain elusive. To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca(2+ channel (LCC and ryanodine receptors (RyRs in aortic stenosis rat models of compensated (CHT and decompensated (DHT hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of "intermolecular failure." Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca(2+ release, visualized as "Ca(2+ spikes," became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca(2+ transients in CHT. These data suggested that, within a certain limit, termed the "stability margin," mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of "hidden" intermolecular failure in CHT has important clinical implications.

  14. Expression and localization of ryanodine receptors in the frog semicircular canal.

    Science.gov (United States)

    Perin, Paola; Botta, Laura; Tritto, Simona; Laforenza, Umberto

    2012-01-01

    Several experiments suggest an important role for store-released Ca²⁺ in hair cell organs: drugs targeting IP₃ and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flat mounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyRα in both ampulla and canal arm and RyRβ in the ampulla only.

  15. Expression and Localization of Ryanodine Receptors in the Frog Semicircular Canal

    Directory of Open Access Journals (Sweden)

    Paola Perin

    2012-01-01

    Full Text Available Several experiments suggest an important role for store-released Ca2+ in hair cell organs: drugs targeting IP3 and ryanodine (RyRs receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types and flat mounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyRα in both ampulla and canal arm and RyRβ in the ampulla only.

  16. Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer.

    Science.gov (United States)

    Mahalingam, Mohana; Girgenrath, Tanya; Svensson, Bengt; Thomas, David D; Cornea, Razvan L; Fessenden, James D

    2014-09-02

    Ryanodine receptors (RyRs) release Ca(2+) to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Sarcoplasmic Reticulum Ca2+ Cycling Protein Phosphorylation in a Physiologic Ca2+ Milieu Unleashes a High-Power, Rhythmic Ca2+ Clock in Ventricular Myocytes: Relevance to Arrhythmias and Bio-Pacemaker Design

    Science.gov (United States)

    Sirenko, Syevda; Maltsev, Victor A; Maltseva, Larissa A; Yang, Dongmei; Lukyanenko, Yevgeniya; Vinogradova, Tatiana; Jones, Larry; Lakatta, Edward G.

    2014-01-01

    Basal phosphorylation of sarcoplasmic reticulum (SR) Ca2+ proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca2+ releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca2+ sparks. We tested the hypothesis that in a physiologic Ca2+ milieu, and independent of increased Ca2+ influx, an increase in basal phosphorylation of SR Ca2+ cycling proteins will convert stochastic Ca2+ sparks into periodic, high-power Ca2+ signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca2+ release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca2+], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2. In the absence of the aforementioned perturbations, VM could only generate stochastic local Ca2+ releases of low power and low amplitude, as assessed by confocal Ca2+ imaging and spectral analysis. When the kinetics of Ca2+ pumping into the SR were increased by an increase in PLB phosphorylation (via PDE and PP inhibition or addition of cAMP) or by 2D12, self-organized, “clock-like” local Ca2+ releases, partially synchronized in space and time (Ca2+ wavelets), emerged, and the ensemble of these rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. Thus, a Ca2+ clock is not specific to pacemaker cells, but can also be unleashed in VM when SR Ca2+ cycling increases and spontaneous local Ca2+ release becomes partially synchronized. This unleashed Ca2+ clock that emerges in a physiological Ca2+ milieu in VM has two faces, however: it can provoke ventricular arrhythmias; or if harnessed, can be an important feature

  18. The calcium uptake of the rat heart sarcoplasmic reticulum is altered by dietary lipid.

    Science.gov (United States)

    Taffet, G E; Pham, T T; Bick, D L; Entman, M L; Pownall, H J; Bick, R J

    1993-01-01

    Small amounts of dietary n-3 fatty acids can have dramatic physiological effects, including the reduction of plasma triglycerides and an elevation of cellular eicosapentanoic (EPA) and docosahexanoic acids (DHA) at the expense of arachidonic acid (AA). We investigated the effects of alterations in the fatty acid compositions of cardiac sarcoplasmic reticulum (CSR) produced by dietary manipulation on the calcium pump protein that is required for energy dependent calcium transport. CSR was isolated from rats fed menhaden oil, which is rich in n-3 fatty acids, and from control animals that were given corn oil. Relative to control membranes, those isolated from rats fed menhaden oil, had a lower content of saturated phospholipids, an increased DHA/AA ratio, and an increased ratio of n-3 to n-6 fatty acids. These changes were associated with a 30% decrease in oxalate-facilitated, ATP-dependent calcium uptake and concomitant decreased Ca-ATPase activity in the membranes from the animals fed menhaden oil. In contrast, there was no alteration in active pump sites as measured by phosphoenzyme formation. Thus, the CSR Ca-ATPase function can be altered by dietary interventions that change the composition, and possibly structure, of the phospholipid membranes thereby affecting enzyme turnover.

  19. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins.

    Science.gov (United States)

    Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

    2016-02-01

    Purification of proteinase inhibitor from common carp (Cyprinus carpio) sarcoplasmic proteins resulted in 2.8% yield with purification fold of 111. Two inhibitors, namely inhibitor I and II, exhibited molecular mass of 47 and 52 kDa, respectively, based on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both inhibitors I and II were identified to be alpha-1-proteinase inhibitor (α1-PI) based on LC-MS/MS. They were glycoproteins and molecular mass after peptide-N-glycosidase F treatment was 38 and 45 kDa, respectively. The N-glycosylation sites of both inhibitors were determined to be at N214 and N226. The inhibitors specifically inhibited trypsin. The common carp α1-PI showed high thermal stability with denaturation temperatures of 65.43 and 73.31 °C, which were slightly less than those of ovomucoid. High stability toward NaCl was also evident up to 3M. The common carp α1-PI effectively reduced autolytic degradation of bigeye snapper surimi at the concentration as low as 0.025%.

  20. Design and characterization of self-assembled fish sarcoplasmic protein-alginate nanocomplexes.

    Science.gov (United States)

    Stephansen, Karen; Mattebjerg, Maria; Wattjes, Jasper; Milisavljevic, Ana; Jessen, Flemming; Qvortrup, Klaus; Goycoolea, Francisco M; Chronakis, Ioannis S

    2015-05-01

    Macrostructures based on natural polymers are subject to large attention, as the application range is wide within the food and pharmaceutical industries. In this study we present nanocomplexes (NCXs) made from electrostatic self-assembly between negatively charged alginate and positively charged fish sarcoplasmic proteins (FSP), prepared by bulk mixing. A concentration screening revealed that there was a range of alginate and FSP concentrations where stable NCXs with similar properties were formed, rather than two exact concentrations. The size of the NCXs was 293 ± 3 nm, and the zeta potential was -42 ± 0.3 mV. The NCXs were stable in water, gastric buffer, intestinal buffer and HEPES buffered glycose, and at all pH values from 2 to 9 except pH 3, where they aggregated. When proteolytic enzymes were present in the buffer, the NCXs were degraded. Only at high concentrations the NCXs caused a decreased viability in HeLa and U2OS cell lines. The simple processing procedure and the high stability of the NCXs, makes them excellent candidates for use in the food and pharmaceutical industry. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Calcium handling by the sarcoplasmic reticulum during oscillatory contractions of skinned skeletal muscle fibres.

    Science.gov (United States)

    Szentesi, P; Zaremba, R; Stienen, G J

    1998-08-01

    Isometric ATP consumption and force were investigated in mechanically skinned fibres from iliofibularis muscle of Xenopus laevis. Measurements were performed at different [Ca2+], in the presence and absence of caffeine (5 nM). In weakly Ca2+-buffered solutions without caffeine, spontaneous oscillations in force and ATPase activity occurred. The repetition frequency was [Ca2+]-and temperature-dependent. The Ca2+ threshold (+/- SEM) for the oscillations corresponded to a pCa of 6.5 +/- 0.1. The maximum ATP consumption associated with calcium uptake by the sarcoplasmic reticulum (SR) reached during the oscillations was similar to the activity under steady-state conditions at saturating calcium concentrations in the presence of caffeine. Maximum activity was reached when the force relaxation was almost complete. The calculated amount of Ca2+ taken up by the SR during a complete cycle corresponded to 5.4 +/ 0.4 mmol per litre cell volume. In strongly Ca2+-buffered solutions, caffeine enhanced the calcium sensitivity of the contractile apparatus and, at low calcium concentrations, SR Ca uptake. These results suggest that when the SR is heavily loaded by net Ca uptake, there is a massive calcium-induced calcium release. Subsequent net Ca uptake by the SR then gives rise to the periodic nature of the calcium transient.

  2. Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Sjøgaard, G; Madsen, Klavs

    2000-01-01

    to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which......The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate...... during the first 0.5-1 h the metabolic state recovered to resting levels, and a slow phase from 1-3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca(2+) release rate was closely correlated to +dF/dt during the slow phase of recovery (r(2) = 0.51; P

  3. Vanadate oligoanions interact with the proton ejection by the Ca2+ pump of sarcoplasmic reticulum.

    Science.gov (United States)

    Aureliano, M; Madeira, V M

    1994-11-30

    Decameric vanadate differs from other oligomeric vanadate species in inhibiting Ca2+ uptake and H+ ejection promoted by sarcoplasmic reticulum ATPase. A decavanadate solution, 2 mM in total vanadium, containing about 200 microM decameric species, inhibits by about 50% the uptake of Ca2+ and by 75% the H+ ejection, whereas 2 mM nominal monovanadate slightly increases the uptake of Ca2+ and inhibits the ejection of H+ by 25%. Moreover, decavanadate linearly increases the Ca2+/H+ ratio, whereas monovanadate mimicks decavanadate behavior only at concentrations up to 1.2 mM. For higher concentrations of monovanadate, this effect is reversed probably due to the formation of metavanadates, namely tetravandate. It is concluded that Ca2+ uptake is tightly coupled to proton ejection through molecular events that are sensitive to the interaction of vanadate species. Apparently, the stoichiometry is variable and modulated by molecular events involved in vanadate interaction suggesting alterations in the energetic coupling associated with Ca2+ translocation.

  4. Disrupted Junctional Membrane Complexes and Hyperactive Ryanodine Receptors Following Acute Junctophilin Knockdown in Mice

    Science.gov (United States)

    van Oort, Ralph J.; Garbino, Alejandro; Wang, Wei; Dixit, Sayali S.; Landstrom, Andrew P.; Gaur, Namit; De Almeida, Angela C.; Skapura, Darlene G.; Rudy, Yoram; Burns, Alan R.; Ackerman, Michael J.; Wehrens, Xander H.T.

    2011-01-01

    Background Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum (SR) within junctional membrane complexes (JMCs). Whereas previous studies revealed a loss of JMCs and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in JMC formation and the Ca2+-induced Ca2+ release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. Methods and Results To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of JMCs and increased variability in the plasmalemma-SR distance. Conclusions Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific shRNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca2+ release in the heart. PMID:21339484

  5. Chloroform extract of hog barn dust modulates skeletal muscle ryanodine receptor calcium-release channel (RyR1)

    Science.gov (United States)

    Tian, Chengju; Shao, Chun Hong; Fenster, Danielle S.; Mixan, Mark; Romberger, Debra J.; Toews, Myron L.

    2010-01-01

    Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca2+-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBDorg, were air-dried and studied further. In competition assays, HBDorg dose-dependently displaced [3H]ryanodine from binding sites on RyR1 with an IC50 of 1.5 ± 0.1 μg/ml (Ki = 0.4 ± 0.0 μg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBDorg exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C2C12 myotubes, addition of HBD triggered a rise in intracellular Ca2+ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers. PMID:20576841

  6. Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes

    Science.gov (United States)

    Petrovič, Pavol; Valent, Ivan; Cocherová, Elena; Pavelková, Jana

    2015-01-01

    The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05–0.1-µM) calcium concentrations but only slightly at high (0.2–0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases. PMID:26009544

  7. Blockage of the Ryanodine Receptor via Azumolene Does Not Prevent Mechanical Ventilation-Induced Diaphragm Atrophy.

    Directory of Open Access Journals (Sweden)

    Erin E Talbert

    Full Text Available Mechanical ventilation (MV is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3. In this regard, leakage of calcium through the ryanodine receptor (RyR1 in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy.

  8. Blockage of the Ryanodine Receptor via Azumolene Does Not Prevent Mechanical Ventilation-Induced Diaphragm Atrophy.

    Science.gov (United States)

    Talbert, Erin E; Smuder, Ashley J; Kwon, Oh Sung; Sollanek, Kurt J; Wiggs, Michael P; Powers, Scott K

    2016-01-01

    Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS) from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3). In this regard, leakage of calcium through the ryanodine receptor (RyR1) in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ) would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy.

  9. A mechanism for sudden infant death syndrome (SIDS): Stress-induced leak via ryanodine receptors

    Science.gov (United States)

    Tester, David J.; Dura, Miroslav; Carturan, Elisa; Reiken, Steven; Wronska, Anetta; Marks, Andrew R.; Ackerman, Michael J.

    2012-01-01

    BACKGROUND Sudden infant death syndrome (SIDS) is the leading cause of postneonatal mortality in the United States. Mutations in the RyR2-encoded cardiac ryanodine receptor cause the highly lethal catecholaminergic polymorphic ventricular tachycardia (CPVT1) in the young. OBJECTIVE The purpose of this study was to determine the spectrum and prevalence of RyR2 mutations in a large cohort of SIDS cases. METHODS Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, a targeted mutational analysis of RyR2 was performed on genomic DNA isolated from frozen necropsy tissue on 134 unrelated cases of SIDS (57 females, 77 males; 83 white, 50 black, 1 Hispanic; average age = 2.7 months). RyR2 mutations were engineered by site-directed mutagenesis, heterologously expressed in HEK293 cells, and functionally characterized using single-channel recordings in planar lipid bilayers. RESULTS Overall, two distinct and novel RyR2 mutations were identified in two cases of SIDS. A 6-month-old black female hosted an R2267H missense mutation, and a 4-week-old white female infant harbored a S4565R mutation. Both nonconservative amino acid substitutions were absent in 400 reference alleles, involved conserved residues, and were localized to key functionally significant domains. Under conditions that simulate stress [Protein Kinase A (PKA) phosphorylation] during diastole (low activating [Ca2+]), SIDS-associated RyR2 mutant channels displayed a significant gain-of-function phenotype consistent with the functional effect of previously characterized CPVT-associated RyR2 mutations. CONCLUSIONS Here we report a novel pathogenic mechanism for SIDS, whereby SIDS-linked RyR2 mutations alter the response of the channels to sympathetic nervous system stimulation such that during stress the channels become “leaky” and thus potentially trigger fatal cardiac arrhythmias. PMID:17556193

  10. Differential expression of the cardiac ryanodine receptor in normal and arrhythmogenic right ventricular cardiomyopathy canine hearts.

    Science.gov (United States)

    Meurs, Kathryn M; Lacombe, Veronique A; Dryburgh, Keith; Fox, Philip R; Reiser, Peter R; Kittleson, Mark D

    2006-08-01

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a form of cardiomyopathy characterized by ventricular tachyarrhythmias and a fibrofatty infiltrate that is believed to preferentially affect the right ventricle. Mutations in the cardiac ryanodine receptor (RyR2) gene have been identified in some human families with a unique form of ARVC, ARVC2. Although the RyR2 has significant importance in excitation-contraction coupling across the ventricles, mutations in the gene encoding for it appear to have the greatest impact on the right ventricle in ARVC2. Using a canine model (boxer), the RyR2 protein and message RNA in the right ventricle, left ventricle and interventricular septum from normal dogs and dogs with ARVC were investigated by immunoblotting and real time PCR. The cardiac RyR2 message and protein expression were differentially expressed across the cardiac walls in the normal heart, with the lowest concentration expressed in the right ventricle (P < 0.05). The message and protein expression of the RyR2 were reduced in all chambers in the canine model of ARVC. We propose that the increased susceptibility of the right ventricle to ARVC may be associated with the lower baseline protein concentration of RyR2 in the normal right ventricle compared to the left ventricle and interventricular septum and that all three areas are equally affected in this canine model of ARVC. Using this naturally occurring model of canine ARVC, we may have provided new insights into the pathogenesis of this cardiomyopathy.

  11. Insecticidal activities of chiral N-trifluoroacetyl sulfilimines as potential ryanodine receptor modulators.

    Science.gov (United States)

    Zhou, Sha; Gu, Yucheng; Liu, Ming; Wu, Changchun; Zhou, Sha; Zhao, Yu; Jia, Zhehui; Wang, Baolei; Xiong, Lixia; Yang, Na; Li, Zhengming

    2014-11-19

    This is the first report of novel chiral N-trifluoroacetyl sulfilimines during research for new environmentally benign and ecologically safe novel insecticides with new modes of action. Four series of phthalamides containing 20 new structures were designed, synthesized, and evaluated against oriental armyworm (Pseudaletia separata Walker) and diamondback moth (Plutella xylostella (L.)) for their insecticidal activities. The target compounds were established by corresponding (1)H NMR, HRMS (or elemental analysis), X-ray diffraction analysis, and optical polarimetry. Introduction of chiral N-trifluoroacetyl sulfiliminyl moieties into the new scaffolds showed that some target compounds possessed impressive activities as commercial flubendiamide. These N-trifluoroacetyl sulfilimines exhibited the sequence of activity against oriental armyworm as (Sc, Ss) ≥ (Sc, Rs) ≫ (Rc, Rs) > (Rc, Ss), in which the chiral carbon influenced the activities stronger than sulfur. For diamondback moth, compounds If, IIa, and IIc exhibited even stronger activity than flubendiamide; especially If displayed a death rate of 100% at 10(-6) mg L(-1), much better than that of flubendiamide (0% at 10(-4) mg L(-1)). Comparative molecular field analysis calculation indicated that stereoisomers with Sc configurations containing more electronegative group as COCF3 are favorable to the insecticidal activity. The present work demonstrated that chiral N-trifluoroacetyl sulfilimines can be considered as potential insect ryanodine receptor modulators. From the standpoint of molecular design, it was concluded that the conventional second methyl group in the aliphatic amido side chain of dicarboxamide might not be a requisite in our research on novel sulfiliminyl insecticides.

  12. Chiral dicarboxamide scaffolds containing a sulfiliminyl moiety as potential ryanodine receptor activators.

    Science.gov (United States)

    Zhou, Sha; Jia, Zhehui; Xiong, Lixia; Yan, Tao; Yang, Na; Wu, Guiping; Song, Haibin; Li, Zhengming

    2014-07-09

    To search for new environmentally benign insecticides with high activity, low toxicity, and low residue, novel chiral configurations introduced into dicarboxamide scaffolds containing N-cyano sulfiliminyl moieties were first studied. Four series of phthalamides with sulfur-containing side chains were designed, synthesized, and evaluated against oriental armyworm (Pseudaletia separata Walker) and diamondback moth (Plutella xylostella (L.)) for their insecticidal activities. All structures were characterized by (1)H NMR, (13)C NMR, and HRMS (or elemental analysis), and their configurations were confirmed by optical polarimetry. The biological assessment indicated that some title compounds exhibited significant insecticidal activities. For oriental armyworm, these stereoisomers exerted different impacts on biological activity following the sequence (Sc, Ss) ≥ (Sc, Rs) ≫ (Rc, Ss) > (Rc, Rs), and carbon chirality influenced the activities more strongly than sulfur. Compounds Ia and IIa reached as high an activity as commercial flubendiamide, with LC50 values of 0.0504 and 0.0699 mg L(-1), respectively, lower than that of flubendiamide (0.1230 mg L(-1)). For diamondback moth, the sequence of activity was (Sc, Ss) > (Sc, Rs), and the sulfur chirality influenced the activities more greatly than carbon. Compound IIe exhibited even higher activity than flubendiamide, whereas Ie and Ic,d reached the activity of the latter. The results indicated that the improvement of insecticidal activity probably required a coordination of both carbon and sulfur chirality. Comparative molecular field analysis calculation indicated that stereoisomers with Sc configurations containing strong electron-withdrawing groups such as as CN are important in maintaining the high activity. The chiral scaffolds containing the N-cyano sulfiliminyl moiety are also essential for high larvicidal activity. Some title compounds could be considered as potential candidates for ryanodine receptor activators.

  13. Ryanodine myopathies without central cores--clinical, histopathologic, and genetic description of three cases.

    Science.gov (United States)

    Rocha, João; Taipa, Ricardo; Melo Pires, Manuel; Oliveira, Jorge; Santos, Rosário; Santos, Manuela

    2014-08-01

    Mutations in ryanodine receptor 1 gene (RYR1) are frequent causes of myopathies. They classically present with central core disease; however, clinical variability and histopathologic overlap are being increasingly recognized. Patient 1 is a 15-year-old girl with mild proximal, four-limb weakness from age 5, presenting with a progressive scoliosis starting at age 10. Patient 2 is an 18-year-old girl with progressively worsening muscle hypotrophy and mild proximal, four-limb weakness. She developed a rapidly progressive scoliosis from age 11 and needed surgical treatment at age 14 years. Patient 3 is an 11-year-old boy with moderate proximal limb weakness and progressive neck flexor weakness, first noticed at age 2. Muscle biopsies revealed type 1 fiber predominance (Patients 1 and 2) or abnormal type 1 fiber uniformity (Patient 3). Different RYR1 variants were identified in all patients. In Patients 1 and 3, these changes were validated as being pathogenic. These patients illustrate early-onset, progressive myopathies with predominant axial involvement. Histopathologic findings were abnormal but not specific for a diagnosis, particularly central core myopathy. Genetic testing helped broaden the range of phenotypes included in the RYR1-related myopathies. Our patients reinforce the need to recognize the broad histopathologic variability of RYR1-related myopathies and sometimes lack of pathognomonic findings that may reduce the diagnostic threshold of this disease. We suggest that the predominance of type 1 fibers and involvement of axial muscles may be an important element to consider the RYR1 gene as candidate. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Directory of Open Access Journals (Sweden)

    Shweta A Raina

    Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  15. FRET-based trilateration of probes bound within functional ryanodine receptors.

    Science.gov (United States)

    Svensson, Bengt; Oda, Tetsuro; Nitu, Florentin R; Yang, Yi; Cornea, Iustin; Chen-Izu, Ye; Fessenden, James D; Bers, Donald M; Thomas, David D; Cornea, Razvan L

    2014-11-04

    To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

  16. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Science.gov (United States)

    Raina, Shweta A; Tsai, Jeffrey; Samsó, Montserrat; Fessenden, James D

    2012-01-01

    Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) insertions within the ryanodine receptor type 1 (RyR1), a large intracellular Ca(2+) release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK)-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  17. Matching of sarcoplasmic reticulum and contractile properties in rat fast- and slow-twitch muscle fibres.

    Science.gov (United States)

    Trinh, Huong H; Lamb, Graham D

    2006-07-01

    1. The twitch characteristics (fast-twitch or slow-twitch) of skeletal muscle fibres are determined not only by the contractile apparatus properties of the fibre, but also by the time-course of Ca2+ release and re-uptake by the sarcoplasmic reticulum (SR). The present study examined, in individual fibres from non-transforming muscle of the rat, whether particular SR properties are matched to the contractile apparatus properties of the fibre, in particular in the case of fibres with fast-twitch contractile apparatus located in a slow-twitch muscle, namely the soleus. 2. Force was recorded in single, mechanically skinned fibres from extensor digitorum longus (EDL), gastrocnemius, peroneus longus and soleus muscles. Using repeated cycles in which the SR was emptied of all releasable Ca2+ and then reloaded, it was possible to determine the relative amount of Ca2+ present in the SR endogenously, the maximum SR capacity and the rate of Ca2+ loading. The sensitivity of the contractile apparatus to Ca2+ and Sr2+ was used to classify the fibres as fast-twitch (FT), slow-twitch (ST) or mixed (< 3% of the fibres examined) and thereby identify the likely troponin C and myosin heavy chain types present. 3. There was no significant difference in SR properties between the groups of FT fibres obtained from the four different muscles, including soleus. Despite some overlap in the SR properties of individual fibres between the FT and ST groups, the properties of the FT fibres in all four muscles studied were significantly different from those of the ST and mixed fibres. 4. In general, in FT fibres the SR had a larger capacity and the endogenous Ca2+ content was a relatively lower percentage of maximum compared with ST fibres. Importantly, in terms of their SR properties, FT fibres from soleus muscle more closely resembled FT fibres from other muscles than they did ST fibres from soleus muscle.

  18. Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity.

    Directory of Open Access Journals (Sweden)

    Yael Yaniv

    Full Text Available BACKGROUND: Mitochondria dynamically buffer cytosolic Ca(2+ in cardiac ventricular cells and this affects the Ca(2+ load of the sarcoplasmic reticulum (SR. In sinoatrial-node cells (SANC the SR generates periodic local, subsarcolemmal Ca(2+ releases (LCRs that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+-Ca(2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP. OBJECTIVE: To determine if mitochondrial Ca(2+ (Ca(2+ (m, cytosolic Ca(2+ (Ca(2+ (c-SR-Ca(2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity. RESULTS: Inhibition of mitochondrial Ca(2+ influx into (Ru360 or Ca(2+ efflux from (CGP-37157 decreased [Ca(2+](m to 80 ± 8% control or increased [Ca(2+](m to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+ influx or efflux, the SR Ca(2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+ signal were highly correlated with the change in the SR Ca(2+ load (r(2 = 0.97. Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control in response to changes in [Ca(2+](m were predicted by concurrent changes in LCR period (r(2 = 0.84. CONCLUSION: A change in SANC Ca(2+ (m flux translates into a change in the AP firing rate by effecting changes in Ca(2+ (c and SR Ca(2+ loading, which affects the characteristics of spontaneous SR Ca(2+ release.

  19. Voltage-dependent modulation of cardiac ryanodine receptors (RyR2 by protamine.

    Directory of Open Access Journals (Sweden)

    Paula L Diaz-Sylvester

    Full Text Available It has been reported that protamine (>10 microg/ml blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02-20 microg/ml added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (V(m; SR lumen-cytosol = 0 mV, protamine induced conductance transitions to several intermediate states (substates as well as full block of RyR2. At V(m>10 mV, the substate with the highest level of conductance was predominant. Increasing V(m from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate had the same magnitude at 0 and +80 mV despite the approximately 3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin. A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

  20. Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin.

    Science.gov (United States)

    Giuffrida, M G; Villalta, D; Mistrello, G; Amato, S; Asero, R

    2014-09-01

    Little is known about the prevalence and clinical relevance of sensitization to shrimp allergens other than tropomyosin. We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients. Sera from 40 shrimp-allergic patients underwent the detection of IgE specific for arginine kinase (rPen m 2) and sarcoplasmic calcium-binding protein (rPen m 4) by ISAC 112 Microarray platform and immunoblot analysis. A high molecular weight shrimp allergen was identified by N-terminal amino acid sequencing. IgE to rPen m 2 and rPen m 4 were found in 4/40 (10%) and 6/40 (15%) sera, respectively; two sera reacted to both allergens. Clinically, 6/8 Pen m 2 and/or Pen m 4 reactors experienced severe allergies to shrimp. On immunoblot, 4/6 rPen m 4-positive sera showed IgE reactivity at about 20 kDa, whereas no rPen m 2-positive serum reacted at about 40 kDa. Nineteen (47%) sera showed IgE reactivity at molecular weights > 60 kDa. Such profile was not associated with IgE reactivity to rPen m 2 or rPen m 4. N-terminal amino acid sequencing of the high molecular weight allergen led to the identification of hemocyanin. Shrimp arginine kinase and sarcoplasmic calcium-binding protein are minor allergens sensitizing only 10%-15% of Italian shrimp-allergic patients, but are clinically relevant. Hemocyanin is a clinically relevant high molecular weight shrimp allergen possibly cross-reacting to house dust mite.

  1. CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency dependent acceleration of relaxation and Ca2+ current facilitation

    OpenAIRE

    Picht, Eckard; DeSantiago, Jaime; Huke, Sabine; Kaetzel, Marcia A.; Dedman, John R.; Bers, Donald M.

    2006-01-01

    Cardiac Ca2+/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca2+ current (ICa) facilitation, enhanced sarcoplasmic reticulum (SR) Ca2+ release and frequency dependent acceleration of relaxation (FDAR) via enhanced SR Ca2+ uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaM-KII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide...

  2. NAADP-mediated Ca2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist

    Science.gov (United States)

    Dammermann, Werner; Zhang, Bo; Nebel, Merle; Cordiglieri, Chiara; Odoardi, Francesca; Kirchberger, Tanja; Kawakami, Naoto; Dowden, James; Schmid, Frederike; Dornmair, Klaus; Hohenegger, Martin; Flügel, Alexander; Guse, Andreas H.; Potter, Barry V. L.

    2009-01-01

    The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca2+ signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca2+ mobilization by d-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca2+ entry. BZ194 specifically and effectively blocked NAADP-stimulated [3H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca2+ mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca2+ mobilization, such as nuclear translocation of “nuclear factor of activated T cells” (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4+ effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases. PMID:19541638

  3. Activation of presynaptic and postsynaptic ryanodine-sensitive calcium stores is required for the induction of long-term depression at GABAergic synapses in the neonatal rat hippocampus.

    Science.gov (United States)

    Caillard, O; Ben-Ari, Y; Gaïarsa, J L

    2000-09-01

    The role of internal calcium stores in the induction of long-term depression at GABAergic synapses was investigated in the neonatal rat hippocampus. Whole-cell recordings of CA3 pyramidal neurons were performed on hippocampal slices from neonatal (2-4 d old) rats. In control conditions, tetanic stimulation (TS) evoked an NMDA-dependent long-term depression of GABA(A) receptor-mediated postsynaptic responses (LTD(GABA-A)). LTD(GABA-A) was prevented when the cells were loaded with ruthenium red, a blocker of Ca2+-induced Ca2+ release (CICR) stores, whereas loading the cells with heparin, a blocker of IP3-induced Ca2+ release stores, had no effect. The effects of ryanodine, another compound that interferes with CICR stores, were also investigated. Intracellular injection of ryanodine prevented the induction of LTD(GABA-A) only when the TS was preceded by depolarizing pulses that increase intracellular Ca2+ concentration. When applied in the bath, ryanodine prevented the induction of LTD(GABA-A). Altogether, these results suggest that ryanodine acts as a Ca2+-dependent blocker of CICR stores and that the induction of LTD(GABA-A) required the activation of both presynaptic and postsynaptic CICR stores.

  4. Interactions of vanadium(V)-citrate complexes with the sarcoplasmic reticulum calcium pump.

    Science.gov (United States)

    Aureliano, Manuel; Tiago, Teresa; Gândara, Ricardo M C; Sousa, Andrea; Moderno, A; Kaliva, M; Salifoglou, A; Duarte, Rui O; Moura, José J G

    2005-12-01

    Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.

  5. Effects of prior exercise and a low-carbohydrate diet on muscle sarcoplasmic reticulum function during cycling in women.

    Science.gov (United States)

    Duhamel, T A; Green, H J; Perco, J G; Ouyang, J

    2006-09-01

    The effects of exercise and diet on sarcoplasmic reticulum Ca(2+)-cycling properties in female vastus lateralis muscle were investigated in two groups of women following four different conditions. The conditions were 4 days of a low-carbohydrate (Lo CHO) and glycogen-depleting exercise plus a Lo CHO diet (Ex + Lo CHO) (experiment 2) and 4 days of normal CHO (Norm CHO) and glycogen-depleting exercise plus Norm CHO (Ex + Norm CHO) (experiment 1). Peak aerobic power (Vo2peak)) was 38.1 +/- 1.4 (SE); n = 9 and 35.6 +/- 1.4 ml.kg(-1).min(-1); n = 9, respectively. Sarcoplasmic reticulum properties measured in vitro in homogenates (micromol.g protein(-1).min(-1)) indicated exercise-induced reductions (P 30, 60 min > fatigue), Ca(2+) uptake (0 > 30 > 60 min, fatigue), and Ca(2+) release, both phase 1 (0, 30 > 60 min, fatigue) and phase 2 (0 > 30, 60 min, fatigue; 30 min > fatigue) in Norm CHO. Exercise was without effect in altering the Hill slope (n(H)), defined as the slope of relationship between Ca(2+)-ATPase activity and Ca(2+) concentration. No differences were observed between Norm CHO and Ex+Norm CHO. Compared with Norm CHO, Lo CHO resulted in a lower (P cycling and that, with the exception of Ca(2+) release, a glycogen-depleting session of exercise before Lo CHO can reverse the effects.

  6. Synthesis, Larvicidal Activities and Antifungal Activities of Novel Chlorantraniliprole Derivatives and Their Target in the Ryanodine Receptor

    Directory of Open Access Journals (Sweden)

    Qichao Chen

    2015-03-01

    Full Text Available In order to identify novel chlorantraniliprole derivatives as potential insecticides or fungicides, 25 analogues of chlorantraniliprole were synthesized. The insecticidal activities against oriental armyworm and the antifungal activities against five typical fungi of these derivatives were tested. Compounds 2u, 2x and 2y exhibited good activities against oriental armyworm, especially compounds 2u and 2x which showed higher larvicidal activities than indoxacarb. Moreover, all of the tested compounds exhibited activities against five typical fungi. The Ki values of all synthesized compounds were calculated using AutoDock4. The relationship between the Ki values and the results of insecticidal activities against oriental armyworm further indicated that the membrane-spanning domain protein of the ryanodine receptor might contain chlorantraniliprole binding sites.

  7. Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate.

    Science.gov (United States)

    Fraqueza, Gil; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-02-01

    Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca(2+)-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca(2+)-ATPase, with the following IC(50) values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb(10)), is the strongest P-type enzyme inhibitor, after decavanadate (V(10)). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca(2+)-ATPase stoichiometry. Furthermore, V(10) binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H(2)VO(4)(2-); V(1)) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca(2+)-ATPase, with a dissociation constant, k(d) of 1 μM(-1). The interaction of decavanadate V(10)O(28)(6-) (V(10)) with Ca(2+)-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb(10)O(28)(6-) (Nb(10)), whereas no significant effects were

  8. Sarcoplasmic reticulum calcium release in frog skeletal muscle fibres estimated from Arsenazo III calcium transients.

    Science.gov (United States)

    Baylor, S M; Chandler, W K; Marshall, M W

    1983-01-01

    Single twitch fibres, dissected from frog muscle, were injected with the metallochromic dye Arsenazo III. Changes in dye-related absorbance measured at 650 or 660 nm were used to estimate the time course of myoplasmic free [Ca2+] following either action potential stimulation or voltage-clamp depolarization (temperature, 15-17 degrees C). The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 microns. The effect appeared to be less marked in H2O Ringer than in D2O Ringer, where a reduction of about 40% was observed in going from 3.0 microns to 3.7-3.9 microns. In fibres heavily injected with dye (1.5-2.2 mM-dye) at least 0.1 mM-Ca2+ was complexed with Arsenazo III following a single action potential, implying that at least 0.1 mM-Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm. Computer simulations were carried out to estimate the flux of Ca2+ between the s.r. and myoplasm (in fibres containing no more that 0.8 mM-dye). The amounts and time courses of Ca2+ bound to the Ca2+-regulatory sites on troponin and to the Ca2+, Mg2+ sites on parvalbumin were estimated from the free [Ca2+] wave form and the law of mass action. In the computations the total myoplasmic [Ca2+] was taken as the total amount of Ca2+ existing either as free ion or as ion complexed with dye, troponin or parvalbumin. The time derivative of total myoplasmic [Ca2+] was used as an estimate of net Ca2+ flux (release minus uptake) from the s.r. into myoplasm. Rate constants for formation of cation: receptor complex were taken from published values. For the Ca2+-regulatory sites on troponin, three sets of rate constants, corresponding to two values of dissociation constant (0.2 and 2 microM) were used. Each set of three simulations was carried out both with and without parvalbumin. The simulations show that following action potential stimulation, 0.2-0.3 mM-Ca2+ enters the myoplasm from the s.r. The wave form of s.r. Ca2

  9. The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham.

    Science.gov (United States)

    Gallego, Marta; Mora, Leticia; Concepción Aristoy, M; Toldrá, Fidel

    2016-04-01

    The aim of this work was to quantify changes in the abundance of the major sarcoplasmic proteins throughout the ham dry-curing process by using a label-free mass spectrometry methodology based on the measurement of mass spectral peak intensities obtained from the extracted ion chromatogram. For this purpose, extraction of sarcoplasmic proteins was followed by trypsin digestion and analysis by nanoliquid chromatography coupled to tandem mass spectrometry (Q/TOF) for the identification and relative quantification of sarcoplasmic proteins through individual quantification of trypsinised peptides. In total, 20 proteins, including 12 glycolytic enzymes, were identified and quantified. The accuracy of the protocol was based on MS/MS replicates, and beta-lactoglobulin protein was used to normalise data and correct possible variations during sample preparation or LC-MS/MS analysis. Mass spectrometry-based proteomics provides precise identification and quantification of proteins in comparison with traditional methodologies based on gel electrophoresis, especially in the case of overlapping proteins. Moreover, the label-free approach used in this study proved to be a simple, fast, reliable method for evaluating proteolytic degradation of sarcoplasmic proteins during the processing of dry-cured ham.

  10. The changes of cardioelectrical activity of rat with myocardial infarction receiving sarcoplasmic reticulum Ca2+-ATPase gene modified bone marrow stem cell transplantation by microelectrode array technology

    Institute of Scientific and Technical Information of China (English)

    范平

    2012-01-01

    Objective Therapy effects and cardiac electrical activity comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) gene modified BMSCs transplantation after acute myocardial infarction(AMI) in rats.Methods Rats with AMI were divided

  11. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

    Science.gov (United States)

    Ruiz, A; Matute, C; Alberdi, E

    2010-01-01

    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  12. S4153R is a gain-of-function mutation in the cardiac Ca(2+) release channel ryanodine receptor associated with catecholaminergic polymorphic ventricular tachycardia and paroxysmal atrial fibrillation.

    Science.gov (United States)

    Zhabyeyev, Pavel; Hiess, Florian; Wang, Ruiwu; Liu, Yingjie; Wayne Chen, S R; Oudit, Gavin Y

    2013-08-01

    Mutations in ryanodine receptor 2 (RYR2) gene can cause catecholaminergic polymorphic ventricular tachycardia (CPVT). The novel RYR2-S4153R mutation has been implicated as a cause of CPVT and atrial fibrillation. The mutation has been functionally characterized via store-overload-induced Ca(2+) release (SOICR) and tritium-labelled ryanodine ([(3)H]ryanodine) binding assays. The S4153R mutation enhanced propensity for spontaneous Ca(2+) release and reduced SOICR threshold but did not alter Ca(2+) activation of [(3)H]ryanodine binding, a common feature of other CPVT gain-of-function RYR2 mutations. We conclude that the S4153R mutation is a gain-of-function RYR2 mutation associated with a clinical phenotype characterized by both CPVT and atrial fibrillation.

  13. Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2002-01-01

    Temperature dependence of Ca2+-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degreesC. Whether the break point arises as a result of temperature dependent changes...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2.......5) between 60 and 250 muM. Results indicated that interaction between SR Ca2+-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated. (C) 2002 Elsevier Science Inc. All rights reserved....

  14. Role of the dysfunctional ryanodine receptor - Na(+)-Ca(2+)exchanger axis in progression of cardiovascular diseases: What we can learn from pharmacological studies?

    Science.gov (United States)

    Acsai, Károly; Ördög, Balázs; Varró, András; Nánási, Péter P

    2016-05-15

    Abnormal Ca(2+)homeostasis is often associated with chronic cardiovascular diseases, such as hypertension, heart failure or cardiac arrhythmias, and typically contributes to the basic ethiology of the disease. Pharmacological targeting of cardiac Ca(2+)handling has great therapeutic potential offering invaluable options for the prevention, slowing down the progression or suppression of the harmful outcomes like life threatening cardiac arrhythmias. In this review we outline the existing knowledge on the involvement of malfunction of the ryanodine receptor and the Na(+)-Ca(2+)exchanger in disturbances of Ca(2+)homeostasis and discuss important proof of concept pharmacological studies targeting these mechanisms in context of hypertension, heart failure, atrial fibrillation and ventricular arrhythmias. We emphasize the promising results of preclinical studies underpinning the potential benefits of the therapeutic strategies based on ryanodine receptor or Na(+)-Ca(2+)exchanger inhibition.

  15. Pre-Slaughter Stress Affects Ryanodine Receptor Protein Gene Expression and the Water-Holding Capacity in Fillets of the Nile Tilapia.

    Directory of Open Access Journals (Sweden)

    Elenice S R Goes

    Full Text Available Current study evaluated the effect of pre-slaughter stress on serum cortisol levels, pH, colorimetry, water-holding capacity (WHC and gene expression of ryanodine receptors (RyR1 and RyR3 in the Nile tilapia. A 3x4 factorial scheme experiment was conducted comprising three densities (100, 200, 400 kg/m³ with four transportation times (60, 120, 180, and 240 minutes.Transportation times alone reduced cortisol levels up to 180 minutes, followed by increased WHC and mRNA expression, RyR1 and RyR3 (200 kg/m³ density. No effect of density x transportation time interacted on the evaluated parameters. Results provided the first evidence that pre-slaughter stress affected ryanodine gene expression receptors and, consequently, the water-holding capacity in tilapia fillets.

  16. Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors.

    Directory of Open Access Journals (Sweden)

    Paola Llanos

    Full Text Available Glucose-stimulated insulin secretion (GSIS from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]. Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC, which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.

  17. 重症肌无力患者血清中Ryanodine受体抗体对其症状学的评估作用%Serum ryanodine receptor antibody on the assessment of clinical symptoms in patients with myasthenia gravis

    Institute of Scientific and Technical Information of China (English)

    张祥; 乔健; 吕传真

    2005-01-01

    BACKGROUND: Myasthenia gravis (MG) patients with thymoma were often neglected in clinical work and delayed the therapy.OBJECTIVE: To investigate the significance of the Ryanodine receptor (RyR) antibody on the assessment of MG.DESIGN: A case analysis.SETTING: Institute of neurology in a hospital of a university.PARTICIPANTS: This experiment was carried out in the Institute of Neurology, Fudan University from June 1999 to March 2002. There were 66 MG patients with thymoma(MGT group), 98 MG patients with non-thymoma (NTMG group), 50 patients with non-myasthenia gravis(NMG) and 123 normal persons (NC group).METHODS: Sarcoplasmic reticulum(SR) abounded in RyR was extracted with differential centrifugation, in order to establish a detecting system of ELISA-RyR-RyR antibody (RyR-ab).MAIN OUTCOME MEASURES: The levels of RyR-ab in serum of researched subjects.RESULTS: Positive rate of RyR-ab in MGT group was higher than that in NTMG and NMG groups(P < 0.01), moreover, the sensitivity and the specificity were 81.8% and 94.5% respectively. The positive rates of MGT groups with different thymus histology were no significant difference(P> 0.05). Ages, clinical scores and levels of acetylcholine receptor antibody (AchR-ab) in patients with positive RyR-ab were higher than those in patients with negative RyR-ab( P < 0.01 ) in MG group. The levels of RyR-ab was positive correlated with the severities of clinical symptoms in MG patients, especially the patients in MGT group( r = 0. 626, P < 0.01) . And among the different histological types of MGT, thymoma of epithelioid cells has the highest correlation coefficient ( r = 0. 592, P < 0. 01).CONCLUSION: The detection of RyR-ab has better sensitivity and specificity for the diagnosis of MGT and the levels of RyR-ab is positive correlatied with the severities of MG patients.%背景:临床工作中对伴胸腺瘤重症肌无力患者仍存在漏诊,从而贻误治疗.目的:探讨Ryanodine受体(ryanodine receptor,RyR

  18. Role of Mitochondrial Enzymes and Sarcoplasmic ATPase in Cardioprotection Mediated by Aqueous Extract of Desmodium gangeticum (L) DC Root on Ischemic Reperfusion Injury.

    Science.gov (United States)

    Kurian, G A; Paddikkala, J

    2010-11-01

    The present study investigate the protective effect of aqueous root extract of Desmodium gangeticum in preserving mitochondrial and sarcoplasmic ATPase during ischemia reperfusion injury. The isolated rat hearts in both drug and control group were subjected to warm ischemia (37°), followed by reperfusion with the Langendorff perfusion system. The aqueous root extract of Desmodium gangeticum (L) at a dose of 50 mg/kg body weight was found to be effective in the rat heart for the management of ischemic reperfusion injury. Physiological parameters were significantly (PDesmodium gangeticum treated rat heart. These results suggest that Desmodium gangeticum aqueous root extract can preserve the mitochondrial and sarcoplasmic ATPase in the myocardium, resulting in the improvement of cardiac function after ischemia reperfusion injury.

  19. Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+- dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium

    OpenAIRE

    1988-01-01

    We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneo...

  20. Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

    Science.gov (United States)

    Picariello, Gianluca; De Martino, Alessandra; Mamone, Gianfranco; Ferranti, Pasquale; Addeo, Francesco; Faccia, Michele; Spagnamusso, Salvatore; Di Luccia, Aldo

    2006-03-20

    In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate

  1. Ryanodine receptor genes of the rice stem borer, Chilo suppressalis: Molecular cloning, alternative splicing and expression profiling.

    Science.gov (United States)

    Peng, Y C; Sheng, C W; Casida, John E; Zhao, C Q; Han, Z J

    2017-01-01

    The ryanodine receptor (RyR) of the calcium release channel is the main target of anthranilic and phthalic diamide insecticides which have high selective insecticidal activity relative to mammalian toxicity. In this study, the full-length cDNA of Chilo suppressalis RyR (CsRyR) was isolated and characterized. The CsRyR mRNA has an open reading frame (ORF) of 15,387bp nucleotides, which encodes 5128 amino acids with GenBank ID: KR088972. Comparison of protein sequences showed that CsRyR shared high identities with other insects of 77-96% and lower identity to mammals and nematodes with only 42-45%. One alternative splicing site (KENLG) unique to Lepidoptera was found and two exclusive exons of CsRyR (I /II) were revealed. Spatial and temporal expression of CsRyR mRNA was at the highest relative level in 3rd instar larvae and head (including brain and muscle), and at the lowest expression level in egg and fat body. The expression levels of whole body CsRyR mRNA were increased remarkably after injection of 4th instar larvae with chlorantraniliprole at 0.004 to 0.4μg/g. This structural and functional information on CsRyR provides the basis for further understanding the selective action of chlorantraniliprole and possibly other diamide insecticides.

  2. The foot structure from the type 1 ryanodine receptor is required for functional coupling to store-operated channels.

    Science.gov (United States)

    Sampieri, Alicia; Diaz-Muñoz, Mauricio; Antaramian, Anaid; Vaca, Luis

    2005-07-01

    In the present study we have explored structural determinants of the functional interaction between skeletal muscle ryanodine receptor (RyR1) and transient receptor potential channel 1 (TRPC1) channels expressed in Chinese hamster ovary cells. We have illustrated a functional interaction between TRPC1 channels and RyR1 for the regulation of store-operated calcium entry (SOCE) initiated after releasing calcium from a caffeine-sensitive intracellular calcium pool. RNA interference experiments directed to reduce the amount of TRPC1 protein indicate that RyR1 associates to at least two different types of store-operated channels (SOCs), one dependent and one independent of TRPC1. In contrast, bradykinin-induced SOCE is completely dependent on the presence of TRPC1 protein, as we have previously illustrated. Removing the foot structure from RyR1 results in normal caffeine-induced release of calcium from internal stores but abolishes the activation of SOCE, indicating that this structure is require for functional coupling to SOCs. The footless RyR1 protein shows a different cellular localization when compared with wild type RyR1. The later protein shows a higher percentage of colocalization with FM-464, a marker of plasma membrane. The implications of the foot structure for the functional and physical coupling to TRPC and SOCs is discussed.

  3. A structural model of the pore-forming region of the skeletal muscle ryanodine receptor (RyR1.

    Directory of Open Access Journals (Sweden)

    Srinivas Ramachandran

    2009-04-01

    Full Text Available Ryanodine receptors (RyRs are ion channels that regulate muscle contraction by releasing calcium ions from intracellular stores into the cytoplasm. Mutations in skeletal muscle RyR (RyR1 give rise to congenital diseases such as central core disease. The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Here, we report a structural model of the pore-forming region of RyR1. Molecular dynamics simulations show high ion binding to putative pore residues D4899, E4900, D4938, and D4945, which are experimentally known to be critical for channel conductance and selectivity. We also observe preferential localization of Ca(2+ over K(+ in the selectivity filter of RyR1. Simulations of RyR1-D4899Q mutant show a loss of preference to Ca(2+ in the selectivity filter as seen experimentally. Electrophysiological experiments on a central core disease mutant, RyR1-G4898R, show constitutively open channels that conduct K(+ but not Ca(2+. Our simulations with G4898R likewise show a decrease in the preference of Ca(2+ over K(+ in the selectivity filter. Together, the computational and experimental results shed light on ion conductance and selectivity of RyR1 at an atomistic level.

  4. Basal bioenergetic abnormalities in skeletal muscle from ryanodine receptor malignant hyperthermia-susceptible R163C knock-in mice.

    Science.gov (United States)

    Giulivi, Cecilia; Ross-Inta, Catherine; Omanska-Klusek, Alicja; Napoli, Eleonora; Sakaguchi, Danielle; Barrientos, Genaro; Allen, Paul D; Pessah, Isaac N

    2011-01-07

    Malignant hyperthermia (MH) and central core disease in humans have been associated with mutations in the skeletal ryanodine receptor (RyR1). Heterozygous mice expressing the human MH/central core disease RyR1 R163C mutation exhibit MH when exposed to halothane or heat stress. Considering that many MH symptoms resemble those that could ensue from a mitochondrial dysfunction (e.g. metabolic acidosis and hyperthermia) and that MH-susceptible mice or humans have a higher than normal cytoplasmic Ca(2+) concentration at rest, we evaluated the role of mitochondria in skeletal muscle from R163C compared with wild type mice under basal (untriggered) conditions. R163C skeletal muscle exhibited a significant increase in matrix Ca(2+), increased reactive oxygen species production, lower expression of mitochondrial proteins, and higher mtDNA copy number. These changes, in conjunction with lower myoglobin and glycogen contents, Myh4 and GAPDH transcript levels, GAPDH activity, and lower glucose utilization suggested a switch to a compromised bioenergetic state characterized by both low oxidative phosphorylation and glycolysis. The shift in bioenergetic state was accompanied by a dysregulation of Ca(2+)-responsive signaling pathways regulated by calcineurin and ERK1/2. Chronically elevated resting Ca(2+) in R163C skeletal muscle elicited the maintenance of a fast-twitch fiber program and the development of insulin resistance-like phenotype as part of a metabolic adaptation to the R163C RyR1 mutation.

  5. Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions.

    Science.gov (United States)

    Wong, Joseph; Baddeley, David; Bushong, Eric A; Yu, Zeyun; Ellisman, Mark H; Hoshijima, Masahiko; Soeller, Christian

    2013-06-04

    We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²⁺ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.

  6. Fluorescence probe study of Ca2+-dependent interactions of calmodulin with calmodulin-binding peptides of the ryanodine receptor.

    Science.gov (United States)

    Gangopadhyay, Jaya Pal; Grabarek, Zenon; Ikemoto, Noriaki

    2004-10-22

    We have used a highly environment-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan) to study the interaction between calmodulin (CaM) and a CaM-binding peptide of the ryanodine receptor (CaMBP) and its sub-fragments F1 and F4. Badan was attached to the Thr34Cys mutant of CaM (CaM-badan). Ca(2+) increase in a physiological range of Ca(2+) (0.1-2 microM) produced about 40 times increase in the badan fluorescence. Upon binding to CaMBP, the badan fluorescence of apo-CaM showed a small increase at a slow rate; whereas that of Ca-CaM showed a large decrease at a very fast rate. Upon binding of CaM to the badan-labeled CaMBP, the badan fluorescence showed a small and slow increase at low Ca(2+), and a large and fast increase at high Ca(2+). Thus, the badan probe attached to CaM Cys(34) can be used to monitor conformational changes occurring not only in CaM, but also those in the CaM-CaMBP interface. Based on our results we propose that both the interaction interface and the global conformation of the CaM-CaMBP complex are altered by calcium.

  7. Stable expression and functional characterisation of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides.

    Science.gov (United States)

    Troczka, Bartlomiej J; Williams, Alan J; Williamson, Martin S; Field, Linda M; Lüemmen, Peter; Davies, T G Emyr

    2015-10-01

    Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella, a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding.

  8. Mediation of autophagic cell death by type 3 ryanodine receptor (RyR3 in adult hippocampal neural stem cells

    Directory of Open Access Journals (Sweden)

    Kyung Min eChung

    2016-05-01

    Full Text Available Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs and IP3 receptors (IP3Rs, the main Ca2+ release channels located in endoplasmic reticulum (ER membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN cells utilizing an insulin withdrawal model of autophagic cell death. Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs — especially RyR3 — were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished autophagic cell death of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology.

  9. HIV-1 Tat activates neuronal ryanodine receptors with rapid induction of the unfolded protein response and mitochondrial hyperpolarization.

    Directory of Open Access Journals (Sweden)

    John P Norman

    Full Text Available Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1 is ultimately refractory to highly active antiretroviral therapy (HAART because of failure of complete virus eradication in the central nervous system (CNS, and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR, followed by the unfolded protein response (UPR and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.

  10. Amyloid β production is regulated by β2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

    Science.gov (United States)

    Bussiere, Renaud; Lacampagne, Alain; Reiken, Steven; Liu, Xiaoping; Scheuerman, Valerie; Zalk, Ran; Martin, Cécile; Checler, Frederic; Marks, Andrew R; Chami, Mounia

    2017-06-16

    Alteration of ryanodine receptor (RyR)-mediated calcium (Ca(2+)) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca(2+) release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca(2+) levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca(2+) signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca(2+) leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca(2+) leakage may be a therapeutic approach to treat AD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

    Science.gov (United States)

    Di Croce, D; Trinks, P W; Grifo, M B; Takara, D; Sánchez, G A

    2015-11-01

    The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

  12. Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

    Science.gov (United States)

    Chen, L; Sumbilla, C; Lewis, D; Zhong, L; Strock, C; Kirtley, M E; Inesi, G

    1996-05-01

    Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

  13. Luteolin improves cardiac dysfunction in heart failure rats by regulating sarcoplasmic reticulum Ca2+-ATPase 2a

    Science.gov (United States)

    Hu, Wenjing; Xu, Tongda; Wu, Pei; Pan, Defeng; Chen, Junhong; Chen, Jing; Zhang, Buchun; Zhu, Hong; Li, Dongye

    2017-01-01

    We previously found that luteolin (Lut) appeared to improve the contractility of cardiomyocytes during ischemia/reperfusion in rats. The enhancement was associated with the alteration in sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a). This finding prompted us to consider if the mechanism worked in heart failure (HF). We studied the regulation of SERCA2a by Lut in failing cardiomyocytes and intact heart of rats. Improvement of contractility and the mechanisms centered on SERCA2a were studied in isolated cardiomyocytes and intact heart. We found that Lut significantly improved contractility and Ca2+ transients, ameliorated expression, activity and stability of SERCA2a and upregulated expression of small ubiquitin-related modifier (SUMO) 1, which is a newfound SERCA2a regulator. Lut also increased phosphorylation of protein kinase B (Akt), phospholaban (PLB) and sumoylation of SERCA2a, specificity protein 1 (Sp1). Transcriptions of SUMO1 and SERCA2a were concurrently increased. Inhibition of posphatidylinositol 3 kinase/Akt (PI3K/Akt) pathway and SERCA2a activity both markedly abolished Lut-induced benefits in vitro and in vivo. Lut upregulated the expression ratio of Bcl-2/Bax, caspase-3/cleaved-Caspase3. Meanwhile, Lut ameliorated the myocardium fibrosis of HF. These discoveries provide an important potential therapeutic strategy that Lut targeted SERCA2a SUMOylation related to PI3K/Akt-mediated regulations on rescuing the dysfunction of HF.

  14. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    Science.gov (United States)

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica

    2011-07-01

    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression.

  15. Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle.

    Science.gov (United States)

    Du, G G; Ashley, C C; Lea, T J

    1994-12-01

    Thapsigargin has been reported to inhibit ATP-dependent Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca2+ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca2+ content of the SR. The SR was first depleted of Ca2+ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 microM was required to inhibit Ca2+ loading by 50%. In contrast, another SR Ca2+ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 microM and total inhibition at 50 microM. When cyclopiazonic acid (100 microM) was applied after, rather than during, Ca2+ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 microM), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca2+ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.

  16. Influence of inorganic phosphate and pH on sarcoplasmic reticular ATPase in skinned muscle fibres of Xenopus laevis.

    Science.gov (United States)

    Stienen, G J; Papp, Z; Zaremba, R

    1999-08-01

    1. The influence of 30 mM inorganic phosphate (Pi) and pH (6.2-7.4) on the rate of ATP utilization was determined in mechanically skinned bundles of myofibrils from the iliofibularis muscle of Xenopus laevis at approximately 5 C. 2. BDM (2,3-butanedione monoxime; 10 mM) depressed isometric force production and actomyosin (AM) ATPase activity equally. Therefore sarcoplasmic reticular (SR) ATPase activity could be determined by extrapolation of the total ATPase activity to zero force. 3. The SR ATPase activity without added Pi at pH 7.1 was 42 +/- 2 % of the total ATPase activity. Addition of 30 mM Pi reduced SR ATPase activity slightly, by 9 +/- 5 %, and depressed force by 62 +/- 2 % and AM ATPase activity by 21 +/- 6 %. 4. At pH 6.2, force, SR ATPase activity and AM ATPase activity were reduced by 21 +/- 5, 61 +/- 5 and 10 +/- 4 % of their respective values at pH 7.1. 5. The SR ATPase activity at 30 mM Pi and pH 6.2 was reduced markedly to 20 +/- 6 % of the value under control conditions, suggesting that the maximum rate of Ca2+ uptake during muscle fatigue was strongly depressed. This reduction was larger than expected on the basis of the effects of Pi and pH alone.

  17. Cellular mechanisms of reduced sarcoplasmic reticulum Ca2+ content in L-thyroxin-induced rat ventricular hypertrophy

    Institute of Scientific and Technical Information of China (English)

    Lai-jing SONG; Guan-lei WANG; Jie LIU; Qin-ying QIU; Jing-hua OU; Yong-yuan GUAN

    2008-01-01

    Aim:To examine how the sarcoplasmic reticulum (SR) Ca2+ content changes and the underlying mechanism in L-thyroxin-induced cardiac hypertrophy. Methods:Echocardiography was used to confirm the establishment of the cardiac hypertro-phy model. The confocal microscopy and fluorescent indicator Fluo-3 was ap-plied to examine the intracellular Ca2+ concentration ([Ca2+]I), the Ca2+ sparks, and the caffeine-induced Ca2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca2+-ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. Results:L-thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca2+ transient in the Ca2+-free solution was detected. The spontaneous Ca2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca2+]I increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca2+-ATPase was decreased in the hypertrophied ventricle tissue. Conclusion:The results suggested that the reduced SR Ca2+ content may be associated with an increased Ca2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca2+ handling during hypertrophy in hyperthyroidism rats.

  18. Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study.

    Science.gov (United States)

    Picard, Martin; Dahmane, Tassadite; Garrigos, Manuel; Gauron, Carole; Giusti, Fabrice; le Maire, Marc; Popot, Jean-Luc; Champeil, Philippe

    2006-02-14

    Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca2+-ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics ("Gulliver" effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle.

  19. Luteolin Exerts Cardioprotective Effects through Improving Sarcoplasmic Reticulum Ca2+-ATPase Activity in Rats during Ischemia/Reperfusion In Vivo

    Directory of Open Access Journals (Sweden)

    Changsheng Nai

    2015-01-01

    Full Text Available The flavonoid luteolin exists in many types of fruits, vegetables, and medicinal herbs. Our previous studies have demonstrated that luteolin reduced ischemia/reperfusion (I/R injury in vitro, which was related with sarcoplasmic reticulum Ca2+-ATPase (SERCA2a activity. However, the effects of luteolin on SERCA2a activity during I/R in vivo remain unclear. To investigate whether luteolin exerts cardioprotective effects and to monitor changes in SERCA2a expression and activity levels in vivo during I/R, we created a myocardial I/R rat model by ligating the coronary artery. We demonstrated that luteolin could reduce the myocardial infarct size, lactate dehydrogenase release, and apoptosis during I/R injury in vivo. Furthermore, we found that luteolin inhibited the I/R-induced decrease in SERCA2a activity in vivo. However, neither I/R nor luteolin altered SERCA2a expression levels in myocardiocytes. Moreover, the PI3K/Akt signaling pathway played a vital role in this mechanism. In conclusion, the present study has confirmed for the first time that luteolin yields cardioprotective effects against I/R injury by inhibiting the I/R-induced decrease in SERCA2a activity partially via the PI3K/Akt signaling pathway in vivo, independent of SERCA2a protein level regulation. SERCA2a activity presents a novel biomarker to assess the progress of I/R injury in experimental research and clinical applications.

  20. An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity.

    Science.gov (United States)

    Takano, Kyoko; Liu, Dan; Tarpey, Patrick; Gallant, Esther; Lam, Alex; Witham, Shawn; Alexov, Emil; Chaubey, Alka; Stevenson, Roger E; Schwartz, Charles E; Board, Philip G; Dulhunty, Angela F

    2012-10-15

    Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca(2+) release channels. These RyR proteins play a major role in the regulation of Ca(2+) signaling in many cells. Utilizing exome capture and deep sequencing of genes on the X-chromosome, we have identified a mutation in CLIC2 (c.303C>G, p.H101Q) which is associated with X-linked intellectual disability (ID), atrial fibrillation, cardiomegaly, congestive heart failure (CHF), some somatic features and seizures. Functional studies of the H101Q variant indicated that it stimulated rather than inhibited the action of RyR channels, with channels remaining open for longer times and potentially amplifying Ca(2+) signals dependent on RyR channel activity. The overly active RyRs in cardiac and skeletal muscle cells and neuronal cells would result in abnormal cardiac function and trigger post-synaptic pathways and neurotransmitter release. The presence of both cardiomegaly and CHF in the two affected males and atrial fibrillation in one are consistent with abnormal RyR2 channel function. Since the dysfunction of RyR2 channels in the brain via 'leaky mutations' can result in mild developmental delay and seizures, our data also suggest a vital role for the CLIC2 protein in maintaining normal cognitive function via its interaction with RyRs in the brain. Therefore, our patients appear to suffer from a new channelopathy comprised of ID, seizures and cardiac problems because of enhanced Ca(2+) release through RyRs in neuronal cells and cardiac muscle cells.

  1. Molecular cloning, characterization and expression profiling of a ryanodine receptor gene in Asian corn borer, Ostrinia furnacalis (Guenee.

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    Li Cui

    Full Text Available Ryanodine receptor (RyR Ca(2+ release channel is the target of diamide insecticides, which show selective insecticidal activity against lepidopterous insects. To study the molecular mechanisms underlying the species-specific action of diamide insecticides, we have cloned and characterized the entire cDNA sequence of RyR from Ostrinia furnacalis (named as OfRyR. The OfRyR mRNA has an Open Reading Frame of 15324 bp nucleotides and encodes a 5108 amino acid polypeptide that displays 79-97% identity with other insects RyR proteins and shows the greatest identity with Cnaphalocrocis medinalis RyR (97%. Quantitative real-time PCR showed that the OfRyR was expressed at the lowest level in egg and the highest level in adult. The relative expression level of OfRyR in first, third and fifth-instar larva were 1.28, 1.19 and 1.99 times of that in egg. Moreover, two alternative splicing sites were identified in the OfRyR gene. One pair of mutually exclusive exons (a/b were present in the central part of the predicted SPRY domain, and an optional exon (c was located between the third and fourth RyR domains. Diagnostic PCR demonstrated that exons a and b existed in all developmental stages of OfRyR cDNA, but exon c was not detected in the egg cDNA. And the usage frequencies of these exons showed a significant difference between different developmental stages. These results provided the crucial basis for the functional expression of OfRyR and for the discovery of compound with potentially selective insect activtity.

  2. N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins*

    Science.gov (United States)

    Girgenrath, Tanya; Mahalingam, Mohana; Svensson, Bengt; Nitu, Florentin R.; Cornea, Razvan L.; Fessenden, James D.

    2013-01-01

    We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca2+ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure. PMID:23585572

  3. N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

    Science.gov (United States)

    Girgenrath, Tanya; Mahalingam, Mohana; Svensson, Bengt; Nitu, Florentin R; Cornea, Razvan L; Fessenden, James D

    2013-05-31

    We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

  4. Effects of nifedipine and ryanodine on adrenergic neurogenic contractions of rat vas deferens: evidence for a pulse-to-pulse change in Ca2+ sources.

    OpenAIRE

    Bültmann, R; von Kügelgen, I; Starke, K

    1993-01-01

    1. The effects of nifedipine and ryanodine on the adrenergic component of neurogenic contractions of the rat isolated vas deferens were studied in an attempt to identify the sources of Ca2+ mediating the contraction. The tissue was electrically stimulated by single pulses or pairs of widely spaced pulses. The purinergic component of contraction was suppressed by the presence of 300 microM suramin. 2. In Mg(2+)-free medium, nifedipine (0.01-10 microM) reduced the first and, to a greater extent...

  5. Ecstacy-induced delayed rhabdomyolysis and neuroleptic malignant syndrome in a patient with a novel variant in the ryanodine receptor type 1 gene.

    Science.gov (United States)

    Russell, T; Riazi, S; Kraeva, N; Steel, A C; Hawryluck, L A

    2012-09-01

    We present the case of a 20-year-old woman who developed rhabdomyolysis, disseminated intravascular coagulopathy and multi-organ failure induced by ecstasy. Following initial improvement, she developed delayed rhabdomyolysis then haloperidol-induced neuroleptic malignant syndrome, which was treated with a total of 50 mg.kg(-1) dantrolene. Subsequent genetic testing revealed a novel potentially pathogenic variant in the ryanodine receptor type 1 gene. However, caffeine-halothane contracture testing of the patient's mother who carried the same gene variant was negative for malignant hyperthermia.

  6. Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.

    Science.gov (United States)

    Cantilina, T; Sagara, Y; Inesi, G; Jones, L R

    1993-08-15

    Vesicular fragments of skeletal (LSR) and cardiac (CSR) sarcoplasmic reticulum were compared with the aim of defining the effect of a monoclonal phospholamban (Pl) antibody (Ab). The Pl Ab has no effect on LSR, while enhancing the Ca2+ transport rates of CSR at Ca2+ concentrations below saturation. We found no direct effect of the Pl Ab on Ca2+ binding by the ATPase in the absence of ATP. Equilibrium measurements of Ca2+ binding yield positively cooperative isotherms which are best fit with a two-interacting sites equation. LSR and CSR display nearly identical affinities for Ca2+, and no effect of the Pl Ab is observed. Taking advantage of a stable CrATP-enzyme complex, we demonstrated that the stoichiometric ratio of occluded Ca2+ to catalytic sites is 2 in either LSR or CSR and that the addition of Pl Ab does not affect the Ca2+ concentration dependence of Ca2+ occluded after equilibration of the system. Interestingly, the cooperative interaction between the two Ca2+ sites is lost in the occluded state, with only one of the two sites acquiring lumenal exposure. The concentration dependence of Ca2+ inhibition of CSR ATPase phosphorylation with Pi is also unaffected by the Pl Ab. Contrary to the lack of Pl Ab effect on reactions measured at equilibrium, enhancement of phosphorylated intermediate formation by the Pl Ab is obtained in kinetic experiments in which nonsaturating Ca2+ and ATP are added to CSR preincubated with EGTA. Therefore, Ab binding to Pl reduces specifically the activation energy for a slow transition triggered by Ca2+ binding, with consequent enhancement of overall kinetics under conditions enhancing the rate-limiting contribution of this transition.

  7. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

    Directory of Open Access Journals (Sweden)

    Lu Fang-hao

    2010-06-01

    Full Text Available Abstract Communication between the SR (sarcoplasmic reticulum, SR and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM. Although it has been demonstrated that CaR (calcium sensing receptor activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re, the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  8. Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation.

    Science.gov (United States)

    Lu, Fang-hao; Tian, Zhiliang; Zhang, Wei-hua; Zhao, Ya-jun; Li, Hu-lun; Ren, Huan; Zheng, Hui-shuang; Liu, Chong; Hu, Guang-xia; Tian, Ye; Yang, Bao-feng; Wang, Rui; Xu, Chang-qing

    2010-06-17

    Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

  9. Decrease in sarcoplasmic reticulum calcium content, not myofilament function, contributes to muscle twitch force decline in isolated cardiac trabeculae

    Science.gov (United States)

    Milani-Nejad, Nima; Brunello, Lucia; Gyorke, Sándor; Janssen, Paul M.L.

    2014-01-01

    We set out to determine the factors responsible for twitch force decline in isolated intact rat cardiac trabeculae. The contractile force of trabeculae declined over extended periods of isometric twitch contractions. The force-frequency relationship within the frequency range of 4–8 Hz, at 37 °C, became more positive and the frequency optimum shifted to higher rates with this decline in baseline twitch tensions. The post-rest potentiation (37 °C), a phenomenon highly dependent on calcium handling mechanisms, became more pronounced with decrease in twitch tensions. We show that the main abnormality during muscle run-down was not due to a deficit in the myofilaments; maximal tension achieved using a K+ contracture protocol was either unaffected or only slightly decreased. Conversely, the sarcoplasmic reticulum (SR) calcium content, as assessed by rapid cooling contractures (from 27 °C to 0 °C), decreased, and had a close association with the declining twitch tensions (R2 ~ 0.76). SR Ca2+-ATPase, relative to Na+/Ca2+ exchanger activity, was not altered as there was no significant change in paired rapid cooling contracture ratios. Furthermore, confocal microscopy detected no abnormalities in the overall structure of the cardiomyocytes and t-tubules in the cardiac trabeculae (~23 °C). Overall, the data indicates that the primary mechanism responsible for force run-down in multi-cellular cardiac preparations is a decline in the SR calcium content and not the maximal tension generation capability of the myofilaments. PMID:25056841

  10. Phospholamban Modulates the Functional Coupling between Nucleotide Domains in Ca-ATPase Oligomeric Complexes in Cardiac Sarcoplasmic Reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.; Yao, Qing; Soares, Thereza A.; Squier, Thomas C.; Bigelow, Diana J.

    2009-03-24

    Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys514 with fluorescein-5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon PKA-dependent phosphorylation of PLB, a second-order dependence between enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamido-fluorescein (IAF) bound to Cys674 within the N- or P-domains respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 ± 2 Å decrease in the proximity between FITC sites within the N-domain and a 9 ± 3 Å increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.

  11. Pressure effects on the interactions of the sarcoplasmic reticulum calcium transport enzyme with calcium and dinitrophenyl phosphate.

    Science.gov (United States)

    Hasselbach, W

    1988-01-01

    The effect of hydrostatic pressure on the calcium-dependent hydrolysis of dinitrophenyl phosphate by the sarcoplasmic calcium transport enzyme has been studied. The magnesium dinitrophenyl phosphate complex is the true substrate of the enzyme (K = 7000 M-1) by which it is hydrolyzed at 20 degrees C with a turnover rate of 4 s-1. Activation by calcium ions occurs between 0.1 and 1 microM as observed for ATP hydrolysis. The activation volume of the enzyme saturated with both ligands exhibits pronounced pressure-dependence, rising from 25 ml/mol at atmospheric pressure to 80 ml/mol at 100 MPa. The apparent binding volumes for magnesium dinitrophenyl phosphate and calcium are likewise pressure-dependent. The volume changes connected with the binding of magnesium dinitrophenyl phosphate is quite small approaching zero at 100 MPa. The apparent binding volume for calcium greatly increases with pressure from 35 ml/mol at atmospheric pressure to 150 ml/mol at 70 MPa. A nearly constant binding volume of approximately 40 ml/mol results if the effect of pressure on the respective rate constants that contribute to the apparent binding constant, is taken into account. The pressure-dependence of enzyme activity at subsaturating calcium concentrations yields an activation volume of 250 ml/mol related to the rate of calcium binding indicating the occurrence of a transient large volume expansion of the enzyme complex. The volume changes observed for the calcium-dependent interaction of the enzyme with magnesium dinitrophenyl phosphate well agree with that found for magnesium p-nitrophenyl phosphate (W. Hasselbach and L. Stephan,Z. Naturforsch. 42 c, 641-652 (1987)) indicating that the found volume changes are intrinsic properties of the transport enzyme, independent of the respective energy donor.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres.

    Science.gov (United States)

    Kabbara, A A; Stephenson, D G

    1994-10-01

    The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Role of SERCA and the sarcoplasmic reticulum calcium content on calcium waves propagation in rat ventricular myocytes.

    Science.gov (United States)

    Salazar-Cantú, Ayleen; Pérez-Treviño, Perla; Montalvo-Parra, Dolores; Balderas-Villalobos, Jaime; Gómez-Víquez, Norma L; García, Noemí; Altamirano, Julio

    2016-08-15

    In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

  14. Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Topp, M R; Blasie, J K

    1983-11-08

    The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.

  15. Single-channel properties of the sarcoplasmic reticulum calcium-release channel in slow- and fast-twitch muscles of Rhesus monkeys.

    Science.gov (United States)

    Bastide, B; Mounier, Y

    1998-08-01

    RyR1 is the main isoform of ryanodine receptor expressed in fast- and slow-twitch mammalian skeletal muscles although differences in Ca2+-release kinetics and properties have been reported. Single-channel measurements reveal that a large proportion (82%) of Ca2+-release channels measured in slow-twitch muscle preparations have properties similar to those of the Ca2+-release channels of fast-twitch preparations, i.e. the same conductance, an identical sensitivity to caffeine and a bell-shaped Ca2+ activation curve for pCa (-log10[Ca2+]) 7 to 3. A low proportion (18%) of Ca2+-release channels observed in preparations from slow-twitch muscles were characterized by a very high activity level. These channels were not inhibited at a millimolar concentration of Ca2+. Our data suggest that the different properties of Ca2+ release in slow- and fast-twitch muscles might not be related to intrinsic properties of the Ca2+-release channels of each type of muscle but rather to the co-expression of two isoforms of ryanodine receptor and the lower amount of Ca2+-release channels expressed in slow- than in fast-twitch muscles.

  16. The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

    Science.gov (United States)

    Laver, Derek R; Hamada, Tomoyo; Fessenden, James D; Ikemoto, Noriaki

    2007-12-01

    In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

  17. Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin.

    Science.gov (United States)

    Fruen, Bradley R; Balog, Edward M; Schafer, Janet; Nitu, Florentin R; Thomas, David D; Cornea, Razvan L

    2005-01-11

    Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).

  18. Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.

    Directory of Open Access Journals (Sweden)

    James D Fessenden

    Full Text Available The type 1 ryanodine receptor (RyR1 is an intracellular Ca(2+ release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET method using a fused green fluorescent protein (GFP donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33% was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10 tag. A high degree of energy transfer (∼66% indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

  19. Forster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.

    Directory of Open Access Journals (Sweden)

    James D Fessenden

    Full Text Available BACKGROUND: While the static structure of the intracellular Ca(2+ release channel, the ryanodine receptor type 1 (RyR1 has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Förster resonance energy transfer (FRET methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescent probes. METHODOLOGY/PRINCIPAL FINDINGS: A novel site-specific labeling method is presented that targets a FRET acceptor, Cy3NTA to 10-residue histidine (His tags engineered into RyR1. Cy3NTA, comprised of the fluorescent dye Cy3, coupled to two Ni(2+/nitrilotriacetic acid moieties, was synthesized and functionally tested for binding to His-tagged green fluorescent protein (GFP. GFP fluorescence emission and Cy3NTA absorbance spectra overlapped significantly, indicating that FRET could occur (Förster distance = 6.3 nm. Cy3NTA bound to His(10-tagged GFP, quenching its fluorescence by 88%. GFP was then fused to the N-terminus of RyR1 and His(10 tags were placed either at the N-terminus of the fused GFP or between GFP and RyR1. Cy3NTA reduced fluorescence of these fusion proteins by 75% and this quenching could be reversed by photobleaching Cy3, thus confirming GFP-RyR1 quenching via FRET. A His(10 tag was then placed at amino acid position 1861 and FRET was measured from GFP located at either the N-terminus or at position 618 to Cy3NTA bound to this His tag. While minimal FRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position 618 to Cy3NTA at position 1861, thus indicating that these sites are in close proximity to each other. CONCLUSIONS/SIGNIFICANCE: These findings illustrate the potential of this site-specific labeling system for use in future FRET-based experiments to elucidate novel aspects of RyR1

  20. Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.

    Science.gov (United States)

    Fessenden, James D; Mahalingam, Mohana

    2013-01-01

    The type 1 ryanodine receptor (RyR1) is an intracellular Ca(2+) release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

  1. Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform.

    Science.gov (United States)

    Picher, M; Decrouy, A; Rousseau, E

    1996-02-21

    Sarcoplasmic reticulum (SR) K+ channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (gamma(max) = 250 pS; Km = 33 mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (delta Gi) and the effective gating charge (Zs): delta Gi = 0.27 kcal/mol and Zs = -1.19 in 250 mM potassium gluconate (K-gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K+ channel shares several biophysical properties with the cardiac isoform: g = 180 pS, delta Gi = 0.75 kcal/mol, Zs = -1.45 in 150 mM K-gluconate, and a similar sigmoid P(o)/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K+ channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca2+ (0.1 microM-1 mM) or Mg2+ (0.25-1 mM), as well as by cGMP (25-100 microM), lemakalim (1-100 microM), glyburide (up to 10 microM) or charybdotoxin (45-200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K+ channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes.

  2. Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.

    Science.gov (United States)

    Dalton, K A; East, J M; Mall, S; Oliver, S; Starling, A P; Lee, A G

    1998-02-01

    The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase

  3. Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle.

    Science.gov (United States)

    Baylor, S M; Hollingworth, S

    2003-08-15

    Experiments were carried out to compare the amplitude and time course of Ca2+ release from the sarcoplasmic reticulum (SR) in intact slow-twitch and fast-twitch mouse fibres. Individual fibres within small bundles were injected with furaptra, a low-affinity, rapidly responding Ca2+ indicator. In response to a single action potential at 16 degrees C, the peak amplitude and half-duration of the change in myoplasmic free [Ca2+] (Delta[Ca2+]) differed significantly between fibre types (slow-twitch: peak amplitude, 9.4 +/- 1.0 microM (mean +/- S.E.M.); half-duration, 7.7 +/- 0.6 ms; fast-twitch: peak amplitude 18.5 +/- 0.5 microM; half-duration, 4.9 +/- 0.3 ms). SR Ca2+ release was estimated from Delta[Ca2+] with a computational model that calculated Ca2+ binding to the major myoplasmic Ca2+ buffers (troponin, ATP and parvalbumin); buffer concentrations and reaction rate constants were adjusted to reflect fibre-type differences. In response to an action potential, the total concentration of released Ca2+ (Delta[CaT]) and the peak rate of Ca2+ release ((d/dt)Delta[CaT]) differed about 3-fold between the fibre types (slow-twitch: Delta[CaT], 127 +/- 7 microM; (d/dt)Delta[CaT], 70 +/- 6 microM ms-1; fast-twitch: Delta[CaT], 346 +/- 6 microM; (d/dt)Delta[CaT], 212 +/- 4 microM ms-1). In contrast, the half-duration of (d/dt)Delta[CaT] was very similar in the two fibre types (slow-twitch, 1.8 +/- 0.1 ms; fast-twitch, 1.6 +/- 0.0 ms). When fibres were stimulated with a 5-shock train at 67 Hz, the peaks of (d/dt)Delta[CaT] in response to the second and subsequent shocks were much smaller than that due to the first shock; the later peaks, expressed as a fraction of the amplitude of the first peak, were similar in the two fibre types (slow-twitch, 0.2-0.3; fast-twitch, 0.1-0.3). The results support the conclusion that individual SR Ca2+ release units function similarly in slow-twitch and fast-twitch mammalian fibres.

  4. Phosphorylation of anchoring protein by calmodulin protein kinase associated to the sarcoplasmic reticulum of rabbit fast-twitch muscle.

    Science.gov (United States)

    Damiani, E; Sacchetto, R; Margreth, A

    2000-12-09

    Regulatory phosphorylation of phospholamban and of SR Ca(2+)-ATPase SERCA2a isoform by endogenous CaM-K II in slow-twitch skeletal and cardiac sarcoplasmic reticulum (SR) is well documented, but much less is known of the exact functional role of CaM K II in fast-twitch muscle SR. Recently, it was shown that RNA splicing of brain-specific alpha CaM K II, gives rise to a truncated protein (alpha KAP), consisting mainly of the association domain, serving to anchor CaM K II to SR membrane in rat skeletal muscle [Bayer, K.-U., et al. (1998) EMBO J. 19, 5598-5605]. In the present study, we searched for the presence of alpha KAP in sucrose-density purified SR membrane fractions from representative fast-twitch and slow-twitch limb muscles, both of the rabbit and the rat, using immunoblot techniques and antibody directed against the association domain of alpha CaM K II. Putative alpha KAP was immunodetected as a 23-kDa electrophoretic component on SDS-PAGE of the isolated SR from fast-twitch but not from slow-twitch muscle, and was further identified as a specific substrate of endogenous CaM K II, in the rabbit. Immunodetected, (32)P-labeled, non-calmodulin binding protein, behaved as a single 23-kDa protein species under several electrophoretic conditions. The 23-kDa protein, with defined properties, was isolated as a complex with 60-kDa delta CaM K II isoform, by sucrose-density sedimentation analysis. Moreover, we show here that putative alphaKAP, in spite of its inability to bind CaM in ligand blot overlay, co-eluted with delta CaM K II from CaM-affinity columns. That raises the question of whether CaM K II-mediated phosphorylation of alpha KAP and triadin together might be involved in a molecular signaling pathway important for SR Ca(2+)-release in fast-twitch muscle SR.

  5. Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle.

    Science.gov (United States)

    Launikonis, B S; Stephenson, D G

    1997-10-15

    1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 micrograms ml-1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min-1. Saponin concentrations up to 150 micrograms ml-1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 micrograms ml-1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 micrograms ml-1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min-1 in the presence of 150 micrograms ml-1 saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 microM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 microns) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 micrograms ml-1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species

  6. Comparison of the kinetics of calcium transport in vesicular dispersions and oriented multilayers of isolated sarcoplasmic reticulum membranes.

    Science.gov (United States)

    Pierce, D H; Scarpa, A; Trentham, D R; Topp, M R; Blasie, J K

    1983-12-01

    Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions. The slow phase, generally believed to be associated with the translocation of Ca+2, across the membrane profile, appears to be the same process in SR dispersions and multilayers through their virtually identical rate constants

  7. 大鼠 H9c2心肌细胞缺氧/复氧损伤模型肌浆网钙调控相关蛋白表达及当归补血汤的干预作用%Danggui Buxue Decoction on expression of sarcoplasmic reticulum calcium regulatory protein in H9c2 myocardial cell model with hypoxia/reoxygenation damage

    Institute of Scientific and Technical Information of China (English)

    周春刚; 汤加; 李卿; 徐辰; 张志斌

    2016-01-01

    Objective To establish the model of hypoxia / reoxygenation damage in H9c2 rat myocardial cell and observe the expression of myocardial sarcoplasmic reticulum calcium regulatory protein and the effect of Danggui Bux-ue Decoction on calcium overload in H9c2 cells with hypoxia / reoxygenation injury. Methods JC-1 staining was used to detect the mitochondrial membrane potential by flow cytometry,Fluo-3 AM calcium fluorescence probe for the detection of intracellular calcium concentration,The expression level of sarcoplasmic reticulum Ca transport ATP en-zyme(SERCA2a),L type calcium channel(CAV1. 3),ryanodine receptor(RyR1,RyR2),phospholamban(PLB), calsequestrin(CASQ)mRNA were detected by RT-PCR. Results Compared with the normal control group,the early apoptosis rate and intracellular calcium concentration in model group increased significantly(P 0. 05),compared with the model group. Con-clusion There are obvious calcium overload and sarcoplasmic reticulum calcium regulating proteins adjustment disor-der in the model of H9c2 rat myocardial cell with hypoxia / reoxygenation damage,Danggui Buxue Decoction can in-crease the expression of the sarcoplasmic reticulumcalcium regulating protein SERCA2a and CASQ mRNA after reoxy-genation,alleviate calcium overload,and significantly reduce the early apoptosis rate of reoxygenation damage cells.%目的:建立缺氧/复氧大鼠 H9c2心肌细胞损伤模型,观察心肌细胞肌浆网钙调控相关蛋白表达及当归补血汤对 H9c2细胞缺氧/复氧损伤钙超载的影响。方法 JC-1染色流式细胞仪检测细胞线粒体膜电位, Fluo-3 AM 钙离子荧光探针检测细胞内钙离子浓度,RT-PCR 荧光相对定量检测大鼠心肌肌浆网 Ca 转运 ATP酶(SERCA2a)、L 型钙通道(CAV1.3)、兰尼碱受体(RyR1,RyR2)、受磷蛋白(PLB)、肌集钙蛋白( CASQ)的mRNA 表达水平。结果与正常对照组比较,模型组细胞早期凋亡显著增加(P <0.05),细胞内

  8. Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Sebastian Gehlert

    Full Text Available BACKGROUND: While ryanodine receptor 1 (RyR1 critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³ results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01 post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05. pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01 in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and

  9. The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+-binding but not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor

    DEFF Research Database (Denmark)

    Søndergaard, Mads Toft; Liu, Yingjie; Larsen, Kamilla Taunsig

    2017-01-01

    (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+-binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start methionine), which markedly reduces CaM Ca2+-binding...... to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+-release by manipulating the CaM-RyR2 interaction....

  10. Cardiac ryanodine receptor gene (hRyR2) mutation underlying catecholaminergic polymorphic ventricular tachycardia in a Chinese adolescent presenting with sudden cardiac arrest and cardiac syncope

    Institute of Scientific and Technical Information of China (English)

    Ngai-Shing Mok; Ching-Wan Lam; Nai-Chung Fong; Yim-Wo Hui; Yuen-Choi Choi; Kwok-Yin Chan

    2006-01-01

    @@ Sudden cardiac death (SCD) in children and adolescents is uncommon and yet it is devastating for both victim's family and the society.Recently, it was increasingly recognized that SCD in young patients with structurally normal heart may be caused by inheritable primary electrical diseases due to the malfunction of cardiac ion channels, a disease entity known as the ion channelopathies.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a specific form of ion channelopathy which can cause cardiac syncope or SCD in young patients by producing catecholamine-induced bi-directional ventricular tachycardia (BiVT), polymorphic VT and ventricular fibrillation (VF) during physical exertion or emotion.1-7 We reported here an index case of CPVT caused by cardiac ryanodine receptor gene (hRyR2)mutation which presented as cardiac syncope and sudden cardiac arrest in a Chinese adolescent female.

  11. Sound Waves Induce Neural Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells via Ryanodine Receptor-Induced Calcium Release and Pyk2 Activation.

    Science.gov (United States)

    Choi, Yura; Park, Jeong-Eun; Jeong, Jong Seob; Park, Jung-Keug; Kim, Jongpil; Jeon, Songhee

    2016-10-01

    Mesenchymal stem cells (MSCs) have shown considerable promise as an adaptable cell source for use in tissue engineering and other therapeutic applications. The aims of this study were to develop methods to test the hypothesis that human MSCs could be differentiated using sound wave stimulation alone and to find the underlying mechanism. Human bone marrow (hBM)-MSCs were stimulated with sound waves (1 kHz, 81 dB) for 7 days and the expression of neural markers were analyzed. Sound waves induced neural differentiation of hBM-MSC at 1 kHz and 81 dB but not at 1 kHz and 100 dB. To determine the signaling pathways involved in the neural differentiation of hBM-MSCs by sound wave stimulation, we examined the Pyk2 and CREB phosphorylation. Sound wave induced an increase in the phosphorylation of Pyk2 and CREB at 45 min and 90 min, respectively, in hBM-MSCs. To find out the upstream activator of Pyk2, we examined the intracellular calcium source that was released by sound wave stimulation. When we used ryanodine as a ryanodine receptor antagonist, sound wave-induced calcium release was suppressed. Moreover, pre-treatment with a Pyk2 inhibitor, PF431396, prevented the phosphorylation of Pyk2 and suppressed sound wave-induced neural differentiation in hBM-MSCs. These results suggest that specific sound wave stimulation could be used as a neural differentiation inducer of hBM-MSCs.

  12. Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.

    Science.gov (United States)

    Novak, Atara; Barad, Lili; Lorber, Avraham; Gherghiceanu, Mihaela; Reiter, Irina; Eisen, Binyamin; Eldor, Liron; Itskovitz-Eldor, Joseph; Eldar, Michael; Arad, Michael; Binah, Ofer

    2015-08-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca(2+) handling resulting from mutations in the RyR2 or CASQ2 genes. Because CASQ2 and RyR2 are involved in different aspects of the excitation-contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca(²+) abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) from CPVT1 and CPVT2 patients carrying the RyR2(R420Q) and CASQ2(D307H) mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC-CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca(2+) ]i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca(2+) ]i . Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.

  13. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy

    Directory of Open Access Journals (Sweden)

    Ke-Yi Wang

    2015-07-01

    Full Text Available Ryanodine receptors (RyRs play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR, an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action.

  14. A computational model of spatio-temporal cardiac intracellular calcium handling with realistic structure and spatial flux distribution from sarcoplasmic reticulum and t-tubule reconstructions.

    Directory of Open Access Journals (Sweden)

    Michael A Colman

    2017-08-01

    Full Text Available Intracellular calcium cycling is a vital component of cardiac excitation-contraction coupling. The key structures responsible for controlling calcium dynamics are the cell membrane (comprising the surface sarcolemma and transverse-tubules, the intracellular calcium store (the sarcoplasmic reticulum, and the co-localisation of these two structures to form dyads within which calcium-induced-calcium-release occurs. The organisation of these structures tightly controls intracellular calcium dynamics. In this study, we present a computational model of intracellular calcium cycling in three-dimensions (3-D, which incorporates high resolution reconstructions of these key regulatory structures, attained through imaging of tissue taken from the sheep left ventricle using serial block face scanning electron microscopy. An approach was developed to model the sarcoplasmic reticulum structure at the whole-cell scale, by reducing its full 3-D structure to a 3-D network of one-dimensional strands. The model reproduces intracellular calcium dynamics during control pacing and reveals the high-resolution 3-D spatial structure of calcium gradients and intracellular fluxes in both the cytoplasm and sarcoplasmic reticulum. We also demonstrated the capability of the model to reproduce potentially pro-arrhythmic dynamics under perturbed conditions, pertaining to calcium-transient alternans and spontaneous release events. Comparison with idealised cell models emphasised the importance of structure in determining calcium gradients and controlling the spatial dynamics associated with calcium-transient alternans, wherein the probabilistic nature of dyad activation and recruitment was constrained. The model was further used to highlight the criticality in calcium spark propagation in relation to inter-dyad distances. The model presented provides a powerful tool for future investigation of structure-function relationships underlying physiological and pathophysiological

  15. Gel-based phosphoproteomics analysis of sarcoplasmic proteins in postmortem porcine muscle with pH decline rate and time differences

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Karlsson, Anders H

    2011-01-01

    Meat quality development is highly influenced by the pH decline caused by the postmortem (PM) glycolysis. Protein phosphorylation is an important mechanism in regulating the activity of glycometabolic enzymes. Here, a gel-based phosphoproteomic study was performed to analyze the protein...... phosphorylation in sarcoplasmic proteins from three groups of pigs with different pH decline rates from PM 1 to 24¿h. Globally, the fast pH decline group had the highest phosphorylation level at PM 1¿h, but lowest at 24¿h, whereas the slow pH decline group showed the reverse case. The same pattern was also...... observed in most individual bands in 1-DE. The protein phosphorylation levels of 12 bands were significantly affected by the synergy effects of pH and time (p...

  16. Sarcoplasmic reticulum calcium mobilization in right ventricular pressure-overload hypertrophy in the ferret: relationships to diastolic dysfunction and a negative treppe.

    Science.gov (United States)

    Gwathmey, J K; Morgan, J P

    1993-03-01

    In a model of right-ventricular pressure-overload hypertrophy (POH) in the ferret, action potential duration (to 90% repolarization) was found to be significantly longer (228 +/- 11 vs 314 +/- 12 ms) with no change in amplitude (85 +/- 3 vs 85 +/- 2 mV) or resting membrane potential (-79 +/- 1.5 vs -79 +/- 1 mV) for control and POH, respectively. Peak sarcoplasmic reticulum Ca2+ release (expressed as the logarithm of the fractional luminescence, -4.2 +/- 0.1 vs -4.4 +/- 0.3) and resting calcium concentrations (-5.5 +/- 0.1 vs -5.7 +/- 0.1) were not different between the two groups (control vs POH respectively). Muscles from control and POH animals demonstrated a positive force/interval relationship in the presence of physiological extracellular [Ca2+]. However, unlike muscles from control animals, muscles from animals with POH subjected to increasing frequencies of contraction in the presence of increased extracellular [Ca2+] demonstrated further impairment of diastolic relaxation and a negative treppe. Exposure of muscles from POH animals to isoproterenol returned the slowed Ca2+ uptake by the sarcoplasmic reticulum as detected with aequorin to control values, although the relaxation phase of the isometric twitch remained prolonged compared to non-hypertrophied muscles. Exposure to milrinone also abbreviated the time course of the intracellular Ca2+ transient, but did not return it to that seen in normal myocardium. The exposure of non-hypertrophied isolated muscles to caffeine resulted in similar prolongation of the isometric twitch duration to that seen in hypertrophied myocardium. Results of these experiments suggest that impaired muscle relaxation in POH reflects changes at the level of the myofilaments. Thus, although slowed intracellular calcium mobilization contributes to diastolic relaxation abnormalities, it can not be the sole factor responsible for the slowed relaxation as has been suggested.

  17. Inositol-1,4,5-trisphosphate and ryanodine-dependent Ca2+ signaling in a chronic dog model of atrial fibrillation.

    Science.gov (United States)

    Zhao, Zhi-Hong; Zhang, Hai-Cheng; Xu, Yuan; Zhang, Ping; Li, Xue-Bing; Liu, Yuan-Sheng; Guo, Ji-Hong

    2007-01-01

    Ca2+ signaling regulation plays an important role in triggering and/or maintaining atrial fibrillation (AF). Little is known about the relationship of the inositol-1,4,5-triphosphate receptors (InsP3Rs) and ryanodine receptors (RyRs) in left atrium to chronic AF. In this study, we investigated the expression and function of InsP3R1, InsP3R2 and RyR2 in a chronic dog model of AF. AF was induced in 6 dogs by rapid right atrial pacing for 24 weeks, and a sham procedure was performed in 5 dogs (control group). The intact left atrial myocytes were used to examine the expression and function of InsP3Rs, RyRs by BODIPY(O,R) TR-X ryanodine, heparin-fluorescein conjugate, and were stimulated by caffeine, ATP to release Ca2+ through RyRs, InsP3Rs separately. We also assessed the molecular components of left atrial tissue underlying the amount of RyR2, InsP3R1 and InsP3R2 determined by RT-PCR, immunohistochemistry and Western blot analysis. In the chronic AF group, the Ca2+ released through RyRs is not altered, but the Ca2+ released through InsP3Rs increased significantly. RyR2 distributed in cytosol of myocytes, cellular membrane; its expression significantly decreased in AF group compared to controls. InsP3R1 distributed in cytosol, InsP3R2 distributed not only in cytosol, cellular membrane, but also in nuclear envelope and intercalated discs. The InsP3R1 and InsP3R2 expression significantly increased in chronic AF group compared to controls. These results indicated that in a chronic dog model of AF, the expression and function of RyR2 down-regulated; on the contrary, the expression and function of InsP3R1, InsP3R2 up-regulated, and InsP3R2 may be the major InsP3Rs, which regulate intracellular or even intercellular Ca2+ signal transmission.

  18. Characterization of the functional and anatomical differences in the atrial and ventricular myocardium from three species of elasmobranch fishes

    DEFF Research Database (Denmark)

    Larsen, Julie; Bushnell, Peter; Steffensen, John Fleng

    2017-01-01

    We assessed the functional properties in atrial and ventricular myocardium (using isolated cardiac strips) of smooth dogfish (Mustelus canis), clearnose skate (Raja eglanteria), and sandbar shark (Carcharhinus plumbeus) by blocking Ca(2+) release from the sarcoplasmic reticulum (SR) with ryanodine...

  19. Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptor.

    Science.gov (United States)

    Ortiz-Capisano, M Cecilia; Reddy, Mahendranath; Mendez, Mariela; Garvin, Jeffrey L; Beierwaltes, William H

    2013-02-01

    The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

  20. Nitric oxide-induced calcium release: activation of type 1 ryanodine receptor, a calcium release channel, through non-enzymatic posttranslational modification by nitric oxide

    Directory of Open Access Journals (Sweden)

    Sho eKakizawa

    2013-10-01

    Full Text Available Nitric oxide (NO is a typical gaseous messenger involved in a wide range of biological processes. In our classical knowledge, effects of NO are largely achieved by activation of soluble guanylyl cyclase to form cyclic guanosine-3’, 5’-monophosphate. However, emerging evidences have suggested another signaling mechanism mediated by NO: S-nitrosylation of target proteins.S-nitrosylation is a covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH, and categorized into non-enzymatic posttranslational modification of proteins, contrasted to enzymatic posttranslational modification of proteins, such as phosphorylation mediated by various protein kinases.Very recently, we found novel intracellular calcium (Ca2+ mobilizing mechanism, NO-induced Ca2+ release (NICR in cerebellar Purkinje cells. NICR is mediated by type 1 ryanodine receptor (RyR1, a Ca2+ release channel expressed in endoplasmic-reticular membrane. Furthermore, NICR is indicated to be dependent on S-nitrosylation of RyR1, and involved in synaptic plasticity in the cerebellum. In this review, molecular mechanisms and functional significance of NICR, as well as non-enzymatic posttranslational modification of proteins by gaseous signals, are described.

  1. Comprehensive behavioral phenotyping of ryanodine receptor type3 (RyR3 knockout mice: Decreased social contact duration in two social interaction tests

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    Naoki Matsuo

    2009-05-01

    Full Text Available Dynamic regulation of the intracellular Ca2+ concentration is crucial for various neuronal functions such as synaptic transmission and plasticity, and gene expression. Ryanodine receptors (RyRs are a family of intracellular calcium release channels that mediate calcium-induced calcium release (CICR from the endoplasmic reticulum. Among the three RyR isoforms, RyR3 is preferentially expressed in the brain especially in the hippocampus and striatum. To investigate the behavioral effects of RyR3 deficiency, we subjected RyR3 knockout (RyR3-/- mice to a battery of behavioral tests. RyR3-/- mice exhibited significantly decreased social contact duration in two different social interaction tests, where two mice can freely move and make contacts with each other. They also exhibited hyperactivity and mildly impaired prepulse inhibition and latent inhibition while they did not show significant abnormalities in motor function and working and reference memory tests. These results suggest that RyR3 has an important role in locomotor activity and social behavior.

  2. Comprehensive behavioral phenotyping of ryanodine receptor type 3 (RyR3) knockout mice: decreased social contact duration in two social interaction tests.

    Science.gov (United States)

    Matsuo, Naoki; Tanda, Koichi; Nakanishi, Kazuo; Yamasaki, Nobuyuki; Toyama, Keiko; Takao, Keizo; Takeshima, Hiroshi; Miyakawa, Tsuyoshi

    2009-01-01

    Dynamic regulation of the intracellular Ca2+ concentration is crucial for various neuronal functions such as synaptic transmission and plasticity, and gene expression. Ryanodine receptors (RyRs) are a family of intracellular calcium release channels that mediate calcium-induced calcium release from the endoplasmic reticulum. Among the three RyR isoforms, RyR3 is preferentially expressed in the brain especially in the hippocampus and striatum. To investigate the behavioral effects of RyR3 deficiency, we subjected RyR3 knockout (RyR3-/-) mice to a battery of behavioral tests. RyR3-/- mice exhibited significantly decreased social contact duration in two different social interaction tests, where two mice can freely move and make contacts with each other. They also exhibited hyperactivity and mildly impaired prepulse inhibition and latent inhibition while they did not show significant abnormalities in motor function and working and reference memory tests. These results indicate that RyR3 has an important role in locomotor activity and social behavior.

  3. Differential rescue of spatial memory deficits in aged rats by L-type voltage-dependent calcium channel and ryanodine receptor antagonism.

    Science.gov (United States)

    Hopp, S C; D'Angelo, H M; Royer, S E; Kaercher, R M; Adzovic, L; Wenk, G L

    2014-11-01

    Age-associated memory impairments may result as a consequence of neuroinflammatory induction of intracellular calcium (Ca(+2)) dysregulation. Altered L-type voltage-dependent calcium channel (L-VDCC) and ryanodine receptor (RyR) activity may underlie age-associated learning and memory impairments. Various neuroinflammatory markers are associated with increased activity of both L-VDCCs and RyRs, and increased neuroinflammation is associated with normal aging. In vitro, pharmacological blockade of L-VDCCs and RyRs has been shown to be anti-inflammatory. Here, we examined whether pharmacological blockade of L-VDCCs or RyRs with the drugs nimodipine and dantrolene, respectively, could improve spatial memory and reduce age-associated increases in microglia activation. Dantrolene and nimodipine differentially attenuated age-associated spatial memory deficits but were not anti-inflammatory in vivo. Furthermore, RyR gene expression was inversely correlated with spatial memory, highlighting the central role of Ca(+2) dysregulation in age-associated memory deficits.

  4. Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

    Science.gov (United States)

    Delmotte, Philippe; Sieck, Gary C

    2015-02-01

    Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

  5. Effects of pH-treated Fish Sarcoplasmic Proteins on the Functional Properties of Chicken Myofibrillar Protein Gel Mediated by Microbial Transglutaminase.

    Science.gov (United States)

    Hemung, Bung-Orn; Chin, Koo Bok

    2014-01-01

    pH adjustment would be of advantage in improving the water holding capacity of muscle proteins. The objective of this study was to evaluate the addition of fish sarcoplasmic protein (SP) solution, which was adjusted to pH 3.0 or 12.0, neutralized to pH 7.0, and lyophilized to obtain the acid- and alkaline-treated SP samples, on the functional properties of the chicken myofibrillar protein induced by microbial transglutaminase (MTG). The solubility of alkaline-treated SP was higher than that of the acid counterpart; however, those values of the two pH-treated samples were lower than that of normal SP (pproteins (MP) extracted from chicken breast, and incubated with MTG. The shear stresses of MP with acid- and alkaline-treated SP were higher than that of normal SP. The thermal stability of MP mixture reduced upon adding SP, regardless of the pH treatment. The breaking force of MP gels with acid-treated SP increased more than those of alkaline-treated SP, while normal SP showed the highest value. The MP gel lightness increased, but cooking loss reduced, with the addition of SP. Smooth microstructure of the gel surface was observed. These results indicated that adjusting the pH of SP improved the water holding capacity of chicken myofibrillar proteins induced by MTG.

  6. Altered calcium pump and secondary deficiency of gamma-sarcoglycan and microspan in sarcoplasmic reticulum membranes isolated from delta-sarcoglycan knockout mice.

    Science.gov (United States)

    Solares-Pérez, Alhondra; Alvarez, Rocío; Crosbie, Rachelle H; Vega-Moreno, Jesús; Medina-Monares, Joel; Estrada, Francisco J; Ortega, Alicia; Coral-Vazquez, Ramón

    2010-07-01

    Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and delta-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of delta-SG isoforms in TT and SR results in a secondary deficiency of gamma-SG and microSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the delta-SG isoforms may stabilize the expression of gamma-SG and microSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle. Copyright 2010 Elsevier Ltd. All rights reserved.

  7. Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction.

    Science.gov (United States)

    Qin, Fuzhong; Yan, Chen; Patel, Ravish; Liu, Weimin; Dong, Erdan

    2006-05-15

    Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta-adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF.

  8. The Ca(2+)-ATPase of the sarcoplasmic reticulum in skeletal and cardiac muscle. An overview from the very beginning to more recent prospects.

    Science.gov (United States)

    Hasselbach, W

    1998-09-16

    The discovery of the ATP-driven calcium pump in the sarcoplasmic reticulum membranes reaches back to the postwar (World War II) years and would not be possible without the generous support by the American scientific community. It was this community that in pre- and postwar years gave shelter to many European scientists, which in return stimulated scientific development in the United States. These pre- and postwar relations helped to establish the calcium pump as a physiologically relevant mechanism in all kinds of cells. The pump and its counterpart, the calcium release channel, proved to be controlled by various intrinsic mechanisms. Rising hydrogen concentrations as occurring in ischemic muscles switch off pump activity and counteract allosterically caffeine-induced calcium release (CICR). Rising phosphate or the presence of other calcium-precipitating anions, on the other hand, prevents pump inhibition by intraluminal calcium precipitation, which, simultaneously, can increase the quantity of releasable calcium. The inactivation of CICR by removing medium chloride must be considered as a hint of additional mechanisms by which calcium-dependent activity regulation can be modified.

  9. Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

    Science.gov (United States)

    Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter

    2016-01-01

    The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

  10. Is there life in the horny layer? Dihydropyridine and ryanodine receptors in the skin of female and male chickens (Gallus domesticus).

    Science.gov (United States)

    Peltonen, Liisa M; Mänttäri, Satu

    2008-05-01

    Previous findings in pigeons and chickens show that Ca(2+) may be accumulated inside the cornified skin cells and that Ca(2+) microenvironments with a lower- or higher-than-blood concentration may exist in the skin. It has been suggested that the skin may function as a secretory pathway or a reservoir for Ca(2+) recycling. To test this hypothesis, we studied the dermis and epidermis of female and male chickens in vivo to find out whether cellular mechanisms exist for the accumulation, recycling or secretion of Ca(2+). For calcium influx and intracellular Ca(2+) release, respectively, the density of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) was examined, using high-affinity (-)-enantiomers of dihydropyridine and ryanodine labelled with fluorophores. To investigate Ca(2+) utilization in the skin, the systemic and local activity of the enzyme alkaline phosphatase (ALP) and the concentration of ionic Ca(2+) were measured in plasma and in cutaneous extracellular fluid, collected by suction blister technique. We found that both DHPRs and RyRs were present in all skin layers from dermis to horny layer. However, receptor densities were highest in the surface layers. With a basic calcium-rich diet, receptor densities were higher in males, particularly in the dermis and mid-epidermis. After a reduction in the nutritional Ca(2+) input, receptor densities in males decreased to the same level as in females, in which the receptor densities were not affected by the amount of Ca(2+) in the diet or that resulting from coming out of lay. The extracellular concentration of ionic Ca(2+) per se was not found to affect the density of DHPRs and RyRs in the skin. Spatially, RyRs seem to be located in the periphery of the sebokeratinocyte. ALP activity was shown to be lower in the extracellular fluid than in the plasma in both sexes. However, activity in both extracellular domains increased significantly in females that had come out of lay. This was probably

  11. Ryanodine receptors contribute to the induction of nociceptive input-evoked long-term potentiation in the rat spinal cord slice

    Directory of Open Access Journals (Sweden)

    Zhao Zhi-Qi

    2010-01-01

    Full Text Available Abstract Background Our previous study demonstrated that nitric oxide (NO contributes to long-term potentiation (LTP of C-fiber-evoked field potentials by tetanic stimulation of the sciatic nerve in the spinal cord in vivo. Ryanodine receptor (RyR is a downstream target for NO. The present study further explored the role of RyR in synaptic plasticity of the spinal pain pathway. Results By means of field potential recordings in the adult male rat in vivo, we showed that RyR antagonist reduced LTP of C-fiber-evoked responses in the spinal dorsal horn by tetanic stimulation of the sciatic nerve. Using spinal cord slice preparations and field potential recordings from superficial dorsal horn, high frequency stimulation of Lissauer's tract (LT stably induced LTP of field excitatory postsynaptic potentials (fEPSPs. Perfusion of RyR antagonists blocked the induction of LT stimulation-evoked spinal LTP, while Ins(1,4,5P3 receptor (IP3R antagonist had no significant effect on LTP induction. Moreover, activation of RyRs by caffeine without high frequency stimulation induced a long-term potentiation in the presence of bicuculline methiodide and strychnine. Further, in patch-clamp recordings from superficial dorsal horn neurons, activation of RyRs resulted in a large increase in the frequency of miniature EPSCs (mEPSCs. Immunohistochemical study showed that RyRs were expressed in the dorsal root ganglion (DRG neurons. Likewise, calcium imaging in small DRG neurons illustrated that activation of RyRs elevated [Ca2+]i in small DRG neurons. Conclusions These data indicate that activation of presynaptic RyRs play a crucial role in the induction of LTP in the spinal pain pathway, probably through enhancement of transmitter release.

  12. Niflumic acid hyperpolarizes the smooth muscle cells by opening BK(Ca) channels through ryanodine-sensitive Ca(2+) release in spiral modiolar artery.

    Science.gov (United States)

    Li, Li; Ma, Ke-Tao; Zhao, Lei; Si, Jun-Qiang

    2008-12-25

    The mechanism by which niflumic acid (NFA), a Cl(-) channel antagonist, hyperpolarizes the smooth muscle cells (SMCs) of cochlear spiral modiolar artery (SMA) was explored. Guinea pigs were used as subjects and perforated patch clamp and intracellular recording technique were used to observe NFA-induced response of SMC in the acutely isolated SMA preparation. The results showed that bath application of NFA, indanyloxyacetic acid 94 (IAA-94) and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) caused hyperpolarization and evoked outward currents in all cells at low resting potential (RP), but had no effects in cells at high RP. In the low RP SMCs, the average RP was about (-42.47+/-1.38) mV (n=24). Application of NFA (100 mumol/L), IAA-94 (10 mumol/L) and DIDS (200 mumol/L) shifted the RP to (13.7+/-4.3) mV (n=9, P<0.01), (11.4+/-4.2) mV (n=7, P<0.01) and (12.3+/-3.7) mV (n=8, P<0.01), respectively. These drug-induced responses were in a concentration-dependent manner. NFA-induced hyperpolarization and outward current were almost blocked by charybdotoxin (100 nmol/L), iberiotoxin (100 nmol/L), tetraethylammonium (10 mmol/L), BAPTA-AM (50 mumol/L), ryanodine (10 mumol/L) and caffeine (0.1-10 mmol/L), respectively, but not by nifedipine (100 mumol/L), CdCl2 (100 mumol/L) and Ca(2+)-free medium. It is concluded that NFA induces a release of intracellular calcium from the Ca(2+) stores and the released intracellular calcium in turn causes concentration-dependent and reversible hyperpolarization and evokes outward currents in the SMCs of the cochlear SMA via activation of the Ca(2+)-activated potassium channels.

  13. PCB 136 atropselectively alters morphometric and functional parameters of neuronal connectivity in cultured rat hippocampal neurons via ryanodine receptor-dependent mechanisms.

    Science.gov (United States)

    Yang, Dongren; Kania-Korwel, Izabela; Ghogha, Atefeh; Chen, Hao; Stamou, Marianna; Bose, Diptiman D; Pessah, Isaac N; Lehmler, Hans-Joachim; Lein, Pamela J

    2014-04-01

    We recently demonstrated that polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substitutions sensitize ryanodine receptors (RyRs), and this activity promotes Ca²⁺-dependent dendritic growth in cultured neurons. Many ortho-substituted congeners display axial chirality, and we previously reported that the chiral congener PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) atropselectively sensitizes RyRs. Here, we test the hypothesis that PCB 136 atropisomers differentially alter dendritic growth and other parameters of neuronal connectivity influenced by RyR activity. (-)-PCB 136, which potently sensitizes RyRs, enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. The dendrite-promoting activity of (-)-PCB 136 is observed at concentrations ranging from 0.1 to 100 nM and is blocked by pharmacologic RyR antagonism. Neither atropisomer alters axonal growth or cell viability. Quantification of PCB 136 atropisomers in hippocampal cultures indicates that atropselective effects on dendritic growth are not due to differential partitioning of atropisomers into cultured cells. Imaging of hippocampal neurons loaded with Ca²⁺-sensitive dye demonstrates that (-)-PCB 136 but not (+)-PCB 136 increases the frequency of spontaneous Ca²⁺ oscillations. Similarly, (-)-PCB 136 but not (+)-PCB 136 increases the activity of hippocampal neurons plated on microelectrode arrays. These data support the hypothesis that atropselective effects on RyR activity translate into atropselective effects of PCB 136 atropisomers on neuronal connectivity, and suggest that the variable atropisomeric enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure.

  14. The effects of ryanodine receptor (RYR1) mutation on natural killer cell cytotoxicity, plasma cytokines and stress hormones during acute intermittent exercise in pigs.

    Science.gov (United States)

    Ciepielewski, Z M; Stojek, W; Borman, A; Myślińska, D; Pałczyńska, P; Kamyczek, M

    2016-04-01

    Stress susceptibility has been mapped to a single recessive gene, the ryanodine receptor 1 (RYR1) gene or halothane (Hal) gene. Homozygous (Hal(nn)), mutated pigs are sensitive to halothane and susceptible to Porcine Stress Syndrome (PSS). Previous studies have shown that stress-susceptible RYR1 gene mutated homozygotes in response to restraint stress showed an increase in natural killer cell cytotoxicity (NKCC) accompanied by more pronounced stress-related hormone and anti-inflammatory cytokine changes. In order to determine the relationship of a RYR1 gene mutation with NKCC, plasma cytokines and stress-related hormones following a different stress model - exercise - 36 male pigs (representing different genotypes according to RYR1 gene mutation: NN, homozygous dominant; Nn, heterozygous; nn, homozygous recessive) were submitted to an intermittent treadmill walking. During the entire experiment the greatest level of NKCC and the greatest concentrations of interleukin (IL-) 6, IL-10, IL-12, interferon (IFN-)γ and tumor necrosis factor-α and stress-related hormones (adrenaline, prolactin, beta-endorphin) were observed in nn pigs, and the greatest concentration of IL-1 and growth hormone in NN pigs. Immunostimulatory effects of intermittent exercise on NKCC in nn pigs were concomitant with increases in IL-2, IL-12 and IFN-γ, the potent NKCC activators. Our findings suggest that stress-susceptible pigs RYR1 gene mutated pigs develop a greater level of NKCC and cytokine production in response to exercise stress. These results suggest that the heterogeneity of immunological and neuroendocrine response to exercise stress in pigs could be influenced by RYR1 gene mutation.

  15. Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6).

    Science.gov (United States)

    Mitchell, Kathryn J; Lai, F Anthony; Rutter, Guy A

    2003-03-28

    We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.

  16. The use of the indicator fluo-5N to measure sarcoplasmic reticulum calcium in single muscle fibres of the cane toad.

    Science.gov (United States)

    Kabbara, A A; Allen, D G

    2001-07-01

    1. Single fibres from the lumbrical muscles of the cane toad (Bufo marinus) were incubated in fluo-5N AM for 2 h at 35 degrees C in order to load the indicator into the sarcoplasmic reticulum. Fluo-5N is a low-affinity calcium indicator (K(Ca) 90 microM). Successful sarcoplasmic reticulum (SR) loading was indicated by a fluorescence signal that declined during contraction. 2. Confocal microscopy showed that the dye loaded principally in lines perpendicular to the long axis of the fibre that repeated each sarcomere. This is consistent with much of the dye residing in the SR. 3. To establish the site of loading, fibres were exposed to 30 mM caffeine in the presence of 20 microM 2,5-di(tert-butyl)1,4-hydroquinone (TBQ, an SR pump inhibitor) which should release most Ca(2+) from the SR; this procedure reduced the fluorescence to 46 +/- 4 % of the control value. To determine how much indicator was in the myoplasm, fibres were exposed to 100 microg ml(-1) saponin which permeabilizes the surface membrane; saponin treatment reduced the fluorescence to 51 +/- 2 % of the control value. 4. During maximally activated tetani (100 Hz stimulation rate, 22 degrees C) the component of signal from the SR declined by 33 +/- 4 %. During relaxation the SR signal recovered in two phases with time constants of 0.38 +/- 0.14 s and 10.1 +/- 1.7 s. Partially activated tetani (30 Hz stimulation rate) showed a smaller SR signal. Application of the SR Ca(2+) pump inhibitor TBQ slowed the rate of recovery of the SR signal. 5. Muscle fatigue was produced by repeated short tetani until tension was reduced to 50 %. The SR signal during the periods between tetani declined steadily and the SR Ca(2+) signal was eventually reduced to 71 +/- 8 % of the control signal. This signal recovered in two phases when the muscle was rested. An initial phase had a time constant of 1.7 +/- 0.2 s so that by 20 s of recovery the SR Ca(2+) signal was 86 +/- 7 % of control; the second phase was slower and by 5 min the

  17. Short-term effects of β2-AR blocker ICI 118,551 on sarcoplasmic reticulum SERCA2a and cardiac function of rats with heart failure.

    Science.gov (United States)

    Gong, Haibin; Li, Yanfei; Wang, Lei; Lv, Qian; Wang, Xiuli

    2016-09-01

    The study was conducted to examine the effects of ICI 118,551 on the systolic function of cardiac muscle cells of rats in heart failure and determine the molecular mechanism of selective β2-adrenergic receptor (β2-AR) antagonist on these cells. The chronic heart failure model for rats was prepared through abdominal aortic constriction and separate cardiac muscle cells using the collagenase digestion method. The rats were then divided into Sham, HF and HF+ICI 50 nM goups and cultivated for 48 h. β2-AR, Gi/Gs and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) protein expression levels in the cardiac muscle cells were evaluated by western blotting and changes in the systolic function of cardiac muscle cells based on the boundary detection system of contraction dynamics for individual cells was measured. The results showed that compared with the Sham group, the survival rate, percentage of basic contraction and maximum contraction amplitude percentage of cardiac muscle cells with heart failure decreased, Gi protein expression increased while Gs and SERCA2a protein expression decreased. Compared with the HF group, the maximum contraction amplitude percentage of cardiac muscle cells in group HF+ICI 50 nM decreased, the Gi protein expression level increased while the SERCA2a protein expression level decreased. Following the stimulation of Ca(2+) and ISO, the maximum contraction amplitude percentage of cardiac muscle cells in the HF+ICI 50 nM group was lower than that in group HF. This indicated that ICI 118,551 has negative inotropic effects on cardiac muscle cells with heart failure, which may be related to Gi protein. Systolic function of cardiac muscle cells with heart failure can therefore be reduced by increasing Gi protein expression and lowering SERCA2a protein expression.

  18. Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

    Directory of Open Access Journals (Sweden)

    Bassani R.A.

    2003-01-01

    Full Text Available Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A, Na+-Ca2+ exchanger (NCX and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake. To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW. Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM, whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM. The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.

  19. NO donors-relaxation is impaired in aorta from hypertensive rats due to a reduced involvement of K(+) channels and sarcoplasmic reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Bonaventura, Daniella; de Lima, Renata Galvão; da Silva, Roberto Santana; Bendhack, Lusiane Maria

    2011-10-24

    To examine the vasodilatation induce by the NO donors, [Ru(terpy)(bdq)NO](3+) (TERPY) and sodium nitroprusside (SNP), and to compare their effects in aortic rings from hypertensive 2K-1C and normotensive 2K rats. Vascular reactivity was performed in aortic rings pre-contracted with phenylephrine (Phe 100nM). We have analyzed the maximal relaxation (Emax) and potency (pD(2)) of NO donors. Potency of SNP was greater than TERPY in both arterial groups. The vasodilatation induced by TERPY was greater in 2K than in 2K-1C, and it was inhibited by sGC inhibitor ODQ in 2K and in 2K-1C aortic rings. ODQ did not alter the efficacy to SNP, but it reduced its potency in 2K and 2K-1C. The blockade of K(+) channels reduced the potency of TERPY only in aortic rings of 2K. On the other hand, the potency of SNP was reduced in both 2K and 2K-1C. The combination of ODQ and TEA reduced the relaxation induced by TERPY and SNP in 2K and reduced the efficacy to SNP in 2K-1C aortic rings but it had no additional effect on the TERPY relaxation in 2K-1C aortas. The production of cGMP induced by TERPY was greater than that produced by SNP, which was similarly increased in 2K and 2K-1C. Sarcoplasmic reticulum Ca-ATPase inhibition only impaired the relaxation induced by SNP in 2K aortic rings. Taken together, our results provide evidences that in this model of hypertension, impaired K(+) channels activation by TERPY and SERCA activation by SNP may contribute to decreased vasodilatation. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue.

    Science.gov (United States)

    Aureliano, Manuel; Henao, Fernando; Tiago, Teresa; Duarte, Rui O; Moura, J J G; Baruah, Bharat; Crans, Debbie C

    2008-07-07

    The general affinity of the sarcoplasmic reticulum (SR) Ca (2+)-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis( N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca (2+)-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 microM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca (2+)-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca (2+)-ATPase.

  1. Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1

    Science.gov (United States)

    Glukhov, Alexey V.; Kalyanasundaram, Anuradha; Lou, Qing; Hage, Lori T.; Hansen, Brian J.; Belevych, Andriy E.; Mohler, Peter J.; Knollmann, Björn C.; Periasamy, Muthu; Györke, Sandor; Fedorov, Vadim V.

    2015-01-01

    Aims Loss-of-function mutations in Calsequestrin 2 (CASQ2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT patients also exhibit bradycardia and atrial arrhythmias for which the underlying mechanism remains unknown. We aimed to study the sinoatrial node (SAN) dysfunction due to loss of CASQ2. Methods and results In vivo electrocardiogram (ECG) monitoring, in vitro high-resolution optical mapping, confocal imaging of intracellular Ca2+ cycling, and 3D atrial immunohistology were performed in wild-type (WT) and Casq2 null (Casq2−/−) mice. Casq2−/− mice exhibited bradycardia, SAN conduction abnormalities, and beat-to-beat heart rate variability due to enhanced atrial ectopic activity both at baseline and with autonomic stimulation. Loss of CASQ2 increased fibrosis within the pacemaker complex, depressed primary SAN activity, and conduction, but enhanced atrial ectopic activity and atrial fibrillation (AF) associated with macro- and micro-reentry during autonomic stimulation. In SAN myocytes, CASQ2 deficiency induced perturbations in intracellular Ca2+ cycling, including abnormal Ca2+ release, periods of significantly elevated diastolic Ca2+ levels leading to pauses and unstable pacemaker rate. Importantly, Ca2+ cycling dysfunction occurred not only at the SAN cellular level but was also globally manifested as an increased delay between action potential (AP) and Ca2+ transient upstrokes throughout the atrial pacemaker complex. Conclusions Loss of CASQ2 causes abnormal sarcoplasmic reticulum Ca2+ release and selective interstitial fibrosis in the atrial pacemaker complex, which disrupt SAN pacemaking but enhance latent pacemaker activity, create conduction abnormalities and increase susceptibility to AF. These functional and extensive structural alterations could contribute to SAN dysfunction as well as AF in CPVT patients. PMID:24216388

  2. Protein kinase C modulation of the regulation of sarcoplasmic reticular function by protein kinase A-mediated phospholamban phosphorylation in diabetic rats.

    Science.gov (United States)

    Watanuki, Satoko; Matsuda, Naoyuki; Sakuraya, Fumika; Jesmin, Subrina; Hattori, Yuichi

    2004-01-01

    1. The goal of this study was to elucidate the possible mechanisms by which protein kinase A (PKA)-mediated regulation of the sarcoplasmic reticulum (SR) via phospholambin protein phosphorylation is functionally impaired in streptozotocin-induced diabetic rats. 2. Phospholamban (PLB) protein and mRNA levels were 1.3-fold higher in diabetic than in control hearts, while protein expression of cardiac SR Ca(2+)-ATPase (SERCA2a) was unchanged. 3. Basal and isoprenaline-stimulated phosphorylation of PLB at Ser(16) or Thr(17) was unchanged in diabetic hearts. However, stronger immunoreactivity was observed at the basal level in diabetic hearts when antiphosphoserine antibody was used. 4. Basal (32)P incorporation into PLB was significantly higher in diabetic than in control SR vesicles, but the extent of the PKA-mediated increase in PLB phosphorylation was the same in the two groups of vesicles. 5. Stimulation of Ca(2+) uptake by PKA-catalyzed PLB phosphorylation was weaker in diabetic than in control SR vesicles. The PKA-induced increase in Ca(2+) uptake was attenuated when control SR vesicles were preincubated with protein kinase C (PKC). 6. PKC activities were increased by more than two-fold in the membranous fractions from diabetic hearts in comparison with control values, regardless of whether Ca(2+) was present. This was associated with increases in the protein content of PKCdelta, PKCeta, PKCiota, and PKClambda in diabetic membranous fractions. 7. The changes observed in diabetic rats were reversed by insulin therapy. 8. These results suggest that PKA-dependent phosphorylation may incompletely counteract the function of PLB as an inhibitor of SERCA2a activity in diabetes in which PKC expression and activity are enhanced.

  3. 17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3.

    LENUS (Irish Health Repository)

    Muchekehu, Ruth W

    2008-09-01

    We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca2+ ([Ca2+]i) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca2+]i, however exposure of cells to E2 in the presence of the microsomal Ca2+ ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca2+]i compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca2+]i. The E2-induced increase in [Ca2+]i was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100 microM). The estrogen receptor antagonist ICI 182,780 (1 microM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca2+]i from ryanodine-receptor-gated stores. The E2-induced effect on [Ca2+]i could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10 microM), the protein kinase A (PKA) inhibitor Rp-adenosine 3\\

  4. Symmetry-Driven Strategy for the Assembly of the Core Tetracycle of (+)-Ryanodine: Synthetic Utility of a Cobalt-Catalyzed Olefin Oxidation and α-Alkoxy Bridgehead Radical Reaction.

    Science.gov (United States)

    Nagatomo, Masanori; Hagiwara, Koji; Masuda, Kengo; Koshimizu, Masaki; Kawamata, Takahiro; Matsui, Yuki; Urabe, Daisuke; Inoue, Masayuki

    2016-01-01

    Ryanodine (1) is a potent modulator of intracellular calcium release channels, designated as ryanodine receptors. The exceptionally complex molecular architecture of 1 comprises a highly oxygenated pentacyclic system with eleven contiguous stereogenic centers, which makes it a formidable target for organic synthesis. We identified the embedded C2 -symmetric tricyclic substructure within 1. This specific recognition permitted us to design a concise synthetic route to enantiopure tricycle 9 by utilizing a series of pairwise functionalizations. The four tetrasubstituted carbon centers of 9 were effectively constructed by three key reactions, a dearomatizing Diels-Alder reaction, the kinetic resolution of the obtained racemic 14 through asymmetric methanolysis, and the transannular aldol reaction of the eight-membered diketone 10. A new combination of cobalt-catalyzed hydroperoxidation and NfF-promoted elimination enabled conversion of the hindered olefin of 9 into the corresponding ketone, thus realizing the desymmetrization. Finally, the tetrasubstituted carbon was stereospecifically installed by utilizing the α-alkoxy bridgehead radical to deliver the core tetracycle 7 with the six contiguous tetrasubstituted carbon centers. Consequently, the present work not only accomplishes efficient assembly of four out of the five fused rings of 1, but also develops two new powerful methodologies: two-step ketone formation and bridgehead radical reaction.

  5. Effects of CaMKII-mediated phosphorylation of ryanodine receptor type 2 on islet calcium handling, insulin secretion, and glucose tolerance.

    Directory of Open Access Journals (Sweden)

    Sayali S Dixit

    Full Text Available Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca(2+-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca(2+ concentration ([Ca(2+]cyt of β cells. This elevation in [Ca(2+]cyt leads to activation of Ca(2+/calmodulin-dependent protein kinase II (CAMKII, which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2, an intracellular Ca(2+-release channel implicated in Ca(2+-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca(2+ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca(2+]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca(2+ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.

  6. Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling, Insulin Secretion, and Glucose Tolerance

    Science.gov (United States)

    Dixit, Sayali S.; Wang, Tiannan; Manzano, Eiffel John Q.; Yoo, Shin; Lee, Jeongkyung; Chiang, David Y.; Ryan, Nicole; Respress, Jonathan L.; Yechoor, Vijay K.; Wehrens, Xander H. T.

    2013-01-01

    Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca2+-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca2+ concentration ([Ca2+]cyt) of β cells. This elevation in [Ca2+]cyt leads to activation of Ca2+/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca2+-release channel implicated in Ca2+-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca2+ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca2+]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca2+ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes. PMID:23516528

  7. Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.

    Science.gov (United States)

    Fessenden, James D

    2009-10-12

    While the static structure of the intracellular Ca(2+) release channel, the ryanodine receptor type 1 (RyR1) has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Förster resonance energy transfer (FRET) methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescent probes. A novel site-specific labeling method is presented that targets a FRET acceptor, Cy3NTA to 10-residue histidine (His) tags engineered into RyR1. Cy3NTA, comprised of the fluorescent dye Cy3, coupled to two Ni(2+)/nitrilotriacetic acid moieties, was synthesized and functionally tested for binding to His-tagged green fluorescent protein (GFP). GFP fluorescence emission and Cy3NTA absorbance spectra overlapped significantly, indicating that FRET could occur (Förster distance = 6.3 nm). Cy3NTA bound to His(10)-tagged GFP, quenching its fluorescence by 88%. GFP was then fused to the N-terminus of RyR1 and His(10) tags were placed either at the N-terminus of the fused GFP or between GFP and RyR1. Cy3NTA reduced fluorescence of these fusion proteins by 75% and this quenching could be reversed by photobleaching Cy3, thus confirming GFP-RyR1 quenching via FRET. A His(10) tag was then placed at amino acid position 1861 and FRET was measured from GFP located at either the N-terminus or at position 618 to Cy3NTA bound to this His tag. While minimal FRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position 618 to Cy3NTA at position 1861, thus indicating that these sites are in close proximity to each other. These findings illustrate the potential of this site-specific labeling system for use in future FRET-based experiments to elucidate novel aspects of RyR1 structure.

  8. Role of the Calcium-Sensing Receptor in Cardiomyocyte Apoptosis via the Sarcoplasmic Reticulum and Mitochondrial Death Pathway in Cardiac Hypertrophy and Heart Failure

    Directory of Open Access Journals (Sweden)

    Fang-Hao Lu

    2013-05-01

    Full Text Available Aims: Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. Methods and Results: In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso. Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours

  9. A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Tobias Gröβl

    Full Text Available In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

  10. Role of the calcium-sensing receptor in cardiomyocyte apoptosis via the sarcoplasmic reticulum and mitochondrial death pathway in cardiac hypertrophy and heart failure.

    Science.gov (United States)

    Lu, Fang-Hao; Fu, Song-Bin; Leng, Xiaoning; Zhang, Xinying; Dong, Shiyun; Zhao, Ya-Jun; Ren, Huan; Li, Hulun; Zhong, Xin; Xu, Chang-Qing; Zhang, Wei-Hua

    2013-01-01

    Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR) and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR) in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso). Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC) in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours, activation of CaR reduced [Ca(2+)]SR

  11. Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca(2+) release during the quasi-steady level of release in twitch fibers from frog skeletal muscle.

    Science.gov (United States)

    Fénelon, Karine; Lamboley, Cédric R H; Carrier, Nicole; Pape, Paul C

    2012-10-01

    Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits

  12. Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca(2+)-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition.

    Science.gov (United States)

    Fraqueza, Gil; Batista de Carvalho, Luís A E; Marques, M Paula M; Maia, Luisa; Ohlin, C André; Casey, William H; Aureliano, Manuel

    2012-11-07

    Recently we demonstrated that the decavanadate (V(10)) ion is a stronger Ca(2+)-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V(10) interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the present study, we further explore this subject, and we can now show that the decaniobate ion, [Nb(10) = Nb(10)O(28)](6-), is a useful tool in deducing the interaction and the non-competitive Ca(2+)-ATPase inhibition by the decavanadate ion [V(10) = V(10)O(28)](6-). Moreover, decavanadate and vanadate induce protein cysteine oxidation whereas no effects were detected for the decaniobate, tungstate or molybdate ions. The presence of the antioxidant quercetin prevents cysteine oxidation, but not ATPase inhibition, by vanadate or decavanadate. Definitive V(IV) EPR spectra were observed for decavanadate in the presence of sarcoplasmic reticulum Ca(2+)-ATPase, indicating a vanadate reduction at some stage of the protein interaction. Raman spectroscopy clearly shows that the protein conformation changes that are induced by V(10), Nb(10) and vanadate are different from the ones induced by molybdate and tungstate monomer ions. Here, Mo and W cause changes similar to those by phosphate, yielding changes similar to the E1P protein conformation. The putative reduction of vanadium(V) to vanadium(IV) and the non-competitive binding of the V(10) and Nb(10) decametalates may explain the differences in the Raman spectra compared to those seen in the presence of molybdate or tungstate. Putting it all together, we suggest that the ability of V(10) to inhibit the Ca(2+)-ATPase may be at least in part due to the process of vanadate reduction and associated protein cysteine oxidation. These

  13. A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

    Science.gov (United States)

    Größl, Tobias; Hammer, Elke; Bien-Möller, Sandra; Geisler, Anja; Pinkert, Sandra; Röger, Carsten; Poller, Wolfgang; Kurreck, Jens; Völker, Uwe

    2014-01-01

    In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile. PMID:24670775

  14. Changes in sarcoplasmic metabolite concentrations and pH associated with the catch contraction and relaxation of the anterior byssus retractor muscle of Mytilus edulis measured by phosphorus-31 nuclear magnetic resonance.

    Science.gov (United States)

    Ishii, N; Mitsumori, F; Takahashi, K

    1991-06-01

    The sarcoplasmic concentrations of phosphorus metabolites and pH (pHin) were measured in the anterior byssus retractor muscle (ABRM) of Mytilus edulis by 31P nuclear magnetic resonance spectroscopy. During an active contraction induced by 10(-3) acetylcholine, the concentration of arginine phosphate ([Arg-P]in) decreased from the resting value of 7.47 +/- 0.26 (mean +/- SE, n = 8) to 6.67 +/- 0.29 (n = 6) mumol g-1, and that of inorganic phosphate (Pi) consistently increased from 0.84 +/- 0.06 (n = 7) to 1.61 +/- 0.12 (n = 5) mumol g-1. In the 'catch' state following the active contraction, these concentrations were close to their resting levels, indicating that the catch is an inactive state. 5-hydroxytryptamine caused a rapid relaxation of the catch, which was associated with a slight decrease in [Arg-P]in and an increase in pHin by ca 0.2 units. The sarcoplasmic concentration of ATP (mean, 1.6 mumol g-1) did not change throughout the contraction-relaxation cycle.

  15. 姜黄素对心力衰竭兔肌浆网钙泵表达的影响%Effects of curcumin on sarcoplasmic reticulum Ca~(2+) -ATPase in rabbits with heart failure

    Institute of Scientific and Technical Information of China (English)

    张艳; 林国生; 包明威; 武欣迎; 王澈; 杨波

    2010-01-01

    目的 探讨姜黄素对心力衰竭(心衰)兔肌浆网钙泵表达的影响.方法 采用主动脉瓣反流联合腹主动脉缩窄制作慢性心衰家兔模型.随机分为心衰姜黄素组、心衰安慰剂组、对照姜黄素组、对照安慰剂组.8周后计算心脏重量与体重比值,观察超微结构,检测肌浆网钙泵mRNA和蛋白的表达水平及活性.结果 心衰姜黄素组和心衰安慰剂组心脏重量与体重比值均大于对照组(P<0.05);且心衰姜黄素组比值小于心衰安慰剂组(P<0.05).电子显微镜显示心衰姜黄素组的心脏超微结构有所改善.心衰姜黄素组和心衰安慰剂组肌浆网钙泵mRNA、蛋白表达及活性均小于对照组(P<0.05),但心衰姜黄素组均显著高于心衰安慰剂组(P<0.05).结论 姜黄素能在mRNA水平和蛋白水平提高心衰家兔肌浆网钙泵的表达,提高肌浆网钙泵的活性,这可能是姜黄素改善心衰的机制之一.%Objective To investigate the effects of curcumin on sarcoplasmic reticulum Ca~(2+)-ATPase in heart failure rabbits.Methods Rabbit heart failure model was made with aortic regurgitation and abdominal aorta constriction and 40 rabbits were randomly divided into 4 groups including:(1) heart failure treated with curcumin;(2) heart failure treated with placebo;(3) healthy control treated with curcumin and (4) healthy control treated with placebo.All rabbits were administrated with curcumin capsules or placebo capsules 100 mg·kg~(-1)·d~(-1),respectively.All groups were sacrificed after eight weeks.Myocardial ultrastructural organization was detected by transmission electron microscope.RT-PCR and Western blot were used to measure the expression of sarcoplasmic reticulum Ca~(2+)-ATPase in mRNA and protein levels,respectively.Malachite green colorimetric assay was used to evaluate the activity of sarcoplasmic reticulum Ca~(2+) -ATPase.Results All detected parameters were similar between control curcumin group and control placebo

  16. Physicochemical and Functional Characteristics Changes of Catfish Sarcoplasmic Proteins Subjected to pH-Shift Method%pH变化对鲶鱼肌浆蛋白理化和功能特性的影响

    Institute of Scientific and Technical Information of China (English)

    李鹏; 李沛然; 郭耀华; 岳兰昕; 张乃琳; 刘彩虹; 马俪珍

    2014-01-01

    This paper used the sarcoplasmic protein of washing water from catfish (Clarias gariepinus), and subjected to pH-shift method (pH=3.0→7.0, pH=5.0→7.0, pH=7.1→7.0, pH=9.0→7.0, pH=11.0→7.0) as the research object. Each treatment group was analyzed by protein solubility, surface hydrophobicity, total sulfhydryl content, SDS-PAGE, rheological property and the change of thermal denaturation. Results showed, as the environment of pH to the direction of the acidic or alkaline, sarcoplasm protein solubility, surface hydrophobicity and total sulfydryl content had decreased, protein oxiution increased, meanwhile, rheological properties (viscosity) and thermal denaturation temperature improved.%提取鲶鱼肉漂洗液中的肌浆蛋白,用酸或碱以5种方式(①pH=3.0→7.0;②pH=5.0→7.0;③pH=7.1→7.0;④pH=9.0→7.0;⑤pH=11.0→7.0)调节其pH后,研究经过这种pH变化后的肌浆蛋白的蛋白溶解度、表面疏水性、总巯基含量、SDS-PAGE、流变性和热变性等指标的变化,以期为漂洗液中肌浆蛋白的综合利用奠定理论基础。研究结果表明:随着pH向酸性或者碱性的方向逐渐变化,肌浆蛋白溶解度、表面疏水性和总巯基含量不断下降,蛋白氧化加剧,流变学特性(黏度值)和热变性温度不断增加。

  17. Sarcoplasmatic and myofibrillar protein changes caused by acute heat stress in broiler chicken Alterações nas proteínas sarcoplasmáticas e miofibrilares em frangos de corte causadas por estresse térmico agudo

    Directory of Open Access Journals (Sweden)

    Carolina de Castro Santos

    2008-01-01

    Full Text Available Acute heat stress (AHS modifies the structure of myofibrils affecting functional properties of meat, mainly the water holding capacity. This experiment aimed to identify changes in proteolysis and migration between the myofibrillar and sarcoplasmatic fractions due to pre-slaughter AHS. Myofibrillar fragmentation index (MFI, SDS-PAGE, western blot of vinculin (WB and shear force (SF were determined. Six hundred broilers (Gallus gallus were slaughtered in three different days (ST. In each ST, groups of ten animals were placed in transport crates and submitted to AHS (35ºC, 75 - 85% RH for 2 hours. Simultaneously, the non-stressed broilers (NS were kept in thermoneutral environment (22ºC, 83 ± 6.6% RH within the crates in the same density. After slaughter, the breast muscles were kept refrigerated until the withdrawal of all samples (0, 1, 2, 6 and 24 hours after slaughter. Sampling within AHS and NS birds was collected according to lightness value (normal L* 51, except for determination of MFI and SF. The lightness was used later to perform SDS-PAGE and WB analyses. MFI kinetics showed that the fragmentation rate was superior in animals NS, indicating that AHS can harm proteolysis and rate of myofibrillar fragmentation. However, the extent of fragmentation did not change, as well as SF values. SDS-PAGE for Troponin fragments indicated a differentiated pattern between AHS and NS. The WB did not show alterations in vinculin fragmentation. Modifications in sarcoplasmatic fraction are observed in meat with high L*values, independent of environmental condition.O estresse térmico agudo (ET causa alterações na estrutura das miofibrilas, afetando propriedades funcionais da carne, principalmente a capacidade de retenção de água. Identificaram-se mudanças na proteólise e migração entre as frações miofibrilar e sarcoplasmática, decorrentes do ET pré-abate, através do índice de fragmentação miofibrilar (MFI, SDS-PAGE para troponina (SDS

  18. 桃小食心虫鱼尼丁受体基因克隆及表达模式分析%Molecular Cloning and Expression Profiling of a Ryanodine Receptor Gene in the Peach Fruit Moth (Carposina sasakii)

    Institute of Scientific and Technical Information of China (English)

    孙丽娜; 张怀江; 闫文涛; 马春森; 仇贵生

    2015-01-01

    【目的】二酰胺类杀虫剂的作用靶标鱼尼丁受体(ryanodine receptor, RyR)是目前所知的最大的离子通道蛋白,该受体可控制细胞内Ca2+的释放,对细胞内Ca2+的稳定起着关键作用。克隆获得桃小食心虫鱼尼丁受体基因(CsRyR)全长序列,进一步解析该基因在桃小食心虫(Carposinasasakii)各发育阶段的表达模式。【方法】通过同源序列比对的方法,利用cDNA末端快速扩增技术(RACE)获得该基因的全长cDNA序列;应用生物信息学软件对该基因的开放阅读框、编码的氨基酸序列、功能结构域等信息进行预测分析,并基于最大似然法构建该基因与其他昆虫相关基因序列的系统发育树,明确其系统进化关系。分别提取桃小食心虫各发育阶段RNA,以GAPDH为内参基因,应用RT-qPCR技术,解析CsRyR在桃小食心虫各发育阶段(卵、幼虫、蛹和成虫)的表达模式。【结果】桃小食心虫CsRyR的cDNA全序列长度为15766 bp,开放阅读框15405 bp,编码5134个氨基酸。氨基酸序列比对结果显示,CsRyR与脊椎动物RyRs的一致度分别为45%—47%;与秀丽隐杆线虫(Caenorhabditiselegans)RyR的一致度也为46%。在昆虫RyRs中,与鳞翅目一致度为91%—94%,与同翅目、双翅目昆虫一致度均为79%。系统发育树显示该基因编码的蛋白质与鳞翅目夜蛾科和螟蛾科害虫RyRs亲缘关系最近。结构域分析结果显示,CsRyR具有RyR的典型结构域,如位于C-末端的6个跨膜结构域(AA 4467-5029)、释放Ca2+的通道形成基序GVRAGGGIGD、Ca2+结合位点EF-hand和3个ATP结合位点GXGXXG等。二酰胺类杀虫剂可能的作用位点AA 183-290(BmRyR),AA 4610-4655(DmRyR)和4946G(PxRyR)在CsRyR中无特殊性。此外,CsRyR中存在AA 2490-2496 TQAPRPG和5131-5134 SQAK两个基序,在其他物种中均没有发现。RT-qPCR分析结果表明,CsRyR在蛹

  19. Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon beta-adrenergic stimulation in normal and failing hearts.

    Science.gov (United States)

    Xiao, Bailong; Zhong, Guofeng; Obayashi, Masakazu; Yang, Dongmei; Chen, Keyun; Walsh, Michael P; Shimoni, Yakhin; Cheng, Heping; Ter Keurs, Henk; Chen, S R Wayne

    2006-05-15

    We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta

  20. Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon β-adrenergic stimulation in normal and failing hearts

    Science.gov (United States)

    Xiao, Bailong; Zhong, Guofeng; Obayashi, Masakazu; Yang, Dongmei; Chen, Keyun; Walsh, Michael P.; Shimoni, Yakhin; Cheng, Heping; ter Keurs, Henk; Chen, S. R. Wayne

    2006-01-01

    We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before β-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after β-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon β-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon

  1. The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase.

    Science.gov (United States)

    Medda, P; Fassold, E; Hasselbach, W

    1987-06-01

    sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].

  2. Effect of downhill exercise on sarcoplasmic reticulum function in rat skeletal muscle%下坡运动对大鼠骨骼肌肌浆网功能的影响

    Institute of Scientific and Technical Information of China (English)

    陈万; Ruell PA; Thompson MW

    2007-01-01

    To investigate the effect of downhill(eccentric)exercise(ECE)on sarcoplasmic reticulum(SR) Ca2+-ATPase activity,Ca2+ uptake and release in rat skeletal muscle,in terms of both magnitude and time course.In addition,ionophore stimulation was determined to assess vesicle integrity by measuring the ratio of calciumdependent ATPase activities in the presence and absence of A23187.Method:Adult male SD rats were randomly assigned to control and ECE groups.The ECE rats were sacrificed at the Oth,4th,24th,48th,72nd and 144th h following ECE(n=7).The ECE protocol consisted of 90min continuous downhill exercise(-16 deg;15m·min-1).Red vastus muscles were sampled separately for each group and muscle homogenates were prepared.The rates of SR Ca2+-ATPase activity.Ca2+uptake and release were measured in vitro.Result:SR Ca2+uptake was significantly lower(P<0.05)compared with control values[19.25+1.38 nmol·min-1·(mg protein)-1],by 29% and 36% immediately and 4h after ECE,respectively,and remained depressed(P<0.05)24h following ECE.SR Ca2+ release was also significantly lower(P<0.05)compared with control values[31.06±2.36 nmol·min-1·(mg protein)-1],by 37% and 39% immediately and 4h after ECE,respectively,and remained depressed(P<0.05)24h following ECE.SR Ca2+-ATPaseactivity measured with ionophore was 31% lower(P<0.05)4h after ECE,and remained lower(P<0.05)24h following ECE.The ratio of Ca2+-ATPase activities in the presence and absence of A23187 was not significantly changed following ECE,indicating that membrane integrity was not altered by the exercise.Conclusion:The present remits suggest that a bout of low-intensity,prolonged downhill exereise results in a long-lasting depression of SR function that is not fully restored after two days of recovery,which may underlie some muscle functional impairments induced by ECE.These changes could be the results of stress from sarcomere length inhomogeneities during eccentric contractions.%目的:观测研究下坡(离心)

  3. Regulation of ryanodine receptor RyR2 by protein-protein interactions: prediction of a PKA binding site on the N-terminal domain of RyR2 and its relation to disease causing mutations [v1; ref status: indexed, http://f1000r.es/4tw

    Directory of Open Access Journals (Sweden)

    Belinda Nazan Walpoth

    2015-01-01

    Full Text Available Protein-protein interactions are the key processes responsible for signaling and function in complex networks. Determining the correct binding partners and predicting the ligand binding sites in the absence of experimental data require predictive models. Hybrid models that combine quantitative atomistic calculations with statistical thermodynamics formulations are valuable tools for bioinformatics predictions. We present a hybrid prediction and analysis model for determining putative binding partners and interpreting the resulting correlations in the yet functionally uncharacterized interactions of the ryanodine RyR2 N-terminal domain. Using extensive docking calculations and libraries of hexameric peptides generated from regulator proteins of the RyR2 channel, we show that the residues 318-323 of protein kinase A, PKA, have a very high affinity for the N-terminal of RyR2. Using a coarse grained Elastic Net Model, we show that the binding site lies at the end of a pathway of evolutionarily conserved residues in RyR2. The two disease causing mutations are also on this path. The program for the prediction of the energetically responsive residues by the Elastic Net Model is freely available on request from the corresponding author.

  4. Design, Synthesis and Biological Activities of New Ryanodine Receptor Pesticides Based on Ugi Reaction%基于Ugi反应的新型鱼尼丁受体杀虫剂的设计、合成及生物活性

    Institute of Scientific and Technical Information of China (English)

    刘鹏飞; 周莎; 熊丽霞; 于淑晶; 张晓; 李正名

    2012-01-01

    利用Ugi反应设计合成了一系列未见文献报道的α-苯基-α-酰胺基-酰胺类化合物,所有化合物均通过1 H NMR谱、元素分析和高分辨质谱表征确定,初步的生物活性测试结果表明,在浓度为200 mg/L时,化合物7h对粘虫有一定抑制活性;在浓度为50 mg/L时,化合物7q对节果轮纹病菌、化合物7e对小麦赤霉菌有一定的抑菌活性.%Chlorantraniliprole, invented by DuPont company in 2001 , is a new type of insecticide with high efficiency, low toxicity, broad-spectrum inaecticidal activity which act at Ryanodine receptor of target insects. Both flubendiamide and chlorantraniliprole contain two amide groups in different locations of the structure, and this could be of great significance in insecticidal activity. Referring to their structural composition, a series of novel a-phenyl-a-amide-amide compounds was designed and synthesized. We changed chlorantraniliprole' s β-amino-acid-amide to the a-phenyl-a-amide-amide. The Ugi reaction was carried on to uphold diversity in the molecular. A series of compounds was obtained and bio-assayed and their structure-activity relationship was discussed. Also, their structures were characterized by 1 H NMR, elemental analysis and HRMS. The preliminary results of biological activity experiment show that the compounds at 200 mg/L exhibit some insecticidal activity against Mythimna separate(7h) ; the compounds also exhibit some fungicidal activity against Physalos-pora piricola(7q) and Alternaria solani(le).

  5. BIN1 localizes the L-type calcium channel to cardiac T-tubules.

    Directory of Open Access Journals (Sweden)

    Ting-Ting Hong

    2010-02-01

    Full Text Available The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2 to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient

  6. Oxidative stress, mitochondrial damage, and cores in muscle from calsequestrin-1 knockout mice

    OpenAIRE

    2015-01-01

    Background Mutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca 2+) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca 2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic...

  7. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx{sub a}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Chul Won; Kim, Jim Il [Chonnam National Univ., Gwangju (Korea, Republic of); Sato, Kazuki [Fukuoka Women' s Univ., Fukuoka (Japan)

    2013-06-15

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx{sub a}), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca{sup 2+} release from sarcoplasmic reticulum (SR). IpTx{sub a} increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states.

  8. Arabidopsis Clade I TGA Factors Regulate Apoplastic Defences against the Bacterial Pathogen Pseudomonas syringae through Endoplasmic Reticulum-Based Processes

    OpenAIRE

    Lipu Wang; Pierre R Fobert

    2013-01-01

    During the plant immune response, large-scale transcriptional reprogramming is modulated by numerous transcription (co) factors. The Arabidopsis basic leucine zipper transcription factors TGA1 and TGA4, which comprise the clade I TGA factors, have been shown to positively contribute to disease resistance against virulent strains of the bacterial pathogen Pseudomonas syringae . Despite physically interacting with the key immune regulator, NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), f...

  9. 心肺复苏后心功能障碍与心肌内质网Ca2+调控蛋白表达关系的研究%The relationship between sarcoplasmic reticulum Ca2+modulation proteins and postresuscitation myocardial dysfunction

    Institute of Scientific and Technical Information of China (English)

    黄煜; 何庆

    2014-01-01

    Objective To investigate the relationship between sarcoplasmic reticulum Ca2+modulation proteins and postresuscitation myocardial dysfunction. Methods Thirty-eight SPF male Sprague-Dawley (SD) rats were randomly divided into control group(n=12)and cardiac arrest(CA)group(n=26). CA was induced by intravenous bolus of potassium chloride(40μg/g),and cardiopulmonary resuscitation(CPR)was conducted 8 minutes later. No CA was induced in control group except catheter placement for monitoring cardiopulmonary parameters after anesthesia. Invasive hemodynamic parameters were monitored for 1 hour after CPR. Echocardiogram was performed to evaluate cardiac function. Myocardial samples were harvested 5 minutes and 1 hour after restoration of spontaneous circulation (ROSC),and sarcoplasmic reticulum Ca2+ ATPase (SERCA2a),phosphorylated phospholamban (p-PLB) and rynodine receptor(RyR)were determined by Western Blot. Results ROSC rate of CA group was 92.3%(24/26),and mean recovery time was (68 ±39)seconds. Cardiac function was significantly impaired in CA group at 1 hour after resuscitation, and ejection fraction, fraction shortening (FS), the maximal rate of left ventricular pressure increase/decline (±dp/dt max)were significantly decreased compared with those in control group 〔ejection fraction:0.548±0.060 vs. 0.809±0.043,F=71.692,P=0.000;FS:(34.4±4.4)%vs. (46.0±3.5)%,F=55.443,P=0.000;+dp/dt max(mmHg/s):4 718±743 vs. 7 098±394,P0.05). Conclusions The impairment of the p-PLB is closely related to postresuscitation myocardial dysfunction.%目的探讨心肌内质网Ca2+调控蛋白表达与心肺复苏(CPR)后心功能障碍的关系。方法38只SPF级雄性SD大鼠按随机数字表法分为对照组(12只)和心搏骤停组(26只)。静脉弹丸式注射氯化钾40μg/g诱导心搏骤停,8 min后进行CPR;对照组大鼠仅麻醉后置管并监测指标,不诱导心搏骤停。在复苏后进行有创血流动力学监测1 h,采用超声心动

  10. LPCES对慢性低压缺氧兔颏舌肌肌球蛋白重链和SR Ca2+摄取-释放动力学的影响%Electrical stimulation at lower physiological frequency induces myosin heavy chain isoform transformation and improves sarcoplasmic reticulum Ca2+ uptake/release in genioglossus of rabbits exposed to chronic hypoxia

    Institute of Scientific and Technical Information of China (English)

    刘熙; 刘刚; 张妮; 欧娜; 张鹏

    2011-01-01

    Objective To identify the effect of chronic electrical stimulation at a lower physiological frequency on the expressions of myosin heavy chain (MHC) isoforms and kinetics of sarcoplasmic reticulum (SR) Ca2 + uptake/release in the genioglossus of rabbits exposed to chronic hypoxia. Methods Twenty-four adult rabbits were randomized into control group ( A), chronic hypoxia group ( B ), 2.5 Hz electrical stimulation group (C) and (2.5 + 40) Hz electrical stimulation group (low frequency plus physical frequency, D).After the rabbits from group B, C and D had been fed with free access to food and water in a hypoxia cabin ( simulating 5 000 m altitude) in 10 h a day for 4 weeks, the rabbits in group C and D received electrical stimulation in their genioglossus at a frequency of 2.5 Hz and (2.5 +40) Hz respectively in 10 h per day for 14 d,while those in group B received no electrical stimulation. Expressions of MHC isoforms in the genioglossus of rabbits in 4 groups were detected by Western blotting, and Fura-2 fluorophotometry was used to assay the kinetics changes of SR Ca2 + uptake-release. Restlts The expression level of MHC l a was significantly higher while that of MHC I was significantly lower in group B than that in group A (P < 0.05 ). Meanwhile,the genioglossus SR Ca2+ uptake/release velocity in group B was significantly decreased compared with that in group A ( P < 0. 05 ). The expression levels of MHC Ⅱ a and MHC I in group C and D after electrical stimulation were significantly higher, while those of MHC Ⅱ b, especially in group D, were significantly lower than those in group B (P < 0.05 ). The genioglossus SR Ca2+ uptake/release velocity in group C and D, especially in group D, was significantly increased compared with that in group B ( P < 0.05 ). No significant difference was found in expression levels of MHC Ⅱ a and MHC I between group C and D after electrical stimulation ( P > 0.05). Conclusion MHC Ⅱb in the genioglossus of rabbits with

  11. Nitric oxide affects sarcoplasmic calcium release in skeletal myotubes.

    NARCIS (Netherlands)

    Heunks, L.M.A.; Machiels, H.A.; Dekhuijzen, P.N.R.; Prakash, Y.S.; Sieck, G.C.

    2001-01-01

    In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces

  12. Ion pathways in the sarcoplasmic reticulum Ca2+-ATPase.

    Science.gov (United States)

    Bublitz, Maike; Musgaard, Maria; Poulsen, Hanne; Thøgersen, Lea; Olesen, Claus; Schiøtt, Birgit; Morth, J Preben; Møller, Jesper Vuust; Nissen, Poul

    2013-04-12

    The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane ion transporter belonging to the P(II)-type ATPase family. It performs the vital task of re-sequestering cytoplasmic Ca(2+) to the sarco/endoplasmic reticulum store, thereby also terminating Ca(2+)-induced signaling such as in muscle contraction. This minireview focuses on the transport pathways of Ca(2+) and H(+) ions across the lipid bilayer through SERCA. The ion-binding sites of SERCA are accessible from either the cytoplasm or the sarco/endoplasmic reticulum lumen, and the Ca(2+) entry and exit channels are both formed mainly by rearrangements of four N-terminal transmembrane α-helices. Recent improvements in the resolution of the crystal structures of rabbit SERCA1a have revealed a hydrated pathway in the C-terminal transmembrane region leading from the ion-binding sites to the cytosol. A comparison of different SERCA conformations reveals that this C-terminal pathway is exclusive to Ca(2+)-free E2 states, suggesting that it may play a functional role in proton release from the ion-binding sites. This is in agreement with molecular dynamics simulations and mutational studies and is in striking analogy to a similar pathway recently described for the related sodium pump. We therefore suggest a model for the ion exchange mechanism in P(II)-ATPases including not one, but two cytoplasmic pathways working in concert.

  13. Solution-blown nanofiber mats from fish sarcoplasmic protein

    DEFF Research Database (Denmark)

    Sett, S.; Boutrup Stephansen, Karen; Yarin, A.L.

    2016-01-01

    that the production rate of solution-blowing was increased 30-fold in relation to electrospinning. Overall, this study reveals FSP as an interesting biopolymeric alternative to synthetic polymers, and the introduction of FSP to nylon 6 provides a composite with controlled properties....

  14. Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy.

    Science.gov (United States)

    Paran, Christopher W; Zou, Kai; Ferrara, Patrick J; Song, Haowei; Turk, John; Funai, Katsuhiko

    2015-12-01

    Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) that contributes to abnormal Ca(2+) homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition.

  15. Muscle Dysfunction in Androgen Deprivation: Role of Ryanodine Receptor

    Science.gov (United States)

    2016-11-01

    were up-regulated after castration. Alteration of antioxidant proteins can cause oxidative stress to cells because the balance of reactive oxygen...associated with oxidation and nytrosilation of RyR1, depletion of calstabin1, and dysmorphic and hypertrophied mitochondria . Preliminary data in mice...superoxide production by using the cell permeable fluorescent indicator MitoSOX Red. *Completion of this task will give expertise in cellular

  16. Ryanodine receptors as pharmacological targets for heart disease

    Institute of Scientific and Technical Information of China (English)

    Marco SANTONASTASI; Xander H T WEHRENS

    2007-01-01

    Calcium release from intracellular stores plays an important role in the regulationof muscle contraction and electrical signals that determine the heart rhythm. Theryanodine receptor (RyR) is the major calcium (Ca2+) release channel required forexcitation-contraction coupling in the heart. Recent studies have demonstratedthat RyR are macromolecular complexes comprising of 4 pore-forming channelsubunits, each of which is associated with regulatory subunits. Clinical andexperimental studies over the past 5 years have provided compelling evidencethat intracellular Ca2+release channels play a pivotal role in the development ofcardiac arrhythmias and heart failure. Changes in the channel regulation andsubunit composition are believed to cause diastolic calcium leakage from thesarcoplasmic reticulum, which could trigger arrhythmias and weaken cardiaccontractility. Therefore, cardiac RyR have emerged as potential therapeutic tar-gets for the treatment of heart disease. Consequently, there is a strong desire toidentify and/or develop novel pharmacological agents that may target these Ca2+signaling pathways. Pharmacological agents known to modulate RyR in the heart,and their potential application towards the treatment of heart disease are dis-cussed in this review.

  17. Effect of exercise on gene expression of calcium modulin in myocardial sarcoplasmic reticulum of diabetic rat%运动对1型糖尿病大鼠心肌肌浆网钙调控蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    江卫华; 罗达亚; 余乐涵; 段荣; 万福生

    2008-01-01

    目的 观察运动对1型糖尿病大鼠心肌肌浆网钙调控蛋白表达的影响,并探讨其机制.方法 选用健康Spragne-Dawley大鼠,随机分为正常对照组、运动对照组、糖尿病组和糖尿病+运动组,每组10只.用腹腔注射链脲佐菌素(55 mg/ks)法复制糖尿病模型,糖尿病+运动组大鼠在糖尿病模型建成后第4天开始跑台运动,于运动第4周末心脏采血,制备血清.采用放射免疫法测定血清胰岛素水平,采用RT-PCR法检测肌浆网Ca结果-ATP酶(SERCA)、磷酸受纳蛋白(PLB)和Ryanodine受体-2(RyR2)mRNA的表达,采用Western-blotting法检测SERCA2、PLB蛋白的表达.结果 与正常对照组相比,糖尿病组血糖、糖化血清蛋白及低密度脂蛋白水平升高,胰岛素和高密度脂蛋白水平降低,心肌SERCA2、PLB、RyR2 mRNA和SERCA2、PLB蛋白表达差异无统计学意义;糖尿病+运动组血糖水平升高,胰岛素水平降低,心肌SERCA2、PLB和RyR2的mRNA表达水平提高,SERCA2、PLB蛋白表达量增加,差异有统计学意义(P -ATPase (SERCA2) and phaspholamban (PLB). Results The level of biochemical indicator of exercisegroup is not affected when comparing with that of the control group, but significantly changed in diabetic group ( P <0. 01 ) ; The level of blood glucose, insulin, blood fat and glycosylated serum protein were ameliorated in diabetic rats inthe exercise training group. No significant changes in mRNA level of SERCA2, PLB and ryanodine receptor type 2(RYR2) were observed between control and diabetic group, the same to protein expression of SERCA2 and PLB. Butexpression of calcium modulin mRNA was significantly increased in exercise group and diabetic rats in the exercisetraining group comparing with that of the control and diabetic groups ( P < 0.01 ), the same to protein expression ofSERCA2 and PLB. Conclusion Exercise exerted good protective effects on the myocardial injury with 1 type diabetesrat, which might attribute to the

  18. Evidence against inhibition of sarcoplasmic reticulum Ca2 + -pump as mechanism of H202-induced contraction of rat aorta%肌浆网钙泵的抑制不参与过氧化氢诱导的大鼠主动脉收缩

    Institute of Scientific and Technical Information of China (English)

    沈建中; 郑秀凤; 魏尔清; 關超然

    2001-01-01

    目的:研究肌浆网钙泵抑制是否参与H2O2诱导的大 鼠主动脉收缩反应。方法:离体主动脉环张力实验 比较H2O2及钙泵特异性抑制剂环匹阿尼酸(CPA)缩 血管效应及其信号机制的差异。结果:H2O2和CPA 均收缩去内皮主动脉环,但H2O2触发快速短暂相位 相收缩,而CPA诱导缓慢持续的张力相收缩。在无 钙液中,仅CPA 30 μmol/L而非H2O2 30μmol/L预 处理取消苯肾上腺素10 μmol/L缩血管效应。Thap- sigargin 30 μmol/L诱导最大收缩反应时,仅H2O2能 使血管环进一步收缩。另外,P2受体拮抗剂 suramin、RB-2(各100μmol/L)以及多种酶抑制剂包 括PLC、PKC、PLA2、COX和蛋白质酪氨酸激酶均 能抑制H2O2而非CPA诱导的缩血管效应,但2-APB 50μmol/L对两者都有抑制作用。结论:肌浆网钙 泵抑制不是H2O2收缩大鼠去内皮主动脉的机制。%AIM: To test whether inhibition of sarcoplasmic reticulum (SR) Ca2+ -pump is involved in H2O2-induced contraction of endothelium-denuded rat aorta. METH ODS: Isometric tension recording of H2O2 and cyclopia zonic acid (CPA)-induced contractions of rat aortic rings were compared in the absence or presence of various pharmacological tools to discriminate their signaling path ways involved. RESULTS: Both H2O2 and CPA con tracted rat aortic rings, but with different contractile pat terns. H2O2 triggered a fast and phasic contraction, whereas CPA elicited a slow and sustained contraction. In Ca 2 +-free medium, pretreatment of aortic tings with CPA 30 μmol/L but not with H2O2 30 μmol/L nearly abolished phenylephrine (10 μmol/L)-induced contrac tion. In addition, upon the maximal contraction induced by thapsigargin 30 μ r mol/L, H2O2 but not CPA further contracted aortic rings. On the other hand, H2O2 (30 μmoL/L)- but not CPA (10 μmol/L)-induced contraction could be inhibited by suramin and RB-2 (each 100 μmol/L), two P2-purinoceptor antagonists. Further more, although

  19. 尼氟灭酸通过钙库钙释放引起豚鼠耳蜗螺旋动脉平滑肌细胞超极化%Niflumic acid hyperpolarizes the smooth muscle cells by opening BKCa channels through ryanodine-sensitive Ca2+ release in spiral modiolar artery

    Institute of Scientific and Technical Information of China (English)

    李丽; 马克涛; 赵磊; 司军强

    2008-01-01

    about(-42.47±1.38)mV(n=24).Application of NFA(100μmol/L),IAA-94(10μmol/L)and DIDS (200 μmol/L)shifted the RP to(13.7±4.3)mV(n=9,P<0.01),(11.4±4.2)mV(n=.7,P<0.01)and(12.3±3.7)mV(n=8,P<0.01),respectively.These drug-induced responses were in a concentration-dependent manner.NFA-induced hyperpolarization and outward current were almost blocked by charybdotoxin(100 nmol/L),iberiotoxin(100 nmol/L),tetraethylammonium(10 retool/L),BAPTA-AM(50 μmol/L),ryanodine(10μmol/L)andcaffeine(0.1-10mmol/L),respectively,but not by nifedipine(100μmol/L),CdC12(100μmol/L)andCa2+-free medium.It is concluded that NFA induces a release of intracellular calcium from the Ca2+ stores and the released intracellular calcium in turn causes concentration-dependent and reversible hyperpolarization and evokes outward currents in the SMCs of the cochlear SMA via activation of the Ca2+-activated potassium channels.

  20. Superoxide enhances Ca2+ entry through L-type channels in the renal afferent arteriole.

    Science.gov (United States)

    Vogel, Paul A; Yang, Xi; Moss, Nicholas G; Arendshorst, William J

    2015-08-01

    Reactive oxygen species regulate cardiovascular and renal function in health and disease. Superoxide participates in acute calcium signaling in afferent arterioles and renal vasoconstriction produced by angiotensin II, endothelin, thromboxane, and pressure-induced myogenic tone. Known mechanisms by which superoxide acts include quenching of nitric oxide and increased ADP ribosyl cyclase/ryanodine-mediated calcium mobilization. The effect(s) of superoxide on other calcium signaling pathways in the renal microcirculation is poorly understood. The present experiments examined the acute effect of superoxide generated by paraquat on calcium entry pathways in isolated rat afferent arterioles. The peak increase in cytosolic calcium concentration caused by KCl (40 mmol/L) was 99±14 nmol/L. The response to this membrane depolarization was mediated exclusively by L-type channels because it was abolished by nifedipine but was unaffected by the T-type channel blocker mibefradil. Paraquat increased superoxide production (dihydroethidium fluorescence), tripled the peak response to KCl to 314±68 nmol/L (Psuperoxide and not of hydrogen peroxide. Unaffected by paraquat and superoxide was calcium entry through store-operated calcium channels activated by thapsigargin-induced calcium depletion of sarcoplasmic reticular stores. Also unresponsive to paraquat was ryanodine receptor-mediated calcium-induced calcium release from the sarcoplasmic reticulum. Our results provide new evidence that superoxide enhances calcium entry through L-type channels activated by membrane depolarization in rat cortical afferent arterioles, without affecting calcium entry through store-operated entry or ryanodine receptor-mediated calcium mobilization.

  1. 肌浆网钙ATP酶基因转导对慢性心力衰竭犬心肌蛋白质组影响的初步研究%Overexpression of sarcoplasmic reticulum calcium ATPase induced hemodynamic and proteomic changes in a dog model of heart failure

    Institute of Scientific and Technical Information of China (English)

    付治卿; 李小鹰; 刘秀华; 孙胜; 刘涛; 米亚非; 周声安; 叶卫华; 王青松

    2008-01-01

    handling,myofibrils,and energy production in this dog model of chronic heart failure.%目的 分析心肌肌浆网Ca2+-ATP酶(sarcoplasmic reticulum Ca2+ ATPase 2a,SERCA2a)基因转导对慢性心力衰竭(HF)犬心肌蛋白质组的影响,探讨SERCA2a基因转导改善心功能的机制.方法 快速右心室起搏建立HF犬模型并随机分为HF组、HF+绿色荧光蛋白(enhanced green fluorescent pmtein,EGFP)组、HF+SERCA2a组.后两组分别向心肌内注射携带EGFP和SERCA2a基因的rAAV载体.于基因转导30 d时停止起搏后进行超声心动图和血流动力学检查并制备心室肌双向电泳蛋白样品和心肌双向电泳图谱,图像分析软件分析蛋白表达差异点,MALDI-TOF-MS数据库搜索鉴定蛋白质.结果 基因转导30 d时,HF+SERCA2a组犬的症状、超声心动图和血流动力学指标与HF+EGFP组相比有显著好转(P<0.05);与对照组相比差异无统计学意义(P>0.05).挑选SERCA2a基因转导后表达量发生明显改变的10个蛋白点进行分析,经质谱鉴定分别为心肌收缩相关蛋白、线粒体能量代谢酶类和应激相关蛋白.结论 以rAAV为载体介导SERCA2a基因转导能够改善HF犬心脏的收缩和舒张功能,其可能的机制是恢复了心肌收缩相关蛋白正常表型或正常表达量,增加了心肌能量的产生,改变了应激相关蛋白的表达.

  2. Differences in Ca2+-management between the ventricle of two species of neotropical teleosts: the jeju, Hoplerythrinus unitaeniatus (Spix & Agassiz, 1829, and the acara, Geophagus brasiliensis (Quoy & Gaimard, 1824

    Directory of Open Access Journals (Sweden)

    Monica Jones Costa

    Full Text Available This study analyzed the physiological role of the cardiac sarcoplasmic reticulum (SR of two neotropical teleosts, the jeju, Hoplerythrinus unitaeniatus (Erythrinidae, and the acara, Geophagus brasiliensis (Cichlidae. While the in vivo heart frequency (fH - bpm of acara (79.6 ± 6.6 was higher than that of the jeju (50.3 ± 2.7, the opposite was observed for the ventricular inotropism (Fc - mN/mm² at 12 bpm (acara = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67. A 5 min diastolic pause resulted in a strong potentiation of Fc (≅ 90% of strips from jeju, which was completely abolished by ryanodine. Ryanodine also resulted in a ≅ 20% decrease in the Fc developed by strips from jeju at both subphysiological (12 bpm and physiological (in vivo frequencies. However, this effect of ryanodine reducing the Fc from jeju was completely compensated by adrenaline increments (10-9 and 10-6 M. In contrast, strips from acara were irresponsive to ryanodine, irrespective of the stimulation frequency, and increases in adrenaline concentration (to 10-9 and 10-6 M further increased Fc. These results reinforce the hypothesis of the functionality of the SR as a common trait in neotropical ostariophysian (as jeju, while in acanthopterygians (as acara it seems to be functional mainly in 'athletic' species.

  3. Threephasic contractions in mammalian myocardium. Isolation and identification of a late component. Implications for rested-state contractions.

    Science.gov (United States)

    Schüttler, K; Szymanski, G

    1989-01-01

    More than 100 years after Bowditch's discovery of the "Treppe" phenomenon the mechanism of the striking rate and rhythm dependence of cardiac contraction is not unequivocally established. Beside sophisticated techniques biphasic contractions seemed to be a promising approach. There was soon a general agreement that the first component of biphasic contractions is activated by Ca released from the sarcoplasmic reticulum. About the second component prevails confusion: It does not directly reflect the Ca current, but is somehow related to that. In addition, involvement of Na-Ca exchange is supposed. We produced threephasic contractions by the use of isoproterenol (0.5-.4 microM) in guinea pig papillary muscle. Either by the application of ryanodine (1 microM) or after long periods of rest a ryanodine resistant late component of contraction could be isolated. On reducing the transmembrane Na gradient either by reducing [Na]o or by experimental manoeuvres expected to increase [Na]i. (.5 mM [K]o; 2-8 microM ouabain) this component was found to be increased. The evidence suggests that this component is substantially activated by Ca entering the cell via the sarcolemmal Na-Ca exchange. The ryanodine sensitive first component of threephasic contractions was found to be sensitive to caffeine, isoproterenol and changes in stimulation as well. These findings support the view that it may be generated by Ca induced Ca release from the sarcoplasmic reticulum. The pharmacological properties of the second and third ryanodine sensitive component and its dependence on the stimulation pattern were similar to those of the aftercontraction. They were found to be sensitive to procaine, verapamil, Mg, Ni and isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Characterization of the functional and anatomical differences in the atrial and ventricular myocardium from three species of elasmobranch fishes: smooth dogfish (Mustelus canis), sandbar shark (Carcharhinus plumbers), and clearnose skate (Raja eglanteria)

    DEFF Research Database (Denmark)

    Larsen, Julie; Bushnell, Peter; Steffensen, John;

    2016-01-01

    We assessed the functional properties in atrial and ventricular myocardium (using isolated cardiac strips) of smooth dogfish (Mustelus canis), clearnose skate (Raja eglanteria), and sandbar shark (Carcharhinus plumbeus) by blocking Ca2+ release from the sarcoplasmic reticulum (SR) with ryanodine...... positive first derivative (i.e., contractility), and increased time to 50 % relaxation in atrial tissue from smooth dogfish at 30 °C. It also increased times to peak force and half relaxation in clearnose skate atrial and ventricular tissue at both temperatures, but only in atrial tissue from sandbar shark...

  5. Characterization and temporal development of cores in a mouse model of malignant hyperthermia

    OpenAIRE

    2009-01-01

    Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca2+ release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1Y522S/WT), the mutation causes SR Ca2+ ...

  6. Doxorubicin-induced vasomotion and [Ca~(2+)]_i elevation in vascular smooth muscle cells from C57BL/6 mice

    Institute of Scientific and Technical Information of China (English)

    Bing SHEN; Chun-ling YE; Kai-he YE; Lan ZHUANG; Jia-hua JIANG

    2009-01-01

    Aim: To explore the action of doxorubicin on vascular smooth muscle cells.Methods: Isometric tension of denuded or intact thoracic aortic vessels was recorded and [Ca~(2+)]_i in isolated aortic smooth muscle cells was measured by using Fluo-3.Results: Doxorubicin induced phasic and tonic contractions in denuded vessels and increased levels of [Ca~(2+)]_i in single muscle cells. Treatment with 10 μmol/L ryanodine had no effect on basal tension, but it did abolish doxorubicin-induced phasic contraction. Treatment with 10 mmol/L caffeine induced a transient phasic contraction only, and the effect was not significantly altered by ryanodine, the omission of extracellular Ca~(2+) or both. Phenylephrine induced rhythmic contraction (RC) in intact vessels. Treatment with 100 μmol/L doxorubicin enhanced RC amplitude, but 1 mmol/L doxorubicin abolished RC, with an increase in maximal tension. Caffeine at 100 μmol/L increased the frequency of the RC only. In the presence of 100 μmol/L caffeine, however, 100 μmol/L doxorubicin abolished the RC and decreased its maximal tension. Treatment with 10 μmol/L ryanodine abolished the RC, with an increase in the maximal tension. In Ca~(2+)-free solution, doxorubicin induced a transient [Ca~(2+)]_i increase that could be abolished by ryanodine pretreatment in single muscle cells. The doxorubicin-induced increase in [Ca~(2+)]_i was suppressed by nifedipine and potentiated by ryanodine and cha-rybdotoxin.Conclusion: Doxorubicin not only releases Ca~(2+) from the sarcoplasmic reticulum but also promotes the entry of extracellular Ca~(2+) into vascular smooth muscle cells.

  7. The impact of extracellular and intracellular Ca2+ on ethanol-induced smooth muscle contraction

    Institute of Scientific and Technical Information of China (English)

    Naciye YAKTUBAY DONDAS; Mahir KAPLAN; Derya KAYA; Ergin SiNGiRiK

    2009-01-01

    Aim:To evaluate the impact of extracellular and intracellular Ca~(2+) on contractions induced by ethanol in smooth muscle.Methods: Longitudinal smooth muscle strips were prepared from the gastric fundi of mice. The contractions of smooth muscle strips were recorded with an isometric force displacement transducer.Results: Ethanol (164 mmol/L) produced reproducible contractions in isolated gastric fundal strips of mice. Although lidocaine (50 and 100 μmol/L), a local anesthetic agent, and hexamethonium (100 and 500 μmol/L), a ganglionic blocking agent, failed to affect these contractions, verapamil (1-50 μmol/L) and nifedipine (1-50 μmol/L), selective blockers of L-type Ca~(2+) channels, significantly inhibited the contractile responses of ethanol. Using a Ca~(2+)-free medium nearly eliminated these contractions in the same tissue. Ryanodine (1-50 μmol/L) and ruthenium red (10-100 μmol/L), selective blockers of intracellular Ca~(2+) channels/ryanodine receptors; cyclopiazonic acid (CPA; 1-10 μmol/L), a selective inhibitor of sarcoplasmic reticulum (SR) Ca~(2+)-ATPase; and caffeine (0.5-5 mmol/L), a depleting agent of intracellular Ca~(2+) stores, significantly inhibited the contractile responses induced by ethanol. In addition, the com-bination of caffeine (5 mmol/L) plus CPA (10 μmol/L), and ryanodine (10 μmol/L) plus CPA (10 μmol/L), caused further inhibition of contractions in response to ethanol. This inhibition was significantly different from those associated with caffeine, ryanodine or CPA. Furthermore the combination of caffeine (5 mmol/L), ryanodine (10 μmol/L) and CPA(10 μmol/L) eliminated the contractions induced by ethanol in isolated gastric fundal strips of mice.Conclusion: Both extracellular and intracellular Ca~(2+) may have important roles in regulating contractions induced by ethanol in the mouse gastric fundus.

  8. Correlation between oxidative stress and alteration of intracellular calcium handling in isoproterenol-induced myocardial infarction.

    Science.gov (United States)

    Díaz-Muñoz, Mauricio; Alvarez-Pérez, Marco Antonio; Yáñez, Lucía; Vidrio, Susana; Martínez, Lidia; Rosas, Gisele; Yáñez, Mario; Ramírez, Sotero; de Sánchez, Victoria Chagoya

    2006-09-01

    Myocardial Ca(2+) overload and oxidative stress are well documented effects associated to isoproterenol (ISO)-induced myocardial necrosis, but information correlating these two issues is scarce. Using an ISO-induced myocardial infarction model, 3 stages of myocardial damage were defined: pre-infarction (0-12 h), infarction (12-24 h) and post-infarction (24-96 h). Alterations in Ca(2+) homeostasis and oxidative stress were studied in mitochondria, sarcoplasmic reticulum and plasmalemma by measuring the Ca(2+) content, the activity of Ca(2+) handling proteins, and by quantifying TBARs, nitric oxide (NO) and oxidative protein damage (changes in carbonyl and thiol groups). Free radicals generated system, antioxidant enzymes and oxidative stress (GSH/GSSG ratio) were also monitored at different times of ISO-induced cardiotoxicity. The Ca(2+) overload induced by ISO was counterbalanced by a diminution in the ryanodine receptor activity and the Na(+)-Ca(+2) exchanger as well as by the increase in both calcium ATPases activities (vanadate- and thapsigargine-sensitive) and mitochondrial Ca(2+) uptake during pre-infarction and infarction stages. Pro-oxidative reactions and antioxidant defences during the 3 stages of cardiotoxicity were observed, with maximal oxidative stress during the infarction. Significant correlations were found among pro-oxidative reactions with plasmalemma and sarcoplasmic reticulum Ca(2+) ATPases, and ryanodine receptor activities at the onset and development of ISO-induced infarction. These findings could be helpful in the design of antioxidant therapies in this pathology.

  9. RyR2 modulates a Ca2+-activated K+ current in mouse cardiac myocytes.

    Directory of Open Access Journals (Sweden)

    Yong-Hui Mu

    Full Text Available In cardiomyocytes, Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs binds to and activates RyR2 channels, resulting in subsequent Ca2+ release from the sarcoplasmic reticulum (SR and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca2+-activated K+ channels (SK channels to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca2+ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca2+-activated K+ current (IK,Ca recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2, or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA (p<0.05, p<0.01, respectively. The activation of RyR2 by caffeine increased the IK,Ca in the cardiac cells (p<0.05, p<0.01, respectively. We further analyzed the effect of RyR2 knockdown on IK,Ca and Ca2+ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA exhibited a significant decrease in IK,Ca (p<0.05 and [Ca2+]i fluorescence intensity (p<0.01. An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca2+ release is responsible for the activation and modulation of SK channels in cardiac myocytes.

  10. Evidence that nitric oxide is a non-adrenergic non-cholinergic inhibitory neurotransmitter in the circular muscle of the mouse distal colon: a study on the mechanism of nitric oxide-induced relaxation.

    Science.gov (United States)

    Nishiyama, Kazuhiro; Azuma, Yasu-Taka; Shintaku, Kazuma; Yoshida, Natsuho; Nakajima, Hidemitsu; Takeuchi, Tadayoshi

    2014-01-01

    The gastrointestinal tract is composed of outer longitudinal muscle layers and inner circular muscle layers. Nitric oxide (NO), carbon monoxide (CO), and ATP play major roles as non-adrenergic non-cholinergic (NANC) inhibitory neurotransmitters in the longitudinal muscle of the mouse distal colon, whereas it is unclear which NANC inhibitory neurotransmitters are in its circular muscle. We investigated the electric field stimulation (EFS)-induced relaxations in the circular smooth muscle of the distal colon under NANC conditions. In the experiments in which N(ω)-nitro-L-arginine, an inhibitor of NO synthase, was added, the EFS-induced relaxation decreased in a concentration-dependent manner and finally vanished. In contrast, CO, purinergic receptor ligands, and peptidergic substances do not play major roles as NANC neurotransmitters in the circular muscle of the mouse distal colon. ODQ, an inhibitor of soluble guanylate cyclase, strongly attenuated EFS-induced relaxation. Ryanodine, a Ca(2+) release modulator at the sarcoplasmic reticulum, strongly attenuated EFS-induced relaxation as well. Relaxation induced by NOR-1, which generates NO, was inhibited by ODQ and ryanodine. Next, we performed experiments that simultaneously measured tension and the cytoplasmic Ca(2+) concentration ([Ca(2+)]cyt). NOR-1 decreased the tension and [Ca(2+)]cyt levels in the circular muscle. ODQ and ryanodine strongly attenuated the NOR-1-induced change in both tension and [Ca(2+)]cyt levels. In this study, we demonstrate that NO functions as a NANC inhibitory neurotransmitter in the circular muscle obtained from the mouse distal colon.

  11. Cellular mechanisms for the slow phase of the Frank-Starling response.

    Science.gov (United States)

    Bluhm, W F; Sung, D; Lew, W Y; Garfinkel, A; McCulloch, A D

    1998-01-01

    Following a step increase in sarcomere length, isometric cardiac muscle tension increases instantaneously by the Frank-Starling mechanism. In isolated papillary muscle and myocytes, there is an additional significant rise in developed tension over the following 15 min due to an unknown mechanism. This slow change in tension could not be explained by mechanical heterogeneity of the muscle preparations or by an increase in myofilament sensitivity to Ca2+. The slow change in tension was not dependent on sarcoplasmic reticulum Ca2+ loading assessed with rapid cooling contractures, and was not significantly altered by sarcoplasmic reticulum Ca2+ depletion (ryanodine) or inhibition of sarcoplasmic reticulum Ca2+ reuptake (cyclopiazonic acid). We used the Luo-Rudy ionic model of the ventricular myocyte together with a model of the length-dependent myofilament activation by Ca2+ to examine the effects of step changes in the parameters of sarcolemmal ion fluxes as possible mechanisms for the slow change in stress. The slow increase in tension was simulated by step changes in the Na+-K+ pump or Na+ leak currents, suggesting that the slow change in stress may be caused by length induced changes in Na+ fluxes. The model also predicted a slow increase in the magnitude of the initial repolarization during phase 1 of the action potential. The combination of experimental and computational models used in this investigation represents a valuable technique in elucidating the cellular mechanisms of fundamental processes in cardiac excitation-contraction coupling.

  12. Temperature dependence of cardiac sarcoplasmic reticulum and sarcolemma in the ventricle of catfish (Clarias gariepinus

    Directory of Open Access Journals (Sweden)

    El-Sabry Abu-Amra

    2015-10-01

    The sarcolemmal Ca2+ contribution of activator Ca2+ was greater at a test temperature of 30 °C as assessed by verapamil. Whereas the SR-Ca2+ contribution was higher at 20 and 30 °C and a frequency rate of 0.2 and 0.4 Hz as assessed by caffeine and adrenaline, respectively. Bradykinin potentiating factor (BPF7 which was isolated from jelly fish (Cassiopea andromeda decreased the cardiac force developed at a frequency rate of 0.2 Hz and a temperature of 20 °C, whereas it increased the force developed at frequency rates of 0.2 and 0.4 Hz at 30 °C. These results indicate that BPF7 may act like verapamil in reducing the cardiac force through blocking the sarcolemmal Ca2+ channels at low temperature and like adrenaline in an increase of the cardiac force developed at warm temperature and the high frequency rate through stimulation of SR-Ca2+ activator. Therefore, this study indicates that the sarcolemmal Ca2+ influx and the SR-Ca2+ release contributors of activator Ca2+ for cardiac force development in the catfish heart were significantly greater at warm temperature and at the pacing frequency rates of 0.2 and 0.4 Hz as assessed by verapamil, adrenaline, caffeine and BPF7. However, the relative contribution of the sarcolemmal Ca2+ influx in the development of cardiac force in the catfish heart was greater than that of SR-Ca2+ release.

  13. Role of glycogen availability in sarcoplasmic reticulum Ca2+ kinetics in human skeletal muscle

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Nielsen, Joachim; Saltin, Bengt

    2011-01-01

    Glucose is stored as glycogen in skeletal muscle. The importance of glycogen as a fuel during exercise has been recognized since the 1960s; however, little is known about the precise mechanism that relates skeletal muscle glycogen to muscle fatigue. We show that low muscle glycogen is associated...... that links energy utilization, i.e. muscle contraction, with the energy content in the muscle, thereby inhibiting a detrimental depletion of the muscle energy store....

  14. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    Science.gov (United States)

    Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

    2012-05-01

    3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.

  15. Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium-transport enzyme.

    Science.gov (United States)

    Ronzani, N; Stephan, L; Hasselbach, W

    1991-10-01

    The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium-transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding. The vanadate-free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7-14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure-binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure-binding relationship indicates that the description of vanadate binding requires at least a two-step reaction sequence. The volume increments which predominate at lower pressure values, range from 200-400 ml.mol-1 depending on the composition of the reaction medium containing 5 microM and 20 microM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20-40 ml.mol-1. Vanadate binding is reduced in the presence of 30 microM calcium, and simultaneously both binding volumes are diminished by 100 ml.mol-1 and 20 ml.mol-1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.

  16. Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

    Science.gov (United States)

    Kjelstrup, Signe; de Meis, Leopoldo; Bedeaux, Dick; Simon, Jean-Marc

    2008-11-01

    We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca(2+) in the Ca(2+)-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca(2+)-uptake, and heat up during ATP synthesis and Ca(2+)-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca(2+)-ATPases, not only act as Ca(2+)-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

  17. ARRHYTHMOGENIC CALMODULIN MUTATIONS AFFECT THE ACTIVATION AND TERMINATION OF CARDIAC RYANODINE RECEPTOR MEDIATED CA2+ RELEASE

    DEFF Research Database (Denmark)

    Søndergaard, Mads Toft; Chazin, Walter J.; Chen, Wayne S.R.;

    We recently identified the first two human missense mutations in a calmodulin (CaM) gene (CALM1) and linked these to catecholaminergic polymorphic ventricular tachycardia (CPVT) and sudden cardiac death in young individuals1. More CaM mutations have since been identified in CALM1 and also......M in the presence of RyR2 CaMBD. The D95V, N97S and D129G mutations lowered the affinity of Ca2+ binding of the C-lobe of CaM, to apparent KDs of ~ 140, 150, and 4000 nM, respectively, consistent with the critical role of these residues in Ca2+ binding to the C-lobe. Thus, we suggest that these mutations may shift...... to an apo-CaM binding state during diastole, leading to dysregulation of RyR2 mediated Ca2+ release. Despite the pronounced impact on RyR2 mediated Ca2+ release, the N-lobe N53I mutation only imposed a small lowering of the N-lobe Ca2+ affinity (KD ~1200 nM). Thus, the RyR2 mediated Ca2+ release is either...

  18. Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice

    National Research Council Canada - National Science Library

    Shan, Jian; Kushnir, Alexander; Betzenhauser, Matthew J; Reiken, Steven; Li, Jingdong; Lehnart, Stephan E; Lindegger, Nicolas; Mongillo, Marco; Mohler, Peter J; Marks, Andrew R

    2010-01-01

    During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output...

  19. Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating

    Science.gov (United States)

    Mukherjee, Saptarshi; Thomas, N. Lowri; Williams, Alan J.

    2016-01-01

    The flow of ions through membrane channels is precisely regulated by gates. The architecture and function of these elements have been studied extensively, shedding light on the mechanisms underlying gating. Recent investigations have focused on ion occupancy of the channel’s selectivity filter and its ability to alter gating, with most studies involving prokaryotic K+ channels. Some studies used large quaternary ammonium blocker molecules to examine the effects of altered ionic flux on gating. However, the absence of blocking events that are visibly distinct from closing events in K+ channels makes unambiguous interpretation of data from single channel recordings difficult. In this study, the large K+ conductance of the RyR2 channel permits direct observation of blocking events as distinct subconductance states and for the first time demonstrates the differential effects of blocker molecules on channel gating. This experimental platform provides valuable insights into mechanisms of blocker-induced modulation of ion channel gating. PMID:27703263

  20. FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1

    OpenAIRE

    Raina, Shweta A.; Jeffrey Tsai; Montserrat Samsó; Fessenden, James D.

    2012-01-01

    Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) inse...

  1. Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation.

    Science.gov (United States)

    Ajmat, M T; Bonilla, F; Zelarayán, L; Bühler, M I

    2011-05-01

    Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.

  2. Sevoflurane protects ventricular myocytes from Ca2+ paradox-mediated Ca2+ overload by blocking the activation of transient receptor potential canonical channels.

    Science.gov (United States)

    Kojima, Akiko; Kitagawa, Hirotoshi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi; Nosaka, Shuichi

    2011-09-01

    Volatile anesthetics produce cardioprotective action by attenuating cellular Ca2+ overload. The Ca2+ paradox is an important model for studying the mechanisms associated with Ca2+ overload-mediated myocardial injury, and was recently found to be mediated by Ca2+ entry through the transient receptor potential canonical channels upon Ca2+ repletion. This study investigated the effect of sevoflurane on cellular mechanisms underlying the Ca2+ paradox. The Ca2+ paradox was examined in fluo-3 or mag-fluo-4-loaded mouse ventricular myocytes using confocal laser scanning microscope, upon Ca2+ repletion after 15 min of Ca2+ depletion in the absence and presence of sevoflurane. The Ca2+ paradox was evoked in approximately 65% of myocytes upon Ca2+ repletion, as determined by an abrupt elevation of cytosolic Ca2+ accompanied by hypercontracture. The Ca2+ paradox was significantly suppressed by sevoflurane administered for 3 min before and during Ca2+ repletion (Post) or during Ca2+ depletion and repletion (Postlong), and Postlong was more beneficial than Post application. The sarcoplasmic reticulum Ca2+ levels gradually decreased during Ca2+ depletion, and the Ca2+ paradox was readily evoked in myocytes with reduced sarcoplasmic reticulum Ca2+ levels. Postlong but not Post application of sevoflurane prevented decrease in sarcoplasmic reticulum Ca2+ levels by blocking Ca2+ leak through ryanodine receptors. Whole cell patch-clamp recordings revealed that sevoflurane rapidly blocked thapsigargin-induced transient receptor potential canonical currents. Sevoflurane protects ventricular myocytes from Ca2+ paradox-mediated Ca2+ overload by blocking transient receptor potential canonical channels and by preventing the decrease in sarcoplasmic reticulum Ca2+ levels, which is associated with less activation of transient receptor potential canonical channels.

  3. Novel Junctophilin-2 Mutation A405S Is Associated With Basal Septal Hypertrophy and Diastolic Dysfunction

    Directory of Open Access Journals (Sweden)

    Ann P. Quick, BA

    2017-02-01

    Full Text Available Junctophilin-2 (JPH2 is a structural calcium (Ca2+ handling protein, which approximates the cardiomyocyte transverse tubules (TTs to the sarcoplasmic reticulum. This facilitates communication of the voltage-gated Ca2+ channel and the ryanodine receptor RyR2. A human patient with hypertrophic cardiomyopathy was positive for a JPH2 mutation substituting alanine-405—located within the alpha helix domain—with a serine (A405S. Using a novel mouse echocardiography plane, we found that mice bearing this JPH2 mutation developed increased subvalvular septal thickness. Cardiomyocytes from the septa of these mice displayed irregular TTs and abnormal Ca2+ handling including increased SERCA activity.

  4. T-tubule biogenesis and triad formation in skeletal muscle and implication in human diseases

    Directory of Open Access Journals (Sweden)

    Al-Qusairi Lama

    2011-07-01

    Full Text Available Abstract In skeletal muscle, the excitation-contraction (EC coupling machinery mediates the translation of the action potential transmitted by the nerve into intracellular calcium release and muscle contraction. EC coupling requires a highly specialized membranous structure, the triad, composed of a central T-tubule surrounded by two terminal cisternae from the sarcoplasmic reticulum. While several proteins located on these structures have been identified, mechanisms governing T-tubule biogenesis and triad formation remain largely unknown. Here, we provide a description of triad structure and plasticity and review the role of proteins that have been linked to T-tubule biogenesis and triad formation and/or maintenance specifically in skeletal muscle: caveolin 3, amphiphysin 2, dysferlin, mitsugumins, junctophilins, myotubularin, ryanodine receptor, and dihydhropyridine Receptor. The importance of these proteins in triad biogenesis and subsequently in muscle contraction is sustained by studies on animal models and by the direct implication of most of these proteins in human myopathies.

  5. RYR2 sequencing reveals novel missense mutations in a Kazakh idiopathic ventricular tachycardia study cohort.

    Directory of Open Access Journals (Sweden)

    Ainur Akilzhanova

    Full Text Available Channelopathies, caused by disturbed potassium or calcium ion management in cardiac myocytes are a major cause of heart failure and sudden cardiac death worldwide. The human ryanodine receptor 2 (RYR2 is one of the key players tightly regulating calcium efflux from the sarcoplasmic reticulum to the cytosol and found frequently mutated (T; p.D4631V in a CPVT patient and a novel rare variant (c5428G>C; p.V1810L of uncertain significance in a patient with VT of idiopathic origin which we suggest represents a low-penetrance or susceptibility variant. In addition we identified a known variant previously associated with arrhythmogenic right ventricular dysplasia type2 (ARVD2. Combining sets of prediction scores and reference databases appeared fundamental to predict the pathogenic potential of novel and rare missense variants in populations where genotype data are rare.

  6. Role of NOD1 in Heart Failure Progression via Regulation of Ca(2+) Handling.

    Science.gov (United States)

    Val-Blasco, Almudena; Piedras, María Jose G M; Ruiz-Hurtado, Gema; Suarez, Natalia; Prieto, Patricia; Gonzalez-Ramos, Silvia; Gómez-Hurtado, Nieves; Delgado, Carmen; Pereira, Laetitia; Benito, Gemma; Zaragoza, Carlos; Domenech, Nieves; Crespo-Leiro, María Generosa; Vasquez-Echeverri, Daniel; Nuñez, Gabriel; Lopez-Collazo, Eduardo; Boscá, Lisardo; Fernández-Velasco, María

    2017-01-31

    Heart failure (HF) is a complex syndrome associated with a maladaptive innate immune system response that leads to deleterious cardiac remodeling. However, the underlying mechanisms of this syndrome are poorly understood. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is a newly recognized innate immune sensor involved in cardiovascular diseases. This study evaluated the role of NOD1 in HF progression. NOD1 was examined in human failing myocardium and in a post-myocardial infarction (PMI) HF model evaluated in wild-type (wt-PMI) and Nod1(-/-) mice (Nod1(-/-)-PMI). The NOD1 pathway was up-regulated in human and murine failing myocardia. Compared with wt-PMI, hearts from Nod1(-/-)-PMI mice had better cardiac function and attenuated structural remodeling. Ameliorated cardiac function in Nod1(-/-)-PMI mice was associated with prevention of Ca(2+) dynamic impairment linked to HF, including smaller and longer intracellular Ca(2+) concentration transients and a lesser sarcoplasmic reticulum Ca(2+) load due to a down-regulation of the sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase pump and by augmented levels of the Na(+)/Ca(2+) exchanger. Increased diastolic Ca(2+) release in wt-PMI cardiomyocytes was related to hyperphosphorylation of ryanodine receptors, which was blunted in Nod1(-/-)-PMI cardiomyocytes. Pharmacological blockade of NOD1 also prevented Ca(2+) mishandling in wt-PMI mice. Nod1(-/-)-PMI mice showed significantly fewer ventricular arrhythmias and lower mortality after isoproterenol administration. These effects were associated with lower aberrant systolic Ca(2+) release and with a prevention of the hyperphosphorylation of ryanodine receptors under isoproterenol administration in Nod1(-/-)-PMI mice. NOD1 modulated intracellular Ca(2+) mishandling in HF, emerging as a new target for HF therapy. Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  7. Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia.

    Science.gov (United States)

    Willis, B Cicero; Pandit, Sandeep V; Ponce-Balbuena, Daniela; Zarzoso, Manuel; Guerrero-Serna, Guadalupe; Limbu, Bijay; Deo, Makarand; Camors, Emmanuel; Ramirez, Rafael J; Mironov, Sergey; Herron, Todd J; Valdivia, Héctor H; Jalife, José

    2016-06-14

    In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca(2+) dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2(R4496C+/Cx40eGFP)), we tested whether PC intracellular Ca(2+) ([Ca(2+)]i) dysregulation results from a constitutive [Na(+)]i surplus relative to VMs. Simultaneous optical mapping of voltage and [Ca(2+)]i in CPVT hearts showed that spontaneous Ca(2+) release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca(2+) imaging, early and delayed afterdepolarizations trailed spontaneous Ca(2+) release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca(2+) load, measured by caffeine-induced Ca(2+) transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na(+)]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na(+)/Ca(2+) exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na(+)]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca(2+) release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na(+)]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca(2+) spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na(+)]i played a central role. In CPVT mice, the constitutive [Na(+)]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs. © 2016 The Authors.

  8. Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia

    Science.gov (United States)

    Willis, B. Cicero; Pandit, Sandeep V.; Ponce-Balbuena, Daniela; Zarzoso, Manuel; Guerrero-Serna, Guadalupe; Limbu, Bijay; Deo, Makarand; Camors, Emmanuel; Ramirez, Rafael J.; Mironov, Sergey; Herron, Todd J.; Valdivia, Héctor H.

    2016-01-01

    Background— In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca2+ dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2R4496C+/Cx40eGFP), we tested whether PC intracellular Ca2+ ([Ca2+]i) dysregulation results from a constitutive [Na+]i surplus relative to VMs. Methods and Results— Simultaneous optical mapping of voltage and [Ca2+]i in CPVT hearts showed that spontaneous Ca2+ release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca2+ imaging, early and delayed afterdepolarizations trailed spontaneous Ca2+ release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca2+ load, measured by caffeine-induced Ca2+ transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na+]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na+/Ca2+ exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na+]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca2+ release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na+]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca2+ spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na+]i played a central role. Conclusions— In CPVT mice, the constitutive [Na+]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs. PMID:27169737

  9. Temperature sensitivity of cardiac function in pelagic fishes with different vertical mobilities: yellowfin tuna (Thunnus albacares), bigeye tuna (Thunnus obesus), mahimahi (Coryphaena hippurus), and swordfish (Xiphias gladius).

    Science.gov (United States)

    Galli, Gina L J; Shiels, Holly A; Brill, Richard W

    2009-01-01

    We measured the temperature sensitivity, adrenergic sensitivity, and dependence on sarcoplasmic reticulum (SR) Ca(2+) of ventricular muscle from pelagic fishes with different vertical mobility patterns: bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), and mahimahi (Coryphaena hippurus) and a single specimen from swordfish (Xiphias gladius). Ventricular muscle from the bigeye tuna and mahimahi exhibited a biphasic response to an acute decrease in temperature (from 26 degrees to 7 degrees C); twitch force and kinetic parameters initially increased and then declined. The magnitude of this response was larger in the bigeye tuna than in the mahimahi. Under steady state conditions at 26 degrees C, inhibition of SR Ca(2+) release and reuptake with ryanodine and thapsigargin decreased twitch force and kinetic parameters, respectively, in the bigeye tuna only. However, the initial inotropy associated with decreasing temperature was abolished by SR inhibition in both the bigeye tuna and the mahimahi. Application of adrenaline completely reversed the effects of ryanodine and thapsigargin, but this effect was diminished at cold temperatures. In the yellowfin tuna, temperature and SR inhibition had minor effects on twitch force and kinetics, while adrenaline significantly increased these parameters. Limited data suggest that swordfish ventricular muscle responds to acute temperature reduction, SR inhibition, and adrenergic stimulation in a manner similar to that of bigeye tuna ventricular muscle. In aggregate, our results show that the temperature sensitivity, SR dependence, and adrenergic sensitivity of pelagic fish hearts are species specific and that these differences reflect species-specific vertical mobility patterns.

  10. Alterations in T-tubule and dyad structure in heart disease: challenges and opportunities for computational analyses.

    Science.gov (United States)

    Poláková, Eva; Sobie, Eric A

    2013-05-01

    Compelling recent experimental results make clear that sub-cellular structures are altered in ventricular myocytes during the development of heart failure, in both human samples and diverse experimental models. These alterations can include, but are not limited to, changes in the clusters of sarcoplasmic reticulum (SR) Ca(2+)-release channels, ryanodine receptors, and changes in the average distance between the cell membrane and ryanodine receptor clusters. In this review, we discuss the potential consequences of these structural alterations on the triggering of SR Ca(2+) release during excitation-contraction coupling. In particular, we describe how mathematical models of local SR Ca(2+) release can be used to predict functional changes resulting from diverse modifications that occur in disease states. We review recent studies that have used simulations to understand the consequences of sub-cellular structural changes, and we discuss modifications that will allow for future modelling studies to address unresolved questions. We conclude with a discussion of improvements in both experimental and mathematical modelling techniques that will be required to provide a stronger quantitative understanding of the functional consequences of changes in sub-cellular structure in heart disease.

  11. Effect of K201, a novel antiarrhythmic drug on calcium handling and arrhythmogenic activity of pulmonary vein cardiomyocytes

    Science.gov (United States)

    Chen, Y-J; Chen, Y-C; Wongcharoen, W; Lin, C-I; Chen, S-A

    2007-01-01

    Background and purpose: Pulmonary veins are the most important focus for the generation of atrial fibrillation. Abnormal calcium homeostasis with ryanodine receptor dysfunction may underlie the arrhythmogenic activity in pulmonary veins. The preferential ryanodine receptor stabilizer (K201) possesses antiarrhythmic effects through calcium regulation. The purpose of this study was to investigate the effects of K201 on the arrhythmogenic activity and calcium regulation of pulmonary vein cardiomyocytes. Experimental approach: The ionic currents and intracellular calcium were studied in isolated single cardiomyocytes from rabbit pulmonary vein before and after the administration of K201, by the whole-cell patch clamp and indo-1 fluorimetric ratio techniques. Key results: K201 (0.1, 0.3, 1 μM) reduced the firing rates in pulmonary vein cardiomyocytes, decreased the amplitudes of the delayed afterdepolarizations and prolonged the action potential duration. K201 decreased the L-type calcium currents, Na+/Ca2+ exchanger currents, transient inward currents and calcium transients. K201 (1 μM, but not 0.1 μM or 0.3 μM) also reduced the sarcoplasmic reticulum calcium content. Moreover, both the pretreatment and administration of K201 (0.3 μM) decreased the isoprenaline (10 nM)-induced arrhythmogenesis in pulmonary veins. Conclusions and implications: K201 reduced the arrhythmogenic activity of pulmonary vein cardiomyocytes and attenuated the arrhythmogenicity induced by isoprenaline. These findings may reveal the anti-arrhythmic potential of K201. PMID:17994112

  12. Phosphatidylinositol 3-kinase inhibition restores Ca2+ release defects and prolongs survival in myotubularin-deficient mice

    Science.gov (United States)

    Kutchukian, Candice; Lo Scrudato, Mirella; Tourneur, Yves; Poulard, Karine; Vignaud, Alban; Berthier, Christine; Allard, Bruno; Lawlor, Michael W.; Buj-Bello, Ana; Jacquemond, Vincent

    2016-01-01

    Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation–contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear. During normal skeletal muscle EC coupling, transverse (t) tubule depolarization triggers sarcoplasmic reticulum (SR) Ca2+ release through ryanodine receptor channels gated by conformational coupling with the t-tubule voltage-sensing dihydropyridine receptors. We report that MTM1 deficiency is associated with a 60% depression of global SR Ca2+ release over the full range of voltage sensitivity of EC coupling. SR Ca2+ release in the diseased fibers is also slower than in normal fibers, or delayed following voltage activation, consistent with the contribution of Ca2+-gated ryanodine receptors to EC coupling. In addition, we found that SR Ca2+ release is spatially heterogeneous within myotubularin-deficient muscle fibers, with focally defective areas recapitulating the global alterations. Importantly, we found that pharmacological inhibition of phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity rescues the Ca2+ release defects in isolated muscle fibers and increases the lifespan and mobility of XLMTM mice, providing proof of concept for the use of PtdIns 3-kinase inhibitors in myotubular myopathy and suggesting that unbalanced PtdIns 3-kinase activity plays a critical role in the pathological process. PMID:27911767

  13. Effects of eugenol on resting tension of rat atria.

    Science.gov (United States)

    Olivoto, R R; Damiani, C E N; Kassouf Silva, I; Lofrano-Alves, M S; Oliveira, M A; Fogaça, R T H

    2014-04-01

    In cardiac and skeletal muscle, eugenol (μM range) blocks excitation-contraction coupling. In skeletal muscle, however, larger doses of eugenol (mM range) induce calcium release from the sarcoplasmic reticulum. The effects of eugenol are therefore dependent on its concentration. In this study, we evaluated the effects of eugenol on the contractility of isolated, quiescent atrial trabeculae from male Wistar rats (250-300 g; n=131) and measured atrial ATP content. Eugenol (1, 3, 5, 7, and 10 mM) increased resting tension in a dose-dependent manner. Ryanodine [100 µM; a specific ryanodine receptor (RyR) blocker] and procaine (30 mM; a nonspecific RyR blocker) did not block the increased resting tension induced by eugenol regardless of whether extracellular calcium was present. The myosin-specific inhibitor 2,3-butanedione monoxime (BDM), however, reversed the increase in resting tension induced by eugenol. In Triton-skinned atrial trabeculae, in which all membranes were solubilized, eugenol did not change resting tension, maximum force produced, or the force vs pCa relationship (pCa=-log [Ca2+]). Given that eugenol reduced ATP concentration, the increase in resting tension observed in this study may have resulted from cooperative activation of cardiac thin filaments by strongly attached cross-bridges (rigor state).

  14. Beat-to-beat cycle length variability of spontaneously beating guinea pig sinoatrial cells: relative contributions of the membrane and calcium clocks.

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    Massimiliano Zaniboni

    Full Text Available The heartbeat arises rhythmically in the sino-atrial node (SAN and then spreads regularly throughout the heart. The molecular mechanism underlying SAN rhythm has been attributed by recent studies to the interplay between two clocks, one involving the hyperpolarization activated cation current If (the membrane clock, and the second attributable to activation of the electrogenic NaCa exchanger by spontaneous sarcoplasmic releases of calcium (the calcium clock. Both mechanisms contain, in principle, sources of beat-to-beat cycle length variability, which can determine the intrinsic variability of SAN firing and, in turn, contribute to the heart rate variability. In this work we have recorded long sequences of action potentials from patch clamped guinea pig SAN cells (SANCs perfused, in turn, with normal Tyrode solution, with the If inhibitor ivabradine (3 µM, then back to normal Tyrode, and again with the ryanodine channels inhibitor ryanodine (3 µM. We have found that, together with the expected increase in beating cycle length (+25%, the application of ivabradine brought about a significant and dramatic increase in beat-to-beat cycle length variability (+50%. Despite the similar effect on firing rate, ryanodine did not modify significantly beat-to-beat cycle length variability. Acetylcholine was also applied and led to a 131% increase of beating cycle length, with only a 70% increase in beat-to-beat cycle length variability. We conclude that the main source of inter-beat variability of SANCs firing rate is related to the mechanism of the calcium clock, whereas the membrane clock seems to act in stabilizing rate. Accordingly, when the membrane clock is silenced by application of ivabradine, stochastic variations of the calcium clock are free to make SANCs beating rhythm more variable.

  15. Involvement of inositol 1,4,5-trisphosphate in nicotinic calcium responses in dystrophic myotubes assessed by near-plasma membrane calcium measurement.

    Science.gov (United States)

    Basset, Olivier; Boittin, François-Xavier; Dorchies, Olivier M; Chatton, Jean-Yves; van Breemen, Cornelis; Ruegg, Urs T

    2004-11-05

    In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.

  16. Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging.

    Science.gov (United States)

    Umanskaya, Alisa; Santulli, Gaetano; Xie, Wenjun; Andersson, Daniel C; Reiken, Steven R; Marks, Andrew R

    2014-10-21

    Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca(2+) transients, decreased intracellular Ca(2+) leak and increased sarcoplasmic reticulum (SR) Ca(2+) load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca(2+) release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (Po). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca(2+) leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.

  17. Modeling Catecholaminergic Polymorphic Ventricular Tachycardia using Induced Pluripotent Stem Cell-derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Atara Novak

    2012-07-01

    Full Text Available Catecholaminergic polymorphic ventricular tachycardia (CPVT is an inherited arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias induced by physical or emotional stress, in the absence structural heart abnormalities. The arrhythmias may cause syncope or degenerate into cardiac arrest and sudden death which usually occurs during childhood. Recent studies have shown that CPVT is caused by mutations in the cardiac ryanodine receptor type 2 (RyR2 or calsequestrin 2 (CASQ2 genes. Both proteins are key contributors to the intracellular Ca2+ handling process and play a pivotal role in Ca2+ release from the sarcoplasmic reticulum to the cytosol during systole. Although the molecular pathogenesis of CPVT is not entirely clear, it was suggested that the CPVT mutations promote excessive sarcoplasmic reticulum Ca2+ leak, which initiates delayed afterdepolarizations (DADs and triggered arrhythmias in cardiac myocytes. The recent breakthrough discovery of induced pluripotent stem cells (iPSC generated from somatic cells (e.g. fibroblasts, keratinocytes now enables researches to investigate mutated cardiomyocytes generated from the patient’s iPSC. To this end, in the present article we review recent studies on CPVT iPSC-derived cardiomyocytes, thus demonstrating in the mutated cells catecholamine-induced DADs and triggered arrhythmias.

  18. [3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

    Energy Technology Data Exchange (ETDEWEB)

    Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome

    2002-06-14

    Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

  19. Membrane potential and Ca2+ concentration dependence on pressure and vasoactive agents in arterial smooth muscle: A model.

    Science.gov (United States)

    Karlin, Arthur

    2015-07-01

    Arterial smooth muscle (SM) cells respond autonomously to changes in intravascular pressure, adjusting tension to maintain vessel diameter. The values of membrane potential (Vm) and sarcoplasmic Ca(2+) concentration (Ca(in)) within minutes of a change in pressure are the results of two opposing pathways, both of which use Ca(2+) as a signal. This works because the two Ca(2+)-signaling pathways are confined to distinct microdomains in which the Ca(2+) concentrations needed to activate key channels are transiently higher than Ca(in). A mathematical model of an isolated arterial SM cell is presented that incorporates the two types of microdomains. The first type consists of junctions between cisternae of the peripheral sarcoplasmic reticulum (SR), containing ryanodine receptors (RyRs), and the sarcolemma, containing voltage- and Ca(2+)-activated K(+) (BK) channels. These junctional microdomains promote hyperpolarization, reduced Ca(in), and relaxation. The second type is postulated to form around stretch-activated nonspecific cation channels and neighboring Ca(2+)-activated Cl(-) channels, and promotes the opposite (depolarization, increased Ca(in), and contraction). The model includes three additional compartments: the sarcoplasm, the central SR lumen, and the peripheral SR lumen. It incorporates 37 protein components. In addition to pressure, the model accommodates inputs of α- and β-adrenergic agonists, ATP, 11,12-epoxyeicosatrienoic acid, and nitric oxide (NO). The parameters of the equations were adjusted to obtain a close fit to reported Vm and Ca(in) as functions of pressure, which have been determined in cerebral arteries. The simulations were insensitive to ± 10% changes in most of the parameters. The model also simulated the effects of inhibiting RyR, BK, or voltage-activated Ca(2+) channels on Vm and Ca(in). Deletion of BK β1 subunits is known to increase arterial-SM tension. In the model, deletion of β1 raised Ca(in) at all pressures, and these

  20. Skeletal muscle myofibrillar and sarcoplasmic protein synthesis rates are affected differently by altitude-induced hypoxia in native lowlanders

    DEFF Research Database (Denmark)

    Holm, Lars; Haslund, Mads Lyhne; Robach, Paul

    2010-01-01

    As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O(2). With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-(13)C-...

  1. Low molecular weight peptides derived from sarcoplasmic proteins produced by an autochthonous starter culture in a beaker sausage model

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    Constanza M. López

    2015-06-01

    Significance: The selection of a specific autochthonous starter culture guarantees the hygiene and typicity of fermented sausages. The identification of new peptides as well as new target proteins by means of peptidomics represents a significant step toward the elucidation of the role of microorganisms in meat proteolysis. Moreover, these peptides may be further used as biomarkers capable to certify the use of the applied autochthonous starter culture described here.

  2. Cardiac function improved by sarcoplasmic reticulum Ca2+-ATPase overexpression in a heart failure model induced by chronic myocardial ischemia

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    Wei XIN

    2011-04-01

    Full Text Available Objective Chronic myocardial ischemia(CMI has become an important cause of heart failure(HF.The aim of present study was to examine the effects of Sarco-endoplasmic reticulum calcium ATPase(SERCA2a gene transfer in HF model in large animal induced by CMI.Methods HF was reproduced in minipigs by ligating the initial segment of proximal left anterior descending(LAD coronary artery with an ameroid constrictor to produce progressive vessel occlusion and ischemia.After confirmation of myocardial perfusion defect and cardiac function impairment by SPECT and echocardiography in the model,animals were divided into 4 groups: HF group;HF+enhanced green fluorescent protein(EGFP group;HF+SERCA2a group;and sham operation group as control.rAAV1-EGFP and rAAV1-SERCA2a(1×1012 vg for each animal were directly and intramyocardially injected to the animals of HF+EGFP and HF+SERCA2a groups.Sixty days after the gene transfer,the expression of SERCA2a at the protein level was examined by Western blotting and immunohistochemistry,the changes in cardiac function were determined by echocardiographic and hemodynamic analysis,and the changes in serum inflammatory and neuro-hormonal factors(including BNP,TNF-a,IL-6,ET-1 and Ang II were determined by radioimmunoassay.Results Sixty days after gene transfer,LVEF,Ev/Av and ±dp/dtmax increased significantly(P < 0.05,along with an increase of SERCA2a protein expression in the ischemic myocardium(PP < 0.05,accompanied by a significant decrease of inflammatory and neural-hormonal factors(PP < 0.05 in HF+SERCA2a group as compared with HF/HF+EGFP group.Conclusions Overexpression of SERCA2a may significantly improve the cardiac function of the ischemic myocardium of HF model induced by CMI and reverse the activation of neural-hormonal factors,implying that it has a potential therapeutic significance in CMI related heart failure.

  3. Improvement in cardiac function after sarcoplasmic reticulum Ca2+-ATPase gene transfer in a beagle heart failure model

    Institute of Scientific and Technical Information of China (English)

    MI Ya-fei; LI Xiao-ying; TANG Li-jiang; LU Xiao-chun; FU Zhi-qing; YE Wei-hua

    2009-01-01

    Background Heart failure (HF) is a major cause of morbidity and mortality worldwide, but current treatment modalities cannot reverse the underlying pathological state of the heart. Gene-based therapies are emerging as promising therapeutic modalities in HF patients. Our previous studies have shown that recombinant adeno-associated viral (rAAV) gene transfer of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) can be effective in treating rats with chronic heart failure (CHF). The aim of this study was to examine the effects of SERCA2a gene transfer in a large HF animal model.Methods HF was induced in beagles by rapid right ventricular pacing (230 beats/min) for 30 days. A reduced rate ventricular pacing (180 beats/min) was continued for another 30 days. The beagles were assigned to four groups: (a) control group (n=4); (b) HF group (n=4); (c) enhanced green fluorescent protein group (n=4); and (d) SERCA2.a group (n=4). rAAVl-EGFP (lx1012 μg) and rAAVl-SERCA2a (lx1012 μg) were delivered intramyocardially. SERCA2.a expression was assessed by Western blotting and immunohistochemistry.Results Following 30 days of SERCA2a gene transfer in HF beagles its protein expression was significantly higher than in the HF group than in the control group (P <0.05). Heart function improved along with the increase in SERCA2a expression. Left ventricular systolic function significantly improved, including the ejection fraction, left ventricular systolic pressure, maximal rate of rise of left ventricular pressure (+dp/dtmax), and the maximal rate of decline of left ventricular pressure (-dp/dtmax) (P <0.05). Left ventricular end-diastole pressure significantly decreased (P <0.05). The expression of SERCA2a in the myocardial tissue was higher in the SERCA2a group than in the HF group (P<0.05). Conclusions Intramyocardial injection of rAAVl-SERCA2a can improve the cardiac function in beagles induced with HE We expect further studies on SERCA2a's long-term safety, efficacy, dosage and the optimization before using it in humans with HF.

  4. S100A1: A Regulator of Striated Muscle Sarcoplasmic Reticulum Ca2+ Handling, Sarcomeric, and Mitochondrial Function

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    Mirko Völkers

    2010-01-01

    S100A1 has further been detected at different sites within the cardiac sarcomere indicating potential roles in myofilament function. More recently, a study reported a mitochondrial location of S100A1 in cardiomyocytes. Additionally, normalizing the level of S100A1 protein by means of viral cardiac gene transfer in animal heart failure models resulted in a disrupted progression towards cardiac failure and enhanced survival. This brief review is confined to the physiological and pathophysiological relevance of S100A1 in cardiac and skeletal muscle Ca2+ handling with a particular focus on its potential as a molecular target for future therapeutic interventions.

  5. Ameliorated stress related proteins are associated with improved cardiac function by sarcoplasmic reticulum calcium ATPase gene transfer in heart failure

    Institute of Scientific and Technical Information of China (English)

    Zhi-Qing Fu; Xiao-Ying Li; Xiao-Chun Lu; Ya-Fei Mi; Tao Liu; Wei-Hua Ye

    2012-01-01

    Background Previous studies showed that overexpression of sarco-endoplasmic reticulum calcium ATPase (SERCA2a) in a variety of heart failure (HF) models was associated with greatly enhanced cardiac performance. However, it still undefined the effect of SERCA2a overexpression on the systemic inflammatory response and neuro-hormonal factors. Methods A rapid right ventricular pacing model of experimental HF was used in beagles. Then the animals underwent recombinant adeno-associated virus 1 (rAAV1) mediated gene transfection by direct intra-myocardium injection. HF animals were randomized to receive the SERCA2a gene, enhanced green fluorescent protein (control) gene, or equivalent phosphate buffered saline. Thirty days after gene delivery, the cardiac function was evaluated by echocardiographic testing. The protein level of SERCA2a was measured by western blotting. The proteomic analysis of left ventricular (LV) sample was determined using two-dimensional (2-D) gel electrophoresis and MALDI-TOF-MS. The serum levels of the systemic inflammatory and neuro-hormonal factors were assayed using radioimmunoassay kits. Results The cardiac function improved after SERCA- 2a gene transfer due to the significantly increased SERCA2a protein level. Beagles treated with SERCA2a had significantly decreased serum levels of the inflammatory markers (interleukin-6 and tumor necrosis factor-α) and neuro-hormonal factors (brain natriuretic peptide, endothelin-1 and angiotensin Ⅱ) compared with HF animals. The myocardial proteomic analysis showed that haptoglobin heavy chain, heat shock protein (alpha-crystallin-related, B6) were down-regulated, and galectin-1 was up-regulated in SERCA2a group compared with HF group, companied by up-regulated contractile proteins and NADH dehydrogenase. Conclusions These findings demonstrate that regional intramyocardial injections of rAAV1-SERCA2a vectors may improve global LV function, correlating with reverse activation of the systemic inflammatory, excessive neuroendocrine factors and the stress-associated myocardial proteins, suggesting that the beneficial effects of SERCA2a gene transfer may involve the attenuation of stress-associated reaction.

  6. Structural dynamics and topology of phosphorylated phospholamban homopentamer reveal its role in the regulation of calcium transport in sarcoplasmic reticulum

    Science.gov (United States)

    Vostrikov, Vitaly V.; Mote, Kaustubh R.; Verardi, Raffaello; Veglia, Gianluigi

    2013-01-01

    Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), thereby regulating cardiac diastole. In membranes, PLN assembles into homopentamers that in both the phosphorylated and non-phosphorylated states have been proposed to form ion-selective channels. Here, we determined the structure of the phosphorylated pentamer using a combination of solution and solid-state nuclear magnetic resonance methods. We found that the pinwheel architecture of the homopentamer is preserved upon phosphorylation, with each monomer having an L-shaped conformation of each monomer. The TM domains form a hydrophobic pore of approximately 24 Å long, and 2 Å in diameter, which is inconsistent with canonical Ca2+ selective channels. Phosphorylation, however, enhances the conformational dynamics of the cytoplasmic region of PLN, causing the partial unwinding of the amphipathic helix. We propose that PLN oligomers act as storage for active monomers, keeping SERCA function within a physiological window. PMID:24207128

  7. Effects of Preslaughter Stress Levels on the Post-mortem Sarcoplasmic Proteomic Profile of Gilthead Seabream Muscle

    DEFF Research Database (Denmark)

    Silva, Tomé Santos; Cordeiro, Odete D; Matos, Elisabete D.

    2012-01-01

    affects these post-mortem processes. For the experiment, two groups of gilthead seabream (n = 5) were subjected to distinct levels of preslaughter stress, with three muscle samples being taken from each fish. Proteins were extracted from the muscle samples, fractionated, and separated by 2DE. Protein......Fish welfare is an important concern in aquaculture, not only due to the ethical implications but also for productivity and quality-related reasons. The purpose of this study was to track soluble proteome expression in post-mortem gilthead seabream muscle and to observe how preslaughter stress...... been hastened by preslaughter stress, confirming that it induces clear post-mortem changes in the muscle proteome of gilthead seabream....

  8. Temperature and Ca2+-dependence of the sarcoplasmic reticulum Ca2(+)-ATPase in haddock, salmon, rainbow trout and zebra cichlid

    DEFF Research Database (Denmark)

    Godiksen, Helene; Jessen, Flemming

    2002-01-01

    in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca2+-dependence of SR Ca2+-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo, salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma...... nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degreesC, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the. temperature area, 6-14 degreesC. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca2...

  9. Doublet discharge stimulation increases sarcoplasmic reticulum Ca2+ release and improves performance during fatiguing contractions in mouse muscle fibres.

    Science.gov (United States)

    Cheng, Arthur J; Place, Nicolas; Bruton, Joseph D; Holmberg, Hans-Christer; Westerblad, Håkan

    2013-08-01

    Double discharges (doublets) of motor neurones at the onset of contractions increase both force and rate of force development during voluntary submaximal contractions. The purpose of this study was to examine the role of doublet discharges on force and myoplasmic free [Ca(2+)] ([Ca(2+)]i) during repeated fatiguing contractions, using a stimulation protocol mimicking the in vivo activation pattern during running. Individual intact fibres from the flexor digitorum brevis muscle of mice were stimulated at 33°C to undergo 150 constant-frequency (five pulses at 70 Hz) or doublet (an initial, extra pulse at 200 Hz) contractions at 300 ms intervals. In the unfatigued state, doublet stimulation resulted in a transient (∼10 ms) approximate doubling of [Ca(2+)]i, which was accompanied by a greater force-time integral (∼70%) and peak force (∼40%) compared to constant frequency contractions. Moreover, doublets markedly increased force-time integral and peak force during the first 25 contractions of the fatiguing stimulation. In later stages of fatigue, addition of doublets increased force production but the increase in force production corresponded to only a minor portion of the fatigue-induced reduction in force. In conclusion, double discharges at the onset of contractions effectively increase force production, especially in early stages of fatigue. This beneficial effect occurs without additional force loss in later stages of fatigue, indicating that the additional energy cost induced by doublet discharges to skeletal muscle is limited.

  10. Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

    DEFF Research Database (Denmark)

    Laursen, Mette; Bublitz, Maike; Moncoq, Karine;

    2009-01-01

    Abstract: We have determined the structure of the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) in an E2.P-i-like form stabilized as a complex with MgF42-, an ATP analog, adenosine 5'-(beta,gamma-methylene) triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.......5 angstrom resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which...... is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing...

  11. Cyclopiazonic Acid Is Complexed to a Divalent Metal Ion When Bound to the Sarcoplasmic Reticulum Ca2+-ATPase

    DEFF Research Database (Denmark)

    Laursen, Mette; Bublitz, Maike; Moncoq, Karine

    2009-01-01

    .5 angstrom resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which...... is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca2+-ATPases, e. g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing...

  12. Defective sarcoplasmic reticulum-mitochondria communication in aged heart and its effect on ischemia and reperfusion injury

    OpenAIRE

    Fernández Sanz, Celia

    2015-01-01

    Las alteraciones mitocondriales están vinculadas a la mayor vulnerabilidad de padecer enfermedades durante el envejecimiento. La edad avanzada es un factor determinante de la incidencia y gravedad de la cardiopatía isquémica. Estudios preclínicos sugieren la existencia de un daño celular intrínseco, por mecanismos no del todo establecidos, que contribuye a un incremento de la susceptibilidad del miocardio senescente al daño isquémico. Esta tesis investiga el papel de la comunicación mitoco...

  13. Defective sarcoplasmic reticulum-mitochondria communication in aged heart and its effect on ischemia and reperfusion injury

    OpenAIRE

    Fernández Sanz, Celia; Meseguer Navarro, Anna

    2015-01-01

    Las alteraciones mitocondriales están vinculadas a la mayor vulnerabilidad de padecer enfermedades durante el envejecimiento. La edad avanzada es un factor determinante de la incidencia y gravedad de la cardiopatía isquémica. Estudios preclínicos sugieren la existencia de un daño celular intrínseco, por mecanismos no del todo establecidos, que contribuye a un incremento de la susceptibilidad del miocardio senescente al daño isquémico. Esta tesis investiga el papel de la comunicación mitocondr...

  14. Skeletal muscle myofibrillar and sarcoplasmic protein synthesis rates are affected differently by altitude-induced hypoxia in native lowlanders

    DEFF Research Database (Denmark)

    Holm, Lars; Haslund, Mads Lyhne; Robach, Paul;

    2010-01-01

    and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041±0.018 at sea-level to 0.080±0.018%·hr(-1) (p0.05). Trends...

  15. Effects of free oxygen radicals on Ca2+ release mechanisms in the sarcoplasmic reticulum of scallop (Pecten jacobaeus) adductor muscle.

    Science.gov (United States)

    Burlando, B; Viarengo, A; Pertica, M; Ponzano, E; Orunesu, M

    1997-08-01

    In vitro oxyradical effects on SR Ca2+ regulation were studied by using a SR-containing cell-free preparation from scallop (Pecten jacobaeus) adductor muscle. Ca2+ variations were fluorimetrically detected after incubation with Fluo-3 in the presence of ATP. Exposure to Fe3+/ascorbate produced dose-dependent Ca2+ release from SR vesicles, eventually leading to massive Ca2+ loss. Exposure to hypoxanthine/xanthine oxidase also caused Ca2+ release but at a much slower rate. Pre-incubations with catalase or with the hydroxyl radical scavenger KMBA led to a significant decrease in the Fe3+/ascorbate-induced Ca2+ release rate and to a delay of massive Ca2+ loss. Pre-incubations with GSH or DTT strongly reduced the Ca2+ release caused by Fe3+/ascorbate and, moreover, they prevented massive Ca2+ loss from SR vesicles. Addition of GSH or DTT after Fe3+/ascorbate promptly reduced the Ca2+ release rate and delayed massive Ca2+ release. Pre-incubation with the SR Ca2+ channel blocker ruthenium red strongly reduced the Ca2+ release caused by Fe3+/ascorbate, and also prevented massive Ca2+ loss. In the presence of ruthenium red, Fe3+/ascorbate treatments followed by Ca2+ addition revealed that Ca2+ uptake inhibition was slower than Ca2+ release. Taken together, data showed that free radicals and, in particular, hydroxyl radicals, affected the scallop SR Ca2+ regulation. This mainly occurred through Ca2+ channel opening, most likely triggered by sulfhydryl oxidation, which eventually led to massive Ca2+ release from SR vesicles. The demonstration of a specific effect of oxyradicals on SR Ca2+ channels is in line with their possible involvement in cell signaling.

  16. Developmental exposure to polychlorinated biphenyls (PCBs) interferes with experience-dependent dendritic plasticity and ryanodine receptor expression in weanling rats.

    Science.gov (United States)

    BACKGROUND: Neurodevelopmental disorders are associated with altered patterns of neuronal connectivity. A critical determinant of neuronal connectivity is the dendritic morphology of individual neurons, which is shaped by experience. The identification of environmental exposures ...

  17. Differences in Ca2+-management between the ventricle of two species of neotropical teleosts: the jeju, Hoplerythrinus unitaeniatus (Spix & Agassiz, 1829, and the acara, Geophagus brasiliensis (Quoy & Gaimard, 1824

    Directory of Open Access Journals (Sweden)

    Monica Jones Costa

    2009-09-01

    Full Text Available This study analyzed the physiological role of the cardiac sarcoplasmic reticulum (SR of two neotropical teleosts, the jeju, Hoplerythrinus unitaeniatus (Erythrinidae, and the acara, Geophagus brasiliensis (Cichlidae. While the in vivo heart frequency (fH - bpm of acara (79.6 ± 6.6 was higher than that of the jeju (50.3 ± 2.7, the opposite was observed for the ventricular inotropism (Fc - mN/mm² at 12 bpm (acara = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67. A 5 min diastolic pause resulted in a strong potentiation of Fc (≅ 90% of strips from jeju, which was completely abolished by ryanodine. Ryanodine also resulted in a ≅ 20% decrease in the Fc developed by strips from jeju at both subphysiological (12 bpm and physiological (in vivo frequencies. However, this effect of ryanodine reducing the Fc from jeju was completely compensated by adrenaline increments (10-9 and 10-6 M. In contrast, strips from acara were irresponsive to ryanodine, irrespective of the stimulation frequency, and increases in adrenaline concentration (to 10-9 and 10-6 M further increased Fc. These results reinforce the hypothesis of the functionality of the SR as a common trait in neotropical ostariophysian (as jeju, while in acanthopterygians (as acara it seems to be functional mainly in 'athletic' species.O presente estudo analisou o papel fisiológico desempenhado pelo retículo sarcoplasmático (RS de duas espécies de teleósteos neotropicais, o jeju, Hoplerythrinus unitaeniatus (Erythrinidae, e o acará, Geophagus brasiliensis (Cichlidae. Enquanto a frequência cardíaca registrada in vivo (fH - bpm para o acará (79.6 ± 6.6 foi superior àquela observada para o jeju (50.3 ± 2.7, resposta inversa foi verificada para o inotropismo ventricular (Fc - mN/mm² na frequência de estimulação de 12 bpm (acará = 28.66 ± 1.86 vs. jeju = 36.09 ± 1.67. Uma pausa diastólica de 5 min resultou em uma expressiva potenciação da Fc (≅ 90% das tiras de jeju, a qual foi

  18. Caveolae and propofol effects on airway smooth muscle

    Science.gov (United States)

    Grim, K. J.; Abcejo, A. J.; Barnes, A.; Sathish, V.; Smelter, D. F.; Ford, G. C.; Thompson, M. A.; Prakash, Y. S.; Pabelick, C. M.

    2012-01-01

    Background The i.v. anaesthetic propofol produces bronchodilatation. Airway relaxation involves reduced intracellular Ca2+ ([Ca2+]i) in airway smooth muscle (ASM) and lipid rafts (caveolae), and constitutional caveolin proteins regulate [Ca2+]i. We postulated that propofol-induced bronchodilatation involves caveolar disruption. Methods Caveolar fractions of human ASM cells were tested for propofol content. [Ca2+]i responses of ASM cells loaded with fura-2 were performed in the presence of 10 µM histamine with and without clinically relevant concentrations of propofol (10 and 30 μM and intralipid control). Effects on sarcoplasmic reticulum (SR) Ca2+ release were evaluated in zero extracellular Ca2+ using the blockers Xestospongin C and ryanodine. Store-operated Ca2+ entry (SOCE) after SR depletion was evaluated using established techniques. The role of caveolin-1 in the effect of propofol was tested using small interference RNA (siRNA) suppression. Changes in intracellular signalling cascades relevant to [Ca2+]i and force regulation were also evaluated. Results Propofol was present in ASM caveolar fractions in substantial concentrations. Exposure to 10 or 30 µM propofol form decreased [Ca2+]i peak (but not plateau) responses to histamine by ∼40%, an effect persistent in zero extracellular Ca2+. Propofol effects were absent in caveolin-1 siRNA-transfected cells. Inhibition of ryanodine receptors prevented propofol effects on [Ca2+]i, while propofol blunted [Ca2+]i responses to caffeine. Propofol reduced SOCE, an effect also prevented by caveolin-1 siRNA. Propofol effects were associated with decreased caveolin-1 expression and extracellular signal-regulated kinase phosphorylation. Conclusions These novel data suggest a role for caveolae (specifically caveolin-1) in propofol-induced bronchodilatation. Due to its lipid nature, propofol may transiently disrupt caveolar regulation, thus altering ASM [Ca2+]i. PMID:22542538

  19. Mechanism for Triggered Waves in Atrial Myocytes.

    Science.gov (United States)

    Shiferaw, Yohannes; Aistrup, Gary L; Wasserstrom, J Andrew

    2017-08-08

    Excitation-contraction coupling in atrial cells is mediated by calcium (Ca) signaling between L-type Ca channels and Ryanodine receptors that occurs mainly at the cell boundary. This unique architecture dictates essential aspects of Ca signaling under both normal and diseased conditions. In this study we apply laser scanning confocal microscopy, along with an experimentally based computational model, to understand the Ca cycling dynamics of an atrial cell subjected to rapid pacing. Our main finding is that when an atrial cell is paced under Ca overload conditions, Ca waves can then nucleate on the cell boundary and propagate to the cell interior. These propagating Ca waves are referred to as "triggered waves" because they are initiated by L-type Ca channel openings during the action potential. These excitations are distinct from spontaneous Ca waves originating from random fluctuations of Ryanodine receptor channels, and which occur after much longer waiting times. Furthermore, we argue that the onset of these triggered waves is a highly nonlinear function of the sarcoplasmic reticulum Ca load. This strong nonlinearity leads to aperiodic response of Ca at rapid pacing rates that is caused by the complex interplay between paced Ca release and triggered waves. We argue further that this feature of atrial cells leads to dynamic instabilities that may underlie atrial arrhythmias. These studies will serve as a starting point to explore the nonlinear dynamics of atrial cells and will yield insights into the trigger and maintenance of atrial fibrillation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. Cellular mechanism for spontaneous calcium oscillations in astrocytes

    Institute of Scientific and Technical Information of China (English)

    Tong-fei WANG; Chen ZHOU; Ai-hui TANG; Shi-qiang WANG; Zhen CHAI

    2006-01-01

    Aim: To determine the Ca2+ source and cellular mechanisms of spontaneous Ca2+ oscillations in hippocampal astrocytes. Methods: The cultured cells were loaded with Fluo-4 AM, the indicator of intracellular Ca2+, and the dynamic Ca2+ transients were visualized with confocal laser-scanning microscopy. Results: The spontaneous Ca2+ oscillations in astrocytes were observed first in co-cultured hippocampal neurons and astrocytes. These oscillations were not affected by tetrodotoxin (TTX) treatment and kept up in purity cultured astrocytes. The spontaneous Ca2+ oscillations were not impacted after blocking the voltage-gated Ca2+ channels or ethylenediamine tetraacetic acid (EDTA) bathing, indicating that intracellular Ca2+ elevation was not the result of extracellular Ca2+ influx. Furthermore, the correlation between the spontaneous Ca2+ oscillations and the Ca2+ store in endoplasmic reticulum (ER) were investigated with pharmacological experiments. The oscillations were: 1) enhanced when cells were exposed to both low Na+ (70 mmol/L) and high Ca2+ (5 mmol/L) solution, and eliminated completely by 2 μmol/L thapsigargin, a blocker of sarcoplasmic reticulum Ca2+-ATPase; and 2) still robust after the application with either 50 μmol/L ryanodine or 400 μmol/L tetracaine, two specific antagonists of ryanodine receptors, but depressed in a dose-dependent manner by 2-APB, an InsP3 receptors (InsP3R) blocker. Conclusion: InsP3R-induced ER Ca2+ release is an important cellular mechanism for the initiation of spontaneous Ca2+ oscillation in hippocampal astrocytes.

  1. Modified cytoplasmic Ca2+ sequestration contributes to spinal cord injury-induced augmentation of nerve-evoked contractions in the rat tail artery.

    Science.gov (United States)

    Al Dera, Hussain; Callaghan, Brid P; Brock, James A

    2014-01-01

    In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca2+ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.

  2. Extracellular Ca(2+) entry and mobilization of inositol trisphosphate-dependent Ca(2+) stores modulate histamine and electrical field stimulation induced contractions of the guinea-pig prostate.

    Science.gov (United States)

    Lam, Michelle; Kerr, Karen; Ventura, Sabatino; Exintaris, Betty

    2011-09-01

    This investigation aimed to examine the source of Ca(2+) mobilization that leads to the contractile response to either exogenously added histamine (1 μM-1mM) or electrical field stimulation (10Hz, 0.5ms, 60V). Removal of extracellular Ca(2+) by removal of Ca(2+) from the bathing medium reduced histamine (1mM) induced responses by 34% and responses induced by electrical field stimulation by 94%. Similarly, blockade of L-type Ca(2+) channels by nifedipine (1 μM) reduced histamine (1mM) induced responses by 43% and responses induced by electrical field stimulation by 77%. Application of cyclopiazonic acid (CPA) (10 μM) to inhibit Ca(2+) reuptake to the sarcoplasmic reticulum enhanced both histamine-induced and electrical field stimulation induced responses to a small degree, while the addition of the inosotol triphosphate (IP(3)) receptor antagonist, 2-aminophenoxyethane borane (2-APB) (100 μM) inhibited histamine induced responses by 70% and electrical field stimulation induced responses by 57%. Ryanodine (1 μM) did not affect contractile responses to either histamine or electrical field stimulation, either in the absence or presence of 2-APB (100 μM). During both histamine and electrical field stimulation induced contractions, prostate smooth muscle generates IP(3) receptor mediated Ca(2+) release in conjunction with Ca(2+) entry from the extracellular environment. Ryanodine receptors on the other hand, appear not to play a role in this physiological mechanism. 2011 Published by Elsevier Ltd. All rights reserved.

  3. Characterization of the functional and anatomical differences in the atrial and ventricular myocardium from three species of elasmobranch fishes: smooth dogfish (Mustelus canis), sandbar shark (Carcharhinus plumbeus), and clearnose skate (Raja eglanteria).

    Science.gov (United States)

    Larsen, Julie; Bushnell, Peter; Steffensen, John; Pedersen, Morten; Qvortrup, Klaus; Brill, Richard

    2017-02-01

    We assessed the functional properties in atrial and ventricular myocardium (using isolated cardiac strips) of smooth dogfish (Mustelus canis), clearnose skate (Raja eglanteria), and sandbar shark (Carcharhinus plumbeus) by blocking Ca(2+) release from the sarcoplasmic reticulum (SR) with ryanodine and thapsigargin and measuring the resultant changes in contraction-relaxation parameters and the force-frequency relationship at 20 °C and 30 °C. We also examined ultrastructural differences with electron microscopy. In tissues from smooth dogfish, net force (per cross-sectional area) and measures of the speeds of contraction and relaxation were all higher in atrial than ventricular myocardium at both temperatures. Atrial-ventricular differences were evident in the other two species primarily in measures of the rates of contraction and relaxation. Ryanodine-thapsigargin treatment reduced net force and its maximum positive first derivative (i.e., contractility), and increased time to 50 % relaxation in atrial tissue from smooth dogfish at 30 °C. It also increased times to peak force and half relaxation in clearnose skate atrial and ventricular tissue at both temperatures, but only in atrial tissue from sandbar shark at 30 °C; indicating that SR involvement in excitation-contraction (EC) coupling is species- and temperature-specific in elasmobranch fishes, as it is in teleost fishes. Atrial and ventricular myocardium from all three species displayed a negative force-frequency relationship, but there was no evidence that SR involvement in EC coupling was influenced by heart rate. SR was evident in electron micrographs, generally located in proximity to mitochondria and intercalated discs, and to a lesser extent between the myofibrils; with mitochondria being more numerous in ventricular than atrial myocardium in all three species.

  4. Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation–contraction coupling supramolecular complex

    Science.gov (United States)

    Lopez, Rubén J.; Mosca, Barbara; Treves, Susan; Maj, Marcin; Bergamelli, Leda; Calderon, Juan C.; Bentzinger, C. Florian; Romanino, Klaas; Hall, Michael N.; Rüegg, Markus A.; Delbono, Osvaldo; Caputo, Carlo; Zorzato, Francesco

    2016-01-01

    The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]–ryanodine and 3[H]–PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation–contraction (E–C) coupling is affected by mTORC1 signalling. PMID:25431931

  5. Advance in clinical application of dantrolene%丹曲林临床应用的研究进展

    Institute of Scientific and Technical Information of China (English)

    王艳萍; 马民玉

    2009-01-01

    丹曲林是目前惟一被证实治疗恶性高热(malignant hyperthermia,MH)的特效药物.丹曲林药动学特性为二房室模型,血浆消除半衰期约为10 h,稳态分布容积为26.14L.最近的研究证实ryanodine受体(RYR)为丹曲林的结合部位,阻断该受体抑制Ca2+从肌浆网内释放,从而抑制肌肉收缩和其他的钙依赖胞质代谢过程是其分子作用基础.丹曲林除了用于治疗恶性高热外,临床上还可用于抗精神病药物恶性综合征(neuroleptic malignant syndrome,NMS)、痉挛状态和摇头丸(3,4-亚甲基二氧去氧麻黄碱,MDMA)中毒等的治疗.%Dantolene is the only available drug which has been proven effective and specific for the treatment of malignant hyperthermia. The pharmacokinetics profile of dantrolene is adequately described by a two-compartment model. The plasma elimina-tion half-life time is about 10 h and distribution volume at steady-state is 26.14 L. Recent studies have identified that ryanodine re-ceptor is dantrolene-binding site. Inhibition of ryanodine receptor, blockade of calcium release from the sarcoplasmic reticulum and inhibition of muscle contraction and other calcium-dependent cytoplasmic metabolic processes is thought to be fundamental in the molecular action of dantrolene. Dantrolene has been used for the treatment of neuroleptic malignant syndrome, spasticity and Ecstasy intoxication besides malignant hyperthermia.

  6. Modified cytoplasmic Ca2+ sequestration contributes to spinal cord injury-induced augmentation of nerve-evoked contractions in the rat tail artery.

    Directory of Open Access Journals (Sweden)

    Hussain Al Dera

    Full Text Available In rat tail artery (RTA, spinal cord injury (SCI increases nerve-evoked contractions and the contribution of L-type Ca2+ channels to these responses. In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.

  7. beta-Adrenergic activation reveals impaired cardiac calcium handling at early stage of diabetes.

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    op den Buijs, Jorn; Miklós, Zsuzsanna; van Riel, Natal A W; Prestia, Christina M; Szenczi, Orsolya; Tóth, András; Van der Vusse, Ger J; Szabó, Csaba; Ligeti, László; Ivanics, Tamás

    2005-01-21

    Cardiac function is known to be impaired in diabetes. Alterations in intracellular calcium handling have been suggested to play a pivotal role. This study aimed to test the hypothesis that beta-adrenergic activation can reveal the functional derangements of intracellular calcium handling of the 4-week diabetic heart. Langendorff perfused hearts of 4-week streptozotocin-induced diabetic rats were subjected to the beta-adrenoceptor agonist isoproterenol. Cyclic changes in [Ca(2+)](i) levels were measured throughout the cardiac cycle using Indo-1 fluorescent dye. Based on the computational analysis of the [Ca(2+)](i) transient the kinetic parameters of the sarcoplasmic reticulum Ca(2+)-ATPase and the ryanodine receptor were determined by minimizing the squared error between the simulated and the experimentally obtained [Ca(2+)](i) transient. Under unchallenged conditions, hemodynamic parameters were comparable between control and diabetic hearts. Isoproterenol administration stimulated hemodynamic function to a greater extent in control than in diabetic hearts, which was exemplified by more pronounced increases in rate of pressure development and decline. Under unchallenged conditions, [Ca(2+)](i) amplitude and rate of rise and decline of [Ca(2+)](i) as measured throughout the cardiac cycle were comparable between diabetic and control hearts. Differences became apparent under beta-adrenoceptor stimulation. Upon beta-activation the rate-pressure product showed a blunted response, which was accompanied by a diminished rise in [Ca(2+)](i) amplitude in diabetic hearts. Computational analysis revealed a reduced function of the sarcoplasmic reticulum Ca(2+)-ATPase and Ca(2+)-release channel in response to beta-adrenoceptor challenge. Alterations in Ca(2+)(i) handling may play a causative role in depressed hemodynamic performance of the challenged heart at an early stage of diabetes.

  8. Obesity induces upregulation of genes involved in myocardial Ca2+ handling

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    A.P. Lima-Leopoldo

    2008-07-01

    Full Text Available Obesity is a complex multifactorial disorder that is often associated with cardiovascular diseases. Research on experimental models has suggested that cardiac dysfunction in obesity might be related to alterations in myocardial intracellular calcium (Ca2+ handling. However, information about the expression of Ca2+-related genes that lead to this abnormality is scarce. We evaluated the effects of obesity induced by a high-fat diet in the expression of Ca2+-related genes, focusing the L-type Ca2+ channel (Cacna1c, sarcolemmal Na+/Ca2+ exchanger (NCX, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a, ryanodine receptor (RyR2, and phospholamban (PLB mRNA in rat myocardium. Male 30-day-old Wistar rats were fed a standard (control or high-fat diet (obese for 15 weeks. Obesity was defined as increased percent of body fat in carcass. The mRNA expression of Ca2+-related genes in the left ventricle was measured by RT-PCR. Compared with control rats, the obese rats had increased percent of body fat, area under the curve for glucose, and leptin and insulin plasma concentrations. Obesity also caused an increase in the levels of SERCA2a, RyR2 and PLB mRNA (P < 0.05 but did not modify the mRNA levels of Cacna1c and NCX. These findings show that obesity induced by high-fat diet causes cardiac upregulation of Ca2+ transport_related genes in the sarcoplasmic reticulum.

  9. Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

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    Vinogradova, Tatiana M.; Lakatta, Edward G.

    2009-01-01

    Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

  10. CaV3.2 Channels and the Induction of Negative Feedback in Cerebral Arteries

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    Harraz, Osama F.; Abd El-Rahman, Rasha R.; Bigdely-Shamloo, Kamran; Wilson, Sean M.; Brett, Suzanne E.; Romero, Monica; Gonzales, Albert L.; Earley, Scott; Vigmond, Edward J.; Nygren, Anders; Menon, Bijoy K.; Mufti, Rania E.; Watson, Tim; Starreveld, Yves; Furstenhaupt, Tobias; Muellerleile, Philip R.; Kurjiaka, David T.; Kyle, Barry D.; Braun, Andrew P.; Welsh, Donald G.

    2015-01-01

    Rationale T-type (CaV3.1/CaV3.2) Ca2+ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective This study tested whether CaV3.2 channels mediate a negative feedback response by triggering Ca2+ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca2+-activated K+ channels. Methods and Results Micromolar Ni2+, an agent that selectively blocks CaV3.2 but not CaV1.2/CaV3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in CaV3.2−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, CaV3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca2+ influx through CaV3.2 could repetitively activate ryanodine receptor, inducing discrete Ca2+-induced Ca2+ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca2+ imaging and perforated patch clamp electrophysiology demonstrated that Ni2+ suppressed Ca2+ sparks and consequently spontaneous transient outward K+ currents, large-conductance Ca2+-activated K+ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni2+. Key experiments on human cerebral arteries indicate that CaV3.2 is present and drives a comparable response to moderate constriction. Conclusions These findings indicate for the first time that CaV3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca2+ sparks, enabling large

  11. Local calcium signals induced by hyper-osmotic stress in mammalian skeletal muscle cells.

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    Apostol, Simona; Ursu, Daniel; Lehmann-Horn, Frank; Melzer, Werner

    2009-01-01

    Strenuous activitiy of skeletal muscle leads to temporary osmotic dysbalance and isolated skeletal muscle fibers exposed to osmotic stress respond with characteristic micro-domain calcium signals. It has been suggested that osmotic stress targets transverse tubular (TT) dihydropyridine receptors (DHPRs) which normally serve as voltage-dependent activators of Ca release via ryanodine receptor (RyR1s) of the sarcoplasmic reticulum (SR). Here, we pursued this hypothesis by imaging the response to hyperosmotic solutions in both mouse skeletal muscle fibers and myotubes. Ca fluctuations in the cell periphery of fibers exposed to osmotic stress were accompanied by a substantial dilation of the peripheral TT. The Ca signals were completely inhibited by a conditioning depolarization that inactivates the DHPR. Dysgenic myotubes, lacking the DHP-receptor-alpha1-subunit, showed strongly reduced, yet not completely inhibited activity when stimulated with solutions of elevated tonicity. The results point to a modulatory, even though not essential, role of the DHP receptor for osmotic stress-induced Ca signals in skeletal muscle.

  12. Imaging atrial arrhythmic intracellular calcium in intact heart.

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    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R

    2013-11-01

    Abnormalities in intracellular Ca(2+) signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca(2+) in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca(2+) imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca(2+) activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca(2+) waves and Ca(2+) alternans. Moreover, we applied this strategy to analyze Ca(2+) signals in the hearts of WT and knock-in mice harboring a 'leaky' type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca(2+) leak increases the susceptibility to Ca(2+) alternans and Ca(2+) waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca(2+) leak via RyR2 by acute treatment with S107 reduced both Ca(2+) alternans and Ca(2+) waves, and prevented atrial arrhythmias.

  13. Triadin/Junctin double null mouse reveals a differential role for Triadin and Junctin in anchoring CASQ to the jSR and regulating Ca(2+ homeostasis.

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    Simona Boncompagni

    Full Text Available Triadin (Tdn and Junctin (Jct are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ and ryanodine receptor (RyRs at the junctional sarcoplasmic reticulum (jSR. However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null, Jct (Jct-null or both (Tdn/Jct-null, we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca(2+ imaging and Ca(2+ selective microelectrodes we found that changes in e-c coupling, SR Ca(2+content and resting [Ca(2+] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca(2+ regulation than Jct/CASQ association.

  14. Central core disease

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    Jungbluth Heinz

    2007-05-01

    Full Text Available Abstract Central core disease (CCD is an inherited neuromuscular disorder characterised by central cores on muscle biopsy and clinical features of a congenital myopathy. Prevalence is unknown but the condition is probably more common than other congenital myopathies. CCD typically presents in infancy with hypotonia and motor developmental delay and is characterized by predominantly proximal weakness pronounced in the hip girdle; orthopaedic complications are common and malignant hyperthermia susceptibility (MHS is a frequent complication. CCD and MHS are allelic conditions both due to (predominantly dominant mutations in the skeletal muscle ryanodine receptor (RYR1 gene, encoding the principal skeletal muscle sarcoplasmic reticulum calcium release channel (RyR1. Altered excitability and/or changes in calcium homeostasis within muscle cells due to mutation-induced conformational changes of the RyR protein are considered the main pathogenetic mechanism(s. The diagnosis of CCD is based on the presence of suggestive clinical features and central cores on muscle biopsy; muscle MRI may show a characteristic pattern of selective muscle involvement and aid the diagnosis in cases with equivocal histopathological findings. Mutational analysis of the RYR1 gene may provide genetic confirmation of the diagnosis. Management is mainly supportive and has to anticipate susceptibility to potentially life-threatening reactions to general anaesthesia. Further evaluation of the underlying molecular mechanisms may provide the basis for future rational pharmacological treatment. In the majority of patients, weakness is static or only slowly progressive, with a favourable long-term outcome.

  15. The contribution of pathways initiated via the Gq\\11 G-protein family to atrial fibrillation.

    Science.gov (United States)

    Tinker, Andrew; Finlay, Malcom; Nobles, Muriel; Opel, Aaisha

    2016-03-01

    Atrial fibrillation is the commonest cardiac arrhythmia and leads to significant clinical morbidity and mortality. It has a complex pathophysiology but is often initiated by atrial ectopic beats and because of atrial remodelling once it occurs it can become established. Thus therapeutic interventions designed to prevent the initial occurrence of the arrhythmia are particularly needed. At the cellular level, these ectopic beats arise because of abnormal calcium release events from the sarcoplasmic reticulum leading to an inward current mediated by the sodium-calcium exchanger. There has been considerable interest in this over the last few years largely focused on the ryanodine receptor and related signalling pathways. However, atrial myocytes also possess a well-developed inositol trisphosphate (IP3) dependent calcium release system and this has been less studied. In this review we focus on pathways and molecules that couple via the Gq\\11 family of G-proteins including regulators of G-protein signalling that may influence IP3 mediated calcium release and atrial fibrillation.

  16. Cartap hydrochloride poisoning: A clinical experience

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    Hari K Boorugu

    2012-01-01

    Full Text Available Cartap hydrochloride, a nereistoxin analog, is a commonly used low toxicity insecticide. We describe a patient who presented to the emergency department with alleged history of ingestion of Cartap hydrochloride as an act of deliberate self-harm. The patient was managed conservatively. To our knowledge this is the first case report of Cartap hydrochloride suicidal poisoning. Cartap toxicity has been considered to be minimal, but a number of animal models have shown significant neuromuscular toxicity resulting in respiratory failure. It is hypothesized that the primary effect of Cartap hydrochloride is through inhibition of the [ 3 H]-ryanodine binding to the Ca 2+ release channel in the sarcoplasmic reticulum in a dose-dependent manner and promotion of extracellular Ca 2+ influx and induction of internal Ca 2+ release. This results in tonic diaphragmatic contraction rather than paralysis. This is the basis of the clinical presentation of acute Cartap poisoning as well as the treatment with chelators namely British Anti Lewisite and sodium dimercaptopropane sulfonate.

  17. 3D structure of muscle dihydropyridine receptor

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    Montserrat Samsó

    2015-01-01

    Full Text Available Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR and the ryanodine receptor (RyR1 mediates the quasi‐instantaneous conversion from T‐tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR. The DHPR has several key modules required for EC coupling: the voltage sensors and II‐III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II‐III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

  18. Antioxidant Treatment Reduces Formation of Structural Cores and Improves Muscle Function in RYR1Y522S/WT Mice

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    Antonio Michelucci

    2017-01-01

    Full Text Available Central core disease (CCD is a congenital myopathy linked to mutations in the ryanodine receptor type 1 (RYR1, the sarcoplasmic reticulum Ca2+ release channel of skeletal muscle. CCD is characterized by formation of amorphous cores within muscle fibers, lacking mitochondrial activity. In skeletal muscle of RYR1Y522S/WT knock-in mice, carrying a human mutation in RYR1 linked to malignant hyperthermia (MH with cores, oxidative stress is elevated and fibers present severe mitochondrial damage and cores. We treated RYR1Y522S/WT mice with N-acetylcysteine (NAC, an antioxidant provided ad libitum in drinking water for either 2 or 6 months. Our results show that 2 months of NAC treatment starting at 2 months of age, when mitochondrial and fiber damage was still minimal, (i reduce formation of unstructured and contracture cores, (ii improve muscle function, and (iii decrease mitochondrial damage. The beneficial effect of NAC treatment is also evident following 6 months of treatment starting at 4 months of age, when structural damage was at an advanced stage. NAC exerts its protective effect likely by lowering oxidative stress, as supported by the reduction of 3-NT and SOD2 levels. This work suggests that NAC administration is beneficial to prevent mitochondrial damage and formation of cores and improve muscle function in RYR1Y522S/WT mice.

  19. Mapping of a further malignant hyperthermia susceptibility locus to chromosome 3q13.1

    Energy Technology Data Exchange (ETDEWEB)

    Sudbrak, R.; Deufel, T. [Universitaet Muenster (Germany); Procaccio, V.; Lunardi, J.; Klausnitzer, M.; Mueller, C.R.; Hartung, E.J.; Curran, J.L.; Stewart, A.D. [and others

    1995-03-01

    Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disease for which MH susceptibility (MHS) is transmitted as an autosomal dominant trait. A potentially life-threatening MH crisis is triggered by exposure to commonly used inhalational anesthetics and depolarizing muscle relaxants. The first malignant hyperthermia susceptibility locus (MHS1) was identified on human chromosome 19{sub q}13.1, and evidence has been obtained that defects in the gene for the calcium-release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor; RYR1) can cause some forms of MH. However, MH has been shown to be genetically heterogeneous, and additional loci on chromosomes 17q and 7q have been suggested. In a collaborative search of the human genome with polymorphic microsatellite markers, we now found linkage of the MHS phenotype, as assessed by the European in vitro contracture test protocol, to markers defining a 1-cM interval on chromosome 3q13.1. A maximum multipoint lod score of 3.22 was obtained in a single German pedigree with classical MH, and none of the other pedigrees investigated in this study showed linkage to this region. Linkage to both MHS1/RYR1 and putative loci on chromosome 17q and 7q were excluded. This study supports the view that considerable genetic heterogeneity exists in MH. 47 refs., 2 figs., 3 tabs.

  20. Mechanisms of excitation-contraction uncoupling relevant to activity-induced muscle fatigue.

    Science.gov (United States)

    Lamb, Graham D

    2009-06-01

    If the free [Ca2+] in the cytoplasm of a skeletal muscle fiber is raised substantially for a period of seconds to minutes or to high levels just briefly, it leads to disruption of the normal excitation-contraction (E-C) coupling process and a consequent long-lasting decrease in force production. It appears that the disruption to the coupling occurs at the triad junction, where the voltage-sensor molecules (dihydropyridine receptors) normally interact with and open the Ca2+ release channels (ryanodine receptors) in the adjacent sarcoplasmic reticulum (SR). This disruption results in inadequate release of SR Ca2+ upon stimulation. Such E-C uncoupling may underlie the long-duration low-frequency fatigue that can occur after various types of exercise, as well as possibly being a contributing factor to the muscle weakness in certain muscle diseases. The process or processes causing the disruption of the coupling between the voltage sensors and the release channels is not known with certainty, but might be associated with structural changes at the triad junction, possibly caused by activation of the Ca2+-dependent protease, micro-calpain.

  1. Enhanced Ca²+ influx through cardiac L-type Ca²+ channels maintains the systolic Ca²+ transient in early cardiac atrophy induced by mechanical unloading.

    Science.gov (United States)

    Schwoerer, A P; Neef, S; Broichhausen, I; Jacubeit, J; Tiburcy, M; Wagner, M; Biermann, D; Didié, M; Vettel, C; Maier, L S; Zimmermann, W H; Carrier, L; Eschenhagen, T; Volk, T; El-Armouche, A; Ehmke, H

    2013-12-01

    Cardiac atrophy as a consequence of mechanical unloading develops following exposure to microgravity or prolonged bed rest. It also plays a central role in the reverse remodelling induced by left ventricular unloading in patients with heart failure. Surprisingly, the intracellular Ca(2+) transients which are pivotal to electromechanical coupling and to cardiac plasticity were repeatedly found to remain unaffected in early cardiac atrophy. To elucidate the mechanisms underlying the preservation of the Ca(2+) transients, we investigated Ca(2+) cycling in cardiomyocytes from mechanically unloaded (heterotopic abdominal heart transplantation) and control (orthotopic) hearts in syngeneic Lewis rats. Following 2 weeks of unloading, sarcoplasmic reticulum (SR) Ca(2+) content was reduced by ~55 %. Atrophic cardiac myocytes also showed a much lower frequency of spontaneous diastolic Ca(2+) sparks and a diminished systolic Ca(2+) release, even though the expression of ryanodine receptors was increased by ~30 %. In contrast, current clamp recordings revealed prolonged action potentials in endocardial as well as epicardial myocytes which were associated with a two to fourfold higher sarcolemmal Ca(2+) influx under action potential clamp. In addition, Cav1.2 subunits which form the pore of L-type Ca(2+) channels (LTCC) were upregulated in atrophic myocardium. These data suggest that in early cardiac atrophy induced by mechanical unloading, an augmented sarcolemmal Ca(2+) influx through LTCC fully compensates for a reduced systolic SR Ca(2+) release to preserve the Ca(2+) transient. This interplay involves an electrophysiological remodelling as well as changes in the expression of cardiac ion channels.

  2. Aerobic interval training partly reverse contractile dysfunction and impaired Ca2+ handling in atrial myocytes from rats with post infarction heart failure.

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    Anne Berit Johnsen

    Full Text Available BACKGROUND: There is limited knowledge about atrial myocyte Ca(2+ handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca(2+ handling in rats with post-infarction heart failure (HF and to examine whether aerobic interval training could reverse a potential dysfunction. METHODS AND RESULTS: Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01 and time to relaxation was prolonged (p<0.01 in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca(2+ amplitude, decreased SR Ca(2+ content, and slower Ca(2+ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca(2+ ATPase activity, increased Na(+/Ca(2+-exchanger activity and increased diastolic Ca(2+ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca(2+ handling in HF. CONCLUSION: Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca(2+ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.

  3. Disease causing mutations of calcium channels.

    Science.gov (United States)

    Lorenzon, Nancy M; Beam, Kurt G

    2008-01-01

    Calcium ions play an important role in the electrical excitability of nerve and muscle, as well as serving as a critical second messenger for diverse cellular functions. As a result, mutations of genes encoding calcium channels may have subtle affects on channel function yet strongly perturb cellular behavior. This review discusses the effects of calcium channel mutations on channel function, the pathological consequences for cellular physiology, and possible links between altered channel function and disease. Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca(2+) ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca(2+) stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores.

  4. Cardiac BIN1 (cBIN1) is a regulator of cardiac contractile function and an emerging biomarker of heart muscle health.

    Science.gov (United States)

    Zhou, Kang; Hong, Tingting

    2017-03-01

    In recent decades, a cardiomyocyte membrane scaffolding protein bridging integrator 1 (BIN1) has emerged as a critical multifunctional regulator of transverse-tubule (t-tubule) function and calcium signaling in cardiomyocytes. Encoded by a single gene with 20 exons that are alternatively spliced, more than ten BIN1 protein isoforms are expressed with tissue and disease specificity. The recently discovered cardiac alternatively spliced isoform BIN1 (cBIN1 or BIN1+13+17)plays a crucial role in organizing membrane microfolds within cardiac t-tubules. These cBIN1-induced microfolds form functional dyad microdomains by trafficking L-type calcium channels (LTCC) to t-tubule membrane and recruiting ryanodine receptors (RyR) to junctional sarcoplasmic reticulum membrane. When cBIN1 is transcriptionally reduced as occurs in heart failure, cBIN1-microfolds are disrupted and fail to form LTCC and RyR couplons. As a result, impaired dyad formation limits excitation-contraction coupling thus cardiac contractility, and accumulation of orphaned leaky RyRs outside of dyads increases ventricular arrhythmias. Reduced myocardial BIN1 in heart failure is also detectable at the blood level, and plasma BIN1 level predicts heart failure progression and future arrhythmias in cardiomyopathy patients. Here we will review the recent progress in BIN1-related cardiomyocyte biology studies and discuss the diagnostic and predictive values of cBIN1 in future clinical use.

  5. Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies

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    Caroline Ramspacher

    2015-06-01

    Full Text Available Desminopathies belong to a family of muscle disorders called myofibrillar myopathies that are caused by Desmin mutations and lead to protein aggregates in muscle fibers. To date, the initial pathological steps of desminopathies and the impact of desmin aggregates in the genesis of the disease are unclear. Using live, high-resolution microscopy, we show that Desmin loss of function and Desmin aggregates promote skeletal muscle defects and alter heart biomechanics. In addition, we show that the calcium dynamics associated with heart contraction are impaired and are associated with sarcoplasmic reticulum dilatation as well as abnormal subcellular distribution of Ryanodine receptors. Our results demonstrate that desminopathies are associated with perturbed excitation-contraction coupling machinery and that aggregates are more detrimental than Desmin loss of function. Additionally, we show that pharmacological inhibition of aggregate formation and Desmin knockdown revert these phenotypes. Our data suggest alternative therapeutic approaches and further our understanding of the molecular determinants modulating Desmin aggregate formation.

  6. Transient Receptor Potential Channels Contribute to Pathological Structural and Functional Remodeling After Myocardial Infarction

    Science.gov (United States)

    Davis, Jennifer; Correll, Robert N.; Trappanese, Danielle M.; Hoffman, Nicholas E.; Troupes, Constantine D.; Berretta, Remus M.; Kubo, Hajime; Madesh, Muniswamy; Chen, Xiongwen; Gao, Erhe; Molkentin, Jeffery D.; Houser, Steven R.

    2014-01-01

    Rationale The cellular and molecular basis for post myocardial infarction (MI) structural and functional remodeling is not well understood. Objective To determine if Ca2+ influx through transient receptor potential (canonical) (TRPC) channels contributes to post-MI structural and functional remodeling. Methods and Results TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca2+ entry. Cardiac myocyte specific expression of a dominant negative (dn: loss of function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes (AFMs) to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 over expression in AFMs induced calcineurin (Cn)-Nuclear Factor of Activated T cells (NFAT) mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in AFMs increased rested state contractions and increased spontaneous sarcoplasmic reticulum (SR) Ca2+ sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady state pacing, likely due to enhanced SR Ca2+ leak. Conclusions Ca2+ influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function. PMID:25047165

  7. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation*♦

    Science.gov (United States)

    Nebel, Merle; Schwoerer, Alexander P.; Warszta, Dominik; Siebrands, Cornelia C.; Limbrock, Ann-Christin; Swarbrick, Joanna M.; Fliegert, Ralf; Weber, Karin; Bruhn, Sören; Hohenegger, Martin; Geisler, Anne; Herich, Lena; Schlegel, Susan; Carrier, Lucie; Eschenhagen, Thomas; Potter, Barry V. L.; Ehmke, Heimo; Guse, Andreas H.

    2013-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca2+ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca2+ signals sensitive to inhibitors of both acidic Ca2+ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca2+ transients were recorded both as increases of the free cytosolic Ca2+ concentration and as decreases of the sarcoplasmic luminal Ca2+ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca2+ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy. PMID:23564460

  8. Effect of weak static magnetic fields on the development of cultured skeletal muscle cells.

    Science.gov (United States)

    Surma, Sergei V; Belostotskaya, Galina B; Shchegolev, Boris F; Stefanov, Vasily E

    2014-12-01

    We studied the effect produced on the development and functional activity of skeletal muscle cells from newborn Wistar rats in primary culture by weak static magnetic fields (WSMF; 60-400 µT) with a high capacity of penetrating the biological media. To reduce the impact of external magnetic fields, cells were cultured at 37 °C in a multilayered shielding chamber with the attenuation coefficient equal to 160. WSMF inside the chamber was created by a circular permanent magnet. We found that the application of WSMF with the magnetic field strength only a few times that of the geomagnetic field can accelerate the development of skeletal muscle cells, resulting in the formation of multinuclear hypertrophied myotubes. WSMF was shown to induce 1.5- to 3.5-fold rise in the concentration of intracellular calcium [Ca(2+)]i due to the release of Ca(2+) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyR), which increases in the maturation of myotubes. We also found that fully differentiated myotubes at late stages of development were less sensitive to WSMF, manifesting a gradual decrease in the frequency of contractions. However, myotubes at the stage when electromechanical coupling was forming dramatically reduced the frequency of contractions during the first minutes of their exposure to WSMF.

  9. Caffeine induces cardiomyocyte hypertrophy via p300 and CaMKII pathways.

    Science.gov (United States)

    Shi, Liang; Xu, Hao; Wei, Jinhong; Ma, Xingfeng; Zhang, Jianbao

    2014-09-25

    Caffeine is commonly utilized to trigger intracellular calcium in cardiomyocyte. It is well accepted that caffeine could induce cardiac arrhythmia, but it is not clear with regard of its impacts on the cardiac function. This article presents a recent study concerning the effects of caffeine on the cardiomyocyte hypertrophy and the associated signal pathway. The experimental results showed that the total protein contents, the surface area of cardiomyocyte and β-myosin heavy chain (β-MHC) expression increased in ventricular myocytes of neonatal Sprague-Dawley (SD) rats after 24h caffeine incubation. It is also observed that the basal intracellular calcium (Ca(2+)) level has increased, while the amplitude of Ca(2+) oscillation and Ca(2+) content have decreased in sarcoplasmic reticulum (SR). The caffeine-induced myocyte enhancer factor-2 (MEF2) expression and hypertrophy can be completely abolished by the inhibition of cardiac ryanodine receptor (RyR2), as well as KN93 and curcumin treatments. Meanwhile, the amplitude of Ca(2+) oscillation and the Ca(2+) content of SR in the completely-inhibited group have reached the physiological level. These results suggest that the caffeine-induced cardiomyocyte hypertrophy established the connection between Ca(2+) release from SR and cytosol that activates CaMKII and p300, which in turn enhances the expression of MEF2 that promotes cardiomyocyte hypertrophy.

  10. Changes in intracellular calcium concentration influence beat-to-beat variability of action potential duration in canine ventricular myocytes.

    Science.gov (United States)

    Kistamas, K; Szentandrassy, N; Hegyi, B; Vaczi, K; Ruzsnavszky, F; Horvath, B; Banyasz, T; Nanasi, P P; Magyar, J

    2015-02-01

    The aim of the present work was to study the influence of changes in intracellular calcium concentration ([Ca(2+)]i) on beat-to-beat variability (short term variability, SV) of action potential duration (APD) in isolated canine ventricular cardiomyocytes. Series of action potentials were recorded from enzymatically isolated canine ventricular cells using conventional microelectrode technique. Drug effects on SV were evaluated as relative SV changes determined by plotting the drug-induced changes in SV against corresponding changes in APD and comparing these data to the exponential SV-APD function obtained with inward and outward current injections. Exposure of myocytes to the Ca(2+) chelator BAPTA-AM (5 μM) decreased, while Ca(2+) ionophore A23187 (1 μM) increased the magnitude of relative SV. Both effects were primarily due to the concomitant changes in APD. Relative SV was reduced by BAPTA-AM under various experimental conditions including pretreatment with veratridine, BAY K8644, dofetilide or E-4031. Contribution of transient changes of [Ca(2+)]i due to Ca(2+) released from the sarcoplasmic reticulum (SR) was studied using 10 μM ryanodine and 1 μM cyclopiazonic acid: relative SV was reduced by both agents. Inhibition of the Na(+)-Ca(2+) exchanger by 1 μM SEA0400 increased relative SV. It is concluded that elevation of [Ca(2+)]i increases relative SV significantly. More importantly, Ca(2+) released from the SR is an important component of this effect.

  11. 5-Hydroxytryptamino-induced calcium sparks in cultured rat stomach fundus smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Xiaoling; (张小玲); YAN; Hongtao; (阎宏涛); YAN; Yang; (闫炀)

    2003-01-01

    With a new fluorescence probe of Ca2+, STDIn-AM, 5-hydroxytryptamino (5-HT)-induced spontaneous calcium release events (calcium sparks) in cultured rat stomach fundus smooth muscle cells (SFSMC) are investigated by laser scanning confocal microscope. The mechanisms of initiation of Ca2+ sparks, propagating Ca2+ waves and their relation to E-C coupling are discussed. After the extracellular [Ca2+] is increased to 10 mmol/L, addition of 5-HT causes hot spots throughout the cytoplasm, which is brighter near the plasmalemma. The amplitude of the event is at least two times greater than the standard deviation of fluorescence intensity fluctuations measured in the neighboring region and the duration of the Ca2+ signal is over 100 ms. The results suggest that 5-HT acts by the way of 5-HT2 receptors on SFSMC, then through 5-HT2 receptors couples IP3/Ca2+ and DG/PKC double signal transduction pathways to cause Ca2+ release from intracellular Ca2+ stores and followed Ca2+ influx possibly through calcium release-activated calcium influx. The acceptor of activated 5-HT2 can also cause membrane depolarization, which then stimulates the L-type Ca2+ channels leading to Ca2+ influx. Thenthe local Ca2+ entry mentioned above activates ryanodine-sensitive Ca2+ releasechannels (RyR) on sarcoplasmic reticulum (SR) to cause local Ca2+ release events (Ca2+ sparks) through calcium-induced calcium release (CICR).

  12. Computational modeling and numerical methods for spatiotemporal calcium cycling in ventricular myocytes

    Directory of Open Access Journals (Sweden)

    Michael eNivala

    2012-05-01

    Full Text Available Intracellular calcium (Ca cycling dynamics in cardiac myocytes is regulated by a complex network of spatially distributed organelles, such as sarcoplasmic reticulum (SR, mitochondria, and myofibrils. In this study, we present a mathematical model of intracellular Ca cycling and numerical and computational methods for computer simulations. The model consists of a coupled Ca release unit (CRU network, which includes a SR domain and a myoplasm domain. Each CRU contains 10 L-type Ca channels and 100 ryanodine receptor channels, with individual channels simulated stochastically using a varient of Gillespie’s method, modified here to handle time-dependent transition rates. Both the SR domain and the myoplasm domain in each CRU are modeled by 5x5x5 voxels to maintain proper Ca diffusion. Advanced numerical algorithms implemented on graphical processing units were used for fast computational simulations. For a myocyte containing 100x20x10 CRUs, a one-second heart time simulation takes about 10 minutes of machine time on a single NVIDIA Tesla C2050. Examples of simulated Ca cycling dynamics, such as Ca sparks, Ca waves, and Ca alternans, are shown.

  13. Pitavastatin-attenuated cardiac dysfunction in mice with dilated cardiomyopathy via regulation of myocardial calcium handling proteins

    Directory of Open Access Journals (Sweden)

    Hu Wei

    2014-03-01

    Full Text Available C57BL/6 mice with dilated cardiomyopathy (DCM were randomly divided to receive placebo or pitavastatin at a dose of 1 or 3 mg kg-1d-1. After 8 weeks treatment, mice with dilated cardiomyopathy developed serious cardiac dysfunction characterized by significantly enhanced left ventricular end-diastolic diameter (LVIDd, decreased left ventricular ejection fraction (LVEF as well as left ventricular short axis fractional shortening (LVFS, accompanied with enlarged cardiomyocytes, and increased plasma levels of N-terminal pro-B type natriuretic peptide (NT-proBNP and plasma angiotensin II (AngII concentration. Moreover, myocardium sarcoplasmic reticulum Ca2+ pump (SERCA-2 activity was decreased. The ratio of phosphorylated phospholamban (PLB to total PLB decreased significantly with the down-regulation of SERCA- -2a and ryanodine receptor (RyR2 expression. Pitavastatin was found to ameliorate the cardiac dysfunction in mice with dilated cardiomyopathy by reversing the changes in the ratios of phosphorylated PLB to total PLB, SERCA-2a and RyR2 via reducing the plasma AngII concentration and the expressions of myocardium angiotensin II type 1 receptor (AT1R and protein kinase C (PKCb2. The possible underlying mechanism might be the regulation of myocardial AT1R-PKCb2-Ca2+ handling proteins.

  14. Compensatory up-regulation of cardiac SR Ca2+-pump by heat-shock counteracts SR Ca2+-channel activation by ischemia/reperfusion.

    Science.gov (United States)

    O'Brien, P J; Li, G O; Locke, M; Klabunde, R E; Ianuzzo, C D

    1997-08-01

    We tested the hypothesis that heat-shock protected myocardial Ca2+-cycling by sarcoplasmic reticulum from ischemia and reperfusion (I/R) injury. Twenty-four hours after increasing body temperature to 42 degrees C for 15 min, rat hearts were isolated, Langendorff-perfused, and subjected to 30 min ischemia then 30 min reperfusion. Left ventricles were homogenized and their ionized Ca2+ concentration monitored with indo- during Ca2+-uptake in the presence and absence of the Ca2+-release channel (CRC) modulator ryanodine. Tissue content of heat-shock protein 72 (HSP 72) was analyzed. Exposure to I/R resulted in a 37% enhancement of CRC activity but no effect on Ca2+-pumping activity, resulting in 25% decreased net Ca2+-uptake activity. Pre-exposure to heat-shock resulted in a 10-fold increase in HSP 72, and a 25% enhancement of maximal Ca2+-pumping activity which counteracted the effect of I/R on CRC and net Ca2+-uptake activities. This protection of SR Ca2+-cycling was associated with partial protection of myocardial physiological performance. Net Ca2+-uptake activity was correlated with the left ventricular developed pressure and its rate of change. We conclude that one of the mechanisms by which heat-shock protects myocardium from I/R injury is to upregulate SR Ca2+-pumping activity to counteract the enhanced SR Ca2+-release produced by I/R.

  15. Ivabradine protects against ventricular arrhythmias in acute myocardial infarction in the rat.

    Science.gov (United States)

    Mackiewicz, Urszula; Gerges, Joseph Y; Chu, Sandy; Duda, Monika; Dobrzynski, Halina; Lewartowski, Bohdan; Mączewski, Michał

    2014-06-01

    Ventricular arrhythmias are an important cause of mortality in the acute myocardial infarction (MI). To elucidate effect of ivabradine, pure heart rate (HR) reducing drug, on ventricular arrhythmias within 24 h after non-reperfused MI in the rat. ECG was recorded for 24 h after MI in untreated and ivabradine treated rats and episodes of ventricular tachycardia/fibrillation (VT/VF) were identified. Forty-five minutes and twenty-four hours after MI epicardial monophasic action potentials (MAPs) were recorded, cardiomyocyte Ca(2+) handling was assessed and expression and function of ion channels were studied. Ivabradine reduced average HR by 17%. Combined VT/VF incidence and arrhythmic mortality were higher in MI versus MI + Ivabradine rats. MI resulted in (1) increase of Ca(2+) sensitivity of ryanodine receptors 24 h after MI; (2) increase of HCN4 expression in the left ventricle (LV) and funny current (IF) in LV cardiomyocytes 24 h after MI, and (3) dispersion of MAP duration both 45 min and 24 h after MI. Ivabradine partially prevented all these three potential proarrhythmic effects of MI. Ivabradine is antiarrhythmic in the acute MI in the rat. Potential mechanisms include prevention of: diastolic Ca(2+)-leak from sarcoplasmic reticulum, upregulation of IF current in LV and dispersion of cardiac repolarization. Ivabradine could be an attractive antiarrhythmic agent in the setting of acute MI.

  16. Mechanisms underlying the impaired contractility of diabetic cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    Marie-Louise; Ward; David; J; Crossman

    2014-01-01

    Cardiac dysfunction is a well-known consequence of diabetes,with sustained hyperglycaemia leading to the development of a cardiomyopathy that is independent of cardiovascular disease or hypertension.Animal models of diabetes are commonly used to study the pathophysiology of diabetic cardiomyopathy,with the hope that increased knowledge will lead ultimately to better therapeutic strategies being developed.At physiological temperature,left ventricular trabeculae isolated from the streptozotocin rat model of type 1 diabetes showed decreased stress and prolonged relaxation,but with no evidence that decreased contractility was a result of altered myocardial Ca2+handling.Although sarcoplasmic reticulum(SR)Ca2+reuptake appeared slower in diabetic trabeculae,it was offset by an increase in actionpotential duration,thereby maintaining SR Ca2+content and favouring increased contraction force.Frequency analysis of t-tubule distribution by confocal imaging of ventricular tissue labeled with wheat germ agglutinin or ryanodine receptor antibodies showed a reduced T-power for diabetic tissue,but the differences were minor in comparison to other models of heart failure.The contractile dysfunction appeared to be the result of disrupted F-actin in conjunction with the increased typeⅠcollagen,with decreased myofilament Ca2+sensitivity contributing to the slowed relaxation.

  17. Cartap hydrochloride poisoning: A clinical experience.

    Science.gov (United States)

    Boorugu, Hari K; Chrispal, Anugrah

    2012-01-01

    Cartap hydrochloride, a nereistoxin analog, is a commonly used low toxicity insecticide. We describe a patient who presented to the emergency department with alleged history of ingestion of Cartap hydrochloride as an act of deliberate self-harm. The patient was managed conservatively. To our knowledge this is the first case report of Cartap hydrochloride suicidal poisoning. Cartap toxicity has been considered to be minimal, but a number of animal models have shown significant neuromuscular toxicity resulting in respiratory failure. It is hypothesized that the primary effect of Cartap hydrochloride is through inhibition of the [(3)H]-ryanodine binding to the Ca(2+) release channel in the sarcoplasmic reticulum in a dose-dependent manner and promotion of extracellular Ca(2+) influx and induction of internal Ca(2+) release. This results in tonic diaphragmatic contraction rather than paralysis. This is the basis of the clinical presentation of acute Cartap poisoning as well as the treatment with chelators namely British Anti Lewisite and sodium dimercaptopropane sulfonate.

  18. Association of cardiac injury with iron-increased oxidative and nitrative modifications of the SERCA2a isoform of sarcoplasmic reticulum Ca(2+)-ATPase in diabetic rats.

    Science.gov (United States)

    Li, Xueli; Li, Wenliang; Gao, Zhonghong; Li, Hailing

    2016-08-01

    The role of iron in the etiology of diabetes complications is not well established. Thus, this study was performed to test whether the iron-induced increase of oxidative/nitrative damage is involved in SERCA2a-related diabetic heart complication. Four randomly divided groups of rats were used: normal control group; iron overload group; diabetes group, and diabetic plus iron overload group. Iron supplementation stimulated cardiomyocyte hypertrophy and led to an increase in cardiac protein carbonyls, nitrotyrosine (3-NT) formation, and iNOS protein expression, thus resulting in abnormal myocardium calcium homeostasis of diabetic rats. The levels of SECA2a oxidation/nitration were significantly increased in the iron overload diabetic rats, along with a decrease in SECA2a expression and activity. In order to elucidate the possible role of iron in SERCA2a dysfunction, the effects of iron (Fe(3+) or hemin) on peroxynitrite (ONOO(-)) induced SERCA2a oxidation and nitration were further investigated in vitro. It was found that tyrosine nitration played more important role in SERCA2a inactivation than thiol oxidation. These results present a potential mechanism in which iron exacerbates the diabetes-induced oxidative/nitrative modification of SERCA2a, which may cause functional deficits in the myocyte associated with diabetic cardiac dysfunction. Our findings may help to further understand the role of iron in the pathogenesis of diabetic complications.

  19. Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed; Gaster, Michel

    2013-01-01

    lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E₁-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²⁺ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C......-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin......, regardless of the presence of glucose. CONCLUSIONS AND IMPLICATIONS: NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²⁺-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel...

  20. Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin and thapsigargin analogs

    DEFF Research Database (Denmark)

    Winther, Anne-Marie Lund; Liu, Huizhen; Sonntag, Yonathan;

    2010-01-01

    transmembrane segments to the putative N-terminal Ca(2+) entry pathway. The long chain analogs provide a rational basis for the localization of the linker, the presence of which is necessary for enabling prostate-specific antigen to cleave peptide-conjugated prodrugs targeting SERCA of cancer cells (Denmeade, S....... R., Jakobsen, C. M., Janssen, S., Khan, S. R., Garrett, E. S., Lilja, H., Christensen, S. B., and Isaacs, J. T. (2003) J. Natl. Cancer Inst. 95, 990-1000). Our study demonstrates the usefulness of a simple in vitro system to test and direct development toward the formulation of new Tg derivatives...

  1. Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca2+-ATPase: quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition

    OpenAIRE

    Fraqueza, Gil; de Carvalho, Luís A. E. Batista; Marques, M. Paula M.; Maia, Luisa; Ohlin, C. André; Casey, William H.; Aureliano, M.

    2012-01-01

    Recently we demonstrated that the decavanadate (V10) ion is a stronger Ca2+-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V10 interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the p...

  2. Effects of type 1 diabetes, sprint training and sex on skeletal muscle sarcoplasmic reticulum Ca2+ uptake and Ca2+-ATPase activity.

    Science.gov (United States)

    Harmer, A R; Ruell, P A; Hunter, S K; McKenna, M J; Thom, J M; Chisholm, D J; Flack, J R

    2014-02-01

    Calcium cycling is integral to muscle performance during the rapid muscle contraction and relaxation of high-intensity exercise. Ca(2+) handling is altered by diabetes mellitus, but has not previously been investigated in human skeletal muscle. We investigated effects of high-intensity exercise and sprint training on skeletal muscle Ca(2+) regulation among men and women with type 1 diabetes (T1D, n = 8, 3F, 5M) and matched non-diabetic controls (CON, n = 8, 3F, 5M). Secondarily, we examined sex differences in Ca(2+) regulation. Subjects undertook 7 weeks of three times-weekly cycle sprint training. Before and after training, performance was measured, and blood and muscle were sampled at rest and after high-intensity exercise. In T1D, higher Ca(2+)-ATPase activity (+28%) and Ca(2+) uptake (+21%) than in CON were evident across both times and days (P women across both times and days. Intense exercise did not alter Ca(2+)-ATPase activity in T1D or CON. However, sex differences were evident: Ca(2+)-ATPase was reduced with exercise among men but increased among women across both days (time × sex interaction, P Sprint training reduced Ca(2+)-ATPase (-8%, P Sprint training reduced Ca(2+)-ATPase in T1D and CON. Sex differences in Ca(2+)-ATPase activity were evident and may be linked with fibre type proportion differences.

  3. Rol de la comunicación retículo sarcoplasmático- mitocondria en la apoptosis del corazón prediabetico

    OpenAIRE

    Marilen, Federico; Sommese, Leandro; Zanuzzi, Carolina Natalia; Portiansky, Enrique Leo; Wehrens, Xaander; Mattiazzi, Alicia; Palomeque, Julieta

    2016-01-01

    La apoptosis cardiaca es uno de los fenómenos más importantes en la transición hacia la insuficiencia cardiaca (IC), y la mitocondria es el intermediario más frecuente de apoptosis desencadenada por los estímulos que generalmente coexisten en la evolución hacia la IC (factores neurohumorales, especies reactivas del oxígeno [ROS], etc). Por otro lado, la IC ocurre más frecuentemente en personas con diabetes de tipo 2 (DMT2) que en la población general. La DMT2 es precedida por un estado de pre...

  4. Monitorização da actividade bioelectroquímica da bomba de cálcio do retículo sarcoplasmático

    OpenAIRE

    Guerreiro, Miguel

    2003-01-01

    Dissertação de lic., Bioquímica, Faculdade Ciências e Tecnologia da Universidade do Algarve, 2003 Neste trabalho foram isoladas vesículas contendo uma proteína, a Ca2+ATPase, a partir do músculo esquelético de Halobatrachus dydactilus. Essas vesículas foram testadas quanto à sua capacidade de ejecção de protões, acoplada à translocação de Ca2+ (que por sua vez está acoplada à hidrólise de ATP), utilizando um novo método mais rápido e mais preciso de monitorização. O novo sis...

  5. Mapping the interactions between ATP and the sarcoplasmic reticulum Ca 2 + -ATPase with ATP and ATP analogs studied by Fourier transform infrared spectroscopy

    OpenAIRE

    Liu, Man

    2004-01-01

    Die Infrarotspektroskopie in Verbindung mit photoaktivierbaren Substraten wurde zur Untersuchung von Substrat-Protein-Wechselwirkungen eingesetzt. Dabei wurden Konformationsänderungen der Ca2+-ATPase des Sarkoplasmatischen Retikulums bei Bindung des Nukleotids, der Phosphorylierung der ATPase und der Hydrolyse des Phosphoenzyms beobachtet. Verwender wurden das native Substrat ATP und seine Analoga ADP, AMPPNP, 2'-deoxyATP, 3'-deoxyATP, ITP, AMP, Pyrophosphat, Ribosetriphosphat und TNP-AMP beo...

  6. [Endoplasmic-mitochondrial Ca(2+)-functional unit: dependence of respiration of secretory cells on activity of ryanodine- and IP3 - sensitive Ca(2+)-channels].

    Science.gov (United States)

    Velykopols'ka, O Iu; Man'ko, B O; Man'ko, V V

    2012-01-01

    Using Clark oxygen electrode, dependence of mitochondrial functions on Ca(2+)-release channels activity of Chironomus plumosus L. larvae salivary glands suspension was investigated. Cells were ATP-permeabilized in order to enable penetration of exogenous oxidative substrates. Activation of plasmalemmal P2X-receptors (as well as P2Y-receptors) per se does not modify the endogenous respiration of salivary gland suspension. That is, Ca(2+)-influx from extracellular medium does not influence functional activity of mitochondria, although they are located along the basal part of the plasma membrane. Activation of RyRs intensifies endogenous respiration and pyruvate-malate-stimulated respiration, but not succinate-stimulated respiration. Neither activation of IP3Rs (via P2Y-receptors activation), nor their inhibition alters endogenous respiration. Nevertheless, IP3Rs inhibition by 2-APB intensifies succinate-stimulated respiration. All abovementioned facts testify that Ca2+, released from stores via channels, alters functional activity of mitochondria, and undoubtedly confirm the existence of endoplasmic-mitochondrial Ca(2+)-functional unit in Ch. plumosus larvae salivary glands secretory cells. In steady state of endoplasmic-mitochondrial Ca(2+)-functional unit the spontaneous activity of IP3Rs is observed; released through IP3Rs, Ca2+ is accumulated in mitochondria via uniporter and modulates oxidative processes. Activation of RyRs induces the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to the active state, which is required to intensify cell respiration and oxidative phosphorylation. As expected, the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to inactivated state (i. e. inhibition of Ca(2+)-release channels at excessive [Ca2+]i) limits the duration of signal transduction, has protective nature and prevents apoptosis.

  7. Post-Pacing Abnormal Repolarization in Catecholaminergic Polymorphic Ventricular Tachycardia Associated with a Mutation in the Cardiac Ryanodine Receptor Gene (RyR2)

    Science.gov (United States)

    Nof, Eyal; Belhassen, Bernard; Arad, Michael; Bhuiyan, Zahurul A.; Antzelevitch, Charles; Rosso, Raphael; Fogelman, Rami; Luria, David; Eli-Ani, Dalia; Mannens, Marcel M.A.M.; Viskin, Sami; Eldar, Michael; Wilde, Arthur A.M.; Glikson, Michael

    2011-01-01

    Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value. Objective We present a CPVT family in which marked post-pacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of RyR2 mutation carriers. Methods The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic ECG testing with epinephrine, adenosine and flecainide. Non-invasive clinical tests were normal in 10 patients evaluated, except for both epinephrine and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in LQTS as well as RyR2. Results Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent post-pacing QT changes. Conclusions M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked post-pacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family. PMID:21699856

  8. The effects of local anesthetics on intracellular Ca2+ release from ryanod ine-sensitive Ca2+ stores in gerbil hippocampal neurons%局麻药对蒙古鼠海马细胞内ryanodine敏感的钙离子释放的影响

    Institute of Scientific and Technical Information of China (English)

    陈俊峰; 徐伟人; 蒋豪

    2002-01-01

    Objective To examine the effects of procaine and lidocaine on intracellular Ca2+ release from sarcoplasmic reticulum ryanodine-sensitive Ca2+ stores. Methods The experiment was performed on hippocampal slices from 60-80g male Mongoliang erbils. Levels of intracellular Ca2+ concentration in the slices were me asured by microfluorometry. The slices were perfused with 50 mmol/L KCl contai ning medium for 30 seconds. Then, the medium was switched to physiological med ium. After 5 min of incubation, the slice was perfused with 20 mmol/L caffeine containing physiology medium for 2 min. Following incubation, the slice was s uperfused with physiological medium until the end of the experiment. The effects of procaine and lidocanin (100 μmol/L) on caffeine-evoked Ca2+ release were evaluated by adding them to the medium after high K+ medium perfusion. Results Caffeine induced a marked increase in intracellular Ca2+ concentration which was then decreased 12% upon the addition of procaine (P<0.05); however, lidocaine, did not induce a similar inhibitory reaction. Conclusion Procaine inhibits ryanodine-receptor mediated Ca2+ release from intracell ular Ca2+ stores, while lidocaine may inhibit Ca2+ release through o ther mechanisms.%目的观察普鲁卡因和利多卡因对细胞内织网内ryanodine 敏感的钙离子释放的影响.方法采用60-80 g 的蒙古鼠海马切片,用显微荧光法测定细胞内钙离子浓度.将切片用含50 mmo l/L KCl的人工脑脊液灌注30秒,然后用人工脑脊液灌注,5分钟后用含20 mmol/L 咖啡因的脑脊液灌注2分钟, 然后换为人工脑脊液直到实验结束.将100 umol/L 的普鲁卡因或利多卡因分别加入到高钾后的灌注液中观察它们对钙离子释放的影响.结果咖啡因可致细胞内钙离子浓度的明显增高, 而普鲁卡因可使这种增高减少12%,而利多卡因则无此抑制作用.结论普鲁卡因可抑制ryanodine 受体介导的细胞内钙离子释放,而利多卡因则可

  9. Effects of Weak Magnetic Field on Ca2+ Modulation of Skeletal Muscle Sarcoplamic Reticulum%弱磁场对骨骼肌肌质网钙调控作用的研究

    Institute of Scientific and Technical Information of China (English)

    刘仁臣; 吴永刚; 程和平; 谢国秋; 陆静; 夏子奂

    2011-01-01

    目的:探讨弱磁场对提取的骨骼肌肌质网系(SR)Ca2+转运、钙泵(Ca2 - Mg2+ -ATPase)及钙释放通道(RyR)活性的影响,从分子水平和细胞信号系统的角度来解释生物电磁效应.方法:利用动态光谱法检测0.4 mT弱磁场辐照过的SR Ca2+转运、Ca2 -ATPase活性,还原型辅酶(NADH)的氧化初速率和超氧(O2)产率,以及用同位素标记方法检测[3H] -Ryanodine与RyR的平衡结合度.结果:弱磁场辐照引起SR的Ca2+摄取功能和Ca2 -ATPase的活性明显下降,Ca2+释放和[3H] -Ryanodine平衡结合度上升,同时上调了NADH的氧化初速率和O2的产率.结论:提示0.4 mT弱磁场辐照30 min对SR Ca2 -ATPase活性有明显抑制,对RyR有一定的激活效果.%Objective: Discuss the effects of weak magnetic field on isolated sarcoplasmic reticulum (SR) Ca2+ modulation, calcium pump (Ca2+-Mg2+-ATPase) and Ca2 + release channel (Ryanodine Receptor, RyR) activity, so as to explain the mechanism of biological effects caused by electromagnetic fields in terms of molecular level and signal transduction system. Methods;The Ca + modulation and Ca + -ATPase activity, NADH oxidation initial rates and superoxide production of SR exposed to 0. 4 mT weak magnetic field ( MF) were investigated with dynamic Ca2 * dye spectrum method, [3 H ] -Ryanodine binding assay was investigated by isotope Labeling. Results: Weak MF exposure decreased SR Ca + uptake and Ca2 + -ATPase activity obviously, increased SR Ca2 + release and [3 H ] -Rryanodine binding, up-regulated the initial rates of NADH oxidation and the production of superoxide. Conclusion: It is indicated that 0. 4 mT weak magnetic field exposure for 30 min inhibited Ca2 + -ATPase activity and promoted the RyR channel function.

  10. The Cl− channel blocker niflumic acid releases Ca2+ from an intracellular store in rat pulmonary artery smooth muscle cells

    Science.gov (United States)

    Cruickshank, Stuart F; Baxter, Lynne M; Drummond, Robert M

    2003-01-01

    The effect of the Cl− channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine-induced increase in [Ca2+]i. These Cl− channel blockers also increased the half-time (t1/2) to peak for the caffeine-induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca2+ in studies involving these Cl− channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl− channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR. PMID:14623766

  11. The Cl(-) channel blocker niflumic acid releases Ca(2+) from an intracellular store in rat pulmonary artery smooth muscle cells.

    Science.gov (United States)

    Cruickshank, Stuart F; Baxter, Lynne M; Drummond, Robert M

    2003-12-01

    The effect of the Cl- channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine-induced increase in [Ca2+]i. These Cl- channel blockers also increased the half-time (t1/2) to peak for the caffeine-induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca2+ in studies involving these Cl- channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl- channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR.

  12. Gene dose influences cellular and calcium channel dysregulation in heterozygous and homozygous T4826I-RYR1 malignant hyperthermia-susceptible muscle.

    Science.gov (United States)

    Barrientos, Genaro C; Feng, Wei; Truong, Kim; Matthaei, Klaus I; Yang, Tianzhong; Allen, Paul D; Lopez, José R; Pessah, Isaac N

    2012-01-20

    Malignant hyperthermia susceptibility (MHS) is primarily conferred by mutations within ryanodine receptor type 1 (RYR1). Here we address how the MHS mutation T4826I within the S4-S5 linker influences excitation-contraction coupling and resting myoplasmic Ca(2+) concentration ([Ca(2+)](rest)) in flexor digitorum brevis (FDB) and vastus lateralis prepared from heterozygous (Het) and homozygous (Hom) T4826I-RYR1 knock-in mice (Yuen, B. T., Boncompagni, S., Feng, W., Yang, T., Lopez, J. R., Matthaei, K. I., Goth, S. R., Protasi, F., Franzini-Armstrong, C., Allen, P. D., and Pessah, I. N. (2011) FASEB J. doi:22131268). FDB responses to electrical stimuli and acute halothane (0.1%, v/v) exposure showed a rank order of Hom ≫ Het ≫ WT. Release of Ca(2+) from the sarcoplasmic reticulum and Ca(2+) entry contributed to halothane-triggered increases in [Ca(2+)](rest) in Hom FDBs and elicited pronounced Ca(2+) oscillations in ∼30% of FDBs tested. Genotype contributed significantly elevated [Ca(2+)](rest) (Hom > Het > WT) measured in vivo using ion-selective microelectrodes. Het and Hom oxygen consumption rates measured in intact myotubes using the Seahorse Bioscience (Billerica, MA) flux analyzer and mitochondrial content measured with MitoTracker were lower than WT, whereas total cellular calpain activity was higher than WT. Muscle membranes did not differ in RYR1 expression nor in Ser(2844) phosphorylation among the genotypes. Single channel analysis showed highly divergent gating behavior with Hom and WT favoring open and closed states, respectively, whereas Het exhibited heterogeneous gating behaviors. [(3)H]Ryanodine binding analysis revealed a gene dose influence on binding density and regulation by Ca(2+), Mg(2+), and temperature. Pronounced abnormalities inherent in T4826I-RYR1 channels confer MHS and promote basal disturbances of excitation-contraction coupling, [Ca(2+)](rest), and oxygen consumption rates. Considering that both Het and Hom T4826I-RYR1 mice are

  13. Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

    Science.gov (United States)

    Zahanich, Ihor; Sirenko, Syevda G; Maltseva, Larissa A; Tarasova, Yelena S; Spurgeon, Harold A; Boheler, Kenneth R; Stern, Michael D; Lakatta, Edward G; Maltsev, Victor A

    2011-01-01

    There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be

  14. The skeletal L-type Ca(2+) current is a major contributor to excitation-coupled Ca(2+) entry.

    Science.gov (United States)

    Bannister, Roger A; Pessah, Isaac N; Beam, Kurt G

    2009-01-01

    The term excitation-coupled Ca(2+) entry (ECCE) designates the entry of extracellular Ca(2+) into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca(2+) entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 microM), and is permeable to Mn(2+). Here, we have examined the effects of these agents on the L-type Ca(2+) current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La(3+), and Gd(3+)) and dantrolene all inhibited the L-type Ca(2+) current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 microM. Like ECCE, the L-type Ca(2+) channel displays permeability to Mn(2+) in the absence of external Ca(2+) and produces a Ca(2+) current that persists during prolonged ( approximately 10-second) depolarization. This current appears to contribute to the Ca(2+) transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca(2+); (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR alpha(1S) pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca(2+)-free solution and were unaffected by Ca(2+) removal; and (3) after block of SR Ca(2+) release by 200 microM ryanodine, normal myotubes still displayed a large Ca(2+) transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca(2+) entry attributed to ECCE.

  15. The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry

    Science.gov (United States)

    Bannister, Roger A.; Pessah, Isaac N.; Beam, Kurt G.

    2009-01-01

    The term excitation-coupled Ca2+ entry (ECCE) designates the entry of extracellular Ca2+ into skeletal muscle cells, which occurs in response to prolonged depolarization or pulse trains and depends on the presence of both the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor in the sarcoplasmic reticulum (SR) membrane. The ECCE pathway is blocked by pharmacological agents that also block store-operated Ca2+ entry, is inhibited by dantrolene, is relatively insensitive to the DHP antagonist nifedipine (1 μM), and is permeable to Mn2+. Here, we have examined the effects of these agents on the L-type Ca2+ current conducted via the DHPR. We found that the nonspecific cation channel antagonists (2-APB, SKF 96356, La3+, and Gd3+) and dantrolene all inhibited the L-type Ca2+ current. In addition, complete (>97%) block of the L-type current required concentrations of nifedipine >10 μM. Like ECCE, the L-type Ca2+ channel displays permeability to Mn2+ in the absence of external Ca2+ and produces a Ca2+ current that persists during prolonged (∼10-second) depolarization. This current appears to contribute to the Ca2+ transient observed during prolonged KCl depolarization of intact myotubes because (1) the transients in normal myotubes decayed more rapidly in the absence of external Ca2+; (2) the transients in dysgenic myotubes expressing SkEIIIK (a DHPR α1S pore mutant thought to conduct only monovalent cations) had a time course like that of normal myotubes in Ca2+-free solution and were unaffected by Ca2+ removal; and (3) after block of SR Ca2+ release by 200 μM ryanodine, normal myotubes still displayed a large Ca2+ transient, whereas no transient was detectable in SkEIIIK-expressing dysgenic myotubes. Collectively, these results indicate that the skeletal muscle L-type channel is a major contributor to the Ca2+ entry attributed to ECCE. PMID:19114636

  16. Effects of cartap on isolated mouse phrenic nerve diaphragm and its related mechanism.

    Science.gov (United States)

    Liao, J W; Kang, J J; Liu, S H; Jeng, C R; Cheng, Y W; Hu, C M; Tsai, S F; Wang, S C; Pang, V F

    2000-06-01

    Cartap, a nereistoxin analogue pesticide, is reported to have no irritation to eyes in rabbits. However, we have demonstrated recently that cartap could actually cause acute death in rabbits via ocular exposure. Our preliminary study with isolated mouse phrenic nerve diaphragms has shown that instead of neuromuscular blockade, cartap caused muscular contracture. The objective of the study was to examine the effect of cartap on the neuromuscular junction in more detail and to investigate its possible underlying mechanism with isolated mouse phrenic nerve diaphragms and sarcoplasmic reticulum (SR) vesicles. Cartap or nereistoxin at various concentrations was added in the organ bath with isolated mouse phrenic nerve diaphragm and both nerve- and muscle-evoked twitches were recorded. Instead of blocking the neuromuscular transmission as nereistoxin did, cartap caused contracture in stimulated or quiescent isolated mouse phrenic nerve diaphragm. Both the cartap-induced muscular contracture force and the time interval to initiate the contracture were dose-dependent. The contracture induced by cartap was not affected by the pretreatment of the diaphragm with the acetylcholine receptor blocker alpha-bungarotoxin; the Na(+) channel blocker tetrodotoxin; or various Ca(2+) channel blockers, NiCl(2), verapamil, and nifedipine. On the contrary, the contracture was significantly inhibited when the diaphragm was pretreated with ryanodine or EGTA containing Ca(2+)-free Krebs solution or in combination. This suggested that both internal and extracellular Ca(2+) might participate in cartap-induced skeletal muscle contracture. Moreover, cartap inhibited the [(3)H]-ryanodine binding to the Ca(2+) release channel of SR in a dose-dependent manner. Additionally, cartap could induce a significant reduction in Ca(2+)-ATPase activity of SR vesicles at a relatively high dose. The results suggested that cartap might cause the influx of extracellular Ca(2+) and the release of internal Ca(2

  17. UniProt search blastx result: AK288850 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK288850 J090075H22 Q92736|RYR2_HUMAN Ryanodine receptor 2 (Cardiac muscle-type rya...nodine receptor) (RyR2) (RYR-2) (Cardiac muscle ryanodine receptor-calcium release channel) (hRYR-2) - Homo sapiens (Human) 0 ...

  18. Low-Level Vagus Nerve Stimulation Reverses Cardiac Dysfunction and Subcellular Calcium Handling in Rats With Post-Myocardial Infarction Heart Failure.

    Science.gov (United States)

    Zhang, Yunhe; Chen, Ao; Song, Lei; Li, Min; Luo, Zhangyuan; Zhang, Wenzan; Chen, Yingmin; He, Ben

    2016-05-25

    Vagus nerve stimulation (VNS), targeting the imbalanced autonomic nervous system, is a promising therapeutic approach for chronic heart failure (HF). Moreover, calcium cycling is an important part of cardiac excitation-contraction coupling (ECC), which also participates in the antiarrhythmic effects of VNS. We hypothesized that low-level VNS (LL-VNS) could improve cardiac function by regulation of intracellular calcium handling properties. The experimental HF model was established by ligation of the left anterior descending coronary artery (LAD). Thirty-two male Sprague-Dawley rats were divided into 3 groups as follows; control group (sham operated without coronary ligation, n = 10), HF-VNS group (HF rats with VNS, n = 12), and HF-SS group (HF rats with sham nerve stimulation, n = 10). After 8 weeks of treatment, LL-VNS significantly improved left ventricular ejection fraction (LVEF) and attenuated myocardial interstitial fibrosis in the HF-VNS group compared with the HF-SS group. Elevated plasma norepinephrine and dopamine, but not epinephrine, were partially reduced by LL-VNS. Additionally, LL-VNS restored the protein and mRNA levels of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), Na(+)-Ca(2+) exchanger 1 (NCX1), and phospholamban (PLB) whereas the expression of ryanodine receptor 2 (RyR2) as well as mRNA level was unaffected. Thus, our study results suggest that the improvement of cardiac performance by LL-VNS is accompanied by the reversal of dysfunctional calcium handling properties including SERCA2a, NCX1, and PLB which may be a potential molecular mechanism of VNS for HF.

  19. Ouabain facilitates cardiac differentiation of mouse embryonic stem cells through ERK1/2 pathway

    Institute of Scientific and Technical Information of China (English)

    Yee-ki LEE; Kwong-man NG; Wing-hon LAI; Cornelia MAN; Deborah K LIEU; Chu-pak LAU; Hung-fat TSE; Chung-wah SIU

    2011-01-01

    Aim:To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs).Methods:Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 μmol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging.Results:Compared with control, mESCs treated with ouabain (20 μmol/L) yielded a significantly higher percentage of cardiomyocytesand significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and β-MHC. The α1 and 2- isoforms Na+/K+ -ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 μmol/L) inhibited cardiac differentiation while ouabain (20 μmol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, includ ing ryanodine receptor (RyR2) and sacroplasmic recticulum Ca2+ ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs.ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca2+ imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca2+ transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR).Conclusion:Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.

  20. Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.

    Science.gov (United States)

    Jayasinghe, Isuru D; Munro, Michelle; Baddeley, David; Launikonis, Bradley S; Soeller, Christian

    2014-10-06

    Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle.

  1. Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+ handling.

    Directory of Open Access Journals (Sweden)

    Christopher J Traynham

    Full Text Available Nitric oxide (NO and superoxide (O(2 (- are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O(2 (.- must exist at defined levels. Unfortunately, the NO and O(2 (.- levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc. leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O(2 (.- while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2-2-3,4-dihydro-2H-pyrrole 1-oxide (EMEPO. We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6 and NOS1 knockout (NOS1(-/- mice, a known model of NO/O(2 (.- imbalance, and incubated with EMEPO. EMEPO significantly reduced O(2 (.- (lucigenin-enhanced chemiluminescence and elevated NO (DAF-FM diacetate levels in NOS1(-/- myocytes. Furthermore, EMEPO increased NOS1(-/- myocyte basal contraction (Ca(2+ transients, Fluo-4AM; shortening, video-edge detection, the force-frequency response and the contractile response to β-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca(2+ leak/load relationship and phospholamban Serine16 phosphorylation (Western blot. We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1(-/- myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O(2 (.- levels may have therapeutic potential in the treatment of various cardiomyopathies.

  2. Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Zhenghang Zhao

    Full Text Available Recent studies have suggested that mitochondria may play important roles in the Ca(2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+ flux can regulate the generation of Ca(2+ waves (CaWs and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+ (Cai (2+ was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR Ca(2+ release and CaWs were induced in the presence of high (4 mM external Ca(2+ (Cao (2+. The protonophore carbonyl cyanide p-(trifluoromethoxyphenylhydrazone (FCCP reversibly raised basal Cai (2+ levels even after depletion of SR Ca(2+ in the absence of Cao (2+ , suggesting Ca(2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m or Ru360 (a mitochondrial Ca(2+ uniporter inhibitor, but not by oligomycin (an ATP synthase inhibitor or iodoacetic acid (a glycolytic inhibitor, excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+ release and uptake exquisitely control the local Ca(2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.

  3. Detergent resistant membrane fractions are involved in calcium signaling in Müller glial cells of retina.

    Science.gov (United States)

    Krishnan, Gopinath; Chatterjee, Nivedita

    2013-08-01

    Compartmentalization of the plasma membrane into lipid microdomains promotes efficient cellular processes by increasing local molecular concentrations. Calcium signaling, either as transients or propagating waves require integration of complex macromolecular machinery. Calcium waves represent a form of intercellular signaling in the central nervous system and the retina. We hypothesized that the mechanism for calcium waves would require effector proteins to aggregate at the plasma membrane in lipid microdomains. The current study shows that in Müller glia of the retina, proteins involved in calcium signaling aggregate in detergent resistant membranes identifying rafts and respond by redistributing on stimulation. We have investigated Purinoreceptor-1 (P2Y1), Ryanodine receptor (RyR), and Phospholipase C (PLC-β1). P2Y1, RyR and PLC-β1, redistribute from caveolin-1 and flotillin-1 positive fractions on stimulation with the agonists, ATP, 2MeS-ATP and Thapsigargin, an inhibitor of sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA). Redistribution is absent on treatment with cyclopiazonic acid, another SERCA inhibitor. Disruption of rafts by removing cholesterol cause proteins involved in this machinery to redistribute and change agonist-induced calcium signaling. Cholesterol depletion from raft lead to increase in time to peak of calcium levels in agonist-evoked calcium signals in all instances, as seen by live imaging. This study emphasizes the necessity of a sub-population of proteins to cluster in specialized lipid domains. The requirement for such an organization at the raft-like microdomains may have implications on intercellular communication in the retina. Such concerted interaction at the rafts can regulate calcium dynamics and could add another layer of complexity to calcium signaling in cells.

  4. Cell model of catecholaminergic polymorphic ventricular tachycardia reveals early and delayed afterdepolarizations.

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    Kirsi Kujala

    Full Text Available BACKGROUND: Induced pluripotent stem cells (iPSC provide means to study the pathophysiology of genetic disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT is a malignant inherited ion channel disorder predominantly caused by mutations in the cardiac ryanodine receptor (RyR2. In this study the cellular characteristics of CPVT are investigated and whether the electrophysiological features of this mutation can be mimicked using iPSC -derived cardiomyocytes (CM. METHODOLOGY/PRINCIPAL FINDINGS: Spontaneously beating CMs were differentiated from iPSCs derived from a CPVT patient carrying a P2328S mutation in RyR2 and from two healthy controls. Calcium (Ca(2+ cycling and electrophysiological properties were studied by Ca(2+ imaging and patch-clamp techniques. Monophasic action potential (MAP recordings and 24h-ECGs of CPVT-P2328S patients were analyzed for the presence of afterdepolarizations. We found defects in Ca(2+ cycling and electrophysiology in CPVT CMs, reflecting the cardiac phenotype observed in the patients. Catecholaminergic stress led to abnormal Ca(2+ signaling and induced arrhythmias in CPVT CMs. CPVT CMs also displayed reduced sarcoplasmic reticulum (SR Ca(2+ content, indicating leakage of Ca(2+ from the SR. Patch-clamp recordings of CPVT CMs revealed both delayed afterdepolarizations (DADs during spontaneous beating and in response to adrenaline and also early afterdepolarizations (EADs during spontaneous beating, recapitulating the changes seen in MAP and 24h-ECG recordings of patients carrying the same mutation. CONCLUSIONS/SIGNIFICANCE: This cell model shows aberrant Ca(2+ cycling characteristic of CPVT and in addition to DADs it displays EADs. This cell model for CPVT provides a platform to study basic pathology, to screen drugs, and to optimize drug therapy.

  5. Molecular aspects of calcium signalling at the crossroads of unikont and bikont eukaryote evolution--the ciliated protozoan Paramecium in focus.

    Science.gov (United States)

    Plattner, Helmut

    2015-03-01

    The ciliated protozoan, Paramecium tetraurelia has a high basic Ca(2+) leakage rate which is counteracted mainly by export through a contractile vacuole complex, based on its V-type H(+)-ATPase activity. In addition Paramecium cells dispose of P-type Ca(2+)-ATPases, i.e. a plasmamembrane and a sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (PMCA, SERCA). Antiporter systems are to be expected, as inferred from indirect evidence. Among the best known cytosolic Ca(2+)-binding proteins, calmodulin activates Ca(2+) influx channels in the somatic cell membrane, but inactivates Ca(2+) influx channels in cilia, where it, thus, ends ciliary reversal induced by depolarization via channels in the somatic cell membrane. Centrin inactivates Ca(2+) signals after stimulation by its high capacity/low affinity binding sites, whereas its high affinity sites regulate some other functions. Cortical Ca(2+) stores (alveolar sacs) are activated during stimulated trichocyst exocytosis and thereby mediate store-operated Ca(2+) entry (SOCE). Ca(2+) release channels (CRCs) localised to alveoli and underlying SOCE are considered as Ryanodine receptor-like proteins (RyR-LPs) which are members of a CRC family with 6 subfamilies. These also encompass genuine inositol 1,4,5-trisphosphate receptors (IP3Rs) and intermediates between the two channel types. All IP3R/RyR-type CRCs possess six carboxyterminal transmembrane domains (TMD), with a pore domain between TMD 5 and 6, endowed with a characteristic selectivity filter. There are reasons to assume a common ancestor molecule for such channels and diversification further on in evolution. The distinct distribution of specific CRCs in the different vesicles undergoing intracellular trafficking suggests constitutive formation of very locally restricted Ca(2+) signals during vesicle-vesicle interaction. In summary, essential steps of Ca(2+) signalling already occur at this level of evolution, including an unexpected multitude of CRCs. For dis

  6. Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus.

    Science.gov (United States)

    Clyman, Ronald I; Waleh, Nahid; Kajino, Hiroki; Roman, Christine; Mauray, Francoise

    2007-10-01

    Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to Ca(2+) entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular Ca(2+) and is unaffected by inhibitors of sarcoplasmic reticulum (SR) Ca(2+) release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated Ca(2+) entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both Ca(2+)-dependent and Ca(2+)-independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process Ca(2+). Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated Ca(2+) entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent Ca(2+) entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of Ca(2+) entry through reverse-mode Na(+)/Ca(2+) exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia.

  7. Impaired P2X signalling pathways in renal microvascular myocytes in genetic hypertension

    KAUST Repository

    Gordienko, Dmitri V.

    2014-12-16

    Aims P2X receptors (P2XRs) mediate sympathetic control and autoregulation of renal circulation triggering preglomerular vasoconstriction, which protects glomeruli from elevated pressures. Although previous studies established a casual link between glomerular susceptibility to hypertensive injury and decreased preglomerular vascular reactivity to P2XR activation, the mechanisms of attenuation of the P2XR signalling in hypertension remained unknown. We aimed to analyse molecular mechanisms of the impairment of P2XR signalling in renal vascular smooth muscle cells (RVSMCs) in genetic hypertension. Methods and results We compared the expression of pertinent genes and P2XR-linked Ca2+ entry and Ca2+ release mechanisms in RVSMCs of spontaneously hypertensive rats (SHRs) and their normotensive controls, Wistar Kyoto (WKY) rats. We found that, in SHR RVSMCs, P2XR-linked Ca2+ entry and Ca2+ release from the sarcoplasmic reticulum (SR) are both significantly reduced. The former is due to down-regulation of the P2X1 subunit. The latter is caused by a decrease of the SR Ca2+ load. The SR Ca2+ load reduction is caused by attenuated Ca2+ uptake via down-regulated sarco-/endoplasmic reticulum Ca2+-ATPase 2b and elevated Ca2+ leak from the SR via ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors. Spontaneous activity of these Ca2+-release channels is augmented due to up-regulation of RyR type 2 and elevated IP3 production by up-regulated phospholipase C-β1. Conclusions Our study unravels the cellular and molecular mechanisms of attenuation of P2XR-mediated preglomerular vasoconstriction that elevates glomerular susceptibility to harmful hypertensive pressures. This provides an important impetus towards understanding of the pathology of hypertensive renal injury.

  8. Heat and chemical stress modulate the expression of the alpha-RYR gene in broiler chickens.

    Science.gov (United States)

    Ziober, I L; Paião, F G; Marchi, D F; Coutinho, L L; Binneck, E; Nepomuceno, A L; Shimokomaki, M

    2010-06-29

    The biological cause of Pork Stress syndrome, which leads to PSE (pale, soft, exudative) meat, is excessive release of Ca(2+) ions, which is promoted by a genetic mutation in the ryanodine receptors (RyR) located in the sarcoplasmic reticulum of the skeletal muscle cells. We examined the relationship between the formation of PSE meat under halothane treatment and heat stress exposure in chicken alphaRYR hot spot fragments. Four test groups were compared: 1) birds slaughtered without any treatment, i.e., the control group (C); 2) birds slaughtered immediately after halothane treatment (H); 3) birds slaughtered immediately after heat stress treatment (HS), and 4) birds exposed to halothane and to heat stress (H+HS), before slaughtering. Breast muscle mRNA was extracted, amplified by RT-PCR, and sequenced. PSE meat was evaluated using color determination (L* value). The most common alteration was deletion of a single nucleotide, which generated a premature stop codon, resulting in the production of truncated proteins. The highest incidence of nonsense transcripts came with exposure to halothane; 80% of these abnormal transcripts were detected in H and H+HS groups. As a consequence, the incidence of abnormal meat was highest in the H+HS group (66%). In HS, H, and C groups, PSE meat developed in 60, 50, and 33% of the samples, respectively. Thus, halothane apparently modulates alphaRYR gene expression in this region, and synergically with exposure to heat stress, causes Avian Stress syndrome, resulting in PSE meat in broiler chickens.

  9. Inhibitors of arachidonate-regulated calcium channel signaling suppress triggered activity induced by the late sodium current.

    Science.gov (United States)

    Wolkowicz, Paul; Umeda, Patrick K; Sharifov, Oleg F; White, C Roger; Huang, Jian; Mahtani, Harry; Urthaler, Ferdinand

    2014-02-05

    Disturbances in myocyte calcium homeostasis are hypothesized to be one cause for cardiac arrhythmia. The full development of this hypothesis requires (i) the identification of all sources of arrhythmogenic calcium and (ii) an understanding of the mechanism(s) through which calcium initiates arrhythmia. To these ends we superfused rat left atria with the late sodium current activator type II Anemonia sulcata toxin (ATXII). This toxin prolonged atrial action potentials, induced early afterdepolarization, and provoked triggered activity. The calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 (N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphon-amide) suppressed ATXII triggered activity but its inactive congener KN-92 (2-[N-(4-methoxy benzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) did not. Neither drug affected normal atrial contractility. Calcium entry via L-type channels or calcium leakage from sarcoplasmic reticulum stores are not critical for this type of ectopy as neither verapamil ((RS)-2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl]-(methyl)amino}-2-prop-2-ylpentanenitrile) nor ryanodine affected ATXII triggered activity. By contrast, inhibitors of the voltage independent arachidonate-regulated calcium (ARC) channel and the store-operated calcium channel specifically suppressed ATXII triggered activity without normalizing action potentials or affecting atrial contractility. Inhibitors of cytosolic calcium-dependent phospholipase A2 also suppressed triggered activity suggesting that this lipase, which generates free arachidonate, plays a key role in ATXII ectopy. Thus, increased left atrial late sodium current appears to activate atrial Orai-linked ARC and store operated calcium channels, and these voltage-independent channels may be unexpected sources for the arrhythmogenic calcium that underlies triggered activity.

  10. Characterization and temporal development of cores in a mouse model of malignant hyperthermia.

    Science.gov (United States)

    Boncompagni, Simona; Rossi, Ann E; Micaroni, Massimo; Hamilton, Susan L; Dirksen, Robert T; Franzini-Armstrong, Clara; Protasi, Feliciano

    2009-12-22

    Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca(2+) release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1(Y522S/WT)), the mutation causes SR Ca(2+) leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1(Y522S/WT) knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1(Y522S/WT) mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2-4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1(Y522S/WT) mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca(2+) sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.

  11. The GSTM2 C-Terminal Domain Depresses Contractility and Ca2+ Transients in Neonatal Rat Ventricular Cardiomyocytes

    Science.gov (United States)

    Hewawasam, Ruwani P.; Liu, Dan; Casarotto, Marco G.; Board, Philip G.; Dulhunty, Angela F.

    2016-01-01

    The cardiac ryanodine receptor (RyR2) is an intracellular ion channel that regulates Ca2+ release from the sarcoplasmic reticulum (SR) during excitation–contraction coupling in the heart. The glutathione transferases (GSTs) are a family of phase II detoxification enzymes with additional functions including the selective inhibition of RyR2, with therapeutic implications. The C-terminal half of GSTM2 (GSTM2C) is essential for RyR2 inhibition, and mutations F157A and Y160A within GSTM2C prevent the inhibitory action. Our objective in this investigation was to determine whether GSTM2C can enter cultured rat neonatal ventricular cardiomyocytes and influence contractility. We show that oregon green-tagged GSTM2C (at 1 μM) is internalized into the myocytes and it reduces spontaneous contraction frequency and myocyte shortening. Field stimulation of myocytes evoked contraction in the same percentage of myocytes treated either with media alone or media plus 15 μM GSTM2C. Myocyte shortening during contraction was significantly reduced by exposure to 15 μM GSTM2C, but not 5 and 10 μM GSTM2C and was unaffected by exposure to 15 μM of the mutants Y160A or F157A. The amplitude of the Ca2+ transient in the 15 μM GSTM2C - treated myocytes was significantly decreased, the rise time was significantly longer and the decay time was significantly shorter than in control myocytes. The Ca2+ transient was not altered by exposure to Y160A or F157A. The results are consistent with GSTM2C entering the myocytes and inhibiting RyR2, in a manner that indicates a possible therapeutic potential for treatment of arrhythmia in the neonatal heart. PMID:27612301

  12. The missing link in the mystery of normal automaticity of cardiac pacemaker cells.

    Science.gov (United States)

    Lakatta, Edward G; Vinogradova, Tatiana M; Maltsev, Victor A

    2008-03-01

    Earlier studies of the initiating event of normal automaticity of the heart's pacemaker cells, inspired by classical quantitative membrane theory, focused upon ion currents (IK, I f) that determine the maximum diastolic potential and the early phase of the spontaneous diastolic depolarization (DD). These early DD events are caused by the prior action potential (AP) and essentially reflect a membrane recovery process. Events following the recovery process that ignite APs have not been recognized and remained a mystery until recently. These critical events are linked to rhythmic intracellular signals initiated by Ca2+ clock (i.e., sarcoplasmic reticulum [SR] cycling Ca2+). Sinoatrial cells, regardless of size, exhibit intense ryanodine receptor (RyR), Na+/Ca2+ exchange (NCX)-1, and SR Ca2+ ATPase-2 immunolabeling and dense submembrane NCX/RyR colocalization; Ca2+ clocks generate spontaneous stochastic but roughly periodic local subsarcolemmal Ca2+ releases (LCR). LCRs generate inward currents via NCX that exponentially accelerate the late DD. The timing and amplitude of LCR/I NCX-coupled events control the timing and amplitude of the nonlinear terminal DD and therefore ultimately control the chronotropic state by determining the timing of the I CaL activation that initiates the next AP. LCR period is precisely controlled by the kinetics of SR Ca2+ cycling, which, in turn, are regulated by 1) the status of protein kinase A-dependent phosphorylation of SR Ca2+ cycling proteins; and 2) membrane ion channels ensuring the Ca2+ homeostasis and therefore the Ca2+ available to Ca2+ clock. Thus, the link between early DD and next AP, missed in earlier studies, is ensured by a precisely physiologically regulated Ca2+ clock within pacemaker cells that integrates multiple Ca2+-dependent functions and rhythmically ignites APs during late DD via LCRs-I NCX coupling.

  13. Life and death of a cardiac calcium spark.

    Science.gov (United States)

    Stern, Michael D; Ríos, Eduardo; Maltsev, Victor A

    2013-09-01

    Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition-a phenomenon we dub "spark metastability." Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.

  14. Junctophilin-2 Expression Silencing Causes Cardiocyte Hypertrophy and Abnormal Intracellular Calcium-Handling

    Science.gov (United States)

    Landstrom, Andrew P.; Kellen, Cherisse A.; Dixit, Sayali S.; van Oort, Ralph J.; Garbino, Alejandro; Weisleder, Noah; Ma, Jianjie; Wehrens, Xander H.T.; Ackerman, Michael J.

    2011-01-01

    Background Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca2+) signaling in cardiac myocytes. Down-regulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca2+ channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of down-regulation of JPH2 expression on intracellular Ca2+-handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca2+-handling in a pro-hypertrophic manner. Methods and Results JPH2 expression was reduced in flash frozen human cardiac tissue procured from patients with HCM compared to ostensibly healthy traumatic death victims. Partial silencing of JPH2 expression in HL-1 cells by a small interfering RNA probe targeted to murine JPH2 mRNA (shJPH2) resulted in myocyte hypertrophy and increased expression of known markers of cardiac hypertrophy. While expression levels of major Ca2+-handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca2+ transient amplitudes that were insensitive to LTCC activation with JPH2 knock-down. Further, reduced caffeine-triggered SR store Ca2+ levels were observed with potentially increased total Ca2+ stores. Spontaneous Ca2+ oscillations were elicited at a higher extracellular [Ca2+] and with decreased frequency in JPH2 knock-down cells. Conclusions Our results show that JPH2 levels are reduced in patients with HCM. Reduced JPH2 expression results in reduced excitation-contraction coupling gain as well as altered Ca2+ homeostasis which may be associated with pro-hypertrophic remodeling. PMID:21216834

  15. Atrial myocyte function and Ca2+ handling is associated with inborn aerobic capacity.

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    Anne Berit Johnsen

    Full Text Available Although high aerobic capacity is associated with effective cardiac function, the effect of aerobic capacity on atrial function, especially in terms of cellular mechanisms, is not known. We aimed to investigate whether rats with low inborn maximal oxygen uptake (VO2 max had impaired atrial myocyte contractile function when compared to rats with high inborn VO2 max.Atrial myocyte function was depressed in Low Capacity Runners (LCR relative to High Capacity Runners (HCR which was associated with impaired Ca(2+ handling. Fractional shortening was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation while time to 50% relengthening was 43% prolonged and 55% prolonged, respectively. Differences in Ca(2+ amplitude and diastolic Ca(2+ level were observed at 5 Hz stimulation where Ca(2+ amplitude was 70% lower and diastolic Ca(2+ level was 11% higher in LCR rats. Prolonged time to 50% Ca(2+ decay was associated with reduced sarcoplasmic reticulum (SR Ca(2+ ATPase function in LCR (39%. Na(+/Ca(2+ exchanger activity was comparable between the groups. Diastolic SR Ca(2+ leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca(2+-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca(2+ release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca(2+-signal onset.This data demonstrates that contrasting for low or high aerobic capacity leads to diverse functional and structural remodelling of atrial myocytes, with impaired contractile function in LCR compared to HCR rats.

  16. Identification and characterization of calcium sparks in cardiomyocytes derived from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Guang Qin Zhang

    Full Text Available INTRODUCTION: Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs-derived cardiomyocytes (hiPSC-CMs may display unique structural and functional properties than mature adult cardiomyocytes. METHODS AND RESULTS: Ca2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM and full duration at half maximum (FDHM of spontaneous Ca2+ sparks. CONCLUSIONS: For the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.

  17. Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle.

    Science.gov (United States)

    Park, S; Scheffler, T L; Gunawan, A M; Shi, H; Zeng, C; Hannon, K M; Grant, A L; Gerrard, D E

    2009-01-01

    Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced GLUT-4 expression in skeletal muscle.

  18. T-tubule disruption promotes calcium alternans in failing ventricular myocytes: mechanistic insights from computational modeling.

    Science.gov (United States)

    Nivala, Michael; Song, Zhen; Weiss, James N; Qu, Zhilin

    2015-02-01

    In heart failure (HF), T-tubule (TT) disruption contributes to dyssynchronous calcium (Ca) release and impaired contraction, but its role in arrhythmogenesis remains unclear. In this study, we investigate the effects of TT disruption and other HF remodeling factors on Ca alternans in ventricular myocytes using computer modeling. A ventricular myocyte model with detailed spatiotemporal Ca cycling modeled by a coupled Ca release unit (CRU) network was used, in which the L-type Ca channels and the ryanodine receptor (RyR) channels were simulated by random Markov transitions. TT disruption, which removes the L-type Ca channels from the associated CRUs, results in "orphaned" RyR clusters and thus provides increased opportunity for spark-induced Ca sparks to occur. This effect combined with other HF remodeling factors promoted alternans by two distinct mechanisms: 1) for normal sarco-endoplasmic reticulum Ca ATPase (SERCA) activity, alternans was caused by both CRU refractoriness and coupling. The increased opportunity for spark-induced sparks by TT disruption combined with the enhanced CRU coupling by Ca elevation in the presence or absence of increased RyR leakiness facilitated spark synchronization on alternate beats to promote Ca alternans; 2) for down-regulated SERCA, alternans was caused by the sarcoplasmic reticulum (SR) Ca load-dependent mechanism, independent of CRU refractoriness. TT disruption and increased RyR leakiness shifted and steepened the SR Ca release-load relationship, which combines with down-regulated SERCA to promote Ca alternans. In conclusion, the mechanisms of Ca alternans for normal and down-regulated SERCA are different, and TT disruption promotes Ca alternans by both mechanisms, which may contribute to alternans at different stages of HF.

  19. αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca(2+) -handling proteins and endoplasmic reticulum stress in mice.

    Science.gov (United States)

    Jiao, Qibin; Sanbe, Atsushi; Zhang, Xingwei; Liu, Jun-Ping; Minamisawa, Susumu

    2014-08-01

    Mutations of αB-crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin-related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28 weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28 weeks) and examined Ca(2+) -handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28- versus 10-week-old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28-week-old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein, inositol requirement 1 and X-box binding protein 1 were increased significantly in 28- versus 10-week-old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild-type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca(2+) regulation, leading to ageing-related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.

  20. Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

    Energy Technology Data Exchange (ETDEWEB)

    Borko, Ľubomír; Bauerová-Hlinková, Vladena, E-mail: vladena.bauerova@savba.sk; Hostinová, Eva; Gašperík, Juraj [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia); Beck, Konrad [Cardiff University School of Dentistry, Heath Park, Cardiff CF14 4XY Wales (United Kingdom); Lai, F. Anthony [Cardiff University School of Medicine, Cardiff CF14 4XN Wales (United Kingdom); Zahradníková, Alexandra, E-mail: vladena.bauerova@savba.sk [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia); Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava (Slovakia); Ševčík, Jozef, E-mail: vladena.bauerova@savba.sk [Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava (Slovakia)

    2014-11-01

    X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

  1. The effect of acidosis on the interval-force relation and mechanical restitution in ferret papillary muscle.

    Science.gov (United States)

    McCall, E; Orchard, C H

    1991-01-01

    1. The effect of a respiratory acidosis on the interval-force relation and on mechanical restitution was investigated in ferret papillary muscles. 2. Acidosis (pH 6.85) decreased developed force over a range of stimulation frequencies (1.0.06 Hz); the percentage decrease was greatest at the lowest stimulation frequencies. Qualitatively similar effects of acidosis on developed force were observed in the presence of the sarcoplasmic reticulum (SR) inhibitor ryanodine. 3. Mechanical restitution curves were constructed by interpolating extra-systoles at different test intervals following a train of steady-state beats. Mechanical restitution in ferret papillary muscle was triphasic: an initial, rapid, exponential increase in force with test intervals to 2 s, a further increase with test intervals between 60 and 90 s and then a slow decline, with a plateau at about 30 min (0.33 Hz, 30 degrees C). 4. Acidosis slowed the initial phase of mechanical restitution. The degree of slowing depended on the steady-state stimulation frequency, being greatest at low frequencies. 5. Inhibition of the SR abolished the initial phase of mechanical restitution, suggesting that this phase depends on Ca2+ release from the SR. 6. The strength of the first contraction after the extra-systole varied inversely with the size of the extra-systole under all conditions studied. 7. It is concluded that acidosis may inhibit the SR by altering the time required for Ca2+ recycling between contractions. This effect may alter Ca2+ release from the SR during acidosis, and may underlie the mechanical alternans (the alternation of small and large contractions) that can occur during acidosis.

  2. A human pluripotent stem cell model of catecholaminergic polymorphic ventricular tachycardia recapitulates patient-specific drug responses

    Directory of Open Access Journals (Sweden)

    Marcela K. Preininger

    2016-09-01

    Full Text Available Although β-blockers can be used to eliminate stress-induced ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT, this treatment is unsuccessful in ∼25% of cases. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs generated from these patients have potential for use in investigating the phenomenon, but it remains unknown whether they can recapitulate patient-specific drug responses to β-blockers. This study assessed whether the inadequacy of β-blocker therapy in an individual can be observed in vitro using patient-derived CPVT iPSC-CMs. An individual with CPVT harboring a novel mutation in the type 2 cardiac ryanodine receptor (RyR2 was identified whose persistent ventricular arrhythmias during β-blockade with nadolol were abolished during flecainide treatment. iPSC-CMs generated from this patient and two control individuals expressed comparable levels of excitation-contraction genes, but assessment of the sarcoplasmic reticulum Ca2+ leak and load relationship revealed intracellular Ca2+ homeostasis was altered in the CPVT iPSC-CMs. β-adrenergic stimulation potentiated spontaneous Ca2+ waves and unduly frequent, large and prolonged Ca2+ sparks in CPVT compared with control iPSC-CMs, validating the disease phenotype. Pursuant to the patient's in vivo responses, nadolol treatment during β-adrenergic stimulation achieved negligible reduction of Ca2+ wave frequency and failed to rescue Ca2+ spark defects in CPVT iPSC-CMs. In contrast, flecainide reduced both frequency and amplitude of Ca2+ waves and restored the frequency, width and duration of Ca2+ sparks to baseline levels. By recapitulating the improved response of an individual with CPVT to flecainide compared with β-blocker therapy in vitro, these data provide new evidence that iPSC-CMs can capture basic components of patient-specific drug responses.

  3. Mechanochemotransduction during cardiomyocyte contraction is mediated by localized nitric oxide signaling.

    Science.gov (United States)

    Jian, Zhong; Han, Huilan; Zhang, Tieqiao; Puglisi, Jose; Izu, Leighton T; Shaw, John A; Onofiok, Ekama; Erickson, Jeffery R; Chen, Yi-Je; Horvath, Balazs; Shimkunas, Rafael; Xiao, Wenwu; Li, Yuanpei; Pan, Tingrui; Chan, James; Banyasz, Tamas; Tardiff, Jil C; Chiamvimonvat, Nipavan; Bers, Donald M; Lam, Kit S; Chen-Izu, Ye

    2014-03-18

    Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca(2+) dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca(2+) transient, which enhanced contractility to counter mechanical load but also caused spontaneous Ca(2+) sparks during diastole that could be arrhythmogenic. The increases in the Ca(2+) transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2(+) in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2(+) sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca(2+) sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca(2+) sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2(+) sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress-induced Ca(2+) dysregulation, arrhythmias, and cardiomyopathy.

  4. Mitochondrial calcium overload is a key determinant in heart failure.

    Science.gov (United States)

    Santulli, Gaetano; Xie, Wenjun; Reiken, Steven R; Marks, Andrew R

    2015-09-08

    Calcium (Ca2+) released from the sarcoplasmic reticulum (SR) is crucial for excitation-contraction (E-C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca2+ is essential for optimal function of these organelles. However, Ca2+ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca2+ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca2+ leak causes mitochondrial Ca2+ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca2+ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca2+ leak, we found that leaky RyR2 channels result in mitochondrial Ca2+ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca2+ overload and dysfunction in HF.

  5. CaMKIId overexpression in hypertrophy and heart failure: cellular consequences for excitation-contraction coupling

    Directory of Open Access Journals (Sweden)

    Maier L.S.

    2005-01-01

    Full Text Available Ca/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta is the predominant isoform in the heart. During excitation-contraction coupling (ECC CaMKII phosphorylates several Ca-handling proteins including ryanodine receptors (RyR, phospholamban, and L-type Ca channels. CaMKII expression and activity have been shown to correlate positively with impaired ejection fraction in the myocardium of patients with heart failure and CaMKII has been proposed to be a possible compensatory mechanism to keep hearts from complete failure. However, in addition to these acute effects on ECC, CaMKII was shown to be involved in hypertrophic signaling, termed excitation-transcription coupling (ETC. Thus, animal models have shown that overexpression of nuclear isoform CaMKIIdeltaB can induce myocyte hypertrophy. Recent study from our laboratory has suggested that transgenic overexpression of the cytosolic isoform CaMKIIdeltaC in mice causes severe heart failure with altered intracellular Ca handling and protein expression leading to reduced sarcoplasmic reticulum (SR Ca content. Interestingly, the frequency of diastolic spontaneous SR Ca release events (or opening of RyR was greatly enhanced, demonstrating increased diastolic SR Ca leak. This was attributed to increased CaMKII-dependent RyR phosphorylation, resulting in increased and prolonged openings of RyR since Ca spark frequency could be reduced back to normal levels by CaMKII inhibition. This review focuses on acute and chronic effects of CaMKII in ECC and ETC. In summary, CaMKII overexpression can lead to heart failure and CaMKII-dependent RyR hyperphosphorylation seems to be a novel and important mechanism in ECC due to SR Ca leak which may be important in the pathogenesis of heart failure.

  6. Isoprenaline enhances local Ca2+ release in cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    Jian-xin SHEN

    2006-01-01

    Aim: Contraction of cardiac myocytes is controlled by the generation and amplification of intracellular Ca2+ signals. The key step of this process is the coupling between sarcolemma L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). β-Adrenergic stimulation is an important regulatory mechanism for this coupling process. But the details underlied the global level, which require local Ca2+ release study are still unclear. The present study is to explore the effects of β-adrenergic stimulation on local Ca2+ release. Methods: Using confocal microscopy combined with loose-seal patch-clamp approaches, effects of isoprenaline (1 μmol·L-1), a β-adrenergic agonist, on local SR Ca2+ release triggered by Ca2+ influx through LCCs in intact rat cardiac myocytes were investigated. Results: Isoprenaline increased the intensity of ensemble averaged local Ca2+ transients, the peak of which displayed a typical bell-shaped voltage-dependence over the membrane voltages ranging from ~-40mV to ~+35mV. Further analysis showed that this enhancement could be explained by the increased coupling fidelity (which refers the increased probability of RyRs activation upon depolarization), and the increased amplitude of evoked Ca2+ sparks (due to more Ca2+ releases through local RyRs). In addition, isoprenaline decreased the first latency, which displayed a typical "U"-shaped voltage-dependence, showing the available acceleration and synchronization of β-adrenergic stimulation on intracellular calcium release. Conclusions: Isoprenaline enhances local Ca2+ release in cardiac myocytes. These results underscore the importance of regulation of β-adrenergic stimulation on local intermolecular signals between LCCs and RyRs in heart cells.

  7. 正性肌力药物作用靶点的研究进展%Progress in research of the targets for inotropic agents

    Institute of Scientific and Technical Information of China (English)

    徐毅; 罗卓卡; 黄霏霏; 李雪华; 陈龙

    2012-01-01

    Inotropic drugs affect cardiac muscle contraction and serve as a major clinical treatment of heart failure. The efficacy and terminal survival rate after inotropic drug therapy depend on the targets ( or the mechanisms of action) in cardiomyocytes. The targets mediating cardiac muscle contraction include many biomolecules, such as β-adrenergic receptor, phosphodiesterase (PDE) , L-type calcium channel, protein kinase (PKA) , PKC, protein phosphatase, Na + -Ca + exchanger ( NCX) , Na+-K+ -ATPase, sarcoplasmic reticulum calcium adenosine triphosphatase 2a (SERCA2a) , phospholamban (PLB) , ryanodine receptor (RyRs) , inositol triphosphate receptor (IP3 receptor) , contractile protein related to calcium sensitizer. This paper reviewed the characteristics of the targets to cardiac muscle contractility for new and currently marketed inotropic drugs.%正性肌力药物能影响心肌收缩力,是治疗心力衰竭的主要药物之一,用于治疗心力衰竭的药物疗效及用药患者终期生存率取决于作用靶点.与心肌收缩力有关的靶点包括:β肾上腺素受体、磷酸二酯酶、L-型钙通道、蛋白激酶A、蛋白激酶C、蛋白磷酸酶、Na+ -Ca2交换体、Na+ -K+ -ATP酶、肌浆网钙泵、受磷蛋白、兰尼碱受体、三磷酸肌醇受体、与钙增敏剂相关的心肌收缩蛋白等.文中对心肌收缩力相关的作用靶点进行综述,分析临床现今使用的和未来的正性肌力药物应具备的靶点特征.

  8. β-Adrenergic modulation of skeletal muscle contraction: key role of excitation-contraction coupling.

    Science.gov (United States)

    Cairns, Simeon P; Borrani, Fabio

    2015-11-01

    Our aim is to describe the acute effects of catecholamines/β-adrenergic agonists on contraction of non-fatigued skeletal muscle in animals and humans, and explain the mechanisms involved. Adrenaline/β-agonists (0.1-30 μm) generally augment peak force across animal species (positive inotropic effect) and abbreviate relaxation of slow-twitch muscles (positive lusitropic effect). A peak force reduction also occurs in slow-twitch muscles in some conditions. β2 -Adrenoceptor stimulation activates distinct cyclic AMP-dependent protein kinases to phosphorylate multiple target proteins. β-Agonists modulate sarcolemmal processes (increased resting membrane potential and action potential amplitude) via enhanced Na(+) -K(+) pump and Na(+) -K(+) -2Cl(-) cotransporter function, but this does not increase force. Myofibrillar Ca(2+) sensitivity and maximum Ca(2+) -activated force are unchanged. All force potentiation involves amplified myoplasmic Ca(2+) transients consequent to increased Ca(2+) release from sarcoplasmic reticulum (SR). This unequivocally requires phosphorylation of SR Ca(2+) release channels/ryanodine receptors (RyR1) which sensitize the Ca(2+) -induced Ca(2+) release mechanism. Enhanced trans-sarcolemmal Ca(2+) influx through phosphorylated voltage-activated Ca(2+) channels contributes to force potentiation in diaphragm and amphibian muscle, but not mammalian limb muscle. Phosphorylation of phospholamban increases SR Ca(2+) pump activity in slow-twitch fibres but does not augment force; this process accelerates relaxation and may depress force. Greater Ca(2+) loading of SR may assist force potentiation in fast-twitch muscle. Some human studies show no significant force potentiation which appears to be related to the β-agonist concentration used. Indeed high-dose β-agonists (∼0.1 μm) enhance SR Ca(2+) -release rates, maximum voluntary contraction strength and peak Wingate power in trained humans. The combined findings can explain how adrenaline

  9. FKBP12 deficiency reduces strength deficits after eccentric contraction-induced muscle injury

    Science.gov (United States)

    Corona, Benjamin T.; Rouviere, Clement; Hamilton, Susan L.; Ingalls, Christopher P.

    2008-01-01

    Strength deficits associated with eccentric contraction-induced muscle injury stem, in part, from excitation-contraction uncoupling. FKBP12 is a 12-kDa binding protein known to bind to the skeletal muscle sarcoplasmic reticulum Ca2+ release channel [ryanodine receptor (RyR1)] and plays an important role in excitation-contraction coupling. To assess the effects of FKBP12 deficiency on muscle injury and recovery, we measured anterior crural muscle (tibialis anterior and extensor digitorum longus muscles) strength in skeletal muscle-specific FKBP12-deficient and wild-type (WT) mice before and after a single bout of 150 eccentric contractions, as well as before and after the performance of six injury bouts. Histological damage of the tibialis anterior muscle was assessed after injury. Body weight and peak isometric and eccentric torques were lower in FKBP12-deficient mice compared with WT mice. There were no differences between FKBP12-deficient and WT mice in preinjury peak isometric and eccentric torques when normalized to body weight, and no differences in the relative decreases in eccentric torque with a single or multiple injury bouts. After a single injury bout, FKBP12-deficient mice had less initial strength deficits and recovered faster (especially females) than WT mice, despite no differences in the degree of histological damage. After multiple injury bouts, FKBP12-deficient mice recovered muscle strength faster than WT mice and exhibited significantly less histological muscle damage than WT mice. In summary, FKBP12 deficiency results in less initial strength deficits and enhanced recovery from single (especially females) and repeated bouts of injury than WT mice. PMID:18511525

  10. Cardio-protection by Ginkgo biloba extract 50 in rats with acute myocardial infarction is related to Na⁺-Ca²⁺ exchanger.

    Science.gov (United States)

    Liu, Ai-Hua; Bao, Yi-Min; Wang, Xing-Yu; Zhang, Zhi-Xiong

    2013-01-01

    Ginkgo biloba has been used for medical purposes for centuries in traditional Chinese medicine. Ginkgo biloba extract 50 (GBE50) is a new standardized GBE product that matches the standardized German product as EGb761. This paper is aimed at studying the cardio-protection effects of GBE50 Salvia miltiorrhiza on myocardial function, area at risk, myocardial ultra-structure, and expression of calcium handling proteins in rat ischemic myocardium. Myocardium ischemia was induced by the left anterior descending (LAD) coronary artery occlusion and myocardial function was recorded by a transducer advanced into the left ventricle on a computer system. In vitro myocardial infarction was measured by 2,3,5-triphenyltetrazolium chloride (TTC) and Evans blue staining of heart sections. Morphological change was evaluated by electric microscopy and Western blotting was used for protein expression. Hemodynamic experiments in vivo showed that postischemic cardiac contractile function was reduced in ischemic rats. Salvia miltiorrhiza (7.5 g/kg/d×7) and Ginkgo biloba extract 50 (GBE50) (100 mg/kg/d×7) improved post-schemic cardiac diastolic dysfunction while not affecting the systolic function. In hearts of GBE50 group and Salvia miltiorrhiza (SM) group, the area at risk was significantly reduced and myocardial structure was better-preserved. Moreover, Na⁺-Ca²⁺ exchanger (NCX) expression increase and sarcoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2), LTCC, and ryanodine receptor 2 (RyR2) expression decreases were smaller than those in ischemia group. There was a significant difference between the GBE50 and ischemia group in NCX expression. GBE50 could improve recovery in contractile function and prevent myocardium from ischemia damage, which may be caused by attenuating the abnormal expression of NCX.

  11. AhV_aPA-induced vasoconstriction involves the IP₃Rs-mediated Ca²⁺ releasing.

    Science.gov (United States)

    Zeng, Fuxing; Zou, Zhisong; Niu, Liwen; Li, Xu; Teng, Maikun

    2013-08-01

    AhV_aPA, the acidic PLA₂ purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K⁺-induced contraction with an EC₅₀ in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca²⁺ releasing from Ca²⁺ store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca²⁺ release from SR is related to the activation of inositol trisphosphate receptors (IP₃Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with heparin, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA₂ and provide a novel pathogenesis of A. halys pallas venom. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Colchicine modulates calcium homeostasis and electrical property of HL-1 cells.

    Science.gov (United States)

    Lu, Yen-Yu; Chen, Yao-Chang; Kao, Yu-Hsun; Lin, Yung-Kuo; Yeh, Yung-Hsin; Chen, Shih-Ann; Chen, Yi-Jen

    2016-06-01

    Colchicine is a microtubule disruptor that reduces the occurrence of atrial fibrillation (AF) after an operation or ablation. However, knowledge of the effects of colchicine on atrial myocytes is limited. The aim of this study was to determine if colchicine can regulate calcium (Ca(2+) ) homeostasis and attenuate the electrical effects of the extracellular matrix on atrial myocytes. Whole-cell clamp, confocal microscopy with fluorescence, and western blotting were used to evaluate the action potential and ionic currents of HL-1 cells treated with and without (control) colchicine (3 nM) for 24 hrs. Compared with control cells, colchicine-treated HL-1 cells had a longer action potential duration with smaller intracellular Ca(2+) transients and sarcoplasmic reticulum (SR) Ca(2+) content by 10% and 47%, respectively. Colchicine-treated HL-1 cells showed a smaller L-type Ca(2+) current, reverse mode sodium-calcium exchanger (NCX) current and transient outward potassium current than control cells, but had a similar ultra-rapid activating outward potassium current and apamin-sensitive small-conductance Ca(2+) -activated potassium current compared with control cells. Colchicine-treated HL-1 cells expressed less SERCA2a, total, Thr17-phosphorylated phospholamban, Cav1.2, CaMKII, NCX, Kv1.4 and Kv1.5, but they expressed similar levels of the ryanodine receptor, Ser16-phosphorylated phospholamban and Kv4.2. Colchicine attenuated the shortening of the collagen-induced action potential duration in HL-1 cells. These findings suggest that colchicine modulates the atrial electrical activity and Ca(2+) regulation and attenuates the electrical effects of collagen, which may contribute to its anti-AF activity.

  13. Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle.

    Science.gov (United States)

    De La Fuente, Sergio; Fernandez-Sanz, Celia; Vail, Caitlin; Agra, Elorm J; Holmstrom, Kira; Sun, Junhui; Mishra, Jyotsna; Williams, Dewight; Finkel, Toren; Murphy, Elizabeth; Joseph, Suresh K; Sheu, Shey-Shing; Csordás, György

    2016-10-28

    Control of myocardial energetics by Ca(2+) signal propagation to the mitochondrial matrix includes local Ca(2+) delivery from sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane (IMM) Ca(2+) uniporter (mtCU). mtCU activity in cardiac mitochondria is relatively low, whereas the IMM surface is large, due to extensive cristae folding. Hence, stochastically distributed mtCU may not suffice to support local Ca(2+) transfer. We hypothesized that mtCU concentrated at mitochondria-SR associations would promote the effective Ca(2+) transfer. mtCU distribution was determined by tracking MCU and EMRE, the proteins essential for channel formation. Both proteins were enriched in the IMM-outer mitochondrial membrane (OMM) contact point submitochondrial fraction and, as super-resolution microscopy revealed, located more to the mitochondrial periphery (inner boundary membrane) than inside the cristae, indicating high accessibility to cytosol-derived Ca(2+) inputs. Furthermore, MCU immunofluorescence distribution was biased toward the mitochondria-SR interface (RyR2), and this bias was promoted by Ca(2+) signaling activity in intact cardiomyocytes. The SR fraction of heart homogenate contains mitochondria with extensive SR associations, and these mitochondria are highly enriched in EMRE. Size exclusion chromatography suggested for EMRE- and MCU-containing complexes a wide size range and also revealed MCU-containing complexes devoid of EMRE (thus disabled) in the mitochondrial but not the SR fraction. Functional measurements suggested more effective mtCU-mediated Ca(2+) uptake activity by the mitochondria of the SR than of the mitochondrial fraction. Thus, mtCU "hot spots" can be formed at the cardiac muscle mitochondria-SR associations via localization and assembly bias, serving local Ca(2+) signaling and the excitation-energetics coupling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Drug: D02347 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available odine and IP3 receptors Ryanodine receptor (RYR) ryanodine receptor (RYR1) [HSA:6261] [KO:K...skeletal muscle] Same as: C06939 ATC code: M03CA01 ryanodine receptor antagonist [HSA:6261 6262 6263] [KO:K0...e D02347 Dantrolene (USAN/INN) Target-based classification of drugs [BR:br08310] Ion channels Ryan...04961] Dantrolene [ATC:M03CA01] D02347 Dantrolene (USAN/INN) ryanodine receptor (RYR2) [HSA:6262] [KO:K04962...] Dantrolene [ATC:M03CA01] D02347 Dantrolene (USAN/INN) ryanodine receptor (RYR3)

  15. Effects of Ultrasonic Treatment on the Physico-chemical Properties and Structure of Chicken Sarcoplasmic Proteins%超声波对鸡肉肌浆蛋白理化性质和结构的影响

    Institute of Scientific and Technical Information of China (English)

    涂宗财; 马达; 王辉; 张露; 沙小梅; 常海霞; 梁百惠; 周华璐

    2013-01-01

    以鸡肉肌浆蛋白为原料,采用超声波对其进行处理,利用紫外、荧光谱仪、SDS-PAGE等分析肌浆蛋白理化性质和结构的变化.结果表明:超声功率和超声时间对肌浆蛋白的乳化性、起泡性、表面疏水性、内源荧光等均有较大的影响.随超声功率和超声时间的增加,肌浆蛋白的乳化性降低,乳化稳定性和起泡稳定性均呈先上升后下降,起泡性随超声功率的增加先下降后上升,而超声时间对其影响相反.超声波处理可使肌浆蛋白的表面疏水性和内源荧光强度增加,但其紫外光谱和分子质量无明显影响,说明超声波处理不会导致其肽键断裂.

  16. Modificaciones postraduccionales de los canales liberadores de Ca2+ (RyR2) del retículo sarcoplasmático (RS) en la evolución hacia la insuficiencia cardíaca (IC)

    OpenAIRE

    Becerra, R.; Mundiña-Weilenmann, Cecilia; Rinaldi, Gustavo; Mattiazzi, Alicia; Said, Matilde; Vittone, Leticia

    2012-01-01

    Observaciones experimentales sugieren que modificaciones postraduccionales, como alteraciones del estado redox o fosforilación de los RyR2, podrían contribuir a un manejo alterado del Ca2+ del RS en la IC. La actividad de los RyR2 puede regularse por las especies reactivas del oxígeno y del nitrógeno (ROS/RNS), tanto reversible como irreversiblemente, alterando su función.

  17. 收缩潜伏期骨骼肌肌浆网的超微结构变化%Ultrastructural Change of Sarcoplasmic Reticulum in Skeletal Muscle during Contraction-Latency

    Institute of Scientific and Technical Information of China (English)

    邰艳红; 杨勇骥; 宋田斌; 汤莹; 吴越

    2000-01-01

    采用双向红外线探测--计算机控制的电刺激与超低温快速冷冻固定同步技术对收缩潜伏期的骨骼肌作实时固定,采用透射电镜对电刺激后0.8 ms,5.6 ms,8.4 ms及静息期的骨骼肌超微结构变化进行了对比研究,发现在骨骼肌收缩潜伏期(小于10 ms)内,肌浆网内的Ca2+从面向T-管的肌浆网前端流出.同时发现肌膜与肌纤维间的间隙大大变窄,肌浆网前端的膜产生两个圆孔,而静息时的骨骼肌却无上述改变.这些发现对揭示骨骼肌兴奋-收缩偶联的发生机制有重大意义.

  18. 鱼尼丁受体类新型杀虫剂氯虫酰胺的研究概述%Summary of chlorantraniliprole-a new type of ryanodines receptor insecticide

    Institute of Scientific and Technical Information of China (English)

    刘熠; 王国胜

    2009-01-01

    作用于鱼尼丁受体的氯虫酰胺是一种高效、低毒、作用机制独特的新型杀虫剂.其通过诱导鱼尼丁受体调解细胞内ca2+释放,从而表现出杀虫作用.本文介绍了鱼尼丁受体类杀虫剂的研究进展以及对氯虫酰胺的基本性能,作用机理,合成方法和开发进展等方面的研究情况作了概述.

  19. STUDY ON EXPRESSION OF CALMODULIN GENE IN THE PATIENTS WITH SECONDARY RIGHT VENTRICULAR HYPERTROPHY%继发性右室肥厚患者钙调蛋白基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    钟明; 张运; 张薇; 卞继峰; 卜培莉; 耿昭; 钟敬泉; 赵静

    2001-01-01

    探讨继发性右室肥厚(RVH)患者心肌细胞内Ca2+超负荷及钙调蛋白mRNA和蛋白 质表达的变化。方法:采用三电极直流等离子体原子发射直读光电光谱法测定心肌细胞内Ca2+含量;采用RT-PCR和Western bbt法测定RVH患者和正常对照钙调蛋白mRNA和蛋白质表达的变化。结果:与正常对照组相比,RVH患者心肌细胞内Ca2+含量提高3倍以上〔(1025.67±414.71)ug/ml vs(270.86±109.24)μg/ml,P0.05);Ca2+ -ATPase的蛋白质表达明显低于对照组(0.78±0.09 vs 1.03±0.03,P0.05)。结论:RVH患者SR Ca2+ -ATPase mRNA和蛋白质表达减低及细胞膜L型Ca2+ 通道mRNA转录减低是导致心肌细胞内Ca2+ 超负荷和RVH发生的主要分子生物学机制。%To elucidate molecular mechanisms of the changes of calmodulin gene expression when Ca2+ is overload in cardic muscle cell of patients with secondary right ventricular hypertrophy(RVH). Methods: Ten patients with secondary RVH and 6 control subjects were randomly selected. The content of calci um was assayed by spectrocomparator. The mRNA amounts of these calmodulin genes including L-type calcium channel, sarcoplasmic reticular(SR) Ca2+ -ATPase, ryanodine receptor, calsequestrin and phospholamban were de tected by reverse transcription-polymerase chain reaction(RT-PCR) and normalized to the mRNA levels of actin. The protein levels of SR Ca2+ -ATPase and phospholamban were analyzed by Western blot analysis. Results:The content of calcium increased significantly in patients with RVH. RT-PCR analyses showed that the steady-state level of mRNA encoding the SR Ca2 +-ATPase and cardiac L-type calcium channel decreased significantly in pa tients with RVH(0.66±0.28 vs 1.09±0.11, P<0.05,0.72±0.21 vs 1.08±0.14, P<0.05 ). In contrast, no alterations at the mRNA level for SR phospholamban, ryanodine receptor,calsequestrin were observed in pa tients with RVH compared with those in control subjects. Protein level of SR Ca2+ -ATPase

  20. Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload.

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    Haifei Zhang

    Full Text Available Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF. Although abnormal sarcoplasmic reticulum (SR function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca2+-handling. We investigated the effects of ascending aortic banding (AoB on Ca2+-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham. Myocytes were either labelled for ryanodine receptor (RyR or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001. RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca2+-release at the cell edge whereas Ca2+ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na+/Ca2+ exchanger, SR Ca2+ ATPase or phospholamban. Mathematical modeling indicated that [Ca2+]i transients at the cell center were accounted for by simple centripetal diffusion of Ca2+ released at the cell edge. In contrast, caffeine (10 mM induced Ca2+ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca2+-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca2+ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca2+-transients following rapid-pacing (4 Hz was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy