Sample records for cardiac myosins

  1. Preparation of human cardiac anti-myosin: a review

    The present communication is a review of the physicochemical characterization and immunological properties of myosin isolated from the cardiac muscle, the production of monoclonal antibody anti-myosin, the radiolabeling of this antibody and its applications as radiopharmaceuticals to imaging myocardial infarcts. The classical example of radioimmunologic diagnosis of non malignant tissues is the detection of myocardial infarction by radiolabeled antibodies to myosin. (author)

  2. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)


    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  3. Cardiac myosin heavy chain transition under altered thyroid status

    Arnoštová, Petra; Jedelský, P.; Soukup, Tomáš; Žurmanová, Jitka

    Geneva: Swiss Society for Neuroscience, 2008. s. 125.3-125.3. ISBN 92-990014-3-X. [FENS. Forum of European Neuroscience /6./. 12.07.2008-16.07.2008, Geneva] Grant ostatní: Myores(XE) 511978 Institutional research plan: CEZ:AV0Z50110509 Source of funding: R - rámcový projekt EK Keywords : spo2 * cardiac myosin heavy chain * transition * thyroid status Subject RIV: ED - Physiology

  4. Canine cardiac myosin with special referrence to pressure overload cardiac hypertrophy. I. Subunit composition.

    Siemankowski, R F; Dreizen, P


    In studies of myosin from left and right ventricles of normal hearts and hypertrophic hearts at 5 weeks and 13 weeks after aortic banding, polyacrylamide gel electrophoresis shows intermediate molecular weight components which derive from heavy chains fragmented in the presence of dodecyl sulfate. The proportion of degraded heavy chains is greater in myosin from hypertrophic hearts than normal hearts, with comparable degradation in left and right ventricle myosin. The observed fragmentation of myosin results from proteolysis due to contaminant proteases or a thermally activated, heat-stable nonenzymatic process, or both. The susceptibility of heavy chains to crude myofibrillar proteases differs in normal and hypertrophic cardiac myosin; however, the kinetics of tryptic digestion are identical for both myosins. With precautions to minimize proteolytic artifacts on dodecyl sulfate-polyacrylamide gel electrophoresis, preparations of myosin from left and right ventricles of normal and hypertrophic hearts exhibit comparable subunit composition, with approximately molar ratios of heavy chains, light chain L1, and light chain L2. Comparable stoichiometry for the light chain fraction is determined by high speed sedimentation equilibrium at pH 11 and direct fractionation of the different cardiac myosins. We do not confirm reports (e.g. Wikman-Coffelt, J., Fenner, C., Smith, A., and Mason, D. T. (1975) J. Biol. Chem. 250, 1257-1262) of different proportions of light chains in left and right ventricle myosin of normal and hypertrophic canine hearts. The light chains display microheterogeneity, with L1 generating two isoelectric variants and L2 generating two major and two minor variants, but identical mobilities and isoelectric values are obtained in the different myosin preparations. PMID:152317

  5. Doxorubicin-induced carbonylation and degradation of cardiac myosin binding protein C promote cardiotoxicity

    Aryal, Baikuntha; Jeong, Jinsook; Rao, V. Ashutosh


    Doxorubicin is one of the most successful anticancer agents. However, 10–30% of all treated patients experience a dose-limiting cardiac adverse event. Oxidative stress is partly responsible for the cardiotoxicity because the heart does not possess required antioxidant mechanisms. Protein oxidation by carbonylation is irreversible and marks proteins for loss of function and degradation. Using proteomics and MS, we identified and investigated cardiac myosin binding protein (MyBPC) as being sele...

  6. Double heterozygosity for mutations in the β-myosin heavy chain and in the cardiac myosin binding protein C genes in a family with hypertrophic cardiomyopathy

    Richard, P.; Isnard, R.; Carrier, L.; Dubourg, O.; Donatien, Y.; Mathieu, B.; Bonne, G.; Gary, F; Charron, P; HAGEGE, A.; Komajda, M; Schwartz, K.; Hainque, B


    Familial hypertrophic cardiomyopathy is a genetically heterogeneous autosomal dominant disease, caused by mutations in several sarcomeric protein genes. So far, seven genes have been shown to be associated with the disease with the β-myosin heavy chain (MYH7) and the cardiac myosin binding protein C (MYBPC3) genes being the most frequently involved.
We performed electrocardiography (ECG) and echocardiography in 15 subjects with hypertrophic cardiomyopathy from a French Caribbean family. Genet...

  7. Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

    Winkelmann, Donald A.; Forgacs, Eva; Miller, Matthew T.; Stock, Ann M.


    Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

  8. Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation.

    Homburger, Julian R; Green, Eric M; Caleshu, Colleen; Sunitha, Margaret S; Taylor, Rebecca E; Ruppel, Kathleen M; Metpally, Raghu Prasad Rao; Colan, Steven D; Michels, Michelle; Day, Sharlene M; Olivotto, Iacopo; Bustamante, Carlos D; Dewey, Frederick E; Ho, Carolyn Y; Spudich, James A; Ashley, Euan A


    Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease. PMID:27247418

  9. Preparation of monoclonal antibodies against cardiac myosin and some radiolabelling studies

    Monoclonal antibodies were raised against myosin, a specific indicator of myocardial infarction and labelled with 125I and 99mTc. Human cardiac myosin was isolated from normal human heart and was used for raising the monoclonal antibodies by the hybridoma technique. Antibody producing clones were identified by ELISA and cloning was done by the limiting dilution technique. Of the 13 clones obtained, 4 were deemed suitable for further studies. The antibodies were grown in ascites, purified, isotyped and their cross reactions with other forms of myosin were estimated. All the clones showed negligible cross reaction with rabbit myosin, but reacted to different extents with bovine skeletal myosin. The most avid antibody Mab-4G4 was chosen for further labelling studies. Mab-4G4 was labelled with 125I using different oxidising agents such as iodogen, chloramine-T and lactoperoxidase. Purified radioiodinated antibody with radiochemical purity >95% could be obtained by gel filtration. Immunoreactivity was retained as tested by binding to myosin immobilised on a solid support. Mab-4G4 was also labelled with 99mTc using stannous tartrate as the reducing agent. Radiolabelling yield was ∼60%, the purity was >95% and the immunoreactivity was retained. Both the labelled preparations were tested for bio-distribution in normal and infarcted rats. The activity accumulation in the infarcted region was ∼ 1.5 and 3.5 times as that in normal heart muscle for 125I and 99mTc labelled Mab-4G4 respectively. The major problem with the iodinated antibody was the in vivo deiodination resulting in very high percentage of activity in the thyroid. Although the fraction of the total activity associated with the infarcted heart is not very impressive, the fact that the activities with the infarcted and normal hearths are significantly different is heartening. With further optimisation of labelling and use of F(ab)'2 fragments, better delineation of the infarct sites is aspired. (author)

  10. Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism

    Mun, Ji Young; Previs, Michael J.; Yu, Hope Y.; Gulick, James; Tobacman, Larry S.; Beck Previs, Samantha; Robbins, Jeffrey; Warshaw, David M.; Craig, Roger


    Myosin-binding protein C (MyBP-C) is a component of myosin filaments, one of the two sets of contractile elements whose relative sliding is the basis of muscle contraction. In the heart, MyBP-C modulates contractility in response to cardiac stimulation; mutations in MyBP-C lead to cardiac disease. The mechanism by which MyBP-C modulates cardiac contraction is not understood. Using electron microscopy and a light microscopic assay for filament sliding, we demonstrate that MyBP-C binds to the o...

  11. Missense mutation of the {beta}-cardiac myosin heavy-chain gene in hypertrophic cardiomyopathy

    Arai, Shoichi; Matsuoka, Rumiko; Hirayama, Kenji; Sakurai, Hisanao [Heart Inst. of Japan, Tokyo (Japan)] [and others


    Hypertrophic cardiomyopathy occurs as an autosomal dominant familial disorder or as a sporadic disease without familial involvement. We describe a missense mutation of the {beta}-cardiac myosin heavy chain (MHC) gene, a G to T transversion (741 Gly{r_arrow}Trp) identified by direct sequencing of exon 20 in four individuals affected with familial hypertrophic cardiomyopathy. Three individuals with sporadic hypertrophic cardiomyopathy, whose parents are clinically and genetically unaffected, had sequence variations of exon 34 of the {alpha}-cardiac MHC gene (a C to T transversion, 1658 Asp{r_arrow}Asp, resulting in FokI site polymorphism), of intron 33 of the {alpha}-cardiac MHC gene (a G to A and an A to T transversion), and also of intron 14 of the {beta}-cardiac MHC gene (a C to T transversion in a patient with Noonan syndrome). Including our case, 30 missense mutations of the {beta}-cardiac MHC gene in 49 families have been reported thus far worldwide. Almost all are located in the region of the gene coding for the globular head of the molecule, and only one mutation was found in both Caucasian and Japanese families. Missense mutations of the {Beta}-cardiac MHC gene in hypertrophic cardiomyopathy may therefore differ according to race. 29 refs., 6 figs., 3 tabs.

  12. Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules

    Sung, Jongmin; Nag, Suman; Mortensen, Kim I.; Vestergaard, Christian L.; Sutton, Shirley; Ruppel, Kathleen; Flyvbjerg, Henrik; Spudich, James A.


    Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using `harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.

  13. Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities.

    Chang, Audrey N; Mahajan, Pravin; Knapp, Stefan; Barton, Hannah; Sweeney, H Lee; Kamm, Kristine E; Stull, James T


    The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties. PMID:27325775

  14. Harmonic Force Spectroscopy Reveals a Force-Velocity Curve from a Single Human Beta Cardiac Myosin Motor

    Sung, Jongmin; Nag, Suman; Vestergaard, Christian L.; Mortensen, Kim; Flyvbjerg, Henrik; Spudich, James


    in thin filaments in the sarcomere, cycling between a strongly bound state (force producing state) and a weakly bound state (relaxed state). Huxley and Simmons have previously proposed that the transition from the strong to the weak interaction can be modulated by an external load, i.e., the...... transition is slow under high load and fast under low load. We use a new, simple method we call "harmonic force spectroscopy" to extract a load-velocity relationship from a single human beta cardiac myosin II motor (S1). With a dual-beam optical trap, we hold an actin dumbbell over a single myosin molecule...... from a single human beta cardiac myosin S1. We also compare load-velocity curves for wild-type motors with load-velocity curves of mutant forms that cause hypertrophic or dilated-cardiomyopathy (HCM or DCM), in order to understand the effects of mutations on the contractile cycle at the single molecule...

  15. The Hypertrophic Cardiomyopathy Myosin Mutation R453C Alters ATP Binding and Hydrolysis of Human Cardiac β-Myosin*

    Bloemink, Marieke; Deacon, John; Langer, Stephen; Vera, Carlos; Combs, Ariana; Leinwand, Leslie; Geeves, Michael A.


    The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long α-helix, helix O, and to Switch-2 via the fifth strand of the central β-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecula...

  16. Novel control of cardiac myofilament response to calcium by S-glutahionylation at specific sites of myosin binding protein C



    Full Text Available Our previous studies demonstrated a relation between glutathionylation of cardiac myosin binding protein C and diastolic dysfunction in a hypertensive mouse model stressed by treatment with salt, deoxycorticosterone acetate, and unilateral nephrectomy. Although these results strongly indicated an important role for S-glutathionylation of myosin binding protein C as a modifier of myofilament function, indirect effects of other post-translational modifications may have occurred. Moreover, we did not determine the sites of thiol modification by glutathionylation. To address these issues, we developed an in vitro method to mimic the in situ S-glutathionylation of myofilament proteins and determined direct functional effects and sites of oxidative modification employing Western blotting and mass spectrometry. We induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers with oxidized glutathione (GSSG. Immuno-blotting results revealed increased glutathionylation with GSSG treatment of a protein band around 140 kDa. Using tandem mass spectrometry, we identified the 140 kDa band as cardiac myosin binding protein C and determined the sites of glutathionylation to be at cysteines 655, 479, and 627. Determination of the relation between Ca2+-activation of myofibrillar acto-myosin ATPase rate demonstrated an increased Ca2+-sensitivity induced by the S-glutathionylation. Force generating skinned fiber bundles also showed an increase in Ca-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol. Our data demonstrate that a specific and direct effect of S-glutathionylation of myosin binding protein C is a significant increase in myofilament Ca2+-sensitivity. Our data also provide new insights into the functional significance of oxidative modification of myosin binding protein C and the potential role of domains not previously considered to

  17. Effect of nuclear factor antisense oligonucleotide on cardiac muscle myosin isoenzymes and cytokines in rat models of chronic heart failure

    Objective: To investigate the effect of nuclear factor kappa B (NF-κB) antisense oligonucleotide (AS-ON) on cardiac muscle myosin isoenzymes (MI) and serum cytokines (TNF-α, IL-1β, Fas) expressions in rat models of chronic heart failure. Methods: Wistar rat models of chronic heart failure were prepared with abdominal aorta constriction. Half of the models were treated with intrapericardial injection of 0.5ml AS-ON at the time of model preparation. Control rats were given intrapericardial injection of normal saline. Non-invasive echocardiographic study or invasive hemodynamic studies with sacrifice of the animal and procurement of left ventricular cardiac muscle for examination of myosin isoenzymes with SDS-PAGE were performed on 10 models each eveny two weeks until six months after establishment of the models. Inner canthus blood aspiration for determination of serum cytokines (TNF -α and IL-1β with RIA and Fas with ELISA) were done at the same time. Results: In the models without AS-ON treatment, cardiac function was deterioated somewhat at 3 months and frank cardiac failure was apparent at 6 months. In the AS-OD treated models, carbiac function parameters were much better, with lower TNF-α, IL-1β and Fas levels as well as less V1→V3 shift in myosin isoenzymes. Conclusion: Intrapericardial injection of AS-ON was of great benefit in prevention of development of cardiac failure in the rat models with abdominal aorta constriction, probably throngh maintainence of normal cytokines network as well as inbibition of V1 →V3 shift of myosin isoenzymes. (authors)

  18. Molecular pathology of familial hypertrophic cardiomyopathy caused by mutations in the cardiac myosin binding protein C gene.

    Yu, B.; French, J. A.; Carrier, L.; Jeremy, R W; McTaggart, D R; Nicholson, M R; Hambly, B; Semsarian, C; Richmond, D R; Schwartz, K.; Trent, R.J.


    DNA studies in familial hypertrophic cardiomyopathy (FHC) have shown that it is caused by mutations in genes coding for proteins which make up the muscle sarcomere. The majority of mutations in the FHC genes result from missense changes, although one of the most recent genes to be identified (cardiac myosin binding protein C gene, MYBPC3) has predominantly DNA mutations which produce truncated proteins. Both dominant negative and haploinsufficiency models have been proposed to explain the mol...

  19. Synthesised genes of VH and VL of single-chain antibody of human cardiac myosin heavy chain

    Objective: To synthesize genes of the heavy chain variable region (VH) and light chain variable region (VL) of single-chain antibody of human cardiac myosin heavy chain for the development of myocardial imaging agents: single-chain antibody of human cardiac myosin heavy chain. Methods: To extracted total RNA of the anti-HCMHC McAb hybridoma using TRIZOL reagent, synthesize the first-strand cDNA, using this first-strand cDNA as template, with specific primers, DNA polymerase and four single nucleotides, amplify the genes of the heavy chain variable region (VH) and light chain variable region (VL) by PCR. To identify products of each step and study relationship between RNA stability and storage temperature and optimize cycle selection temperature with MgCl2 concentration. Results: The purity of the first-strand cDNA reached 95%, PCR products by agarose gel electrophoresis showed a single band with a bright swimming, molecular weight of VH and VL genes were 340bp and 320bp, which was consistent with literature reports. Conclusion: Synthesized genes of VH and VL, laid the foundation for the development of myocardial imaging agents: single-chain antibody of human cardiac myosin heavy chain. (authors)

  20. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

    Muthu, Priya; Wang, Li; Yuan, Chen-Ching; Kazmierczak, Katarzyna; Huang, Wenrui; Hernandez, Olga M.; Kawai, Masataka; Irving, Thomas C.; Szczesna-Cordary, Danuta (IIT); (Iowa); (Miami-MED)


    The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-{Delta}43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing ({approx} 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I{sub 1,1}/I{sub 1,0}, indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-{Delta}43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-{Delta}43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

  1. Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

    Petra Arnostova


    Full Text Available The expression of two cardiac myosin heavy chain (MyHC isoforms in response to the thyroid status was studied in left ventricles (LVs of Lewis rats. Major MyHC isoform in euthyroid and hyperthyroid LVs had a higher mobility on SDS-PAGE, whereas hypothyroid LVs predominantly contained a MyHC isoform with a lower mobility corresponding to that of the control soleus muscle. By comparing the MyHC profiles obtained under altered thyroid states together with the control soleus, we concluded that MyHCα was represented by the lower band with higher mobility and MyHCβ by the upper band. The identity of these two bands in SDS-PAGE gels was confirmed by western blot and mass spectrometry. Thus, in contrast to the literature data, we found that the MyHCα possessed a higher mobility rate than the MyHCβ isoform. Our data highlighted the importance of the careful identification of the MyHCα and MyHCβ isoforms analyzed by the SDS-PAGE.

  2. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.

    Stathopoulou, Konstantina; Wittig, Ilka; Heidler, Juliana; Piasecki, Angelika; Richter, Florian; Diering, Simon; van der Velden, Jolanda; Buck, Friedrich; Donzelli, Sonia; Schröder, Ewald; Wijnker, Paul J M; Voigt, Niels; Dobrev, Dobromir; Sadayappan, Sakthivel; Eschenhagen, Thomas; Carrier, Lucie; Eaton, Philip; Cuello, Friederike


    Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure. PMID:26839380

  3. The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

    Kazmierczak, Katarzyna; Xu, Yuanyuan; Jones, Michelle; Guzman, Georgianna; Hernandez, Olga M.; Kerrick, W. Glenn L.; Szczesna-Cordary, Danuta


    To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43 amino acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle and the ELC pro...

  4. Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myfibrils from rainbow trout and freshwater turtle

    Haagensen, L.; Jensen, D.H.; Gesser, Hans


    The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the...... pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with phosphocreatine. The steady-state concentration of ADP in the solution was varied through the activity of pyruvate kinase added to the solution. For rainbow trout...... myofibrils at a high pyruvate kinase activity, creatine kinase competed for ADP but did not influence the total ATPase activity. When the ADP concentration was elevated within the physiological range by lowering the pyruvate kinase activity, creatine kinase competed efficiently and increased the ATPase...

  5. Cardiac myosin binding protein C and MAP-kinase activating death domain-containing gene polymorphisms and diastolic heart failure.

    Cho-Kai Wu

    Full Text Available OBJECTIVE: Myosin binding protein C (MYBPC3 plays a role in ventricular relaxation. The aim of the study was to investigate the association between cardiac myosin binding protein C (MYBPC3 gene polymorphisms and diastolic heart failure (DHF in a human case-control study. METHODS: A total of 352 participants of 1752 consecutive patients from the National Taiwan University Hospital and its affiliated hospital were enrolled. 176 patients diagnosed with DHF confirmed by echocardiography were recruited. Controls were matched 1-to-1 by age, sex, hypertension, diabetes, renal function and medication use. We genotyped 12 single nucleotide polymorphisms (SNPs according to HapMap Han Chinese Beijing databank across a 40 kb genetic region containing the MYBPC3 gene and the neighboring DNA sequences to capture 100% of haplotype variance in all SNPs with minor allele frequencies ≥ 5%. We also analyzed associations of these tagging SNPs and haplotypes with DHF and linkage disequilibrium (LD structure of the MYBPC3 gene. RESULTS: In a single locus analysis, SNP rs2290149 was associated with DHF (allele-specific p = 0.004; permuted p = 0.031. The SNP with a minor allele frequency of 9.4%, had an odds ratio 2.14 (95% CI 1.25-3.66; p = 0.004 for the additive model and 2.06 for the autosomal dominant model (GG+GA : AA, 95% CI 1.17-3.63; p = 0.013, corresponding to a population attributable risk fraction of 12.02%. The haplotypes in a LD block of rs2290149 (C-C-G-C was also significantly associated with DHF (odds ratio 2.10 (1.53-2.89; permuted p = 0.029. CONCLUSIONS: We identified a SNP (rs2290149 among the tagging SNP set that was significantly associated with early DHF in a Chinese population.

  6. Myomegalin is a novel A-kinase anchoring protein involved in the phosphorylation of cardiac myosin binding protein C

    Riedemann Johann


    Full Text Available Abstract Background Cardiac contractility is regulated by dynamic phosphorylation of sarcomeric proteins by kinases such as cAMP-activated protein kinase A (PKA. Efficient phosphorylation requires that PKA be anchored close to its targets by A-kinase anchoring proteins (AKAPs. Cardiac Myosin Binding Protein-C (cMyBPC and cardiac troponin I (cTNI are hypertrophic cardiomyopathy (HCM-causing sarcomeric proteins which regulate contractility in response to PKA phosphorylation. Results During a yeast 2-hybrid (Y2H library screen using a trisphosphorylation mimic of the C1-C2 region of cMyBPC, we identified isoform 4 of myomegalin (MMGL as an interactor of this N-terminal cMyBPC region. As MMGL has previously been shown to interact with phosphodiesterase 4D, we speculated that it may be a PKA-anchoring protein (AKAP. To investigate this possibility, we assessed the ability of MMGL isoform 4 to interact with PKA regulatory subunits R1A and R2A using Y2H-based direct protein-protein interaction assays. Additionally, to further elucidate the function of MMGL, we used it as bait to screen a cardiac cDNA library. Other PKA targets, viz. CARP, COMMD4, ENO1, ENO3 and cTNI were identified as putative interactors, with cTNI being the most frequent interactor. We further assessed and confirmed these interactions by fluorescent 3D-co-localization in differentiated H9C2 cells as well as by in vivo co-immunoprecipitation. We also showed that quantitatively more interaction occurs between MMGL and cTNI under β-adrenergic stress. Moreover, siRNA-mediated knockdown of MMGL leads to reduction of cMyBPC levels under conditions of adrenergic stress, indicating that MMGL-assisted phosphorylation is requisite for protection of cMyBPC against proteolytic cleavage. Conclusions This study ascribes a novel function to MMGL isoform 4: it meets all criteria for classification as an AKAP, and we show that is involved in the phosphorylation of cMyBPC as well as cTNI, hence MMGL

  7. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D


    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  8. The A31P missense mutation in cardiac myosin binding protein C alters protein structure but does not cause haploinsufficiency.

    van Dijk, Sabine J; Bezold Kooiker, Kristina; Mazzalupo, Stacy; Yang, Yuanzhang; Kostyukova, Alla S; Mustacich, Debbie J; Hoye, Elaine R; Stern, Joshua A; Kittleson, Mark D; Harris, Samantha P


    Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function. PMID:26777460

  9. Increased specificity in human cardiac-myosin radioimmunoassay utilizing two monoclonal antibodies in a double sandwich assay

    An immunoradiometric assay that simultaneously measured two different epitopes on the same molecule was devised to differential between cardiac- and skeletal-myosin light chains. Three monoclonal antibodies were examined that were 100% (lC5), 25% (2B9) and 17% (4F10) cross reactive, respectively, between the two antigens. One antibody of the pair to be studied was immobilized to cyanogen bromide-activated Sepharose 4B while the other was iodinated with 125I using the lactoperoxidase method. The antigen was mixed with the immobilized antibody, the labeled antibody was added and the precipitate then washed and counted in a gamma counter. When both antibodies of the pair to be studied (immobilized and labeled) were the same (2B9), no radioactivity above background was bound to the precipitate, indicating that the second antibody could not bind to an already occupied epitope. When two different antibodies were employed, the specificity of the assay increased over that of a single antibody. The cross reactivity of a pair approximated the product of the cross reactivities of the individual antibodies. Thus, lC5 and 2B9 were 25% cross reactive together, lC5 and 4F10 17% cross reactive, and 2B9 and 4F10 4.3% cross reactive. (author)

  10. Changes in myosin isozyme expression during cardiac hypertrophy in hyperthyroid rabbits.

    Seiden, D; Srivatsan, M; Navidad, P A


    The myosin isozyme distribution in the left ventricle and in the interventricular septum of rabbits was studied after 3, 7, 11, 14 and 21 days of L-thyroxine (500 micrograms/kg/day) administration. Histochemical procedures were employed to identify V1 and V3 by their Ca2+ ATPase activity and their proportions were quantified through polyacrylamide gel electrophoresis. In the left ventricle, the subepicardium was the first to show the shift from V3 to V1, followed by the subendocardium. The intermediate region became heterogeneous by 11 days and remained so until 21 days. The right subendocardial and the intermediate regions of the interventricular septum were heterogeneous in the normal rabbit and hyperthyroidism resulted in a shift from V3 to V1 in both the right and left subendocardial regions of the septum. Like the left ventricle, the intermediate region of the interventricular septum remained heterogeneous. Localized accumulations of collagen were seen in all regions of the left ventricle and interventricular septum. From these results we conclude that in thyrotoxic myocardial hypertrophy the isozymic shift from V3 to V1 is progressive, region-specific and is directly correlated with the period of hyperthyroidism in the first 2 weeks. Prolonged hyperthyroidism results in localized accumulation of collagen which does not exhibit any regional specificity. PMID:2528881

  11. Electrophoretic Mobility of Cardiac Myosin Heavy Chain Isoforms Revisited: Application of MALDI TOF/TOF Analysis

    Arnoštová, P.; Jedelsky, P. L.; Soukup, Tomáš; Žurmanová, J.


    Roč. 2011, - (2011), e634253. ISSN 1110-7243 R&D Projects: GA AV ČR IAAX01110901; GA ČR(CZ) GA304/08/0256 Institutional research plan: CEZ:AV0Z50110509 Keywords : cardiac MyHC isoforms * MyHC isoform mobility * effect of thyroid hormones * mass spectrometry * SDS-PAGE and western blot Subject RIV: ED - Physiology Impact factor: 2.436, year: 2011

  12. Assessment of myocardial damage in hypertrophic cardiomyopathy using cardiac enzymes, myosin light chain and myocardial scintigraphy

    To assess myocardial damage in hypertrophic cardiomyopathy (HCM), CPK-MB, %LDH 1, myoglobin (Mb), and myosin light chain (MLC) were determined in 45 HCM patients. Of these patients, 10 also underwent Tl-201 myocardial scintigraphy and In-111-antimyosin antibody (In-111 Fab-DTPA)(In-AM) myocardial scintigraphy. MLC was 0.56±0.55 ng/ml. An increase in CPK-MB, %LDH 1, and Mb was seen in 6 (14%), 19 (44%), and 7 (18%) patients, respectively. There was no correlation between MLC and any of CPK-MB, %LDH1 or Mb. Perfusion defects were seen on Tl-201 myocardial scintigrams in 4 patients. All of these patients had diffuse tracer uptake on In-AM myocardial scintigrams. The degree of In-AM uptake was not correlated with MLC; however, of 4 patients with intense In-AM uptake, 3 had perfusion defects on Tl-201 myocardial scintigrams and decreased left ventricular function. In 3 patients in whom CPK-MB and %LDH 1 were increased but MLC was not increased, diffuse tracer uptake was seen on In-AM myocardial scintigrams. Because diffuse uptake of In-AM was seen in spite of the lack of increased MLC, In-111-Fab-DTPA is likely to be incorporated by the myocardial damaged cells, as well as necrotic cells. HCM seems to be associated with a high likelihood of myocardial damage. Integrated assessment of myocardial damage is required, including an increase of MLC, CPK-MB, %LDH 1, and Mb, perfusion defects on Tl-201 scintigrams, and tracer uptake on In-AM scintigrams. (N.K.)

  13. Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

    Li Wang

    Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

  14. Isolation of cardiac myosin light-chain isotypes by chromatofocusing. Comparison of human cardiac atrial light-chain 1 and foetal ventricular light-chain 1.

    Vincent, N D; Cummins, P


    Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients using ion-exchange resins and buffers with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of delta pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2* light chains. The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207-212]. The latter three residues are known to be rich in the N-termini of cardiac and

  15. Genotype-phenotype correlation between the cardiac myosin binding protein C mutation A31P and hypertrophic cardiomyopathy in a cohort of Maine Coon cats

    Granström, S; Godiksen, M T N; Christiansen, M;


    OBJECTIVES: A missense mutation (A31P) in the cardiac myosin binding protein C gene has been associated with hypertrophic cardiomyopathy (HCM) in Maine Coon cats. The aim of this study was to investigate the effect of A31P on development of HCM, myocardial diastolic dysfunction detected by color...... tissue Doppler imaging and occurrence of cardiac death during longitudinal follow-up in a cohort of Maine Coon cats. ANIMALS: The original cohort comprised 282 cats (158 of wild-type genotype, 99 heterozygous for A31P and 25 homozygous for A31P). METHODS: Prospective longitudinal study including...... echocardiography and registration of survival. RESULTS: The median age at the initial examination was 1.7 years (range, 0.8-9.2 years) and 6.4% (18/282) of the cats were diagnosed with HCM. One hundred sixty-five cats were eligible for echocardiographic re-examination, and during an average follow-up period of 2...

  16. Indium-111 myosin-specific antibodies and technetium-99m pyrophosphate in the detection of acute cardiac rejection of transplanted hearts

    111In-labelled myosin-specific antibodies were evaluated as an indicator of early changes in acute rejection in a rat heart heterotopic transplant model. Uptake of antibodies was measured in allograft and isograft hearts of animals undergoing different regimens of cyclosporine treatment and compared with the uptake of technetium 99m pyrophosphate. The data were correlated with histological estimation of the severity of myocyte necrosis and sign of early rejection (venous cuffing and endocardial inflammation, indicators of perivascular infiltrate and intermyocyte extension, respectively). Myocyte necrosis in transplanted hearts was reflected by increases in technetium 99m pyrophosphate accumulation (r=0.88) but was poorly correlated with labelled antibody uptake (r=0.58). There was no positive correlation between the degree of early cardiac rejection and uptake of either of the radiopharmaceuticals: accumulation of the labeled antibodies paradoxically declined with increased histological severity scores, whereas that of technetium 99m pyrophosphate remained unchanged. Cyclosporine treatment augmented the uptake of labelled antibodies in transplanted hearts. This may be due to alterations in plasma membrane permeability brought about by the drug, resulting in a rise in antibody binding to intracellular myosin. (orig.)

  17. Genotype phenotype correlations of cardiac beta-myosin heavy chain mutations in Indian patients with hypertrophic and dilated cardiomyopathy

    Rai, Taranjit Singh; Ahmad, Shamim; Bahl, Ajay; Ahuja, Monica; Ahluwalia, Tarun Veer Singh; Singh, Balvinder; Talwar, K K; Khullar, Madhu

    The aim of the current study was to determine the frequency of mutations in the beta-myosin heavy chain gene (MYH7) in a cohort of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) and their families, and to investigate correlations between genotype and phenotype. About 130...... consecutive patients diagnosed with HCM or DCM (69 with HCM and 61 with DCM) attending the cardiology clinic of Post Graduate Institute of Medical Education and Research were screened for mutations in the MYH7 gene. The control group for genetic studies consisted of 100 healthy subjects. We report 14...... mutations in 6 probands (5 probands in HCM and 1 proband in DCM) and their family members. Out of these 6 mutations, 3 are new and are being reported for the first time. One known mutation (p.Gly716Arg) was found to be "de novo" which resulted in severe asymmetric septal hypertrophy (31 mm) and resulted in...

  18. A Positive GATA Element and a Negative Vitamin D Receptor-Like Element Control Atrial Chamber-Specific Expression of a Slow Myosin Heavy-Chain Gene during Cardiac Morphogenesis

    Wang, Gang Feng; Nikovits, William; Schleinitz, Mark; Stockdale, Frank E.


    We have used the slow myosin heavy chain (MyHC) 3 gene to study the molecular mechanisms that control atrial chamber-specific gene expression. Initially, slow MyHC 3 is uniformly expressed throughout the tubular heart of the quail embryo. As cardiac development proceeds, an anterior-posterior gradient of slow MyHC 3 expression develops, culminating in atrial chamber-restricted expression of this gene following chamberization. Two cis elements within the slow MyHC 3 gene promoter, a GATA-bindi...

  19. Heavy and light roles: myosin in the morphogenesis of the heart

    England, Jennifer; Loughna, Siobhan


    Myosin is an essential component of cardiac muscle, from the onset of cardiogenesis through to the adult heart. Although traditionally known for its role in energy transduction and force development, recent studies suggest that both myosin heavy-chain and myosin lightchain proteins are required for a correctly formed heart. Myosins are structural proteins that are not only expressed from early stages of heart development, but when mutated in humans they may give rise to congeni...

  20. Radioimmunoassay of myosin heavy beta chains in human serum for the evaluation of the size of myocardial infarction: correlation with myocardial Tl-201 SPECT and cardiac angioscintigraphy

    To determine the relationship between serum levels of myosin heavy beta chains assessed by an IRMA technique and other radionuclide and enzymatic parameters in the evaluation of the size of myocardial infarction, we studied 22 patients with acute myocardial infarction. Blood samples taken daily between 1st to 13th day of evolution allow the determination of peak and integral of myosine release that showed a good correlation (p<0.01) with myocardial underperfusion score in T1-201 SPECT, left ventricular ejection fractions at 1st day and at the pre-discharge study, just as CPK peak. This new assay is an interesting mean to evaluate the size of myocardial infarction

  1. The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T on the muscle length-mediated recruitment of crossbridges is modified divergently by α- and β-myosin heavy chain.

    Gollapudi, Sampath K; Chandra, Murali


    Hypertrophic cardiomyopathy mutations in cardiac troponin T (TnT) lead to sudden cardiac death. Augmented myofilament Ca(2+) sensitivity is a common feature in TnT mutants, but such observations fail to provide a rational explanation for severe cardiac phenotypes. To better understand the mutation-induced effect on the cardiac phenotype, it is imperative to determine the effects on dynamic contractile features such as the muscle length (ML)-mediated activation against α- and β-myosin heavy chain (MHC) isoforms. α- and β-MHC are not only differentially expressed in rodent and human hearts, but they also modify ML-mediated activation differently. Mouse analog of human TnTR94L (TnTR97L) or wild-type TnT was reconstituted into de-membranated muscle fibers from normal (α-MHC) and transgenic (β-MHC) mouse hearts. TnTR97L augmented myofilament Ca(2+) sensitivity by a similar amount in α- and β-MHC fibers. However, TnTR97L augmented the negative impact of strained crossbridges on other crossbridges (γ) by 22% in α-MHC fibers, but attenuated γ by 21% in β-MHC fibers. TnTR97L decreased the magnitude of ML-mediated recruitment of crossbridges (ER) by 37% in α-MHC fibers, but increased ER by 35% in β-MHC fibers. We provide a mechanistic basis for the TnTR97L-induced effects in α- and β-MHC fibers and discuss the relevance to human hearts. PMID:26792537

  2. Mammalian myosin-18A, a highly divergent myosin.

    Guzik-Lendrum, Stephanie; Heissler, Sarah M; Billington, Neil; Takagi, Yasuharu; Yang, Yi; Knight, Peter J; Homsher, Earl; Sellers, James R


    The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells. PMID:23382379

  3. Myosin heavy chain gene expression in human heart failure.

    Nakao, K; Minobe, W.; Roden, R; Bristow, M R; Leinwand, L A


    Two isoforms of myosin heavy chain (MyHC), alpha and beta, exist in the mammalian ventricular myocardium, and their relative expression is correlated with the contractile velocity of cardiac muscle. Several pathologic stimuli can cause a shift in the MyHC composition of the rodent ventricle from alpha- to beta-MyHC. Given the potential physiological consequences of cardiac MyHC isoform shifts, we determined MyHC gene expression in human heart failure where cardiac contractility is impaired si...

  4. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

    Hariadi, R. F.; Sommese, R. F.; Adhikari, A. S.; Taylor, R. E.; Sutton, S.; Spudich, J. A.; Sivaramakrishnan, S.


    The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

  5. Genetics Home Reference: myosin storage myopathy

    ... myosin rod cause myosin storage myopathy via multiple mechanisms. Proc Natl Acad Sci U S A. 2009 Apr ... PubMed Tajsharghi H, Oldfors A. Myosinopathies: pathology and mechanisms. Acta Neuropathol. 2013 Jan;125(1):3-18. ...

  6. Myosin is involved in postmitotic cell spreading


    We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading....

  7. Effect of trimetazidine and verapamil on the cardiomyopathic hamster myosin phenotype

    D'hahan, Nathalie; Taouil, Karima; Janmot, Chantal; Morel, Jean-Emile


    In this study we investigated whether long-term trimetazidine (anti-ischaemic drug) therapy alters the ventricular myosin heavy chain (MHC) isoform composition in a model of cardiomyopathy.MHC isoforms were analysed in the native state by electrophoresis in a pyrophosphate buffer. Myosin isoform patterns were studied in cardiac muscle from cardiomyopathic hamsters (CMH) of the BIO 14 : 6 strain during the time course of the disease and compared with those of healthy golden hamsters (F1B). The...

  8. Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

    Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bártoli, Fulvia; Salazar, Leiría; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl


    Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that my...

  9. Revisiting Myosin Families Through Large-scale Sequence Searches Leads to the Discovery of New Myosins.

    Pasha, Shaik Naseer; Meenakshi, Iyer; Sowdhamini, Ramanathan


    Myosins are actin-based motor proteins involved in many cellular movements. It is interesting to study the evolutionary patterns and the functional attributes of various types of myosins. Computational search algorithms were performed to identify putative myosin members by phylogenetic analysis, sequence motifs, and coexisting domains. This study is aimed at understanding the distribution and the likely biological functions of myosins encoded in various taxa and available eukaryotic genomes. We report here a phylogenetic analysis of around 4,064 myosin motor domains, built entirely from complete or near-complete myosin repertoires incorporating many unclassified, uncharacterized sequences and new myosin classes, with emphasis on myosins from Fungi, Haptophyta, and other Stramenopiles, Alveolates, and Rhizaria (SAR). The identification of large classes of myosins in Oomycetes, Cellular slime molds, Choanoflagellates, Pelagophytes, Eustigmatophyceae, Fonticula, Eucoccidiorida, and Apicomplexans with novel myosin motif variants that are conserved and thus presumably functional extends our knowledge of this important family of motor proteins. This work provides insights into the distribution and probable function of myosins including newly identified myosin classes. PMID:27597808

  10. Myosin II Dynamics during Embryo Morphogenesis

    Kasza, Karen


    During embryonic morphogenesis, the myosin II motor protein generates forces that help to shape tissues, organs, and the overall body form. In one dramatic example in the Drosophila melanogaster embryo, the epithelial tissue that will give rise to the body of the adult animal elongates more than two-fold along the head-to-tail axis in less than an hour. This elongation is accomplished primarily through directional rearrangements of cells within the plane of the tissue. Just prior to elongation, polarized assemblies of myosin II accumulate perpendicular to the elongation axis. The contractile forces generated by myosin activity orient cell movements along a common axis, promoting local cell rearrangements that contribute to global tissue elongation. The molecular and mechanical mechanisms by which myosin drives this massive change in embryo shape are poorly understood. To investigate these mechanisms, we generated a collection of transgenic flies expressing variants of myosin II with altered motor function and regulation. We found that variants that are predicted to have increased myosin activity cause defects in tissue elongation. Using biophysical approaches, we found that these myosin variants also have decreased turnover dynamics within cells. To explore the mechanisms by which molecular-level myosin dynamics are translated into tissue-level elongation, we are using time-lapse confocal imaging to observe cell movements in embryos with altered myosin activity. We are utilizing computational approaches to quantify the dynamics and directionality of myosin localization and cell rearrangements. These studies will help elucidate how myosin-generated forces control cell movements within tissues. This work is in collaboration with J. Zallen at the Sloan-Kettering Institute.

  11. Myosinome: a database of myosins from select eukaryotic genomes to facilitate analysis of sequence-structure-function relationships.

    Syamaladevi, Divya P; Sunitha, Margaret S; Kalaimathy, S; Reddy, Chandrashekar C; Iftekhar, Mohammed; Pasha, Shaik N; Sowdhamini, R


    Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms (Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from PMID:23189029

  12. Myosin motor isoforms direct specification of actomyosin function by tropomyosins

    Clayton, Joseph E.; Pollard, Luther W.; Murray, George G.; Lord, Matthew


    Myosins and tropomyosins represent two cytoskeletal proteins that often work together with actin filaments in contractile and motile cellular processes. While the specialized role of tropomyosin in striated muscle myosin-II regulation is well characterized, its role in non-muscle myosin regulation is poorly understood. We previously showed that fission yeast tropomyosin (Cdc8p) positively regulates myosin-II (Myo2p) and myosin-V (Myo52p) motors. To understand the broader implications of this ...

  13. Thiol groups of gizzard myosin heavy chains

    Bailin, G.


    Proteolysis of phosphorylated and /sup 3/H-labeled dinitrophenylated chicken gizzard myosin with trypsin released major fragments of M/sub r/ 25,000, 50,000 and 66,000 in a 1:1 ratio. They contained 57% of the dinitrophenyl (N/sub 2/ph) group bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The fragments of M/sub r/ 25,000 and M/sub r/ 66,000 were dinitrophenylated predominantly when the K/sup +/-ATPase activity was inhibited. Thiolysis of phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol removed 60% and 25% of the N/sub 2/ph group from the N-terminal and M/sub r/ 66,000 fragments of the heavy chain, respectively, when 48% of the K/sup +/-ATPase activity was restored. Papain proteolysis of the tryptic digest of modified myosin released a C-terminal segment from the fragment of M/sub r/ 66,000 and it contained most of the remaining label. Proteolysis of /sup 3/H-labeled dinitrophenylated myosin alone resulted in the same digestion pattern but less of the label was bound to the heavy chain fragments. In this case, restoration of enzymic activity occurred in thiolyzed dinitrophenylated myosin when the N/sub 2/ph group was removed from the light chains, predominantly. Conformational changes in gizzard myosin, mediated by phosphorylation, altered the reactivity of the thiols in specific fragments of the heavy chain. Thiol groups of the N- and C-terminal heavy chain regions are involved in maintaining the ATPase activity of myosin.

  14. Clinical assessment of serum myosin light chain I in patients with dilated cardiomyopathy

    Serum cardiac myosin light chain I (LCI) levels were quantitated using a radioimmunoassay kit in patients suspected of dilated cardiomyopathy (DCM). In this study, 55 patients were evaluated between 1986 and 1991. They were composed of 40 males and 15 females, and their age was 27-75 years (51±11 years). The patients with renal dysfunction were excluded due to their serum creatinine levels (>2.0 mg/dl). After cardiac catheterization, endomyocardial biopsy and echocardiography, 44 patients were diagnosed as DCM, 2 as ischemic heart disease, 2 as chronic myocarditis, 1 as restrictive cardiomyopathy, 1 as dilated hypertrophic cardiomyopathy, 1 as cardiac amyloidosis, 2 as myopathy, 1 as polymyositis and 1 as hypothyroidism. Only two patients with DCM had elevated LCI. Besides, two patients with myopathy or hypothyroidism had elevated LCI. In the follow-up, one patient died suddenly 6 months later and another showed normal value of LCI four years later. LCI elevation in DCM was not related to either the severity of heart failure or cardiac function and it showed no finding of 201Tl myocardial defect or elevated CPK. The mechanism for elevated LCI in myopathy is related to a crossreaction with myosin light chain in the skeletal muscle. In hypothyroidism, it may be related to decreased clearance of normal LCI concentration or increased myosin light chain from damaged skeletal muscle. In conclusion, it is evident that the measurement of LCI is not helpful in clinical assessment of patients with DCM, but may be useful in detection of secondary cardiomyopathy. (author)

  15. Myosin Specific-T Lymphocytes Mediated Myocardial Inflammation in Adoptive Transferred Rats

    Jin Zhang; Yuhua Liao; Xiang Cheng; Jing Chen; Peng Chen; Xiang Gao; Zhengjenny Zhang


    Myosin specific-T lymphocytes might mediate myocardial inflammation and remodeling after AMI. Myosinactivated or unactivated T lymphocytes in vitro were transferred into naǐve syngeneic rats, respectively. T lymphocyte infiltration and myocyte apoptosis were explored by the H&E and TUNNEL. Proteins and mRNA levels of cytokines (IL-1β, IL-6 and TNF-α) in myocardium were determined by RT-PCR and immunohistochemistry. T lymphocyte infiltration was evidently observed after one week of activated T cell transfer. The expressions of cytokines were elevated markedly one week later. The myocyte apoptosis occurred after T lymphocyte infiltration in myocardium. Our findings suggest that cardiac myosin activated-T lymphocytes may mediate myocardial inflammation and remodeling.

  16. Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

    D.V. Vassallo


    Full Text Available Lead (Pb2+ poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM to the bath. Changes in rate of stimulation (0.1-1.5 Hz, relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM, and the effect of isoproterenol (20 ng/mL were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

  17. Atrial and ventricular myosin heavy-chain expression in the developing chicken heart: Strengths and limitations of non-radioactive in situ hybridization

    S. Somi; A.T.J. Klein; A.C. Houweling; J.M. Ruijter; A.A.M. Buffing; A.F.M. Moorman; M.J.B. van den Hoff


    Myosin heavy-chain (MHC) isoforms are major structural components of the contractile apparatus of the heart muscle. Their spatio-temporal patterns of expression have been used as a tool to dissect cardiac development and differentiation. Although extensively investigated, controversy still exists co

  18. Purification and characterization of myosin from wheat mitochondria


    Myosin was purified from wheat mitochondria using DE-52 anion exchange chromatography and Sephacryl S-300 gel ffitration. The molecular weight of its heavy chain is about 210 ku, similar to that of muscle myosin Ⅱ (205 ku),and it could be recognized by the polyclonal antibodies against human skeletal muscle myosin Ⅱ. The ATPase activity of the mitochondrial myosin stimulated by F-actin from chicken muscle is 202.5 nmoles Pi/min @ mg. The mitochondrial myosin could be activated by Ca2+ and was not inhibited by Ca2+ at high concentration. The results demonstrate that the myosin of wheat mitochondria shares some similarities with the skeletal muscle myosin Ⅱ.

  19. Myosin rods are a source of second harmonic generation signals in skeletal muscle

    Schürmann, Sebastian; Weber, Cornelia; Fink, Rainer H. A.; Vogel, Martin


    Intrinsic second harmonic generation (SHG) signals can be used to visualize the three-dimensional structure of cardiac and skeletal muscle with high spatial resolution. Fluorescence labeling of complementary sarcomeric proteins, e.g. actin, indicates that the observed SHG signals arise from the myosin filaments. Recently, the myosin rod domain or LMM - light meromyosin - has been reported to be the dominant source of this SHG signal. However, to date, mostly negative and indirect evidence has been presented to support this assumption. Here, we show, to our knowledge, the first direct evidences that strong SHG signals can be obtained from synthetic paracrystals. These rod shaped filaments are formed from purified LMM. SDS-PAGE protein analysis confirmed that the LMM crystals lack myosin head domains. Some regions of the LMM paracrystals produce a strong SHG signal whereas others did not. The SHG signals were recorded with a laser-scanning microscope (Leica SP2). A ps laser tuned to 880 nm was used to excite the sample through an 63x objective of 1.2 NA. In order to visualize the synthetic filaments - in addition to SHG imaging -, the LMM was labeled with the fluorescent marker 5-IAF. We were able to observe filaments of 1 to 50 μm in length and of up to 5 μm in diameter. In conclusion, we can show that the myosin rod domain (LMM) is a dominant source for intrinsic SHG signals. There seems, however, a signal dependence on the paracrystals' morphology. This dependence is being investigated.

  20. Cardiac cell proliferation assessed by EdU, a novel analysis of cardiac regeneration.

    Zeng, Bin; Tong, Suiyang; Ren, Xiaofeng; Xia, Hao


    Emerging evidence suggests that mammalian hearts maintain the capacity for cardiac regeneration. Rapid and sensitive identification of cardiac cellular proliferation is prerequisite for understanding the underlying mechanisms and strategies of cardiac regeneration. The following immunologically related markers of cardiac cells were analyzed: cardiac transcription factors Nkx2.5 and Gata 4; specific marker of cardiomyocytes TnT; endothelial cell marker CD31; vascular smooth muscle marker smooth muscle myosin IgG; cardiac resident stem cells markers IsL1, Tbx18, and Wt1. Markers were co-localized in cardiac tissues of embryonic, neonatal, adult, and pathological samples by 5-ethynyl-2'-deoxyuridine (EdU) staining. EdU was also used to label isolated neonatal cardiomyocytes in vitro. EdU robustly labeled proliferating cells in vitro and in vivo, co-immunostaining with different cardiac cells markers. EdU can rapidly and sensitively label proliferating cardiac cells in developmental and pathological states. Cardiac cell proliferation assessed by EdU is a novel analytical tool for investigating the mechanism and strategies of cardiac regeneration in response to injury. PMID:25480318

  1. Peptide growth factors can provoke "fetal" contractile protein gene expression in rat cardiac myocytes.

    Parker, T G; Packer, S E; Schneider, M. D.


    Cardiac-specific gene expression is intricately regulated in response to developmental, hormonal, and hemodynamic stimuli. To test whether cardiac muscle might be a target for regulation by peptide growth factors, the effect of three growth factors on the actin and myosin gene families was investigated by Northern blot analysis in cultured neonatal rat cardiac myocytes. Transforming growth factor-beta 1 (TGF beta 1, 1 ng/ml) and basic fibroblast growth factor (FGF, 25 ng/ml) elicited changes ...

  2. Antiparallel coiled-coil-mediated dimerization of myosin X.

    Lu, Qing; Ye, Fei; Wei, Zhiyi; Wen, Zilong; Zhang, Mingjie


    Processive movements of unconventional myosins on actin filaments generally require motor dimerization. A commonly accepted myosin dimerization mechanism is via formation of a parallel coiled-coil dimer by a stretch of amino acid residues immediately carboxyl-terminal to the motor's lever-arm domain. Here, we discover that the predicted coiled-coil region of myosin X forms a highly stable, antiparallel coiled-coil dimer (anti-CC). Disruption of the anti-CC either by single-point mutations or by replacement of the anti-CC with a parallel coiled coil with a similar length compromised the filopodial induction activity of myosin X. We further show that the anti-CC and the single α-helical domain of myosin X are connected by a semirigid helical linker. The anti-CC-mediated dimerization may enable myosin X to walk on both single and bundled actin filaments. PMID:23012428

  3. Nuclear myosin I regulates cell membrane tension

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Dzijak, Rastislav; Pastorek, Lukáš; Rohožková, Jana; Malohlava, Jakub; Hozák, Pavel


    Plasma membrane tension is an important feature that determines the cell shape and influences processes such as cell motility, spreading, endocytosis and exocytosis. Unconventional class 1 myosins are potent regulators of plasma membrane tension because they physically link the plasma membrane with adjacent cytoskeleton. We identified nuclear myosin 1 (NM1) - a putative nuclear isoform of myosin 1c (Myo1c) - as a new player in the field. Although having specific nuclear functions, NM1 localizes predominantly to the plasma membrane. Deletion of NM1 causes more than a 50% increase in the elasticity of the plasma membrane around the actin cytoskeleton as measured by atomic force microscopy. This higher elasticity of NM1 knock-out cells leads to 25% higher resistance to short-term hypotonic environment and rapid cell swelling. In contrast, overexpression of NM1 in wild type cells leads to an additional 30% reduction of their survival. We have shown that NM1 has a direct functional role in the cytoplasm as a dynamic linker between the cell membrane and the underlying cytoskeleton, regulating the degree of effective plasma membrane tension. PMID:27480647

  4. The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation.

    Fusi, Luca; Huang, Zhe; Irving, Malcolm


    In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads. PMID:26287630

  5. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

    Cyr Janet L


    Full Text Available Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3 in its neck region; we identified a fourth IQ domain (IQ4, located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail each bound 2–3 calmodulin molecules. At a physiologically relevant temperature (25°C and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 μM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25°C; one calmodulin was retained if 5 μM free calmodulin was present. Conclusions We inferred from these data that at 25°C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.

  6. Enhanced force generation by smooth muscle myosin in vitro.

    VanBuren, P; Work, S S; Warshaw, D.M.


    To determine whether the apparent enhanced force-generating capabilities of smooth muscle relative to skeletal muscle are inherent to the myosin cross-bridge, the isometric steady-state force produced by myosin in the in vitro motility assay was measured. In this assay, myosin adhered to a glass surface pulls on an actin filament that is attached to an ultracompliant (50-200 nm/pN) glass microneedle. The number of myosin cross-bridge heads able to interact with a length of actin filament was ...

  7. Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments

    Sobieszek, Apolinary; Sarg, Bettina; Seow, Chun Y.; Lindner, Herbert


    Phosphorylation of myosin by myosin light chain kinase (MLCK) is essential for smooth muscle contraction. In this study we show that caldesmon (CaD) is also phosphorylated in vitro by MLCK. The phosphorylation is calcium- and calmodulin (CaM)-dependent and requires a MLCK concentration close to that found in vivo. On average, approximately 2 mol Pi per mol of CaD are incorporated at Thr-626 and Thr-693, with additional partial phosphorylation at Ser-658 and Ser-702. The phosphorylation rate f...

  8. Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy


    Polyclonal antisera have been raised against purified Acanthamoeba myosin II and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of Acanthamoeba myosin IC. These antisera are specific for their respective antigens as determined by immunoblotting after SDS-PAGE of total cell lysates. By using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. Myosin II occurred in the cell cytopla...

  9. Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

    Reiser, Peter J; Bicer, Sabahattin; Chen, Qun; Zhu, Ling; Quan, Ning


    Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style. PMID:19648394

  10. A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila.

    Achal, Madhulika; Trujillo, Adriana S; Melkani, Girish C; Farman, Gerrie P; Ocorr, Karen; Viswanathan, Meera C; Kaushik, Gaurav; Newhard, Christopher S; Glasheen, Bernadette M; Melkani, Anju; Suggs, Jennifer A; Moore, Jeffrey R; Swank, Douglas M; Bodmer, Rolf; Cammarato, Anthony; Bernstein, Sanford I


    An "invariant proline" separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138-43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc(5), an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue. PMID:27107639

  11. Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

    Pulcastro, Hannah C; Awinda, Peter O; Breithaupt, Jason J; Tanner, Bertrand C W


    Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles. PMID:26763941

  12. Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments.

    Kampourakis, Thomas; Sun, Yin-Biao; Irving, Malcolm


    Contraction of heart muscle is triggered by calcium binding to the actin-containing thin filaments but modulated by structural changes in the myosin-containing thick filaments. We used phosphorylation of the myosin regulatory light chain (cRLC) by the cardiac isoform of its specific kinase to elucidate mechanisms of thick filament-mediated contractile regulation in demembranated trabeculae from the rat right ventricle. cRLC phosphorylation enhanced active force and its calcium sensitivity and altered thick filament structure as reported by bifunctional rhodamine probes on the cRLC: the myosin head domains became more perpendicular to the filament axis. The effects of cRLC phosphorylation on thick filament structure and its calcium sensitivity were mimicked by increasing sarcomere length or by deleting the N terminus of the cRLC. Changes in thick filament structure were highly cooperative with respect to either calcium concentration or extent of cRLC phosphorylation. Probes on unphosphorylated myosin heads reported similar structural changes when neighboring heads were phosphorylated, directly demonstrating signaling between myosin heads. Moreover probes on troponin showed that calcium sensitization by cRLC phosphorylation is mediated by the thin filament, revealing a signaling pathway between thick and thin filaments that is still present when active force is blocked by Blebbistatin. These results show that coordinated and cooperative structural changes in the thick and thin filaments are fundamental to the physiological regulation of contractility in the heart. This integrated dual-filament concept of contractile regulation may aid understanding of functional effects of mutations in the protein components of both filaments associated with heart disease. PMID:27162358

  13. Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

    Reddy, A. S.; Day, I. S.


    BACKGROUND: Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants. RESULTS: Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication. CONCLUSIONS: Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.

  14. Myosins and cell dynamics in cellular slime molds.

    Yumura, Shigehiko; Uyeda, Taro Q P


    Myosin is a mechanochemical transducer and serves as a motor for various motile activities such as cell migration, cytokinesis, maintenance of cell shape, phagocytosis, and morphogenesis. Nonmuscle myosin in vivo does not either stay static at specific subcellular regions or construct highly organized structures, such as sarcomere in skeletal muscle cells. The cellular slime mold Dictyostelium discoideum is an ideal "model organism" for the investigation of cell movement and cytokinesis. The advantages of this organism prompted researchers to carry out pioneering cell biological, biochemical, and molecular genetic studies on myosin II, which resulted in elucidation of many fundamental features of function and regulation of this most abundant molecular motor. Furthermore, recent molecular biological research has revealed that many unconventional myosins play various functions in vivo. In this article, how myosins are organized and regulated in a dynamic manner in Dictyostelium cells is reviewed and discussed. PMID:12722951

  15. Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

    Vu, N.D.; Wagner, P.D.


    Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of /sup 32/P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca/sup 2 +/- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation.

  16. Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform

    Yi Ting


    Full Text Available Abstract It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG and that exposure of zinc ion (Zn2+ to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after β-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% β-myosin heavy chain expression in the heart. β-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our

  17. Myosin-I Isozymes in Neonatal Rodent Auditory and Vestibular Epithelia

    Dumont, Rachel A.; Zhao, Yi-Dong; Holt, Jeffrey R.; Bähler, Martin; Gillespie, Peter G.


    Myosin isozymes are essential for hair cells, the sensory cells of the inner ear. Because a myosin-I subfamily member may mediate adaptation of mechanoelectrical transduction, we examined expression of all eight myosin-I isozymes in rodent auditory and vestibular epithelia. Using RT-PCR, we found prominent expression of three isozymes, Myo1b (also known as myosin-Ia or myr 1), Myo1c (myosin-Ib or myr 2), and Myo1e (myr 3). By contrast, Myo1a (brush-border myosin-I), Myo1d (myosin lg or myr 4)...

  18. Different subcellular localizations and functions of Arabidopsis myosin VIII

    Belausov Eduard


    Full Text Available Abstract Background Myosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB. Results In transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain, fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding. Conclusion Taken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity.

  19. ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease.

    Sequeira, Vasco; Najafi, Aref; Wijnker, Paul J M; Dos Remedios, Cristobal G; Michels, Michelle; Kuster, Diederik W D; van der Velden, Jolanda


    Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin-myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca(2+). We investigated whether ADP-stimulated force development (without Ca(2+)) can be used to reveal changes in actin-myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7mut, MYBPC3mut) and thin-filament (TNNT2mut, TNNI3mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCMsmn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCMsmn, and MYH7mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2mut sample corrected the abnormal actin-myosin blockade. In MYBPC3trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C's role in actin-myosin regulation. In the presence of Ca(2+), ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca(2+), pathologic [ADP] and low PKA-phosphorylation, high actin-myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies. PMID:26621701

  20. Antiparallel coiled-coil–mediated dimerization of myosin X

    Lu, Qing; Ye, Fei; Wei, Zhiyi; Wen, Zilong; Zhang, Mingjie


    Processive movements of unconventional myosins on actin filaments generally require motor dimerization. A commonly accepted myosin dimerization mechanism is via formation of a parallel coiled-coil dimer by a stretch of amino acid residues immediately carboxyl-terminal to the motor’s lever-arm domain. Here, we discover that the predicted coiled-coil region of myosin X forms a highly stable, antiparallel coiled-coil dimer (anti-CC). Disruption of the anti-CC either by single-point mutations or ...

  1. Rod phosphorylation favors folding in a catch muscle myosin.

    Castellani, L; Cohen, C


    Myosin from a molluscan catch muscle is unusual in being phosphorylated in the rod by an endogenous heavy chain kinase. The overall structure of the molecule resembles that of other muscle myosins, although the tail is somewhat longer (approximately equal to 1700 A). At low ionic strength the unphosphorylated molecules associate in filaments that display a striking axial repeat of 145 A. Phosphorylation of the rod enhances myosin solubility in the range of NaCl between 0.05 and 0.15 M. Depend...

  2. Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

    Cadete, Virgilio J. J.; Sawicka, Jolanta; Jaswal, Jagdip; Lopaschuk, Gary D.; Schulz, Richard; Szczesna-Cordary, Danuta; Sawicki, Grzegorz


    Summary Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia. PMID:22564771

  3. Expression of non-muscle type myosin heavy polypeptide 9 (MYH9 in mammalian cells

    T Takubo


    Full Text Available Myosin is a functional protein associated with cellular movement, cell division, muscle contraction and other functions. Members of the myosin super-family are distinguished from the myosin heavy chains that play crucial roles in cellular processes. Although there are many studies of myosin heavy chains in this family, there are fewer on non-muscle myosin heavy chains than of muscle myosin heavy chains. Myosin is classified as type I (myosin I or type II (myosin II. Myosin I, called unconventional myosin or mini-myosin, has one head, while myosin II, called conventional myosin, has two heads. We transfected myosin heavy polypeptide 9 (MYH9 into HeLa cells as a fusion protein with enhanced green fluorescent protein (EGFP and analyzed the localization and distribution of MYH9 in parallel with those of actin and tubulin. The results indicate that MYH9 colocalizes with actin stress fibers. Since it has recently been shown by genetic analysis that autosomal dominant giant platelet syndromes are MYH9-related disorders, our development of transfected EGFP-MYH9 might be useful for predicting the associations between the function of actin polymerization, the MYH9 motor domain, and these disorders.

  4. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus

    Venit, Tomáš; Dzijak, Rastislav; Rohožková, Jana; Kalendová, Alžběta; Hozák, Pavel

    Debrecen : University of Debrecen, 2013. s. 45-45. [Wilhelm Bernard Workshop on the cell nucleus /23./. 19.08.2013-24.08.2013, Debrecen] R&D Projects: GA ČR(CZ) GD204/09/H084; GA ČR GAP305/11/2232; GA TA ČR TE01020118; GA MŠk LH12143 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : atomic force microscopy * cell membrane * myosin 1C * NM1 * nuclear myosin I * myosin knock-out Subject RIV: EB - Genetics ; Molecular Biology

  5. Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

    Anabelle S. Cornachione


    Full Text Available Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

  6. Still and rotating myosin clusters determine cytokinetic ring constriction.

    Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Kruse, Karsten; Riveline, Daniel


    The cytokinetic ring is essential for separating daughter cells during division. It consists of actin filaments and myosin motors that are generally assumed to organize as sarcomeres similar to skeletal muscles. However, direct evidence is lacking. Here we show that the internal organization and dynamics of rings are different from sarcomeres and distinct in different cell types. Using micro-cavities to orient rings in single focal planes, we find in mammalian cells a transition from a homogeneous distribution to a periodic pattern of myosin clusters at the onset of constriction. In contrast, in fission yeast, myosin clusters rotate prior to and during constriction. Theoretical analysis indicates that both patterns result from acto-myosin self-organization and reveals differences in the respective stresses. These findings suggest distinct functional roles for rings: contraction in mammalian cells and transport in fission yeast. Thus self-organization under different conditions may be a generic feature for regulating morphogenesis in vivo. PMID:27363521

  7. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    Bekyarova, T. I.; Reedy, M C; Baumann, B. A. J.; Tregear, R T; Ward, A; Krzic, U.; Prince, K.M.; Perz-Edwards, R. J.; Reconditi, M.; Gore, D.; Irving, T C; Reedy, M K


    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the “steric blocking” mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca2+ with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence...

  8. Internal Motility in Stiffening Actin-Myosin Networks

    Uhde, Joerg; Keller, Manfred; Sackmann, Erich; Parmeggiani, Andrea; Frey, Erwin


    We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and sug...

  9. Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

    Chen, Xuesong; Pavlish, Kristin; Benoit, Joseph N.


    A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or β-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylat...

  10. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments


    Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the hea...

  11. Cardiac troponin: an emerging cardiac biomarker in animal health

    Vishal V. Undhad

    Full Text Available Analysis of cardiac troponin I (cTn I and T (cTnT are considered the “gold standard” for the non-invasive diagnosis of myocardial injury in human and animals. It has replaced traditionally used cardiac biomarkers such as myoglobin, lactate dehydrogenase (LDH, creatine kinase (CK and CK-MB due to its high sensitivity and specificity for the detection of myocardial injury. Cardiac troponins are proteins that control the calcium-mediated interaction between actin and myosin, allowing contraction at the sarcomere level. Concentration of the cTn can be correlated microscopic lesion and loss of immunolabeling in myocardium damage. Troponin concentration remains elevated in blood for 1-2wks so that wide window is available for diagnosis of myocardial damage. The cTn test has >95% specificity and sensitivity and test is less time consuming (10 to 15 minutes and less costly (INR 200 to INR 500. [Vet. World 2012; 5(8.000: 508-511

  12. Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.

    Alamo, Lorenzo; Qi, Dan; Wriggers, Willy; Pinto, Antonio; Zhu, Jingui; Bilbao, Aivett; Gillilan, Richard E; Hu, Songnian; Padrón, Raúl


    Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles. PMID:26851071

  13. Transplantation of 5-azacytidine treated cardiac fibroblasts improves cardiac function of infarct hearts in rats

    TANG Cheng-chun; MA Gan-shan; CHEN Ji-yuan


    Background Cellular cardiomyoplasty by transplantation of various cell types has been investigated as potential treatments for the improvement of cardiac function after myocardial injury. A major barrier for the clinical application of cell transplantation is obtaining sufficiently large quantities of suitable cells. AIIogeneic cellular cardiomyoplasty may provide an alternative source of abundant, transplantable, myogenic cells by in vitro manipulation of cardiac fibroblasts using chemicals including 5-azacytidine. This study evaluated cardiomyogenic differentiation of cardiac fibroblasts, their survival in myocardial scar tissue, and the effect of the implanted cells on heart function.Methods Primary cardiac fibroblasts from neonatal rats were treated with 5-azacytidine (10 μmol/L) or control.Treatment of 5-azacytidine caused myogenic differentiation of cultured cardiac fibroblasts, as defined by elongation and fusion into multinucleated myotubes with sarcomeric structures as identified by electron microscopy, and positive immunostaining for cardiac specific proteins, troponin I and β-myosin heavy chain (β-MHC) and the gap junction protein connexin 43. The myogenic cells (1.0x106) were transplanted into the infarcted myocardium 2 weeks after coronary artery occlusion.Results By 1 month after transplantation, the converted fibroblasts gave rise to a cluster of cardiac-like muscle cells that in the hearts occupied a large part of the scar with positive immunostaining for the myogenic proteins troponin I and β-MHC. Engrafted cells also expressed the gap junction protein connexin 43 in a disorganized manner. There was no positive staining in the control hearts treated with injections of culture medium. Heart function was evaluated at 6 weeks after myocardial injury with echocardiographic and hemodynamic measurements. Improvement in cardiac function was seen in the hearts transplanted with the 5-azacytidine-treated cardiac fibroblasts which was absent in the

  14. Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

    Lametsch, Marianne Lund; Luxford, Catherine; Skibsted, Leif Horsfelt; Davies, Michael Jonathan


    result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple...... thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation on...

  15. Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides

    Cyr Janet L; Gillespie Peter G


    Abstract Background Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. Results In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2–4 μM at normal ionic strength; the interaction with an IQ4 peptide was much...

  16. Two Regions of the Tail Are Necessary for the Isoform-specific Functions of Nonmuscle Myosin IIB

    Sato, Masaaki K.; Takahashi, Masayuki; Yazawa, Michio


    To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing a...

  17. Cardiac-specific miRNA in cardiogenesis, heart function, and cardiac pathology (with focus on myocardial infarction).

    Chistiakov, Dimitry A; Orekhov, Alexander N; Bobryshev, Yuri V


    Cardiac miRNAs (miR-1, miR133a, miR-208a/b, and miR-499) are abundantly expressed in the myocardium. They play a central role in cardiogenesis, heart function and pathology. While miR-1 and miR-133a predominantly control early stages of cardiogenesis supporting commitment of cardiac-specific muscle lineage from embryonic stem cells and mesodermal precursors, miR-208 and miR-499 are involved in the late cardiogenic stages mediating differentiation of cardioblasts to cardiomyocytes and fast/slow muscle fiber specification. In the heart, miR-1/133a control cardiac conductance and automaticity by regulating all phases of the cardiac action potential. miR-208/499 located in introns of the heavy chain myosin genes regulate expression of sarcomeric contractile proteins. In cardiac pathology including myocardial infarction (MI), expression of cardiac miRNAs is markedly altered that leads to deleterious effects associated with heart wounding, arrhythmia, increased apoptosis, fibrosis, hypertrophy, and tissue remodeling. In acute MI, circulating levels of cardiac miRNAs are significantly elevated making them to be a promising diagnostic marker for early diagnosis of acute MI. Great cardiospecific capacity of these miRNAs is very helpful for enhancing regenerative properties and survival of stem cell and cardiac progenitor transplants and for reprogramming of mature non-cardiac cells to cardiomyocytes. PMID:27056419

  18. Involvement of myosin in intracellular motility and cytomorphogenesis in Micrasterias.

    Oertel, Anke; Holzinger, Andreas; Lütz-Meindl, Ursula


    Myosin was detected on Western blots of Micrasterias denticulata extracts by use of antibodies from different sources. Inhibitors with different targets of the actomyosin system, such as the myosin ATPase-blockers N-ethylmaleimide (NEM) and 2,3-butanedione monoxime (BDM), or the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapine (ML7), had similar effects on intracellular motility during cell development in the green alga Micrasterias, thus pointing towards a participation of myosin in these processes. The drugs markedly altered the mode of postmitotic nuclear migration, slowed down cytoplasmic streaming, changed cell pattern development and prevented normal chloroplast distribution and spreading into the growing semicell. In addition, an increase and dilatations in ER cisternae and marked morphological changes of the Golgi system were observed by transmission electron microscopy after exposure of growing cells to BDM. Neither BDM nor ML7 exhibited any effect on the distribution or arrangement of the cortical F-actin network nor on the F-actin basket around the nucleus, characteristic of untreated growing Micrasterias cells (J Cell Sci 107 (1994) 1929). This is particularly interesting since BDM caused disintegration of the microtubule system co-localized to the F-actin cage during normal nuclear migration. Together with the fact that other microtubules not connected to the F-actin system remained uninfluenced by BDM, this observation is evidence of an integrative function of myosin between the cytoskeleton elements. PMID:14642529

  19. Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

    Hanson Maureen R


    Full Text Available Abstract Background Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2, which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins. Results We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1, myosin XI-6 (At MYA2, myosin XI-8 (At XI-B, myosin XI-15 (At XI-I, myosin XI-16 (At XI-J and myosin XI-17 (At XI-K were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2, previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria. Conclusion 6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and

  20. Photocleavage of myosin subfragment 1 by vanadate

    The heavy chain of myosin's subfragment 1 (S1) was cleaved at two distinct sites (termed V1 and V2) after irradiation with UV light in the presence of millimolar concentrations of vanadate and in the absence of nucleotides or divalent metals. The V1 site cleavage appeared to be identical with the previously described active site cleavage at serine-180, which is effected by irradiation of a photomodified form of the S1-MgADP-Vi complex. The V2 site was cleaved specifically, without cleavage at the V1 site, first by formation of the light-stable S1-Co2+ADP-Vi complex at the active site and then by irradiation in the presence of millimolar vanadate. By gel electrophoresis, the V2 site was localized to a region about 20 kDa from the COOH terminus of the S1 heavy chain. From the results of tryptic digestion experiments, the COOH-terminal V2 cleavage peptide appeared to contain lysine-636 in the linker region between the 50- and 20-kDa tryptic peptides of the heavy chain. This site appeared to be the same site cleaved by irradiation of S1 (not complexed with Co2+ADP-Vi) in the presence of millimolar vanadate as previously described. Cleavage at the V2 site was inhibited by Co2+ but was not significantly affected by the presence of nucleotides or Mg2+ ions. Tris buffer significantly inhibited V2 cleavage. From the results of UV-visible absorption, 51V NMR, and frozen-solution EPR spectral experiments, it was concluded that irradiation with UV light reduced vanadate +5 to the +4 oxidation state, which was then protected from rapid reoxidation by O2 by complexation with the Tris buffer. The relatively stable reduced form or forms of vanadium were not competent to cleave S1 at either the V1 or the V2 site

  1. Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

    Batters, Christopher; Ellrich, Heike; Helbig, Constanze; Woodall, Katy Anna; Hundschell, Christian; Brack, Dario; Veigel, Claudia


    Myosin XXI is the only myosin isoform expressed in the Leishmania parasite. The myosin-XXI homozygous knockout is lethal, and a reduction in expression levels leads to loss of endocytosis and affects other intracellular trafficking processes. In this paper we show that myosin XXI can adopt a monomeric or dimeric state. The states are determined by calmodulin binding to an IQ motif that, when bound, prevents dimerization of a coiled-coil motif. In the monomeric state the motor binds phospholip...

  2. Review: The ATPase mechanism of myosin and actomyosin.

    Geeves, Michael A


    Myosins are a large family of molecular motors that use the common P-loop, Switch 1 and Switch 2 nucleotide binding motifs to recognize ATP, to create a catalytic site than can efficiently hydrolyze ATP and to communicate the state of the nucleotide pocket to other allosteric binding sites on myosin. The energy of ATP hydrolysis is used to do work against an external load. In this short review I will outline current thinking on the mechanism of ATP hydrolysis and how the energy of ATP hydrolysis is coupled to a series of protein conformational changes that allow a myosin, with the cytoskeleton track actin, to operate as a molecular motor of distinct types; fast movers, processive motors or strain sensors. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 483-491, 2016. PMID:27061920

  3. Myosin IIA deficient cells migrate efficiently despite reduced traction forces at cell periphery

    Melissa H. Jorrisch


    Cell motility is a cornerstone of embryogenesis, tissue remodeling and repair, and cancer cell invasion. It is generally thought that migrating cells grab and exert traction force onto the extracellular matrix in order to pull the cell body forward. While previous studies have shown that myosin II deficient cells migrate efficiently, whether these cells exert traction forces during cell migration in the absence of the major contractile machinery is currently unknown. Using an array of micron-sized pillars as a force sensor and shRNA specific to each myosin II isoform (A and B, we analyzed how myosin IIA and IIB individually regulate cell migration and traction force generation. Myosin IIA and IIB localized preferentially to the leading edge where traction force was greatest, and the trailing edge, respectively. When individual myosin II isoforms were depleted by shRNA, myosin IIA deficient cells lost actin stress fibers and focal adhesions, whereas myosin IIB deficient cells maintained similar actin organization and focal adhesions as wild-type cells. Interestingly, myosin IIA deficient cells migrated faster than wild-type or myosin IIB deficient cells on both a rigid surface and a pillar array, yet myosin IIA deficient cells exerted significantly less traction force at the leading edge than wild-type or myosin IIB deficient cells. These results suggest that, in the absence of myosin IIA mediated force-generating machinery, cells move with minimal traction forces at the cell periphery, thus demonstrating the remarkable ability of cells to adapt and migrate.

  4. Cryo-atomic force microscopy of smooth muscle myosin.

    Y. Zhang; Shao, Z; Somlyo, A. P.; Somlyo, A V


    The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low p...

  5. Nonmuscle myosin dependent synthesis of type I collagen

    Cai, Le; Fritz, Dillon; Stefanovic, Lela; Stefanovic, Branko


    Type I collagen is the most abundant protein in human body synthesized in all tissues as the heterotrimer of two α1(I) and one α2(I) polypeptides. Here we show that intact nonmuscle myosin filaments are required for synthesis of heterotrimeric type I collagen. Conserved 5′ stem-loop in collagen α1(I) and α2(I) mRNAs binds RNA binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle my...

  6. Internal Motility in Stiffening Actin-Myosin Networks

    Uhde, J; Sackmann, E; Parmeggiani, A; Frey, E; Uhde, Joerg; Keller, Manfred; Sackmann, Erich; Parmeggiani, Andrea; Frey, Erwin


    We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and suggest a "zipping" mechanism to explain the filament motion in the vicinity of the sol-gel transition.

  7. Cardiac rehabilitation

    ... attack or other heart problem. You might consider cardiac rehab if you have had: Heart attack Coronary heart disease (CHD) Heart failure Angina (chest pain) Heart or heart valve surgery Heart transplant Procedures such as angioplasty and stenting In some ...

  8. Cardiac Rehabilitation

    Cardiac rehabilitation (rehab) is a medically supervised program to help people who have A heart attack Angioplasty or coronary artery bypass grafting for coronary heart disease A heart valve repair or replacement A ...

  9. Cardiac sarcoidosis

    Costello BT; Nadel J.; Taylor AJ


    Benedict T Costello,1,2 James Nadel,3 Andrew J Taylor,1,21Department of Cardiovascular Medicine, The Alfred Hospital, 2Baker IDI Heart and Diabetes Research Institute, Melbourne, VIC, 3School of Medicine, University of Notre Dame, Sydney, NSW, Australia Abstract: Cardiac sarcoidosis is a rare but life-threatening condition, requiring a high degree of clinical suspicion and low threshold for investigation to make the diagnosis. The cardiac manifestations include heart failure, conducting syst...

  10. Ouabain induces cardiac remodeling in rats independent of blood pressure

    Xing JIANG; Yan-ping REN; Zhuo-ren L(U)


    Aim: To investigate the ouabain's effects on cardiac remodeling in rats. Methods:Male Sprague-Dawley rats were treated with ouabain. Systolic blood pressure(SBP) was recorded weekly. After 4 and 6 weeks, echocardiography were performed,hemodynamic parameters were measured by invasive cardiac catheterization,changes in cardiac ultrastructure were analyzed using transmission electron microscopy, the collagen fraction of the left ventricle was assessed with Picrosirius red stain, and RT-PCR was applied to evaluate the mRNA level of myosin heavy chain-α and-β in the left ventricle. Results: Having been treated with ouabain for 4 weeks, there was no significant difference in the mean SBP of the two groups.However, left ventricular hypertrophy, myocardial ultrastructure deterioration,and extracellular matrix remodeling were induced by ouabain treatment; meanwhile,cardiac systolic and diastolic performance were both worsened. Moreover, the cardiac MHC-β mRNA was upregulated by ouabain treatment, whereas MHC-αmRNA was downregulated. After 4 weeks, the mean SBP in the ouabain group began to increase and was significantly higher than that in control group after 6 weeks (P<0.01); the rats' cardiac structure and function were worsened.Conclusion: These results suggested that ouabain induces alterations in cardiac structure and function, and the effects happened before the increase of blood pressure. The results indicated that ouabain induced cardiac remodeling in rats independent of blood pressure.

  11. Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus

    Venit, Tomáš; Dzijak, Rastislav; Kalendová, Alžběta; Kahle, Michal; Rohožková, Jana; Schmidt, V.; Rülicke, T.; Rathkolb, B.; Hans, W.; Bohla, A.; Eickelberg, O.; Stoeger, T.; Wolf, E.; Yildirim, A.Ö.; Gailus-Durner, V.; Fuchs, H.; de Angelis, M.H.; Hozák, Pavel


    Roč. 8, č. 4 (2013), e61406. E-ISSN 1932-6203 R&D Projects: GA ČR GAP305/11/2232; GA TA ČR TE01020022; GA MŠk LH12143; GA ČR(CZ) GD204/09/H084 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : nuclear myosin * myosin isoforms * cell nucleus Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  12. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Mansson, Alf; Kocer, Armagan


    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies.

  13. Calcium and cargoes as regulators of myosin 5a activity

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins

  14. Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

    Khalid Amari


    Full Text Available Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP and to a delay in the MP accumulation in plasmodesmata (PD. The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

  15. Model of Rho-Mediated Myosin Recruitment to the Cleavage Furrow during Cytokinesis

    Veksler, Alexander; Vavylonis, Dimitrios


    The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During cytokinesis, the myosin attached to the cell's cortex progressively disassembles at the flanking regions and concentrates in the equator [1]. This recruitment depends on myosin motor activity and activation by Rho proteins. Central spindle and astral microtubules establish a spatial pattern of differential Rho activity [2]. We propose a reaction-diffusion model for the dynamics of myosin and Rho proteins during cytokinesis. In the model, the mitotic spindle activates Rho at the equator. Active Rho promotes, in a switch-like manner, myosin assembly into cortical minifilaments. Mechanical stress by cortical myosin causes disassembly of myosin minifilaments and deactivates Rho. Our results explain both the recruitment of myosin to the cleavage furrow and the observed damped myosin oscillations in the cell's flanking regions [1]. Spatial extent, period and decay rate of myosin oscillations are calculated. Various regimes of myosin recruitment are predicted. [1] Zhou & Wang, Mol. Biol. Cell 19:318 (2008) [2] Murthy & Wadsworth, J. Cell Sci. 121:2350 (2008)

  16. Structural Basis of Cargo Recognition by Unconventional Myosins in Cellular Trafficking.

    Li, Jianchao; Lu, Qing; Zhang, Mingjie


    Unconventional myosins are a superfamily of actin-based molecular motors playing diverse roles including cellular trafficking, mechanical supports, force sensing and transmission, etc. The variable neck and tail domains of unconventional myosins function to bind to specific cargoes including proteins and lipid vesicles and thus are largely responsible for the diverse cellular functions of myosins in vivo. In addition, the tail regions, together with their cognate cargoes, can regulate activities of the motor heads. This review outlines the advances made in recent years on cargo recognition and cargo binding-induced regulation of the activity of several unconventional myosins including myosin-I, V, VI and X in cellular trafficking. We approach this topic by describing a series of high-resolution structures of the neck and tail domains of these unconventional myosins either alone or in complex with their specific cargoes, and by discussing potential implications of these structural studies on cellular trafficking of these myosin motors. PMID:26842936

  17. Antimyosin imaging in cardiac transplant rejection

    Johnson, L.L.; Cannon, P.J. (Department of Medicine, College of Physicians and Surgeons, Columbia University, New York (United States))


    Fab fragments of antibodies specific for cardiac myosin have been labeled with indium-111 and injected intravenously into animals and into patients with heart transplants. The antibodies, developed by Khaw, Haber, and co-workers, localize in cardiac myocytes that have been damaged irreversibly by ischemia, myocarditis, or the rejection process. After clearance of the labeled antibody from the cardiac blood pool, planar imaging or single photon emission computed tomography is performed. Scintigrams reveal the uptake of the labeled antimyosin in areas of myocardium undergoing transplant rejection. In animal studies, the degree of antimyosin uptake appears to correlate significantly with the degree of rejection assessed at necropsy. In patients, the correlation between scans and pathologic findings from endomyocardial biopsy is not as good, possibly because of sampling error in the endomyocardial biopsy technique. The scan results at 1 year correlate with either late complications (positive) or benign course (negative). Current limitations of the method include slow blood clearance, long half-life of indium-111, and hepatic uptake. Overcoming these limitations represents a direction for current research. It is possible that from these efforts a noninvasive approach to the diagnosis and evaluation of cardiac transplantation may evolve that will decrease the number of endomyocardial biopsies required to evaluate rejection. This would be particularly useful in infants and children. 31 references.

  18. Antimyosin imaging in cardiac transplant rejection

    Fab fragments of antibodies specific for cardiac myosin have been labeled with indium-111 and injected intravenously into animals and into patients with heart transplants. The antibodies, developed by Khaw, Haber, and co-workers, localize in cardiac myocytes that have been damaged irreversibly by ischemia, myocarditis, or the rejection process. After clearance of the labeled antibody from the cardiac blood pool, planar imaging or single photon emission computed tomography is performed. Scintigrams reveal the uptake of the labeled antimyosin in areas of myocardium undergoing transplant rejection. In animal studies, the degree of antimyosin uptake appears to correlate significantly with the degree of rejection assessed at necropsy. In patients, the correlation between scans and pathologic findings from endomyocardial biopsy is not as good, possibly because of sampling error in the endomyocardial biopsy technique. The scan results at 1 year correlate with either late complications (positive) or benign course (negative). Current limitations of the method include slow blood clearance, long half-life of indium-111, and hepatic uptake. Overcoming these limitations represents a direction for current research. It is possible that from these efforts a noninvasive approach to the diagnosis and evaluation of cardiac transplantation may evolve that will decrease the number of endomyocardial biopsies required to evaluate rejection. This would be particularly useful in infants and children. 31 references

  19. Cardiac CT

    Dewey, Marc [Charite - Universitaetsmedizin Berlin (Germany). Inst. fuer Radiologie


    Computed tomography of the heart has become a highly accurate diagnostic modality that is attracting increasing attention. This extensively illustrated book aims to assist the reader in integrating cardiac CT into daily clinical practice, while also reviewing its current technical status and applications. Clear guidance is provided on the performance and interpretation of imaging using the latest technology, which offers greater coverage, better spatial resolution, and faster imaging. The specific features of scanners from all four main vendors, including those that have only recently become available, are presented. Among the wide range of applications and issues to be discussed are coronary artery bypass grafts, stents, plaques, and anomalies, cardiac valves, congenital and acquired heart disease, and radiation exposure. Upcoming clinical uses of cardiac CT, such as plaque imaging and functional assessment, are also explored. (orig.)

  20. Cardiac echinococcosis

    Ivanović-Krstić Branislava A.


    Full Text Available Cardiac hydatid disease is rare. We report on an uncommon hydatid cyst localized in the right ventricular wall, right atrial wall tricuspid valve left atrium and pericard. A 33-year-old woman was treated for cough, fever and chest pain. Cardiac echocardiograpic examination revealed a round tumor (5.8 x 4 cm in the right ventricular free wall and two smaller cysts behind that tumor. There were cysts in right atrial wall and tricuspidal valve as well. Serologic tests for hydatidosis were positive. Computed tomography finding was consistent with diagnosis of hydatid cyst in lungs and right hylar part. Surgical treatment was rejected due to great risk of cardiac perforation. Medical treatment with albendazole was unsuccessful and the patient died due to systemic hydatid involvement of the lungs, liver and central nervous system.

  1. Cardiac sarcoidosis

    Smedema, J.P.; Zondervan, P.E.; van Hagen, P.; ten Cate, F.J.; Bresser, P.; Doubell, A.F.; Pattynama, P.; Hoogsteden, H.C.; Balk, A.H.M.M.


    Sarcoidosis is a multi-system granulomatous disorder of unknown aetiology. Symptomatic cardiac involvement occurs in approximately 5% of patients. The prevalence of sarcoidosis in the Netherlands is unknown, but estimated to be approximately 20 per 100,000 population (3200 patients). We report on five patients who presented with different manifestations of cardiac sarcoidosis, and give a brief review on the current management of this condition. Magnetic Resonance Imaging (MRI) can be of great help in diagnosing this condition as well as in the follow-up of the response to therapy. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:25696121

  2. Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

    Drescher Uwe


    Full Text Available Abstract Background In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A. It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. Results In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK and myosin light chain kinase (MLCK, which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. Conclusions Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

  3. Quantitative determination of type I myosin heavy chain in bovine muscle with anti myosin monoclonal antibodies.

    Picard, B; Leger, J; Robelin, J


    Bovine type I muscle fibers were characterized by enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for slow myosin heavy chains (MHC 1). Two bovine muscles, the Masseter and Cutaneus trunci, were analyzed by different complementary techniques: electrophoresis, immunoblotting and immunohistiology. The results showed that the two muscles have extreme characteristics. The Masseter contains only slow MHC and the Cutaneus trunci is composed solely of rapid MHC (MHC 2a and 2b). A standard for this ELISA was obtained by mixing the two muscles and was used as a reference in the determination of the percentage of MHC 1 in a given muscle. In this study, the Longissimus thoracis of 27 Charolais cattle were examined. The different conditions under which assays were carried out were described and the accuracy of the measurement was calculated. In view of the results, ELISA was chosen for the analysis of muscle fiber types in large numbers of animal specimens. This technique could be used in several research projects to study the muscle characteristics that determine beef quality. PMID:22061628

  4. Cardiac Pacemakers

    A complete survey of physiological biophysical,clinical and engineering aspects of cardiac facing,including the history and an assessment of possible future developments.Among the topics studied are: pacemakers, energy search, heart stimulating with pacemakers ,mathematical aspects of the electric cardio stimulation chronic, pacemaker implants,proceeding,treatment and control

  5. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K. (IIT); (EMBL); (Scripps); (Duke); (Prince); (FSU); (MRC); (U. Florence)


    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  6. Mode coupling points to functionally important residues in myosin II.

    Varol, Onur; Yüret, Deniz; Erman, Burak; Kabakçıoğlu, Alkan


    Relevance of mode coupling to energy/information transfer during protein function, particularly in the context of allosteric interactions is widely accepted. However, existing evidence in favor of this hypothesis comes essentially from model systems. We here report a novel formal analysis of the near-native dynamics of myosin II, which allows us to explore the impact of the interaction between possibly non-Gaussian vibrational modes on fluctutational dynamics. We show that an information-theo...

  7. "Slow" myosins in vertebrate skeletal muscle. An immunofluorescence study


    Specific antisera were raised in rabbits against column-purified myosins from a slow avian muscle, the chicken anterior latissimus dorsi (ALD), and a slow-twitch mammalian muscle, the guinea pig soleus (SOL). The antisera were labeled with fluorescein and applied to sections of muscles from various vertebrae species. Two distinct categories of the slow fibers were identified on the basis of their differential reactivity with the two antisera. Fibers stained by anti-ALD appear to correspond in...

  8. The IQ domain drives nuclear translocation of Nuclear myosin I

    Dzijak, Rastislav; Kahle, Michal; Přidalová, Jarmila; Moško, Tibor; Hozák, Pavel

    St Andrews : Wilhelm Bernhard Workshop, 2007. ---. [Wilhelm Bernhard Workshop on the Cell Nucleus /20./. 27.08.2007-31.08.2007, St. Andrews] R&D Projects: GA MŠk LC545; GA ČR(CZ) GA204/07/1592; GA ČR GD204/05/H023 Institutional research plan: CEZ:AV0Z50520514 Keywords : nucleus * nuclear myosin * calmodulin Subject RIV: EB - Genetics ; Molecular Biology

  9. Smooth muscle myosin: a high force-generating molecular motor.

    VanBuren, P; Guilford, W. H.; Kennedy, G.; Wu, J.; Warshaw, D.M.


    Smooth muscle generates as much force per cross sectional area of muscle as skeletal muscle with only one-fifth the myosin content. Although this apparent difference could be explained at the tissue or cellular level, it is possible that at the molecular level smooth muscle cross-bridges generate greater average force than skeletal muscle cross-bridges. To test this hypothesis, we used an in vitro motility assay (VanBuren et al., 1994) in which either chicken thiophosphorylated gizzard smooth...

  10. Covalent immobilization of myosin for in-vitro motility of actin

    Ellis Bagga; Sunita Kumari; Rajesh Kumar; Rakesh Kumar; R P Bajpai; Lalit M Bharadwaj


    The present study reports the covalent immobilization of myosin on glass surface and in-vitro motility of actin-myosin biomolecular motor. Myosin was immobilized on poly-L-lysine coated glass using heterobifunctional cross linker EDC and characterized by AFM. The in-vitro motility of actin was carried out on the immobilized myosin. It was observed that velocity of actin over myosin increases with increasing actin concentration (0.4-1.0 mg/ml) and was found in the range of 0.40-3.25 m/s. The motility of actin-myosin motor on artificial surfaces is of immense importance for developing nanodevices for healthcare and engineering applications.

  11. Computational characterization of the mutation impact on domain C5 of Myosin Binding Protein C

    Guardiani, Carlo; Cecconi, Fabio; Livi, Roberto


    Three mutations of domain C5 of Myosin Binding Protein C are involved in Familial Hypertrophic Cardiomyopathy. We assess their impact through Molecular Dynamics simulations within the framework of a native-centric coarse-grained model. We characterize the clinical relevance of a mutation by: the extent of temperature shift it induces in the unfolding transition, the increase of the kinetic unfolding rates with respect to the wild type, and by \\Fgr-value analysis. Further analysis of folding stages based on the evolution of native contact probabilities reveals an entropy-driven pathway originating in the protein region close to Res115 and ending up in the area of Res28. The mutation of the former residue thus appears to be responsible for an early interruption of the folding process, leaving the protein largely unstructured and yielding a serious impairment of cardiac function. Mut28, on the contrary, thwarts a late stage of folding when the protein is almost completely native-like, leading to a mild phenotype. A bio-informatic analisys of the long and destabilizing CD loop finally shows an excess of negative charge and a low hydrophobicity indicating a possible classification as a natively unfolded sequence. Accordingly, the folding mechanism is suggested to be coupled with binding with a specific ligand.

  12. Stretch activates myosin light chain kinase in arterial smooth muscle

    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol [32P]phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the [32P]phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in [32P]phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively

  13. Stretch activates myosin light chain kinase in arterial smooth muscle

    Barany, K.; Rokolya, A.; Barany, M. (Univ. of Illinois, Chicago (USA))


    Stretching of porcine carotid arterial muscle increased the phosphorylation of the 20 kDa myosin light chain from 0.23 to 0.68 mol (32P)phosphate/mol light chain, whereas stretching of phorbol dibutyrate treated muscle increased the phosphorylation from 0.30 to 0.91 mol/mol. Two-dimensional gel electrophoresis followed by two-dimensional tryptic phosphopeptide mapping was used to identify the enzyme involved in the stretch-induced phosphorylation. Quantitation of the (32P)phosphate content of the peptides revealed considerable light chain phosphorylation by protein kinase C only in the phorbol dibutyrate treated arterial muscle, whereas most of the light chain phosphorylation was attributable to myosin light chain kinase. Upon stretch of either the untreated or treated muscle, the total increment in (32P)phosphate incorporation into the light chain could be accounted for by peptides characteristic for myosin light chain kinase catalyzed phosphorylation, demonstrating that the stretch-induced phosphorylation is caused by this enzyme exclusively.

  14. Cardiac rhabdomyosarcoma

    Chlumský, Jaromír; Holá, Dana; Hlaváček, Karel; Michal, Michal; Švec, Alexander; Špatenka, Jaroslav; Dušek, Jan


    Cardiac sarcoma is a very rare neoplasm and is difficult to diagnose. The case of a 51-year-old man with a left atrial tumour, locally recurrent three months after its surgical removal, is presented. Computed tomography showed metastatic spread to the lung parenchyma. On revised histology, the mass extirpated was a sarcoma. Because of the metastatic spread, further therapy was symptomatic only; the patient died 15 months after the first manifestation of his problems. Immunohistochemical stain...

  15. Cardiac Calcification

    Morteza Joorabian


    Full Text Available There is a spectrum of different types of cardiac"ncalcifications with the importance and significance"nof each type of cardiac calcification, especially"ncoronary artery calcification. Radiologic detection of"ncalcifications within the heart is quite common. The"namount of coronary artery calcification correlates"nwith the severity of coronary artery disease (CAD."nCalcification of the aortic or mitral valve may indicate"nhemodynamically significant valvular stenosis."nMyocardial calcification is a sign of prior infarction,"nwhile pericardial calcification is strongly associated"nwith constrictive pericarditis. A spectrum of different"ntypes of cardiac calcifications (linear, annular,"ncurvilinear,... could be seen in chest radiography and"nother imaging modalities. So a carful inspection for"ndetection and reorganization of these calcifications"nshould be necessary. Numerous modalities exist for"nidentifying coronary calcification, including plain"nradiography, fluoroscopy, intravascular ultrasound,"nMRI, echocardiography, and conventional, helical and"nelectron-beam CT (EBCT. Coronary calcifications"ndetected on EBCT or helical CT can be quantifie,"nand a total calcification score (Cardiac Calcification"nScoring may be calculated. In an asymptomatic"npopulation and/or patients with concomitant risk"nfactors like diabetes mellitus, determination of the"npresence of coronary calcifications identifies the"npatients at risk for future myocardial infarction and"ncoronary artery disease. In patients without coronary"ncalcifications, future cardiovascular events could"nbe excluded. Therefore, detecting and recognizing"ncalcification related to the heart on chest radiography"nand other imaging modalities such as fluoroscopy, CT"nand echocardiography may have important clinical"nimplications.

  16. Structural basis for myopathic defects engendered by alterations in the myosin rod

    Cammarato, Anthony; Li, Xiaochuan; Reedy, Mary C.; Lee, Chi F.; Lehman, William; Bernstein, Sanford I


    While mutations in the myosin S1 motor domain can directly disrupt the generation and transmission of force along myofibrils and lead to myopathy, the mechanism whereby mutations in the myosin rod influence mechanical function is less clear. Here, we used a combination of various imaging techniques and molecular dynamics simulations to test the hypothesis that perturbations in the myosin rod can disturb normal sarcomeric uniformity and, like motor domain lesions, would influence force product...

  17. Myosin 5a controls insulin granule recruitment during late-phase secretion.

    Ivarsson, Rosita; Jing, Xing-Jun; Waselle, Laurent; Regazzi, Romano; Renström, Erik


    We have examined the importance of the actin-based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS-1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single-cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac-2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single-cell exocytosis. ...

  18. Myosin light chain kinase and Src control membrane dynamics in volume recovery from cell swelling

    Barfod, Elisabeth T.; Moore, Ann L.; Van de Graaf, Benjamin G.; Steven D Lidofsky


     The expansion of the plasma membrane, which occurs during osmotic swelling of epithelia, must be retrieved for volume recovery, but the mechanisms are unknown. Here we have identified myosin light chain kinase (MLCK) as a regulator of membrane internalization in response to osmotic swelling in a model liver cell line. On hypotonic exposure, we found that there was time-dependent phosphorylation of the MLCK substrate myosin II regulatory light chain. At the sides of the cell, MLCK and myosin ...

  19. A myosin activator improves actin assembly and sarcomere function of human-induced pluripotent stem cell-derived cardiomyocytes with a troponin T point mutation.

    Broughton, K M; Li, J; Sarmah, E; Warren, C M; Lin, Y-H; Henze, M P; Sanchez-Freire, V; Solaro, R J; Russell, B


    We have investigated cardiac myocytes derived from human-induced pluripotent stem cells (iPSC-CMs) from two normal control and two family members expressing a mutant cardiac troponin T (cTnT-R173W) linked to dilated cardiomyopathy (DCM). cTnT is a regulatory protein of the sarcomeric thin filament. The loss of this basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable platform for understanding clinically relevant mutations in sarcomeric proteins; however, there are important questions to be addressed with regard to myocyte adaptation that we model here by plating iPSC-CMs on softer substrates (100 kPa) to create a more physiologic environment during recovery and maturation of iPSC-CMs after thawing from cryopreservation. During the first week of culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening, actin content, and actin assembly dynamics were depressed in CMs from the severely affected mutant at 1 wk of culture, but by 2 wk differences were less apparent. Sarcomeric troponin and myosin isoform composition were fetal/neonatal. Furthermore, the troponin complex, reconstituted with wild-type cTnT or recombinant cTnT-R173W, depressed the entry of cross-bridges into the force-generating state, which can be reversed by the myosin activator omecamtiv mecarbil. Therapeutic doses of this drug increased both contractility and the content of F-actin in the mutant iPSC-CMs. Collectively, our data suggest the use of a myosin activation reagent to restore function within patient-specific iPSC-CMs may aid in understanding and treating this familial DCM. PMID:27199119

  20. Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes

    Heuck, Alexander; Du, Tung-Gia; Jellbauer, Stephan; Richter, Klaus; Kruse, Claudia; Jaklin, Sigrun; Müller, Marisa; Buchner, Johannes; Jansen, Ralf-Peter; Niessing, Dierk


    Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing f...

  1. Arabidopsis myosin XI-K localizes to the motile endomembrane vesicles associated with F-actin

    Valera V. Peremyslov


    Full Text Available Plant myosins XI were implicated in cell growth, F-actin organization, and organelle transport, with myosin XI-K being a critical contributor to each of these processes. However, subcellular localization of myosins and the identity of their principal cargoes remain poorly understood. Here, we generated a functionally competent, fluorescent protein-tagged, myosin XI-K, and investigated its spatial distribution within Arabidopsis cells. This myosin was found to associate primarily not with larger organelles (e.g., Golgi as was broadly assumed, but with endomembrane vesicles trafficking along F-actin. Subcellular localization and fractionation experiments indicated that the nature of myosin-associated vesicles is organ- and cell type-specific. In leaves, a large proportion of these vesicles aligned and co-fractionated with a motile ER subdomain. In roots, non-ER vesicles were a dominant myosin cargo. Myosin XI-K showed a striking polar localization at the tips of growing, but not mature, root hairs. These results strongly suggest that a major mechanism whereby myosins contribute to plant cell physiology is vesicle transport, and that this activity can be regulated depending on the growth phase of a cell.

  2. Differential patterns of myosin Va expression during the ontogenesis of the rat hippocampus

    L.S. Brinn


    Full Text Available Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.

  3. Myosin individualized: single nucleotide polymorphisms in energy transduction

    Wieben Eric D


    Full Text Available Abstract Background Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC. Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism. Results An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain. Conclusions Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these

  4. Harmonic Force Spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules

    Sung, Jongmin; Nag, Suman; Mortensen, Kim;


    Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using ‘harmonic force spectroscopy’. In th...

  5. Cardiac conduction system

    The cardiac conduction system is a group of specialized cardiac muscle cells in the walls of the heart that send signals ... to contract. The main components of the cardiac conduction system are the SA node, AV node, bundle ...

  6. Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

    C.C.R. Souza


    Full Text Available Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL. In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

  7. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 00C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain

  8. Pioglitazone reverses down-regulation of cardiac PPARγ expression in Zucker diabetic fatty rats

    Peroxisome proliferator-activated receptor-γ (PPARγ) plays a critical role in peripheral glucose homeostasis and energy metabolism, and inhibits cardiac hypertrophy in non-diabetic animal models. The functional role of PPARγ in the diabetic heart, however, is not fully understood. Therefore, we analyzed cardiac gene expression, metabolic control, and cardiac glucose uptake in male Zucker diabetic fatty rats (ZDF fa/fa) and lean ZDF rats (+/+) treated with the high affinity PPARγ agonist pioglitazone or placebo from 12 to 24 weeks of age. Hyperglycemia, hyperinsulinemia, and hypertriglyceridemia as well as lower cardiac PPARγ, glucose transporter-4 and α-myosin heavy chain expression levels were detected in diabetic ZDF rats compared to lean animals. Pioglitazone increased body weight and improved metabolic control, cardiac PPARγ, glut-4, and α-MHC expression levels in diabetic ZDF rats. Cardiac [18F]fluorodeoxyglucose uptake was not detectable by micro-PET studies in untreated and pioglitazone treated ZDF fa/fa rats but was observed after administration of insulin to pioglitazone treated ZDF fa/fa rats. PPARγ agonists favorably affect cardiac gene expression in type-2 diabetic rats via activation and up-regulation of cardiac PPARγ expression whereas improvement of impaired cardiac glucose uptake in advanced type-2 diabetes requires co-administration of insulin

  9. Dynamics of the Coiled-Coil Unfolding Transition of Myosin Rod Probed by Dissipation Force Spectrum

    Taniguchi, Yukinori; Khatri, Bhavin S.; Brockwell, David J.; Paci, Emanuele; Kawakami, Masaru


    The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic f...

  10. Laing early onset distal myopathy: slow myosin defect with variable abnormalities on muscle biopsy

    P.J. Lamont; B. Udd; F.L. Mastaglia; M. de Visser; P. Hedera; T. Voit; L.R. Bridges; V. Fabian; A. Rozemuller; N.G. Laing


    Background: Laing early onset distal myopathy (MPD1) is an autosomal dominant myopathy caused by mutations within the slow skeletal muscle fibre myosin heavy chain gene, MYH7 It is allelic with myosin storage myopathy, with the commonest form of familial hypertrophic cardiomyopathy, and with one for

  11. Localization of Myosin and Actin in the Pelage and Whisker Hair Follicles of Rat

    The combined effects of myosin II and actin enable muscle and nonmuscle cells to generate forces required for muscle contraction, cell division, cell migration, cellular morphological changes, the maintenance of cellular tension and polarity, and so on. However, except for the case of muscle contraction, the details are poorly understood. We focus on nonmuscle myosin and actin in the formation and maintenance of hair and skin, which include highly active processes in mammalian life with respect to the cellular proliferation, differentiation, and movement. The localization of nonmuscle myosin II and actin in neonatal rat dorsal skin, mystacial pad, hair follicles, and vibrissal follicles was studied by immunohistochemical technique to provide the basis for the elucidation of the roles of these proteins. Specificities of the antibodies were verified by using samples from the relevant tissues and subjecting them to immunoblotting test prior to morphological analyses. The myosin and actin were abundant and colocalized in the spinous and granular layers but scarce in the basal layer of the dorsal and mystacial epidermis. In hair and vibrissal follicles, nonmuscle myosin and actin were colocalized in the outer root sheath and some hair matrix cells adjoining dermal papillae. In contrast, most areas of the inner root sheath and hair matrix appeared to comprise very small amounts of myosin and actin. Hair shaft may comprise significant myosin during the course of its keratinization. These results suggest that the actin-myosin system plays a part in cell movement, differentiation, protection and other key functions of skin and hair cells

  12. My oh my(osin): Insights into how auditory hair cells count, measure, and shape

    Pollock, Lana M.; Chou, Shih-Wei; McDermott, Brian M., Jr.


    The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle’s morphology and hearing.

  13. Intra-axonal myosin and actin in nerve regeneration.

    McQuarrie, Irvine G; Lund, Linda M


    A focused review of sciatic nerve regeneration in the rat model, based on research conducted by the authors, is presented. We examine structural proteins carried distally in the axon by energy-requiring motor enzymes, using protein chemistry and molecular biology techniques in combination with immunohistochemistry. Relevant findings from other laboratories are cited and discussed. The general conclusion is that relatively large amounts of actin and tubulin are required to construct a regenerating axon and that these materials mainly originate in the parent axon. The motor enzymes that carry these proteins forward as macromolecules include kinesin and dynein but probably also include myosin. PMID:19927086

  14. Molecular regulation of skeletal muscle myosin heavy chain isoforms

    Brown, David M.


    Research investigating the regulation of muscle fibre type has traditionally been conducted in vivo, analyzing global changes at a whole muscle level. Broadly, this thesis aimed to explore more “molecular” approaches, utilizing molecular and cell biology to understand the expression and regulation of myosin heavy chain (MyHC) isoforms as an indicator of muscle fibre composition. The mRNA expression profile of six MyHC isoform genes during C2C12 myogenesis was elucidated to reveal that the...

  15. The energetics of allosteric regulation of ADP release from myosin heads.

    Jackson, Del R; Baker, Josh E


    Myosin molecules are involved in a wide range of transport and contractile activities in cells. A single myosin head functions through its ATPase reaction as a force generator and as a mechanosensor, and when two or more myosin heads work together in moving along an actin filament, the interplay between these mechanisms contributes to collective myosin behaviors. For example, the interplay between force-generating and force-sensing mechanisms coordinates the two heads of a myosin V molecule in its hand-over-hand processive stepping along an actin filament. In muscle, it contributes to the Fenn effect and smooth muscle latch. In both examples, a key force-sensing mechanism is the regulation of ADP release via interhead forces that are generated upon actin-myosin binding. Here we present a model describing the mechanism of allosteric regulation of ADP release from myosin heads as a change, DeltaDeltaG(-D), in the standard free energy for ADP release that results from the work, Deltamicro(mech), performed by that myosin head upon ADP release, or DeltaDeltaG(-D) = Deltamicro(mech). We show that this model is consistent with previous measurements for strain-dependent kinetics of ADP release in both myosin V and muscle myosin II. The model makes explicit the energetic cost of accelerating ADP release, showing that acceleration of ADP release during myosin V processivity requires approximately 4 kT of energy whereas the energetic cost for accelerating ADP release in a myosin II-based actin motility assay is only approximately 0.4 kT. The model also predicts that the acceleration of ADP release involves a dissipation of interhead forces. To test this prediction, we use an in vitro motility assay to show that the acceleration of ADP release from both smooth and skeletal muscle myosin II correlates with a decrease in interhead force. Our analyses provide clear energetic constraints for models of the allosteric regulation of ADP release and provide novel, testable insights

  16. Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution.

    Shimada, Masato; Takai, Eisuke; Ejima, Daisuke; Arakawa, Tsutomu; Shiraki, Kentaro


    Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0 M NaCl at 25 °C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution. PMID:25445683

  17. Life without double-headed non-muscle myosin II motor proteins

    Venkaiah eBetapudi


    Full Text Available Non-muscle myosin II motor proteins (myosin IIA, myosin IIB, and myosin IIC belong to a class of molecular motor proteins that are known to transduce cellular free-energy into biological work more efficiently than man-made combustion engines. Nature has given a single myosin II motor protein for lower eukaryotes and multiple for mammals but none for plants in order to provide impetus for their life. These specialized nanomachines drive cellular activities necessary for embryogenesis, organogenesis, and immunity. However, these multifunctional myosin II motor proteins are believed to go awry due to unknown reasons and contribute for the onset and progression of many autosomal-dominant disorders, cataract, deafness, infertility, cancer, kidney, neuronal, and inflammatory diseases. Many pathogens like HIV, Dengue, hepatitis C, and Lymphoma viruses as well as Salmonella and Mycobacteria are now known to take hostage of these dedicated myosin II motor proteins for their efficient pathogenesis. Even after four decades since their discovery, we still have a limited knowledge of how these motor proteins drive cell migration and cytokinesis. We need to enrich our current knowledge on these fundamental cellular processes and develop novel therapeutic strategies to fix mutated myosin II motor proteins in pathological conditions. This is the time to think how to relieve the hijacked myosins from pathogens in order to provide a renewed impetus for patients’ life. Understanding how to steer these molecular motors in proliferating and differentiating stem cells will improve stem cell based-therapeutics development. Given the plethora of cellular activities non-muscle myosin motor proteins are involved in, their importance is apparent for human life.

  18. Cardiac MRI in Athletes

    Luijkx, T.


    Cardiac magnetic resonance imaging (CMR) is often used in athletes to image cardiac anatomy and function and is increasingly requested in the context of screening for pathology that can cause sudden cardiac death (SCD). In this thesis, patterns of cardiac adaptation to sports are investigated with C

  19. Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function.

    Mogami, K; O'Donnell, P T; Bernstein, S I; Wright, T.R.; Emerson, C P


    Mutations of the myosin heavy-chain (MHC) gene of Drosophila melanogaster were identified among a group of dominant flightless and recessive lethal mutants (map position 2-52, 36A8-B1,2). One mutation is a 0.1-kilobase deletion in the 5' region of the MHC gene and reduces MHC protein in the leg and thoracic muscles of heterozygotes to levels found in 36AC haploids. Three mutations are insertions of 8-to 10-kilobase DNA elements within the MHC gene and produce truncated MHC transcripts. Hetero...

  20. Minimum energy reaction profiles for ATP hydrolysis in myosin.

    Grigorenko, Bella L; Kaliman, Ilya A; Nemukhin, Alexander V


    The minimum energy reaction profiles corresponding to two possible reaction mechanisms of adenosine triphosphate (ATP) hydrolysis in myosin are computed in this work within the framework of the quantum mechanics-molecular mechanics (QM/MM) method by using the same partitioning of the model system to the QM and MM parts and the same computational protocol. On the first reaction route, one water molecule performs nucleophilic attack at the phosphorus center P(γ) from ATP while the second water molecule in the closed protein cleft serves as a catalytic base assisted by the Glu residue from the myosin salt bridge. According to the present QM/MM calculations consistent with the results of kinetic studies this reaction pathway is characterized by a low activation energy barrier about 10 kcal/mol. The computed activation energy barrier for the second mechanism, which assumes the penta-coordinated oxyphosphorane transition state upon involvement of single water molecule in the reaction, is considerably higher than that for the two-water mechanism. PMID:21839658

  1. Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

    Nie, Wei

    The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

  2. Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium.

    Uyeda, T Q; Yumura, S


    The cellular slime mold Dictyostelium discoideum is amenable to biochemical, cell biological, and molecular genetic analyses, and offers a unique opportunity for multifaceted approaches to dissect the mechanism of cytokinesis. One of the important questions that are currently under investigation using Dictyostelium is to understand how cleavage furrows or contractile rings are assembled in the equatorial region. Contractile rings consist of a number of components including parallel filaments of actin and myosin II. Phenotypic analyses and in vivo localization studies of cells expressing mutant myosin IIs have demonstrated that myosin II's transport to and localization at the equatorial region does not require regulation by phosphorylation of myosin II, specific amino acid sequences of myosin II, or the motor activity of myosin II. Rather, the transport appears to depend on a myosin II-independent flow of cortical cytoskeleton. What drives the flow of cortical cytoskeleton is still elusive. However, a growing number of mutants that affect assembly of contractile rings have been accumulated. Analyses of these mutations, identification of more cytokinesis-specific genes, and information deriving from other experimental systems, should allow us to understand the mechanism of contractile ring formation and other aspects of cytokinesis. PMID:10816252

  3. Approaches to myosin modelling in a two-phase flow model for cell motility

    Kimpton, L. S.; Whiteley, J. P.; Waters, S. L.; Oliver, J. M.


    A wide range of biological processes rely on the ability of cells to move through their environment. Mathematical models have been developed to improve our understanding of how cells achieve motion. Here we develop models that explicitly track the cell's distribution of myosin within a two-phase flow framework. Myosin is a small motor protein which is important for contracting the cell's actin cytoskeleton and enabling cell motion. The two phases represent the actin network and the cytosol in the cell. We start from a fairly general description of myosin kinetics, advection and diffusion in the two-phase flow framework, then identify a number of sub-limits of the model that may be relevant in practice, two of which we investigate further via linear stability analyses and numerical simulations. We demonstrate that myosin-driven contraction of the actin network destabilizes a stationary steady state leading to cell motion, but that rapid diffusion of myosin and rapid unbinding of myosin from the actin network are stabilizing. We use numerical simulation to investigate travelling-wave solutions relevant to a steadily gliding cell and we consider a reduction of the model in which the cell adheres strongly to the substrate on which it is crawling. This work demonstrates that a number of existing models for the effect of myosin on cell motility can be understood as different sub-limits of our two-phase flow model.

  4. Involvement of myosin light-chain kinase in endothelial cell retraction

    Wysolmerski, R.B.; Lagunoff, D. (Saint Louis Univ. School of Medicine, MO (USA))


    Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylation of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.

  5. Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.

    Batters, Christopher; Woodall, Katy A; Toseland, Christopher P; Hundschell, Christian; Veigel, Claudia


    The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle. PMID:22718767

  6. Quantitation of the distribution and flux of myosin-II during cytokinesis

    Cavet Guy


    Full Text Available Abstract Background During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring. Results The fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex. Conclusion The analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis.

  7. Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction

    A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10 mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 deg. C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement

  8. Characterizations of myosin essential light chain’s N-terminal truncation mutant Δ43 in transgenic mouse papillary muscles by using tension transients in response to sinusoidal length alterations

    Wang, Li; Muthu, Priya; Szczesna-Cordary, Danuta; Kawai, Masataka


    Cross-bridge kinetics were studied at 20 °C in cardiac muscle strips from transgenic (Tg) mice expressing N-terminal 43 amino acid truncation mutation (Δ43) of myosin essential light chain (ELC), and the results were compared to those from Tg-wild type (WT) mice. Sinusoidal length changes were applied to activated skinned papillary muscle strips to induce tension transients, from which two exponential processes were deduced to characterize the cross-bridge kinetics. Their two rate constants w...

  9. Cardiac perception and cardiac control. A review.

    Carroll, D


    The evidence regarding specific cardiac perception and discrimination, and its relationship to voluntary cardiac control, is critically reviewed. Studies are considered in three sections, depending on the method used to assess cardiac perception: questionnaire assessment, discrimination procedures, and heartbeat tracking. The heartbeat tracking procedure would appear to suffer least from interpretative difficulties. Recommendations are made regarding the style of analysis used to assess heartbeat perception in such tracking tasks. PMID:348240

  10. The SAH domain extends the functional length of the myosin lever

    Baboolal, TG; Sakamoto, T.; Forgacs, E; White, HD; Jackson, SM; Takagi, Y.; Farrow, RE; Molloy, JE; Knight, PJ; Sellers, JS; Peckham, M.


    Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to...

  11. Melanophilin and myosin Va track the microtubule plus end on EB1

    Wu, Xufeng S.; Tsan, Grace L.; Hammer, John A.


    In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end–tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp–microtubule...

  12. D-loop of Actin Differently Regulates the Motor Function of Myosins II and V*

    Kubota, Hiroaki; Mikhailenko, Sergey V; Okabe, Harumi; Taguchi, Hideki; Ishiwata, Shin'ichi


    To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on m...

  13. Cooperative folding of muscle myosins: I. Mechanical model

    Caruel, Matthieu; Truskinovsky, Lev


    Mechanically induced folding of passive cross-linkers is a fundamental biological phenomenon. A typical example is a conformational change in myosin II responsible for the power-stroke in skeletal muscles. In this paper we present an athermal perspective on such folding by analyzing the simplest purely mechanical prototype: a parallel bundle of bi-stable units attached to a common backbone. We show that in this analytically transparent model, characterized by a rugged energy landscape, the ground states are always highly coherent, single-phase configurations. We argue that such cooperative behavior, ensuring collective conformational change, is due to the dominance of long- range interactions making the system non-additive. The detailed predictions of our model are in agreement with experimentally observed non-equivalence of fast force recovery in skeletal muscles loaded in soft and hard devices. Some features displayed by the model are also recognizable in the behavior of other biological systems with passiv...

  14. Myosin Vc Is Specialized for Transport on a Secretory Superhighway.

    Sladewski, Thomas E; Krementsova, Elena B; Trybus, Kathleen M


    A hallmark of the well-studied vertebrate class Va myosin is its ability to take multiple steps on actin as a single molecule without dissociating, a feature called "processivity." Therefore, it was surprising when kinetic and single-molecule assays showed that human myosin Vc (MyoVc) was not processive on single-actin filaments [1-3]. We explored the possibility that MyoVc is processive only under conditions that resemble its biological context. Recently, it was shown that zymogen vesicles are transported on actin "superhighways" composed of parallel actin cables nucleated by formins from the plasma membrane [4]. Loss of these cables compromises orderly apical targeting of vesicles. MyoVc has been implicated in transporting secretory vesicles to the apical membrane [5]. We hypothesized that actin cables regulate the processive properties of MyoVc. We show that MyoVc is unique in taking variable size steps, which are frequently in the backward direction. Results obtained with chimeric constructs implicate the lever arm/rod of MyoVc as being responsible for these properties. Actin bundles allow single MyoVc motors to move processively. Remarkably, even teams of MyoVc motors require actin bundles to move continuously at physiological ionic strength. The irregular stepping pattern of MyoVc, which may result from flexibility in the lever arm/rod of MyoVc, appears to be a unique structural adaptation that allows the actin track to spatially restrict the activity of MyoVc to specialized actin cables in order to co-ordinate and target the final stages of vesicle secretion. PMID:27498562

  15. What Is Cardiac Rehabilitation?

    ANSWERS by heart Treatments + Tests What Is Cardiac Rehabilitation? A cardiac rehabilitation (rehab) program takes place in a hospital or ... special help in making lifestyle changes. During your rehabilitation program you’ll… • Have a medical evaluation to ...

  16. Identification and characterization of the Bombyx mori myosin II essential light chain and its effect in BmNPV infection

    L Hao


    Full Text Available Myosin, as a type of molecular motor, is mainly involved in muscle contraction. Recently, myosin research has made considerable progress. However, the function of Bombyx mori myosin remains unclear. In this study, we cloned the BmMyosin II essential light chain (BmMyosin II ELC gene from a cDNA library of silkworm, which had an open reading frame (ORF of 444 bp encoding 147 amino acids (about 16 kDa. After analyzing their sequences, BmMyosin II ELC was similar to the ELCs of 27 other Myosin II types, which contained EFh domain that bound Ca2+. In addition, 28 sequences had five motifs, motifs 1 and 3 were relatively conserved. We constructed two vectors with BmMyosin to transfect MGC803 or BmN, monolayer wound healing of cells indicated they can promote cell migration successfully. For three fifth instar silkworms, Bm306, BmNB, BmBC8, we mainly analyzed the change of BmMyosin II ELC from transcription and translation after infecting with nucleopolyhedrovirus (BmNPV. We found that gene expression of resistant strains were higher than susceptible strains at 12 h, while the result of the translation level was opposite that of the transcription level. Through in vitro protein interactions, we found BmMyosin II ELC can interact with BmNPV ubiquitin.

  17. Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad.

    Ono, Kanako; Ono, Shoichiro


    The myoepithelial sheath in the somatic gonad of the nematodeCaenorhabditis eleganshas nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle-like contractile apparatuses, it has a striated muscle-like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath. PMID:26864628

  18. Therapeutic Inhibition of miR-208a Improves Cardiac Function and Survival During Heart Failure

    Montgomery, Rusty L.; Hullinger, Thomas G.; Semus, Hillary M.; Dickinson, Brent A.; Seto, Anita G.; Lynch, Joshua M.; Stack, Christianna; Latimer, Paul A.; Olson, Eric N.; van Rooij, Eva


    Background Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown. Methods and Results Here, we show that systemic delivery of an antisense oligonucleotide induces potent and sustained silencing of miR-208a in the heart. Therapeutic inhibition of miR-208a by subcutaneous delivery of antimiR-208a during hypertension-induced heart failure in Dahl hypertensive rats dose-dependently prevents pathological myosin switching and cardiac remodeling while improving cardiac function, overall health, and survival. Transcriptional profiling indicates that antimiR-208a evokes prominent effects on cardiac gene expression; plasma analysis indicates significant changes in circulating levels of miRNAs on antimiR-208a treatment. Conclusions These studies indicate the potential of oligonucleotide-based therapies for modulating cardiac miRNAs and validate miR-208 as a potent therapeutic target for the modulation of cardiac function and remodeling during heart disease progression. PMID:21900086

  19. Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers

    Prochniewicz, Ewa; Lowe, Dawn A.; Spakowicz, Daniel J; Higgins, LeeAnn; O'Conor, Kate; Thompson, LaDora V.; Deborah A Ferrington; Thomas, David D.


    To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with d...

  20. Sequential Myosin Phosphorylation Activates Tarantula Thick Filament via a Disorder-Order Transition

    Espinoza-Fonseca, L Michel; Alamo, Lorenzo; Pinto, Antonio; Thomas, David D.; Padrón, Raúl


    Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscle and a secondary (modulatory) role in striated muscle, which is regulated by Ca2+ via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying f...

  1. Identification of recombinant phages containing sequences from different rat myosin heavy chain genes.

    Nudel, U.; Katcoff, D; Carmon, Y; Zevin-Sonkin, D; Levi, Z; Shaul, Y; Shani, M.; Yaffe, D.


    The construction and identification of a recombinant plasmid containing a cDNA insert which hybridizes specifically to myosin heavy chain mRNA is described. The plasmid was used as a probe to screen a rat genomic library for recombinant phages containing myosin heavy chain sequences. Six clones with approximately 15 k bp inserts each were isolated. Digestion with several restriction enzymes and hybridization of the fractionated DNA with the plasmid probe showed that the clones contained 3 dif...

  2. Targeting a Dynamic Protein–Protein Interaction: Fragment Screening against the Malaria Myosin A Motor Complex

    Douse, Christopher H; Vrielink, Nina; Wenlin, Zhang; Cota, Ernesto; Tate, Edward W


    Motility is a vital feature of the complex life cycle of Plasmodium falciparum, the apicomplexan parasite that causes human malaria. Processes such as host cell invasion are thought to be powered by a conserved actomyosin motor (containing myosin A or myoA), correct localization of which is dependent on a tight interaction with myosin A tail domain interacting protein (MTIP) at the inner membrane of the parasite. Although disruption of this protein–protein interaction represents an attractive...


    Sciote, James J.; Morris, Terence J.; Horton, Michael J.; Brandon, Carla A.; Rosen, Clark


    Myosin description in human laryngeal muscles is incomplete, but evidence suggests the presence of type I, IIA, IIX, and tonic myosin heavy chain (MHC) fibers. This study describes the unloaded shortening velocity (V0) of chemically skinned laryngeal muscle fibers measured by the slack test method in relation to MHC content. Skeletal fibers from human laryngeal and limb muscle biopsy specimens were obtained for determination of V0, and subsequently, glycerol–sodium dodecyl sulfate–polyacrylam...

  4. Changes in myosin S1 orientation and force induced by a temperature increase

    Griffiths, Peter J.; Bagni, Maria A; Colombini, Barbara; Amenitsch, Heinz; Bernstorff, Sigrid; Ashley, Christopher C.; Cecchi, Giovanni


    Force generation in myosin-based motile systems is thought to result from an angular displacement of the myosin subfragment 1 (S1) tail domain with respect to the actin filament axis. In muscle, raised temperature increases the force generated by S1, implying a greater change in tail domain angular displacement. We used time-resolved x-ray diffraction to investigate the structural corollary of this force increase by measuring M3 meridional reflection intensity during sinu...

  5. Contraction due to microtubule disruption is associated with increased phosphorylation of myosin regulatory light chain.

    Kolodney, M S; Elson, E L


    Microtubules have been proposed to function as rigid struts which oppose cellular contraction. Consistent with this hypothesis, microtubule disruption strengthens the contractile force exerted by many cell types. We have investigated alternative explanation for the mechanical effects of microtubule disruption: that microtubules modulate the mechanochemical activity of myosin by influencing phosphorylation of the myosin regulatory light chain (LC20). We measured the force produced by a populat...

  6. Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

    Zubov, Eugene O.; Nikolaeva, Olga P.; Kurganov, Boris I.; Dmitrii I. Levitsky; Markov, Denis I.


    We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calor...

  7. Atomic force microscopy reveals differences in cell membrane properties in nuclear myosin I mutant

    Venit, Tomáš; Rohožková, Jana; Kalendová, Alžběta; Hozák, Pavel

    Praha : ČSMS, 2013. s. 25-25. [Mikroskopie 2013. 13.05.2013-14.05.2013, Lednice] R&D Projects: GA ČR GAP305/11/2232; GA TA ČR TE01020118; GA MŠk LH12143 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : atomic force microscopy * cell membrane * myosin 1C * NM1 * nuclear myosin I

  8. Nucleotide Dependent Intrinsic Fluorescence Changes of W29 and W36 in Smooth Muscle Myosin

    van Duffelen, Marilyn; Chrin, Lynn R.; Berger, Christopher L.


    The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin. In this study, an MDE containing all the endogen...

  9. Strong Binding of Myosin Heads Stretches and Twists the Actin Helix

    Tsaturyan, Andrey K.; Koubassova, Natalia; Ferenczi, Michael A.; Narayanan, Theyencheri; Roessle, Manfred; Bershitsky, Sergey Y.


    Calculation of the size of the power stroke of the myosin motor in contracting muscle requires knowledge of the compliance of the myofilaments. Current estimates of actin compliance vary significantly introducing uncertainty in the mechanical parameters of the motor. Using x-ray diffraction on small bundles of permeabilized fibers from rabbit muscle we show that strong binding of myosin heads changes directly the actin helix. The spacing of the 2.73-nm meridional x-ray reflection increased by...

  10. Diffuse infiltrative cardiac tuberculosis

    We present the cardiac magnetic resonance images of an unusual form of cardiac tuberculosis. Nodular masses in a sheet-like distribution were seen to infiltrate the outer myocardium and pericardium along most of the cardiac chambers. The lesions showed significant resolution on antitubercular therapy

  11. Actin-myosin network is required for proper assembly of influenza virus particles

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail:; Nagata, Kyosuke, E-mail:


    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  12. Nonmuscle Myosin II helps regulate synaptic vesicle mobility at the Drosophila neuromuscular junction

    Qiu Xinping


    Full Text Available Abstract Background Although the mechanistic details of the vesicle transport process from the cell body to the nerve terminal are well described, the mechanisms underlying vesicle traffic within nerve terminal boutons is relatively unknown. The actin cytoskeleton has been implicated but exactly how actin or actin-binding proteins participate in vesicle movement is not clear. Results In the present study we have identified Nonmuscle Myosin II as a candidate molecule important for synaptic vesicle traffic within Drosophila larval neuromuscular boutons. Nonmuscle Myosin II was found to be localized at the Drosophila larval neuromuscular junction; genetics and pharmacology combined with the time-lapse imaging technique FRAP were used to reveal a contribution of Nonmuscle Myosin II to synaptic vesicle movement. FRAP analysis showed that vesicle dynamics were highly dependent on the expression level of Nonmuscle Myosin II. Conclusion Our results provide evidence that Nonmuscle Myosin II is present presynaptically, is important for synaptic vesicle mobility and suggests a role for Nonmuscle Myosin II in shuttling vesicles at the Drosophila neuromuscular junction. This work begins to reveal the process by which synaptic vesicles traverse within the bouton.

  13. Actin-myosin network is required for proper assembly of influenza virus particles

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network

  14. Myosin molecule packing within the vertebrate skeletal muscle thick filaments. A complete bipolar model.

    Skubiszak, Ludmila; Kowalczyk, Leszek


    Computer modelling related to the real dimensions of both the whole filament and the myosin molecule subfragments has revealed two alternative modes for myosin molecule packing which lead to the head disposition similar to that observed by EM on the surface of the cross-bridge zone of the relaxed vertebrate skeletal muscle thick filaments. One of the modes has been known for three decades and is usually incorporated into the so-called three-stranded model. The new mode differs from the former one in two aspects: (1) myosin heads are grouped into asymmetrical cross-bridge crowns instead of symmetrical ones; (2) not the whole myosin tail, but only a 43-nm C-terminus of each of them is straightened and near-parallel to the filament axis, the rest of the tail is twisted. Concurrent exploration of these alternative modes has revealed their influence on the filament features. The parameter values for the filament models as well as for the building units depicting the myosin molecule subfragments are verified by experimental data found in the literature. On the basis of the new mode for myosin molecule packing a complete bipolar structure of the thick filament is created. PMID:12545190

  15. Thyroid hormone regulates expression of a transfected human. alpha. -myosin heavy-chain fusion gene in fetal rat heart cells

    Tsika, R.W.; Bahl, J.J.; Morkin, E. (Univ. of Arizona College of Medicine, Tucson (USA)); Leinwand, L.A. (Albert Einstein College of Medicine, Bronx, NY (USA))


    The rat {alpha}-myosin heavy-chain ({alpha}-MHC) gene is regulated by 3,5,3{prime}-triiodo-L-thyronine (T{sub 3}) in ventricular myocardium and is constitutively expressed in atrial tissue. Less is known about regulation of the human gene, but conservation of sequences in the 5{prime}-flanking region between the rat and human {alpha}-MHC genes suggests that the human gene may be regulated similarly. Accordingly, T{sub 3}-responsiveness and tissue-specific expression of human and rat {alpha}-MHC/chloramphenicol acetyltransferase fusion constructs have been compared in rat fetal heart cells, L{sub 6}E{sub 9} myoblasts and myotubes, 3T3 fibroblasts, and HeLa cells. Transient transfection assays revealed a complex series of cis-regulatory elements in the 5{prime}-flanking sequences in the human genes, including a basal promoter element with canonical TATAA and CAAT sequences, two positive regulatory element(s), and two negative regulatory-elements, which markedly diminished both constitutive and T{sub 3}-inducible activity. Interestingly, the human gene seemed to contain a proximal thyroid-hormone response element(s) not found in the rat gene. The authors propose that interactions among the thyroid hormone responsive elements and other cis-acting elements in the human {alpha}-MHC 5{prime}-flanking sequences may be sufficient to explain the characteristic features of expression of this gene in cardiac tissues.

  16. SNPs within the beta myosin heavy chain (MYH7 and the pyruvate kinase muscle (PKM2 genes in horse

    Vincenzo Russo


    Full Text Available Two highly expressed skeletal muscle genes (the MYH7 gene encoding the myosin heavy chain slow/β-cardiac isoform and the PKM2 gene encoding the pyruvate kinase muscle isoforms were investigated with the objective to identify DNA markers in horses. A panel of DNA samples from different horse breeds was analysed using a PCR-single strand conformation polymorphism (SSCP approach. Four and two alleles were identified for the MYH7 and PKM2 loci, respectively. Mendelian inheritance of alleles of the two investigated genes was confirmed analysing horse families. Sequencing of PCR products obtained from the MYH7 and PKM2 genes made it possible to characterise two SSCP alleles for each gene. The polymorphisms found in the MYH7 and PKM2 genes were further studied in 61 and 68 horses of three (Italian Heavy Draught Horse, Italian Saddler and Murgese and five (Franches-Montagnes, Haflinger, Italian Heavy Draught Horse, Murgese and Standardbred breeds, respectively. Allele frequencies of the two loci varied among the considered breeds. The SNPs discovery in MYH7 and PKM2 genes makes it possible to locate new molecular markers to ECA1. The identified markers could be used in association analysis with performance traits in horses.

  17. Cardiac tumours in children

    Parsons Jonathan M


    Full Text Available Abstract Cardiac tumours are benign or malignant neoplasms arising primarily in the inner lining, muscle layer, or the surrounding pericardium of the heart. They can be primary or metastatic. Primary cardiac tumours are rare in paediatric practice with a prevalence of 0.0017 to 0.28 in autopsy series. In contrast, the incidence of cardiac tumours during foetal life has been reported to be approximately 0.14%. The vast majority of primary cardiac tumours in children are benign, whilst approximately 10% are malignant. Secondary malignant tumours are 10–20 times more prevalent than primary malignant tumours. Rhabdomyoma is the most common cardiac tumour during foetal life and childhood. It accounts for more than 60% of all primary cardiac tumours. The frequency and type of cardiac tumours in adults differ from those in children with 75% being benign and 25% being malignant. Myxomas are the most common primary tumours in adults constituting 40% of benign tumours. Sarcomas make up 75% of malignant cardiac masses. Echocardiography, Computing Tomography (CT and Magnetic Resonance Imaging (MRI of the heart are the main non-invasive diagnostic tools. Cardiac catheterisation is seldom necessary. Tumour biopsy with histological assessment remains the gold standard for confirmation of the diagnosis. Surgical resection of primary cardiac tumours should be considered to relieve symptoms and mechanical obstruction to blood flow. The outcome of surgical resection in symptomatic, non-myxomatous benign cardiac tumours is favourable. Patients with primary cardiac malignancies may benefit from palliative surgery but this approach should not be recommended for patients with metastatic cardiac tumours. Surgery, chemotherapy and radiotherapy may prolong survival. The prognosis for malignant primary cardiac tumours is generally extremely poor.

  18. Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5.

    Elisa Belian

    Full Text Available Adult cardiac stem cells (CSCs express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT, and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains. MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized.(1 GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2 Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3 Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

  19. Modelling the effect of myosin X motors on filopodia growth

    Wolff, Katrin; Evans, Martin R; Goryachev, Andrew B; Marenduzzo, Davide


    We present a numerical simulation study of the dynamics of filopodial growth in the presence of active transport by myosin X motors. We employ both a microscopic agent-based model, which captures the stochasticity of the growth process, and a continuum mean-field theory which neglects fluctuations. We show that in the absence of motors, filopodia growth is overestimated by the continuum mean-field theory. Thus fluctuations slow down the growth, especially when the protrusions are driven by a small number (10 or less) of F-actin fibres, and when the force opposing growth (coming from membrane elasticity) is large enough. We also show that, with typical parameter values for eukaryotic cells, motors are unlikely to provide an actin transport mechanism which enhances filopodial size significantly, unless the G-actin concentration within the filopodium greatly exceeds that of the cytosol bulk. We explain these observations in terms of order-of-magnitude estimates of diffusion-induced and advection-induced growth o...

  20. Modelling the effect of myosin X motors on filopodia growth

    We present a numerical simulation study of the dynamics of filopodial growth in the presence of active transport by myosin X motors. We employ both a microscopic agent-based model, which captures the stochasticity of the growth process, and a continuum mean-field theory which neglects fluctuations. We show that in the absence of motors, filopodia growth is overestimated by the continuum mean-field theory. Thus fluctuations slow down the growth, especially when the protrusions are driven by a small number (10 or less) of F-actin fibres, and when the force opposing growth (coming from membrane elasticity) is large enough. We also show that, with typical parameter values for eukaryotic cells, motors are unlikely to provide an actin transport mechanism which enhances filopodial size significantly, unless the G-actin concentration within the filopodium greatly exceeds that of the cytosol bulk. We explain these observations in terms of order-of-magnitude estimates of diffusion-induced and advection-induced growth of a bundle of Brownian ratchets. (paper)

  1. Absence of platelet phenotype in mice lacking the motor protein myosin Va.

    Matthew T Harper

    Full Text Available BACKGROUND: The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. METHODOLOGY/PRINCIPAL FINDINGS: Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a(-/- platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin α(IIbβ(3 activation, Ca(2+ signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a(-/- platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. CONCLUSION: Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets.

  2. Stimulating endogenous cardiac regeneration

    Amanda eFinan


    Full Text Available The healthy adult heart has a low turnover of cardiac myocytes. The renewal capacity, however, is augmented after cardiac injury. Participants in cardiac regeneration include cardiac myocytes themselves, cardiac progenitor cells, and peripheral stem cells, particularly from the bone marrow compartment. Cardiac progenitor cells and bone marrow stem cells are augmented after cardiac injury, migrate to the myocardium, and support regeneration. Depletion studies of these populations have demonstrated their necessary role in cardiac repair. However, the potential of these cells to completely regenerate the heart is limited. Efforts are now being focused on ways to augment these natural pathways to improve cardiac healing, primarily after ischemic injury but in other cardiac pathologies as well. Cell and gene therapy or pharmacological interventions are proposed mechanisms. Cell therapy has demonstrated modest results and has passed into clinical trials. However, the beneficial effects of cell therapy have primarily been their ability to produce paracrine effects on the cardiac tissue and recruit endogenous stem cell populations as opposed to direct cardiac regeneration. Gene therapy efforts have focused on prolonging or reactivating natural signaling pathways. Positive results have been demonstrated to activate the endogenous stem cell populations and are currently being tested in clinical trials. A potential new avenue may be to refine pharmacological treatments that are currently in place in the clinic. Evidence is mounting that drugs such as statins or beta blockers may alter endogenous stem cell activity. Understanding the effects of these drugs on stem cell repair while keeping in mind their primary function may strike a balance in myocardial healing. To maximize endogenous cardiac regeneration,a combination of these approaches couldameliorate the overall repair process to incorporate the participation ofmultiple cell players.

  3. Cardiac remodeling and physical training post myocardial infarction

    Michael; A; Garza; Emily; A; Wason; John; Q; Zhang


    After myocardial infarction(MI), the heart undergoes extensive myocardial remodeling through the accumulation of fibrous tissue in both the infarcted and noninfarcted myocardium, which distorts tissue structure, increases tissue stiffness, and accounts for ventricular dysfunction. There is growing clinical consensus that exercise training may beneficially alter the course of post-MI myocardial remodeling and improve cardiac function. This review summarizes the present state of knowledge regarding the effect of post-MI exercise training on infarcted hearts. Due to the degree of difficulty to study a viable human heart at both protein and molecular levels, most of the detailed studies have been performed by using animal models. Although there are some negative reports indicating that post-MI exercise may further cause deterioration of the wounded hearts, a growing body of research from both human and animal experiments demonstrates that post-MI exercise may beneficially alter the course of wound healing and improve cardiac function. Furthermore, the improved function is likely due to exercise training-induced mitigation of reninangiotensin-aldosterone system, improved balance between matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1, favorable myosin heavy chain isoform switch, diminished oxidative stress, enhanced antioxidant capacity, improved mitochondrial calcium handling, and boosted myocardial angiogenesis. Additionally, meta-analyses revealed that exercise-based cardiac rehabilitation has proven to be effective, and remains one of the least expensive therapies for both the prevention and treatment of cardiovascular disease, and prevents re-infarction.

  4. Preoperative cardiac risk management

    Vidaković Radosav; Poldermans Don; Nešković Aleksandar N.


    Approximately 100 million people undergo noncardiac surgery annually worldwide. It is estimated that around 3% of patients undergoing noncardiac surgery experience a major adverse cardiac event. Although cardiac events, like myocardial infarction, are major cause of perioperative morbidity or mortality, its true incidence is difficult to assess. The risk of perioperative cardiac complications depends mainly on two conditions: 1) identified risk factors, and 2) the type of the surgical p...

  5. Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

    Soon-Jeong JEONG; Su-Gwan KIM; Jiyun YOO; Mi-Young HAN; Joo-Cheol PARK; Heung-Joong KIM; Seong Soo KANG; Baik-Dong CHOI; Moon-Jin JEONG


    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells.Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins.The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ interacted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway,was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  6. Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation.

    Morel, J E; D'hahan, N; Taouil, K; Francin, M; Aguilar, A; Dalbiez, J P; Merah, Z; Grussaute, H; Hilbert, B; Ollagnon, F; Selva, G; Piot, F


    The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb). PMID:9548927

  7. The Role of Dietary Protein Intake and Resistance Training on Myosin Heavy Chain Expression

    Willoughby Darryn S


    Full Text Available Abstract During resistance training the muscle undergoes many changes. Possibly the most profound and significant changes are those that occur in the muscles contractile proteins. Increases in these contractile proteins are one of the primary factors contributing to myofibrillar hypertrophy. The most abundant muscle protein is myosin, which comprises 25% of the total muscle protein. Due to the large amount of skeletal muscle that is composed of myosin, changes in this fiber may have profound effects on skeletal muscle size and strength. The myosin molecule is made up of 6 subunits, 2 very large heavy chains, and 4 smaller light chains. The myosin heavy chain (MHC accounts for 25–30% of all muscle proteins making its size an important factor in skeletal muscle growth. In conjunction with resistance training, dietary protein intake must be adequate to illicit positive adaptations. Although many studies have evaluated the role of dietary protein intake on skeletal muscle changes, few have evaluated the MHC specifically. Research has clearly defined the need for dietary protein and resistance training to facilitate positive changes in skeletal muscle. The purpose of this review was to evaluate the current literature on the effects of dietary protein and resistance training on the expression of the myosin heavy chain.

  8. Internal dynamics of F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

    Various biological functions related to cell motility are driven by the interaction between the partner proteins, actin and myosin. To obtain insights into how this interaction occurs, the internal dynamics of F-actin and myosin subfragment-1 (S1) were characterized by the quasielastic neutron scattering measurements on the solution samples of F-actin and S1. Contributions of the internal motions of the proteins to the scattering spectra were separated from those of the global macromolecular diffusion. Analysis of the spectra arising from the internal dynamics showed that the correlation times of the atomic motions were about two times shorter for F-actin than for S1, suggesting that F-actin fluctuates more rapidly than S1. It was also shown that the fraction of the immobile atoms is larger for S1 than for F-actin. These results suggest that F-actin actively facilitates the binding of myosin by utilizing the more frequent conformational fluctuations than those of S1. - Highlights: • We studied the internal dynamics of F-actin and myosin S1 by neutron scattering. • The correlation times of the atomic motions were smaller for F-actin than for S1. • The fraction of the immobile atoms was also smaller for F-actin than for S1. • Our results suggest that mobility of atoms in F-actin is higher than that in S1. • We propose that high flexibility of F-actin facilitates the binding of myosin

  9. Transportation of nanoscale cargoes by myosin propelled actin filaments.

    Malin Persson

    Full Text Available Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached" or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  10. The MEF2A gene is excluded as a candidate in 3 families with cardiomyopathies that are not linked to myosin

    Bachinski, L.L.; Abchee, A. [Baylor College of Medicine, Houston, TX (United States); Krahe, R. [Dupont Institute, Wilmington, DE (United States)] [and others


    Inherited cardiomyopathies, as exemplified by hypertrophic cardiomyopathy (HCM) and idiopathic dilated cardiomyopathy (DCM), are genetically heterogeneous disorders of the myocardium which serve as paradigms for the growth response of the heart to most stimuli. HCM is an autosomal dominant disease shown to be caused by at least 5 different genes, but {beta}-myosin heavy chain (MYH7) is the only one of these genes to be identified to date. No loci have as yet been identified for DCM. MEF2A is one of at least 4 known MEF2 genes which are members of the MADS box family of transcription factors. MEF2A is expressed in cardiac tissue; thus MEF2A is a logical candidate gene for these disorders of the cardiac growth response. We have investigated the possibility of linkage between a trinucleotide repeat polymorphism in the MEF2A gene and these cardiomyopathies is a DCM family and 2 HCM families not linked to MYH7. MEF2A was excluded as a candidate for DCM (LOD of -9.03) and HCM (LODs of-5.43 and -2.44) in these families. No expansion of this trinucleotide repeat was seen in 100 unrelated HCM probands or in the DCM family, which appears to exhibit anticipation.

  11. Inverse interaction between tropomyosin and phosphorylated myosin in the presence or absence of caldesmon

    Ying Zhang; Houli Zhang; Zeyao Tang; Kazuhiro Kohama; Yuan Lin


    In the present study,co-sedimentation assay,intrinsic fluorescence intensity measurement,and Mg2+-ATPase activity analysis were carried out to investigate the direct effect of tropomyosin (TM) on unphosphorylated myosin (UM) or phosphorylated myosin (PM) in the presence or absence of caldesmon (CaD).Results showed that TM significantly decreased the sedimentation,intrinsic fluorescence intensity,and the Mg2+-ATPase activity of PM,but not UM.In the presence of CaD,TM also significantly decreased these parameters irrespective of myosin phosphorylation,suggesting that the interaction between TM and CaD abolished the effects of TM on PM or UM and that there was an inverse interaction between TM and PM,characterized by the decreased PM sedimentation and intrinsic fluorescence intensity.

  12. Twirling motion of actin filaments in gliding assays with non-processive myosin motors

    Vilfan, Andrej


    We present a model study of gliding assays in which actin filaments are moved by non-processive myosin motors. We show that even if the power stroke of the motor protein has no lateral component, the filaments will rotate around their axis while moving over the surface. Notably, the handedness of this twirling motion is opposite from that of the actin filament structure. It stems from the fact that the gliding actin filament has "target zones" where its subunits point towards the surface and are therefore more accessible for myosin heads. Each myosin head has a higher binding probability before it reaches the center of the target zone than afterwards, which results in a left-handed twirling. We present a stochastic simulation and an approximative analytical solution. The calculated pitch of the twirling motion depends on the filament velocity (ATP concentration). It reaches about 400nm for low speeds and increases with higher speeds.

  13. Metastasis-associated protein Mts1 (S100A4) inhibits CK2-mediated phosphorylation and self-assembly of the heavy chain of nonmuscle myosin

    Kriajevska, M; Bronstein, I B; Scott, D J; Tarabykina, S; Fischer-Larsen, M; Issinger, O; Lukanidin, E

    A role for EF-hand calcium-binding protein Mts1 (S100A4) in the phosphorylation and the assembly of myosin filaments was studied. The nonmuscle myosin molecules form bipolar filaments, which interact with actin filaments to produce a contractile force. Phosphorylation of the myosin plays a regula...

  14. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

    Shenping Wu

    Full Text Available Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  15. Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons.

    Li, Lin; Wu, Xiaomei; Yue, Hai-Yuan; Zhu, Yong-Chuan; Xu, Jianhua


    At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling, and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5-40 Hz) was slowed by acute inhibition of MLCK and down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as

  16. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development

    Peremyslov, Valera V.; Cole, Rex A.; Fowler, John E.; Dolja, Valerian V.


    Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development. PMID:26426395

  17. Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

    Valera V Peremyslov

    Full Text Available Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI, cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

  18. BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

    Ming Zhang


    Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

  19. Statistical Thermodynamics for Actin-Myosin Binding: The Crucial Importance of Hydration Effects.

    Oshima, Hiraku; Hayashi, Tomohiko; Kinoshita, Masahiro


    Actomyosin is an important molecular motor, and the binding of actin and myosin is an essential research target in biophysics. Nevertheless, the physical factors driving or opposing the binding are still unclear. Here, we investigate the role of water in actin-myosin binding using the most reliable statistical-mechanical method currently available for assessing biomolecules immersed in water. This method is characterized as follows: water is treated not as a dielectric continuum but as an ensemble of molecules; the polyatomic structures of proteins are taken into consideration; and the binding free energy is decomposed into physically insightful entropic and energetic components by accounting for the hydration effect to its full extent. We find that the actin-myosin binding brings large gains of electrostatic and Lennard-Jones attractive interactions. However, these gains are accompanied by even larger losses of actin-water and myosin-water electrostatic and LJ attractive interactions. Although roughly half of the energy increase due to the losses is cancelled out by the energy decrease arising from structural reorganization of the water released upon binding, the remaining energy increase is still larger than the energy decrease brought by the gains mentioned above. Hence, the net change in system energy is positive, which opposes binding. Importantly, the binding is driven by a large gain of configurational entropy of water, which surpasses the positive change in system energy and the conformational entropy loss occurring for actin and myosin. The principal physical origin of the large water-entropy gain is as follows: the actin-myosin interface is closely packed with the achievement of high shape complementarity on the atomic level, leading to a large increase in the total volume available to the translational displacement of water molecules in the system and a resultant reduction of water crowding (i.e., entropic correlations among water molecules). PMID

  20. Radiation-induced myosin IIA expression stimulates collagen type I matrix reorganization

    Background and purpose: Extracellular matrix (ECM) reorganization critically contributes to breast cancer (BC) progression and radiotherapy response. We investigated the molecular background and functional consequences of collagen type I (col-I) reorganization by irradiated breast cancer cells (BCC). Materials and methods: Radiation-induced (RI) col-I reorganization was evaluated for MCF-7/6, MCF-7/AZ, T47D and SK-BR-3 BCC. Phase-contrast microscopy and a stressed matrix contraction assay were used for visualization and quantification of col-I reorganization. Cell–matrix interactions were assessed by the inhibition of β1 integrin (neutralizing antibody ‘P5D2’) or focal adhesion kinase (FAK; GSK22560098 small molecule kinase inhibitor). The role of the actomyosin cytoskeleton was explored by western blotting analysis of myosin II expression and activity; and by gene silencing of myosin IIA and pharmacological inhibition of the actomyosin system (blebbistatin, cytochalasin D). BCC death was evaluated by propidium iodide staining. Results: We observed a radiation dose-dependent increase of col-I reorganization by BCC. β1 Integrin/FAK-mediated cell–matrix interactions are essential for RI col-I reorganization. Irradiated BCC are characterized by increased myosin IIA expression and myosin IIA-dependent col-I reorganization. Moreover, RI col-I reorganization by BCC is associated with decreased BCC death, as suggested by pharmacological targeting of the β1 integrin/FAK/myosin IIA pathway. Conclusions: Our data indicate the role of myosin IIA in col-I reorganization by irradiated BCC and reciprocal BCC death

  1. Blunt cardiac rupture.

    Martin, T D; Flynn, T C; Rowlands, B J; Ward, R E; Fischer, R P


    Blunt injury to the heart ranges from contusion to disruption. This report comprises 14 patients seen during a 6-year period with cardiac rupture secondary to blunt trauma. Eight patients were injured in automobile accidents, two patients were injured in auto-pedestrian accidents, two were kicked in the chest by ungulates, and two sustained falls. Cardiac tamponade was suspected in ten patients. Five patients presented with prehospital cardiac arrest or arrested shortly after arrival. All underwent emergency department thoracotomy without survival. Two patients expired in the operating room during attempted cardiac repair; both had significant extracardiac injury. Seven patients survived, three had right atrial injuries, three had right ventricular injuries, and one had a left atrial injury. Cardiopulmonary bypass was not required for repair of the surviving patients. There were no significant complications from the cardiac repair. The history of significant force dispersed over a relatively small area of the precordium as in a kicking injury from an animal or steering wheel impact should alert the physician to possible cardiac rupture. Cardiac rupture should be considered in patients who present with signs of cardiac tamponade or persistent thoracic bleeding after blunt trauma. PMID:6708151

  2. Extensibility of the Extended Tail Domain of Processive and Nonprocessive Myosin V Molecules

    Nagy, Attila; Piszczek, Grzegorz; Sellers, James R.


    Myosin V is a single-molecule motor that moves organelles along actin. When myosin V pulls loads inside the cell in a highly viscous environment, the force on the motor is unlikely to be constant. We propose that the tether between the single-molecule motor and the cargo (i.e., the extended tail domain of the molecule) must be able to absorb the sudden mechanical motions of the motor and allow smooth relaxation of the motion of the cargo to a new position. To test this hypothesis, we compared...

  3. Involvement of headless myosin X in the motility of immortalized gonadotropin-releasing hormone neuronal cells

    WANG, JUN-JIE; Fu, Xiu-Qing; Guo, Yu-Guang; Yuan, Lin; Gao, Qian-Qian; Yu, Hua-Li; Shi, Heng-Liang; Wang, Xing-Zhi; Xiong, Wen-Cheng; Zhu, Xiao-Juan


    Myosin X (Myo X), an unconventional myosin with a tail homology 4-band 4.1/ezrin/radixin/moesin (MyTH4-FERM) tail, is expressed ubiquitously in various mammalian tissues. In addition to the full-length Myo X (Myo X FL), a headless form is synthesized in the brain. So far, little is known about the function of this motor-less Myo X. In this study, the role of the headless Myo X was investigated in immortalized gonadotropin-releasing hormone (GnRH) neuronal cells, NLT. NLT cells overexpressing ...

  4. Opening the Arg-Glu salt bridge in myosin: computational study.

    Kaliman, Ilya; Grigorenko, Bella; Shadrina, Maria; Nemukhin, Alexander


    Opening the Arg-Glu salt bridge in myosin, which presumably succeeds the myosin-catalyzed hydrolysis of adenosine triphosphate, was modeled computationally on the basis of the structures corresponding to the enzyme-substrate and enzyme-product complexes found in the quantum mechanics-molecular mechanics simulations. According to the calculations of the potential of mean force, opening the bridge is considerably facilitated upon termination of the chemical reaction, but does not promote egress of inorganic phosphate by the back-door mechanism. PMID:19506754


    Naber, Nariman; Cooke, Roger; Pate, Edward


    We measured the nucleotide turnover rate of myosin in tarantula leg-muscle fibers by observing single turnovers of the fluorescent nucleotide analog, mantATP, as monitored by the decrease in fluorescence when mantATP is replaced by ATP in a chase experiment. We find a multi-exponential process, with approximately two-thirds of the myosin showing a very slow nucleotide turnover time constant, ~30 minutes. This slow-turnover state is termed the super-relaxed state (SRX). If fibers are incubated...

  6. The Rho kinases I and II regulate different aspects of myosin II activity

    Yoneda, Atsuko; Multhaupt, Hinke A B; Couchman, John R


    The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament...... persistent ROCK II and guanine triphosphate-bound RhoA. In contrast, the microfilament cytoskeleton was enhanced by ROCK II down-regulation. Phagocytic uptake of fibronectin-coated beads was strongly down-regulated in ROCK II-depleted cells but not those lacking ROCK I. These effects originated in part from...

  7. Myosin Vc Is a Molecular Motor That Functions in Secretory Granule Trafficking

    Jacobs, Damon T.; Weigert, Roberto; Grode, Kyle D.; Donaldson, Julie G.; Cheney, Richard E.


    Class V myosins are actin-based motor proteins that have critical functions in organelle trafficking. Of the three class V myosins expressed in mammals, relatively little is known about Myo5c except that it is abundant in exocrine tissues. Here we use MCF-7 cells to identify the organelles that Myo5c associates with, image the dynamics of Myo5c in living cells, and test the functions of Myo5c. Endogenous Myo5c localizes to two distinct compartments: small puncta and slender tubules. Myo5c oft...

  8. Specific Myosins Control Actin Organization, Cell Morphology, and Migration in Prostate Cancer Cells

    Katarzyna A. Makowska; Ruth E. Hughes; Kathryn J. White; Claire M. Wells; Michelle Peckham


    We investigated the myosin expression profile in prostate cancer cell lines and found that Myo1b, Myo9b, Myo10, and Myo18a were expressed at higher levels in cells with high metastatic potential. Moreover, Myo1b and Myo10 were expressed at higher levels in metastatic tumors. Using an siRNA-based approach, we found that knockdown of each myosin resulted in distinct phenotypes. Myo10 knockdown ablated filopodia and decreased 2D migration speed. Myo18a knockdown increased circumferential non-mus...

  9. Atomic force microscopy reveals differences in cell membrane properties in nuclear myosin I mutant

    Venit, Tomáš; Kalendová, Alžběta; Petr, Martin; Rohožková, Jana; Hozák, Pavel

    Praha : Society for Histochemistry, 2013. [55th Symposium of the Society for Histochemistry. 11.06.2013-14.06.2013, Praha] R&D Projects: GA ČR(CZ) GD204/09/H084; GA ČR GAP305/11/2232; GA TA ČR TE01020118; GA MŠk LH12143 Institutional research plan: CEZ:AV0Z50520514 Institutional support: RVO:68378050 Keywords : atomic force microscopy * cell membrane * myosin 1C * NM1 * nuclear myosin I Subject RIV: EB - Genetics ; Molecular Biology

  10. Biomaterials for cardiac regeneration

    Ruel, Marc


    This book offers readers a comprehensive biomaterials-based approach to achieving clinically successful, functionally integrated vasculogenesis and myogenesis in the heart. Coverage is multidisciplinary, including the role of extracellular matrices in cardiac development, whole-heart tissue engineering, imaging the mechanisms and effects of biomaterial-based cardiac regeneration, and autologous bioengineered heart valves. Bringing current knowledge together into a single volume, this book provides a compendium to students and new researchers in the field and constitutes a platform to allow for future developments and collaborative approaches in biomaterials-based regenerative medicine, even beyond cardiac applications. This book also: Provides a valuable overview of the engineering of biomaterials for cardiac regeneration, including coverage of combined biomaterials and stem cells, as well as extracellular matrices Presents readers with multidisciplinary coverage of biomaterials for cardiac repair, including ...

  11. Mathematical cardiac electrophysiology

    Colli Franzone, Piero; Scacchi, Simone


    This book covers the main mathematical and numerical models in computational electrocardiology, ranging from microscopic membrane models of cardiac ionic channels to macroscopic bidomain, monodomain, eikonal models and cardiac source representations. These advanced multiscale and nonlinear models describe the cardiac bioelectrical activity from the cell level to the body surface and are employed in both the direct and inverse problems of electrocardiology. The book also covers advanced numerical techniques needed to efficiently carry out large-scale cardiac simulations, including time and space discretizations, decoupling and operator splitting techniques, parallel finite element solvers. These techniques are employed in 3D cardiac simulations illustrating the excitation mechanisms, the anisotropic effects on excitation and repolarization wavefronts, the morphology of electrograms in normal and pathological tissue and some reentry phenomena. The overall aim of the book is to present rigorously the mathematica...

  12. Cardiac hypertrophy and failure--a disease of adaptation. Modifications in membrane proteins provide a molecular basis for arrhythmogenicity.

    Moalic, J M; Charlemagne, D; Mansier, P; Chevalier, B; Swynghedauw, B


    Cardiac hypertrophy is the physiological adaptation of the heart to chronic mechanical overload. Cardiac failure indicates the limits of the process. Cardiac hypertrophy is only one example of biological adaptation and results from the induction of several changes in gene expression, mostly of the fetal type, including those coding for the myosin heavy chain or the alpha-subunit of the Na+,K(+)-ATPase. From a thermodynamic point of view, the decrease in Vmax allows the heart to produce a normal tension at a lower cost. This process results from changes both in the sarcomere and in the expression of certain membrane proteins. The decrease in calcium transient is determined by several changes in membrane proteins that result in a rather fragile equilibrium in terms of calcium homeostasis. Any abnormal input in calcium will have exaggerated detrimental consequences on a hypertrophied myocyte and may cause automaticity and arrhythmias or an exaggerated response to anoxia in terms of compliance. PMID:8485830

  13. [Cardiac evaluation before non-cardiac surgery].

    Menzenbach, Jan; Boehm, Olaf


    Before non-cardiac surgery, evaluation of cardiac function is no frequent part of surgical treatment. European societies of anesthesiology and cardiology published consensus-guidelines in 2014 to present a reasonable approach for preoperative evaluation. This paper intends to differentiate the composite of perioperative risk and to display the guidelines methodical approach to handle it. Features to identify patients at risk from an ageing population with comorbidities, are the classification of surgical risk, functional capacity and risk indices. Application of diagnostic means, should be used adjusted to this risk estimation. Cardiac biomarkers are useful to discover risk of complications or mortality, that cannot be assessed by clinical signs. After preoperative optimization and perioperative cardiac protection, the observation of the postoperative period remains, to prohibit complications or even death. In consideration of limited resources of intensive care department, postoperative ward rounds beyond intensive care units are considered to be an appropriate instrument to avoid or recognize complications early to reduce postoperative mortality. PMID:27479258

  14. Cardiac metabolism and arrhythmias

    Barth, Andreas S.; Tomaselli, Gordon F.


    Sudden cardiac death remains a leading cause of mortality in the Western world, accounting for up to 20% of all deaths in the U.S.1, 2 The major causes of sudden cardiac death in adults age 35 and older are coronary artery disease (70–80%) and dilated cardiomyopathy (10–15%).3 At the molecular level, a wide variety of mechanisms contribute to arrhythmias that cause sudden cardiac death, ranging from genetic predisposition (rare mutations and common polymorphisms in ion channels and structural...

  15. [Cardiac Rehabilitation 2015].

    Hoffmann, Andreas


    The goals of cardiac rehabilitation are (re-)conditioning and secondary prevention in patients with heart disease or an elevated cardiovascular risk profile. Rehabilitation is based on motivation through education, on adapted physical activity, instruction of relaxation techniques, psychological support and optimized medication. It is performed preferably in groups either in outpatient or inpatient settings. The Swiss working group on cardiac rehabilitation provides a network of institutions with regular quality auditing. Positive effects of rehabilitation programs on mortality and morbidity have been established by numerous studies. Although a majority of patients after cardiac surgery are being referred to rehabilitation, these services are notoriously underused after catheter procedures. PMID:26602848

  16. Comprehensive cardiac rehabilitation

    Kruse, Marie; Hochstrasser, Stefan; Zwisler, Ann-Dorthe O;


    OBJECTIVES: The costs of comprehensive cardiac rehabilitation are established and compared to the corresponding costs of usual care. The effect on health-related quality of life is analyzed. METHODS: An unprecedented and very detailed cost assessment was carried out, as no guidelines existed for...... uncertain and may be as high as euro 1.877. CONCLUSIONS: Comprehensive cardiac rehabilitation is more costly than usual care, and the higher costs are not outweighed by a quality of life gain. Comprehensive cardiac rehabilitation is, therefore, not cost-effective....

  17. Morphological Modifications in Myofibrils by Suppressing Tropomyosin 4α in Chicken Cardiac Myocytes.

    Toyota, Naoji; Fujitsuka, Chiaki; Ishibashi, Goushi; S Yoshida, Lucia; Takano-Ohmuro, Hiromi


    Tropomyosin (TPM) localizes along F-actin and, together with troponin T (TnT) and other components, controls calcium-sensitive muscle contraction. The role of the TPM isoform (TPM4α) that is expressed in embryonic and adult cardiac muscle cells in chicken is poorly understood. To analyze the function of TPM4α in myofibrils, the effects of TPM4α-suppression were examined in embryonic cardiomyocytes by small interference RNA transfection. Localization of myofibril proteins such as TPM, actin, TnT, α-actinin, myosin and connectin was examined by immunofluorescence microscopy on day 5 when almost complete TPM4α-suppression occurred in culture. A unique large structure was detected, consisting of an actin aggregate bulging from the actin bundle, and many curved filaments projecting from the aggregate. TPM, TnT and actin were detected on the large structure, but myosin, connectin, α-actinin and obvious myofibril striations were undetectable. It is possible that TPM4α-suppressed actin filaments are sorted and excluded at the place of the large structure. This suggests that TPM4α-suppression significantly affects actin filament, and that TPM4α plays an important role in constructing and maintaining sarcomeres and myofibrils in cardiac muscle. PMID:27118431

  18. Myosin II Motor Activity in the Lateral Amygdala Is Required for Fear Memory Consolidation

    Gavin, Cristin F.; Rubio, Maria D.; Young, Erica; Miller, Courtney; Rumbaugh, Gavin


    Learning induces dynamic changes to the actin cytoskeleton that are required to support memory formation. However, the molecular mechanisms that mediate filamentous actin (F-actin) dynamics during learning and memory are poorly understood. Myosin II motors are highly expressed in actin-rich growth structures including dendritic spines, and we have…

  19. Model of myosin recruitment to the cell equator for cytokinesis: feedback mechanisms and dynamical regimes

    Veksler, Alexander; Vavylonis, Dimitrios


    The formation and constriction of the contractile ring during cytokinesis, the final step of cell division, depends on the recruitment of motor protein myosin to the cell's equatorial region. During animal cell cytokinesis, cortical myosin filaments (MF) disassemble at the flanking regions and concentrate in the equator. This recruitment depends on myosin motor activity and the Rho proteins that regulate MF assembly and disassembly. Central spindle and astral microtubules help establish a spatial pattern of differential Rho activity. We propose a reaction-diffusion model for the dynamics of MF recruitment to the equatorial region. In the model, the central spindle and mechanical stress promote self-reinforcing MF assembly. Negative feedback is introduced by MF-induced recruitment of inhibitor myosin phosphatase. Our model yields various dynamical regimes and explains both the recruitment of MF to the cleavage furrow and the observed damped MF oscillations in the flanking regions, as well as steady MF assembly. Space and time parameters of MF oscillations are calculated. We predict oscillatory relaxation of cortical MF upon removal of locally-applied external stress.

  20. Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active

    Schoenitzer, Veronika [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Eichner, Norbert [Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany); Clausen-Schaumann, Hauke [Munich University of Applied Sciences, Lothstrasse 34, D-80335 Muenchen, Germany, and Center for NanoScience (CeNS), Geschwister-Scholl-Platz 1, D-80539 Muenchen (Germany); Weiss, Ingrid M., E-mail: [INM - Leibniz Institute for New Materials, Biomineralisation Group, Campus D2.2, D-66123 Saarbruecken (Germany); Universitaet Regensburg, Biochemie I, Universitaetsstrasse 31, D-93053 Regensburg (Germany)


    Highlights: Black-Right-Pointing-Pointer Dictyostelium produces the 264 kDa myosin chitin synthase of bivalve mollusc Atrina. Black-Right-Pointing-Pointer Chitin synthase activity releases chitin, partly associated with the cell surface. Black-Right-Pointing-Pointer Membrane extracts of transgenic slime molds produce radiolabeled chitin in vitro. Black-Right-Pointing-Pointer Chitin producing Dictyostelium cells can be characterized by atomic force microscopy. Black-Right-Pointing-Pointer This model system enables us to study initial processes of chitin biomineralization. -- Abstract: Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA{sup -} cell lines are shown.

  1. Suppression of contractile force in muscle fibers by antibody to myosin subfragment 2.

    Lovell, S; Karr, T; Harrington, W F


    Polyclonal antibody directed against the subfragment-2 region of myosin was purified by affinity chromatography. Skinned muscle fibers that had been preincubated with antibody were able to sustain only 7% of the active isometric force generated by control fibers. The effect of antibody on force production could not be accounted for by inhibition of ATP turnover.

  2. Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkia.

    Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in close species. In this study, MLC with the molecular mass of 18kDa was purified from crayfish (Procambarus clarkii) muscle fibrils. Its physicochemical characterization showed that the...

  3. Brush border myosin Ia inactivation in gastric but not endometrial tumors

    Mazzolini, Rocco; Rodrigues, Paulo; Bazzocco, Sarah; Dopeso, Higinio; Ferreira, Ana M.; Mateo-Lozano, Silvia; Andretta, Elena; Woerner, Stefan M.; Alazzouzi, Hafid; Landolfi, Stefania; Hernandez-Losa, Javier; Macaya, Irati; Suzuki, Hiromu; Ramon y Cajal, Santiago; Mooseker, Mark S.; Mariadason, John M.; Gebert, Johannes; Hofstra, Robert M. W.; Reventos, Jaume; Yamamoto, Hiroyuki; Schwartz, Simo; Arango, Diego


    Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MS

  4. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients

    Conti, Antonio


    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS\\'s pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V.

  5. Myosin concentration underlies cell size–dependent scalability of actomyosin ring constriction

    Wright, Graham D.; Leong, Fong Yew; Chiam, Keng-Hwee; Chen, Yinxiao; Jedd, Gregory; Balasubramanian, Mohan K.


    In eukaryotes, cytokinesis is accomplished by an actomyosin-based contractile ring. Although in Caenorhabditis elegans embryos larger cells divide at a faster rate than smaller cells, it remains unknown whether a similar mode of scalability operates in other cells. We investigated cytokinesis in the filamentous fungus Neurospora crassa, which exhibits a wide range of hyphal circumferences. We found that N. crassa cells divide using an actomyosin ring and larger rings constricted faster than smaller rings. However, unlike in C. elegans, the total amount of myosin remained constant throughout constriction, and there was a size-dependent increase in the starting concentration of myosin in the ring. We predict that the increased number of ring-associated myosin motors in larger rings leads to the increased constriction rate. Accordingly, reduction or inhibition of ring-associated myosin slows down the rate of constriction. Because the mechanical characteristics of contractile rings are conserved, we predict that these findings will be relevant to actomyosin ring constriction in other cell types. PMID:22123864

  6. Review: Ras GTPases and myosin: Qualitative conservation and quantitative diversification in signal and energy transduction.

    Mueller, Matthias P; Goody, Roger S


    Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016. PMID:27018658

  7. Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

    Shuji Ueda

    Full Text Available Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion.

  8. Force dependent biotinylation of myosin IIA by α-catenin tagged with a promiscuous biotin ligase.

    Ueda, Shuji; Blee, Alexandra M; Macway, Katherine G; Renner, Derrick J; Yamada, Soichiro


    Tissues and organs undergo constant physical perturbations and individual cells must respond to mechanical forces to maintain tissue integrity. However, molecular interactions underlying mechano-transduction are not fully defined at cell-cell junctions. This is in part due to weak and transient interactions that are likely prevalent in force-induced protein complexes. Using in situ proximal biotinylation by the promiscuous biotin ligase BirA tagged to α-catenin and a substrate stretch cell chamber, we sought to identify force-dependent molecular interactions surrounding α-catenin, an actin regulator at the sites of cadherin mediated cell-cell adhesion. While E-cadherin, β-catenin, vinculin and actin localize with α-catenin at cell-cell contacts in immuno-fluorescent staining, only β-catenin and plakoglobin were biotinylated, suggesting that this proximal biotinylation is limited to the molecules that are in the immediate vicinity of α-catenin. In mechanically stretched samples, increased biotinylation of non-muscle myosin IIA, but not myosin IIB, suggests close spatial proximity between α-catenin and myosin IIA during substrate stretching. This force-induced biotinylation diminished as myosin II activity was inhibited by blebbistatin. Taken together, this promising technique enables us to identify force sensitive complexes that may be essential for mechano-responses in force bearing cell adhesion. PMID:25806963

  9. Molecular Basis of Cardiac Myxomas

    Pooja Singhal


    Full Text Available Cardiac tumors are rare, and of these, primary cardiac tumors are even rarer. Metastatic cardiac tumors are about 100 times more common than the primary tumors. About 90% of primary cardiac tumors are benign, and of these the most common are cardiac myxomas. Approximately 12% of primary cardiac tumors are completely asymptomatic while others present with one or more signs and symptoms of the classical triad of hemodynamic changes due to intracardiac obstruction, embolism and nonspecific constitutional symptoms. Echocardiography is highly sensitive and specific in detecting cardiac tumors. Other helpful investigations are chest X-rays, magnetic resonance imaging and computerized tomography scan. Surgical excision is the treatment of choice for primary cardiac tumors and is usually associated with a good prognosis. This review article will focus on the general features of benign cardiac tumors with an emphasis on cardiac myxomas and their molecular basis.

  10. Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-gamma

    D.S. Reis


    Full Text Available Actin-based motor protein requirements and nitric oxide (NO production are important features of macrophage activity during phagocytosis or microbicidal processes. Different classes of myosins contribute directly or indirectly to phagocytosis by providing mechanical force for phagosome closure or organelle movement. Recent data have shown the presence of myosins IC, II, V and IXb in phagosomes of bone marrow-derived murine macrophages. In our investigation we demonstrated the presence of different classes of myosins in J774 macrophages. We also analyzed the effect of gamma interferon (IFN-gamma, with or without calcium ionophore or cytochalasin B, on myosins as well as on inducible nitric oxide synthase (iNOS expression and NO production. Myosins IC, II, Va, VI and IXb were identified in J774 macrophages. There was an increase of myosin V expression in IFN-gamma-treated cells. iNOS expression was increased by IFN-gamma treatment, while calcium ionophore and cytochalasin B had a negative influence on both myosin and iNOS expression, which was decreased. The increases in NO synthesis were reflected by increased iNOS expression. Macrophages activated by IFN-gamma released significant amounts of NO when compared to control groups. In contrast, NO production by calcium ionophore- and cytochalasin B-treated cells was similar to that of control cells. These results suggest that IFN-gamma is involved in macrophage activation by stimulating protein production to permit both phagocytosis and microbicidal activity.