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Sample records for human cardiac myocytes

  1. De Novo Human Cardiac Myocytes for Medical Research: Promises and Challenges

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    Hamel, Veronique; Cheng, Kang; Liao, Shudan; Lu, Aizhu; Zheng, Yong; Chen, Yawen; Xie, Yucai

    2017-01-01

    The advent of cellular reprogramming technology has revolutionized biomedical research. De novo human cardiac myocytes can now be obtained from direct reprogramming of somatic cells (such as fibroblasts), from induced pluripotent stem cells (iPSCs, which are reprogrammed from somatic cells), and from human embryonic stem cells (hESCs). Such de novo human cardiac myocytes hold great promise for in vitro disease modeling and drug screening and in vivo cell therapy of heart disease. Here, we review the technique advancements for generating de novo human cardiac myocytes. We also discuss several challenges for the use of such cells in research and regenerative medicine, such as the immature phenotype and heterogeneity of de novo cardiac myocytes obtained with existing protocols. We focus on the recent advancements in addressing such challenges.

  2. Protective function of tocilizumab in human cardiac myocytes ischemia reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Hai-Feng Cheng; Yan Feng; Da-Ming Jiang; Kai-Yu Tao; Min-Jian Kong

    2015-01-01

    Objective:To investigate the protective function of tocilizumab in human cardiac myocytes ischemia-reperfusion injury.Methods:The human cardiac myocytes were treated by tocilizumab with different concentrations(1.0 mg/mL, 3.0 mg/mL, 5.0 mg/mL) for 24 h,then cells were cultured in ischemia environment for 24 h and reperfusion environment for 1 h. The MTT and flow cytometry were used to detect the proliferation and apoptosis of human cardiac myocytes, respectively. The mRNA and protein expressions of Bcl-2 and Bax were measured by qRT-PCR and western blot, respectively.Results:Compared to the negative group, pretreated by tocilizumab could significantly enhance the proliferation viability and suppress apoptosis of human cardiac myocytes after suffering ischemia reperfusion injury(P<0.05).The expression of Bcl-2 in tocilizumab treated group were higher thanNC group(P<0.05), while theBax expression were lower(P<0.05).Conclusions:Tocilizumab could significantly inhibit apoptosis and keep the proliferation viability of human cardiac myocytes after suffering ischemia reperfusion injury. Tocilizumab may obtain a widely application in the protection of ischemia reperfusion injury.

  3. Maturation status of sarcomere structure and function in human iPSC-derived cardiac myocytes.

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    Bedada, Fikru B; Wheelwright, Matthew; Metzger, Joseph M

    2016-07-01

    Human heart failure due to myocardial infarction is a major health concern. The paucity of organs for transplantation limits curative approaches for the diseased and failing adult heart. Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) have the potential to provide a long-term, viable, regenerative-medicine alternative. Significant progress has been made with regard to efficient cardiac myocyte generation from hiPSCs. However, directing hiPSC-CMs to acquire the physiological structure, gene expression profile and function akin to mature cardiac tissue remains a major obstacle. Thus, hiPSC-CMs have several hurdles to overcome before they find their way into translational medicine. In this review, we address the progress that has been made, the void in knowledge and the challenges that remain. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  4. Antioxidant Effect of Human Selenium-containing Single-chain Fv in Rat Cardiac Myocytes

    Institute of Scientific and Technical Information of China (English)

    HUO Rui; SHI Yi; XU Jun-jie; YAN Fei; L(U) Shao-wu; SU Jia-ming; DUAN Yu-jing; FAN Jia; NING Bo; CONG Deng-li; YAN Gang-lin; LUO Gui-min; WEI Jing-yan

    2009-01-01

    Reactive oxygen species(ROS) plays a key role in human heart diseases.Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide.In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3),a new mimic of GPX,a model system of hydrogen peroxide(H2O2)-induced rat cardiac myocyte damage was established.The cardiac myocyte damage was characterized in terms of cell viability,lipid peroxidation,cell membrane integrity,and intracellular H2O2 level.The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability,the decline of malondialdehyde(MDA) production,lactate dehydrogenase(LDH) release,and intracellular H2O2 level.So Se-scFvB3 may have a great potential in the treatment of human heart diseases induced by ROS.

  5. Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

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    Bizy, Alexandra; Guerrero-Serna, Guadalupe; Hu, Bin; Ponce-Balbuena, Daniela; Willis, B. Cicero; Zarzoso, Manuel; Ramirez, Rafael J.; Sener, Michelle F.; Mundada, Lakshmi V.; Klos, Matthew; Devaney, Eric J.; Vikstrom, Karen L.; Herron, Todd J.; Jalife, José

    2014-01-01

    Applications of human induced pluripotent stemcell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricularmyocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. PMID:24095945

  6. Expression of androgen-binding protein (ABP) in human cardiac myocytes.

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    Schock, H W; Herbert, Z; Sigusch, H; Figulla, H R; Jirikowski, G F; Lotze, U

    2006-04-01

    Cardiomyocytes are known to be androgen targets. Changing systemic steroid levels are thought to be linked to various cardiac ailments, including dilated cardiomyopathy (DCM). The mode of action of gonadal steroid hormones on the human heart is unknown to date. In the present study, we used high-resolution immunocytochemistry on semithin sections (1 microm thick), IN SITU hybridization, and mass spectrometry to investigate the expression of androgen-binding protein (ABP) in human myocardial biopsies taken from male patients with DCM. We observed distinct cytoplasmic ABP immunoreactivity in a fraction of the myocytes. IN SITU hybridization with synthetic oligonucleotide probes revealed specific hybridization signals in these cells. A portion of the ABP-positive cells contained immunostaining for androgen receptor. With SELDI TOF mass spectrometry of affinity purified tissue extracts of human myocardium, we confirmed the presence of a 50 kDa protein similar to ABP. Our observations provide evidence of an intrinsic expression of ABP in human heart. ABP may be secreted from myocytes in a paracrine manner perhaps to influence the bioavailabity of gonadal steroids in myocardium.

  7. Effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes cultured with high glucose

    Institute of Scientific and Technical Information of China (English)

    LI Xing; LI Mei-rong; GUO Zhi-xin

    2012-01-01

    Background Diabetic cardiomyopathy is the major cause of morbidity and mortality in diabetic patients.Oxidative stress plays an important role in diabetic cardiomyopathy.This study aimed to investigate the effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes (HCM) cultured with high glucose.Methods The cells were assigned to three group: control group,high glucose group and high glucose plus adiponectin group.After culture for 24,48,72 hours,oxidative stress was evaluated by detecting levels of malondialdehyde (MDA)and superoxide dismutase (SOD) in the supernatant of culture media.The expression of p66Shc and Heme oxygenase-1 (HO-1) was detected by real-time polymerase chain reaction (PCR).Flow cytometry was designed to observe and detect cellular apoptosis.Results Our findings showed significant increase in MDA levels and decrease in SOD activity in the high glucose group compared with the control group (P <0.05).However,MDA levels were significantly decreased and SOD activity was significantly increased in the adiponectin group compared with those in the high-glucose group (P <0.05).The mRNA expression of HO-1 in the high glucose group was significantly increased in a time-dependent manner compared with that in the control group (P <0.05).Adiponectin further increased the mRNA expression of HO-1 induced by high glucose in a time-dependent manner (P <0.05).The expression of p66Shc was significantly increased in high glucose group compared with that in the control group (P <0.05).Adiponectin significantly suppressed the upregulation of p66Shc induced by high glucose (P <0.05).The apoptotic rate of cardiomyocytes was significantly increased in the high glucose group compared with that in the control group while the apoptotic rate in the adiponectin group was remarkably declined in comparison with that in the high glucose group.Conclusion Adiponectin reduces high glucose-induced oxidative stress and apoptosis and plays a

  8. Modulation of the transient outward current (Ito) in rat cardiac myocytes and human Kv4.3 channels by mefloquine.

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    Perez-Cortes, E J; Islas, A A; Arevalo, J P; Mancilla, C; Monjaraz, E; Salinas-Stefanon, E M

    2015-10-15

    The antimalarial drug mefloquine, is known to be a potassium channel blocker, although its mechanism of action has not being elucidated and its effects on the transient outward current (Ito) and the molecular correlate, the Kv4.3 channel has not being studied. Here, we describe the mefloquine-induced inhibition of the rat ventricular Ito and of CHO cells co-transfected with human Kv4.3 and its accessory subunit hKChIP2C by whole-cell voltage-clamp. Mefloquine inhibited rat Ito and hKv4.3+KChIP2C currents in a concentration-dependent manner with a limited voltage dependence and similar potencies (IC50=8.9μM and 10.5μM for cardiac myocytes and Kv4.3 channels, respectively). In addition, mefloquine did not affect the activation of either current but significantly modified the hKv4.3 steady-state inactivation and recovery from inactivation. The effects of this drug was compared with that of 4-aminopyridine (4-AP), a well-known potassium channel blocker and its binding site does not seem to overlap with that of 4-AP.

  9. Mechano-chemo-transduction in cardiac myocytes.

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    Chen-Izu, Ye; Izu, Leighton T

    2017-06-15

    The heart has the ability to adjust to changing mechanical loads. The Frank-Starling law and the Anrep effect describe exquisite intrinsic mechanisms the heart has for autoregulating the force of contraction to maintain cardiac output under changes of preload and afterload. Although these mechanisms have been known for more than a century, their cellular and molecular underpinnings are still debated. How does the cardiac myocyte sense changes in preload or afterload? How does the myocyte adjust its response to compensate for such changes? In cardiac myocytes Ca(2+) is a crucial regulator of contractile force and in this review we compare and contrast recent studies from different labs that address these two important questions. The 'dimensionality' of the mechanical milieu under which experiments are carried out provide important clues to the location of the mechanosensors and the kinds of mechanical forces they can sense and respond to. As a first approximation, sensors inside the myocyte appear to modulate reactive oxygen species while sensors on the cell surface appear to also modulate nitric oxide signalling; both signalling pathways affect Ca(2+) handling. Undoubtedly, further studies will add layers to this simplified picture. Clarifying the intimate links from cellular mechanics to reactive oxygen species and nitric oxide signalling and to Ca(2+) handling will deepen our understanding of the Frank-Starling law and the Anrep effect, and also provide a unified view on how arrhythmias may arise in seemingly disparate diseases that have in common altered myocyte mechanics. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

  10. Early Regulation of Profibrotic Genes in Primary Human Cardiac Myocytes by Trypanosoma cruzi.

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    Aniekanabassi N Udoko

    2016-01-01

    Full Text Available The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes. The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1 transcription factor network components (including FOSB, FOS and JUNB, early growth response proteins 1 and 3 (EGR1, EGR3, and cytokines/chemokines (IL5, IL6, IL13, CCL11, which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic

  11. Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

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    Dorota Jeziorowska

    2017-06-01

    Full Text Available Human induced pluripotent stem cells (iPSCs represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D or, more recently, on monolayer culture (2D. We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response or IWP2 (inhibitor of Wnt production. We firstly found that the level of Troponin T (TNNT2 expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies.

  12. Phosphatidylinositol-bisphosphate regulates intercellular coupling in cardiac myocytes

    DEFF Research Database (Denmark)

    Hofgaard, Johannes P; Banach, Kathrin; Mollerup, Sarah

    2008-01-01

    Changes in the lipid composition of cardiac myocytes have been reported during cardiac hypertrophy, cardiomyopathy, and infarction. Because a recent study indicates a relation between low phosphatidylinositol-bisphosphate (PIP(2)) levels and reduced intercellular coupling, we tested the hypothesi...

  13. Signaling Pathways in Cardiac Myocyte Apoptosis

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    Xia, Peng; Liu, Yuening

    2016-01-01

    Cardiovascular diseases, the number 1 cause of death worldwide, are frequently associated with apoptotic death of cardiac myocytes. Since cardiomyocyte apoptosis is a highly regulated process, pharmacological intervention of apoptosis pathways may represent a promising therapeutic strategy for a number of cardiovascular diseases and disorders including myocardial infarction, ischemia/reperfusion injury, chemotherapy cardiotoxicity, and end-stage heart failure. Despite rapid growth of our knowledge in apoptosis signaling pathways, a clinically applicable treatment targeting this cellular process is currently unavailable. To help identify potential innovative directions for future research, it is necessary to have a full understanding of the apoptotic pathways currently known to be functional in cardiac myocytes. Here, we summarize recent progress in the regulation of cardiomyocyte apoptosis by multiple signaling molecules and pathways, with a focus on the involvement of these pathways in the pathogenesis of heart disease. In addition, we provide an update regarding bench to bedside translation of this knowledge and discuss unanswered questions that need further investigation. PMID:28101515

  14. Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

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    Götz Pilarczyk

    2016-01-01

    Full Text Available The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds.

  15. Network Reconstruction and Systems Analysis of Cardiac Myocyte Hypertrophy Signaling*

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    Ryall, Karen A.; Holland, David O.; Delaney, Kyle A.; Kraeutler, Matthew J.; Parker, Audrey J.; Saucerman, Jeffrey J.

    2012-01-01

    Cardiac hypertrophy is managed by a dense web of signaling pathways with many pathways influencing myocyte growth. A quantitative understanding of the contributions of individual pathways and their interactions is needed to better understand hypertrophy signaling and to develop more effective therapies for heart failure. We developed a computational model of the cardiac myocyte hypertrophy signaling network to determine how the components and network topology lead to differential regulation of transcription factors, gene expression, and myocyte size. Our computational model of the hypertrophy signaling network contains 106 species and 193 reactions, integrating 14 established pathways regulating cardiac myocyte growth. 109 of 114 model predictions were validated using published experimental data testing the effects of receptor activation on transcription factors and myocyte phenotypic outputs. Network motif analysis revealed an enrichment of bifan and biparallel cross-talk motifs. Sensitivity analysis was used to inform clustering of the network into modules and to identify species with the greatest effects on cell growth. Many species influenced hypertrophy, but only a few nodes had large positive or negative influences. Ras, a network hub, had the greatest effect on cell area and influenced more species than any other protein in the network. We validated this model prediction in cultured cardiac myocytes. With this integrative computational model, we identified the most influential species in the cardiac hypertrophy signaling network and demonstrate how different levels of network organization affect myocyte size, transcription factors, and gene expression. PMID:23091058

  16. Hyperplasia of myocyte nuclei in long-term cardiac hypertrophy in rats.

    OpenAIRE

    Olivetti, G; R. Ricci; Anversa, P.

    1987-01-01

    In contrast to observations made in the human heart, hyperplasia of myocyte nuclei has never been demonstrated in experimental cardiac hypertrophy. To test the hypothesis that the duration of the mechanical load more than the magnitude of ventricular hypertrophy may be the inciting stimulus for myocyte nuclei hyperplasia, constriction of the pulmonary artery was produced in rats and the hearts were examined 6 mo later. A 76% increase in right ventricular weight was measured. This hypertrophic...

  17. Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes

    OpenAIRE

    Han, Xiao; Liu, Jian-Xun; Xin-zhi LI

    2010-01-01

    Aim: To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes. Methods: Cardiac myocytes were incubated under starvation conditions (GD) for 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, the abundance of LC3-positive fluoresce...

  18. Sensing Cardiac Electrical Activity With a Cardiac Myocyte--Targeted Optogenetic Voltage Indicator

    NARCIS (Netherlands)

    Chang Liao, Mei-Ling; de Boer, Teun P; Mutoh, Hiroki; Raad, Nour; Richter, Claudia; Wagner, Eva; Downie, Bryan R; Unsöld, Bernhard; Arooj, Iqra; Streckfuss-Bömeke, Katrin; Döker, Stephan; Luther, Stefan; Guan, Kaomei; Wagner, Stefan; Lehnart, Stephan E; Maier, Lars S; Stühmer, Walter; Wettwer, Erich; van Veen, Toon; Morlock, Michael M; Knöpfel, Thomas; Zimmermann, Wolfram-Hubertus

    2015-01-01

    RATIONALE: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac

  19. The Frank-Starling mechanism in vertebrate cardiac myocytes.

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    Shiels, Holly A; White, Ed

    2008-07-01

    The Frank-Starling law of the heart applies to all classes of vertebrates. It describes how stretch of cardiac muscle, up to an optimum length, increases contractility thereby linking cardiac ejection to cardiac filling. The cellular mechanisms underlying the Frank-Starling response include an increase in myofilament sensitivity for Ca2+, decreased myofilament lattice spacing and increased thin filament cooperativity. Stretching of mammalian, amphibian and fish cardiac myocytes reveal that the functional peak of the sarcomere length (SL)-tension relationship occurs at longer SL in the non-mammalian classes. These findings correlate with in vivo cardiac function as non-mammalian vertebrates, such as fish, vary stroke volume to a relatively larger extent than mammals. Thus, it seems the length-dependent properties of individual myocytes are modified to accommodate differences in organ function, and the high extensibility of certain hearts is matched by the extensibility of their myocytes. Reasons for the differences between classes are still to be elucidated, however, the structure of mammalian ventricular myocytes, with larger widths and higher levels of passive stiffness than those from other vertebrate classes may be implicated.

  20. Nuclear morphology and deformation in engineered cardiac myocytes and tissues.

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    Bray, Mark-Anthony P; Adams, William J; Geisse, Nicholas A; Feinberg, Adam W; Sheehy, Sean P; Parker, Kevin K

    2010-07-01

    Cardiac tissue engineering requires finely-tuned manipulation of the extracellular matrix (ECM) microenvironment to optimize internal myocardial organization. The myocyte nucleus is mechanically connected to the cell membrane via cytoskeletal elements, making it a target for the cellular response to perturbation of the ECM. However, the role of ECM spatial configuration and myocyte shape on nuclear location and morphology is unknown. In this study, printed ECM proteins were used to configure the geometry of cultured neonatal rat ventricular myocytes. Engineered one- and two-dimensional tissue constructs and single myocyte islands were assayed using live fluorescence imaging to examine nuclear position, morphology and motion as a function of the imposed ECM geometry during diastolic relaxation and systolic contraction. Image analysis showed that anisotropic tissue constructs cultured on microfabricated ECM lines possessed a high degree of nuclear alignment similar to that found in vivo; nuclei in isotropic tissues were polymorphic in shape with an apparently random orientation. Nuclear eccentricity was also increased for the anisotropic tissues, suggesting that intracellular forces deform the nucleus as the cell is spatially confined. During systole, nuclei experienced increasing spatial confinement in magnitude and direction of displacement as tissue anisotropy increased, yielding anisotropic deformation. Thus, the nature of nuclear displacement and deformation during systole appears to rely on a combination of the passive myofibril spatial organization and the active stress fields induced by contraction. Such findings have implications in understanding the genomic consequences and functional response of cardiac myocytes to their ECM surroundings under conditions of disease.

  1. Cardiac myocyte exosomes: stability, HSP60, and proteomics.

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    Malik, Z A; Kott, K S; Poe, A J; Kuo, T; Chen, L; Ferrara, K W; Knowlton, A A

    2013-04-01

    Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at "physiological" concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated.

  2. Nanomaterials for Cardiac Myocyte Tissue Engineering

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    Rodolfo Amezcua

    2016-07-01

    Full Text Available Since their synthesizing introduction to the research community, nanomaterials have infiltrated almost every corner of science and engineering. Over the last decade, one such field has begun to look at using nanomaterials for beneficial applications in tissue engineering, specifically, cardiac tissue engineering. During a myocardial infarction, part of the cardiac muscle, or myocardium, is deprived of blood. Therefore, the lack of oxygen destroys cardiomyocytes, leaving dead tissue and possibly resulting in the development of arrhythmia, ventricular remodeling, and eventual heart failure. Scarred cardiac muscle results in heart failure for millions of heart attack survivors worldwide. Modern cardiac tissue engineering research has developed nanomaterial applications to combat heart failure, preserve normal heart tissue, and grow healthy myocardium around the infarcted area. This review will discuss the recent progress of nanomaterials for cardiovascular tissue engineering applications through three main nanomaterial approaches: scaffold designs, patches, and injectable materials.

  3. Improving cardiac myocytes performance by CNTs platforms

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    Valentina eMartinelli

    2013-09-01

    Full Text Available The application of nanotechnology to the cardiovascular system has increasingly caught scientists’ attention as a potentially powerful tool for the development of new generation devices able to interface, repair or boost the performance of cardiac tissue. Carbon nanotubes (CNTs are considered as promising materials for nanomedicine applications in general and have been recently tested towards excitable cell growth. CNTs are cylindrically shaped structures made up of rolled-up graphene sheets, with unique electrical, thermal and mechanical properties, able to effectively conducting electrical current in electrochemical interfaces. CNTs-based scaffolds have been recently found to support the in vitro growth of cardiac cells: in particular, their ability to improve cardiomyocytes proliferation, maturation and electrical behavior are making CNTs extremely attractive for the development and exploitation of interfaces able to impact on cardiac cells physiology and function.

  4. Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.

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    Xiaojing Sun

    Full Text Available BACKGROUND: An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart. METHODOLOGY/PRINCIPAL FINDINGS: Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ. CONCLUSIONS/SIGNIFICANCE: The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized.

  5. Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes

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    Han, Xiao; Liu, Jian-xun; Li, Xin-zhi

    2011-01-01

    Aim: To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes. Methods: Cardiac myocytes were incubated under starvation conditions (GD) for 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, the abundance of LC3-positive fluorescent dots in cells, cell viability and cellular ATP levels were determined using immunoblotting, immunofluorescence microscopy, MTT assay and luminometer, respectively. Moreover, electron microscopy (EM) and immunofluorescent duel labeling of LC3 and Caspase-8 were used to examine the characteristics of autophagy and apoptosis. Results: Immunoblot analysis showed that the amount of LC3-II in starving cells increased in a time-dependent manner accompanied by increased LC3-positive fluorescence and decreased cell viability and ATP content. Sal B (50 μmol/L) inhibited the increase in LC3-II, reduced the abundance of LC3 immunofluorescence and intensity of Caspase-8 fluorescence, and enhanced cellular viability and ATP levels in myocytes under GD 3 h, regardless of whether chloroquine was present. Conclusion: Autophagy induced by starvation for 3 h led to cell injury. Sal B protected starving cells by blocking the early stage of autophagic flux and inhibiting apoptosis that occurred during autophagy. PMID:21113177

  6. Acquisition of a Quantitative, Stoichiometrically Conserved Ratiometric Marker of Maturation Status in Stem Cell-Derived Cardiac Myocytes

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    Fikru B. Bedada

    2014-10-01

    Full Text Available There is no consensus in the stem cell field as to what constitutes the mature cardiac myocyte. Thus, helping formalize a molecular signature for cardiac myocyte maturation would advance the field. In the mammalian heart, inactivation of the “fetal” TNNI gene, TNNI1 (ssTnI, together in temporal concert with its stoichiometric replacement by the adult TNNI gene product, TNNI3 (cTnI, represents a quantifiable ratiometric maturation signature. We examined the TNNI isoform transition in human induced pluripotent stem cell (iPSC cardiac myocytes (hiPSC-CMs and found the fetal TNNI signature, even during long-term culture. Rodent stem cell-derived and primary myocytes, however, transitioned to the adult TnI profile. Acute genetic engineering of hiPSC-CMs enabled a rapid conversion toward the mature TnI profile. While there is no single marker to denote the mature cardiac myocyte, we propose that tracking the cTnI:ssTnI protein isoform ratio provides a valuable maturation signature to quantify myocyte maturation status across laboratories.

  7. Isoprenaline enhances local Ca2+ release in cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    Jian-xin SHEN

    2006-01-01

    Aim: Contraction of cardiac myocytes is controlled by the generation and amplification of intracellular Ca2+ signals. The key step of this process is the coupling between sarcolemma L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). β-Adrenergic stimulation is an important regulatory mechanism for this coupling process. But the details underlied the global level, which require local Ca2+ release study are still unclear. The present study is to explore the effects of β-adrenergic stimulation on local Ca2+ release. Methods: Using confocal microscopy combined with loose-seal patch-clamp approaches, effects of isoprenaline (1 μmol·L-1), a β-adrenergic agonist, on local SR Ca2+ release triggered by Ca2+ influx through LCCs in intact rat cardiac myocytes were investigated. Results: Isoprenaline increased the intensity of ensemble averaged local Ca2+ transients, the peak of which displayed a typical bell-shaped voltage-dependence over the membrane voltages ranging from ~-40mV to ~+35mV. Further analysis showed that this enhancement could be explained by the increased coupling fidelity (which refers the increased probability of RyRs activation upon depolarization), and the increased amplitude of evoked Ca2+ sparks (due to more Ca2+ releases through local RyRs). In addition, isoprenaline decreased the first latency, which displayed a typical "U"-shaped voltage-dependence, showing the available acceleration and synchronization of β-adrenergic stimulation on intracellular calcium release. Conclusions: Isoprenaline enhances local Ca2+ release in cardiac myocytes. These results underscore the importance of regulation of β-adrenergic stimulation on local intermolecular signals between LCCs and RyRs in heart cells.

  8. Isolation of cardiac myocytes and fibroblasts from neonatal rat pups.

    Science.gov (United States)

    Golden, Honey B; Gollapudi, Deepika; Gerilechaogetu, Fnu; Li, Jieli; Cristales, Ricardo J; Peng, Xu; Dostal, David E

    2012-01-01

    Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FBs) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Both cell types are relatively easy to culture as monolayers and can be manipulated using molecular and pharmacological tools. Because NRVM cease to proliferate after birth, and FBs undergo phenotypic changes and senescence after a few passages in tissue culture, primary cultures of both cell types are required for experiments. Below we describe methods that provide good cell yield and viability of primary cultures of NRVM and FBs from 0 to 3-day-old neonatal rat pups.

  9. Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    Xiao HAN; Jian-xun LIU; Xin-zhi LI

    2011-01-01

    Aim: To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes.Methods: Cardiac myocytes were incubated under starvation conditions (GD) for O, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sat B (50 μmol/L) in the presence or absence of chloro-quine (3 μmol/L) under GD 3 h, the amount of LC3-11, the abundance of LC3-positive fluorescent dots in cells, cell viability and cellular ATP levels were determined using immunoblotting, immunofluorescence microscopy, MTT assay and luminometer, respectively. More-over, electron microscopy (EM) and immunofluorescent duel labeling of LC3 and Caspase-8 were used to examine the characteristics of autophagy and apoptosis.Results: Immunoblot analysis showed that the amount of LC3-11 in starving cells increased in a time-dependent manner accompanied by increased LC3-positive fluorescence and decreased cell viability and ATP content. Sal B (50 μmol/L) inhibited the increase in LC3-11, reduced the abundance of LC3 immunofluorescence and intensity of Caspase-8 fluorescence, and enhanced cellular viability and ATP levels in myocytes under GD 3 h, regardless of whether chloroquine was present.Conclusion: Autophagy induced by starvation for 3 h led to cell injury. Sal B protected starving cells by blocking the early stage of autophagic flux and inhibiting apoptosis that occurred during autophagy.

  10. Magnesium homeostasis in cardiac myocytes of Mg-deficient rats.

    Directory of Open Access Journals (Sweden)

    Michiko Tashiro

    Full Text Available To study possible modulation of Mg(2+ transport in low Mg(2+ conditions, we fed either a Mg-deficient diet or a Mg-containing diet (control to Wistar rats for 1-6 weeks. Total Mg concentrations in serum and cardiac ventricular tissues were measured by atomic absorption spectroscopy. Intracellular free Mg(2+ concentration ([Mg(2+]i of ventricular myocytes was measured with the fluorescent indicator furaptra. Mg(2+ transport rates, rates of Mg(2+ influx and Mg(2+ efflux, were estimated from the rates of change in [Mg(2+]i during Mg loading/depletion and recovery procedures. In Mg-deficient rats, the serum total Mg concentration (0.29±0.026 mM was significantly lower than in control rats (0.86±0.072 mM after 4-6 weeks of Mg deficiency. However, neither total Mg concentration in ventricular tissues nor [Mg(2+]i of ventricular myocytes was significantly different between Mg-deficient rats and control rats. The rates of Mg(2+ influx and efflux were not significantly different in both groups. In addition, quantitative RT-PCR revealed that Mg deficiency did not substantially change mRNA expression levels of known Mg(2+ channels/transporters (TRPM6, TRPM7, MagT1, SLC41A1 and ACDP2 in heart and kidney tissues. These results suggest that [Mg(2+]i as well as the total Mg content of cardiac myocytes, was well maintained even under chronic hypomagnesemia without persistent modulation in function and expression of major Mg(2+ channels/transporters in the heart.

  11. Microfluidic partitioning of the extracellular space around single cardiac myocytes.

    Science.gov (United States)

    Klauke, Norbert; Smith, Godfrey L; Cooper, Jonathan M

    2007-02-01

    This paper describes the partitioning of the extracellular space around an electrically activated single cardiac myocyte, constrained within a microfluidic device. Central to this new method is the production of a hydrophobic gap-structure, which divides the extracellular space into two distinct microfluidic pools. The content of these pools was controlled using a pair of concentric automated pipets (subsequently called "dual superfusion pipet"), each providing the ability to dispense (i.e., the source, inner pipet) and aspirate (the sink, outer pipet) a buffer solution (perfusate) into each of the two pools. For rapid solution switching around the cell, additional dual superfusion pipets were inserted into the microchannel for defined time periods using a piezostepper, enabling us to add a test solution, such as a drug. Three distinct areas of the cell were manipulated, namely, the microfluidic environment, the cellular membrane, and the intracellular space. Planar integrated microelectrodes enabled the electrical stimulation of the cardiomyocyte and the recording of the evoked action potential. The device was mounted on an inverted microscope to allow simultaneous sarcomere length and epifluorescence measurements during evoked electrical activity, including, for example, the response of the stimulated end of the cardiac myocyte in comparison with the untreated cell end.

  12. The timing statistics of spontaneous calcium release in cardiac myocytes.

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    Mesfin Asfaw

    Full Text Available A variety of cardiac arrhythmias are initiated by a focal excitation that disrupts the regular beating of the heart. In some cases it is known that these excitations are due to calcium (Ca release from the sarcoplasmic reticulum (SR via propagating subcellular Ca waves. However, it is not understood what are the physiological factors that determine the timing of these excitations at both the subcellular and tissue level. In this paper we apply analytic and numerical approaches to determine the timing statistics of spontaneous Ca release (SCR in a simplified model of a cardiac myocyte. In particular, we compute the mean first passage time (MFPT to SCR, in the case where SCR is initiated by spontaneous Ca sparks, and demonstrate that this quantity exhibits either an algebraic or exponential dependence on system parameters. Based on this analysis we identify the necessary requirements so that SCR occurs on a time scale comparable to the cardiac cycle. Finally, we study how SCR is synchronized across many cells in cardiac tissue, and identify a quantitative measure that determines the relative timing of SCR in an ensemble of cells. Using this approach we identify the physiological conditions so that cell-to-cell variations in the timing of SCR is small compared to the typical duration of an SCR event. We argue further that under these conditions inward currents due to SCR can summate and generate arrhythmogenic triggered excitations in cardiac tissue.

  13. Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

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    Quan He

    2014-01-01

    Full Text Available Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress.

  14. Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes-A model

    Energy Technology Data Exchange (ETDEWEB)

    Kanani, S. [Institut Genomique Fonctionelle, 141 Rue de la Cardonille, 34396 Montpellier (France); Institut Non Lineaire de Nice, CNRS and Universite de Nice, 1361 route des Lucioles, 06560 Valbonne (France); Pumir, A. [Institut Non Lineaire de Nice, CNRS and Universite de Nice, 1361 route des Lucioles, 06560 Valbonne (France); Laboratoire J.A. Dieudonne, CNRS and Universite de Nice, Parc Valrose, 06108 Nice (France)], E-mail: alain.pumir@unice.fr; Krinsky, V. [Institut Non Lineaire de Nice, CNRS and Universite de Nice, 1361 route des Lucioles, 06560 Valbonne (France)

    2008-01-07

    One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler-Reuter model, as well as with the elaborate dynamic Luo-Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin-Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I{sub K1} channels is low enough. At too high an expression level of I{sub K1} channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I{sub K1} channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I{sub K1} channels observed in ventricular myocytes, both in the Beeler-Reuter and in the dynamic Luo-Rudy models are too high to allow to observe oscillations. With expression levels below {approx}1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I{sub K1} has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

  15. Protective effect of eicosapentaenoic acid on ouabain toxicity in neonatal rat cardiac myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hallaq, H.; Leaf, A. (Harvard Medical School, Boston, MA (USA)); Sellmayer, A. (Univ. Munchen, (Germany)); Smith, T.W. (Brigham and Women' s Hospital, Boston, MA (USA))

    1990-10-01

    Isolated neonatal cardiac myocytes have been utilized as a model for the study of cardiac arrhythmogenic factors. The myocytes respond to the toxic effects of a potent cardiac glycoside, ouabain at 0.1 mM, by an increase in their spontaneous beating rate and a reduction in amplitude of contractions resulting within minutes in a lethal state of contracture. Incubating the isolated myocytes for 3{endash}5 days in culture medium enriched with 5 {mu}M arachidonic acid had no effect on the development of lethal contracture after subsequent exposure to 0.1 mM ouabain. By contrast, incubating the myocytes for 3{endash}5 days with 5 {mu}M eicosapentaenoic acid completely prevented the toxic effects of ouabain at 0.1 mM. No differences in bumetanide-inhibitable {sup 86}Rb flux were observed between the three preparations. However, measurements with fura-2 of cytosolic free calcium levels indicated that control and arachidonic acid-enriched myocytes developed toxic cytosolic calcium concentrations of 845 {plus minus} 29 and 757 {plus minus} 64 nM, respectively, on exposure to 0.1 mM ouabain, whereas in eicosapentaenoic acid-enriched myocytes, physiologic calcium levels were preserved. Incubating the myocytes with eicosapentaenoic acid for 3{endash}5 days resulted in a small reduction of arachidonic acid and a small but significant increase of eicosapentaenoic acid in membrane phospolipids of the myocytes.

  16. Stochastic Alternating Dynamics for Synchronous EAD-Like Beating Rhythms in Cultured Cardiac Myocytes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ning; ZHANG Hui-Min; LIU Zhi-Qiang; DING Xue-Li; YANG Ming-Hao; GU Hua-Guang; REN Wei

    2009-01-01

    Dissolved cardiac myocytes can couple together and generate synchronous beatings in culture. We observed a synchronized early after-depolarization(EAD)-like rhythm in cultured cardiac myocytes and reproduced the experimental observation in a network mathematical model whose dynamics are close to a Hopf bifurcation. The mechanism for this EAD-like rhythm is attributed to noised-induced stochastic alternatings between the focus and the limit cycle. These results provide novel understandings for pathological heart rhythms like the early immature beatings.

  17. Finite element model to study two dimensional unsteady state calcium distribution in cardiac myocytes

    Directory of Open Access Journals (Sweden)

    Kunal Pathak

    2016-09-01

    Full Text Available The calcium signaling plays a crucial role in expansion and contraction of cardiac myocytes. This calcium signaling is achieved by calcium diffusion, buffering mechanisms and influx in cardiac myocytes. The various calcium distribution patterns required for achieving calcium signaling in myocytes are still not well understood. In this paper an attempt has been made to develop a model of calcium distribution in myocytes incorporating diffusion of calcium, point source and excess buffer approximation. The model has been developed for a two dimensional unsteady state case. Appropriate boundary conditions and initial condition have been framed. The finite element method has been employed to obtain the solution. The numerical results have been used to study the effect of buffers and source amplitude on calcium distribution in myocytes.

  18. Effects of ventricular unloading on apoptosis and atrophy of cardiac myocytes.

    Science.gov (United States)

    Schena, Stefano; Kurimoto, Yoshihiko; Fukada, Johji; Tack, Ivan; Ruiz, Phillip; Pang, Manhui; Striker, Liliane J; Aitouche, Abdelouahab; Pham, Si M

    2004-07-01

    Ventricular unloading decreases cardiac ventricular mass. This loss of ventricular mass can be due to either atrophy (a reversible process) or apoptosis (an irreversible process) of the cardiac myocytes. We investigated the effect of ventricular unloading on atrophy and apoptosis of cardiac myocytes, using working and nonworking transplant heart models in rats. ACI rats underwent heterotopic heart transplantation with two different techniques to create working and nonworking cardiac grafts. Cardiac grafts were harvested at different time points after transplantation. TUNEL, caspase-3 assay, and electron microscopy were used to assess the degree of apoptosis while cellular atrophy was estimated by calculation of the cytoplasmic index (CI = mean sectional cytoplasmic area/nucleus). Ventricular mass reduction was more pronounced in nonworking than in working hearts (P atrophy is the primary mechanism that accounts for myocardial weight reduction following ventricular unloading. The inference is that ventricular unloading by ventricular assist devices may not cause permanent loss of cardiac myocytes, thus allowing for functional recovery.

  19. Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase.

    Science.gov (United States)

    Song, Jianliang; Zhang, Xue-Qian; Wang, JuFang; Cheskis, Ellina; Chan, Tung O; Feldman, Arthur M; Tucker, Amy L; Cheung, Joseph Y

    2008-10-01

    Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct

  20. Construction of calcium release sites in cardiac myocytes

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    Alexandra eZahradnikova

    2012-08-01

    Full Text Available Local character of calcium release in cardiac myocytes, as defined by confocal recordings of calcium sparks, implies independent activation of individual calcium release sites based on ryanodine receptor (RyR channel recruitment. We constructed virtual calcium release sites (vCRSs composed of a variable number of RyR channels distributed in clusters in accordance with the experimentally observed cluster size distribution. The vCRSs consisted either of a single virtual calcium release unit, in which all clusters shared a common dyadic space, or of multiple virtual calcium release units containing one cluster each and having separate dyadic spaces. We explored the stochastic behavior of vCRSs to understand the activation and recruitment of RyRs during calcium sparks. RyRs were represented by the published allosteric gating model that included regulation by cytosolic Ca2+ and Mg2+. The interaction of Mg2+ with the RyR Ca2+-binding sites and the refractory period of vCRSs were optimized to accord with the experimentally observed calcium dependence of calcium spark frequency. The Mg2+-binding parameters of RyRs that provided the best description of spark frequency depended on the number of RyRs assembled in the virtual calcium release sites. Adequate inhibitory effect of Mg2+ on the calcium dependence of RyR open probability was achieved if the virtual calcium release sites contained at least three clusters. For the distribution of the number of open RyRs in evoked calcium sparks to correspond to the experimentally observed distribution of spark calcium release fluxes, at least 3 clusters had to share a common virtual calcium release unit, in which ~ 3 RyRs open to form an average spark. These results reconcile the small cluster size and stochastic placement of RyRs in the release sites with the estimates of the amount of RyR protein, volume density of calcium release sites, and the size of calcium release sites in rat cardiac myocytes.

  1. VAMP-1, VAMP-2, and syntaxin-4 regulate ANP release from cardiac myocytes.

    Science.gov (United States)

    Ferlito, Marcella; Fulton, William B; Zauher, Mohamed A; Marbán, Eduardo; Steenbergen, Charles; Lowenstein, Charles J

    2010-11-01

    ANP is a peptide released by cardiac myocytes that regulates blood pressure and natriuresis. However, the molecular mechanisms controlling ANP release from cardiac myocytes are not defined. We now identify three components of the exocytic machinery that regulate ANP release from atrial myocytes. We found that cardiac myocytes express N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment protein (α-SNAP), and SNAP receptors (SNAREs). Additionally we found that specific SNARE molecules, VAMP-1 and VAMP-2, both co-sediment and co-localize with ANP. Also, one SNARE molecule, syntaxin-4, partially co-sediments and partially co-localizes with ANP. Furthermore, these three SNAREs, syntaxin-4 and VAMP-1 and VAMP-2, form a SNARE complex inside cardiac myocytes. Finally, knockdown of VAMP-1, VAMP-2, or syntaxin-4 blocks regulated release of ANP. In contrast, silencing of VAMP-3 did not have an effect on ANP release. Our data suggest that three specific SNAREs regulate cardiac myocyte exocytosis of ANP. Pathways that modify the exocytic machinery may influence natriuresis and blood pressure.

  2. Ionic Remodeling and Direct Effects of Valsartan on Ionic Currentsin Human Atrial Myocytes with Atrial Fibrillation

    Institute of Scientific and Technical Information of China (English)

    Xue Yumei; Wu Shulin; Deng Chunyu; Qian Weimin; Chen Chunbo

    2004-01-01

    Objectives Previous studies demonstrated that angiotensin receptor antagonists had effects on some potassium channels in guinea pig myocytes and cloned channels that expressed in human cardiac myocytes. This study determined the direct effects of Valsartan on I caL, INa, IKur, IK1 and Ito1 in isolated human atrial myocytes. Methods and Results Specimens of right atrial appendage tissue were obtained from 39 patients with coronary artery and valvular heart diseases during cardiopulmonary bypass procedure. Pre- operation cardiac rhythm was sinus (SR)in 19 patients and was atrial fibrillation (AF) in the others. Single atrial myocyte was isolated by enzymatic dissociation with the chunk method. The ionic currents were recorded using the whole cell coffiguration of the voltage clamp technique. ICaL and Ito1 densities in AF patients were significantly lower than those in SR patients by 74% and 60%, respectively, while IK1density was significantly higher by 34% at command potential of - 120 mV. With 10 μmol/L Valsartan, INa density was significantly decreased by 59% in SR patients and by 66% in AF patients. IKur and IKl density were significantly decreased in only AF patients by 31% and23%, respectively. Conclusions Conclusions Decreased IcaL and Itol and increased IKl at hyperpolarizing potentials in AF patients' atrial myocytes may result from the electrophysiological remodeling by AF. Valsartan significantly decreases INa, IK1 and IKur current densities in AF patients' myocyte, but decreases only INa in SR patients' myocyte, suggesting that Valsartan may be beneficial to the recovering of remolded atria.

  3. Cellular redox status determines sensitivity to BNIP3-mediated cell death in cardiac myocytes

    OpenAIRE

    Lee, Youngil; Kubli, Dieter A.; Hanna, Rita A.; Cortez, Melissa Q.; Lee, Hwa-Youn; Miyamoto, Shigeki; Gustafsson, Åsa B.

    2015-01-01

    The atypical BH3-only protein Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) is an important regulator of hypoxia-mediated cell death. Interestingly, the susceptibility to BNIP3-mediated cell death differs between cells. In this study we examined whether there are mechanistic differences in BNIP3-mediated cell death between neonatal and adult cardiac myocytes. We discovered that BNIP3 is a potent inducer of cell death in neonatal myocytes, whereas adult myocytes are remarkably resi...

  4. Cardiac Hypertrophy Involves both Myocyte Hypertrophy and Hyperplasia in Anemic Zebrafish

    OpenAIRE

    Xiaojing Sun; Tiffany Hoage; Ping Bai; Yonghe Ding; Zhenyue Chen; Ruilin Zhang; Wei Huang; Ashad Jahangir; Barry Paw; Yi-Gang Li; Xiaolei Xu

    2009-01-01

    BACKGROUND: An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chroni...

  5. Novel Protective Role of Endogenous Cardiac Myocyte P2X4 Receptors in Heart Failure

    Science.gov (United States)

    Yang, Tiehong; Shen, Jian-bing; Yang, Ronghua; Redden, John; Dodge-Kafka, Kimberly; Grady, James; Jacobson, Kenneth A.; Liang, Bruce T.

    2014-01-01

    Background Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic P2X4R overexpression can protect against HF, but this does not explain the role of native cardiac P2X4R. Our goal is to define the physiological role of endogenous cardiac myocyte P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Methods and Results Mice established with conditional cardiac-specific P2X4R knockout were subjected to left anterior descending coronary artery ligation–induced postinfarct or transverse aorta constriction–induced pressure overload HF. Knockout cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. Knockout hearts showed normal basal cardiac function but depressed contractile performance in postinfarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R coimmunoprecipitated and colocalized with nitric oxide synthase 3 (eNOS) in wild-type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cyclic GMP, NO formation, and were protected from postinfarct and pressure overload HF. Inhibitor of eNOS, L-N5-(1-iminoethyl)ornithine hydrochloride, blocked the salutary effect of cardiac P2X4R overexpression in postinfarct and pressure overload HF as did eNOS knockout. Conclusions This study establishes a new protective role for endogenous cardiac myocyte P2X4R in HF and is the first to demonstrate a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection. PMID:24622244

  6. Slow [Na+]i dynamics impacts arrhythmogenesis and spiral wave reentry in cardiac myocyte ionic model

    Science.gov (United States)

    Krogh-Madsen, Trine; Christini, David J.

    2017-09-01

    Accumulation of intracellular Na+ is gaining recognition as an important regulator of cardiac myocyte electrophysiology. The intracellular Na+ concentration can be an important determinant of the cardiac action potential duration, can modulate the tissue-level conduction of excitation waves, and can alter vulnerability to arrhythmias. Mathematical models of cardiac electrophysiology often incorporate a dynamic intracellular Na+ concentration, which changes much more slowly than the remaining variables. We investigated the dependence of several arrhythmogenesis-related factors on [Na+]i in a mathematical model of the human atrial action potential. In cell simulations, we found that [Na+]i accumulation stabilizes the action potential duration to variations in several conductances and that the slow dynamics of [Na+]i impacts bifurcations to pro-arrhythmic afterdepolarizations, causing intermittency between different rhythms. In long-lasting tissue simulations of spiral wave reentry, [Na+]i becomes spatially heterogeneous with a decreased area around the spiral wave rotation center. This heterogeneous region forms a functional anchor, resulting in diminished meandering of the spiral wave. Our findings suggest that slow, physiological, rate-dependent variations in [Na+]i may play complex roles in cellular and tissue-level cardiac dynamics.

  7. Uptake and metabolism of the novel peptide angiotensin-(1-12 by neonatal cardiac myocytes.

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    Sarfaraz Ahmad

    Full Text Available BACKGROUND: Angiotensin-(1-12 [Ang-(1-12] functions as an endogenous substrate for the productions of Ang II and Ang-(1-7 by a non-renin dependent mechanism. This study evaluated whether Ang-(1-12 is incorporated by neonatal cardiac myocytes and the enzymatic pathways of ¹²⁵I-Ang-(1-12 metabolism in the cardiac myocyte medium from WKY and SHR rats. METHODOLOGY/PRINCIPAL FINDINGS: The degradation of ¹²⁵I-Ang-(1-12 (1 nmol/L in the cultured medium of these cardiac myocytes was evaluated in the presence and absence of inhibitors for angiotensin converting enzymes 1 and 2, neprilysin and chymase. In both strains uptake of ¹²⁵I-Ang-(1-12 by myocytes occurred in a time-dependent fashion. Uptake of intact Ang-(1-12 was significantly greater in cardiac myocytes of SHR as compared to WKY. In the absence of renin angiotensin system (RAS enzymes inhibitors the hydrolysis of labeled Ang-(1-12 and the subsequent generation of smaller Ang peptides from Ang-(1-12 was significantly greater in SHR compared to WKY controls. ¹²⁵I-Ang-(1-12 degradation into smaller Ang peptides fragments was significantly inhibited (90% in WKY and 71% in SHR in the presence of all RAS enzymes inhibitors. Further analysis of peptide fractions generated through the incubation of Ang-(1-12 in the myocyte medium demonstrated a predominant hydrolytic effect of angiotensin converting enzyme and neprilysin in WKY and an additional role for chymase in SHR. CONCLUSIONS/SIGNIFICANCE: These studies demonstrate that neonatal myocytes sequester angiotensin-(1-12 and revealed the enzymes involved in the conversion of the dodecapeptide substrate to biologically active angiotensin peptides.

  8. Photoelectric recording of mechanical responses of cardiac myocytes.

    Science.gov (United States)

    Meyer, R; Wiemer, J; Dembski, J; Haas, H G

    1987-04-01

    A method to monitor contraction of isolated myocytes by transmicroscopic photometry is illustrated. Two photodiodes are mounted inside an inverse microscope used for visual control of a cell. Illumination of one diode varies in proportion to changes in cell length. The contraction signal is amplified in a comparator circuit. Spatial resolution of the device is in the order of 1 micron which corresponds to about 5% of cell shortening in the fully activated state of contraction. The method was tested on isolated myocytes from guinea-pig ventricle. Optical records of contraction in response to action potentials or during voltage clamp compare well with the contractile behavior of multicellular preparations.

  9. Modulation of excitability, membrane currents and survival of cardiac myocytes by N-acylethanolamines.

    Science.gov (United States)

    Voitychuk, Oleg I; Asmolkova, Valentyna S; Gula, Nadiya M; Sotkis, Ganna V; Galadari, Sehamuddin; Howarth, Frank C; Oz, Murat; Shuba, Yaroslav M

    2012-09-01

    N-acylethanolamines (NAE) are endogenously produced lipids playing important roles in a diverse range of physiological and pathological conditions. In the present study, using whole-cell patch clamp technique, we have for the first time investigated the effects of the most abundantly produced NAEs, N-stearoylethanolamine (SEA) and N-oleoylethanolamine (OEA), on electric excitability and membrane currents in cardiomyocytes isolated from endocardial, epicardial, and atrial regions of neonatal rat heart. SEA and OEA (1-10μM) attenuated electrical activity of the myocytes from all regions of the cardiac muscle by hyperpolarizing resting potential, reducing amplitude, and shortening the duration of the action potential. However, the magnitudes of these effects varied significantly depending on the type of cardiac myocyte (i.e., endocardial, epicardial, atrial) with OEA being generally more potent. OEA and to a lesser extent SEA suppressed in a concentration-dependent manner currents through voltage-gated Na(+) (VGSC) and L-type Ca(2+) (VGCC) channels, but induced variable cardiac myocyte type-dependent effects on background K(+) and Cl(-) conductance. The mechanisms of inhibitory action of OEA on cardiac VGSCs and VGCCs involved influence on channels' activation/inactivation gating and partial blockade of ion permeation. OEA also enhanced the viability of cardiac myocytes by reducing necrosis without a significant effect on apoptosis. We conclude that SEA and OEA attenuate the excitability of cardiac myocytes mainly through inhibition of VGSCs and VGCC-mediated Ca(2+) entry. Since NAEs are known to increase during tissue ischemia and infarction, these effects of NAEs may mediate some of their cardioprotective actions during these pathological conditions.

  10. ROLE OF CALCINEURIN IN ANGIOTENSIN II INDUCED CARDIAC MYOCYTE HYPERTROPHY OF RATS

    Institute of Scientific and Technical Information of China (English)

    符民桂; 张继峰; 许松; 庞永政; 刘乃奎; 唐朝枢

    2001-01-01

    Objective. The present study investigated the role of calcineurin in angiotensin II(AngII) induced cardiac myocyte hypertrophy of rats. Method. The primary cardiac myocytes were cultured under the standard conditions. The calcineurin activity in AngII treated cardiomyocytes was tested by using PNPP;protein synethsis rate was assessed by 3H leucine incorporation; atrial natriuretic factor(ANF) Mrna level was determined by Northern blot analysis. Cell viability was estimated by lactate dehydrogenase(LDH) levels in cultured medium and by dyed cell numbers. Result. After stimulation of 10,100 and 1 000nmol/L of AngII, calcineurin activities in the cardiomyocytes were increased by 13% ,57% (P< 0.05) and 228% (P< 0.01) respectively, compared with control group. Cyclosporin A(CsA), a specific inhibitor of calcineurin, markedly inhibited the calcineurin activity and decreased the 3H leucine incorporation in AngII treated cardiomyocytes in a dose dependent manner. It was also found that CsA slightly reduced the Mrna level of ANF gene in AngII stimulated cardiomyocytes. Conclusion. During AngII induced cardiac myocyte hypertrophy, calcineurin signal pathway is activated, and inhibition of the pathway can attenuate AngII induced cardiac myocyte hypertrophy, which suggests that the calcineurin signal pathway may play an important role in AngII induced myocardial hypertrophy of rats.

  11. An Experimental Model Using Cultured Cardiac Myocytes for a Study of the Generation of Premature Ventricular Contractions Under Ultrasound Exposure

    Science.gov (United States)

    Kudo, Nobuki; Yamamoto, Masaya

    2011-09-01

    It is known that use of a contrast agents in echocardiography increases the probability of generation of premature ventricular contractions (PVCs). As a basic study to elucidate the mechanisms and to reduce adverse effects, the generation of PVCs was investigated using cultured cardiac myocytes instead of the intact heart in vivo. Cardiac myocytes were isolated from neonatal rats and cultured on a cover slip. The myocyte sample was exposed to pulsed ultrasound with microbubbles adjacent to the myocytes, and generation of PVCs was examined with ultrasound exposure at various delay times after onset of myocyte contraction. The experimental results showed that generation of PVCs had a stable threshold delay time and that PVCs were generated only when myocytes were exposed to ultrasound with delay times longer than the threshold. The results indicate that the model used in this study is useful for revealing the mechanisms by which PVCs are induced by ultrasound exposure.

  12. Transformation of adult rat cardiac myocytes in primary culture.

    Science.gov (United States)

    Banyasz, Tamas; Lozinskiy, Ilya; Payne, Charles E; Edelmann, Stephanie; Norton, Byron; Chen, Biyi; Chen-Izu, Ye; Izu, Leighton T; Balke, C William

    2008-03-01

    We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

  13. Excitation-Contraction Coupling between Human Atrial Myocytes with Fibroblasts and Stretch Activated Channel Current: A Simulation Study

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    Heqing Zhan

    2013-01-01

    Full Text Available Myocytes have been regarded as the main objectives in most cardiac modeling studies and attracted a lot of attention. Connective tissue cells, such as fibroblasts (Fbs, also play crucial role in cardiac function. This study proposed an integrated myocyte-Isac-Fb electromechanical model to investigate the effect of Fbs and stretch activated ion channel current (Isac on cardiac electrical excitation conduction and mechanical contraction. At the cellular level, an active Fb model was coupled with a human atrial myocyte electrophysiological model (including Isac and a mechanical model. At the tissue level, electrical excitation conduction was coupled with an elastic mechanical model, in which finite difference method (FDM was used to solve the electrical excitation equations, while finite element method (FEM was used for the mechanics equations. The simulation results showed that Fbs and Isac coupling caused diverse effects on action potential morphology during repolarization, depolarized the resting membrane potential of the human atrial myocyte, slowed down wave propagation, and decreased strains in fibrotic tissue. This preliminary simulation study indicates that Fbs and Isac have important implications for modulating cardiac electromechanical behavior and should be considered in future cardiac modeling studies.

  14. Direct differentiation of atrial and ventricular myocytes from human embryonic stem cells by alternating retinoid signals

    Institute of Scientific and Technical Information of China (English)

    Qiangzhe Zhang; Li Chen; Tian Tian; Xin Wang; Pu Li; Jurgen Hescheler; Guangju Ji; Yue Ma; Junjie Jiang; Pengcheng Han; Qi Yuan; Jing Zhang; Xiaoqian Zhang; Yanyan Xu; Henghua Cao; Qingzhang Meng

    2011-01-01

    Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs)are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the panretinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, hut not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiologicai studies indicated that with 64.7 ± 0.88% (mean ± s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca2+ sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin

  15. Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

    Science.gov (United States)

    Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

    1998-01-01

    Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists

  16. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    Energy Technology Data Exchange (ETDEWEB)

    Morton, M.J., E-mail: michael.morton@astrazeneca.com [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)

    2014-09-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  17. The Electrophysiological Effects of Qiliqiangxin on Cardiac Ventricular Myocytes of Rats

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    Yidong Wei

    2013-01-01

    Full Text Available Qiliqiangxin, a Chinese herb, represents the affection in Ca channel function of cardiac myocytes. It is unknown whether Qiliqiangxin has an effect on Na current and K current because the pharmacological actions of this herb’s compound are very complex. We investigated the rational usage of Qiliqiangxin on cardiac ventricular myocytes of rats. Ventricular myocytes were exposed acutely to 1, 10, and 50 mg/L Qiliqiangxin, and whole cell patch-clamp technique was used to study the acute effects of Qiliqiangxin on Sodium current (INa, outward currents delayed rectifier outward K+ current (IK, slowly activating delayed rectifier outward K+ current (IKs, transient outward K+ current (Ito, and inward rectifier K+ current (IK1. Qiliqiangxin can decrease INa by 28.53%±5.98%, and its IC50 was 9.2 mg/L. 10 and 50 mg/L Qiliqiangxin decreased by 37.2%±6.4% and 55.9%±5.5% summit current density of Ito. 10 and 50 mg/L Qiliqiangxin decreased IKs by 15.51%±4.03% and 21.6%±5.6%. Qiliqiangxin represented a multifaceted pharmacological profile. The effects of Qiliqiangxin on Na and K currents of ventricular myocytes were more profitable in antiarrhythmic therapy in the clinic. We concluded that the relative efficacy of Qiliqiangxin was another choice for the existing antiarrhythmic therapy.

  18. Optimisation of a Generic Ionic Model of Cardiac Myocyte Electrical Activity

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    Tianruo Guo

    2013-01-01

    Full Text Available A generic cardiomyocyte ionic model, whose complexity lies between a simple phenomenological formulation and a biophysically detailed ionic membrane current description, is presented. The model provides a user-defined number of ionic currents, employing two-gate Hodgkin-Huxley type kinetics. Its generic nature allows accurate reconstruction of action potential waveforms recorded experimentally from a range of cardiac myocytes. Using a multiobjective optimisation approach, the generic ionic model was optimised to accurately reproduce multiple action potential waveforms recorded from central and peripheral sinoatrial nodes and right atrial and left atrial myocytes from rabbit cardiac tissue preparations, under different electrical stimulus protocols and pharmacological conditions. When fitted simultaneously to multiple datasets, the time course of several physiologically realistic ionic currents could be reconstructed. Model behaviours tend to be well identified when extra experimental information is incorporated into the optimisation.

  19. T3 and cardiac myocyte cell: a theoretical model.

    Science.gov (United States)

    Athanasios, Tsatsaris; Antonios, Baldoukas; Antonios, Loumousiotis; Eustathios, Koukounaris; Maria, Giota; Despina, Perrea

    2013-08-01

    In the last decades, the outstanding role of Thyroid gland in regulating both physiological and pathological operation of cardiovascular system has been acknowledged worldwide. Three main domains of Thyroid function, that is to say, euthyroidism -hyperthyroidism-hypothyroidism, have a direct impact on cardiac response through a variety of mechanisms. Cellular pathways mediate in cardiac contractility, cardiac output, cardiac rhythm, arterial blood pressure and peripheral vessel resistance. Particular biochemical algorithms exist not only between Thyroid hormones' serum concentration and thyroid gland but also between the hormones' serum level and heart muscle genes. These biochemical pathways primarily regulate the appropriate secretion of levothyroxine (T4) and triiodothyronine(T3) via Thyroid- Stimulating-Hormone(TSH) pituitary system, and secondly adjust the cardiac function. In this study, a mathematic model has been developed describing significant aspects of positive or negative feedback mechanisms of THYRO-CARDIAC (THY-CAR) system along with potential applications of novel up-to-date patents in this area of research.

  20. Direct, differential effects of tamoxifen, 4-hydroxytamoxifen, and raloxifene on cardiac myocyte contractility and calcium handling.

    Directory of Open Access Journals (Sweden)

    Michelle L Asp

    Full Text Available Tamoxifen (Tam, a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT, and related drug raloxifene (Ral on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²⁺ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²⁺ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²⁺ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.

  1. Stability of beating frequency in cardiac myocytes by their community effect measured by agarose microchamber chip

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    Yasuda Kenji

    2005-05-01

    Full Text Available Abstract To understand the contribution of community effect on the stability of beating frequency in cardiac myocyte cell groups, the stepwise network formation of cells as the reconstructive approach using the on-chip agarose microchamber cell microcultivation system with photo-thermal etching method was applied. In the system, the shapes of agarose microstructures were changed step by step with photo-thermal etching of agarose-layer of the chip using a 1064-nm infrared focused laser beam to increase the interaction of cardiac myocyte cells during cultivation. First, individual rat cardiac myocyte in each microstructure were cultivated under isolated condition, and then connected them one by one through newly-created microchannels by photo-thermal etching to compare the contribution of community size for the magnitude of beating stability of the cell groups. Though the isolated individual cells have 50% fluctuation of beating frequency, their stability increased as the number of connected cells increased. And finally when the number reached to eight cells, they stabilized around the 10% fluctuation, which was the same magnitude of the tissue model cultivated on the dish. The result indicates the importance of the community size of cells to stabilize their performance for making cell-network model for using cells for monitoring their functions like the tissue model.

  2. FINITE ELEMENT ANALYSIS OF CARDIAC MYOCYTE DEBONDING AND REORIENTATION DURING CYCLIC SUBSTRATE STRETCH EXPERIMENTS

    Institute of Scientific and Technical Information of China (English)

    Tao Tang; Jun Qiu; Meng Zhang; Zhuo Zhuang

    2009-01-01

    The substrate stretch experiment, which is carried out on several kinds of adherent cells, is usually used to catch the physiological variation and morphological response to cyclic substrate deformation. In this paper, stretch loading was exerted on cardiac myocytes cultured on silica substrates using a custom-made substrate stretch device. The effect of stretch on the alignment orientation of cardiac myocytes was studied through morphocytological statistics. Under cyclic stretch stimulus, the long axes of cardiac myocytes oriented perpendicularly to the stretch direction for continuous stretch acting. However, the mechanism underlying these behaviors is not well understood from such in vitro tests. Finite element (FE) model was developed in the analysis to investigate these behaviors. Xu-Needleman formulation was used to define the interaction behavior for contact surfaces between cell and substrate. The role of cell viscoelasticity nature is studied in adherent cell debonding with the substrate and aligning perpendicular to the stretch direction during long time cyclic stretch stimulation. There were four different strain magnitudes considered in the simulation to find out the cell debonding affected by the cyclic strains. The potential role of cyclic strain frequency in regulating cell debonding and alignment was also studied using FE analysis.

  3. Modeling calcium wave based on anomalous subdiffusion of calcium sparks in cardiac myocytes.

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    Xi Chen

    Full Text Available Ca(2+ sparks and Ca(2+ waves play important roles in calcium release and calcium propagation during the excitation-contraction (EC coupling process in cardiac myocytes. Although the classical Fick's law is widely used to model Ca(2+ sparks and Ca(2+ waves in cardiac myocytes, it fails to reasonably explain the full-width at half maximum(FWHM paradox. However, the anomalous subdiffusion model successfully reproduces Ca(2+ sparks of experimental results. In this paper, in the light of anomalous subdiffusion of Ca(2+ sparks, we develop a mathematical model of calcium wave in cardiac myocytes by using stochastic Ca(2+ release of Ca(2+ release units (CRUs. Our model successfully reproduces calcium waves with physiological parameters. The results reveal how Ca(2+ concentration waves propagate from an initial firing of one CRU at a corner or in the middle of considered region, answer how large in magnitude of an anomalous Ca(2+ spark can induce a Ca(2+ wave. With physiological Ca(2+ currents (2pA through CRUs, it is shown that an initial firing of four adjacent CRUs can form a Ca(2+ wave. Furthermore, the phenomenon of calcium waves collision is also investigated.

  4. Effects of Adiponectin on Cell Oxidative Stress and Apoptosis in Human Cardiac Myocytes Cultured with High Glucose%脂联素对高糖环境心肌细胞氧化应激及凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    李美蓉; 李兴

    2012-01-01

    Objective To establish a myocardial cell oxidative stress model by cultivating human cardiac myocytes (HCM) in a high glucose environment, and to observe if there is a protective role of adiponectin (ADPN) on the oxidative stress and apoplosis of myocardial cells, and reveals the protection mechanism of the adiponectin. Methods The in vitro cultured HCM were divided into three groups;the control group, high glucose group, high glucose + adiponectin group, and these cells were cultured 24, 48, 72hours respectively. Then we observed the morphological change of the HCM under the inverted microscope and evaluated the level of superoxide dis-mutase ( SOD) by the xanthineoxidase method. The level of malondialdehyde ( MDA) was detected by benzodiazepines acid method. The expression of adaptin ( P66Shc) and heme oxygenase - 1 ( Ho - 1) in three time points was detected by real - time PCR, and the apoptosis rate of the HCM was tested by the flow cytometry. Results Our findings showed significant increase of MDA levels (P <0.05) and decrease of SOD activity(P<0.05) in the high glucose group compared with the control group. However,in the adiponectin group,MDA levels decreased (P<0.05) and SOD activity increased significantly (P <0.05) compared with those in the high - glucose group and they were time dependent. Compared with the control group,the expression of Ho-1 mRNA and P66Shc mRNA increased in the high -glucose group. The adiponectin group expressed a higher level of Ho - 1 mRNA but a lower level of P66Shc mRNA compared with the glucose group (P < 0.05) and they were time dependent. The apoptosis rate of the HCM in the high - glucose group was higher than the control group; while the apoptotic rate in the adiponectin group declined remarkably compared with the high glucose group. Conclusion The adiponectin has a protective effect on diabetes myocardial cells by upregulating the expression of Ho - 1 mRNA and downregulating the expression of P66Shc mRNA, which can

  5. PARM-1 is an endoplasmic reticulum molecule involved in endoplasmic reticulum stress-induced apoptosis in rat cardiac myocytes.

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    Koji Isodono

    Full Text Available To identify novel transmembrane and secretory molecules expressed in cardiac myocytes, signal sequence trap screening was performed in rat neonatal cardiac myocytes. One of the molecules identified was a transmembrane protein, prostatic androgen repressed message-1 (PARM-1. While PARM-1 has been identified as a gene induced in prostate in response to castration, its function is largely unknown. Our expression analysis revealed that PARM-1 was specifically expressed in hearts and skeletal muscles, and in the heart, cardiac myocytes, but not non-myocytes expressed PARM-1. Immunofluorescent staining showed that PARM-1 was predominantly localized in endoplasmic reticulum (ER. In Dahl salt-sensitive rats, high-salt diet resulted in hypertension, cardiac hypertrophy and subsequent heart failure, and significantly stimulated PARM-1 expression in the hearts, with a concomitant increase in ER stress markers such as GRP78 and CHOP. In cultured cardiac myocytes, PARM-1 expression was stimulated by proinflammatory cytokines, but not by hypertrophic stimuli. A marked increase in PARM-1 expression was observed in response to ER stress inducers such as thapsigargin and tunicamycin, which also induced apoptotic cell death. Silencing PARM-1 expression by siRNAs enhanced apoptotic response in cardiac myocytes to ER stresses. PARM-1 silencing also repressed expression of PERK and ATF6, and augmented expression of CHOP without affecting IRE-1 expression and JNK and Caspase-12 activation. Thus, PARM-1 expression is induced by ER stress, which plays a protective role in cardiac myocytes through regulating PERK, ATF6 and CHOP expression. These results suggested that PARM-1 is a novel ER transmembrane molecule involved in cardiac remodeling in hypertensive heart disease.

  6. Cardiac fibroblasts are predisposed to convert into myocyte phenotype: Specific effect of transforming growth factor. beta

    Energy Technology Data Exchange (ETDEWEB)

    Eghbali, M.; Tomek, R.; Woods, C.; Bhambi, B. (Univ. of Chicago, IL (United States))

    1991-02-01

    Cardiac fibroblasts are mainly responsible for the synthesis of major extracellular matrix proteins in the heart, including fibrillar collagen types I and III and fibronectin. In this report we show that these cells, when stimulated by transforming growth factor {beta}{sub 1} (TGF-{beta}{sub 1}), acquire certain myocyte-specific properties. Cultured cardiac fibroblasts from adult rabbit heart were treated with TGF-{beta}{sub 1}, (10-15 ng/ml) for different periods of time. Northern hybridization analysis of total RNA showed that cells treated with TGF-{beta}{sub 1} became stained with a monoclonal antibody to muscle-specific actin. After treatment of quiescent cells with TGF-{beta}{sub 1}, cell proliferation (as measured by ({sup 3}H)thymidine incorporation) was moderately increased. Cultured cardiac fibroblasts at the subconfluent stage, when exposed to TGF-{beta}{sub 1} in the presence of 10% fetal bovine serum, gave rise to a second generation of slowly growing cells that expressed muscle-specific actin filaments. The findings demonstrate that cardiac fibroblasts can be made to differentiate into cells that display many characteristics of cardiac myocytes. TGF-{beta}{sub 1} seems to be a specific inducer of such conversion.

  7. Calcium Imaging in Pluripotent Stem Cell-Derived Cardiac Myocytes.

    Science.gov (United States)

    Walter, Anna; Šarić, Tomo; Hescheler, Jürgen; Papadopoulos, Symeon

    2016-01-01

    The possibility to generate cardiomyocytes (CMs) from disease-specific induced pluripotent stem cells (iPSCs) is a powerful tool for the investigation of various cardiac diseases in vitro. The pathological course of various cardiac conditions, causatively heterogeneous, often converges into disturbed cellular Ca(2+) cycling. The gigantic Ca(2+) channel of the intracellular Ca(2+) store of CMs, the ryanodine receptor type 2 (RyR2), controls Ca(2+) release and therefore plays a crucial role in Ca(2+) cycling of CMs. In the present protocol we describe ways to measure and analyze global as well as local cellular Ca(2+) release events in CMs derived from a patient carrying a CPVT-causing RyR2 mutation.

  8. Membrane Localization, Caveolin-3 Association and Rapid Actions of Vitamin D Receptor in Cardiac Myocytes

    Science.gov (United States)

    Zhao, Guisheng; Simpson, Robert U.

    2009-01-01

    The active form of vitamin D, 1alpha, 25-Dihydroxyvitamin D3 (1, 25(OH)2D3), mediates both genomic and rapid non-genomic actions in heart cells. We have previously shown that the vitamin D receptor (VDR) is located in the t-tubular structure of cardiomyocytes. Here we show that VDR specifically interacts with Caveolin-3 in the t-tubules and sarcolemma of adult rat cardiac myocytes. Co-Immunoprecipitation studies using VDR antibodies revealed that Caveolin-3 specifically co-precipitates with the VDR and similarly the VDR is co-precipitated with Caveolin-3 antibody. Confocal immunofluorescence microscopy analysis also showed co-localization of VDR and Caveolin-3 in t-tubules and sarcolemma. The non-genomic effects of the functional VDR were studied in electrically stimulated myocytes isolated from adult rat hearts. Sarcomere shortening and re-lengthening were measured in 1, 25 (OH)2D3 treated cardiac myocytes. A 1nM treatment decreased peak shortening within minutes, suggesting a rapid effect through the membrane-bound VDR. This novel finding of the interaction between VDR and Caveolin-3 is fundamentally important in understanding 1, 25(OH)2D3 signal transduction in heart cells and provides further evidence that VDR plays a role in regulation of heart structure and function. PMID:20015453

  9. Bnip3 Binds and Activates p300: Possible Role in Cardiac Transcription and Myocyte Morphology.

    Directory of Open Access Journals (Sweden)

    John W Thompson

    Full Text Available Bnip3 is a hypoxia-regulated member of the Bcl-2 family of proteins that is implicated in apoptosis, programmed necrosis, autophagy and mitophagy. Mitochondria are thought to be the primary targets of Bnip3 although its activities may extend to the ER, cytoplasm, and nucleus. Bnip3 is induced in the heart by ischemia and pressure-overload, and may contribute to cardiomyopathy and heart failure. Only mitochondrial-dependent programmed death actions have been described for Bnip3 in the heart. Here we describe a novel activity of Bnip3 in cultured cardiac myocytes and transgenic mice overexpressing Bnip3 in the heart (Bnip3-TG. In cultured myocytes Bnip3 bound and activated the acetyltransferase p300, increased acetylation of histones and the transcription factor GATA4, and conferred p300 and GATA4-sensitive cellular morphological changes. In intact Bnip3-TG hearts Bnip3 also bound p300 and GATA4 and conferred enhanced GATA4 acetylation. Bnip3-TG mice underwent age-dependent ventricular dilation and heart failure that was partially prevented by p300 inhibition with curcumin. The results suggest that Bnip3 regulates cardiac gene expression and perhaps myocyte morphology by activating nuclear p300 acetyltransferase activity and hyperacetylating histones and p300-selective transcription factors.

  10. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

    Science.gov (United States)

    Inesi, G; Lewis, D; Sumbilla, C; Nandi, A; Strock, C; Huff, K W; Rogers, T B; Johns, D C; Kessler, P D; Ordahl, C P

    1998-03-01

    Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

  11. Nanoscale three-dimensional imaging of the human myocyte.

    Science.gov (United States)

    Sulkin, Matthew S; Yang, Fei; Holzem, Katherine M; Van Leer, Brandon; Bugge, Cliff; Laughner, Jacob I; Green, Karen; Efimov, Igor R

    2014-10-01

    The ventricular human myocyte is spatially organized for optimal ATP and Ca(2+) delivery to sarcomeric myosin and ionic pumps during every excitation-contraction cycle. Comprehension of three-dimensional geometry of the tightly packed ultrastructure has been derived from discontinuous two-dimensional images, but has never been precisely reconstructed or analyzed in human myocardium. Using a focused ion beam scanning electron microscope, we created nanoscale resolution serial images to quantify the three-dimensional ultrastructure of a human left ventricular myocyte. Transverse tubules (t-tubule), lipid droplets, A-bands, and mitochondria occupy 1.8, 1.9, 10.8, and 27.9% of the myocyte volume, respectively. The complex t-tubule system has a small tortuosity (1.04±0.01), and is composed of long transverse segments with diameters of 317±24nm and short branches. Our data indicates that lipid droplets located well beneath the sarcolemma are proximal to t-tubules, where 59% (13 of 22) of lipid droplet centroids are within 0.50μm of a t-tubule. This spatial association could have an important implication in the development and treatment of heart failure because it connects two independently known pathophysiological alterations, a substrate switch from fatty acids to glucose and t-tubular derangement.

  12. Insulin improves cardiac myocytes contractile function recovery in simulated ischemia-reperfusion: Key role of Akt

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bo; ZHANG Haifeng; FAN Qian; MA Xinliang; GAO Feng

    2003-01-01

    The present study examined cardiac myocyte contractile and Ca2+ transient responses to insulin during simulated ischemia/reperfusion (I/R) and furtherinvestigated the role of protein kinase B (Akt) in the insulin- induced inotropic effect. Ventricular myocytes were enzymatically isolated from adult Sprague-Dawley rats and perfused with Tyrode solution while electrically field-stimulated. Simulated I/R was induced by perfusing the cells with chemical anoxic solution including sodium cyanide-sodium lactate for 15 min followed by reperfusion with normal oxygenated Tyrode solution with or without insulin. It is found that insulin only at concentration as high as 10 IU/L could increase cell shortening (16±5%, P < 0.05) in normal myocytes, whereas it concentration-dependently (0.01-10 IU/L) increased the contraction,the velocity of shortening/releng- theningand Ca2+ transient in I/R myocytes. In addition, insulin treatment (1 IU/L) increased Akt phosphorylation of I/R cardiomyocytes by 2.4-fold compared with that of the control (P < 0.01). Most importantly, pretreatment with LY 294002, a specific inhibitor of phosphatidylinositol 3′-kinase (PI3-kinase), significantly inhibited both Akt phosphorylation and the positive inotropic response to insulin in the I/R cardiomyocytes. These results suggest that insulin exerts direct positive inotropic effect by increasing Ca2+ transient of cardiomyocytes, which is enhanced in the pathological condition of I/R. Akt activation plays an important role in the insulin-induced improvement of myocyte contractile function following I/R.

  13. Speckle based configuration for simultaneous in vitro inspection of mechanical contractions of cardiac myocyte cells

    Science.gov (United States)

    Golberg, Mark; Fixler, Dror; Shainberg, Asher; Zlochiver, Sharon; Micó, Vicente; Garcia, Javier; Beiderman, Yevgeny; Zalevsky, Zeev

    2013-04-01

    In this manuscript we propose optical lensless configuration for a remote non-contact measuring of mechanical contractions of vast number of cardiac myocytes. All the myocytes were taken from rats, and the measurements were done in an in vitro mode. The optical method is based on temporal analysis of secondary reflected speckle patterns generated in lensless microscope configuration. The processing involves analyzing the movement and the change in the statistics of the generated secondary speckle patterns that are created on top of the cell culture when it is illuminated by a spot of laser beam. The main advantage of the proposed system is the ability to measure many cells simultaneously (approximately one thousand cells) and to extract the statistical data of their movement at once. The presented experimental results also include investigation the effect of isoproteranol on cells contraction process.

  14. Effects of simulated microgravity on nitric oxide level in cardiac myocytes and its mechanism

    Institute of Scientific and Technical Information of China (English)

    熊江辉; 李莹辉; 聂捷琳

    2003-01-01

    The depression of cardiac contractility induced by space microgravity is an important issue of aerospace medicine research, while its precise mechanism is still unknown. In the present study, we explored effects of simulated microgravity on nitric oxide (NO) level, inducible nitric oxide synthase (iNOS) expression and related regulative mechanism using electron spin resonance (ESR) spectroscopy, immunocytochemistry and in situ hybridization. We found a remarkable increase of NO level and up-regulation of iNOS and iNOS mRNA expression in rat cardiac myocytes under simulated microgravity. Staurosporine (a nonselective protein kinase inhibitor), calphostin C (a selective protein kinase C inhibitor), partially inhibited the effect of simulated microgravity. Thus regulative effect of simulated microgravity on iNOS expression is mediated at least partially via activation of protein kinase C. These results indicate that NO system in cardiac myocytes is sensitive to simulated microgravity and may play an important role in the depression of cardiac contractility induced by simulated microgravity.

  15. Interferon-γ causes cardiac myocyte atrophy via selective degradation of myosin heavy chain in a model of chronic myocarditis.

    Science.gov (United States)

    Cosper, Pippa F; Harvey, Pamela A; Leinwand, Leslie A

    2012-12-01

    Interferon-γ (IFN-γ), a proinflammatory cytokine, has been implicated in the pathogenesis of a number of forms of heart disease including myocarditis and congestive heart failure. In fact, overexpression of IFN-γ in mice causes dilated cardiomyopathy. However, the direct effects of IFN-γ on cardiac myocytes and the mechanism by which it causes cardiac dysfunction have not been described. Here, we present the molecular pathology of IFN-γ exposure and its effect on myofibrillar proteins in isolated neonatal rat ventricular myocytes. Treatment with IFN-γ caused cardiac myocyte atrophy attributable to a specific decrease in myosin heavy chain protein. This selective degradation of myosin heavy chain was not accompanied by a decrease in total protein synthesis or by an increase in total protein degradation. IFN-γ increased both proteasome and immunoproteasome activity in cardiac myocytes and their inhibition blocked myosin heavy chain loss and myocyte atrophy, whereas inhibition of the lysosome or autophagosome did not. Collectively, these results provide a mechanism by which IFN-γ causes cardiac pathology in the setting of chronic inflammatory diseases. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Yong-Hui Mu

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

  17. Toward improved myocardial maturity in an organ-on-chip platform with immature cardiac myocytes.

    Science.gov (United States)

    Sheehy, Sean P; Grosberg, Anna; Qin, Pu; Behm, David J; Ferrier, John P; Eagleson, Mackenzie A; Nesmith, Alexander P; Krull, David; Falls, James G; Campbell, Patrick H; McCain, Megan L; Willette, Robert N; Hu, Erding; Parker, Kevin K

    2017-01-01

    In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration-response to the β-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology

  18. Developmental remodeling and shortening of the cardiac outflow tract involves myocyte programmed cell death.

    Science.gov (United States)

    Watanabe, M; Choudhry, A; Berlan, M; Singal, A; Siwik, E; Mohr, S; Fisher, S A

    1998-10-01

    The embryonic outflow tract is a simple tubular structure that connects the single primitive ventricle with the aortic sac and aortic arch arteries. This structure undergoes a complex sequence of morphogenetic processes to become the portion of the heart that aligns the right and left ventricles with the pulmonary artery and aorta. Abnormalities of the outflow tract are involved in many clinically significant congenital cardiac defects; however, the cellular and molecular processes governing the development of this important structure are incompletely understood. Histologic and tissue-tagging studies indicate that the outflow tract tissues compact and are incorporated predominantly into a region of the right ventricle. The hypothesis tested in the current study was that cell death or apoptosis in the muscular portion of the outflow tract is an important cellular mechanism for outflow tract shortening. The tubular outflow tract myocardium was specifically marked by infecting myocytes of the chicken embryo heart with a recombinant replication-defective adenovirus expressing beta-galactosidase (beta-gal) under the control of the cytomegalovirus promoter. Histochemical detection of the beta -gal-labeled outflow tract myocytes revealed that the tubular structure shortened to become a compact ring at the level of the pulmonic infundibulum over several days of development (stages 25-32, embryonic days 4-8). The appearance of apoptotic cardiomyocytes was correlated with OFT shortening by two histologic assays, TUNEL labeling of DNA fragments and AnnexinV binding. The rise and fall in the number of apoptotic myocytes detected by histologic analyses paralleled the change in activity levels of Caspase-3, a protease in the apoptotic cascade, measured in outflow tract homogenates. These results suggest that the elimination of myocytes by programmed cell death is one mechanism by which the outflow tract myocardium remodels to form the proper connection between the ventricular

  19. Effect of Sodium Tanshinone Ⅱ A Sulfonate on Cardiac Myocyte Hypertrophy and Its Underlying Mechanism

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective:To investigate the effects of sodium tanshinone Ⅱ A sulfonate (STS) on the hypertrophy induced by angiotensin Ⅱ (Ang Ⅱ) in primary cultured neonatal rat cardiac myocytes.Methods:The effect of STS on cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-3,5-phenytetrazoliumromide (MTT) assay.As indexes for cardiocyte hypertrophy,cell size was determined by phase contrast microscopy and protein synthesis rate was measured by 3H-leucine incorporation.The proto-oncogene c-fos mRNA expression of cardiocytes was assessed using reverse transcription polymerase chain reaction (RT-PCR).Results:STS could inhibit cardiocyte hypertrophy,increase the protein synthesis rate and enhance proto-oncogene c-los mRNA expression in cardiocytes induced by Ang Ⅱ (P<0.01),with an effect similar to that of Valsartan,the Ang Ⅱ receptor antagonist.Conclusion:STS can prevent the hypertrophy of cardiac myocytes induced by Ang Ⅱ,which may be related to its inhibition of the expression of proto-oncogene c-fos mRNA.

  20. C-reactive protein inhibits survivin expression via Akt/mTOR pathway downregulation by PTEN expression in cardiac myocytes.

    Directory of Open Access Journals (Sweden)

    Beom Seob Lee

    Full Text Available C-reactive protein (CRP is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

  1. MRP4 and CFTR in the regulation of cAMP and β-adrenergic contraction in cardiac myocytes.

    Science.gov (United States)

    Sellers, Zachary M; Naren, Anjaparavanda P; Xiang, Yang; Best, Philip M

    2012-04-15

    Spatiotemporal regulation of cAMP in cardiac myocytes is integral to regulating the diverse functions downstream of β-adrenergic stimulation. The activities of cAMP phosphodiesterases modulate critical and well-studied cellular processes. Recently, in epithelial and smooth muscle cells, it was found that the multi-drug resistant protein 4 (MRP4) acts as a cAMP efflux pump to regulate intracellular cAMP levels and alter effector function, including activation of the cAMP-stimulated Cl(-) channel, CFTR (cystic fibrosis transmembrane conductance regulator). In the current study we investigated the potential role of MRP4 in regulating intracellular cAMP and β-adrenergic stimulated contraction rate in cardiac myocytes. Cultured neonatal ventricular myocytes were used for all experiments. In addition to wildtype mice, β(1)-, β(2)-, and β(1)/β(2)-adrenoceptor, and CFTR knockout mice were used. MRP4 expression was probed via Western blot, intracellular cAMP was measured by fluorescence resonance energy transfer, while the functional role of MRP4 was assayed via monitoring of isoproterenol-stimulated contraction rate. We found that MRP4 is expressed in mouse neonatal ventricular myocytes. A pharmacological inhibitor of MRP4, MK571, potentiated submaximal isoproterenol-stimulated cAMP accumulation and cardiomyocyte contraction rate via β(1)-adrenoceptors. CFTR expression was critical for submaximal isoproterenol-stimulated contraction rate. Interestingly, MRP4-dependent changes in contraction rate were CFTR-dependent, however, PDE4-dependent potentiation of contraction rate was CFTR-independent. We have shown, for the first time, a role for MRP4 in the regulation of cAMP in cardiac myocytes and involvement of CFTR in β-adrenergic stimulated contraction. Together with phosphodiesterases, MRP4 must be considered when examining cAMP regulation in cardiac myocytes. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Modeling beta-adrenergic control of cardiac myocyte contractility in silico

    Science.gov (United States)

    Saucerman, Jeffrey J.; Brunton, Laurence L.; Michailova, Anushka P.; McCulloch, Andrew D.; McCullough, A. D. (Principal Investigator)

    2003-01-01

    The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

  3. Regulation of excitation-contraction coupling in mouse cardiac myocytes: integrative analysis with mathematical modelling

    Directory of Open Access Journals (Sweden)

    Weckström Matti

    2009-08-01

    normal and defective cellular physiology. We propose that integrative modelling as in the present work is a valuable complement to experiments in understanding the causality within complex biological systems such as cardiac myocytes.

  4. {beta}1-Adrenergic receptor activation induces mouse cardiac myocyte death through both L-type calcium channel-dependent and -independent pathways.

    Science.gov (United States)

    Wang, Wei; Zhang, Hongyu; Gao, Hui; Kubo, Hajime; Berretta, Remus M; Chen, Xiongwen; Houser, Steven R

    2010-08-01

    Cardiac diseases persistently increase the contractility demands of cardiac myocytes, which require activation of the sympathetic nervous system and subsequent increases in myocyte Ca(2+) transients. Persistent exposure to sympathetic and/or Ca(2+) stress is associated with myocyte death. This study examined the respective roles of persistent beta-adrenergic receptor (beta-AR) agonist exposure and high Ca(2+) concentration in myocyte death. Ventricular myocytes (VMs) were isolated from transgenic (TG) mice with cardiac-specific and inducible expression of the beta(2a)-subunit of the L-type Ca(2+) channel (LTCC). VMs were cultured, and the rate of myocyte death was measured in the presence of isoproterenol (ISO), other modulators of Ca(2+) handling and the beta-adrenergic system, and inhibitors of caspases and reactive oxygen species generation. The rate of myocyte death was greater in TG vs. wild-type myocytes and accelerated by ISO in both groups, although ISO did not increase LTCC current (I(Ca-L)) in TG-VMs. Nifedipine, an LTCC antagonist, only partially prevented myocyte death. These results suggest both LTCC-dependent and -independent mechanisms in ISO induced myocyte death. ISO increased the contractility of wild type and TG-VMs by enhancing sarcoplasmic reticulum function and inhibiting sarco(endo)plasmic reticulum Ca(2+)-ATPase, Na(+)/Ca(2+) exchanger, and CaMKII partially protected myocyte from death induced by both Ca(2+) and ISO. Caspase and reactive oxygen species inhibitors did not, but beta(2)-AR activation did, reduce myocyte death induced by enhanced I(Ca-L) and ISO stimulation. Our results suggest that catecholamines induce myocyte necrosis primarily through beta(1)-AR-mediated increases in I(Ca-L), but other mechanisms are also involved in rodents.

  5. Brief Myocardial Ischemia Produces Cardiac Troponin I Release and Focal Myocyte Apoptosis in the Absence of Pathological Infarction in Swine

    Directory of Open Access Journals (Sweden)

    Brian R. Weil, PhD

    2017-04-01

    Full Text Available In a porcine model of brief ischemia leading to reversible stunning in the absence of tissue necrosis, we demonstrated delayed release of cardiac troponin I (cTnI that exceeded the 99th percentile for normal animals 60 min after reperfusion and rose to readily detectable levels 24 h later. Although tissue analysis at 60 min showed no evidence of infarction, TUNEL staining demonstrated isolated myocytes undergoing apoptosis, which was absent after 24 h. These results demonstrate that cTnI elevations occur after ischemia of a duration that is insufficient to produce myocyte necrosis and reflect myocyte injury associated with apoptosis in the absence of pathological evidence of infarction.

  6. Chemical-defined and albumin-free generation of human atrial and ventricular myocytes from human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Fei Pei

    2017-03-01

    Full Text Available Most existing culture media for cardiac differentiation of human pluripotent stem cells (hPSCs contain significant amounts of albumin. For clinical transplantation applications of hPSC-derived cardiomyocytes (hPSC-CMs, culturing cells in an albumin containing environment raises the concern of pathogen contamination and immunogenicity to the recipient patients. In addition, batch-to-batch variation of albumin may cause the inconsistent of hPSC cardiac differentiation. Here, we demonstrated that antioxidants l-ascorbic acid, trolox, N-acetyl-l-cysteine (NAC and sodium pyruvate could functionally substitute albumin in the culture medium, and formulated an albumin-free, chemical-defined medium (S12 medium. We showed that S12 medium could support efficient hPSC cardiac differentiation with significantly improved reproducibility, and maintained long-term culture of hPSC-CMs. Furthermore, under chemical-defined and albumin-free conditions, human-induced pluripotent stem cells (hiPSCs were established, and differentiated into highly homogenous atrial and ventricular myocytes in a scalable fashion with normal electrophysiological properties. Finally, we characterized the activity of three typical cardiac ion channels of those cells, and demonstrated that hPSC-derived ventricular cardiomyocytes (hPSC-vCMs were suitable for drug cardiac safety evaluation. In summary, this simplified, chemical-defined and albumin-free culture medium supports efficient generation and maintaining of hPSC-CMs and facilitates both research and clinical applications of these cells.

  7. PGC-1{alpha} accelerates cytosolic Ca{sup 2+} clearance without disturbing Ca{sup 2+} homeostasis in cardiac myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Min, E-mail: chenminyx@gmail.com [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Yunnan Centers for Diseases Prevention and Control, Kunming 650022 (China); Wang, Yanru [Institute of Molecular Medicine, State Key Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871 (China); Qu, Aijuan [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2010-06-11

    Energy metabolism and Ca{sup 2+} handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1{alpha}) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1{alpha} in cardiac Ca{sup 2+} signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1{alpha} via adenoviral transduction. Our data shows that overexpressing PGC-1{alpha} improved myocyte contractility without increasing the amplitude of Ca{sup 2+} transients, suggesting that myofilament sensitivity to Ca{sup 2+} increased. Interestingly, the decay kinetics of global Ca{sup 2+} transients and Ca{sup 2+} waves accelerated in PGC-1{alpha}-expressing cells, but the decay rate of caffeine-elicited Ca{sup 2+} transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca{sup 2+}-ATPase (SERCA2a), but not Na{sup +}/Ca{sup 2+} exchange (NCX) contribute to PGC-1{alpha}-induced cytosolic Ca{sup 2+} clearance. Furthermore, PGC-1{alpha} induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1{alpha} did not disturb cardiac Ca{sup 2+} homeostasis, because SR Ca{sup 2+} load and the propensity for Ca{sup 2+} waves remained unchanged. These data suggest that PGC-1{alpha} can ameliorate cardiac Ca{sup 2+} cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1{alpha}-calcium handing pathway sheds new light on the role of PGC-1{alpha} in the therapy of cardiac diseases.

  8. Carbon nanotubes instruct physiological growth and functionally mature syncytia: nongenetic engineering of cardiac myocytes.

    Science.gov (United States)

    Martinelli, Valentina; Cellot, Giada; Toma, Francesca Maria; Long, Carlin S; Caldwell, John H; Zentilin, Lorena; Giacca, Mauro; Turco, Antonio; Prato, Maurizio; Ballerini, Laura; Mestroni, Luisa

    2013-07-23

    Myocardial tissue engineering currently represents one of the most realistic strategies for cardiac repair. We have recently discovered the ability of carbon nanotube scaffolds to promote cell division and maturation in cardiomyocytes. Here, we test the hypothesis that carbon nanotube scaffolds promote cardiomyocyte growth and maturation by altering the gene expression program, implementing the cell electrophysiological properties and improving networking and maturation of functional syncytia. In our study, we combine microscopy, biological and electrophysiological methodologies, and calcium imaging, to verify whether neonatal rat ventricular myocytes cultured on substrates of multiwall carbon nanotubes acquire a physiologically more mature phenotype compared to control (gelatin). We show that the carbon nanotube substrate stimulates the induction of a gene expression profile characteristic of terminal differentiation and physiological growth, with a 2-fold increase of α-myosin heavy chain (P carbon nanotubes appear to exert a protective effect against the pathologic stimulus of phenylephrine. Finally, cardiomyocytes on carbon nanotubes demonstrate a more mature electrophysiological phenotype of syncytia and intracellular calcium signaling. Thus, carbon nanotubes interacting with cardiomyocytes have the ability to promote physiological growth and functional maturation. These properties are unique in the current vexing field of tissue engineering, and offer unprecedented perspectives in the development of innovative therapies for cardiac repair.

  9. Transgenic overexpression of Hdac3 in the heart produces increased postnatal cardiac myocyte proliferation but does not induce hypertrophy.

    Science.gov (United States)

    Trivedi, Chinmay M; Lu, Min Min; Wang, Qiaohong; Epstein, Jonathan A

    2008-09-26

    Class I and II histone deacetylases (HDACs) play vital roles in regulating cardiac development, morphogenesis, and hypertrophic responses. Although the roles of Hdac1 and Hdac2, class I HDACs, in cardiac hyperplasia, growth, and hypertrophic responsiveness have been reported, the role in the heart of Hdac3, another class I HDAC, has been less well explored. Here we report that myocyte-specific overexpression of Hdac3 in mice results in cardiac abnormalities at birth. Hdac3 overexpression produces thickening of ventricular myocardium, especially the interventricular septum, and reduction of both ventricular cavities in newborn hearts. Our data suggest that increased thickness of myocardium in Hdac3-transgenic (Hdac3-Tg) mice is due to increased cardiomyocyte hyperplasia without hypertrophy. Hdac3 overexpression inhibits several cyclin-dependent kinase inhibitors, including Cdkn1a, Cdkn1b, Cdkn1c, Cdkn2b, and Cdkn2c. Hdac3-Tg mice did not develop cardiac hypertrophy at 3 months of age, unlike previously reported Hdac2-Tg mice. Further, Hdac3 overexpression did not augment isoproterenol-induced cardiac hypertrophy when compared with wild-type littermates. These findings identify Hdac3 as a novel regulator of cardiac myocyte proliferation during cardiac development.

  10. Towards an integrative computational model of the guinea pig cardiac myocyte

    Directory of Open Access Journals (Sweden)

    Laura Doyle Gauthier

    2012-07-01

    Full Text Available The local control theory of excitation-contraction (EC coupling asserts that regulation of calcium (Ca2+ release occurs at the nanodomain level, where openings of single L-type Ca2+ channels (LCCs trigger openings of small clusters of ryanodine receptors (RyRs co-localized within the dyad. A consequence of local control is that the whole-cell Ca2+ transient is a smooth continuous function of influx of Ca2+ through LCCs. While this so-called graded release property has been known for some time, it’s functional importance to the integrated behavior of the cardiac ventricular myocyte has not been fully appreciated. We previously formulated a biophysically-based model, in which LCCs and RyRs interact via a coarse-grained representation of the dyadic space. The model captures key features of local control using a low-dimensional system of ordinary differential equations. Voltage-dependent gain and graded Ca2+ release are emergent properties of this model by virtue of the fact that model formulation is closely based on the sub-cellular basis of local control. In this current work, we have incorporated this graded release model into a prior model of guinea pig ventricular myocyte electrophysiology, metabolism, and isometric force production. The resulting integrative model predicts the experimentally-observed causal relationship between action potential (AP shape and timing of Ca2+ and force transients, a relationship that is not explained by models lacking the graded release property. Model results suggest that even relatively subtle changes in AP morphology that may result, for example, from remodeling of membrane transporter expression in disease or spatial variation in cell properties, may have major impact on the temporal waveform of Ca2+ transients, thus influencing tissue-level electro-mechanical function.

  11. Mechanism for Muscarinic Inhibitory Regulation of the L-type Ca2 + Current in Cardiac Ventricular Myocytes

    Institute of Scientific and Technical Information of China (English)

    蒋彬; 杨向军; 惠杰; 蒋廷波; 宋建平; 刘志华

    2004-01-01

    @@ Objective The autonomic nervous system plays a key role in regulating cardiac function by modifying heart rate, contractility and impulse. The parasympathetic neurotransmitter acetyl-choline and muscarinic agonist carbachol (Cch) inhibit excitation-contraction coupling in cardiac ventricular myocytes. Muscarinic agonists suppress adenylyl cyclase (AC) acitivity and,by reducing activation of the cAMP/protein kinase A (PKA)cascade, inhibit the L-type Ca2+ current (ICa(L) ). They also increase the content of cGMP by stimulating guanylyl cyclase (GC) activity. The role of nitric oxide (NO)/cGMP in muscarinic inhibition has undergone considerable scrutiny. The role of the NO/cGMP pathway in the inhibition of ICa(L) by Cch was examined in guinea-pig ventricular myocytes.

  12. [Transfection of hypertrophic cardiac myocytes in vitro with (99)Tc(m)-labeled antisense miR208b oligonucleotide].

    Science.gov (United States)

    Wang, Jing; Feng, Huijuan; Ou, Yangwei; Sun, Yungang; Wu, Juqing; Chen, Pan

    2015-08-01

    To test the efficiency of transfecting (99)Tc(m)-labeled anti-miR208b oligonucleotide into early hypertrophic cardiac myocytes in vitro. The anti-oligonucleotide targeting miR208b (AMO) was synthesized and modified with LNA followed by conjugation with N-hydroxysuccinimidyl S-acetyl-meraptoacetyl triglycine (NHS-MAG3) and radiolabeling with (99)Tc(m). NHS-MAG3-LNA-AMO and labeled AMO were purified with Sep-Pak C18 column chromatography, and the former was examined for UV absorption at the 260 nm using Gene Quant DNA/RNA calculator. The labeling efficiency, radiochemical purity, stability and molecular hybridization activity were analyzed. An angiotensin II-induced cell model of hypertrophic cardiac myocytes was transfected with (99)Tc(m)-NHS-MAG3-LNA-AMO via liposome, and the relative expression of miRNA208b and retention ratio of the labeled AMO in early hypertrophic cells were determined. The labeling efficiency and radiochemical purity of the labeled AMO after purification exceeded 84% and 86%, respectively. The radio- chemical purities of the labeled AMO incubated in serum and normal saline for 12 h were both higher than 80%, and the labeled AMO showed a capacity to hybridize with the target gene. In the hypertrophic model of cardiac myocytes, the retention ratio of labeled AMO at 6 h was higher than 20%. The (99)Tc(m)-labeled antisense probe can be efficiently transfected into hypertrophic cardiac myocytes in vitro, which provides an experimental basis for subsequent radionuclide imaging studies.

  13. Down-regulation of replication factor C-40 (RFC40 causes chromosomal missegregation in neonatal and hypertrophic adult rat cardiac myocytes.

    Directory of Open Access Journals (Sweden)

    Hirotaka Ata

    Full Text Available BACKGROUND: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36 and DNA polymerase δ (Pol δ proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied. METHODS: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH for rat chromosome 12. RESULTS: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers. CONCLUSION: Our

  14. Basic methods for monitoring intracellular Ca2+ in cardiac myocytes using Fluo-3.

    Science.gov (United States)

    Bito, Virginie; Sipido, Karin R; Macquaide, Niall

    2015-04-01

    In cardiac myocytes, the physiological increase of intracellular calcium, the [Ca(2+)]i transient, elicited during excitation-contraction coupling typically reaches a peak amplitude of up to 1 µm, from a resting value of ∼100 nm, within 50-100 msec, depending on the species. Various conditions will affect the amplitude and rise time of the [Ca(2+)]i transient and, depending on the nature of the Ca(2+) signals under study, a variety of different probes are available for monitoring changes in intracellular Ca(2+). In this protocol, we focus on Fluo-3, which exists in the cytosol in its salt form K5Fluo-3. This form is practically nonfluorescent in the absence of Ca(2+), but the fluorescence increases dramatically on Ca(2+) binding. Although Fluo-3 is a single excitation-emission dye, it has a number of advantages for investigators, including an ideal dissociation constant (Kd) value and high quantum yield, meaning that it can be used at low concentrations that introduce minimal buffering. Here, we describe the basic setup and methodology for recording the global cytosolic [Ca(2+)]i transient with this probe during simultaneous patch-clamp and whole-cell recording of membrane voltage or of ionic currents under voltage clamp. © 2015 Cold Spring Harbor Laboratory Press.

  15. Vagus nerve stimulation mitigates intrinsic cardiac neuronal and adverse myocyte remodeling postmyocardial infarction.

    Science.gov (United States)

    Beaumont, Eric; Southerland, Elizabeth M; Hardwick, Jean C; Wright, Gary L; Ryan, Shannon; Li, Ying; KenKnight, Bruce H; Armour, J Andrew; Ardell, Jeffrey L

    2015-10-01

    This paper aims to determine whether chronic vagus nerve stimulation (VNS) mitigates myocardial infarction (MI)-induced remodeling of the intrinsic cardiac nervous system (ICNS), along with the cardiac tissue it regulates. Guinea pigs underwent VNS implantation on the right cervical vagus. Two weeks later, MI was produced by ligating the ventral descending coronary artery. VNS stimulation started 7 days post-MI (20 Hz, 0.9 ± 0.2 mA, 14 s on, 48 s off; VNS-MI, n = 7) and was compared with time-matched MI animals with sham VNS (MI n = 7) vs. untreated controls (n = 8). Echocardiograms were performed before and at 90 days post-MI. At termination, IC neuronal intracellular voltage recordings were obtained from whole-mount neuronal plexuses. MI increased left ventricular end systolic volume (LVESV) 30% (P = 0.027) and reduced LV ejection fraction (LVEF) 6.5% (P < 0.001) at 90 days post-MI compared with baseline. In the VNS-MI group, LVESV and LVEF did not differ from baseline. IC neurons showed depolarization of resting membrane potentials and increased input resistance in MI compared with VNS-MI and sham controls (P < 0.05). Neuronal excitability and sensitivity to norepinephrine increased in MI and VNS-MI groups compared with controls (P < 0.05). Synaptic efficacy, as determined by evoked responses to stimulating input axons, was reduced in VNS-MI compared with MI or controls (P < 0.05). VNS induced changes in myocytes, consistent with enhanced glycogenolysis, and blunted the MI-induced increase in the proapoptotic Bcl-2-associated X protein (P < 0.05). VNS mitigates MI-induced remodeling of the ICNS, correspondingly preserving ventricular function via both neural and cardiomyocyte-dependent actions.

  16. Gene Product Expression of Cyclin D2 and p16 During the Transition from Cardiac Myocyte Hyperplasia to Hypertrophy

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The current study was to investigate mRNA expression of cyclin D2 and p16 during the transition from cardiac myocyte hyperplasia to hypertrophy. Cultured cardiac myocytes (CM) and fibroblasts (FC) obtained from 1-day-old Sparague-Dawley rats were used in this study. We have determined (1) hyperplasia by cell growth curve and fluorescence activated cell sorting (FACS); and (2) ultrastructure by electron microscope observation; and (3) expressions of cyclin D2 mRNA and p16 mRNA by using in situ hybridization and image analysis. The results were shown (1) Results of cell growth curve and FACS analysis showed CM could proliferate in the first 3 cultured days (4 days in postnatal development). But the ability decreased quickly, concomitant with the differentiation. (2) The ultrastructure of CM showed the large amount of myofilaments and mitochondrion and FC showed moderate amount of rough endoplasmic reticulum. (3) The expression of cyclin D2 mRNA in 3-, 4-, 5-day CM group was 0.89 times(p<0.05), 0.80 times (p<0.05)and 0.56 times (p<0.01)of that in 1-day group respectively. P16 mRNA in 2-, 3-, 4-, 5-day CM group were 1.63 times(p<0.01),1.72 times(p<0.01),1.99 times (p<0.01)and 2.84 times (p<0.01) of that in 1-day group respectively. It can be concluded that cultured neonatal rat cardiac myocytes could proliferate during the first 3 cultured days, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D2 and p16 have the key roles during the transition from myocyte hyperplasia to hypertrophy.

  17. FAK-related nonkinase attenuates hypertrophy induced by angiotensin-Ⅱ in cultured neonatal rat cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    Jin QIN; Zheng-xiang LIU

    2006-01-01

    Aim: To examine the inhibitory effect of FAK-related nonkinase (FRNK) in cardiac hypertrophy in vitro and investigate the possible mechanisms. Methods: A functional fragment of FRNK cDNA was amplified by reverse transcription-polymerase chain reaction and cloned into the vector pcDNA3.1. Hypertrophy in neonatal rat cardiac myocytes was established with angiotensin-Ⅱ stimulation. The pcDNA3.1-FRNK or pcDNA3.1 was respectively transfected into cardiomyocytes by Lipofectamine 2000. The surface area and mRNA expression of atrial natriuretic peptide (ANP) of myocytes were employed to detect cardiac hypertrophy. NF-κB p65 protein in nuclear extracts, phosphorylation levels of ERK1/2 (p-ERK1/2) and AKT (p-AKT), as well as total ERK1/2, and AKT in variant treated cardiomyocytes were determined by Western blot. Results: Under the stimulation of angiotensin Ⅱ, the surface area of myocytes and levels of ANP mRNA were significantly increased. But transient transfection with pcDNA3.1-FRNK in advance may reduce the surface area and expression of ANP mRNA of hypertrophic myocytes. The protein levels of NF-κB p65 in nuclear extracts and p-ERK1/2, p-AKT in FRNK treated cardiomyocytes were significantly decreased compared with that in angiotensin-Ⅱ induced cardiomyocytes, while different treatments had little effect on total ERK1/2 and AKT. Conclusion: FRNK may inhibit angiotensin-Ⅱ-induced cardiomyocyte hypertrophy via decreasing phosphorylation levels at ERK1/2 and AKT, consequently downregulating nuclear translocation of NF-κB p65.

  18. Contribution of NHE-1 to cell length cardiac shortening of normal and failing rabbit myocytes

    NARCIS (Netherlands)

    M.M.G.J. van Borren; J.G. Zegers; A. Baartscheer; J.H. Ravesloot

    2006-01-01

    At the same intracellular pH (pH(i)) Na+/H+ exchange (NHE-1) fluxes of ventricular myocytes of hypertrophied failing hearts (HFH) are increased. We assessed how NHE-1 affected cell length shortening. pH(i) was measured fluorimetrically in resting and twitching (1 - 3 Hz)normal and HFH rabbit myocyte

  19. Remodeling of the sarcomeric cytoskeleton in cardiac ventricular myocytes during heart failure and after cardiac resynchronization therapy.

    Science.gov (United States)

    Lichter, Justin G; Carruth, Eric; Mitchell, Chelsea; Barth, Andreas S; Aiba, Takeshi; Kass, David A; Tomaselli, Gordon F; Bridge, John H; Sachse, Frank B

    2014-07-01

    Sarcomeres are the basic contractile units of cardiac myocytes. Recent studies demonstrated remodeling of sarcomeric proteins in several diseases, including genetic defects and heart failure. Here we investigated remodeling of sarcomeric α-actinin in two models of heart failure, synchronous (SHF) and dyssynchronous heart failure (DHF), as well as a model of cardiac resynchronization therapy (CRT). We applied three-dimensional confocal microscopy and quantitative methods of image analysis to study isolated cells from our animal models. 3D Fourier analysis revealed a decrease of the spatial regularity of the α-actinin distribution in both SHF and DHF versus control cells. The spatial regularity of α-actinin in DHF cells was reduced when compared with SHF cells. The spatial regularity of α-actinin was partially restored after CRT. We found longitudinal depositions of α-actinin in SHF, DHF and CRT cells. These depositions spanned adjacent Z-disks and exhibited a lower density of α-actinin than in the Z-disk. Differences in the occurrence of depositions between the SHF, CRT and DHF models versus control were significant. Also, CRT cells exhibited a higher occurrence of depositions versus SHF, but not DHF cells. Other sarcomeric proteins did not accumulate in the depositions to the same extent as α-actinin. We did not find differences in the expression of α-actinin protein and its encoding gene in our animal models. In summary, our studies indicate that HF is associated with two different types of remodeling of α-actinin and only one of those was reversed after CRT. We suggest that these results can guide us to an understanding of remodeling of structures and function associated with sarcomeres.

  20. Design-based stereological estimation of the total number of cardiac myocytes in histological sections

    DEFF Research Database (Denmark)

    Brüel, Annemarie; Nyengaard, Jens Randel

    2005-01-01

    with antibodies against cadherin and type IV collagen to visualise the intercalated discs and the myocyte membranes, respectively. Using the physical disector in "local vertical windows" of the serial sections, the average number of nuclei per myocyte was estimated.RESULTS: The total number of myocyte nuclei...... in LV was 34.1 x 10(6) (0.08) (mean (coefficient of variation)), and the mean number of nuclei per myocyte 1.85 (0.03). Combining these estimates the total number of myocytes in LV was calculated to be 18.5 x 10(6) (0.09). CONCLUSIONS: This new method is applicable to a range of experiments focusing...

  1. Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca2+ current in cardiac myocytes.

    Science.gov (United States)

    Wang, Honglan; Kohr, Mark J; Traynham, Christopher J; Ziolo, Mark T

    2009-08-01

    Endothelial nitric oxide synthase (NOS3) regulates the functional response to beta-adrenergic (beta-AR) stimulation via modulation of the L-type Ca(2+) current (I(Ca)). However, the NOS3 signaling pathway modulating I(Ca) is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I(Ca). Myocytes were isolated from NOS3 knockout (NOS3(-/-)) and wildtype (WT) mice. We measured I(Ca) (whole-cell voltage-clamp), and simultaneously measured Ca(2+) transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased beta-AR stimulated (isoproterenol, ISO)-I(Ca), and Ca(2+) transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I(Ca), but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3(-/-) myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I(Ca), or Ca(2+) transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I(Ca), and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I(Ca); YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca(2+) transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I(Ca) and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating beta-AR stimulated I(Ca), while limiting effects on cardiac contraction.

  2. Raisanberine protected pulmonary arterial rings and cardiac myocytes of rats against hypoxia injury by suppressing NADPH oxidase and calcium influx

    Institute of Scientific and Technical Information of China (English)

    Jie GAO; Yi-qun TANG; De-zai DAI; Yu-si CHENG; Guo-lin ZHANG; Can ZHANG; Yin DAI

    2012-01-01

    To investigate the protection of pulmonary arterial rings and cardiac myocytes of rats by raisanberine (RS),a derivative of berberine,against hypoxia injury and to elucidate the action mechanisms.Methods:Adult SD rats were exposed to intermittent hypoxia for 17 d or 28 d.The pulmonary arterial rings were isolated and vascular activity was measured using a transducer and computer-aided system.The difference in the tension produced by phenylephrine in the presence and absence of L-nitroarginine (10 μmol/L) was referred to as the NO bioavailability; the maximum release of NO was assessed by the ratio of the maximal dilatation caused by ACh to those caused by sodium nitroprusside.After the lungs were fixed,the internal and the external diameters of the pulmonary arterioles were measured using a graphic analysis system.Cultured cardiac myocytes from neonatal rats were exposed to H2O2 (10 μmol/L) to mimic hypoxia injury.ROS generation and [Ca2+]i level in the myocytes were measured using DHE and Fluo-3 fluorescence,respectively.Results:Oral administration of RS (80 mg/kg),the NADPH oxidase inhibitor apocynin (APO,80 mg/kg) or Ca2+ channel blocker nifedipine (Nif,10 mg/kg,) significantly alleviated the abnormal increase in the vasoconstriction force and endothelium-related vasodilatation induced by the intermittent hypoxia.The intermittent hypoxia markedly decreased the NO bioavailability and maximal NO release from pulmonary arterial rings,which were reversed by APO or RS administration.However,RS administration did not affect the NO bioavailability and maximal NO release from pulmonary arterial rings of normal rats.RS,Nif or APO administration significantly attenuated the pulmonary arteriole remodeling.Treatment of cultured cardiac myocytes with RS (10 μmol/L) suppressed the ROS generation and [Ca2+]i increase induced by H2O2,which were comparable to those caused by APO (10 μmol/L) or Nif (0.1 μmol/L).Conclusion:Raisanberine relieved hypoxic/oxidant insults to the

  3. A cardiac myocyte-restricted Lin28/let-7 regulatory axis promotes hypoxia-mediated apoptosis by inducing the AKT signaling suppressor PIK3IP1.

    Science.gov (United States)

    Joshi, Shaurya; Wei, Jianqin; Bishopric, Nanette H

    2016-02-01

    The let-7 family of microRNAs (miRs) regulates critical cell functions, including survival signaling, differentiation, metabolic control and glucose utilization. These functions may be important during myocardial ischemia. MiR-let-7 expression is under tight temporal and spatial control through multiple redundant mechanisms that may be stage-, isoform- and tissue-specific. To determine the mechanisms and functional consequences of miR-let-7 regulation by hypoxia in the heart. MiR-let-7a, -7c and -7g were downregulated in the adult mouse heart early after coronary occlusion, and in neonatal rat ventricular myocytes subjected to hypoxia. Let-7 repression did not require glucose depletion, and occurred at a post-transcriptional level. Hypoxia also induced the RNA binding protein Lin28, a negative regulator of let-7. Hypoxia ineither induced Lin28 nor repressed miR-let-7 in cardiac fibroblasts. Both changes were abrogated by treatment with the histone deacetylase inhibitor trichostatin A. Restoration of let-7g to hypoxic myocytes and to ischemia-reperfused mouse hearts in vivo via lentiviral transduction potentiated the hypoxia-induced phosphorylation and activation of Akt, and prevented hypoxia-dependent caspase activation and death. Mechanistically, phosphatidyl inositol 3-kinase interacting protein 1 (Pik3ip1), a negative regulator of PI3K, was identified as a novel target of miR-let-7 by a crosslinking technique showing that miR-let-7g specifically targets Pik3ip1 to the cardiac myocyte Argonaute complex RISC. Finally, in non-failing and failing human myocardium, we found specific inverse relationships between Lin28 and miR-let-7g, and between miR-let-7g and PIK3IP1. A conserved hypoxia-responsive Lin28-miR-let-7-Pik3ip1 regulatory axis is specific to cardiac myocytes and promotes apoptosis during myocardial ischemic injury. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    王世强; 周曾铨; 钱洪

    1999-01-01

    The temperature-dependence of infraeeliular free caleimn (Ca) was investigated in mdo-1 loaded ventricular myocytes from the ral, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-l at different temperatures was calibrated both al pll-tat and at @-stat . and the result demonstrated that the @-stat ralibration should be prettrred . Analysis of the fluoreseent image showed a striking increase of Ca2 as well as spontaneous caleiuni waves in ral cells, indicating an overloaded cakuum. In contrast, cardiac myocytes of the ground sqnirraf were found to keep a constant (Ca2+) without caleium overload regardless of temperature variation. It is be-lieved that understanding of the mechanisms underlying the interccllular caleima homeostasis of hibrernators may lead to solutions of some medical questions .

  5. Protein kinase D selectively targets cardiac troponin I and regulates myofilament Ca2+ sensitivity in ventricular myocytes.

    Science.gov (United States)

    Cuello, Friederike; Bardswell, Sonya C; Haworth, Robert S; Yin, Xiaoke; Lutz, Susanne; Wieland, Thomas; Mayr, Manuel; Kentish, Jonathan C; Avkiran, Metin

    2007-03-30

    Protein kinase D (PKD) is a serine/threonine kinase with emerging myocardial functions; in skinned adult rat ventricular myocytes (ARVMs), recombinant PKD catalytic domain phosphorylates cardiac troponin I at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity. We used adenoviral gene transfer to determine the effects of full-length PKD on protein phosphorylation, sarcomere shortening and [Ca(2+)](i) transients in intact ARVMs. In myocytes transduced to express wild-type PKD, the heterologously expressed enzyme was activated by endothelin 1 (ET1) (5 nmol/L), as reflected by PKD phosphorylation at Ser744/Ser748 (PKC phosphorylation sites) and Ser916 (autophosphorylation site). The ET1-induced increase in cellular PKD activity was accompanied by increased cardiac troponin I phosphorylation at Ser22/Ser23; this measured approximately 60% of that induced by isoproterenol (10 nmol/L), which activates cAMP-dependent protein kinase (PKA) but not PKD. Phosphorylation of other PKA targets, such as phospholamban at Ser16, phospholemman at Ser68 and cardiac myosin-binding protein C at Ser282, was unaltered. Furthermore, heterologous PKD expression had no effect on isoproterenol-induced phosphorylation of these proteins, or on isoproterenol-induced increases in sarcomere shortening and relaxation rate and [Ca(2+)](i) transient amplitude. In contrast, heterologous PKD expression suppressed the positive inotropic effect of ET1 seen in control cells, without altering ET1-induced increases in relaxation rate and [Ca(2+)](i) transient amplitude. Complementary experiments in "skinned" myocytes confirmed reduced myofilament Ca(2+) sensitivity by ET1-induced activation of heterologously expressed PKD. We conclude that increased myocardial PKD activity induces cardiac troponin I phosphorylation at Ser22/Ser23 and reduces myofilament Ca(2+) sensitivity, suggesting that altered PKD activity in disease may impact on contractile function.

  6. Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl

  7. A device for rapid and quantitative measurement of cardiac myocyte contractility

    Science.gov (United States)

    Gaitas, Angelo; Malhotra, Ricky; Li, Tao; Herron, Todd; Jalife, José

    2015-03-01

    Cardiac contractility is the hallmark of cardiac function and is a predictor of healthy or diseased cardiac muscle. Despite advancements over the last two decades, the techniques and tools available to cardiovascular scientists are limited in their utility to accurately and reliably measure the amplitude and frequency of cardiomyocyte contractions. Isometric force measurements in the past have entailed cumbersome attachment of isolated and permeabilized cardiomyocytes to a force transducer followed by measurements of sarcomere lengths under conditions of submaximal and maximal Ca2+ activation. These techniques have the inherent disadvantages of being labor intensive and costly. We have engineered a micro-machined cantilever sensor with an embedded deflection-sensing element that, in preliminary experiments, has demonstrated to reliably measure cardiac cell contractions in real-time. Here, we describe this new bioengineering tool with applicability in the cardiovascular research field to effectively and reliably measure cardiac cell contractility in a quantitative manner. We measured contractility in both primary neonatal rat heart cardiomyocyte monolayers that demonstrated a beat frequency of 3 Hz as well as human embryonic stem cell-derived cardiomyocytes with a contractile frequency of about 1 Hz. We also employed the β-adrenergic agonist isoproterenol (100 nmol l-1) and observed that our cantilever demonstrated high sensitivity in detecting subtle changes in both chronotropic and inotropic responses of monolayers. This report describes the utility of our micro-device in both basic cardiovascular research as well as in small molecule drug discovery to monitor cardiac cell contractions.

  8. Microfluidic systems to examine intercellular coupling of pairs of cardiac myocytes.

    Science.gov (United States)

    Klauke, Norbert; Smith, Godfrey; Cooper, Jonathan M

    2007-06-01

    In this paper we describe a microfluidic environment that enables us to explore cell-to-cell signalling between longitudinally linked primary heart cells. We have chosen to use pairs (or doublets) of cardiac myocyte as a model system, not only because of the importance of cell-cell signalling in the study of heart disease but also because the single cardiomyocytes are both mechanically and electrically active and their synchronous activation due to the intercellular coupling within the doublet can be readily monitored on optical and electrical recordings. Such doublets have specialised intercellular contact structures in the form of the intercalated discs, comprising the adhesive junction (fascia adherens and macula adherens or desmosome) and the connecting junction (known as gap junction). The latter structure enables adjacent heart cells to share ions, second messengers and small metabolites (<1 kDa) between them and thus provides the structural basis for the synchronous (syncytical) behaviour of connected cardiomyocytes. Using the unique environment provided by the microfluidic system, described in this paper, we explore the local ionic conditions that enable the propagation of Ca(2+) waves between two heart cells. We observe that the ability of intracellular Ca(2+) waves to traverse the intercalated discs is dependent on the relative concentrations of diastolic Ca(2+) in the two adjacent cells. These experiments rely upon our ability to independently control both the electrical stimulation of each of the cells (using integrated microelectrodes) and to rapidly change (or switch) the local concentrations of ions and drugs in the extracellular buffer within the microfluidic channel (using a nanopipetting system). Using this platform, it is also possible to make simultaneous optical recordings (including fluorescence and cell contraction) to explore the effect of drugs on one or both cells, within the doublet.

  9. Role of NF-κB in protection of EPO pretreatment on neonatal rat cardiac myocytes with hypoxia/reoxygenation injury

    Institute of Scientific and Technical Information of China (English)

    QIN Chuan; XIAO Ying-bin; ZHONG Qian-jin; CHEN Lin; WANG Xue-feng

    2005-01-01

    Objective:To observe the protective effects of erythropoietin (EPO) pretreatment on cardiac myocyte with hypoxia/reoxygenation (H/R) injury and the role of NF-κB in this effects. Methods:After the H/R model of cardiac myocytes of neonatal rats was established, the cultured cardiac myocytes were divided into 4 groups, including EPO pretreatment group ( EPO 10 U/ml 24 h before H/R), EPO pretreatment + PDTC group( EPO 10 U/ml and PDTC 5 μg/ml 24 h before H/R), PDTC group (PDTC 5 μg /ml 24 h before H/R) and control group. Before and after the H/R, assay of LDH concentration in the culture medium, the survival rate of the myocytes tested by MTT chromatometry and the apoptosis by flow cytometry were undertaken. Activation of NF-κB was determined by EMSA before and after H/R. Results:EPO pretreatment markedly reduced the LDH concentration in the medium, elevated the survival rate of myocytes and inhibited the apoptosis after H/R. Addition of PDTC during the pretreatment abolished the protective effects of EPO pretreatment. NF-κB was markedly activated during EPO pretreatment and PDTC inhibited the activation. However, after H/R, the activity of NF-κB in myocytes with EPO pretreatment was significantly inhibited compared to the other myocytes. Conclusion:NF-κB is significantly activated during EPO pretreatment, but is inhibited after H/R, which is correlated with the protective effects of EPO pretreatment on cardiac myocytes with H/R. This phenomenon can be explained as the negative feedback mechanism of the activation of NF-κB.

  10. Statistical Metamodeling and Sequential Design of Computer Experiments to Model Glyco-Altered Gating of Sodium Channels in Cardiac Myocytes.

    Science.gov (United States)

    Du, Dongping; Yang, Hui; Ednie, Andrew R; Bennett, Eric S

    2016-09-01

    Glycan structures account for up to 35% of the mass of cardiac sodium ( Nav ) channels. To question whether and how reduced sialylation affects Nav activity and cardiac electrical signaling, we conducted a series of in vitro experiments on ventricular apex myocytes under two different glycosylation conditions, reduced protein sialylation (ST3Gal4(-/-)) and full glycosylation (control). Although aberrant electrical signaling is observed in reduced sialylation, realizing a better understanding of mechanistic details of pathological variations in INa and AP is difficult without performing in silico studies. However, computer model of Nav channels and cardiac myocytes involves greater levels of complexity, e.g., high-dimensional parameter space, nonlinear and nonconvex equations. Traditional linear and nonlinear optimization methods have encountered many difficulties for model calibration. This paper presents a new statistical metamodeling approach for efficient computer experiments and optimization of Nav models. First, we utilize a fractional factorial design to identify control variables from the large set of model parameters, thereby reducing the dimensionality of parametric space. Further, we develop the Gaussian process model as a surrogate of expensive and time-consuming computer models and then identify the next best design point that yields the maximal probability of improvement. This process iterates until convergence, and the performance is evaluated and validated with real-world experimental data. Experimental results show the proposed algorithm achieves superior performance in modeling the kinetics of Nav channels under a variety of glycosylation conditions. As a result, in silico models provide a better understanding of glyco-altered mechanistic details in state transitions and distributions of Nav channels. Notably, ST3Gal4(-/-) myocytes are shown to have higher probabilities accumulated in intermediate inactivation during the repolarization and yield a

  11. Enhanced effect of VEGF165 on L-type calcium currents in guinea-pig cardiac ventricular myocytes.

    Science.gov (United States)

    Xing, Wenlu; Gao, Chuanyu; Qi, Datun; Zhang, You; Hao, Peiyuan; Dai, Guoyou; Yan, Ganxin

    2017-01-01

    The mechanisms of vascular endothelial growth factor 165 (VEGF165) on electrical properties of cardiomyocytes have not been fully elucidated. The aim of this study is to test the hypothesis that VEGF165, an angiogenesis-initiating factor, affects L-type calcium currents (ICa,L) and cell membrane potential in cardiac myocytes by acting on VEGF type-2 receptors (VEGFR2). ICa,L and action potentials (AP) were recorded by the whole-cell patch clamp method in isolated guinea-pig ventricular myocytes treated with different concentrations of VEGF165 proteins. Using a VEGFR2 inhibitor, we also tested the receptor of VEGF165 in cardiomyocytes. We found that VEGF165 increased ICa,L in a concentration-dependent manner. SU5416, a VEGFR2 inhibitor, almost completely eliminated VEGF165-induced ICa,L increase. VEGF165 had no significant influence on action potential 90 (APD90) and other properties of AP. We conclude that in guinea-pig ventricular myocytes, ICa,L can be increased by VEGF165 in a concentration-dependent manner through binding to VEGFR2 without causing any significant alteration to action potential duration. Results of this study may further expound the safety of VEGF165 when used in the intervention of heart diseases.

  12. Ca2+/Calmodulin-Dependent Protein Kinase II and Androgen Signaling Pathways Modulate MEF2 Activity in Testosterone-Induced Cardiac Myocyte Hypertrophy

    Directory of Open Access Journals (Sweden)

    Javier Duran

    2017-09-01

    Full Text Available Testosterone is known to induce cardiac hypertrophy through androgen receptor (AR-dependent and -independent pathways, but the molecular underpinnings of the androgen action remain poorly understood. Previous work has shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII and myocyte-enhancer factor 2 (MEF2 play key roles in promoting cardiac myocyte growth. In order to gain mechanistic insights into the action of androgens on the heart, we investigated how testosterone affects CaMKII and MEF2 in cardiac myocyte hypertrophy by performing studies on cultured rat cardiac myocytes and hearts obtained from adult male orchiectomized (ORX rats. In cardiac myocytes, MEF2 activity was monitored using a luciferase reporter plasmid, and the effects of CaMKII and AR signaling pathways on MEF2C were examined by using siRNAs and pharmacological inhibitors targeting these two pathways. In the in vivo studies, ORX rats were randomly assigned to groups that were administered vehicle or testosterone (125 mg⋅kg-1⋅week-1 for 5 weeks, and plasma testosterone concentrations were determined using ELISA. Cardiac hypertrophy was evaluated by measuring well-characterized hypertrophy markers. Moreover, western blotting was used to assess CaMKII and phospholamban (PLN phosphorylation, and MEF2C and AR protein levels in extracts of left-ventricle tissue from control and testosterone-treated ORX rats. Whereas testosterone treatment increased the phosphorylation levels of CaMKII (Thr286 and phospholambam (PLN (Thr17 in cardiac myocytes in a time- and concentration-dependent manner, testosterone-induced MEF2 activity and cardiac myocyte hypertrophy were prevented upon inhibition of CaMKII, MEF2C, and AR signaling pathways. Notably, in the hypertrophied hearts obtained from testosterone-administered ORX rats, both CaMKII and PLN phosphorylation levels and AR and MEF2 protein levels were increased. Thus, this study presents the first evidence indicating that

  13. Recording of calcium transient and analysis of calcium removal mechanisms in cardiac myocytes from rats and ground squirrels

    Institute of Scientific and Technical Information of China (English)

    王世强; 周曾铨; 钱洪

    2000-01-01

    With confocal microscopy, we recorded calcium transients and analyzed calcium removal rate at different temperatures in cardiac myocytes from the rat, a non-hibernator, and the ground squirrel, a hibernator. The results showed a remarkable increase of the diastolic level of calcium transients in the rat but no detectable change in the ground squirrel. Calcium transient of the ground squirrel, compared with that of the rat at the same temperature, had a shorter duration and showed a faster calcium removal. As indicated by the pharmacological effect of cyclopiazonic acid, calcium uptake by sarcoplasmic reticulum (SR) was the major mechanism of calcium removal, and was faster in the ground squirrel than in the rat. Our results confirmed the essential role of SR in hypothermia-tolerant adaptation, and negated the importance of Na-Ca exchange. We postulated the possibility to improve hypothermia-tolerance of the cardiac tissue of non-hibernating mammals.

  14. The adult heart responds to increased workload with physiologic hypertrophy, cardiac stem cell activation, and new myocyte formation.

    Science.gov (United States)

    Waring, Cheryl D; Vicinanza, Carla; Papalamprou, Angela; Smith, Andrew J; Purushothaman, Saranya; Goldspink, David F; Nadal-Ginard, Bernardo; Torella, Daniele; Ellison, Georgina M

    2014-10-14

    It is a dogma of cardiovascular pathophysiology that the increased cardiac mass in response to increased workload is produced by the hypertrophy of the pre-existing myocytes. The role, if any, of adult-resident endogenous cardiac stem/progenitor cells (eCSCs) and new cardiomyocyte formation in physiological cardiac remodelling remains unexplored. In response to regular, intensity-controlled exercise training, adult rats respond with hypertrophy of the pre-existing myocytes. In addition, a significant number (∼7%) of smaller newly formed BrdU-positive cardiomyocytes are produced by the exercised animals. Capillary density significantly increased in exercised animals, balancing cardiomyogenesis with neo-angiogenesis. c-kit(pos) eCSCs increased their number and activated state in exercising vs. sedentary animals. c-kit(pos) eCSCs in exercised hearts showed an increased expression of transcription factors, indicative of their commitment to either the cardiomyocyte (Nkx2.5(pos)) or capillary (Ets-1(pos)) lineages. These adaptations were dependent on exercise duration and intensity. Insulin-like growth factor-1, transforming growth factor-β1, neuregulin-1, bone morphogenetic protein-10, and periostin were significantly up-regulated in cardiomyocytes of exercised vs. sedentary animals. These factors differentially stimulated c-kit(pos) eCSC proliferation and commitment in vitro, pointing to a similar role in vivo. Intensity-controlled exercise training initiates myocardial remodelling through increased cardiomyocyte growth factor expression leading to cardiomyocyte hypertrophy and to activation and ensuing differentiation of c-kit(pos) eCSCs. This leads to the generation of new myocardial cells. These findings highlight the endogenous regenerative capacity of the adult heart, represented by the eCSCs, and the fact that the physiological cardiac adaptation to exercise stress is a combination of cardiomyocyte hypertrophy and hyperplasia (cardiomyocytes and capillaries

  15. Image Processing Techniques for Assessing Contractility in Isolated Neonatal Cardiac Myocytes

    Directory of Open Access Journals (Sweden)

    Carlos Bazan

    2011-01-01

    employed in determining myocyte contractility almost simultaneously with the acquisition of the Ca2+ transient and other correlates of cell contraction. The proposed methodology can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease models, transgeneity, or other common applications of neonatal cardiocytes.

  16. Cardiac myocyte diversity and a fibroblast network in the junctional region of the zebrafish heart revealed by transmission and serial block-face scanning electron microscopy.

    Science.gov (United States)

    Lafontant, Pascal J; Behzad, Ali R; Brown, Evelyn; Landry, Paul; Hu, Norman; Burns, Alan R

    2013-01-01

    The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.

  17. Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy

    KAUST Repository

    Lafontant, Pascal J.

    2013-08-23

    The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherentes junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart. © 2013 Lafontant et al.

  18. Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and fibroblasts.

    Science.gov (United States)

    Nayak, Alok Ranjan; Shajahan, T K; Panfilov, A V; Pandit, Rahul

    2013-01-01

    Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as [Formula: see text], the fibroblast resting-membrane potential, the fibroblast conductance [Formula: see text], and the MF gap-junctional coupling [Formula: see text]. Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as [Formula: see text], and [Formula: see text], and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity [Formula: see text] decreases as a function of [Formula: see text], for zero-sided and one-sided couplings; however, for two-sided coupling, [Formula: see text] decreases initially and then increases as a function of [Formula: see text], and, eventually, we observe that conduction failure occurs for low values of [Formula: see text]. In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling [Formula: see text] or [Formula: see text]. Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac

  19. The mechanism of PDT-induced electrical blockade: the measurement of intracellular Ca 2+ concentration changes in cardiac myocytes

    Science.gov (United States)

    Ito, A.; Hosokawa, S.; Hakomori, S.; Miyoshi, S.; Soejima, K.; Arai, T.

    2008-02-01

    We propose the application of early state photodynamic therapy (PDT) to treatment of atrial fibrillation, which is a kind of arrhythmia characterized by irregular rapid beating of heart. We had demonstrated that our PDT can block the propagation of electrical excitation in cardiac myocytes. However, the mechanism of the PDT-induced electrical blockade was not clear. In order to clarify this mechanism, changes in intracellular Ca 2+ concentration during the PDT with Talaporfin sodium (water soluble photosensitizer) were measured by fluorescence Ca 2+ indicator, fluo-4 AM. The PDT led to the rapid increase of intracellular Ca 2+ concentration and the changes in cell shapes. These results indicated that extracellular Ca 2+ flowed into the cells mediated by cell membrane. Moreover, we found bubble generation in the cells after the PDT. In conclusion, the PDT-induced electrical blockade in myocytes can be caused by cell death following the bubble generation, which is accompanied by the increase in intracellular Ca 2+ concentration due to the cell membrane malfunction with the PDT.

  20. JS-K, a GST-activated nitric oxide donor prodrug, enhances chemo-sensitivity in renal carcinoma cells and prevents cardiac myocytes toxicity induced by Doxorubicin.

    Science.gov (United States)

    Qiu, Mingning; Ke, Longzhi; Zhang, Sai; Zeng, Xin; Fang, Zesong; Liu, Jianjun

    2017-08-01

    Doxorubicin, a highly effective and widely used anthracycline antibiotic in multiple chemotherapy regimens, has been limited by its cardiotoxicity. The aim of this study is to investigate the effect of nitric oxide donor prodrug JS-K on proliferation and apoptosis in renal carcinoma cells and cardiac myocytes toxicity induced by Doxorubicin and to explore possible p53-related mechanism in renal carcinoma cells. The effect of JS-K on anti-cancer activity of Doxorubicin was investigated in renal carcinoma cells via detecting cell proliferation, cytotoxicity, cell death and apoptosis and expressions of apoptotic-related proteins. Effect of p53 on the combination of JS-K and Doxorubicin was determined using p53 inhibitor Pifithrin-α and p53 activator III. Furthermore, the effect of JS-K on cardiac myocytes toxicity of Doxorubicin was investigated in H9c2 (2-1) cardiac myocytes via measuring cell growth, cell death and apoptosis, expressions of proteins involved in apoptosis and intracellular reactive oxygen species. We demonstrated that JS-K could increase Doxorubicin-induced renal carcinoma cell growth suppression and apoptosis and could increase expressions of proteins that are involved in apoptosis. Additionally, Pifithrin-α reversed the promoting effect of JS-K on Doxorubicin-induced renal carcinoma cell apoptosis; conversely, the p53 activator III exacerbated the promoting effect of JS-K on Doxorubicin-induced renal carcinoma cell apoptosis. Furthermore, JS-K protected H9c2 (2-1) cardiac myocytes against Doxorubicin-induced toxicity and decreased Doxorubicin-induced reactive oxygen species production. JS-K enhances the anti-cancer activity of Doxorubicin in renal carcinoma cells by upregulating p53 expression and prevents cardiac myocytes toxicity of Doxorubicin by decreasing oxidative stress.

  1. The human adult cardiomyocyte phenotype

    NARCIS (Netherlands)

    Bird, SD; Doevendans, PA; van Rooijen, MA; de la Riviere, AB; Hassink, RJ; Passier, R; Mummery, CL

    2003-01-01

    Aim: Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. Methods: Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured

  2. Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

    DEFF Research Database (Denmark)

    Jeppesen, Pia Lindgren; Christensen, Gitte Lund; Schneider, Mikael

    2011-01-01

    Background and purpose: The Angiotensin II type 1 receptor (AT(1) R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we asked whether miRNAs might...

  3. Carbon Nanohorns Promote Maturation of Neonatal Rat Ventricular Myocytes and Inhibit Proliferation of Cardiac Fibroblasts: a Promising Scaffold for Cardiac Tissue Engineering

    Science.gov (United States)

    Wu, Yujing; Shi, Xiaoli; Li, Yi; Tian, Lei; Bai, Rui; Wei, Yujie; Han, Dong; Liu, Huiliang; Xu, Jianxun

    2016-06-01

    Cardiac tissue engineering (CTE) has developed rapidly, but a great challenge remains in finding practical scaffold materials for the construction of engineered cardiac tissues. Carbon nanohorns (CNHs) may be a potential candidate due to their special structure and properties. The purpose of this study was to assess the effect of CNHs on the biological behavior of neonatal rat ventricular myocytes (NRVMs) for CTE applications. CNHs were incorporated into collagen to form growth substrates for NRVMs. Transmission electron microscopy (TEM) observations demonstrated that CNHs exhibited a good affinity to collagen. Moreover, it was found that CNH-embedded substrates enhanced adhesion and proliferation of NRVMs. Immunohistochemical staining, western blot analysis, and intracellular calcium transient measurements indicated that the addition of CNHs significantly increased the expression and maturation of electrical and mechanical proteins (connexin-43 and N-cadherin). Bromodeoxyuridine staining and a Cell Counting Kit-8 assay showed that CNHs have the ability to inhibit the proliferation of cardiac fibroblasts. These findings suggest that CNHs can have a valuable effect on the construction of engineered cardiac tissues and may be a promising scaffold for CTE.

  4. Ca(2+ release events in cardiac myocytes up close: insights from fast confocal imaging.

    Directory of Open Access Journals (Sweden)

    Vyacheslav M Shkryl

    Full Text Available The spatio-temporal properties of Ca(2+ transients during excitation-contraction coupling and elementary Ca(2+ release events (Ca(2+ sparks were studied in atrial and ventricular myocytes with ultra-fast confocal microscopy using a Zeiss LSM 5 LIVE system that allows sampling rates of up to 60 kHz. Ca(2+ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca(2+ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca(2+ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca(2+]i. 2-D imaging of Ca(2+ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca(2+ entry through surface membrane Ca(2+ channels and subsequent activation of Ca(2+-induced Ca(2+ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca(2+ entry could be detected that was followed by SR Ca(2+ release after an additional 3 ms delay. Maximum Ca(2+ release was observed 4 ms after the beginning of release. The timing of Ca(2+ entry and release was confirmed by simultaneous [Ca(2+]i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca(2+ release events that fused into a peripheral ring of elevated [Ca(2+]i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca(2+ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca(2+ transient. In summary, ultra-fast confocal imaging allows investigation of Ca(2+ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion.

  5. Automatic quantitative analysis of t-tubule organization in cardiac myocytes using ImageJ.

    Science.gov (United States)

    Pasqualin, Côme; Gannier, François; Malécot, Claire O; Bredeloux, Pierre; Maupoil, Véronique

    2015-02-01

    The transverse tubule system in mammalian striated muscle is highly organized and contributes to optimal and homogeneous contraction. Diverse pathologies such as heart failure and atrial fibrillation include disorganization of t-tubules and contractile dysfunction. Few tools are available for the quantification of the organization of the t-tubule system. We developed a plugin for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. This plugin (TTorg) analyzes raw confocal microscopy images. Analysis options include the whole image, specific regions of the image (cropping), and z-axis analysis of the same image. Batch analysis of a series of images with identical criteria is also one of the options. There is no need to either reorientate any specimen to the horizontal or to do a thresholding of the image to perform analysis. TTorg includes a synthetic "myocyte-like" image generator to test the plugin's efficiency in the user's own experimental conditions. This plugin was validated on synthetic images for different simulated cell characteristics and acquisition parameters. TTorg was able to detect significant differences between the organization of the t-tubule systems in experimental data of mouse ventricular myocytes isolated from wild-type and dystrophin-deficient mice. TTorg is freely distributed, and its source code is available. It provides a reliable, easy-to-use, automatic, and unbiased measurement of t-tubule organization in a wide variety of experimental conditions.

  6. Effect of cardiac glycosides on action potential characteristics and contractility in cat ventricular myocytes: role of calcium overload.

    Science.gov (United States)

    Ruch, Stuart R; Nishio, Manabu; Wasserstrom, J Andrew

    2003-10-01

    There is increasing evidence that cardiac glycosides act through mechanisms distinct from inhibition of the sodium pump but which may contribute to their cardiac actions. To more fully define differences between agents indicative of multiple sites of action, we studied changes in contractility and action potential (AP) configuration in cat ventricular myocytes produced by six cardiac glycosides (ouabain, ouabagenin, dihydroouabain, actodigin, digoxin, and resibufogenin). AP shortening was observed only with ouabain and actodigin. There was extensive inotropic variability between agents, with some giving full inotropic effects before automaticity occurred whereas others produced minimal inotropy before toxicity. AP shortening was not a result of alterations in calcium current or the inward rectifier potassium current, but correlated with an increase in steady-state outward current (Iss), which was sensitive to KB-R7943, a Na+-Ca2+ exchange (NCX) inhibitor. Interestingly, Iss was observed following exposure to ouabain and dihydroouabain, suggesting that an additional mechanism is operative with dihydroouabain that prevents AP shortening. Further investigation into differences in inotropy between ouabagenin, dihydroouabain and ouabain revealed almost identical responses under AP voltage clamp. Thus all agents appear to act on the sodium pump and thereby secondarily increase the outward reverse mode NCX current, but the extent of AP duration shortening and positive inotropy elicited by each agent is limited by development of their toxic actions. The quantitative differences between cardiac glycosides suggest that mechanisms independent of sodium pump inhibition may result from an altered threshold for calcium overload possibly involving direct or indirect effects on calcium release from the sarcoplasmic reticulum.

  7. Characterization of Mg2+-regulated TRPM7-like current in human atrial myocytes

    Directory of Open Access Journals (Sweden)

    Macianskiene Regina

    2012-08-01

    Full Text Available Abstract Background TRPM7 (Transient Receptor Potential of the Melastatin subfamily proteins are highly expressed in the heart, however, electrophysiological studies, demonstrating and characterizing these channels in human cardiomyocytes, are missing. Methods We have used the patch clamp technique to characterize the biophysical properties of TRPM7 channel in human myocytes isolated from right atria small chunks obtained from 116 patients in sinus rhythm during coronary artery and valvular surgery. Under whole-cell voltage-clamp, with Ca2+ and K+ channels blocked, currents were generated by symmetrical voltage ramp commands to potentials between -120 and +80 mV, from a holding potential of -80 mV. Results We demonstrate that activated native current has dual control by intracellular Mg2+ (free-Mg2+ or ATP-bound form, and shows up- or down-regulation by its low or high levels, respectively, displaying outward rectification in physiological extracellular medium. High extracellular Mg2+ and Ca2+ block the outward current, while Gd3+, SpM4+, 2-APB, and carvacrol inhibit both (inward and outward currents. Besides, divalents also permeate the channel, and the efficacy sequence, at 20 mM, was Mg2+>Ni2+>Ca2+>Ba2+>Cd2+ for decreasing outward and Ni2+>Mg2+>Ba2+≥Ca2+>Cd2+ for increasing inward currents. The defined current bears many characteristics of heterologously expressed or native TRPM7 current, and allowed us to propose that current under study is TRPM7-like. However, the time of beginning and time to peak as well steady state magnitude (range from 1.21 to 11.63 pA/pF, ncells/patients = 136/77 of induced TRPM7-like current in atrial myocytes from different patients showed a large variability, while from the same sample of human atria all these parameters were very homogenous. We present new information that TRPM7-like current in human myocytes is less sensitive to Mg2+. In addition, in some myocytes (from 24 out of 77 patients that current

  8. P2X4 receptor–eNOS signaling pathway in cardiac myocytes as a novel protective mechanism in heart failure

    Science.gov (United States)

    Yang, Ronghua; Beqiri, Dardan; Shen, Jian-Bing; Redden, John M.; Dodge-Kafka, Kimberly; Jacobson, Kenneth A.; Liang, Bruce T.

    2014-01-01

    We have demonstrated using immunoprecipitation and immunostaining a novel physical association of the P2X4 receptor (P2X4R), a ligand-gated ion channel, with the cardioprotective, calcium-dependent enzyme endothelial nitric oxide synthase (eNOS). Treatment of murine ventricular myocytes with the P2XR agonist 2-methylthioATP (2-meSATP) to induce a current (mainly Na+) increased the formation of nitric oxide (NO), as measured using a fluorescent probe. Possible candidates for downstream effectors mediating eNOS activity include cyclic GMP and PKG or cellular protein nitrosylation. A cardiac-specific P2X4R overexpressing mouse line was protected from heart failure (HF) with improved cardiac function and survival in post-infarct, pressure overload, and calsequestrin (CSQ) overexpression models of HF. Although the role of the P2X4R in other tissues such as the endothelium and monocytes awaits characterization in tissue-specific KO, cardiac-specific activation of eNOS may be more cardioprotective than an increased activity of global systemic eNOS. The intra-myocyte formation of NO may be more advantageous over NO derived externally from a donor. A small molecule drug stimulating this sarcolemmal pathway or gene therapy-mediated overexpression of the P2X4R in cardiac myocytes may represent a new therapy for both ischemic and pressure overloaded HF. PMID:25750695

  9. P2X4 receptor–eNOS signaling pathway in cardiac myocytes as a novel protective mechanism in heart failure

    Directory of Open Access Journals (Sweden)

    Ronghua Yang

    2015-01-01

    Full Text Available We have demonstrated using immunoprecipitation and immunostaining a novel physical association of the P2X4 receptor (P2X4R, a ligand-gated ion channel, with the cardioprotective, calcium-dependent enzyme endothelial nitric oxide synthase (eNOS. Treatment of murine ventricular myocytes with the P2XR agonist 2-methylthioATP (2-meSATP to induce a current (mainly Na+ increased the formation of nitric oxide (NO, as measured using a fluorescent probe. Possible candidates for downstream effectors mediating eNOS activity include cyclic GMP and PKG or cellular protein nitrosylation. A cardiac-specific P2X4R overexpressing mouse line was protected from heart failure (HF with improved cardiac function and survival in post-infarct, pressure overload, and calsequestrin (CSQ overexpression models of HF. Although the role of the P2X4R in other tissues such as the endothelium and monocytes awaits characterization in tissue-specific KO, cardiac-specific activation of eNOS may be more cardioprotective than an increased activity of global systemic eNOS. The intra-myocyte formation of NO may be more advantageous over NO derived externally from a donor. A small molecule drug stimulating this sarcolemmal pathway or gene therapy-mediated overexpression of the P2X4R in cardiac myocytes may represent a new therapy for both ischemic and pressure overloaded HF.

  10. Inhibition of PKR protects against H2O2-induced injury on neonatal cardiac myocytes by attenuating apoptosis and inflammation

    Science.gov (United States)

    Wang, Yongyi; Men, Min; Xie, Bo; Shan, Jianggui; Wang, Chengxi; Liu, Jidong; Zheng, Hui; Yang, Wengang; Xue, Song; Guo, Changfa

    2016-01-01

    Reactive oxygenation species (ROS) generated from reperfusion results in cardiac injury through apoptosis and inflammation, while PKR has the ability to promote apoptosis and inflammation. The aim of the study was to investigate whether PKR is involved in hydrogen peroxide (H2O2) induced neonatal cardiac myocytes (NCM) injury. In our study, NCM, when exposed to H2O2, resulted in persistent activation of PKR due to NCM endogenous RNA. Inhibition of PKR by 2-aminopurine (2-AP) or siRNA protected against H2O2 induced apoptosis and injury. To elucidate the mechanism, we revealed that inhibition of PKR alleviated H2O2 induced apoptosis companied by decreased caspase3/7 activity, BAX and caspase-3 expression. We also revealed that inhibition of PKR suppressed H2O2 induced NFκB pathway and NLRP3 activation. Finally, we found ADAR1 mRNA and protein expression were both induced after H2O2 treatment through STAT-2 dependent pathway. By gain and loss of ADAR1 expression, we confirmed ADAR1 modulated PKR activity. Therefore, we concluded inhibition of PKR protected against H2O2-induced injury by attenuating apoptosis and inflammation. A self-preservation mechanism existed in NCM that ADAR1 expression is induced by H2O2 to limit PKR activation simultaneously. These findings identify a novel role for PKR/ADAR1 in myocardial reperfusion injury. PMID:27929137

  11. Carbon nanotubes enhance intercalated disc assembly in cardiac myocytes via the β1-integrin-mediated signaling pathway.

    Science.gov (United States)

    Sun, Hongyu; Lü, Shuanghong; Jiang, Xiao-Xia; Li, Xia; Li, Hong; Lin, Qiuxia; Mou, Yongchao; Zhao, Yuwei; Han, Yao; Zhou, Jin; Wang, Changyong

    2015-07-01

    Carbon nanotubes (CNTs) offer a new paradigm for constructing functional cardiac patches and repairing myocardial infarction (MI). However, little is known about how CNTs enhance the mechanical integrity and electrophysiological function of cardiac myocytes. To address this issue, we investigated the regularity and precise mechanism of the influence of CNTs on the assembly of intercalated disc (IDs). Here, single walled CNTs incorporated into collagen substrates were utilized as growth supports for neonatal cardiomyocytes, which enhanced cardiomyocyte adhesion and maturation. Furthermore, through the use of immunohistochemical staining, western blotting, transmission electron microscopy, and intracellular calcium transient measurement, we discovered that the addition of CNTs remarkably increased ID-related protein expression and enhanced ID assembly and functionality. On that basis, we further explored the underlying mechanism for how CNTs enhanced ID assembly through the use of immunohistochemical staining and western blotting. We found that the β1-integrin-mediated signaling pathway mediated CNT-induced upregulation of electrical and mechanical junction proteins. Notably, CNTs remarkably accelerated gap junction formation via activation of the β1-integrin-mediated FAK/ERK/GATA4 pathway. These findings provide valuable insight into the mechanistic effects that CNTs have on neonatal cardiomyocyte performance and will have a significant impact on the future of nanomedical research.

  12. Re-evaluation of the action potential upstroke velocity as a measure of the Na+ current in cardiac myocytes at physiological conditions.

    Directory of Open Access Journals (Sweden)

    Géza Berecki

    Full Text Available BACKGROUND: The SCN5A encoded sodium current (I(Na generates the action potential (AP upstroke and is a major determinant of AP characteristics and AP propagation in cardiac myocytes. Unfortunately, in cardiac myocytes, investigation of kinetic properties of I(Na with near-physiological ion concentrations and temperature is technically challenging due to the large amplitude and rapidly activating nature of I(Na, which may seriously hamper the quality of voltage control over the membrane. We hypothesized that the alternating voltage clamp-current clamp (VC/CC technique might provide an alternative to traditional voltage clamp (VC technique for the determination of I(Na properties under physiological conditions. PRINCIPAL FINDINGS: We studied I(Na under close-to-physiological conditions by VC technique in SCN5A cDNA-transfected HEK cells or by alternating VC/CC technique in both SCN5A cDNA-transfected HEK cells and rabbit left ventricular myocytes. In these experiments, peak I(Na during a depolarizing VC step or maximal upstroke velocity, dV/dt(max, during VC/CC served as an indicator of available I(Na. In HEK cells, biophysical properties of I(Na, including current density, voltage dependent (inactivation, development of inactivation, and recovery from inactivation, were highly similar in VC and VC/CC experiments. As an application of the VC/CC technique we studied I(Na in left ventricular myocytes isolated from control or failing rabbit hearts. CONCLUSIONS: Our results demonstrate that the alternating VC/CC technique is a valuable experimental tool for I(Na measurements under close-to-physiological conditions in cardiac myocytes.

  13. Measurement of Strain in Cardiac Myocytes at Micrometer Scale Based on Rapid Scanning Confocal Microscopy and Non-Rigid Image Registration.

    Science.gov (United States)

    Lichter, J; Li, Hui; Sachse, Frank B

    2016-10-01

    Measurement of cell shortening is an important technique for assessment of physiology and pathophysiology of cardiac myocytes. Many types of heart disease are associated with decreased myocyte shortening, which is commonly caused by structural and functional remodeling. Here, we present a new approach for local measurement of 2-dimensional strain within cells at high spatial resolution. The approach applies non-rigid image registration to quantify local displacements and Cauchy strain in images of cells undergoing contraction. We extensively evaluated the approach using synthetic cell images and image sequences from rapid scanning confocal microscopy of fluorescently labeled isolated myocytes from the left ventricle of normal and diseased canine heart. Application of the approach yielded a comprehensive description of cellular strain including novel measurements of transverse strain and spatial heterogeneity of strain. Quantitative comparison with manual measurements of strain in image sequences indicated reliability of the developed approach. We suggest that the developed approach provides researchers with a novel tool to investigate contractility of cardiac myocytes at subcellular scale. In contrast to previously introduced methods for measuring cell shorting, the developed approach provides comprehensive information on the spatio-temporal distribution of 2-dimensional strain at micrometer scale.

  14. Type 2 diabetes and obesity induce similar transcriptional reprogramming in human myocytes

    DEFF Research Database (Denmark)

    Väremo, Leif; Henriksen, Tora Ida; Scheele, Camilla

    2017-01-01

    BACKGROUND: Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize...... intrinsic properties of myocytes, associated independently with T2D or obesity. METHODS: We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor...... on genome-wide transcription. This setup enabled us to identify intrinsic properties, originating from muscle precursor cells and retained in the corresponding myocytes. Bioinformatic and statistical methods, including differential expression analysis, gene-set analysis, and metabolic network analysis, were...

  15. Alterations in intracellular ionic calcium levels in isolated adult rat cardiac myocytes due to the generation of free radicals

    Energy Technology Data Exchange (ETDEWEB)

    Burton, K.P.; Nazeran, H.; Hagler, H.K. (Univ. of Texas, Dallas, TX (United States))

    1991-03-15

    Oxygen-derived free radical production has been documented to occur on reperfusion of the ischemic myocardium. Intracellular ionic calcium ((Ca{sup ++}){sub i}) levels in isolated adult rat cardiac myocytes (M) exposed to free radicals were evaluated using the fluorescent calcium indicator, fura-2. The effect of different time periods of free radical exposure and the level of extracellular Ca{sup ++} concentration on altering (Ca{sup ++}){sub i} was examined. The free radical generating system (FRGS) utilized consisted of a HEPES buffered physiological salt solution containing 2.3 mM purine, 2.4. {mu}M iron-loaded transferrin and 0.01 U/ml xanthine oxidase. M maintained in HEPES buffer or the HEPES buffer containing purine and iron-loaded transferrin continued to stimulate, exhibited relatively uniform 340/380 ratios and maintained a rod shape for extended time periods. M continuously exposed to the FRGS showed a significant increase in (Ca{sup ++}){sub i}, became unresponsive to stimulation at 31 {plus minus} 7 (SE) min and eventually exhibited contracture. Exposure to the FRGS for 10 min resulted in a response similar to continuous exposure. M exposed to the FRGS for 5 min exhibited regular Ca{sup ++} transients for 55{plus minus}5 min. M exposed to the FRGS for 10 min and maintained in 2.5 mM Ca{sup ++} versus 1.25 mM Ca{sup ++}, accumulated significantly higher (CA{sup ++}){sub i}. Quiescent myocytes continuously exposed to the FRGS also exhibited a significant increase in (Ca{sup ++}){sub i} over time. Thus, a brief period of free radical exposure may induce subsequent damage. Alterations in Ca{sup ++} flux resulting from the generation of free radicals may possibly contribute to the development of Ca{sup ++} overload and myocardial arrhythmias.

  16. Effects of antibiotics on the contractility and Ca2+ transients of rat cardiac myocytes.

    Science.gov (United States)

    Belus, A; White, E

    2001-01-26

    We have compared the effects of streptomycin sulphate, gentamicin sulphate and neomycin sulphate on cell shortening (our index of contractility) and intracellular Ca2+ ([Ca2+](i)) transients of rat ventricular myocytes. All three agents abolished shortening and [Ca2+](i), transients but streptomycin was significantly less potent than the other agents. The IC(50) of streptomycin was 0.37 mM for shortening and 0.78 mM for [Ca2+](i), approximately an order of magnitude greater than equivalent values for gentamicin and neomycin. Gentamicin and streptomycin shortened the action potential duration of most cells but prolonged the action potential duration of others. We therefore conclude that multiple ionic mechanisms affecting action potential duration are modulated by these antibiotics. Our observations are consistent with the negative inotropic effect of antibiotics being caused by a decrease in Ca2+ influx causing a reduction in the [Ca2+](i) transient.

  17. Subcellular heterogeneity of sodium current properties in adult cardiac ventricular myocytes.

    Science.gov (United States)

    Lin, Xianming; Liu, Nian; Lu, Jia; Zhang, Jie; Anumonwo, Justus M B; Isom, Lori L; Fishman, Glenn I; Delmar, Mario

    2011-12-01

    Sodium channel α-subunits in ventricular myocytes (VMs) segregate either to the intercalated disc or to lateral membranes, where they associate with region-specific molecules. To determine the functional properties of sodium channels as a function of their location in the cell. Local sodium currents were recorded from adult rodent VMs and Purkinje cells by using the cell-attached macropatch configuration. Electrodes were placed either in the cell midsection (M) or at the cell end (area originally occupied by the intercalated disc [ID]). Channels were identified as tetrodotoxin (TTX)-sensitive (TTX-S) or TTX-resistant (TTX-R) by application of 100 nM of TTX. Average peak current amplitude was larger in ID than in M and largest at the site of contact between attached cells. TTX-S channels were found only in the M region of VMs and not in Purkinje myocytes. TTX-R channels were found in both M and ID regions, but their biophysical properties differed depending on recording location. Sodium current in rat VMs was upregulated by tumor necrosis factor-alpha. The magnitude of current increase was largest in the M region, but this difference was abolished by application of 100 nM of TTX. Our data suggest that (a) a large fraction of TTX-R (likely Na(v)1.5) channels in the M region of VMs are inactivated at normal resting potential, leaving most of the burden of excitation to TTX-R channels in the ID region; (b) cell-cell adhesion increases functional channel density at the ID; and (c) TTX-S (likely non-Na(v)1.5) channels make a minimal contribution to sodium current under control conditions, but they represent a functional reserve that can be upregulated by exogenous factors. Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  18. Inhibitory effect of antisense oligodeoxynucleotide to p44/p42 MAPK on angiotensin II-induced hypertrophic response in cultured neonatal rat cardiac myocyte

    Institute of Scientific and Technical Information of China (English)

    Shi-qinZHANG; BoDING; Zhao-guiGUO; Yun-xiaLI

    2004-01-01

    AIM: To explore the inhibitory effect of antisense oligonucleotide (ODN) to mitogen activated protein kinase(MAPK) on cardiomyocyte hypertrophy induced by angiotensin Ⅱ (Ang Ⅱ). METHODS: A 17-mer phosphorothioate-protected antisense ODN directed against the initiation of translation sites of the p42 and p44 MAPK isoforms byliposomal transfection was applied to inhibit the translation of p44/p42 MAPK mRNA. The sense and random ODNs to p44/p42MAPK were used as sequence controls. Neonatal cardiac myocytes were exposed to Ang Ⅱ (10nmol/L) for 5 min and then harvested in lysis buffer for the measurement of the activity and the phosphorylated protein content of p44/p42MAPK that were tested by P-81 phosphocellulose filter paper method and Western blotting, respectively. The rate of protein synthesis by [3H]leucine incorporation and the diameter of cell were measured after exposure to Ang Ⅱ for 24 h and 72 h, respectively. RESULTS: In cardiac myocyte Ang Ⅱ increased p44/p42MAPK activity and phosphorylated protein content by 140 % and 699 %, and also increased [3H]leucine incorporation and cell diameter by 40 % and 27 %. c-fos and c-myc mRNAs were induced significantly after exposure to Ang Ⅱ. Antisense ODN to p44/p42MAPK (0.2 μmol/L) reduced Ang Ⅱ-induced MAPK activity by 30 %,and phophorylated MAPK protein expression by 59 % in cardiac myocyte, and inhibited c-fos and c-myc mRNA expression induced by Ang Ⅱ by 44 % and 43 %, respectively. The diameter and the rate of protein synthesis of cardiac myocyte induced by Ang Ⅱ were decreased by 16 % and 22 % after pretreatment with antisense ODN to p44/p42MAPK. CONCLUSION: Antisense ODN to p44/p42 MAPK inhibited the increase of rate of protein synthesis,and the augmentation of cell diameter and expression of c-fos and c-myc mRNA induced by Ang Ⅱ in culturedcardiac myocytes, p44/p42 MAPK played a critical role in the hypertrophic response induced by Ang Ⅱ in cultured neonatal rat cardiac myocytes.

  19. Type 2 diabetes and obesity induce similar transcriptional reprogramming in human myocytes.

    Science.gov (United States)

    Väremo, Leif; Henriksen, Tora Ida; Scheele, Camilla; Broholm, Christa; Pedersen, Maria; Uhlén, Mathias; Pedersen, Bente Klarlund; Nielsen, Jens

    2017-05-25

    Skeletal muscle is one of the primary tissues involved in the development of type 2 diabetes (T2D). The close association between obesity and T2D makes it difficult to isolate specific effects attributed to the disease alone. Therefore, here we set out to identify and characterize intrinsic properties of myocytes, associated independently with T2D or obesity. We generated and analyzed RNA-seq data from primary differentiated myotubes from 24 human subjects, using a factorial design (healthy/T2D and non-obese/obese), to determine the influence of each specific factor on genome-wide transcription. This setup enabled us to identify intrinsic properties, originating from muscle precursor cells and retained in the corresponding myocytes. Bioinformatic and statistical methods, including differential expression analysis, gene-set analysis, and metabolic network analysis, were used to characterize the different myocytes. We found that the transcriptional program associated with obesity alone was strikingly similar to that induced specifically by T2D. We identified a candidate epigenetic mechanism, H3K27me3 histone methylation, mediating these transcriptional signatures. T2D and obesity were independently associated with dysregulated myogenesis, down-regulated muscle function, and up-regulation of inflammation and extracellular matrix components. Metabolic network analysis identified that in T2D but not obesity a specific metabolite subnetwork involved in sphingolipid metabolism was transcriptionally regulated. Our findings identify inherent characteristics in myocytes, as a memory of the in vivo phenotype, without the influence from a diabetic or obese extracellular environment, highlighting their importance in the development of T2D.

  20. Variations in Local Calcium Signaling in Adjacent Cardiac Myocytes of the Intact Mouse Heart Detected with Two-Dimensional Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Karin P Hammer

    2015-01-01

    Full Text Available Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart.Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 µm by 315 µm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length.Image registration from multiple sequential Ca transients allowed reconstruction of high temporal resolution (2.4 ±1.3ms local CaT in 2D image sets (N= 4 hearts, n= 8 regions. During steady state stimulation, spatial Ca gradients were homogeneous within cells in both directions and independent of distance between measured points. Variation in CaT amplitudes was similar across the short and the long side of neighboring cells. Variations in TAU and TTP were similar in both directions. Isoproterenol enhanced the CaT but not the overall pattern of spatial heterogeneities.Here we detected and analyzed local Ca signals in intact mouse hearts with high temporal and spatial resolution, taking into account 2D arrangement of the cells. We observed significant differences in the variation of CaT amplitude along the long and short axis of cardiac myocytes. Variations of Ca signals between neighboring cells may contribute to the substrate of cardiac remodeling.

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

    OpenAIRE

    1988-01-01

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

  2. Patterns of evolution of myocyte damage after human heart transplantation detected by indium-111 monoclonal antimyosin

    Energy Technology Data Exchange (ETDEWEB)

    Ballester-Rodes, M.; Carrio-Gasset, I.; Abadal-Berini, L.; Obrador-Mayol, D.; Berna-Roqueta, L.; Caralps-Riera, J.M.

    1988-09-15

    The indium-111 labeled Fab fragment of antimyosin monoclonal antibody was used to study cardiac rejection and the time course of myocyte damage after transplantation. Fifty-three studies were performed in 21 patients, 17 men and 4 women, aged 19 to 54 years (mean 37 +/- 8), from 7 to 40 months after transplantation. Repeat studies were available in 8, and 10 were studied after the first year of transplantation. A heart-to-lung ratio was used for quantitation of uptake (normal 1.46 +/- 0.04). Differences between absent (1.69 +/- 0.29) and moderate (1.90 +/- 0.36) rejection were significant (p less than 0.03). Antimyosin ratio at 1 to 3 months (1.89 +/- 0.35) differed from that at greater than 12 months (1.65 +/- 0.2) (p less than 0.01). Repeat studies revealed a decrease in antimyosin ratio in 5 patients with uneventful clinical course; 2 had persistent activity after transplantation and suffered heart failure from rejection. After 1 year of transplantation uptake was within normal limits in 7 of 10 patients, and high uptake was associated with vascular rejection in 1. Because they can define evolving patterns of myocardial lesion activity, antimyosin studies could be useful both in patient management and in concentrating resources for those patients who most require them. The heart-to-lung ratio is suggested to monitor sequentially the degree of myocyte damage after transplantation.

  3. Comparative study of cellular kinetics of reporter probe [{sup 131}I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: lxl730724@hotmail.com; Yin Xiaohua; Wang Ruihua; Liu Ying [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China) and Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: zhyx1229@163.com

    2009-02-15

    Aim: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyluracil) was labeled with {sup 131}I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [{sup 131}I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry. Results: FAU could be labeled with {sup 131}I, and the labeling efficiency and radiochemical purity rates were 53.82{+-}2.05% and 94.85{+-}1.76%, respectively. Time-dependent increase of the accumulation of [{sup 131}I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55{+-}0.37% and 2.09{+-}0.34%, respectively. Greater uptakes of [{sup 131}I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene

  4. Regional electroporation of single cardiac myocytes in a focused electric field.

    Science.gov (United States)

    Klauke, Norbert; Smith, Godfrey; Cooper, Jonathan M

    2010-01-15

    There is now a significant interest in being able to locate single cells within geometrically defined regions of a microfluidic chip and to gain intracellular access through the local electroporation of the cell membrane. This paper describes the microfabrication of electroporation devices which can enable the regional electroporation of adult ventricular myocytes, in order to lower the local electrical resistance of the cell membrane. Initially three different devices, designed to suit the characteristic geometry of the cardiomyocyte, were investigated (all three designs serve to focus the electric field to selected regions of the cell). We demonstrate that one of these three devices revealed the sequence of cellular responses to field strengths of increasing magnitudes, namely, cell contraction, hypercontraction, and lysis. This same device required a reduced threshold voltage for each of these events, including in particular membrane breakdown. We were not only able to show the gradual regional increase in the electric conductivity of the cell membrane but were also able to avoid changes in the local intra- and extracellular pH (by preventing the local generation of protons at the electrode surface, as a consequence of the reduced threshold voltage). The paper provides evidence for new strategies for achieving robust and reproducible regional electroporation, a technique which, in future, may be used for the insertion of large molecular weight molecules (including genes) as well as for on-chip voltage clamping of the primary adult cardiomyocyte.

  5. Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes.

    Directory of Open Access Journals (Sweden)

    Jeremy Kim

    Full Text Available We recently characterized an autocrine renin angiotensin system (RAS in canine heart. Activation of Angiotensin II Type 1 Receptors (AT1Rs induced electrical remodeling, including inhibition of the transient outward potassium current Ito, prolongation of the action potential (AP, increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and Ito in isolated myocytes. Application of angiotensin II (A2 for 2 or more hours reduced Ito magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of Ito (Ito,fast and IK,slow appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2, which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT1R mediated regulation of Ito in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

  6. β-adrenergic effects on cardiac myofilaments and contraction in an integrated rabbit ventricular myocyte model.

    Science.gov (United States)

    Negroni, Jorge A; Morotti, Stefano; Lascano, Elena C; Gomes, Aldrin V; Grandi, Eleonora; Puglisi, José L; Bers, Donald M

    2015-04-01

    A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca(2+) transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by β-adrenergic stimulation (β-AS). β-AS effect on sarcoplasmic reticulum Ca(2+) handling, Ca(2+), K(+) and Cl(-) currents, and Na(+)/K(+)-ATPase properties was included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca(2+) sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental β-AS responses on action potentials and Ca(2+) transient amplitude and kinetics. It also replicated the behavior of force-Ca(2+), release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The β-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing β-AS effects on L-type Ca(2+) channels or phospholamban limited Ca(2+) transients and contractile responses in parallel, while blocking phospholemman and K(+) channel (IKs) effects enhanced Ca(2+) and inotropy. Removal of β-AS effects from XBCa enhanced contractile force while decreasing peak Ca(2+) (due to greater Ca(2+) buffering), but had less effect on shortening. Conversely, preventing β-AS effects on XBcy preserved Ca(2+) transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of β-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. β-AS effects on XBCa and XBcy

  7. Sequential dissection of multiple ionic currents in single cardiac myocytes under action potential-clamp.

    Science.gov (United States)

    Banyasz, Tamas; Horvath, Balazs; Jian, Zhong; Izu, Leighton T; Chen-Izu, Ye

    2011-03-01

    The cardiac action potential (AP) is shaped by myriad ionic currents. In this study, we develop an innovative AP-clamp Sequential Dissection technique to enable the recording of multiple ionic currents in the single cell under AP-clamp. This new technique presents a significant step beyond the traditional way of recording only one current in any one cell. The ability to measure many currents in a single cell has revealed two hitherto unknown characteristics of the ionic currents in cardiac cells: coordination of currents within a cell and large variation of currents between cells. Hence, the AP-clamp Sequential Dissection method provides a unique and powerful tool for studying individual cell electrophysiology.

  8. Difference of Sodium Currents between Pediatric and Adult Human Atrial Myocytes: Evidence for Developmental Changes of Sodium Channels

    Directory of Open Access Journals (Sweden)

    Benzhi Cai, Xiaoqin Mu, Dongmei Gong, Shulin Jiang, Jianping Li, Qingxin Meng, Yunlong Bai, Yanju Liu, Xinyue Wang, Xueying Tan, Baofeng Yang, Yanjie Lu

    2011-01-01

    Full Text Available Voltage-gated calcium currents and potassium currents were shown to undergo developmental changes in postnatal human and animal cardiomocytes. However, so far, there is no evidence whether sodium currents also presented the developmental changes in postnatal human atrial cells. The aim of this study was to observe age-related changes of sodium currents between pediatric and adult atrial myocytes. Human atrial myocytes were acutely isolated and the whole-cell patch clamp technique was used to record sodium currents isolated from pediatric and adult atrial cardiomocytes. The peak amplitude of sodium currents recorded in adult atrial cells was significantly larger than that in pediatric atrial myocytes. However, there was no significant difference of the activation voltage for peak sodium currents between two kinds of atrial myocytes. The time constants for the activation and inactivation of sodium currents were smaller in adult atria than pediatric atria. The further study revealed that the voltage-dependent inactivation of sodium currents were more slow in adult atrial cardiomyocytes than pediatric atrial cells. A significant difference was also observed in the recovery process of sodium channel from inactivation. In summary, a few significant differences were demonstrated in sodium currents characteristics between pediatric and adult atrial myocytes, which indicates that sodium currents in human atria also undergo developmental changes.

  9. Diabetic cardiomyopathy: from the pathophysiology of the cardiac myocytes to current diagnosis and management strategies

    Directory of Open Access Journals (Sweden)

    Christina Voulgari

    2010-10-01

    Full Text Available Christina Voulgari, Dimitrios Papadogiannis, Nicholas TentolourisFirst Department of Propaedeutic and Internal Medicine, Athens University Medical School, Laiko General Hospital, Athens, GreeceAbstract: Diabetic cardiomyopathy (DCM, although a distinct clinical entity, is also a part of the diabetic atherosclerosis process. It may be independent of the coexistence of ischemic heart disease, hypertension, or other macrovascular complications. Its pathological substrate is characterized by the presence of myocardial damage, reactive hypertrophy, and intermediary fibrosis, structural and functional changes of the small coronary vessels, disturbance of the management of the metabolic cardiovascular load, and cardiac autonomic neuropathy. These alterations make the diabetic heart susceptible to ischemia and less able to recover from an ischemic attack. Arterial hypertension frequently coexists with and exacerbates cardiac functioning, leading to the premature appearance of heart failure. Classical and newer echocardiographic methods are available for early diagnosis. Currently, there is no specific treatment for DCM; targeting its pathophysiological substrate by effective risk management protects the myocardium from further damage and has a recognized primary role in its prevention. Its pathophysiological substrate is also the objective for the new therapies and alternative remedies.Keywords: cardiovascular disease, atherosclerosis, cardiac autonomic neuropathy, echocardiography, treatment strategies

  10. Differential extracellular signal-regulated kinases 1 and 2 activation by the angiotensin type 1 receptor supports distinct phenotypes of cardiac myocytes

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Schneider, Mikael;

    2007-01-01

    The angiotensin II (AngII) type 1 receptor (AT(1)R) is a seven-transmembrane receptor well established to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. The biological importance of this, however, remains...... that phosphorylates p90 Ribosomal S6 Kinase, a ubiquitous and versatile mediator of ERK1/2 signal transduction. Moreover, the beta-arrestin2-dependent ERK1/2 signal supports intact proliferation of cardiac myocytes. In contrast to G(q)-activated ERK1/2, and in keeping with its failure to translocate to the nucleus......, the beta-arrestin2-scaffolded pool of ERK1/2 does not phosphorylate the transcription factor Elk-1, induces no increased transcription of the immediate-early gene c-Fos, and does not entail myocyte hypertrophy. These results clearly demonstrate the biological significance of differential signalling...

  11. [Structural and metabolic aspects of heterogenicity of the ventricular cardiac myocytes in coronary insufficiency].

    Science.gov (United States)

    Gavrish, A S; Blagodarov, V N; Verbitskiĭ, V V; Danilishina, M V

    2003-01-01

    The paper shows comparative analysis of structural-metabolic changes of the ventricular cardiac hystiocytes during transitional restriction of blood supply, which leads to relapsing myocardium hypoperfusion, ischemia and even to cardiac infarction. We have established that an increase of the myocardium arrhythmogenicity, damage of myocardium electrophysiological activity and coronary deficiency are caused by pathologically intensified structural-metabolic heterogeneity of cardiomyocites. The chronic ischemic heart disease is worsening the situation by gradual accumulation of the metastructure changes of heart's working cells. It is possible to connect adaptable hypertrophy of cardiomyocites with compensatory redistribution of loading among them, what is accompanied with the deep reorganization of their metabolism and metastructure under the influence of neurohumoral factors, inadequate transport-trophic maintenance of the function, nonequal edema and fibrosis of the interstitium. The data obtained describe the formation of "prereadiness" of the myocardium to the fatal rhythm disturbance determined by interconnected structural-metabolic changes. Each of these pathological changes separately doesn't induce an impairment of myocardium electric stability but represents a necessary part in origin and development of the pathological process.

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

    Science.gov (United States)

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

    2016-01-01

    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.

  13. Liganded peroxisome proliferator-activated receptors (PPARs) preserve nuclear histone deacetylase 5 levels in endothelin-treated Sprague-Dawley rat cardiac myocytes.

    Science.gov (United States)

    Zhang, Haining; Shao, Zongjun; Alibin, Caroline P; Acosta, Crystal; Anderson, Hope D

    2014-01-01

    Ligand activation of peroxisome proliferator-activated receptors (PPARs) prevents cardiac myocyte hypertrophy, and we previously reported that diacylglycerol kinase zeta (DGKζ) is critically involved. DGKζ is an intracellular lipid kinase that catalyzes phosphorylation of diacylglycerol; by attenuating DAG signaling, DGKζ suppresses protein kinase C (PKC) and G-protein signaling. Here, we investigated how PPAR-DGKζ signaling blocks activation of the hypertrophic gene program. We focused on export of histone deacetylase 5 (HDAC5) from the nucleus, a key event during hypertrophy, since crosstalk occurs between PPARs and other members of the HDAC family. Using cardiac myocytes isolated from Sprague-Dawley rats, we determined that liganded PPARs disrupt endothelin-1 (ET1)-induced nuclear export of HDAC5 in a manner that is dependent on DGKζ. When DGKζ-mediated PKC inhibition was circumvented using a constitutively-active PKCε mutant, PPARs failed to block ET1-induced nuclear retention of HDAC5. Liganded PPARs also prevented (i) activation of protein kinase D (the downstream effector of PKC), (ii) HDAC5 phosphorylation at 14-3-3 protein chaperone binding sites (serines 259 and 498), and (iii) physical interaction between HDAC5 and 14-3-3, all of which are consistent with blockade of nucleo-cytoplasmic shuttling of HDAC5. Finally, the ability of PPARs to prevent neutralization of HDAC5 activity was associated with transcriptional repression of hypertrophic genes. This occurred by first, reduced MEF2 transcriptional activity and second, augmented deacetylation of histone H3 associated with hypertrophic genes expressing brain natriuretic peptide, β-myosin heavy chain, skeletal muscle α-actin, and cardiac muscle α-actin. Our findings identify spatial regulation of HDAC5 as a target for liganded PPARs, and to our knowledge, are the first to describe a mechanistic role for nuclear DGKζ in cardiac myocytes. In conclusion, these results implicate modulation of HDAC5

  14. Effects of NIP-141 on K currents in human atrial myocytes.

    Science.gov (United States)

    Seki, Akiko; Hagiwara, Nobuhisa; Kasanuki, Hiroshi

    2002-01-01

    A novel benzopyran derivative, NIP-141, effectively terminates experimental atrial fibrillation in canine hearts by prolonging atrial refractoriness. However, the effects of this drug on human atrial myocytes are unknown. This experiment evaluated the effects of NIP-141 on K currents in isolated human atrial myocytes using a whole-cell voltage-clamp method. NIP-141 inhibited the transient outward current (I(to)) and the ultra-rapid delayed rectifier K current (I(Kur)), each in a dose-dependent manner, with half-maximal inhibition concentrations of 16.3 microM and 5.3 microM, respectively (n = 5). NIP-141 inhibited both K currents in a voltage- and use-independent fashion, and it preferentially blocked them in the open state and dissociated rapidly from the channel. Because both K currents contribute significantly to the repolarization of the atrial action potential, these findings suggest that NIP-141 may terminate atrial fibrillation by prolonging action potential duration.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Athletic Cardiac Adaptation in Males Is a Consequence of Elevated Myocyte Mass.

    Science.gov (United States)

    McDiarmid, Adam K; Swoboda, Peter P; Erhayiem, Bara; Lancaster, Rosalind E; Lyall, Gemma K; Broadbent, David A; Dobson, Laura E; Musa, Tarique A; Ripley, David P; Garg, Pankaj; Greenwood, John P; Ferguson, Carrie; Plein, Sven

    2016-04-01

    Cardiac remodeling occurs in response to regular athletic training, and the degree of remodeling is associated with fitness. Understanding the myocardial structural changes in athlete's heart is important to develop tools that differentiate athletic from cardiomyopathic change. We hypothesized that athletic left ventricular hypertrophy is a consequence of increased myocardial cellular rather than extracellular mass as measured by cardiovascular magnetic resonance. Forty-five males (30 athletes and 15 sedentary age-matched healthy controls) underwent comprehensive cardiovascular magnetic resonance studies, including native and postcontrast T1 mapping for extracellular volume calculation. In addition, the 30 athletes performed a maximal exercise test to assess aerobic capacity and anaerobic threshold. Participants were grouped by athleticism: untrained, low performance, and high performance (O2max 60 mL/kg per min, respectively). In athletes, indexed cellular mass was greater in high- than low-performance athletes 60.7±7.5 versus 48.6±6.3 g/m(2); Pcellular compartment while the extracellular volume becomes relatively smaller: a difference which becomes more marked as left ventricular mass increases. Athletic remodeling, both on a macroscopic and cellular level, is associated with the degree of an individual's fitness. Cardiovascular magnetic resonance ECV quantification may have a future role in differentiating athlete's heart from change secondary to cardiomyopathy. © 2016 The Authors.

  17. The Scaffold Protein Muscle A-Kinase Anchoring Protein β Orchestrates Cardiac Myocyte Hypertrophic Signaling Required for the Development of Heart Failure

    Science.gov (United States)

    Kritzer, Michael D.; Li, Jinliang; Passariello, Catherine L.; Gayanilo, Marjorie; Thakur, Hrishikesh; Dayan, Joseph; Dodge-Kafka, Kimberly; Kapiloff, Michael S.

    2014-01-01

    Background Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAPβ signalosomes to pathological remodeling and heart failure in vivo remains unknown. Methods and Results Using conditional, cardiac myocyte–specific gene deletion, we now demonstrate that mAKAPβ expression in mice is important for the cardiac hypertrophy induced by pressure overload and catecholamine toxicity. mAKAPβ targeting prevented the development of heart failure associated with long-term transverse aortic constriction, conferring a survival benefit. In contrast to 29% of control mice (n=24), only 6% of mAKAPβ knockout mice (n=31) died in the 16 weeks of pressure overload (P=0.02). Accordingly, mAKAPβ knockout inhibited myocardial apoptosis and the development of interstitial fibrosis, left atrial hypertrophy, and pulmonary edema. This improvement in cardiac status correlated with the attenuated activation of signaling pathways coordinated by the mAKAPβ scaffold, including the decreased phosphorylation of protein kinase D1 and histone deacetylase 4 that we reveal to participate in a new mAKAP signaling module. Furthermore, mAKAPβ knockout inhibited pathological gene expression directed by myocyte-enhancer factor-2 and nuclear factor of activated T-cell transcription factors that associate with the scaffold. Conclusions mAKAPβ orchestrates signaling that regulates pathological cardiac remodeling in mice. Targeting of the underlying physical architecture of signaling networks, including mAKAPβ signalosome formation, may constitute an effective therapeutic strategy for the prevention and treatment of pathological remodeling and heart failure. PMID:24812305

  18. Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

    Institute of Scientific and Technical Information of China (English)

    Bei ZHOU; Li-jun WU; Shin-ichi TASHIRO; Satoshi ONODERA; Fumiaki UCHIUMI; Takashi IKEJIMA

    2007-01-01

    Aim: To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.Methods: The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.Results: The protective effects of silibinin were significantly sup-pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously,the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expres-sion of phosphorylated ERK was increased by silibinin, the expression of phos-phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphory-lated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.Conclusion: Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.

  19. Pre-Conditioning with CDP-Choline Attenuates Oxidative Stress-Induced Cardiac Myocyte Death in a Hypoxia/Reperfusion Model

    Science.gov (United States)

    González-Pacheco, Héctor; Méndez-Domínguez, Aurelio; Hernández, Salomón; López-Marure, Rebeca; Vazquez-Mellado, Maria J.; Aguilar, Cecilia; Rocha-Zavaleta, Leticia

    2014-01-01

    Background. CDP-choline is a key intermediate in the biosynthesis of phosphatidylcholine, which is an essential component of cellular membranes, and a cell signalling mediator. CDP-choline has been used for the treatment of cerebral ischaemia, showing beneficial effects. However, its potential benefit for the treatment of myocardial ischaemia has not been explored yet. Aim. In the present work, we aimed to evaluate the potential use of CDP-choline as a cardioprotector in an in vitro model of ischaemia/reperfusion injury. Methods. Neonatal rat cardiac myocytes were isolated and subjected to hypoxia/reperfusion using the coverslip hypoxia model. To evaluate the effect of CDP-choline on oxidative stress-induced reperfusion injury, the cells were incubated with H2O2 during reperfusion. The effect of CDP-choline pre- and postconditioning was evaluated using the cell viability MTT assay, and the proportion of apoptotic and necrotic cells was analyzed using the Annexin V determination by flow cytometry. Results. Pre- and postconditioning with 50 mg/mL of CDP-choline induced a significant reduction of cells undergoing apoptosis after hypoxia/reperfusion. Preconditioning with CDP-choline attenuated postreperfusion cell death induced by oxidative stress. Conclusion. CDP-choline administration reduces cell apoptosis induced by oxidative stress after hypoxia/reperfusion of cardiac myocytes. Thus, it has a potential as cardioprotector in ischaemia/reperfusion-injured cardiomyocytes. PMID:24578622

  20. Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

    Science.gov (United States)

    Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

    2016-07-01

    First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

  1. PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation.

    Science.gov (United States)

    Mendez, Mariela; LaPointe, Margot C

    2005-05-01

    Upon induction of cyclooxygenase-2 (COX-2), neonatal ventricular myocytes (VMs) mainly synthesize prostaglandin E2 (PGE2). The biological effects of PGE2 are mediated through four different G protein-coupled receptor (GPCR) subtypes (EP(1-4)). We have previously shown that PGE2 stimulates cAMP production and induces hypertrophy of VMs. Because the EP4 receptor is coupled to adenylate cyclase and increases in cAMP, we hypothesized that PGE2 induces hypertrophic growth of cardiac myocytes through a signaling cascade that involves EP4-cAMP and activation of protein kinase A (PKA). To test this, we used primary cultures of VMs and measured [3H]leucine incorporation into total protein. An EP4 antagonist was able to partially block PGE2 induction of protein synthesis and prevent PGE2-dependent increases in cell surface area and activity of the atrial natriuretic factor promoter, which are two other indicators of hypertrophic growth. Surprisingly, a PKA inhibitor had no effect. In other cell types, G protein-coupled receptor activation has been shown to transactivate the epidermal growth factor receptor (EGFR) and result in p42/44 mitogen-activated protein kinase (MAPK) activation and cell growth. Immunoprecipitation of myocyte lysates demonstrated that the EGFR was rapidly phosphorylated by PGE2 in VMs, and the EP4 antagonist blocked this. In addition, the selective EGFR inhibitor AG-1478 completely blocked PGE2-induced protein synthesis. We also found that PGE2 rapidly phosphorylated p42/44 MAPK, which was inhibited by the EP4 antagonist and by AG-1478. Finally, the p42/44 MAPK inhibitor PD-98053 (25 micromol/l) blocked PGE2-induced protein synthesis. Altogether, we believe these are the first data to suggest that PGE2 induces protein synthesis in cardiac myocytes in part via activation of the EP4 receptor and subsequent activation of p42/44 MAPK. Activation of p42/44 MAPK is independent of the common cAMP-PKA pathway and involves EP4-dependent transactivation of EGFR.

  2. Cardiac Myocyte De Novo DNA Methyltransferases 3a/3b Are Dispensable for Cardiac Function and Remodeling after Chronic Pressure Overload in Mice.

    Directory of Open Access Journals (Sweden)

    Thomas G Nührenberg

    Full Text Available Recent studies reported altered DNA methylation in failing human hearts. This may suggest a role for de novo DNA methylation in the development of heart failure. Here, we tested whether cardiomyocyte-specific loss of de novo DNA methyltransferases Dnmt3a and Dnmt3b altered cardiac function and remodeling after chronic left ventricular pressure overload.Mice with specific ablation of Dnmt3a and Dnmt3b expression in cardiomyocytes were generated by crossing floxed Dnmt3afl and Dnmt3bfl alleles with mice expressing Cre recombinase under control of the atrial myosin light chain gene promoter. The efficacy of combined Dnmt3a/3b ablation (DKO was characterized on cardiomyocyte-specific genomic DNA and mRNA levels. Cardiac phenotyping was carried out without (sham or with left ventricular pressure overload induced by transverse aortic constriction (TAC. Under similar conditions, cardiac genome-wide transcriptional profiling was performed and DNA methylation levels of promoters of differentially regulated genes were assessed by pyrosequencing.DKO cardiomyocytes showed virtual absence of targeted Dnmt3a and Dnmt3b mRNA transcripts. Cardiac phenotyping revealed no significant differences between DKO and control mice under sham and TAC conditions. Transcriptome analyses identified upregulation of 44 and downregulation of 9 genes in DKO as compared with control sham mice. TAC mice showed similar changes with substantial overlap of regulated genes compared to sham. Promoters of upregulated genes were largely unmethylated in DKO compared to control mice.The absence of cardiac pathology in the presence of the predicted molecular phenotype suggests that de novo DNA methylation in cardiomyocytes is dispensable for adaptive mechanisms after chronic cardiac pressure overload.

  3. Steady-state solutions of cell volume in a cardiac myocyte model elaborated for membrane excitation, ion homeostasis and Ca2+ dynamics.

    Science.gov (United States)

    Cha, Chae Young; Noma, Akinori

    2012-08-21

    The cell volume continuously changes in response to varying physiological conditions, and mechanisms underlying volume regulation have been investigated in both experimental and theoretical studies. Here, general formulations concerning cell volume change are presented in the context of developing a comprehensive cell model which takes Ca(2+) dynamics into account. Explicit formulas for charge conservation and steady-state volumes of the cytosol and endoplasmic reticulum (ER) are derived in terms of membrane potential, amount of ions, Ca(2+)-bound buffer molecules, and initial cellular conditions. The formulations were applied to a ventricular myocyte model which has plasma-membrane Ca(2+) currents with dynamic gating mechanisms, Ca(2+)-buffering reactions with diffusive and non-diffusive buffer proteins, and Ca(2+) uptake into or release from the sarcoplasmic reticulum (SR) accompanied by compensatory cationic or anionic currents through the SR membrane. Time-dependent volume changes in cardiac myocytes induced by varying extracellular osmolarity or by action potential generation were successfully simulated by the novel formulations. Through application of bifurcation analysis, the existence and uniqueness of steady-state solutions of the cell volume were validated, and contributions of individual ion channels and transporters to the steady-state volume were systematically analyzed. The new formulas are consistent with previous fundamental theory derived from simple models of minimum compositions. The new formulations may be useful for examination of the relationship between cell function and volume change in other cell types.

  4. Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Guangqin; FU Yu; YANG Dongmei; HAO Xuemei; BAI Shuhua; TANG Yiqun; Edward G LAKATTA; WU Caihong; CHENG Heping

    2004-01-01

    Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sarcoplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCl2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of ~10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.

  5. Chloride current in mammalian cardiac myocytes. Novel mechanism for autonomic regulation of action potential duration and resting membrane potential

    OpenAIRE

    1990-01-01

    The properties of the autonomically regulated chloride current (ICl) were studied in isolated guinea pig ventricular myocytes. This current was elicited upon exposure to isoproterenol (ISO) and reversed upon concurrent exposure to acetylcholine (ACh). ICl was time independent and exhibited outward rectification. The responses to ISO and ACh could be blocked by propranolol and atropine, respectively, and ICl was also elicited by forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, and 3-iso...

  6. Exercise training and detraining modify the morphological and mechanical properties of single cardiac myocytes obtained from spontaneously hypertensive rats.

    Science.gov (United States)

    Carneiro-Júnior, M A; Pelúzio, M C G; Silva, C H O; Amorim, P R S; Silva, K A; Souza, M O; Castro, C A; Roman-Campos, D; Prímola-Gomes, T N; Natali, A J

    2010-11-01

    We determined the effects of exercise training and detraining on the morphological and mechanical properties of left ventricular myocytes in 4-month-old spontaneously hypertensive rats (SHR) randomly divided into the following groups: sedentary for 8 weeks (SED-8), sedentary for 12 weeks (SED-12), treadmill-running trained for 8 weeks (TRA, 16 m/min, 60 min/day, 5 days/week), and treadmill-running trained for 8 weeks followed by 4 weeks of detraining (DET). At sacrifice, left ventricular myocytes were isolated enzymatically, and resting cell length, width, and cell shortening after stimulation at a frequency of 1 Hz (~25°C) were measured. Cell length was greater in TRA than in SED-8 (161.30 ± 1.01 vs 156.10 ± 1.02 μm, P exercise training or detraining. Cell length to width ratio was higher in TRA than in SED-8 (8.50 ± 0.08 vs 8.22 ± 0.10, P Exercise training did not affect cell shortening, which was unchanged with detraining. TRA cells exhibited higher maximum velocity of shortening than SED-8 (102.01 ± 4.50 vs 82.01 ± 5.30 μm/s, P exercise training affected left ventricle remodeling in SHR towards eccentric hypertrophy, which remained after detraining. It also improved single left ventricular myocyte contractile function, which was reversed by detraining.

  7. Effects of adding intravenous nicorandil to standard therapy on cardiac sympathetic nerve activity and myocyte dysfunction in patients with acute decompensated heart failure

    Energy Technology Data Exchange (ETDEWEB)

    Kasama, Shu [Gunma University Graduate School of Medicine, Department of Medicine and Biological Science (Cardiovascular Medicine), Maebashi, Gunma (Japan); Cardiovascular Hospital of Central Japan (Kitakanto Cardiovascular Hospital), Department of Cardiovascular Medicine, Gunma (Japan); Toyama, Takuji; Funada, Ryuichi; Takama, Noriaki; Koitabashi, Norimichi; Kurabayashi, Masahiko [Gunma University Graduate School of Medicine, Department of Medicine and Biological Science (Cardiovascular Medicine), Maebashi, Gunma (Japan); Ichikawa, Shuichi [Cardiovascular Hospital of Central Japan (Kitakanto Cardiovascular Hospital), Department of Cardiovascular Medicine, Gunma (Japan); Suzuki, Yasuyuki; Matsumoto, Naoya [Nihon University School of Medicine, Department of Cardiology, Tokyo (Japan); Sato, Yuichi [Health Park Clinic, Department of Imaging, Takasaki, Gunma (Japan)

    2015-04-01

    Nicorandil, an adenosine triphosphate-sensitive potassium channel opener, improves cardiac sympathetic nerve activity (CSNA) in ischemic heart disease or chronic heart failure. However, its effects on CSNA and myocyte dysfunction in acute heart failure (AHF) remain unclear. We investigated the effects of adding intravenous nicorandil to standard therapy on CSNA and myocyte dysfunction in AHF. We selected 70 patients with mild to moderate nonischemic AHF who were treated with standard conventional therapy soon after admission. Thirty-five patients were assigned to additionally receive intravenous nicorandil (4-12 mg/h; group A), whereas the remaining patients continued their current drug regimen (group B). Delayed total defect score (TDS), delayed heart to mediastinum count (H/M) ratio, and washout rate (WR) were determined by {sup 123}I-metaiodobenzylguanidine (MIBG) scintigraphy within 3 days of admission and 4 weeks later. High sensitivity troponin T (hs-TnT) level was also measured at the same time points. After treatment, MIBG scintigraphic parameters significantly improved in both groups. However, the extent of the changes in these parameters in group A significantly exceeded the extent of the changes in group B [TDS -11.3 ± 4.3 in group A vs -4.0 ± 6.0 in group B (p < 0.01); H/M ratio 0.31 ± 0.16 vs 0.14 ± 0.16 (p < 0.01); WR -13.8 ± 7.8 % vs -6.1 ± 8.9 % (p < 0.01)]. The hs-TnT level decreased significantly from 0.052 ± 0.043 to 0.041 ± 0.033 ng/ml (p < 0.05) in group A, but showed no significant change in group B. Moreover, in both groups, no relationships between the extent of changes in MIBG parameters and hs-TnT level were observed. Adding intravenous nicorandil to standard therapy provides additional benefits for CSNA and myocyte dysfunction over conventional therapy alone in AHF patients. Furthermore, the mechanisms of improvement in CSNA and myocyte dysfunction after nicorandil treatment in AHF patients were distinct. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Jerry Curran

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

  9. Stearoyl-CoA desaturase-1 (SCD1 augments saturated fatty acid-induced lipid accumulation and inhibits apoptosis in cardiac myocytes.

    Directory of Open Access Journals (Sweden)

    Hiroki Matsui

    Full Text Available Mismatch between the uptake and utilization of long-chain fatty acids in the myocardium leads to abnormally high intracellular fatty acid concentration, which ultimately induces myocardial dysfunction. Stearoyl-Coenzyme A desaturase-1 (SCD1 is a rate-limiting enzyme that converts saturated fatty acids (SFAs to monounsaturated fatty acids. Previous studies have shown that SCD1-deficinent mice are protected from insulin resistance and diet-induced obesity; however, the role of SCD1 in the heart remains to be determined. We examined the expression of SCD1 in obese rat hearts induced by a sucrose-rich diet for 3 months. We also examined the effect of SCD1 on myocardial energy metabolism and apoptotic cell death in neonatal rat cardiac myocytes in the presence of SFAs. Here we showed that the expression of SCD1 increases 3.6-fold without measurable change in the expression of lipogenic genes in the heart of rats fed a high-sucrose diet. Forced SCD1 expression augmented palmitic acid-induced lipid accumulation, but attenuated excess fatty acid oxidation and restored reduced glucose oxidation. Of importance, SCD1 substantially inhibited SFA-induced caspase 3 activation, ceramide synthesis, diacylglycerol synthesis, apoptotic cell death, and mitochondrial reactive oxygen species (ROS generation. Experiments using SCD1 siRNA confirmed these observations. Furthermore, we showed that exposure of cardiac myocytes to glucose and insulin induced SCD1 expression. Our results indicate that SCD1 is highly regulated by a metabolic syndrome component in the heart, and such induction of SCD1 serves to alleviate SFA-induced adverse fatty acid catabolism, and eventually to prevent SFAs-induced apoptosis.

  10. The Myocyte Expression of Adiponectin Receptors and PPARδ Is Highly Coordinated and Reflects Lipid Metabolism of the Human Donors

    Directory of Open Access Journals (Sweden)

    Anna-Maria Ordelheide

    2011-01-01

    Full Text Available Muscle lipid oxidation is stimulated by peroxisome proliferator-activated receptor (PPAR δ or adiponectin receptor signalling. We studied human myocyte expression of the PPARδ and adiponectin receptor genes and their relationship to lipid parameters of the donors. The mRNA levels of the three adiponectin receptors, AdipoR1, AdipoR2, and T-cadherin, were highly interrelated (r≥0.91. However, they were not associated with GPBAR1, an unrelated membrane receptor. In addition, the adiponectin receptors were positively associated with PPARδ expression (r≥0.75. However, they were not associated with PPARα. Using stepwise multiple linear regression analysis, PPARδ was a significant determinant of T-cadherin (P=.0002. However, pharmacological PPARδ activation did not increase T-cadherin expression. The myocyte expression levels of AdipoR1 and T-cadherin were inversely associated with the donors' fasting plasma triglycerides (P<.03. In conclusion, myocyte expression of PPARδ and the adiponectin receptors are highly coordinated, and this might be of relevance for human lipid metabolism in vivo.

  11. Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

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    Salem, K A; Adrian, T E; Qureshi, M A; Parekh, K; Oz, M; Howarth, F C

    2012-12-01

    There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0

  12. Exercise training and detraining modify the morphological and mechanical properties of single cardiac myocytes obtained from spontaneously hypertensive rats

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    M.A. Carneiro-Júnior

    2010-11-01

    Full Text Available We determined the effects of exercise training and detraining on the morphological and mechanical properties of left ventricular myocytes in 4-month-old spontaneously hypertensive rats (SHR randomly divided into the following groups: sedentary for 8 weeks (SED-8, sedentary for 12 weeks (SED-12, treadmill-running trained for 8 weeks (TRA, 16 m/min, 60 min/day, 5 days/week, and treadmill-running trained for 8 weeks followed by 4 weeks of detraining (DET. At sacrifice, left ventricular myocytes were isolated enzymatically, and resting cell length, width, and cell shortening after stimulation at a frequency of 1 Hz (~25°C were measured. Cell length was greater in TRA than in SED-8 (161.30 ± 1.01 vs 156.10 ± 1.02 μm, P < 0.05, 667 vs 618 cells, respectively and remained larger after detraining. Cell width and volume were unaffected by either exercise training or detraining. Cell length to width ratio was higher in TRA than in SED-8 (8.50 ± 0.08 vs 8.22 ± 0.10, P < 0.05 and was maintained after detraining. Exercise training did not affect cell shortening, which was unchanged with detraining. TRA cells exhibited higher maximum velocity of shortening than SED-8 (102.01 ± 4.50 vs 82.01 ± 5.30 μm/s, P < 0.05, 70 cells per group, with almost complete regression after detraining. The maximum velocity of relengthening was higher in TRA cells than in SED-8 (88.20 ± 4.01 vs70.01 ± 4.80 μm/s, P < 0.05, returning to sedentary values with detraining. Therefore, exercise training affected left ventricle remodeling in SHR towards eccentric hypertrophy, which remained after detraining. It also improved single left ventricular myocyte contractile function, which was reversed by detraining.

  13. Fetal reprogramming and senescence in hypoplastic left heart syndrome and in human pluripotent stem cells during cardiac differentiation.

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    Gaber, Naila; Gagliardi, Mark; Patel, Pranali; Kinnear, Caroline; Zhang, Cindy; Chitayat, David; Shannon, Patrick; Jaeggi, Edgar; Tabori, Uri; Keller, Gordon; Mital, Seema

    2013-09-01

    Hypoplastic left heart syndrome (HLHS) is a severe cardiac malformation characterized by left ventricle (LV) hypoplasia and abnormal LV perfusion and oxygenation. We studied hypoxia-associated injury in fetal HLHS and human pluripotent stem cells during cardiac differentiation to assess the effect of microenvironmental perturbations on fetal cardiac reprogramming. We studied LV myocardial samples from 32 HLHS and 17 structurally normal midgestation fetuses. Compared with controls, the LV in fetal HLHS samples had higher nuclear expression of hypoxia-inducible factor-1α but lower angiogenic growth factor expression, higher expression of oncogenes and transforming growth factor (TGF)-β1, more DNA damage and senescence with cell cycle arrest, fewer cardiac progenitors, myocytes and endothelial lineages, and increased myofibroblast population (P cells (SMCs) had less DNA damage compared with endothelial cells and myocytes. We recapitulated the fetal phenotype by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation. DNA damage was prevented by treatment with a TGF-β1 inhibitor (P cells). The hypoplastic LV in fetal HLHS samples demonstrates hypoxia-inducible factor-1α up-regulation, oncogene-associated cellular senescence, TGF-β1-associated fibrosis and impaired vasculogenesis. The phenotype is recapitulated by subjecting human pluripotent stem cells to hypoxia during cardiac differentiation and rescued by inhibition of TGF-β1. This finding suggests that hypoxia may reprogram the immature heart and affect differentiation and development.

  14. Components of the interleukin-33/ST2 system are differentially expressed and regulated in human cardiac cells and in cells of the cardiac vasculature.

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    Demyanets, Svitlana; Kaun, Christoph; Pentz, Richard; Krychtiuk, Konstantin A; Rauscher, Sabine; Pfaffenberger, Stefan; Zuckermann, Andreas; Aliabadi, Arezu; Gröger, Marion; Maurer, Gerald; Huber, Kurt; Wojta, Johann

    2013-07-01

    Interleukin-33 (IL-33) is a recently described member of the IL-1 family of cytokines, which was identified as a ligand for the ST2 receptor. Components of the IL-33/ST2 system were shown to be expressed in normal and pressure overloaded human myocardium, and soluble ST2 (sST2) has emerged as a prognostic biomarker in myocardial infarction and heart failure. However, expression and regulation of IL-33 in human adult cardiac myocytes and fibroblasts was not tested before. In this study we found that primary human adult cardiac fibroblasts (HACF) and human adult cardiac myocytes (HACM) constitutively express nuclear IL-33 that is released during cell necrosis. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-1β significantly increased both IL-33 protein and IL-33 mRNA expression in HACF and HACM as well as in human coronary artery smooth muscle cells (HCASMC). The nuclear factor-κB (NF-κB) inhibitor dimethylfumarate inhibited TNF-α- and IL-1β-induced IL-33 production as well as nuclear translocation of p50 and p65 NF-κB subunits in these cells. Mitogen-activated protein/extracellular signal-regulated kinase inhibitor U0126 abrogated TNF-α-, IFN-γ-, and IL-1β-induced and Janus-activated kinase inhibitor I reduced IFN-γ-induced IL-33 production. We detected IL-33 mRNA in human myocardial tissue from patients undergoing heart transplantation (n=27) where IL-33 mRNA levels statistically significant correlated with IFN-γ (r=0.591, p=0.001) and TNF-α (r=0.408, p=0.035) mRNA expression. Endothelial cells in human heart expressed IL-33 as well as ST2 protein. We also reveal that human cardiac and vascular cells have different distribution patterns of ST2 isoforms (sST2 and transmembrane ST2L) mRNA expression and produce different amounts of sST2 protein. Both human macrovascular (aortic and coronary artery) and heart microvascular endothelial cells express specific mRNA for both ST2 isoforms (ST2L and sST2) and are a source for sST2 protein, whereas

  15. Sudden cardiac death and inherited channelopathy: the basic electrophysiology of the myocyte and myocardium in ion channel disease.

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    Martin, Claire A; Matthews, Gareth D K; Huang, Christopher L-H

    2012-04-01

    Mutations involving cardiac ion channels result in abnormal action potential formation or propagation, leading to cardiac arrhythmias. Despite the large impact on society of sudden cardiac death resulting from such arrhythmias, understanding of the underlying cellular mechanism is poor and clinical risk stratification and treatment consequently limited. Basic research using molecular techniques, as well as animal models, has proved extremely useful in improving our knowledge of inherited arrhythmogenic syndromes. This offers the practitioner tools to accurately diagnose rare disorders and provides novel markers for risk assessment and a basis for new strategies of treatment.

  16. Profile of L-type Ca2+ current and Na+/Ca2+ exchange current during cardiac action potential in ventricular myocytes

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    Banyasz, Tamas; Horvath, Balazs; Jian, Zhong; Izu, Leighton T.; Chen-Izu, Ye

    2011-01-01

    Objective The L-type Ca2+ current (ICa,L) and the Na+/Ca2+ exchange current (INCX) are major inward currents that shape the cardiac action potential (AP). Previously, the profile of these currents during AP was determined from voltage-clamp experiments that used Ca2+ buffer. In this study, we aimed to obtain direct experimental measurement of these currents during cardiac AP with Ca2+ cycling. Method A newly developed AP-clamp sequential dissection method was used to record ionic currents in guinea pig ventricular myocytes under a triad of conditions: using the cell’s own AP as the voltage command, using internal and external solutions that mimic the cell’s ionic composition and, importantly, no exogenous Ca2+ buffer was used. Results The nifedipine-sensitive current (INIFE), which is composed of ICa,L and INCX, revealed hitherto unreported features during AP with Ca2+ cycling in the cell. We identified two peaks in the current profile followed by a long residual current extending beyond the AP, coinciding with a residual depolarization. The second peak and the residual current become apparent only when Ca2+ is not buffered. Pharmacological dissection of INIFE using SEA0400 shows that ICa,L is dominant during phase-1&2 whereas INCX contributes significantly to the inward current at phase-3&4 of AP. Conclusion These data provide the first direct experimental visualization of ICa,L and INCX during cardiac AP and Ca2+ cycle. The residual current reported here can serve as a potential substrate for afterdepolarizations when increased under pathologic conditions. PMID:21884673

  17. β-Adrenergic stimulation increases Cav3.1 activity in cardiac myocytes through protein kinase A.

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    Yingxin Li

    Full Text Available The T-type Ca(2+ channel (TTCC plays important roles in cellular excitability and Ca(2+ regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs from Cav3.1 double transgenic (TG mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca(2+ current (I(Ca-T(3.1. I(Ca-T(3.1 was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO and a cyclic AMP analog (dibutyryl-cAMP significantly increased I(Ca-T(3.1 as well as I(Ca-L in TG-VMs at both physiological and room temperatures. The ISO effect on I(Ca-L and I(Ca-T in TG myocytes was blocked by H89, a PKA inhibitor. I(Ca-T was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of I(Ca-T in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. I(Ca-T in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR system. In conclusion, β-adrenergic stimulation increases I(Ca-T(3.1 in cardiomyocytes(, which is mediated by the cAMP/PKA pathway. The upregulation of I(Ca-T(3.1 by the β-adrenergic system could play important roles in cellular functions involving Cav3.1.

  18. β-Adrenergic stimulation increases Cav3.1 activity in cardiac myocytes through protein kinase A.

    Science.gov (United States)

    Li, Yingxin; Wang, Fang; Zhang, Xiaoying; Qi, Zhao; Tang, Mingxin; Szeto, Christopher; Li, Ying; Zhang, Hongyu; Chen, Xiongwen

    2012-01-01

    The T-type Ca(2+) channel (TTCC) plays important roles in cellular excitability and Ca(2+) regulation. In the heart, TTCC is found in the sinoatrial nodal (SAN) and conduction cells. Cav3.1 encodes one of the three types of TTCCs. To date, there is no report regarding the regulation of Cav3.1 by β-adrenergic agonists, which is the topic of this study. Ventricular myocytes (VMs) from Cav3.1 double transgenic (TG) mice and SAN cells from wild type, Cav3.1 knockout, or Cav3.2 knockout mice were used to study β-adrenergic regulation of overexpressed or native Cav3.1-mediated T-type Ca(2+) current (I(Ca-T(3.1))). I(Ca-T(3.1)) was not found in control VMs but was robust in all examined TG-VMs. A β-adrenergic agonist (isoproterenol, ISO) and a cyclic AMP analog (dibutyryl-cAMP) significantly increased I(Ca-T(3.1)) as well as I(Ca-L) in TG-VMs at both physiological and room temperatures. The ISO effect on I(Ca-L) and I(Ca-T) in TG myocytes was blocked by H89, a PKA inhibitor. I(Ca-T) was detected in control wildtype SAN cells but not in Cav3.1 knockout SAN cells, indicating the identity of I(Ca-T) in normal SAN cells is mediated by Cav3.1. Real-time PCR confirmed the presence of Cav3.1 mRNA but not mRNAs of Cav3.2 and Cav3.3 in the SAN. I(Ca-T) in SAN cells from wild type or Cav3.2 knockout mice was significantly increased by ISO, suggesting native Cav3.1 channels can be upregulated by the β-adrenergic (β-AR) system. In conclusion, β-adrenergic stimulation increases I(Ca-T(3.1)) in cardiomyocytes(,) which is mediated by the cAMP/PKA pathway. The upregulation of I(Ca-T(3.1)) by the β-adrenergic system could play important roles in cellular functions involving Cav3.1.

  19. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

    Science.gov (United States)

    Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

    2016-01-01

    Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation.

  20. Serum Positive for the Autoantibody against the β1-Adrenoceptor from Chinese Patients with Congestive Heart Failure Decreases Iss in Mouse Cardiac Myocytes

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    Yuan-yuan Wang

    2011-01-01

    Full Text Available Autoantibodies targeting the β1-adrenergic receptor (AAB-β1 display agonist-like effects, which may have a pathogenic role in the progression of heart failure. Here, we used the electrophysiological recordings to explore the effects of AAB-β1-positive serum from Chinese patients with heart failure on the activity of the peak transient outward potassium current (Ito and the end 50 ms steady-state potassium current (Iss in mouse cardiac myocytes. We found that the AAB-β1-positive serum had no effect on the activity of Ito, but it produced a decrease in the currents of Iss. A low concentration of positive serum (1/100 had a small inhibitory effect on Iss. However, positive serum at 1 : 10, 1 : 20, and 1 : 50 significantly decreased Iss. The concentration-dependence analysis showed that the EC50 of AAB-β1-positive serum was 1/60.24 and its nH was 2.86. It indicated that the AAB-β1 could inhibit Iss in mouse cardiomyocyte in a concentration-dependent manner.

  1. Neonatal multiorgan failure due to ACAD9 mutation and complex I deficiency with mitochondrial hyperplasia in liver, cardiac myocytes, skeletal muscle, and renal tubules.

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    Leslie, Nancy; Wang, Xinjian; Peng, Yanyan; Valencia, C Alexander; Khuchua, Zaza; Hata, Jessica; Witte, David; Huang, Taosheng; Bove, Kevin E

    2016-03-01

    Complex I deficiency causes Leigh syndrome, fatal infant lactic acidosis, and neonatal cardiomyopathy. Mutations in more than 100 nuclear DNA and mitochondrial DNA genes miscode for complex I subunits or assembly factors. ACAD9 is an acyl-CoA dehydrogenase with a novel function in assembly of complex I; biallelic mutations cause progressive encephalomyopathy, recurrent Reye syndrome, and fatal cardiomyopathy. We describe the first autopsy in fatal neonatal lethal lactic acidosis due to mutations in ACAD9 that reduced complex I activity. We identified mitochondrial hyperplasia in cardiac myocytes, diaphragm muscle, and liver and renal tubules in formalin-fixed, paraffin-embedded tissue using immunohistochemistry for mitochondrial antigens. Whole-exome sequencing revealed compound heterozygous variants in the ACAD9 gene: c.187G>T (p.E63*) and c.941T>C (p.L314P). The nonsense mutation causes late infantile lethality; the missense variant is novel. Autopsy-derived fibroblasts had reduced complex I activity (53% of control) with normal activity in complexes II to IV, similar to reported cases of ACAD9 deficiency.

  2. Effect of intracellular Ca2+ and action potential duration on L-type Ca2+ channel inactivation and recovery from inactivation in rabbit cardiac myocytes.

    Science.gov (United States)

    Altamirano, Julio; Bers, Donald M

    2007-07-01

    Ca(2+) current (I(Ca)) recovery from inactivation is necessary for normal cardiac excitation-contraction coupling. In normal hearts, increased stimulation frequency increases force, but in heart failure (HF) this force-frequency relationship (FFR) is often flattened or reversed. Although reduced sarcoplasmic reticulum Ca(2+)-ATPase function may be involved, decreased I(Ca) availability may also contribute. Longer action potential duration (APD), slower intracellular Ca(2+) concentration ([Ca(2+)](i)) decline, and higher diastolic [Ca(2+)](i) in HF could all slow I(Ca) recovery from inactivation, thereby decreasing I(Ca) availability. We measured the effect of different diastolic [Ca(2+)](i) on I(Ca) inactivation and recovery from inactivation in rabbit cardiac myocytes. Both I(Ca) and Ba(2+) current (I(Ba)) were measured. I(Ca) decay was accelerated only at high diastolic [Ca(2+)](i) (600 nM). I(Ba) inactivation was slower but insensitive to [Ca(2+)](i). Membrane potential dependence of I(Ca) or I(Ba) availability was not affected by [Ca(2+)](i) <600 nM. Recovery from inactivation was slowed by both depolarization and high [Ca(2+)](i). We also used perforated patch with action potential (AP)-clamp and normal Ca(2+) transients, using various APDs as conditioning pulses for different frequencies (and to simulate HF APD). Recovery of I(Ca) following longer APD was increasingly incomplete, decreasing I(Ca) availability. Trains of long APs caused a larger I(Ca) decrease than short APD at the same frequency. This effect on I(Ca) availability was exacerbated by slowing twitch [Ca(2+)](i) decline by approximately 50%. We conclude that long APD and slower [Ca(2+)](i) decline lead to cumulative inactivation limiting I(Ca) at high heart rates and might contribute to the negative FFR in HF, independent of altered Ca(2+) channel properties.

  3. Anandamide reduces intracellular Ca2+ concentration through suppression of Na+/Ca2+ exchanger current in rat cardiac myocytes.

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    Qian Li

    Full Text Available PURPOSE: Anandamide, one of the endocannabinoids, has been reported to exhibit cardioprotective properties, particularly in its ability to limit the damage produced by ischemia reperfusion injury. However, the mechanisms underlying the effect are not well known. This study is to investigate whether anandamide alter Na(+/Ca(2+ exchanger and the intracellular free Ca(2+ concentration ([Ca(2+]i. METHODS: Na(+/Ca(2+ exchanger current (I(NCX was recorded and analysed by using whole-cell patch-clamp technique and [Ca(2+]i was measured by loading myocytes with the fluorescent Ca(2+ indicator Fura-2/AM. RESULTS: We found that I(NCX was enhanced significantly after perfusion with simulated ischemic external solution; [Ca(2+]i was also significantly increased by simulated ischemic solution. The reversal potential of I(NCX was shifted to negative potentials in simulated ischemic external solution. Anandamide (1-100 nM failed to affect I(NCX and [Ca(2+]i in normal solution. However, anandamide (1-100 nM suppressed the increase in INCX in simulated ischemic external solution concentration-dependently and normalized INCX reversal potential. Furthermore, anandamide (100 nM significantly attenuated the increase in [Ca(2+]i in simulated ischemic solution. Blocking CB1 receptors with the specific antagonist AM251 (500 nM failed to affect the effects of anandamide on I(NCX and [Ca(2+]i in simulated ischemic solution. CB2 receptor antagonist AM630 (100 nM eliminated the effects of anandamide on I(NCX and [Ca(2+]i in simulated ischemic solution, and CB2 receptor agonist JWH133 (100 nM simulated the effects of anandamide that suppressed the increase in I(NCX and [Ca(2+]i in simulated ischemic solution. In addition, pretreatment with the Gi/o-specific inhibitor pertussis toxin (PTX, 500 ng/ml eliminated the effects of anandamide and JWH133 on I(NCX in simulated ischemic solution. CONCLUSIONS: Collectively, these findings suggest that anandamide suppresses calcium

  4. Cell volume control in phospholemman (PLM) knockout mice: do cardiac myocytes demonstrate a regulatory volume decrease and is this influenced by deletion of PLM?

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    Bell, James R; Lloyd, David; Curl, Claire L; Delbridge, Lea M D; Shattock, Michael J

    2009-03-01

    In addition to modulatory actions on Na+-K+-ATPase, phospholemman (PLM) has been proposed to play a role in cell volume regulation. Overexpression of PLM induces ionic conductances, with 'PLM channels' exhibiting selectivity for taurine. Osmotic challenge of host cells overexpressing PLM increases taurine efflux and augments the cellular regulatory volume decrease (RVD) response, though a link between PLM and cell volume regulation has not been studied in the heart. We recently reported a depressed cardiac contractile function in PLM knockout mice in vivo, which was exacerbated in crystalloid-perfused isolated hearts, indicating that these hearts were osmotically challenged. To address this, the present study investigated the role of PLM in osmoregulation in the heart. Isolated PLM wild-type and knockout hearts were perfused with a crystalloid buffer supplemented with mannitol in a bid to prevent perfusate-induced cell swelling and maintain function. Accordingly, and in contrast to wild-type control hearts, contractile function was improved in PLM knockout hearts with 30 mM mannitol. To investigate further, isolated PLM wild-type and knockout cardiomyocytes were subjected to increasing hyposmotic challenges. Initial validation studies showed the IonOptix video edge-detection system to be a simple and accurate 'real-time' method for tracking cell width as a marker of cell size. Myocytes swelled equally in both genotypes, indicating that PLM, when expressed at physiological levels in cardiomyocytes, is not essential to limit water accumulation in response to a hyposmotic challenge. Interestingly, freshly isolated adult cardiomyocytes consistently failed to mount RVDs in response to cell swelling, adding to conflicting reports in the literature. A proposed perturbation of the RVD response as a result of the cell isolation process was not restored, however, with short-term culture in either adult or neonatal cardiomyocytes.

  5. The inhibition of N-glycosylation of glycoprotein 130 molecule abolishes STAT3 activation by IL-6 family cytokines in cultured cardiac myocytes.

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    Reo Matsuo

    Full Text Available Interleukin-6 (IL-6 family cytokines play important roles in cardioprotection against pathological stresses. IL-6 cytokines bind to their specific receptors and activate glycoprotein 130 (gp130, a common receptor, followed by further activation of STAT3 and extracellular signal-regulated kinase (ERK1/2 through janus kinases (JAKs; however the importance of glycosylation of gp130 remains to be elucidated in cardiac myocytes. In this study, we examined the biological significance of gp130 glycosylation using tunicamycin (Tm, an inhibitor of enzyme involved in N-linked glycosylation. In cardiomyocytes, the treatment with Tm completely replaced the glycosylated form of gp130 with its unglycosylated one. Tm treatment inhibited leukemia inhibitory factor (LIF-mediated activation of STAT3 and ERK1/2. Similarly, IL-11 failed to activate STAT3 and ERK1/2 in the presence of Tm. Interestingly, Tm inhibited the activation of JAKs 1 and 2, without influencing the expression of suppressor of cytokine signalings (SOCSs and protein-tyrosine phosphatase 1B (PTP1B, which are endogenous inhibitors of JAKs. To exclude the possibility that Tm blocks LIF and IL-11 signals by inhibiting the glycosylation of their specific receptors, we investigated whether the stimulation with IL-6 plus soluble IL-6 receptor (sIL-6R could transduce their signals in Tm-treated cardiomyocytes and found that this stimulation was unable to activate the downstream signals. Collectively, these findings indicate that glycosylation of gp130 is essential for signal transduction of IL-6 family cytokines in cardiomyocytes.

  6. A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct

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    Valarmathi, Mani T.; Fuseler, John W.; Davis, Jeffrey M.; Price, Robert L.

    2017-01-01

    Organ tissue engineering, including cardiovascular tissues, has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Attempts to provide oxygen and nutrients to the cells contained in the biomaterial constructs have had varying degrees of success. The aim of this current study is to develop a three-dimensional (3-D) model of vascularized cardiac tissue to examine the concurrent temporal and spatial regulation of cardiomyogenesis in the context of postnatal de novo vasculogenesis during stem cell cardiac regeneration. In order to achieve the above aim, we have developed an in vitro 3-D functional vascularized cardiac muscle construct using human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human mesenchymal stem cells (hMSCs). First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions. In this milieu, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed neo-angiogenesis and neo-cardiomyogenesis. Thus, our unique 3-D co-culture system provided us the apt in vitro functional vascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty. PMID:28194397

  7. Differential effects of central and peripheral fat tissues on the delayed rectifier K(+) outward currents in cardiac myocytes.

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    Lee, Kun-Tai; Tang, Paul Wei-Hua; Tsai, Wei-Chung; Liu, I-Hsin; Yen, Hsueh-Wei; Voon, Wen-Chol; Wu, Bin-Nan; Sheu, Sheng-Hsiung; Lai, Wen-Ter

    2013-01-01

    The amount of fat tissue is associated with an increasing incidence of cardiac arrhythmias. The purpose of this study was to investigate effects of adipocytokines from different body fat on delayed rectifier K(+) outward currents (IK). H9c2 cells were treated with adipocytokine-free medium (the Adipo-free group) and with adipocytokines from epicardial (central fat group) and limb (peripheral fat group) rat fat tissues. IK, as well as expressions of Kv2.1 and Kv2.1 mRNA in H9c2 cells, were measured and compared between different groups. IK measured in H9c2 cells immediately after treatment with adipocytokines were not significantly different from those treated with adipocytokine-free medium. After H9c2 cells were treated with adipocytokines for 18 h, IK were significantly decreased in the peripheral and central fat groups in comparison with the Adipo-free group. Compared with the peripheral fat group, IK were more significantly decreased in the central fat group. Expressions of Kv2.1 and Kv2.1 mRNA in H9c2 cells were not significantly different among the three groups. Adipocytokines significantly decreased IK in H9c2 cells, and IK was more prominently decreased by adipocytokines from epicardial fat than from limb fat tissues. The decrease in IK by adipocytokines may partially contribute to the mechanisms of arrhythmogenesis by fat tissues. Copyright © 2013 S. Karger AG, Basel.

  8. Quantification of exponential Na+ current activation in N-bromoacetamide-treated cardiac myocytes of guinea-pig.

    Science.gov (United States)

    Mitsuiye, T; Noma, A

    1993-01-01

    1. The activation kinetics of the Na+ current was investigated in single ventricular cells of the guinea-pig heart using an improved oil-gap voltage clamp method. The inactivation of the current was removed by an intracellular application of N-bromoacetamide (NBA) for less than 1 min. Although the NBA treatment slightly decreased the peak amplitudes (81.7 +/- 13.4% of control, n = 15), the Na+ current remained stable after the removal of inactivation. 2. On depolarization, the activation of Na+ current took an exponential time course after the capacitive current decreased to 5% of its peak amplitude (40-100 microseconds after the pulse onset). The time course of deactivation, recorded on repolarization from 1.2 ms depolarization, was also a single exponential. 3. The time constants of activation and deactivation were almost identical when compared at a given test potential within a range of -50 to -30 mV. These findings indicate that the cardiac Na+ current activation is determined by m1 kinetics, or one rate-limiting step. 4. At potentials negative to -60 mV, the deactivation was complete, and its time constant decreased e-fold per 20.3 +/- 1.8 mV hyperpolarization (n = 7). 5. The degree of steady-state activation (m(infinity)) was fitted to a Boltzmann equation with a slope factor of 7.4 +/- 0.3 mV and a half-maximum potential of -33.3 +/- 0.8 mV (n = 8). 6. Rate constants for the rate-limiting activation step between a closed state and an open state (alpha m, beta m), were determined from m(infinity) and tau m over a potential range between -100 and +50 mV. On a logarithmic scale, beta m-1 was a linear function of the membrane potential over the range -100 and -30 mV. 7. Fitting the newly determined activation kinetics to the rising phase of the action potential indicated that the activation kinetics in the present study is relevant to the physiological action potential. The density of the Na+ channels thus obtained was 1075 +/- 186 pF-1 (n = 6). 8. The

  9. Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes.

    Science.gov (United States)

    Wang, Huan; Wang, Hong-Fei; Wang, Chen; Chen, Yu-Fang; Ma, Rong; Xiang, Ji-Zhou; Du, Xin-Ling; Tang, Qiang

    2016-10-15

    In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell.

  10. I(f) blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes.

    Science.gov (United States)

    Scheruebel, Susanne; Koyani, Chintan N; Hallström, Seth; Lang, Petra; Platzer, Dieter; Mächler, Heinrich; Lohner, Karl; Malle, Ernst; Zorn-Pauly, Klaus; Pelzmann, Brigitte

    2014-07-01

    Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.

  11. Interactions between inflammatory signals and the progesterone receptor in regulating gene expression in pregnant human uterine myocytes

    Science.gov (United States)

    Lee, Yun; Sooranna, Suren R; Terzidou, Vasso; Christian, Mark; Brosens, Jan; Huhtinen, Kaisa; Poutanen, Matti; Barton, Geraint; Johnson, Mark R; Bennett, Phillip R

    2012-01-01

    The absence of a fall in circulating progesterone levels has led to the concept that human labour is associated with ‘functional progesterone withdrawal’ caused through changes in the expression or function of progesterone receptor (PR). At the time of labour, the human uterus is heavily infiltrated with inflammatory cells, which release cytokines to create a ‘myometrial inflammation’ via NF-κB activation. The negative interaction between NF-κB and PR, may represent a mechanism to account for ‘functional progesterone withdrawal’ at term. Conversely, PR may act to inhibit NF-κB function and so play a role in inhibition of myometrial inflammation during pregnancy. To model this inter-relationship, we have used small interfering (si) RNA-mediated knock-down of PR in human pregnant myocytes and whole genome microarray analysis to identify genes regulated through PR. We then activated myometrial inflammation using IL-1β stimulation to determine the role of PR in myometrial inflammation regulation. Through PR-knock-down, we found that PR regulates gene networks involved in myometrial quiescence and extracellular matrix integrity. Activation of myometrial inflammation was found to antagonize PR-induced gene expression, of genes normally upregulated via PR. We found that PR does not play a role in repression of pro-inflammatory gene networks induced by IL-1β and that only MMP10 was significantly regulated in opposite directions by IL-1β and PR. We conclude that progesterone acting through PR does not generally inhibit myometrial inflammation. Activation of myometrial inflammation does cause ‘functional progesterone withdrawal’ but only in the context of genes normally upregulated via PR. PMID:22435466

  12. Increased tolerance to stress in cardiac expressed gain-of-function of adenosine triphosphate-sensitive potassium channel subunit Kir6.1.

    Science.gov (United States)

    Henn, Matthew C; Janjua, M Burhan; Zhang, Haixia; Kanter, Evelyn M; Makepeace, Carol M; Schuessler, Richard B; Nichols, Colin G; Lawton, Jennifer S

    2016-12-01

    The adenosine triphosphate-sensitive potassium (KATP) channel opener diazoxide (DZX) prevents myocyte volume derangement and reduced contractility secondary to stress. KATP channels are composed of pore-forming (Kir6.1 or Kir6.2) and regulatory (sulfonylurea receptor, SUR1 or SUR2) subunits. Gain of function (GOF) of Kir6.1 subunits has been implicated in cardiac pathology in Cantu syndrome in humans (cardiomegaly, lymphedema, and pericardial effusions). We hypothesized that GOF of Kir6.1 subunits would result in altered myocyte response to stress. Isolated cardiac myocytes from wild type (WT) and transgenic Kir6.1GOF mice were exposed to Tyrode's physiologic solution for 20 min, test solution (Tyrode's or stress [hyperkalemic cardioplegia {CPG, known myocyte stress}] +/- KATP channel opener DZX), followed by Tyrode's for 20 min. Myocyte volume and contractility were measured and compared. WT myocytes demonstrated significant swelling in response to stress, but significantly less swelling was seen in Kir6.1GOF myocytes. DZX prevented swelling secondary to CPG in WT but resulted in a nonsignificant reduction in swelling in Kir6.1GOF myocytes. Both WT and Kir6.1GOF myocytes demonstrated a reduction in contractility during stress, although this was only significant in Kir6.1GOF myocytes. DZX was not associated with an improvement in contractility in Kir6.1GOF myocytes following stress. Similar to previous results in Kir6.1(-/-) myocytes, Kir6.1GOF myocytes demonstrate resistance (less volume derangement) to stress of cardioplegia. Understanding the role of Kir6.1 in myocyte response to stress may aid in the treatment of patients with Cantu syndrome and warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Dynamical mechanisms of phase-2 early afterdepolarizations in human ventricular myocytes: insights from bifurcation analyses of two mathematical models.

    Science.gov (United States)

    Kurata, Yasutaka; Tsumoto, Kunichika; Hayashi, Kenshi; Hisatome, Ichiro; Tanida, Mamoru; Kuda, Yuhichi; Shibamoto, Toshishige

    2017-01-01

    Early afterdepolarization (EAD) is known as a cause of ventricular arrhythmias in long QT syndromes. We theoretically investigated how the rapid (IKr) and slow (IKs) components of delayed-rectifier K(+) channel currents, L-type Ca(2+) channel current (ICaL), Na(+)/Ca(2+) exchanger current (INCX), Na(+)-K(+) pump current (INaK), intracellular Ca(2+) (Cai) handling via sarcoplasmic reticulum (SR), and intracellular Na(+) concentration (Nai) contribute to initiation, termination, and modulation of phase-2 EADs, using two human ventricular myocyte models. Bifurcation structures of dynamical behaviors in model cells were explored by calculating equilibrium points, limit cycles (LCs), and bifurcation points as functions of parameters. EADs were reproduced by numerical simulations. The results are summarized as follows: 1) decreasing IKs and/or IKr or increasing ICaL led to EAD generation, to which mid-myocardial cell models were especially susceptible; the parameter regions of EADs overlapped the regions of stable LCs. 2) Two types of EADs (termination mechanisms), IKs activation-dependent and ICaL inactivation-dependent EADs, were detected; IKs was not necessarily required for EAD formation. 3) Inhibiting INCX suppressed EADs via facilitating Ca(2+)-dependent ICaL inactivation. 4) Cai dynamics (SR Ca(2+) handling) and Nai strongly affected bifurcations and EAD generation in model cells via modulating ICaL, INCX, and INaK Parameter regions of EADs, often overlapping those of stable LCs, shifted depending on Cai and Nai in stationary and dynamic states. 5) Bradycardia-related induction of EADs was mainly due to decreases in Nai at lower pacing rates. This study demonstrates that bifurcation analysis allows us to understand the dynamical mechanisms of EAD formation more profoundly.

  14. LIF is a contraction-induced myokine stimulating human myocyte proliferation

    DEFF Research Database (Denmark)

    Broholm, Christa; Laye, Matthew J; Brandt, Claus

    2011-01-01

    Background: The cytokine leukemia inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of myoblasts. We hypothesized that LIF is a contraction-induced myokine functioning in an autocrine fashion to activate gene regulation of human muscle satellite cell proliferation....... Methods: Skeletal muscle LIF expression, regulation and action were examined in two models: 1) Young men performing a bout of heavy resistance exercise of the quadriceps muscle and 2) cultured primary human satellite cells. Results: Resistance exercise induced a 9-fold increase in LIF mRNA content...... in skeletal muscle, but LIF was not detectable in plasma of the subjects. However, electrically stimulated cultured human myotubes produced and secreted LIF, suggesting that LIF is a myokine with local effects. The well-established exercise-induced signaling molecules PI3K, Akt and mTor contributed...

  15. The angiotensin type 1 receptor activates extracellular signal-regulated kinases 1 and 2 by G protein-dependent and -independent pathways in cardiac myocytes and langendorff-perfused hearts

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Schneider, Mikael

    2007-01-01

    effects of ERK1/2 activity, differential activation of the AT(1)R in its native cellular context could have important biological and pharmacological implications. To examine if AT(1)R activates ERK1/2 by G protein-independent mechanisms in the heart, we used the [Sar(1), Ile(4), Ile(8)]-AngII ([SII] Ang......II) analogue in native preparations of cardiac myocytes and beating hearts. We found that [SII] AngII does not activate G(q)-coupling, yet stimulates the beta-arrestin2-dependent ERK1/2. The G(q)-activated pool of ERK1/2 rapidly translocates to the nucleus, while the beta-arrestin2-scaffolded pool remains...... in the cytosol. Similar biased agonism was achieved in Langendorff-perfused hearts, where both agonists elicit ERK1/2 phosphorylation, but [SII] AngII induces neither inotropic nor chronotropic effects....

  16. Cardiac tissue engineering: state of the art.

    Science.gov (United States)

    Hirt, Marc N; Hansen, Arne; Eschenhagen, Thomas

    2014-01-17

    The engineering of 3-dimensional (3D) heart muscles has undergone exciting progress for the past decade. Profound advances in human stem cell biology and technology, tissue engineering and material sciences, as well as prevascularization and in vitro assay technologies make the first clinical application of engineered cardiac tissues a realistic option and predict that cardiac tissue engineering techniques will find widespread use in the preclinical research and drug development in the near future. Tasks that need to be solved for this purpose include standardization of human myocyte production protocols, establishment of simple methods for the in vitro vascularization of 3D constructs and better maturation of myocytes, and, finally, thorough definition of the predictive value of these methods for preclinical safety pharmacology. The present article gives an overview of the present state of the art, bottlenecks, and perspectives of cardiac tissue engineering for cardiac repair and in vitro testing.

  17. Changes in Intracellular Na+ following Enhancement of Late Na+ Current in Virtual Human Ventricular Myocytes

    Science.gov (United States)

    Giles, Wayne R.

    2016-01-01

    The slowly inactivating or late Na+ current, INa-L, can contribute to the initiation of both atrial and ventricular rhythm disturbances in the human heart. However, the cellular and molecular mechanisms that underlie these pro-arrhythmic influences are not fully understood. At present, the major working hypothesis is that the Na+ influx corresponding to INa-L significantly increases intracellular Na+, [Na+]i; and the resulting reduction in the electrochemical driving force for Na+ reduces and (may reverse) Na+/Ca2+ exchange. These changes increase intracellular Ca2+, [Ca2+]i; which may further enhance INa-L due to calmodulin-dependent phosphorylation of the Na+ channels. This paper is based on mathematical simulations using the O’Hara et al (2011) model of baseline or healthy human ventricular action potential waveforms(s) and its [Ca2+]i homeostasis mechanisms. Somewhat surprisingly, our results reveal only very small changes (≤ 1.5 mM) in [Na+]i even when INa-L is increased 5-fold and steady-state stimulation rate is approximately 2 times the normal human heart rate (i.e. 2 Hz). Previous work done using well-established models of the rabbit and human ventricular action potential in heart failure settings also reported little or no change in [Na+]i when INa-L was increased. Based on our simulations, the major short-term effect of markedly augmenting INa-L is a significant prolongation of the action potential and an associated increase in the likelihood of reactivation of the L-type Ca2+ current, ICa-L. Furthermore, this action potential prolongation does not contribute to [Na+]i increase. PMID:27875582

  18. Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Green, Charlotte Jane; Pedersen, Maria; Pedersen, Bente K;

    2011-01-01

    To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes....

  19. Elevated NF-κB activation is conserved in human myocytes cultured from obese type 2 diabetic patients and attenuated by AMP-activated protein kinase

    DEFF Research Database (Denmark)

    Green, Charlotte Jane; Pedersen, Maria; Pedersen, Bente K

    2011-01-01

    To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes.......To examine whether the inflammatory phenotype found in obese and diabetic individuals is preserved in isolated, cultured myocytes and to assess the effectiveness of pharmacological AMP-activated protein kinase (AMPK) activation upon the attenuation of inflammation in these myocytes....

  20. Changes in human atrial myocyte dimension among different ages%不同年龄人心房肌细胞的形态观察

    Institute of Scientific and Technical Information of China (English)

    孙秀梅; 胡小琴; 苏俊武; 潘世伟; 刘迎龙

    2001-01-01

    AIM: To observe the changes in right atrial myocyte dimensionswith myocardial development. METHODS: Cell length, width, length/width and cross-sectional area were measured in right atrial myocytes isolated from 12 weaning [ages 0.4-5 years, (1.8±1.3) years, n=103 cells], 10 young [6-11 years, (9.3±1.8) years, n=104 cells], 13 adult [30-56 years, (43.7±11.5) years, n=110 cells] human hearts. RESULTS: Cell length, width, length/width and cross-sectional area, at weanling group were (70.2±1.3) μm, (8.0±0.2) μm, (9.0±0.2) and (50.9±2.6) μm2, respectively, at young group were (93.5±1.6) μm, (11.7±0.3) μm, 8.1±0.2, (109.7±5.8) μm2, and (100.9±2.2) μm, (12.1±0.3) μm, 8.5±0.2, (119.0±5.5) μm2 for adult group. Clearly, young myocyte lenght, width, cross-sectional area were greater than that of myocytes at weanling group(P<0.01) but adult myocytes length/width and cross-sectional area increase slightly compared with young group. The length/width ratio has no significant change through myocyte maturity, which is between 8-9. CONCLUSIONS: Our data suggest that myocyte dimensions expect length/width ratio have increased progressively with maturity, the length/width ratio is preserved in atrial myocyte during the period of normal myocyte growth from weanling to adulthood, and these changes in myocyte dimensions are similar with ventricular myocytes changes from other mammalian species.%的:观察不同年龄人心房肌细胞的形态变化。方法:取右心房组织块进行酶解,得到单个细胞。分别观察小儿组(12例0.4-5岁,平均1.8岁±1.3岁,n=103个细胞)、儿童组(10例6-11岁,平均9.3岁±1.8岁,n=104)、成年组(13例30-56岁,平均43.7岁±11.5岁,n=110)病人心房肌细胞的长、宽(直径)、长/宽比和细胞横截面积(±s)。结果:细胞长(μm)小儿组为70.2±1.3,儿童组为93.5±1.6,成年组为100.8±2.2,细胞宽(μm)分别为8.0±0.2、11.7±0.3、12.0±0.3

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

    Science.gov (United States)

    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

    2016-05-01

    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. © FASEB.

  2. MCI-154对大鼠心肌细胞的变力作用%Inotropic effects of MCI-154 on rat cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    陈还珍; 崔香丽; 赵画晨; 赵录英; 吕吉元; 吴博威

    2004-01-01

    Calcium sensitizers exert positive inotropic effects without increasing intracellular Ca2+. Thus, they avoid the undesired effects of Ca2+ overload such as arrhythmias and cell injury, but most of them may impair myocyte relaxation. However, MCI-154, also a calcium sensitizer, has no impairment to cardiomyocyte relaxation. To clarify the underlying mechanisms, we examined the effects of MCI-154 on Ca2+ transient and cell contraction using ion imaging system, and its influence on L-type Ca2+ current and Na+/Ca2+ exchange current with patch clamp technique in rat ventricular myocytes as well. The results showed that: (1) MCI-154 (1~100 μmol/L) had no effect on L-type Ca2+ current; (2) MCI-154 concentration-dependently increased cell shortening from 5.00± 1.6 μm of control to 6.2± 1.6 μm at 1 μmol/L,8.7±1.6 μm at 10 μmol/L and 14.0±1.4 μm at 100 μmol/L, respectively, with a slight increase in Ca2+ transient amplitude and an abbreviation of Ca2+ transient restore kinetics assessed by time to 50% restore (TR50) and time to 90% restore (TR90); (3) MCI-154 dosedependently increased the electrogenic Na+/Ca2+ exchange current both in the inward and the outward directions in rat ventricular myocytes. These results indicate that MCI-154 exerted a positive inotropic action without impairing myocyte relaxation. The stimulation of inward Na+/Ca2+ exchange current may accelerate the Ca2+ effiux, leading to abbreviations of TR50 and TR90 in rat myocytes. The findings suggest that the improvement by MCI-154 of myocyte relaxation is attributed to the forward mode of Na+/Ca2+ exchange.%钙增敏剂具有正性肌力作用,同时不增加细胞内钙浓度,因此可避免导致心律失常和最终心肌细胞死亡的钙超载.然而大部分钙增敏剂对心肌舒张功能有损害作用.MCI-154是一种钙增敏剂,但不损害舒张功能.为阐明其变力作用机制,我们应用离子成像技术研究了MCI-154对分离的单个大鼠心室肌细胞钙瞬变和

  3. Cardiac, respiratory, and locomotor coordination during walking in humans.

    Science.gov (United States)

    Niizeki, K; Kawahara, K; Miyamoto, Y

    1996-01-01

    Interactions between locomotor, respiratory, and cardiac rhythms were investigated in human subjects (n = 11) walking on a treadmill. Investigation of the phase relationship between heart rate and gait signals revealed that cardiac rhythms were entrained to locomotor rhythms when both frequencies were close to an integer ratio. Coherence spectra were estimated between heartbeat fluctuation, respiratory, and gait signals, and their magnitudes were evaluated. The results suggest that the respiratory-induced fluctuation in heartbeat would vary depending on the strength of the cardiolocomotor coupling. The synchronization tends to occur for one or two specific phases in an individual subject, but there was some variation among subjects. When the subjects voluntarily synchronized their cadence with the cardiac rhythm, the heart rate and blood pressure varied depending on the phase lag within a cardiac cycle. The coordination of locomotor and cardiac rhythms is discussed.

  4. Regulation of the instantaneous inward rectifier and the delayed outward rectifier potassium channels by Captopril and Angiotensin II via the Phosphoinositide-3 kinase pathway in volume-overload-induced hypertrophied cardiac myocytes

    Science.gov (United States)

    Alvin, Zikiar; Laurence, Graham G.; Coleman, Bernell R.; Zhao, Aiqiu; Hajj-Moussa, Majd; Haddad, Georges E.

    2011-01-01

    Summary Background Early development of cardiac hypertrophy may be beneficial but sustained hypertrophic activation leads to myocardial dysfunction. Regulation of the repolarizing currents can be modulated by the activation of humoral factors, such as angiotensin II (ANG II) through protein kinases. The aim of this work is to assess the regulation of IK and IK1 by ANG II through the PI3-K pathway in hypertrophied ventricular myocytes. Material/Methods Cardiac eccentric hypertrophy was induced through volume-overload in adult male rats by aorto-caval shunt (3 weeks). After one week half of the rats were given captopril (2 weeks; 0.5 g/l/day) and the other half served as control. The voltage-clamp and western blot techniques were used to measure the delayed outward rectifier potassium current (IK) and the instantaneous inward rectifier potassium current (IK1) and Akt activity, respectively. Results Hypertrophied cardiomyocytes showed reduction in IK and IK1. Treatment with captopril alleviated this difference seen between sham and shunt cardiomyocytes. Acute administration of ANG II (10−6M) to cardiocytes treated with captopril reduced IK and IK1 in shunts, but not in sham. Captopril treatment reversed ANG II effects on IK and IK1 in a PI3-K-independent manner. However in the absence of angiotensin converting enzyme inhibition, ANG II increased both IK and IK1 in a PI3-K-dependent manner in hypertrophied cardiomyocytes. Conclusions Thus, captopril treatment reveals a negative effect of ANG II on IK and IK1, which is PI3-K independent, whereas in the absence of angiotensin converting enzyme inhibition IK and IK1 regulation is dependent upon PI3-K. PMID:21709626

  5. Liberación de endotelina-1 por angiotensina ll en miocitos cardíacos aislados Angiotensin II-induced endothelin-1 release in cardiac myocytes

    Directory of Open Access Journals (Sweden)

    María C. Villa-Abrille

    2006-06-01

    Full Text Available Muchos de los efectos de la angiotensina II (Ang II son mediados en realidad por la acción de endotelina (ET endógena liberada y/o producida en respuesta a la Ang II. En este trabajo evaluamos la interacción Ang II/ET-1, sus consecuencias en la contractilidad cardíaca y el papel de las especies reactivas del oxígeno (EROs. Se usaron cardiomiocitos aislados de gato. La Ang II, 1 nM, produjo un efecto inotrópico positivo (EIP de 31.8±3.8% que fue cancelado por inhibición de los receptores AT1, de los receptores de ET, del intercambiador Na+/H+ (NHE, del modo inverso del intercambiador Na+/Ca2+ (NCX o por el secuestro de EROs. La Ang II, 100 nM, produjo un EIP de 70.5±7.6% que fue cancelado por inhibición de los receptores AT1 y bloqueado en parte por inhibición de los receptores de ET, del NHE, del modo inverso del NCX o por el secuestro de EROs. La Ang II, 1 nM, incrementó el ARNm de la preproET-1 lo cual fue anulado por el bloqueo de los receptores AT1. Los resultados permiten concluir que el EIP de la Ang II es debido a la acción de la ET-1 endógena liberada/formada por la Ang II. La ET-1 produce: estimulación del NHE, activación del modo inverso del NCX y un consecuente EIP. Dentro de esta cascada también participarían los EROs.Many of the effects thought to be due to angiotensin II (Ang II are due to the release/formation of endothelin (ET. We tested whether Ang II elicits its positive inotropic effect (PIE by the action of endogenous ET-1 and the role played by the reactive oxygen species (ROS in this mechanism. Experiments were performed in cat isolated ventricular myocytes in which sarcomere shortening (SS was measured to asses contractility after pharmacological interventions and the effect of Ang II on inotropism were analyzed. Ang II 1 nM increased SS by 31.8±3.8% (p<0.05. This PIE was cancelled by AT1 receptor blockade, by ET-1 receptors blockade, by Na+/H+ exchanger (NHE inhibition, by reverse mode Na+/Ca2

  6. The benefits of the Atlas of Human Cardiac Anatomy website for the design of cardiac devices.

    Science.gov (United States)

    Spencer, Julianne H; Quill, Jason L; Bateman, Michael G; Eggen, Michael D; Howard, Stephen A; Goff, Ryan P; Howard, Brian T; Quallich, Stephen G; Iaizzo, Paul A

    2013-11-01

    This paper describes how the Atlas of Human Cardiac Anatomy website can be used to improve cardiac device design throughout the process of development. The Atlas is a free-access website featuring novel images of both functional and fixed human cardiac anatomy from over 250 human heart specimens. This website provides numerous educational tutorials on anatomy, physiology and various imaging modalities. For instance, the 'device tutorial' provides examples of devices that were either present at the time of in vitro reanimation or were subsequently delivered, including leads, catheters, valves, annuloplasty rings and stents. Another section of the website displays 3D models of the vasculature, blood volumes and/or tissue volumes reconstructed from computed tomography and magnetic resonance images of various heart specimens. The website shares library images, video clips and computed tomography and MRI DICOM files in honor of the generous gifts received from donors and their families.

  7. The lipid peroxidation product 4-hydroxy-trans-2-nonenal causes protein synthesis in cardiac myocytes via activated mTORC1-p70S6K-RPS6 signaling.

    Science.gov (United States)

    Calamaras, Timothy D; Lee, Charlie; Lan, Fan; Ido, Yasuo; Siwik, Deborah A; Colucci, Wilson S

    2015-05-01

    Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 μM for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value.

  8. PET imaging of human cardiac opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Villemagne, Patricia S.R.; Dannals, Robert F. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Ravert, Hayden T. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Frost, James J. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States)

    2002-10-01

    The presence of opioid peptides and receptors and their role in the regulation of cardiovascular function has been previously demonstrated in the mammalian heart. The aim of this study was to image {mu} and {delta} opioid receptors in the human heart using positron emission tomography (PET). Five subjects (three females, two males, 65{+-}8 years old) underwent PET scanning of the chest with [{sup 11}C]carfentanil ([{sup 11}C]CFN) and [{sup 11}C]-N-methyl-naltrindole ([{sup 11}C]MeNTI) and the images were analyzed for evidence of opioid receptor binding in the heart. Either [{sup 11}C]CFN or [{sup 11}C]MeNTI (20 mCi) was injected i.v. with subsequent dynamic acquisitions over 90 min. For the blocking studies, either 0.2 mg/kg or 1 mg/kg of naloxone was injected i.v. 5 min prior to the injection of [{sup 11}C]CFN and [{sup 11}C]MeNTI, respectively. Regions of interest were placed over the left ventricle, left ventricular chamber, lung and skeletal muscle. Graphical analysis demonstrated average baseline myocardial binding potentials (BP) of 4.37{+-}0.91 with [{sup 11}C]CFN and 3.86{+-}0.60 with [{sup 11}C]MeNTI. Administration of 0.2 mg/kg naloxone prior to [{sup 11}C]CFN produced a 25% reduction in BP in one subject in comparison with baseline values, and a 19% decrease in myocardial distribution volume (DV). Administration of 1 mg/kg of naloxone before [{sup 11}C]MeNTI in another subject produced a 14% decrease in BP and a 21% decrease in the myocardial DV. These results demonstrate the ability to image these receptors in vivo by PET. PET imaging of cardiac opioid receptors may help to better understand their role in cardiovascular pathophysiology and the effect of abuse of opioids and drugs on heart function. (orig.)

  9. Trichostatin A induces a unique set of microRNAs including miR-129-5p that blocks cyclin-dependent kinase 6 expression and proliferation in H9c2 cardiac myocytes.

    Science.gov (United States)

    Majumdar, Gipsy; Raghow, Rajendra

    2016-04-01

    The pan-histone deacetylase inhibitor (HDACI), trichostatin A (TSA), was shown to normalize interleukin-18-induced cardiac hypertrophy in vivo and in vitro; evidently, this occurred via epigenetic mechanisms that profoundly altered cardiac gene expression (Majumdar et al. in, Physiol Genom, 43: 1392, 2011; BMC Genom, 13: 709, 2012). Here, we tested the hypothesis that TSA-induced changes in chromatin architecture also led to altered expression of microRNAs that in turn, contributed to the unique transcriptome of cardiac myocytes exposed to the HDACI. Using miRCURY LNA™ Universal microRNA PCR system, we demonstrate that H9c2 cells exposed to TSA for 6 and 24 h elicited differential expression of 19 and 16 microRNAs, respectively. H9c2 cells incubated in medium-containing 100 nM of TSA elicited a rapid and robust induction of miR-129-5p. Enhanced expression of miR-129-5p was also observed in the hearts of TSA-treated mice. Induction of miR-129-5p in H9c2 cells was accompanied by reduced expression of its direct target, cyclin-dependent kinase 6 (CDK6) that is a key regulator of cell cycle. Using cell division-dependent dilution of Cell Trace™ violet measurements we showed that concomitant induction of miR-129-5p and reduced CDK6 expression were mechanistically involved in TSA-induced inhibition of proliferation of H9c2 cells. Consistent with this scenario, cells expressing an antagomiR of miR-129-5p were resistant to the anti-proliferative actions of TSA. These data indicate that although TSA treatment led to altered expression of several microRNAs, the overarching action of TSA (i.e., inhibition of cell division) in H9c2 cells was achieved via miR-129-5p.

  10. Self-organizing human cardiac microchambers mediated by geometric confinement

    Science.gov (United States)

    Ma, Zhen; Wang, Jason; Loskill, Peter; Huebsch, Nathaniel; Koo, Sangmo; Svedlund, Felicia L.; Marks, Natalie C.; Hua, Ethan W.; Grigoropoulos, Costas P.; Conklin, Bruce R.; Healy, Kevin E.

    2015-07-01

    Tissue morphogenesis and organ formation are the consequences of biochemical and biophysical cues that lead to cellular spatial patterning in development. To model such events in vitro, we use PEG-patterned substrates to geometrically confine human pluripotent stem cell colonies and spatially present mechanical stress. Modulation of the WNT/β-catenin pathway promotes spatial patterning via geometric confinement of the cell condensation process during epithelial-mesenchymal transition, forcing cells at the perimeter to express an OCT4+ annulus, which is coincident with a region of higher cell density and E-cadherin expression. The biochemical and biophysical cues synergistically induce self-organizing lineage specification and creation of a beating human cardiac microchamber confined by the pattern geometry. These highly defined human cardiac microchambers can be used to study aspects of embryonic spatial patterning, early cardiac development and drug-induced developmental toxicity.

  11. Technetium-99m labeled 1-(4-fluorobenzyl)-4-(2-mercapto-2-methyl-4-azapentyl)-4- (2-mercapto-2-methylp ropylamino)-piperidine and iodine-123 metaiodobenzylguanidine for studying cardiac adrenergic function: a comparison of the uptake characteristics in vascular smooth muscle cells and neonatal cardiac myocytes, and an investigation in rats

    Energy Technology Data Exchange (ETDEWEB)

    Samnick, Samuel E-mail: rassam@uniklinik-saarland.de; Scheuer, Claudia; Muenks, Sven; El-Gibaly, Amr M.; Menger, Michael D.; Kirsch, Carl-Martin

    2004-05-01

    In developing technetium-99m-based radioligands for in vivo studies of cardiac adrenergic neurons, we compared the uptake characteristics of the {sup 99m}Tc-labeled 1-(4-fluorobenzyl)-4-(2-mercapto-2-methyl-4-azapentyl)-4- (2-mercapto-2-methylpropylamino)-piperidine ({sup 99m}Tc-FBPBAT) with those of the clinically established meta-[{sup 123}I]iodobenzylguanidine ({sup 123}I-MIBG) in rat vascular smooth muscle cells and neonatal cardiac myocytes. Furthermore, the cardiac and extracardiac uptake of both radiopharmaceuticals was assessed in intact rats and in rats pretreated with various {alpha}- and {beta}-adrenoceptor drugs, and adrenergic reuptake blocking agents. The uptake of {sup 99m}Tc-FBPBAT and {sup 123}I-MIBG into vascular smooth muscle cells and neonatal cardiac myocytes was rapid; more than 85% of the radioactivity accumulation into the cells occurring within the first 3 minutes. Radioactivity uptake after a 60-minute incubation at 37 degree sign C (pH 7.4) varied from 15% to 65% of the total loaded activity per million cells. In all cases, {sup 99m}Tc-FBPBAT showed the higher uptake, relative to {sup 123}I-MIBG, at any given cell concentration. The cellular uptake of {sup 99m}Tc-FBPBAT was lower at 4 degree sign C and 20 degree sign C than at 37 degree sign C. In contrast, the {sup 123}I-MIBG uptake was only slightly temperature dependent. Inhibition experiments confirmed that the cellular uptake of {sup 123}I-MIBG is mediated by the uptake-I carrier, whereas {alpha}{sub 1}- and {beta}{sub 1}-adrenoceptors were predominantly involved in the uptake of {sup 99m}Tc-FBPBAT into the cardiovascular tissues. Biodistribution studies in rats showed that {sup 99m}Tc-FBPBAT accumulated in myocardium after intravenous injection. Radioactivity in rat heart amounted to 2.32% and 1.91% of the injected dose per gram at 15 and 60 minutes postinjection, compared with 3.10% and 2.21% injected dose per gram of tissue (%ID/g) in the experiment with {sup 123}I

  12. Proteome- and transcriptome-driven reconstruction of the human myocyte metabolic network and its use for identification of markers for diabetes

    DEFF Research Database (Denmark)

    Väremo, Leif; Scheele, Camilla; Broholm, Christa

    2015-01-01

    -scale metabolic models (GEMs) provide a network context for the integration of high-throughput data. We generated myocyte-specific RNA-sequencing data and investigated their correlation with proteome data. These data were then used to reconstruct a comprehensive myocyte GEM. Next, we performed a meta......-analysis of six studies comparing muscle transcription in T2D versus healthy subjects. Transcriptional changes were mapped on the myocyte GEM, revealing extensive transcriptional regulation in T2D, particularly around pyruvate oxidation, branched-chain amino acid catabolism, and tetrahydrofolate metabolism...

  13. Satellite cells derived from obese humans with type 2 diabetes and differentiated into myocytes in vitro exhibit abnormal response to IL-6.

    Directory of Open Access Journals (Sweden)

    Camilla Scheele

    Full Text Available Obesity and type 2 diabetes are associated with chronically elevated systemic levels of IL-6, a pro-inflammatory cytokine with a role in skeletal muscle metabolism that signals through the IL-6 receptor (IL-6Rα. We hypothesized that skeletal muscle in obesity-associated type 2 diabetes develops a resistance to IL-6. By utilizing western blot analysis, we demonstrate that IL-6Rα protein was down regulated in skeletal muscle biopsies from obese persons with and without type 2 diabetes. To further investigate the status of IL-6 signaling in skeletal muscle in obesity-associated type 2 diabetes, we isolated satellite cells from skeletal muscle of people that were healthy (He, obese (Ob or were obese and had type 2 diabetes (DM, and differentiated them in vitro into myocytes. Down-regulation of IL-6Rα was conserved in Ob myocytes. In addition, acute IL-6 administration for 30, 60 and 120 minutes, resulted in a down-regulation of IL-6Rα protein in Ob myocytes compared to both He myocytes (P<0.05 and DM myocytes (P<0.05. Interestingly, there was a strong time-dependent regulation of IL-6Rα protein in response to IL-6 (P<0.001 in He myocytes, not present in the other groups. Assessing downstream signaling, DM, but not Ob myocytes demonstrated a trend towards an increased protein phosphorylation of STAT3 in DM myocytes (P = 0.067 accompanied by a reduced SOCS3 protein induction (P<0.05, in response to IL-6 administration. Despite this loss of negative control, IL-6 failed to increase AMPKα2 activity and IL-6 mRNA expression in DM myocytes. There was no difference in fusion capacity of myocytes between cell groups. Our data suggest that negative control of IL-6 signaling is increased in myocytes in obesity, whereas a dysfunctional IL-6 signaling is established further downstream of IL-6Rα in DM myocytes, possibly representing a novel mechanism by which skeletal muscle function is compromised in type 2 diabetes.

  14. Validation of an in vitro contractility assay using canine ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Harmer, A.R., E-mail: alex.harmer@astrazeneca.com; Abi-Gerges, N.; Morton, M.J.; Pullen, G.F.; Valentin, J.P.; Pollard, C.E.

    2012-04-15

    Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ∼ 36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6–8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds. -- Highlights: ► Cardiac contractility is an important physiological function of the heart. ► Assessment of contractility is a logical part of pre-clinical drug safety testing. ► There are limited validated assays that predict effects of compounds on contractility. ► Using dog myocytes, we have developed an in vitro cardiac contractility assay. ► The assay predicted the in vivo contractility with a good level of accuracy.

  15. The effect of Gi-protein inactivation on basal, and β1- and β2AR-stimulated contraction of myocytes from transgenic mice overexpressing the β2-adrenoceptor

    OpenAIRE

    Gong, Haibin; Adamson, Dawn L; Ranu, Hardeep K; Koch, Walter J.; Heubach, Jürgen F; Ravens, Ursula; Zolk, Oliver; Harding, Sian E

    2000-01-01

    The atria and ventricles of transgenic mice (TGβ2) with cardiac overexpression of the human β2-adrenoceptor (β2AR) were initially reported to show maximum contractility in the absence of β-AR stimulation. However, we have previously observed a different phenotype in these mice, with myocytes showing normal contractility but reduced βAR responses. We have investigated the roles of cyclic AMP and Gi in basal and βAR function in these myocytes.ICI 118,551 at inverse agonist concentrations decrea...

  16. Human Engineered Cardiac Tissues Created Using Induced Pluripotent Stem Cells Reveal Functional Characteristics of BRAF-Mediated Hypertrophic Cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Timothy J Cashman

    Full Text Available Hypertrophic cardiomyopathy (HCM is a leading cause of sudden cardiac death that often goes undetected in the general population. HCM is also prevalent in patients with cardio-facio-cutaneous syndrome (CFCS, which is a genetic disorder characterized by aberrant signaling in the RAS/MAPK signaling cascade. Understanding the mechanisms of HCM development in such RASopathies may lead to novel therapeutic strategies, but relevant experimental models of the human condition are lacking. Therefore, the objective of this study was to develop the first 3D human engineered cardiac tissue (hECT model of HCM. The hECTs were created using human cardiomyocytes obtained by directed differentiation of induced pluripotent stem cells derived from a patient with CFCS due to an activating BRAF mutation. The mutant myocytes were directly conjugated at a 3:1 ratio with a stromal cell population to create a tissue of defined composition. Compared to healthy patient control hECTs, BRAF-hECTs displayed a hypertrophic phenotype by culture day 6, with significantly increased tissue size, twitch force, and atrial natriuretic peptide (ANP gene expression. Twitch characteristics reflected increased contraction and relaxation rates and shorter twitch duration in BRAF-hECTs, which also had a significantly higher maximum capture rate and lower excitation threshold during electrical pacing, consistent with a more arrhythmogenic substrate. By culture day 11, twitch force was no longer different between BRAF and wild-type hECTs, revealing a temporal aspect of disease modeling with tissue engineering. Principal component analysis identified diastolic force as a key factor that changed from day 6 to day 11, supported by a higher passive stiffness in day 11 BRAF-hECTs. In summary, human engineered cardiac tissues created from BRAF mutant cells recapitulated, for the first time, key aspects of the HCM phenotype, offering a new in vitro model for studying intrinsic mechanisms and

  17. Bioengineering Human Myocardium on Native Extracellular Matrix

    Science.gov (United States)

    Guyette, Jacques P.; Charest, Jonathan M; Mills, Robert W; Jank, Bernhard J.; Moser, Philipp T.; Gilpin, Sarah E.; Gershlak, Joshua R.; Okamoto, Tatsuya; Gonzalez, Gabriel; Milan, David J.; Gaudette, Glenn R.; Ott, Harald C.

    2015-01-01

    Rationale More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. Objective The objective of this study is to translate previous work to human scale and clinically relevant cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human iPS-derived cardiac myocytes. Methods and Results To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue, showed electrical conductivity, left ventricular pressure development, and metabolic function. Conclusions Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable

  18. Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes

    Science.gov (United States)

    Chorvatova, Alzbeta; Aneba, Swida; Mateasik, Anton; Chorvat, Dusan; Comte, Blandine

    2013-06-01

    Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

  19. Cross-bridge cycling gives rise to spatiotemporal heterogeneity of dynamic subcellular mechanics in cardiac myocytes probed with atomic force microscopy.

    Science.gov (United States)

    Azeloglu, Evren U; Costa, Kevin D

    2010-03-01

    To study how the dynamic subcellular mechanical properties of the heart relate to the fundamental underlying process of actin-myosin cross-bridge cycling, we developed a novel atomic force microscope elastography technique for mapping spatiotemporal stiffness of isolated, spontaneously beating neonatal rat cardiomyocytes. Cells were indented repeatedly at a rate close but unequal to their contractile frequency. The resultant changes in pointwise apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 +/- 5.1 kPa and a diastolic value of 7.8 +/- 4.1 kPa at a representative depth of 400 nm. In cells probed along their major axis, spatiotemporal changes in systolic stiffness displayed a heterogeneous pattern, reflecting the banded sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform apparent modulus of 6.5 +/- 4.8 kPa. This study represents the first quantitative dynamic mechanical mapping of beating cardiomyocytes. The technique provides a means of probing the micromechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes, providing new insights and relating subcellular cardiac structure and function.

  20. 钙调蛋白激酶Ⅱ抑制剂对肥厚心肌细胞的影响%The effects of calmodulin kinase Ⅱ inhibitor on hypertrophic cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    柯俊; 陈锋; 肖幸; 戴木森; 王晓萍; 陈兵; 陈敏; 张存泰

    2012-01-01

    目的 观察钙调蛋白激酶Ⅱ(CaMKⅡ)抑制剂KN-93对肥厚心肌细胞L型钙电流(ICa,L)及细胞内钙离子浓度([Ca2+]i)的影响.方法 选取雌性新西兰大白兔48只,随机(随机数字法)分为4组:假手术组(sham组)、心肌肥厚组(LVH组)、心肌肥厚+KN-93组(KN-93组)、心肌肥厚+KN-92组(KN-92组),每组12只,通过缩窄腹主动脉制备兔心肌肥厚模型,Sham组仅游离腹主动脉未进行缩窄.8周后,采用胶原酶消化法分离单个心肌细胞,应用穿孔膜片钳技术记录L型钙电流(ICa,L);应用钙荧光指示剂Fura-2/AM结合图像分析技术测定各组心肌细胞内[Ca2+]i.结果 8周后,心肌肥厚模型建立成功.在0 mV时LVH组、Sham组的峰值ICa.L分另为(1.38±0.3)nA、(0.87±0.1)nA(P<0.01,n=12),电流密度分别为(6.7±1.0)pA/pF、(6.3 ±0.7)pA/pF(P>0.05,n=12).当KN-92及KN-93在浓度为0.5μmol/L时,可分别使肥厚心肌细胞0 mV时的峰值ICa,L降低(9.4±2.8)%、(10.5±3)%(P>0.05,n=12);当浓度增至1 μmol/L时,其峰值ICa,L降低程度分别为(13.4±3.7)%、(40±4.9)%(P<0.01,n=12).Sham组、LVH组、KN-92组及KN-93组中心肌细胞[Ca2+]i分别为(98.0±12.3)nmol/L、(154.0±26.2)nmol/L、(147.0±29.6)nmol/L和(108.0±21.2)nmol/L.结论 CaMKⅡ特异性抑制剂KN-93可有效抑制肥厚心肌细胞ICa.L,减轻细胞内钙超载,这可能是其抗肥厚心肌室性心律失常发生的主要细胞电生理机制.%Objective To investigate the effect of the calmodulin kinase Ⅱ Inhibitor KN-93 on L-typecalcium current(ICa,L)and intracellular calcium concentration([Ca2+]i)in hypertrophic cardiac myocytes.Methods Forty-eight female New Zealand white rabbits were randomized(random number)into four groups(12 animals in each group):the sham operation group(sham group),the left ventricular hypertrophy group(LVH group),the myocardial hypertrophy + KN-93 group(KN-93 group),and the myocardial hypertrophy + KN-92 group(KN-92 group).Myocardial hypertrophy in

  1. Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

    Science.gov (United States)

    Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

    2016-01-01

    Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

  2. Human torso phantom for imaging of heart with realistic modes of cardiac and respiratory motion

    Science.gov (United States)

    Boutchko, Rostyslav; Balakrishnan, Karthikayan; Gullberg, Grant T; O& #x27; Neil, James P

    2013-09-17

    A human torso phantom and its construction, wherein the phantom mimics respiratory and cardiac cycles in a human allowing acquisition of medical imaging data under conditions simulating patient cardiac and respiratory motion.

  3. Cardiac Progenitor Cell Extraction from Human Auricles

    KAUST Repository

    Di Nardo, Paolo

    2017-02-22

    For many years, myocardial tissue has been considered terminally differentiated and, thus, incapable of regenerating. Recent studies have shown, instead, that cardiomyocytes, at least in part, are slowly substituted by new cells originating by precursor cells mostly embedded into the heart apex and in the atria. We have shown that an elective region of progenitor cell embedding is represented by the auricles, non-contractile atria appendages that can be easily sampled without harming the patient. The protocol here reported describes how from auricles a population of multipotent, cardiogenic cells can be isolated, cultured, and differentiated. Further studies are needed to fully exploit this cell population, but, sampling auricles, it could be possible to treat cardiac patients using their own cells circumventing rejection or organ shortage limitations.

  4. Role of CaMKII in post acidosis arrhythmias: a simulation study using a human myocyte model.

    Science.gov (United States)

    Lascano, Elena C; Said, Matilde; Vittone, Leticia; Mattiazzi, Alicia; Mundiña-Weilenmann, Cecilia; Negroni, Jorge A

    2013-07-01

    Postacidotic arrhythmias have been associated to increased sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. However, the molecular mechanisms underlying these arrhythmias are still unclear. To better understand this process, acidosis produced by CO2 increase from 5% to 30%, resulting in intracellular pH (pHi) change from 7.15 to 6.7, was incorporated into a myocyte model of excitation-contraction coupling and contractility, including acidotic inhibition of L-type Ca(2+) channel (I(CaL)), Na(+)-Ca(2+) exchanger, Ca(2+) release through the SR ryanodine receptor (RyR2) (I(rel)), Ca(2+) reuptake by the SR Ca(2+) ATPase2a (I(up)), Na(+)-K(+) pump, K(+) efflux through the inward rectifier K(+) channel and the transient outward K(+) flow (I(to)) together with increased activity of the Na(+)-H(+) exchanger (I(NHE)). Simulated CaMKII regulation affecting I(rel), I(up), I(CaL), I(NHE) and I(to) was introduced in the model to partially compensate the acidosis outcome. Late Na(+) current increase by CaMKII was also incorporated. Using this scheme and assuming that diastolic Ca(2+) leak through the RyR2 was modulated by the resting state of this channel and the difference between SR and dyadic cleft [Ca(2+)], postacidotic delayed after depolarizations (DADs) were triggered upon returning to normal pHi after 6 min acidosis. The model showed that DADs depend on SR Ca(2+) load and on increased Ca(2+) leak through RyR2. This postacidotic arrhythmogenic pattern relies mainly on CaMKII effect on I(CaL) and I(up), since its individual elimination produced the highest DAD reduction. The model further revealed that during the return to normal pHi, DADs are fully determined by SR Ca(2+) load at the end of acidosis. Thereafter, DADs are maintained by SR Ca(2+) reloading by Ca(2+) influx through the reverse NCX mode during the time period in which [Na(+)]i is elevated.

  5. Fitting membrane resistance along with action potential shape in cardiac myocytes improves convergence: application of a multi-objective parallel genetic algorithm.

    Directory of Open Access Journals (Sweden)

    Jaspreet Kaur

    Full Text Available Fitting parameter sets of non-linear equations in cardiac single cell ionic models to reproduce experimental behavior is a time consuming process. The standard procedure is to adjust maximum channel conductances in ionic models to reproduce action potentials (APs recorded in isolated cells. However, vastly different sets of parameters can produce similar APs. Furthermore, even with an excellent AP match in case of single cell, tissue behaviour may be very different. We hypothesize that this uncertainty can be reduced by additionally fitting membrane resistance (Rm. To investigate the importance of Rm, we developed a genetic algorithm approach which incorporated Rm data calculated at a few points in the cycle, in addition to AP morphology. Performance was compared to a genetic algorithm using only AP morphology data. The optimal parameter sets and goodness of fit as computed by the different methods were compared. First, we fit an ionic model to itself, starting from a random parameter set. Next, we fit the AP of one ionic model to that of another. Finally, we fit an ionic model to experimentally recorded rabbit action potentials. Adding the extra objective (Rm, at a few voltages to the AP fit, lead to much better convergence. Typically, a smaller MSE (mean square error, defined as the average of the squared error between the target AP and AP that is to be fitted was achieved in one fifth of the number of generations compared to using only AP data. Importantly, the variability in fit parameters was also greatly reduced, with many parameters showing an order of magnitude decrease in variability. Adding Rm to the objective function improves the robustness of fitting, better preserving tissue level behavior, and should be incorporated.

  6. 运动性心肌肥厚中PKC信号通路研究进展%Advance of PKC Signaling Pathway in Exercise -induced Cardiac Myocyte Hypertrophy

    Institute of Scientific and Technical Information of China (English)

    柳华; 杨翼

    2011-01-01

    心肌肥厚是心脏受到刺激后的一种代偿反应。心肌的生理性肥厚可提高心脏机能,但病理性肥厚则会引起心血管疾病心律失常发病和死亡,亦是运动员发生猝死的原因之一。明确运动性心肌肥厚的机制,可为保护运动员心脏提供理论依据。目前为止,形成心肌肥厚的信号通路包括PKC、蛋白激酶B(Akt)、钙调神经磷酸酶(CaM)、丝裂原活化蛋白激酶MAPK、酪氨酸激酶受体gp130/STAT、牵拉敏感离子通道以及细胞骨架等。其中PKC途径是调控心肌细胞的生长、分化以及肥厚的过程中信号转导通路的关键环节。心肌细胞可通过Gq/PLC/Ca2+/PKC、PKC/MAPK、PKC/NF-κB等相关途径促使活化晚期反应基因使心肌肥厚。在运动过程中,形成的生理性肥厚主要通过AkffmTOR途径。心肌细胞的机械牵拉亦可以通过PKC信号传导促使肥厚。PKC包括13种亚型,各亚型对心脏的作用不一。PKCa对多次运动后心肌有保护作用;PKCβ1对心肌可能有保护作用,B2则是相反的作用;PKC8有利于心肌细胞的适应;PKCs对心肌的保护作用亦有可能有相反的作用;PKCζ促使心肌肥厚。%Myocardial hypertrophy is a compensatory response after the heart stimulation. Myocardial physio- logic hypertrophy can improve heart function, but pathological hypertrophy may cause cardiovascular disease as onset of arrhythmia and death, and also one of the causes of sudden death in athletes. The clearing mecha- nism of exercise -induced cardiac hypertrophy can provide a theoretical basis for the protection of athlete' s heart. So far, formation of myocardial hypertrophy signaling pathways includes PKC, protein kinase B ( Akt), calcineurin ( CaM), activation of the mitogen - activated protein kinase MAPK, tyrosine kinase re- ceptor gpl30/STAT, and the stretch of sensitive ion channels and cytoskeletal etc.. The PKC pathway is the

  7. Transcriptional profile of isoproterenol-induced cardiomyopathy and comparison to exercise-induced cardiac hypertrophy and human cardiac failure

    Directory of Open Access Journals (Sweden)

    McIver Lauren J

    2009-12-01

    Full Text Available Abstract Background Isoproterenol-induced cardiac hypertrophy in mice has been used in a number of studies to model human cardiac disease. In this study, we compared the transcriptional response of the heart in this model to other animal models of heart failure, as well as to the transcriptional response of human hearts suffering heart failure. Results We performed microarray analyses on RNA from mice with isoproterenol-induced cardiac hypertrophy and mice with exercise-induced physiological hypertrophy and identified 865 and 2,534 genes that were significantly altered in pathological and physiological cardiac hypertrophy models, respectively. We compared our results to 18 different microarray data sets (318 individual arrays representing various other animal models and four human cardiac diseases and identified a canonical set of 64 genes that are generally altered in failing hearts. We also produced a pairwise similarity matrix to illustrate relatedness of animal models with human heart disease and identified ischemia as the human condition that most resembles isoproterenol treatment. Conclusion The overall patterns of gene expression are consistent with observed structural and molecular differences between normal and maladaptive cardiac hypertrophy and support a role for the immune system (or immune cell infiltration in the pathology of stress-induced hypertrophy. Cross-study comparisons such as the results presented here provide targets for further research of cardiac disease that might generally apply to maladaptive cardiac stresses and are also a means of identifying which animal models best recapitulate human disease at the transcriptional level.

  8. Micro and Nano-mediated 3D Cardiac Tissue Engineering

    Science.gov (United States)

    2009-10-01

    13-70% positive for CD34, similar to values and ranges found for both excised and liposuction derived human cells. Also similar to human cells... position , policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public...studies. We examined the effects of substrate stiffness on the beating rate and beating force in embryonic chicken cardiac myocytes. Our results

  9. Preliminary assessment of cardiac short term safety and efficacy of manganese chloride for cardiovascular magnetic resonance in humans

    Directory of Open Access Journals (Sweden)

    Kalaf Jose M

    2011-01-01

    Full Text Available Abstract Background Manganese based agents are intracellular and accumulate inside myocytes allowing for different imaging strategies compared to gadolinium contrasts. While previous agents release manganese very slowly in the circulation, MnCl2 allows for rapid Mn2+ uptake in myocytes, creating a memory effect that can be potentially explored. Data on animal models are very encouraging but the safety and efficacy of this approach in humans has not yet been investigated. Therefore, our objectives were to study the safety and efficacy of a rapid infusion of manganese chloride (MnCl2 for cardiovascular magnetic resonance (CMR in humans. Methods Fifteen healthy volunteers underwent a CMR scan on a 1.5 T scanner. Before the infusion, cardiac function was calculated and images of a short axis mid-ventricular slice were obtained using a 2D and 3D gradient-echo inversion recovery (GRE-IR sequence, a phase-sensitive IR sequence and a single breath-hold segmented IR prepared steady-state precession acquisition for T1 calculations. MnCl2 was infused over three minutes at a total dose of 5 μMol/kg. Immediately after the infusion, and at 15 and 30 minutes later, new images were obtained and cardiac function re-evaluated. Results There was a significant decrease in T1 values compared to baseline, sustained up to 30 minutes after the MnCl2 infusion (pre,839 ± 281 ms; 0 min, 684 ± 99; 15 min, 714 ± 168; 30 min, 706 ± 172, P = 0.003. The 2D and 3D GRE-IR sequence showed the greatest increase in signal-to-noise ratio compared to the other sequences (baseline 6.6 ± 4.2 and 9.7 ± 5.3; 0 min, 11.3 ± 4.1 and 15.0 ± 8.7; 15 min, 10.8 ± 4.0 and 16.9 ± 10.2; 30 min, 10.6 ± 5.2 and 16.5 ± 8.3, P 2 with no major adverse events, despite all reporting transient facial flush. Conclusions In the short term, MnCl2 appears safe for human use. It effectively decreases myocardium T1, maintaining this effect for a relatively long period of time and allowing for the

  10. 利多卡因对家兔左右心室外膜心肌细胞电不均一性的影响%Effects of Lidocaine on Cardiac Electrical Heterogeneity of Left and Right Subepicardial Myocytes in Ventricular Walls of Rabbits

    Institute of Scientific and Technical Information of China (English)

    周军; 王腾; 胡河; 曹红; 秦牧

    2012-01-01

    addition (compared with lidocaine group, P<0. 05 and 0. 01, respectively). Lidocaine shifted upward the I-V curves of In, of both left and right subepicardial myocytes, but lifted outstandingly the I-V curve of Ina of right subepicardial myocytes on the top of all I-V curves. Moreover, lidocaine changed the peak potential of Ina from -20 to -10 mV in left and right subepicardial myocytes. Conclusion: Lidocaine could completely elicited the cardiac electrical heterogeneity of left and right subepicardial myocytes in ventricular walls of rabbits, which attributed to AP and Ina of right subepicardial myocytes which was attenuated more obviously than that of left subepicardial myocytes by administrating lidocaine.%目的:探讨利多卡因对家兔左、右心室心外膜心肌细胞动作电位和钠电流(INa)的影响,阐明利多卡因引起左、右心室心外膜心肌细胞电不均一性机制.方法:酶解法分离家兔单个左、右心室心外膜心肌细胞,全细胞膜片钳技术记录左、右心室心外膜心肌细胞动作电位和INa在应用利多卡因前后变化.结果:在电流钳制下,对照组中的左、右心室心外膜心肌细胞上记录动作电位都具有典型的0至4期的形态,2相平台期有心外膜心肌细胞特有的穹窿样凸起;但在利多卡因组,左、右心室心外膜心肌细胞动作均明显地失去2相平台期穹窿样凸起和高度,右室心外膜心肌细胞动作电位失去2相平台期立即复极,形似三角形,左、右心室心外膜心肌细胞动作电位的幅度(APA)和复极化50%和90%(APD50和APD90)均明显减少(P<0.05),且右室心外膜心肌细胞的APA和APD50以及APD90受利多卡因影响后减小最为严重(P<0.05或0.01).通过电压钳制方式,利多卡因使左、右心室心外膜心肌细胞的INa在各个指令电位下明显减小,而且右室心外膜心肌细胞INa减小的幅度要显著强于左室心外膜心肌细胞INa的减小幅度,当钳制电压为-20 mV

  11. Modes of induced cardiac arrest: hyperkalemia and hypocalcemia - Literature review

    OpenAIRE

    Oliveira,Marcos Aurélio Barboza de; Brandi, Antônio Carlos; dos Santos, Carlos Alberto; Botelho, Paulo Henrique Husseini; Cortez, José Luis Lasso; Braile, Domingo Marcolino

    2014-01-01

    The entry of sodium and calcium play a key effect on myocyte subjected to cardiac arrest by hyperkalemia. They cause cell swelling, acidosis, consumption of adenosine triphosphate and trigger programmed cell death. Cardiac arrest caused by hypocalcemia maintains intracellular adenosine triphosphate levels, improves diastolic performance and reduces oxygen consumption, which can be translated into better protection to myocyte injury induced by cardiac arrest.

  12. Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用%Construction of a lentiviral vector for RNA interference targeting rat cardiac myocytes connexin 43 gene and its effect

    Institute of Scientific and Technical Information of China (English)

    陈立; 钟国强; 涂荣会; 黎庆捷; 何艳

    2013-01-01

    目的 构建缝隙连接蛋白(Cx)43基因shRNA慢病毒载体,并检测其对大鼠心肌细胞Cx43基因的作用.方法 针对Cx43基因序列,设计RNA干扰靶点序列,合成靶序列的双链DNA,接入pGCL-GFP载体,挑选阳性克隆行PCR鉴定及测序.用pHelper1.0和pHelper2.0质粒转染293T细胞,包装产生具备感染能力的慢病毒.以293T细胞中绿色荧光蛋白的细胞数量计算病毒滴度;以最适感染复数感染大鼠心肌细胞,通过荧光显微镜观察感染效率;应用real-time PCR和Western blot法检测大鼠心肌细胞.Cx43 mRNA及Cx43蛋白表达,并评价其抑制效果.结果 经PCR鉴定和测序证实,Cx43慢病毒载体构建正确,其病毒滴度为8×108 TU/mL、转染效率为82.59%.荧光定量real-time PCR法检测Cx43 mRNA的抑制率为96.10%,Western blot法检测Cx43蛋白的抑制率为77.16%.结论 成功构建了Cx43基因shRNA慢病毒载体,其能显著抑制大鼠心肌细胞Cx43基因的表达.%Objective To construct a lentiviral RNAi vector and to detect its effect on connexin 43 (Cx43) gene in rat cardiac myocytes.Methods Short hairpin RNA (shRNA) sequence targeting rat cardiac myocytes Cx43 gene was designed.After synthesis and annealing,the double-stranded oligonucleotides (ds oligo) were connecting to pGC-LV vectors.The positive clones were selected and conducted by PCR identification and sequencing.Then,The viral particles were gen erated by cotransfection of 293T cells with the pGC-lv-Cx43 and two packaging vector (pHelper1.0,pHelper2.0),and the virus titer was determined by counting the number of GFP positive cells.After transfection of lentiviral vector into rat cardiac myocytes with multiplicity of infection (MOI),the level of Cx43 mRNA in rat cardiac myocytes was determined by realtime PCR and the level of Cx43 protein was determined by Western blot assay.The inhibitory effect was evaluated.Results The construction of Cx43 lentiviral vector was confirmed by PCR identification

  13. Toward GPGPU accelerated human electromechanical cardiac simulations.

    Science.gov (United States)

    Vigueras, Guillermo; Roy, Ishani; Cookson, Andrew; Lee, Jack; Smith, Nicolas; Nordsletten, David

    2014-01-01

    In this paper, we look at the acceleration of weakly coupled electromechanics using the graphics processing unit (GPU). Specifically, we port to the GPU a number of components of CHeart--a CPU-based finite element code developed for simulating multi-physics problems. On the basis of a criterion of computational cost, we implemented on the GPU the ODE and PDE solution steps for the electrophysiology problem and the Jacobian and residual evaluation for the mechanics problem. Performance of the GPU implementation is then compared with single core CPU (SC) execution as well as multi-core CPU (MC) computations with equivalent theoretical performance. Results show that for a human scale left ventricle mesh, GPU acceleration of the electrophysiology problem provided speedups of 164 × compared with SC and 5.5 times compared with MC for the solution of the ODE model. Speedup of up to 72 × compared with SC and 2.6 × compared with MC was also observed for the PDE solve. Using the same human geometry, the GPU implementation of mechanics residual/Jacobian computation provided speedups of up to 44 × compared with SC and 2.0 × compared with MC. © 2013 The Authors. International Journal for Numerical Methods in Biomedical Engineering published by John Wiley & Sons, Ltd.

  14. 26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Samaras, Susan E. [Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN (United States); Chen, Billy [Molecular Medicine Program, Department of Medicine, Boston University School of Medicine, Boston, MA (United States); Koch, Stephen R. [Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN (United States); Sawyer, Douglas B.; Lim, Chee Chew [Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, TN (United States); Davidson, Jeffrey M., E-mail: jeff.davidson@vanderbilt.edu [Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN (United States); Research Service, Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. Black-Right-Pointing-Pointer Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. Black-Right-Pointing-Pointer Differential degradation appears related to nuclear vs. sarcolemmal localization. Black-Right-Pointing-Pointer Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

  15. Maturing human pluripotent stem cell-derived cardiomyocytes in human engineered cardiac tissues.

    Science.gov (United States)

    Feric, Nicole T; Radisic, Milica

    2016-01-15

    Engineering functional human cardiac tissue that mimics the native adult morphological and functional phenotype has been a long held objective. In the last 5 years, the field of cardiac tissue engineering has transitioned from cardiac tissues derived from various animal species to the production of the first generation of human engineered cardiac tissues (hECTs), due to recent advances in human stem cell biology. Despite this progress, the hECTs generated to date remain immature relative to the native adult myocardium. In this review, we focus on the maturation challenge in the context of hECTs, the present state of the art, and future perspectives in terms of regenerative medicine, drug discovery, preclinical safety testing and pathophysiological studies. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Cyamemazine metabolites: effects on human cardiac ion channels in-vitro and on the QTc interval in guinea pigs.

    Science.gov (United States)

    Crumb, William; Benyamina, Amine; Arbus, Christophe; Thomas, George P; Garay, Ricardo P; Hameg, Ahcène

    2008-11-01

    Monodesmethyl cyamemazine and cyamemazine sulfoxide, the two main metabolites of the antipsychotic and anxiolytic phenothiazine cyamemazine, were investigated for their effects on the human ether-à-go-go related gene (hERG) channel expressed in HEK 293 cells and on native I(Na), I(Ca), I(to), I(sus) or I(K1) of human atrial myocytes. Additionally, cyamemazine metabolites were compared with terfenadine for their effects on the QT interval in anaesthetized guinea pigs. Monodesmethyl cyamemazine and cyamemazine sulfoxide reduced hERG current amplitude, with IC50 values of 0.70 and 1.53 microM, respectively. By contrast, at a concentration of 1 microM, cyamemazine metabolites failed to significantly affect I(Na), I(to), I(sus) or I(K1) current amplitudes. Cyamemazine sulfoxide had no effect on I(Ca) at 1 microM, while at this concentration, monodesmethyl cyamemazine only slightly (17%), albeit significantly, inhibited I(Ca) current. Finally, cyamemazine metabolites (5 mg kg(-1) i v.) were unable to significantly prolong QTc values in the guinea pig. Conversely, terfenadine (5 mg kg(-1) i.v.) significantly increased QTc values. In conclusion, cyamemazine metabolite concentrations required to inhibit hERG current substantially exceed those necessary to achieve therapeutic activity of the parent compound in humans. Moreover, cyamemazine metabolites, in contrast to terfenadine, do not delay cardiac repolarization in the anaesthetized guinea pig. These non-clinical findings explain the excellent cardiac safety records of cyamemazine during its 30 years of extensive therapeutic use.

  17. Interactions between cardiac, respiratory, and brain activity in humans

    Science.gov (United States)

    Musizza, Bojan; Stefanovska, Aneta

    2005-05-01

    The electrical activity of the heart (ECG), respiratory function and electric activity of the brain (EEG) were simultaneously recorded in conscious, healthy humans. Instantaneous frequencies of the heart beat, respiration and α-waves were then determined from 30-minutes recordings. The instantaneous cardiac frequency was defined as the inverse value of the time interval between two consecutive R-peaks. The instantaneous respiratory frequency was obtained from recordings of the excursions of thorax by application of the Hilbert transform. To obtain the instantaneous frequency of α-waves, the EEG signal recorded from the forehead was first analysed using the wavelet transform. Then the frequency band corresponding to α-waves was extracted and the Hilbert transform applied. Synchronization analysis was performed and the direction of coupling was ascertained, using pairs of instantaneous frequencies in each case. It is shown that the systems are weakly bidirectionally coupled. It was confirmed that, in conscious healthy humans, respiration drives cardiac activity. We also demonstrate from these analyses that α-activity drives both respiration and cardiac activity.

  18. Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

    Science.gov (United States)

    O’Connell, Timothy D.; Jensen, Brian C.; Baker, Anthony J.

    2014-01-01

    Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure. PMID:24368739

  19. Peripheral vasodilatation determines cardiac output in exercising humans

    DEFF Research Database (Denmark)

    Bada, A A; Svendsen, J H; Secher, N H

    2012-01-01

    conditions, because of a parallel decrease in stroke volume (P exercise. Atrial pacing lowered central venous pressure (P ...In dogs, manipulation of heart rate has no effect on the exercise-induced increase in cardiac output. Whether these findings apply to humans remain uncertain, because of the large differences in cardiovascular anatomy and regulation. To investigate the role of heart rate and peripheral...... vasodilatation in the regulation of cardiac output during steady-state exercise, we measured central and peripheral haemodynamics in 10 healthy male subjects, with and without atrial pacing (100–150 beats min(−1)) during: (i) resting conditions, (ii) one-legged knee extensor exercise (24 W) and (iii) femoral...

  20. Distribution of phenotypically disparate myocyte subpopulations in airway smooth muscle.

    Science.gov (United States)

    Halayko, Andrew J; Stelmack, Gerald L; Yamasaki, Akira; McNeill, Karol; Unruh, Helmut; Rector, Edward

    2005-01-01

    Phenotype and functional heterogeneity of airway smooth muscle (ASM) cells in vitro is well known, but there is limited understanding of these features in vivo. We tested whether ASM is composed of myocyte subsets differing in contractile phenotype marker expression. We used flow cytometry to compare smooth muscle myosin heavy chain (smMHC) and smooth muscle-alpha-actin (sm-alpha-actin) abundance in myocytes dispersed from canine trachealis. Based on immunofluorescent intensity and light scatter characteristics (forward and 90 degrees side scatter), 2 subgroups were identified and isolated. Immunoblotting confirmed smMHC and sm-alpha-actin were 10- and 5-fold greater, respectively, in large, elongate myocytes that comprised approximately 60% of total cells. Immunohistochemistry revealed similar phenotype heterogeneity in human bronchial smooth muscle. Canine tracheal myocyte subpopulations isolated by flow cytometry were used to seed primary subcultures. Proliferation of subcultures established with myocytes exhibiting low levels of smMHC and sm-alpha-actin was approximately 2 x faster than subcultures established with ASM cells with a high marker protein content. These studies demonstrate broad phenotypic heterogeneity of myocytes in normal ASM tissue that is maintained in cell culture, as demonstrated by divergent proliferative capacity. The distinct roles of these subgroups could be a key determinant of normal and pathological lung development and biology.

  1. Partial cardiac sympathetic denervation after bilateral thoracic sympathectomy in humans.

    Science.gov (United States)

    Moak, Jeffrey P; Eldadah, Basil; Holmes, Courtney; Pechnik, Sandra; Goldstein, David S

    2005-06-01

    Upper thoracic sympathectomy is used to treat several disorders. Sympathetic nerve fibers emanating from thoracic ganglia innervate the heart. Whether unilateral or bilateral upper thoracic sympathectomy affects cardiac sympathetic innervation in humans in vivo has been unclear. The purpose of this study was to assess whether thoracic sympathectomy decreases cardiac sympathetic innervation, as indicated by positron emission tomographic scanning after intravenous injection of the sympathoneural imaging agent 6-[18F]fluorodopamine. Nine patients with previous upper thoracic sympathectomies (four right-sided, one left-sided, four bilateral) underwent thoracic 6-[18F]fluorodopamine scanning between 1 and 2 hours after injection of the imaging agent. In each case, a low rate of entry of norepinephrine into the arm venous drainage (norepinephrine spillover) verified upper limb sympathectomy. Data were compared with those from the interventricular septum of patients with cardiac sympathetic denervation associated with pure autonomic failure and from normal volunteers. All four patients with bilateral sympathectomy had low septal myocardial 6-[18F]fluorodopamine-derived radioactivity (2,673 +/- 92 nCi-kg/cc-mCi at an average of 89 minutes after injection) compared with normal volunteers (3,634 +/- 311 nCi-kg/cc-mCi at 83 minutes, N = 22, P = .007) and higher radioactivity than in patients with pure autonomic failure (1,320 +/- 300 nCi-kg/cc-mCi at 83 minutes, N = 7, P = .003). Patients with unilateral sympathectomy had normal 6-[18F]fluorodopamine-derived radioactivity (3,971 +/- 337 nCi-kg/cc-mCi at 87 minutes). Bilateral upper thoracic sympathectomy partly decreases cardiac sympathetic innervation density.

  2. Determining a human cardiac pacemaker using fuzzy logic

    Science.gov (United States)

    Varnavsky, A. N.; Antonenco, A. V.

    2017-01-01

    The paper presents a possibility of estimating a human cardiac pacemaker using combined application of nonlinear integral transformation and fuzzy logic, which allows carrying out the analysis in the real-time mode. The system of fuzzy logical conclusion is proposed, membership functions and rules of fuzzy products are defined. It was shown that the ratio of the value of a truth degree of the winning rule condition to the value of a truth degree of any other rule condition is at least 3.

  3. Satellite cells derived from obese humans with type 2 diabetes and differentiated into myocytes in vitro exhibit abnormal response to IL-6

    DEFF Research Database (Denmark)

    Scheele, Camilla; Nielsen, Søren; Broholm, Christa

    2012-01-01

    a resistance to IL-6. By utilizing western blot analysis, we demonstrate that IL-6Rα protein was down regulated in skeletal muscle biopsies from obese persons with and without type 2 diabetes. To further investigate the status of IL-6 signaling in skeletal muscle in obesity-associated type 2 diabetes, we......Obesity and type 2 diabetes are associated with chronically elevated systemic levels of IL-6, a pro-inflammatory cytokine with a role in skeletal muscle metabolism that signals through the IL-6 receptor (IL-6Rα). We hypothesized that skeletal muscle in obesity-associated type 2 diabetes develops...... isolated satellite cells from skeletal muscle of people that were healthy (He), obese (Ob) or were obese and had type 2 diabetes (DM), and differentiated them in vitro into myocytes. Down-regulation of IL-6Rα was conserved in Ob myocytes. In addition, acute IL-6 administration for 30, 60 and 120 minutes...

  4. Mechanical analysis of single myocyte contraction in a 3-D elastic matrix.

    Directory of Open Access Journals (Sweden)

    John Shaw

    Full Text Available BACKGROUND: Cardiac myocytes experience mechanical stress during each heartbeat. Excessive mechanical stresses under pathological conditions cause functional and structural remodeling that lead to heart diseases, yet the precise mechanisms are still incompletely understood. To study the cellular and molecular level mechanotransduction mechanisms, we developed a new 'cell-in-gel' experimental system to exert multiaxial (3-D stresses on a single myocyte during active contraction. METHODS: Isolated myocytes are embedded in an elastic hydrogel to simulate the mechanical environment in myocardium (afterload. When electrically stimulated, the in-gel myocyte contracts while the matrix resists shortening and broadening of the cell, exerting normal and shear stresses on the cell. Here we provide a mechanical analysis, based on the Eshelby inclusion problem, of the 3-D strain and stress inside and outside the single myocyte during contraction in an elastic matrix. RESULTS: (1 The fractional shortening of the myocyte depends on the cell's geometric dimensions and the relative stiffness of the cell to the gel. A slender or softer cell has less fractional shortening. A myocyte of typical dimensions embedded in a gel of similar elastic stiffness can contract only 20% of its load-free value. (2 The longitudinal stress inside the cell is about 15 times the transverse stress level. (3 The traction on the cell surface is highly non-uniform, with a maximum near its ends, showing 'hot spots' at the location of intercalated disks. (4 The mechanical energy expenditure of the myocyte increases with the matrix stiffness in a monotonic and nonlinear manner. CONCLUSION: Our mechanical analyses provide analytic solutions that readily lend themselves to parametric studies. The resulting 3-D mapping of the strain and stress states serve to analyze and interpret ongoing cell-in-gel experiments, and the mathematical model provides an essential tool to decipher and quantify

  5. Human cardiac systems electrophysiology and arrhythmogenesis: iteration of experiment and computation.

    Science.gov (United States)

    Holzem, Katherine M; Madden, Eli J; Efimov, Igor R

    2014-11-01

    Human cardiac electrophysiology (EP) is a unique system for computational modelling at multiple scales. Due to the complexity of the cardiac excitation sequence, coordinated activity must occur from the single channel to the entire myocardial syncytium. Thus, sophisticated computational algorithms have been developed to investigate cardiac EP at the level of ion channels, cardiomyocytes, multicellular tissues, and the whole heart. Although understanding of each functional level will ultimately be important to thoroughly understand mechanisms of physiology and disease, cardiac arrhythmias are expressly the product of cardiac tissue-containing enough cardiomyocytes to sustain a reentrant loop of activation. In addition, several properties of cardiac cellular EP, that are critical for arrhythmogenesis, are significantly altered by cell-to-cell coupling. However, relevant human cardiac EP data, upon which to develop or validate models at all scales, has been lacking. Thus, over several years, we have developed a paradigm for multiscale human heart physiology investigation and have recovered and studied over 300 human hearts. We have generated a rich experimental dataset, from which we better understand mechanisms of arrhythmia in human and can improve models of human cardiac EP. In addition, in collaboration with computational physiologists, we are developing a database for the deposition of human heart experimental data, including thorough experimental documentation. We anticipate that accessibility to this human heart dataset will further human EP computational investigations, as well as encourage greater data transparency within the field of cardiac EP.

  6. Recreating the Cardiac Microenvironment in Pluripotent Stem Cell Models of Human Physiology and Disease.

    Science.gov (United States)

    Atmanli, Ayhan; Domian, Ibrahim John

    2016-12-19

    The advent of human pluripotent stem cell (hPSC) biology has opened unprecedented opportunities for the use of tissue engineering to generate human cardiac tissue for in vitro study. Engineering cardiac constructs that recapitulate human development and disease requires faithful recreation of the cardiac niche in vitro. Here we discuss recent progress in translating the in vivo cardiac microenvironment into PSC models of the human heart. We review three key physiologic features required to recreate the cardiac niche and facilitate normal cardiac differentiation and maturation: the biochemical, biophysical, and bioelectrical signaling cues. Finally, we discuss key barriers that must be overcome to fulfill the promise of stem cell biology in preclinical applications and ultimately in clinical practice.

  7. Modes of induced cardiac arrest: hyperkalemia and hypocalcemia--literature review.

    Science.gov (United States)

    Oliveira, Marcos Aurélio Barboza de; Brandi, Antônio Carlos; Santos, Carlos Alberto dos; Botelho, Paulo Henrique Husseini; Cortez, José Luis Lasso; Braile, Domingo Marcolino

    2014-01-01

    The entry of sodium and calcium play a key effect on myocyte subjected to cardiac arrest by hyperkalemia. They cause cell swelling, acidosis, consumption of adenosine triphosphate and trigger programmed cell death. Cardiac arrest caused by hypocalcemia maintains intracellular adenosine triphosphate levels, improves diastolic performance and reduces oxygen consumption, which can be translated into better protection to myocyte injury induced by cardiac arrest.

  8. Detection of cardiac activity changes from human speech

    Science.gov (United States)

    Tovarek, Jaromir; Partila, Pavol; Voznak, Miroslav; Mikulec, Martin; Mehic, Miralem

    2015-05-01

    Impact of changes in blood pressure and pulse from human speech is disclosed in this article. The symptoms of increased physical activity are pulse, systolic and diastolic pressure. There are many methods of measuring and indicating these parameters. The measurements must be carried out using devices which are not used in everyday life. In most cases, the measurement of blood pressure and pulse following health problems or other adverse feelings. Nowadays, research teams are trying to design and implement modern methods in ordinary human activities. The main objective of the proposal is to reduce the delay between detecting the adverse pressure and to the mentioned warning signs and feelings. Common and frequent activity of man is speaking, while it is known that the function of the vocal tract can be affected by the change in heart activity. Therefore, it can be a useful parameter for detecting physiological changes. A method for detecting human physiological changes by speech processing and artificial neural network classification is described in this article. The pulse and blood pressure changes was induced by physical exercises in this experiment. The set of measured subjects was formed by ten healthy volunteers of both sexes. None of the subjects was a professional athlete. The process of the experiment was divided into phases before, during and after physical training. Pulse, systolic, diastolic pressure was measured and voice activity was recorded after each of them. The results of this experiment describe a method for detecting increased cardiac activity from human speech using artificial neural network.

  9. Cardiac Disease Associated with Human Immunodeficiency Virus Infection.

    Science.gov (United States)

    Bloomfield, Gerald S; Leung, Claudia

    2017-02-01

    Over the last 2 decades human immunodeficiency virus (HIV) infection has become a chronic disease requiring long-term management. Aging, antiretroviral therapy, chronic inflammation, and several other factors contribute to the increased risk of cardiovascular disease in patients infected with HIV. In low-income and middle-income countries where antiretroviral therapy access is limited, cardiac disease is most commonly related to opportunistic infections and end-stage manifestations of HIV/acquired immunodeficiency syndrome, including HIV-associated cardiomyopathy, pericarditis, and pulmonary arterial hypertension. Cardiovascular screening, prevention, and risk factor management are important factors in the management of patients infected with HIV worldwide. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Vasopressin Type 1A Receptor Deletion Enhances Cardiac Contractility, β-Adrenergic Receptor Sensitivity and Acute Cardiac Injury-induced Dysfunction.

    Science.gov (United States)

    Wasilewski, Melissa A; Grisanti, Laurel A; Song, Jianliang; Carter, Rhonda L; Repas, Ashley A; Myers, Valerie D; Gao, Erhe; Koch, Walter J; Cheung, Joseph Y; Feldman, Arthur M; Tilley, Douglas

    2016-09-02

    V1AR expression is elevated in chronic human heart failure and contributes to cardiac dysfunction in animal models, in part via reduced βAR responsiveness.  While cardiac V1AR overexpression and V1AR stimulation are each sufficient to decrease βAR activity, it is unknown whether V1AR inhibition conversely augments βAR responsiveness.  Further, although V1AR has been shown to contribute to chronic progression of heart failure, its impact on cardiac function following acute ischemic injury has not been reported.  Using V1AR KO mice we assessed the impact of V1AR deletion on cardiac contractility at baseline and following ischemic injury, βAR sensitivity and cardiomyocyte responsiveness to βAR stimulation.  Strikingly, baseline cardiac contractility was enhanced in V1AR KO mice and they experienced a greater loss in contractile function than control mice following acute ischemic injury, although the absolute levels of cardiac dysfunction and survival rates did not differ.  Enhanced cardiac contractility in V1AR KO mice was associated with augmented β-blocker sensitivity, suggesting increased basal βAR activity, and indeed levels of left ventricular cAMP, as well as phospholamban and cardiac troponin I phosphorylation were elevated versus control mice.  At the cellular level, myocytes isolated from V1AR KO mice demonstrated increased responsiveness to βAR stimulation consistent with the finding that acute pharmacological V1AR inhibition enhanced βAR-mediated contractility in control myocytes.  Therefore, while V1AR deletion does not protect the heart from the rapid development of cardiac dysfunction following acute ischemic injury, its effects on βAR activity suggest that acute V1AR inhibition could be utilized to promote myocyte contractile performance.

  11. Primitive cardiac cells from human embryonic stem cells.

    Science.gov (United States)

    Hudson, James; Titmarsh, Drew; Hidalgo, Alejandro; Wolvetang, Ernst; Cooper-White, Justin

    2012-06-10

    Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study, we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures, single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells, corresponding to an increased expression of pluripotency markers OCT4 and NANOG, and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed, aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols, with induction of primitive streak cells using bone morphogenetic protein 4 and activin A, followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5, thus indicating the production of large numbers of immature cardiomyocytes (~65,000/cm(2) or ~1.5 per input hESC). This protocol was shown to be effective in HES3, H9, and, to a lesser, extent, MEL1 hESC lines. In addition, we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression, whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation, and potentially for the future treatment of heart failure.

  12. Changes in cardiac phenotype in hypertrophy and failure: From receptor to gene

    NARCIS (Netherlands)

    H.A. van Heugten (Han); J.M.J. Lamers (Jos)

    1997-01-01

    textabstractThe terminally differentiated adult cardiac myocyte cannot undergo cellular division. Growth of the heart in response to chronic hemodynamic overload therefore occurs through hypertrophy of the myocytes. The adaptation of the myocyte during hypertrophy not only involves an increase in ce

  13. Fluorescent Reporters in Human Pluripotent Stem Cells: Contributions to Cardiac Differentiation and Their Applications in Cardiac Disease and Toxicity

    NARCIS (Netherlands)

    Hartogh, den Sabine C.; Passier, Robert

    2016-01-01

    In the last decade, since the first report of induced pluripotent stem cells, the stem cell field has made remarkable progress in the differentiation to specialized cell-types of various tissues and organs, including the heart. Cardiac lineage- and tissue-specific human pluripotent stem cell (hPSC)

  14. Unlocking Barriers to DNA Vaccine Immunogenicity: A Cross-Species Analysis of Cytosolic DNA Sensing in Skeletal Muscle Myocytes

    Science.gov (United States)

    2016-10-01

    Skeletal Muscle Myocytes PRINCIPAL INVESTIGATOR: Dr. Gerasimos J. Zaharatos CONTRACTING ORGANIZATION: Sir Mortimer B. Davis Jewish General Hospital...Immunogenicity: A Cross-Species Analysis of Cytosolic DNA Sensing in Skeletal Muscle Myocytes 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Dr...humans. The present work is testing the hypothesis that skeletal muscle myocytes sense cytosolic DNA and elaborate an inflammatory response to DNA

  15. Effects of 17-methoxyl-7-hydroxy-benzfuranchalcon on Intracellular Calcium Overload in Cultured Cardiac Myocytes of Neonatal Rats Injured by H2O2 and L-type Calcium Current in Isolated Ventricular Myocytes of Mice%17-甲氧基-7-羟基-苯并呋喃查尔酮对心肌细胞内游离钙浓度及L-型钙电流的影响

    Institute of Scientific and Technical Information of China (English)

    李映新; 黄媛恒; 覃斐章; 林兴; 黄仁彬

    2013-01-01

    Objective:To investigate the effects of 17-methoxyl-7-hydroxy-benzfuranchalcon (YLSC) on intracellular calcium overload in cultured cardiac myocytes of neonatal rats injured by H2O2 and L-type calcium current (ICa-L) in isolated ventricular myocytes.Method:The cells of primary cultured cardiac myocytes of neonatal rats were divided into groups:①control group; ② model group:administrated with 0.3 mmol · L-1 H2O2; ③YLSC treated groups:incubation respectively with 100,200,400 μmol ·L-1 YLSC for 24 h,and add 0.3 mmol · L-1 H2O2.Laser confocal microscopy was used with Fluo-3/Am as indicator to detect changes of [Ca2+]i immediately and 15 mintues after H2O2 intervention.Single ventricular cell was obtained enzymatically by Langendorff perfusion apparatus.The wholecell patch clamp was used to record the ICa-L.Result:①The average fluorescence intensity values of the model group treated by H2O2 higher significantly than control group and the fluorescence enhancement ratio was 60.43 % ± 7.75%.The YLSC could decline the intensity values in a does-dependent manner manner,the fluorescence enhancement ratios of low,middle and high does respectively were 38.39% ±13.87%,14.49% ±2.94%,-28.1% ± 1.52%,and all of them were much lower compared with H2O2 group (P < 0.01).②YLSC can up-shifted the current-voltage (Ⅰ-Ⅴ) curves and markedly shifted the steady state activation and inactivation curve of ICa-L to the left.Conclusion:YLSC inhibited the L-type calcium channel,it can significantly reduce the myocardial intracellular Ca2+ overload induced by H2O2.%目的:研究17-甲氧基-7-羟基-苯并呋喃查尔酮(YLSC)对H2O2诱导的心肌细胞内钙超载的拮抗作用及对L型钙电流(ICa-L)的影响.方法:采用SD大鼠乳鼠进行心肌细胞培养,实验分为①正常对照组;②H2O2组:上机前加入终浓度为0.3 mmol·L-1的H2O2;③预先给予低、中、高不同终浓度YLSC药物处理组:分别给予100,200,400 μmol·L-1YLSC

  16. Rate-dependent force, intracellular calcium, and action potential voltage alternans are modulated by sarcomere length and heart failure induced-remodeling of thin filament regulation in human heart failure: A myocyte modeling study.

    Science.gov (United States)

    Zile, Melanie A; Trayanova, Natalia A

    2016-01-01

    Microvolt T-wave alternans (MTWA) testing identifies heart failure patients at risk for lethal ventricular arrhythmias at near-resting heart rates (voltage alternans (APV-ALT), the cellular driver of MTWA. Our goal was to uncover the mechanisms linking APV-ALT and FORCE-ALT in failing human myocytes and to investigate how the link between those alternans was affected by pacing rate and by physiological conditions such as sarcomere length and heart failure induced-remodeling of mechanical parameters. To achieve this, a mechanically-based, strongly coupled human electromechanical myocyte model was constructed. Reducing the sarcoplasmic reticulum calcium uptake current (Iup) to 27% was incorporated to simulate abnormal calcium handling in human heart failure. Mechanical remodeling was incorporated to simulate altered thin filament activation and crossbridge (XB) cycling rates. A dynamical pacing protocol was used to investigate the development of intracellular calcium concentration ([Ca]i), voltage, and active force alternans at different pacing rates. FORCE-ALT only occurred in simulations incorporating reduced Iup, demonstrating that alternans in the intracellular calcium concentration (CA-ALT) induced FORCE-ALT. The magnitude of FORCE-ALT was found to be largest at clinically relevant pacing rates (<110 bpm), where APV-ALT was smallest. We found that the magnitudes of FORCE-ALT, CA-ALT and APV-ALT were altered by heart failure induced-remodeling of mechanical parameters and sarcomere length due to the presence of myofilament feedback. These findings provide important insight into the relationship between heart-failure-induced electrical and mechanical alternans and how they are altered by physiological conditions at near-resting heart rates.

  17. Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential.

    Directory of Open Access Journals (Sweden)

    Francesca Oltolina

    Full Text Available A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM. We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.

  18. 3D engineered cardiac tissue models of human heart disease: learning more from our mice.

    Science.gov (United States)

    Ralphe, J Carter; de Lange, Willem J

    2013-02-01

    Mouse engineered cardiac tissue constructs (mECTs) are a new tool available to study human forms of genetic heart disease within the laboratory. The cultured strips of cardiac cells generate physiologic calcium transients and twitch force, and respond to electrical pacing and adrenergic stimulation. The mECT can be made using cells from existing mouse models of cardiac disease, providing a robust readout of contractile performance and allowing a rapid assessment of genotype-phenotype correlations and responses to therapies. mECT represents an efficient and economical extension to the existing tools for studying cardiac physiology. Human ECTs generated from iPSCMs represent the next logical step for this technology and offer significant promise of an integrated, fully human, cardiac tissue model.

  19. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    . An inefficient post-translational prohormone maturation will also affect the biology of the cardiac natriuretic peptide system. This review aims at summarizing the myocardial synthesis of natriuretic peptides focusing on B-type natriuretic peptide, where new data has disclosed cardiac myocytes as highly...

  20. The immunogenicity of human cardiac valve allografts in vitro and in vivo

    NARCIS (Netherlands)

    F.M.E. Hoekstra (Franciska)

    1999-01-01

    textabstractAllogeneic transplantation has become an accepted method for the replacement of diseased organs and tissues. The concept of cardiac valve transplantation for the replacement of semilunar valves was introduced by Lam et al in 1952'. In about 1960 the first human cardiac valve

  1. Molecular Aspects of Exercise-induced Cardiac Remodeling.

    Science.gov (United States)

    Bernardo, Bianca C; McMullen, Julie R

    2016-11-01

    Exercise-induced cardiac remodeling is typically an adaptive response associated with cardiac myocyte hypertrophy and renewal, increased cardiac myocyte contractility, sarcomeric remodeling, cell survival, metabolic and mitochondrial adaptations, electrical remodeling, and angiogenesis. Initiating stimuli/triggers of cardiac remodeling include increased hemodynamic load, increased sympathetic activity, and the release of hormones and growth factors. Prolonged and strenuous exercise may lead to maladaptive exercise-induced cardiac remodeling including cardiac dysfunction and arrhythmia. In addition, this article describes novel therapeutic approaches for the treatment of heart failure that target mechanisms responsible for adaptive exercise-induced cardiac remodeling, which are being developed and tested in preclinical models.

  2. The interplay between NF-kappaB and E2F1 coordinately regulates inflammation and metabolism in human cardiac cells.

    Directory of Open Access Journals (Sweden)

    Xavier Palomer

    Full Text Available Pyruvate dehydrogenase kinase 4 (PDK4 inhibition by nuclear factor-κB (NF-κB is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα and peroxisome proliferator-activated receptor (PPAR regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α. NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.

  3. Physiological Function and Transplantation of Scaffold-Free and Vascularized Human Cardiac Muscle Tissue

    National Research Council Canada - National Science Library

    K. R. Stevens; K. L. Kreutziger; S. K. Dupras; F. S. Korte; M. Regnier; V. Muskheli; M. B. Nourse; K. Bendixen; H. Reinecke; C. E. Murry; William A. Catterall

    2009-01-01

    Success of human myocardial tissue engineering for cardiac repair has been limited by adverse effects of scaffold materials, necrosis at the tissue core, and poor survival after transplantation due to ischemie injury...

  4. Cardiac stem cell niches

    Directory of Open Access Journals (Sweden)

    Annarosa Leri

    2014-11-01

    Full Text Available The critical role that stem cell niches have in cardiac homeostasis and myocardial repair following injury is the focus of this review. Cardiac niches represent specialized microdomains where the quiescent and activated state of resident stem cells is regulated. Alterations in niche function with aging and cardiac diseases result in abnormal sites of cardiomyogenesis and inadequate myocyte formation. The relevance of Notch1 signaling, gap-junction formation, HIF-1α and metabolic state in the regulation of stem cell growth and differentiation within the cardiac niches are discussed.

  5. Exercise-Induced Cardiac Remodeling: Lessons from Humans, Horses, and Dogs

    Directory of Open Access Journals (Sweden)

    Rob Shave

    2017-02-01

    Full Text Available Physical activity is dependent upon the cardiovascular system adequately delivering blood to meet the metabolic and thermoregulatory demands of exercise. Animals who regularly exercise therefore require a well-adapted heart to support this delivery. The purpose of this review is to examine cardiac structure, and the potential for exercise-induced cardiac remodeling, in animals that regularly engage in strenuous activity. Specifically, we draw upon the literature that has studied the “athlete’s heart” in humans, horses, and dogs, to enable the reader to compare and contrast cardiac remodeling in these three athletic species. The available literature provides compelling evidence for exercise-induced cardiac remodeling in all three species. However, more work is required to understand the influence of species/breed specific genetics and exercise-related hemodynamics, in order to fully understand the impact of exercise on cardiac structure.

  6. Skeletal and cardiac muscle defects in a murine model of Emery-Dreifuss muscular dystrophy.

    Science.gov (United States)

    Grattan, M J; Kondo, C; Thurston, J; Alakija, P; Burke, B J; Stewart, C; Syme, D; Giles, W R

    2005-01-01

    Previous histological findings, physiological data, and behavioral observations on the A-type lamin knockout mouse (Lmna(-/-)) suggest that important aspects of this model resemble the human Emery-Dreifuss muscular dystrophy (EDMD) phenotype. The main goal of our experiments was to study skeletal and cardiac muscle function in this murine model to obtain the semiquantitative data needed for more detailed comparisons with human EDMD defects. Measurements of the mechanical properties of preparations from two different skeletal muscle groups, the soleus and the diaphragm, were made in vitro. In addition, records of the electrocardiogram, and measurements of heart rate variability were obtained; and phasic contractions (unloaded shortening) of enzymatically isolated ventricular myocytes were monitored. Soleus muscles from Lmna(-/-) mice produced less force and work than control preparations. In contrast, force and work production in strips of diaphragm were not changed significantly. Lead II electrocardiograms from conscious, restrained Lmna(-/-) mice revealed slightly decreased heart rates, with significant prolongations of PQ, QRS, and 'QT' intervals compared with those from control recordings. These ECG changes resemble some aspects of the ECG records from humans with EDMD; however, the cardiac phenotype in this Lmna(-/-) mouse model appears to be less well-defined/developed. Ventricular myocytes isolated from Lmna(-/-) mice exhibited impaired contractile responses, particularly when superfused with the beta-adrenergic agonist, isoproterenol (1 microM). This deficit was more pronounced in myocytes isolated from the left ventricle(s) than in myocytes from the right ventricle(s). In summary, tissues from the Lmna(-/-) mouse exhibit a number of skeletal and cardiac muscle deficiencies, some of which are similar to those which have been reported in studies of human EDMD.

  7. Peptidergic innervation of human esophageal and cardiac carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shuang-Hong Lü; Yan Zhou; Hai-Ping Que; Shao-Jun Liu

    2003-01-01

    AIM: To investigate the distribution of neuropeptideimmunoreactive nerve fibers in esophageal and cardiac carcinoma as well as their relationship with tumor cells so as to explore if there is nerve innervation in esophageal and cardiac carcinoma.METHODS: Esophageal and cardiac carcinoma specimens were collected from surgical operation. One part of them were fixed immediately with 4 % paraformaldehyde and then cut with a cryostat into 40-pm-thick sections to perform immunohistochemical analysis. Antibodies of ten kinds of neuropeptide including calcitonin gene-related peptide (CGRP), galanin (GAL), substance P (SP), etc. were used for immunostaining of nerve fibers. The other part of the tumor specimens were cut into little blocks (1 mm3) and cocultured with chick embryo dorsal root ganglia (DRG) to investigate if the tumor blocks could induce the neurons of DRG to extend processes, so as to probe into the possiblereasons for the nerve fibers growing into tumors. RESULTS: Substantial amounts of neuropeptide including GAL-, NPY-, SP-immunoreactive nerve bundles and scattered nerve fibers were distributed in esophageal and cardiac carcinomas. The scattered nerve fibers waved their way among tumor cells and contacted with tumor cells closely. Some of them even encircled tumor cells. There were many varicosities aligned on the nerve fibers like beads. They were also closely related to tumor cells. In the co-culture group, about 63 %and 67 % of DRG co-cultured with esophageal and cardiac tumor blocks respectively extended enormous processes,especially on the side adjacent to the tumor, whereas in the control group (without tumor blocks), no processes grew out.CONCLUSION: Esophageal and cardiac carcinomas may be innervated by peptidergic nerve fibers, and they can induce neurons of DRG to extend processes in vitro.

  8. 3-OST-7 regulates BMP-dependent cardiac contraction.

    Directory of Open Access Journals (Sweden)

    Shiela C Samson

    2013-12-01

    Full Text Available The 3-O-sulfotransferase (3-OST family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4 expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function.

  9. Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

    OpenAIRE

    Bauwens, Celine L.; Toms, Derek; Ungrin, Mark

    2016-01-01

    Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization l...

  10. 1,25 Dihydroxyvitamin D3 Inhibits TGFβ1-Mediated Primary Human Cardiac Myofibroblast Activation.

    Directory of Open Access Journals (Sweden)

    Anna Meredith

    Full Text Available Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av, and investigated the relationship between circulating vitamin D (25(OHD3 and cardiac fibrosis in human myocardial samples.Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OHD3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGFβ1 to induce activation, in the presence or absence of active vitamin D (1,25(OH2D3. Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH2D3 treatment significantly inhibited TGFβ1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH2D3. Treatment with 1,25(OH2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation.Our results demonstrate that active vitamin D can prevent TGFβ1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.

  11. Patterning human stem cells and endothelial cells with laser printing for cardiac regeneration.

    Science.gov (United States)

    Gaebel, Ralf; Ma, Nan; Liu, Jun; Guan, Jianjun; Koch, Lothar; Klopsch, Christian; Gruene, Martin; Toelk, Anita; Wang, Weiwei; Mark, Peter; Wang, Feng; Chichkov, Boris; Li, Wenzhong; Steinhoff, Gustav

    2011-12-01

    Recent study showed that mesenchymal stem cells (MSC) could inhibit apoptosis of endothelial cells in hypoxic condition, increase their survival, and stimulate the angiogenesis process. In this project we applied Laser-Induced-Forward-Transfer (LIFT) cell printing technique and prepared a cardiac patch seeded with human umbilical vein endothelial cells (HUVEC) and human MSC (hMSC) in a defined pattern for cardiac regeneration. We seeded HUVEC and hMSC in a defined pattern on a Polyester urethane urea (PEUU) cardiac patch. On control patches an equal amount of cells was randomly seeded without LIFT. Patches were cultivated in vitro or transplanted in vivo to the infarcted zone of rat hearts after LAD-ligation. Cardiac performance was measured by left ventricular catheterization 8 weeks post infarction. Thereafter hearts were perfused with fluorescein tomato lectin for the assessment of functional blood vessels and stored for histology analyses. We demonstrated that LIFT-derived cell seeding pattern definitely modified growth characteristics of co-cultured HUVEC and hMSC leading to increased vessel formation and found significant functional improvement of infarcted hearts following transplantation of a LIFT-tissue engineered cardiac patch. Further, we could show enhanced capillary density and integration of human cells into the functionally connected vessels of murine vascular system. LIFT-based Tissue Engineering of cardiac patches for the treatment of myocardial infarction might improve wound healing and functional preservation. Copyright © 2011. Published by Elsevier Ltd.

  12. 辛伐他汀预处理在脓毒症大鼠心肌细胞NF-κB 表达中的意义%Significance of simvastatin pretreatment on NF-κB expression in cardiac myocytes of rats with sepsis

    Institute of Scientific and Technical Information of China (English)

    张立涛; 刘洪英; 许宁; 赵鹤龄

    2013-01-01

    目的:观察心肌细胞核转录因子-κB( NF-κB)在脓毒症大鼠心肌细胞的表达,并探讨辛伐他汀预处理对其表达的影响及意义。方法雌性Wistar大鼠60只随机分为3组:正常组,脓毒症组和辛伐他汀组,每组20例。处理方法:应用盲肠结扎穿孔法制造脓毒症模型。辛伐他汀组:辛伐他汀20 mg/kg喂养,每天1次,共2周。2周后辛伐他汀组与脓毒症组大鼠行盲肠结扎穿孔术。术后48 h取3组大鼠心肌组织制成石蜡切片,采用免疫组织化学染色的方法观察心肌细胞NF-κB的蛋白表达。结果免疫组织化学染色显示脓毒症组大鼠心肌细胞NF-κB蛋白表达较正常组明显增加( P <0抖.05),辛伐他汀组大鼠心肌细胞NF-κB 蛋白表达与脓毒症组相比明显减弱( P <0.05),与正常组相比差异有统计学意义( P <0.05)。结论辛伐他汀可以减弱脓毒症大鼠心肌细胞NF-κB的蛋白表达,改善脓毒症时的心脏功能。%Objce tive To investigate the expression of NF-κB in cardiac myocytes of rats with sepsis and to explore the effects of simvastatin pretreatment on expression of NF-κB and its significance .Methods Sixty female Wistar rats were randomly divided into three groups:control group ,sepsis group and simvastatin group ,with 20 rats in each group.The animal models with sepsis were established by cecal ligation and puncture (CLP).The rats in simvastatin group were treated with simvastatin ,20mg/kg,once a day for two weeks before CLP .After two weeks the rats in simvastatin group and sepsis group received CLP .The cardiac tissues of the three groups were collected 48h after CLP, and all the specimens were made into paraffin sections , then the expression levels of NF-κB were detected by immunohistochemistry staining .Resutl s Immunohistochemistry staining showed that the expression levels of NF-κB in sepsis group were significantly higher than those in

  13. Human embryonic stem cells as a model for cardiac gene discovery : from chip to chap

    NARCIS (Netherlands)

    Beqqali, A.

    2008-01-01

    Here we described the use of human embryonic stem cells (hESCs) as a model to obtain insights into commitment to the mesoderm and endoderm lineages and the early steps in human cardiac cell differentiation by means of whole-genome temporal expression profiling. Furthermore, we used it as an approach

  14. Effect of contractile protein alterations on cardiac myofilament function in human heart failure

    NARCIS (Netherlands)

    Narolska, N.A.

    2006-01-01

    The main objective of this thesis was to elucidate the effect of translational and post-translational alterations in contractile proteins occurring during heart failure on contractile function in human cardiac tissue. Isometric force and ATPase activity measurements were performed in skinned human

  15. Capillary/myocyte mismatch in the heart in renal failure--a role for erythropoietin?

    Science.gov (United States)

    Amann, K; Buzello, M; Simonaviciene, A; Miltenberger-Miltenyi, G; Koch, A; Nabokov, A; Gross, M L; Gless, B; Mall, G; Ritz, E

    2000-07-01

    Chronic renal failure is characterized by remodeling of the heart with left ventricular hypertrophy (increasing oxygen demand) and capillary deficit leading to capillary/myocyte mismatch (decreasing oxygen supply). Erythropoietin (Epo) has known angiogenic properties causing endothelial cell activation, migration and sprouting, mediated at least in part via the JAK/STAT (Janus kinase/signal transducers and activators of transcription) pathway. In uraemic cardiac hypertrophy the presence of diminished capillary supply implies that capillary growth does not keep pace with development of hypertrophy. To investigate whether this was due to a deficit of the angiogenic hormone Epo we examined whether Epo levels are altered and whether an increase in haematocrit by administration of rhEpo influences capillary supply, i.e. capillary/myocyte mismatch in experimental renal failure. Male Spraque-Dawley rats were either subjected to partial renal ablation or sham operation. Only modest amounts of renal tissue were removed so that the rats were not anemic. Subgroups of rats received either human (rh)Epo alone or in combination with unspecific antihypertensive treatment (dihydralazine plus furosemide) in order to control the Epo induced rise in blood pressure. Capillary supply was measured stereologically as capillary length per volume myocardium using the orientator method. Capillary length density was reduced by approximately 25% after partial renal ablation (3237+/-601 vs 4293+/-501 mm/mm(3) in controls). It was not statistically different in animals with partial renal ablation+rhEpo+antihypertensive treatment (3620+/-828 mm/mm(3)) compared to partial ablation alone. The study shows that lack of Epo does not cause, or contribute to, the deficit of capillary growth in the hypertrophied left ventricle of rats with renal failure. In addition, a rise in haematocrit is not accompanied by beneficial effects on alterations of cardiovascular structure in experimental renal failure.

  16. Calcium response of KCl-excited populations of ventricular myocytes from the European sea bass (Dicentrarchus labrax): a promising approach to integrate cell-to-cell heterogeneity in studying the cellular basis of fish cardiac performance.

    Science.gov (United States)

    Ollivier, Hélène; Marchant, James; Le Bayon, Nicolas; Servili, Arianna; Claireaux, Guy

    2015-10-01

    Climate change challenges the capacity of fishes to thrive in their habitat. However, through phenotypic diversity, they demonstrate remarkable resilience to deteriorating conditions. In fish populations, inter-individual variation in a number of fitness-determining physiological traits, including cardiac performance, is classically observed. Information about the cellular bases of inter-individual variability in cardiac performance is scarce including the possible contribution of excitation-contraction (EC) coupling. This study aimed at providing insight into EC coupling-related Ca(2+) response and thermal plasticity in the European sea bass (Dicentrarchus labrax). A cell population approach was used to lay the methodological basis for identifying the cellular determinants of cardiac performance. Fish were acclimated at 12 and 22 °C and changes in intracellular calcium concentration ([Ca(2+)]i) following KCl stimulation were measured using Fura-2, at 12 or 22 °C-test. The increase in [Ca(2+)]i resulted primarily from extracellular Ca(2+) entry but sarcoplasmic reticulum stores were also shown to be involved. As previously reported in sea bass, a modest effect of adrenaline was observed. Moreover, although the response appeared relatively insensitive to an acute temperature change, a difference in Ca(2+) response was observed between 12- and 22 °C-acclimated fish. In particular, a greater increase in [Ca(2+)]i at a high level of adrenaline was observed in 22 °C-acclimated fish that may be related to an improved efficiency of adrenaline under these conditions. In conclusion, this method allows a rapid screening of cellular characteristics. It represents a promising tool to identify the cellular determinants of inter-individual variability in fishes' capacity for environmental adaptation.

  17. Cardiac fibroblast-derived extracellular matrix (biomatrix) as a model for the studies of cardiac primitive cell biological properties in normal and pathological adult human heart.

    Science.gov (United States)

    Castaldo, Clotilde; Di Meglio, Franca; Miraglia, Rita; Sacco, Anna Maria; Romano, Veronica; Bancone, Ciro; Della Corte, Alessandro; Montagnani, Stefania; Nurzynska, Daria

    2013-01-01

    Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

  18. Cardiac Fibroblast-Derived Extracellular Matrix (Biomatrix as a Model for the Studies of Cardiac Primitive Cell Biological Properties in Normal and Pathological Adult Human Heart

    Directory of Open Access Journals (Sweden)

    Clotilde Castaldo

    2013-01-01

    Full Text Available Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix, composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

  19. Abaqus/Standard-based quantification of human cardiac mechanical properties

    CERN Document Server

    Genet, Martin; Kuhl, Ellen; Guccione, Julius

    2016-01-01

    Computational modeling can provide critical insight into existing and potential new surgical procedures, medical or minimally-invasive treatments for heart failure, one of the leading causes of deaths in the world that has reached epidemic proportions. In this paper, we present our Abaqus/Standard-based pipeline to create subject-specific left ventricular models. We first review our generic left ventricular model, and then the personalization process based on magnetic resonance images. Identification of subject-specific cardiac material properties is done by coupling Abaqus/Standard to the python optimization library NL-Opt. Compared to previous studies from our group, the emphasis is here on the fully implicit solving of the model, and the two-parameter optimization of the passive cardiac material properties.

  20. Stimulating endogenous cardiac regeneration

    Directory of Open Access Journals (Sweden)

    Amanda eFinan

    2015-09-01

    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.

  1. Mechanical modulation of cardiac microtubules.

    Science.gov (United States)

    White, Ed

    2011-07-01

    Microtubules are a major component of the cardiac myocyte cytoskeleton. Interventions that alter it may influence cardiac mechanical and electrical activity by disrupting the trafficking of proteins to and from the surface membrane by molecular motors such as dynein, which use microtubules as tracks to step along. Free tubulin dimers may transfer GTP to the α-subunits of G-proteins, thus an increase in free tubulin could increase the activity of G-proteins; evidence for and against such a role exists. There is more general agreement that microtubules act as compression-resisting structures within myocytes, influencing visco-elasticity of myocytes and increasing resistance to shortening when proliferated and resisting deformation from longitudinal shear stress. In response to pressure overload, there can be post-translational modifications resulting in more stable microtubules and an increase in microtubule density. This is accompanied by contractile dysfunction of myocytes which can be reversed by microtubule disruption. There are reports of mechanically induced changes in electrical activity that are dependent upon microtubules, but at present, a consensus is lacking on whether disruption or proliferation would be beneficial in the prevention of arrhythmias. Microtubules certainly play a role in the response of cardiac myocytes to mechanical stimulation, the exact nature and significance of this role is still to be fully determined.

  2. Sudden Cardiac Death in Patients with Human Immunodeficiency Virus Infection

    Science.gov (United States)

    Tseng, Zian H.; Secemsky, Eric A.; Dowdy, David; Vittinghoff, Eric; Moyers, Brian; Wong, Joseph K.; Havlir, Diane V.; Hsue, Priscilla Y.

    2012-01-01

    Objectives We sought to determine the incidence and clinical characteristics of sudden cardiac death (SCD) in patients with HIV. Background As the HIV-infected population ages, cardiovascular disease prevalence and mortality are increasing; however, the incidence and features of SCD have not yet been described. Methods Records of 2860 consecutive patients in a public HIV clinic in San Francisco, CA between April 2000 and August 2009 were examined. Identification of deaths, causes of death, and clinical characteristics were obtained by search of the National Death Index and/or clinic records. SCDs were determined using published retrospective criteria: (1) ICD10 code for all cardiac causes of death and (2) circumstances of death meeting WHO criteria. Results Of 230 deaths over 3.7 median years’ follow-up, 30 (13%) met SCD criteria, 131 (57%) were due to AIDS, 25 (11%) other (natural) diseases, and 44 (19%) overdose/suicides/unknown. SCDs accounted for 86% (30/35) of all cardiac deaths. The mean SCD rate was 2.6 per 1,000 person-years (95%CI 1.8-3.8), 4.5-fold higher than expected. SCDs occurred in older patients than AIDS deaths (mean 49.0 vs. 44.9 years, p=0.02). Compared to AIDS and natural deaths combined, SCDs had higher prevalence of prior MI (17% vs. 1%, p<0.0005), cardiomyopathy (23% vs. 3%, p<0.0005), heart failure (30% vs. 9%, p=0.004), and arrhythmias (20% vs. 3%, p=0.003). Conclusions SCDs account for most cardiac and many non-AIDS natural deaths in HIV-infected patients. Further investigation is needed to ascertain underlying mechanisms, which may include inflammation, antiretroviral therapy interruption, and concomitant medications. PMID:22595409

  3. Monocytes increase human cardiac myofibroblast-mediated extracellular matrix remodeling through TGF-β1.

    Science.gov (United States)

    Mewhort, Holly E M; Lipon, Brodie D; Svystonyuk, Daniyil A; Teng, Guoqi; Guzzardi, David G; Silva, Claudia; Yong, V Wee; Fedak, Paul W M

    2016-03-15

    Following myocardial infarction (MI), cardiac myofibroblasts remodel the extracellular matrix (ECM), preventing mechanical complications. However, prolonged myofibroblast activity leads to dysregulation of the ECM, maladaptive remodeling, fibrosis, and heart failure (HF). Chronic inflammation is believed to drive persistent myofibroblast activity; however, the mechanisms are unclear. We assessed the influence of peripheral blood monocytes on human cardiac myofibroblast activity in a three-dimensional (3D) ECM microenvironment. Human cardiac myofibroblasts isolated from surgical biopsies of the right atrium and left ventricle were seeded into 3D collagen matrices. Peripheral blood monocytes were isolated from healthy human donors and cocultured with myofibroblasts. Monocytes increased myofibroblast activity measured by collagen gel contraction (baseline: 57.6 ± 5.9% vs. coculture: 65.2 ± 7.1% contraction; P matrix metalloproteinase 9 compared with baseline (122.9 ± 10.1 pg/ml and 3,496.0 ± 190.4 pg/ml, respectively, vs. 21.5 ± 16.3 pg/ml and 183.3 ± 43.9 pg/ml; P matrix. Peripheral blood monocyte interaction with human cardiac myofibroblasts stimulates myofibroblast activity through release of TGF-β1. These data implicate inflammation as a potential driver of cardiac fibrosis.

  4. PERK signal pathway is involved in hypoxia-induced endoplasmic reti culum stress and apoptosis in cultured cardiac myocytes%PERK通路参与了缺氧心肌细胞的内质网应激及凋亡

    Institute of Scientific and Technical Information of China (English)

    刘春蕾; 李鑫; 李蕊君; 何云云; 何昆仑; 王莉莉

    2012-01-01

    AIM: To invesligale the role of endoplasmic reliculum (ER) slress in the process of hypoxia — induced neonalal ral myocardial injury through PERK signal pathway. METHODS: Neonalal rat cardiac myocyles were randomly divided into conlrol group and hypoxia 1 h, 4 h, 8 h, 12 h and 24 h groups. Cell viability was evaluated by delermi-ning the inlracellular conlenl of ATP. Apoptosis was measured by high — conlenl analysis ( HCA) cell imaging syslem. The prolein levels of GRP78, calreliculin, p — PERK, p — eIF2α, ATF4 and CHOP were delecled by Weslern blolling at differ-ent Lime poinls. The primary cullured neonalal ral cardiac myocyles were Irealed with an agonisl of PERK palhway salu-brinal and the cell apoplosis was observed under hypoxia. RESULTS: In the early phase, hypoxia induced an increase in the expression of calreliculin and GPR78. In the middle phase of hypoxia, the levels of p — PERK, p — eIF2α and ATF4 were increased. In the later phase of hypoxia, increased CHOP level was also observed. Salubrinal effectively prolecled the cardiac myocyles from hypoxic injury. CONCLUSION: Hypoxia aclivales ER slress in cardiac myocyles and also aclivales PERK signal palhway. PERK signaling prolecls cardiac myocyles from hypoxic damage in the early stage and triggers apoplosis of the cells in the laler phase.%目的:观察缺氧对原代培养的Wistar乳鼠心肌细胞的损伤,探讨内质网应激在缺氧心肌损伤发生发展过程中起的作用及PERK通路是否参与其信号转导过程.方法:将原代培养的乳鼠心肌细胞随机分为正常对照组和缺氧1 h、4 h、8 h、12 h、24 h组,通过测定细胞ATP含量反映细胞活力;高内涵分析细胞成像系统检测多参数凋亡;采用免疫细胞化学和蛋白印迹方法检测以内质网为靶点的分子伴侣(GRP78和钙网蛋白)的表达,PERK通路(PERK和eIF2α)的磷酸化水平,以及其下游分子(ATF4和CHOP)在缺氧不同时点蛋白的表达变化特征.采用PERK

  5. The Visible Heart® project and free-access website 'Atlas of Human Cardiac Anatomy'.

    Science.gov (United States)

    Iaizzo, Paul A

    2016-12-01

    Pre- and post-evaluations of implantable cardiac devices require innovative and critical testing in all phases of the design process. The Visible Heart(®) Project was successfully launched in 1997 and 3 years later the Atlas of Human Cardiac Anatomy website was online. The Visible Heart(®) methodologies and Atlas website can be used to better understand human cardiac anatomy, disease states and/or to improve cardiac device design throughout the development process. To date, Visible(®) Heart methodologies have been used to reanimate 75 human hearts, all considered non-viable for transplantation. The Atlas is a unique free-access website featuring novel images of functional and fixed human cardiac anatomies from >400 human heart specimens. Furthermore, this website includes education tutorials on anatomy, physiology, congenital heart disease and various imaging modalities. For instance, the Device Tutorial provides examples of commonly deployed devices that were present at the time of in vitro reanimation or were subsequently delivered, including: leads, catheters, valves, annuloplasty rings, leadless pacemakers and stents. Another section of the website displays 3D models of vasculature, blood volumes, and/or tissue volumes reconstructed from computed tomography (CT) and magnetic resonance images (MRI) of various heart specimens. A new section allows the user to interact with various heart models. Visible Heart(®) methodologies have enabled our laboratory to reanimate 75 human hearts and visualize functional cardiac anatomies and device/tissue interfaces. The website freely shares all images, video clips and CT/MRI DICOM files in honour of the generous gifts received from donors and their families. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For Permissions, please email: journals.permissions@oup.com.

  6. Raf-mediated cardiac hypertrophy in adult Drosophila.

    Science.gov (United States)

    Yu, Lin; Daniels, Joseph; Glaser, Alex E; Wolf, Matthew J

    2013-07-01

    In response to stress and extracellular signals, the heart undergoes a process called cardiac hypertrophy during which cardiomyocytes increase in size. If untreated, cardiac hypertrophy can progress to overt heart failure that causes significant morbidity and mortality. The identification of molecular signals that cause or modify cardiomyopathies is necessary to understand how the normal heart progresses to cardiac hypertrophy and heart failure. Receptor tyrosine kinase (RTK) signaling is essential for normal human cardiac function, and the inhibition of RTKs can cause dilated cardiomyopathies. However, neither investigations of activated RTK signaling pathways nor the characterization of hypertrophic cardiomyopathy in the adult fly heart has been previously described. Therefore, we developed strategies using Drosophila as a model to circumvent some of the complexities associated with mammalian models of cardiovascular disease. Transgenes encoding activated EGFR(A887T), Ras85D(V12) and Ras85D(V12S35), which preferentially signal to Raf, or constitutively active human or fly Raf caused hypertrophic cardiomyopathy as determined by decreased end diastolic lumen dimensions, abnormal cardiomyocyte fiber morphology and increased heart wall thicknesses. There were no changes in cardiomyocyte cell numbers. Additionally, activated Raf also induced an increase in cardiomyocyte ploidy compared with control hearts. However, preventing increases in cardiomyocyte ploidy using fizzy-related (Fzr) RNAi did not rescue Raf-mediated cardiac hypertrophy, suggesting that Raf-mediated polyploidization is not required for cardiac hypertrophy. Similar to mammals, the cardiac-specific expression of RNAi directed against MEK or ERK rescued Raf-mediated cardiac hypertrophy. However, the cardiac-specific expression of activated ERK(D334N), which promotes hyperplasia in non-cardiac tissues, did not cause myocyte hypertrophy. These results suggest that ERK is necessary, but not sufficient, for

  7. Raf-mediated cardiac hypertrophy in adult Drosophila

    Directory of Open Access Journals (Sweden)

    Lin Yu

    2013-07-01

    In response to stress and extracellular signals, the heart undergoes a process called cardiac hypertrophy during which cardiomyocytes increase in size. If untreated, cardiac hypertrophy can progress to overt heart failure that causes significant morbidity and mortality. The identification of molecular signals that cause or modify cardiomyopathies is necessary to understand how the normal heart progresses to cardiac hypertrophy and heart failure. Receptor tyrosine kinase (RTK signaling is essential for normal human cardiac function, and the inhibition of RTKs can cause dilated cardiomyopathies. However, neither investigations of activated RTK signaling pathways nor the characterization of hypertrophic cardiomyopathy in the adult fly heart has been previously described. Therefore, we developed strategies using Drosophila as a model to circumvent some of the complexities associated with mammalian models of cardiovascular disease. Transgenes encoding activated EGFRA887T, Ras85DV12 and Ras85DV12S35, which preferentially signal to Raf, or constitutively active human or fly Raf caused hypertrophic cardiomyopathy as determined by decreased end diastolic lumen dimensions, abnormal cardiomyocyte fiber morphology and increased heart wall thicknesses. There were no changes in cardiomyocyte cell numbers. Additionally, activated Raf also induced an increase in cardiomyocyte ploidy compared with control hearts. However, preventing increases in cardiomyocyte ploidy using fizzy-related (Fzr RNAi did not rescue Raf-mediated cardiac hypertrophy, suggesting that Raf-mediated polyploidization is not required for cardiac hypertrophy. Similar to mammals, the cardiac-specific expression of RNAi directed against MEK or ERK rescued Raf-mediated cardiac hypertrophy. However, the cardiac-specific expression of activated ERKD334N, which promotes hyperplasia in non-cardiac tissues, did not cause myocyte hypertrophy. These results suggest that ERK is necessary, but not sufficient, for Raf

  8. Raf-mediated cardiac hypertrophy in adult Drosophila

    Science.gov (United States)

    Yu, Lin; Daniels, Joseph; Glaser, Alex E.; Wolf, Matthew J.

    2013-01-01

    SUMMARY In response to stress and extracellular signals, the heart undergoes a process called cardiac hypertrophy during which cardiomyocytes increase in size. If untreated, cardiac hypertrophy can progress to overt heart failure that causes significant morbidity and mortality. The identification of molecular signals that cause or modify cardiomyopathies is necessary to understand how the normal heart progresses to cardiac hypertrophy and heart failure. Receptor tyrosine kinase (RTK) signaling is essential for normal human cardiac function, and the inhibition of RTKs can cause dilated cardiomyopathies. However, neither investigations of activated RTK signaling pathways nor the characterization of hypertrophic cardiomyopathy in the adult fly heart has been previously described. Therefore, we developed strategies using Drosophila as a model to circumvent some of the complexities associated with mammalian models of cardiovascular disease. Transgenes encoding activated EGFRA887T, Ras85DV12 and Ras85DV12S35, which preferentially signal to Raf, or constitutively active human or fly Raf caused hypertrophic cardiomyopathy as determined by decreased end diastolic lumen dimensions, abnormal cardiomyocyte fiber morphology and increased heart wall thicknesses. There were no changes in cardiomyocyte cell numbers. Additionally, activated Raf also induced an increase in cardiomyocyte ploidy compared with control hearts. However, preventing increases in cardiomyocyte ploidy using fizzy-related (Fzr) RNAi did not rescue Raf-mediated cardiac hypertrophy, suggesting that Raf-mediated polyploidization is not required for cardiac hypertrophy. Similar to mammals, the cardiac-specific expression of RNAi directed against MEK or ERK rescued Raf-mediated cardiac hypertrophy. However, the cardiac-specific expression of activated ERKD334N, which promotes hyperplasia in non-cardiac tissues, did not cause myocyte hypertrophy. These results suggest that ERK is necessary, but not sufficient, for

  9. Human Cardiac Tissue Engineering: From Pluripotent Stem Cells to Heart Repair

    Science.gov (United States)

    Jackman, Christopher P.; Shadrin, Ilya Y.; Carlson, Aaron L.; Bursac, Nenad

    2014-01-01

    Engineered cardiac tissues hold great promise for use in drug and toxicology screening, in vitro studies of human physiology and disease, and as transplantable tissue grafts for myocardial repair. In this review, we discuss recent progress in cell-based therapy and functional tissue engineering using pluripotent stem cell-derived cardiomyocytes and we describe methods for delivery of cells into the injured heart. While significant hurdles remain, notable advances have been made in the methods to derive large numbers of pure human cardiomyocytes, mature their phenotype, and produce and implant functional cardiac tissues, bringing the field a step closer to widespread in vitro and in vivo applications. PMID:25599018

  10. Expression and assembly of active human cardiac troponin in Escherichia coli.

    Science.gov (United States)

    Lassalle, Michael W

    2013-02-01

    Cardiomyopathy-related mutations in human cardiac troponin subunits, including troponin C (hcTnC), troponin I (hcTnI), and troponin T (hcTnT), are well-documented. Recently, it has been recognised that human cardiac troponin (hcTn) is a sophisticated allosteric system. Therefore, the effect of drugs on this protein complex should be studied with assembled hcTn rather than a short fragment of a subunit or the subunit itself. Here, we describe the expression and assembly of active hcTn in Escherichia coli, a novel method that is rapid and simple, and produces large amounts of functional hcTn.

  11. Persistence length of human cardiac α-tropomyosin measured by single molecule direct probe microscopy.

    Directory of Open Access Journals (Sweden)

    Campion K P Loong

    Full Text Available α-Tropomyosin (αTm is the predominant tropomyosin isoform in adult human heart and constitutes a major component in Ca²+-regulated systolic contraction of cardiac muscle. We present here the first direct probe images of WT human cardiac αTm by atomic force microscopy, and quantify its mechanical flexibility with three independent analysis methods. Single molecules of bacterially-expressed human cardiac αTm were imaged on poly-lysine coated mica and their contours were analyzed. Analysis of tangent-angle (θ(s correlation along molecular contours, second moment of tangent angles (, and end-to-end length (L(e-e distributions respectively yielded values of persistence length (L(p of 41-46 nm, 40-45 nm, and 42-52 nm, corresponding to 1-1.3 molecular contour lengths (L(c. We also demonstrate that a sufficiently large population, with at least 100 molecules, is required for a reliable L(p measurement of αTm in single molecule studies. Our estimate that L(p for αTm is only slightly longer than L(c is consistent with a previous study showing there is little spread of cooperative activation into near-neighbor regulatory units of cardiac thin filaments. The L(p determined here for human cardiac αTm perhaps represents an evolutionarily tuned optimum between Ca²+ sensitivity and cooperativity in cardiac thin filaments and likely constitutes an essential parameter for normal function in the human heart.

  12. 1a,25(OH)2 D3对心肌细胞生物钟基因表达的影响%Effect of 1a,25(OH)2 D3 on circadian clock gene expression in cardiac myocytes

    Institute of Scientific and Technical Information of China (English)

    陈玉梅; 范邁; 李杏; 李华; 吴丽洁; 张奇军; 程子杰; 钱玲梅

    2016-01-01

    目的:探讨1a,25(OH)2 D3对心肌细胞生物钟基因 Bmal1 mRNA、Per2 mRNA 和 Rev-erba mRNA表达的影响。方法分离培养7日龄 SD 大鼠乳鼠心肌细胞,并用免疫荧光法鉴定。原代心肌细胞培养72 h后,设置1a,25(OH)2 D35个终浓度梯度即0 nmol/L、1 nmol/L、10 nmol/L、50 nmol/L 和100 nmol/L 处理心肌细胞2 h,然后提取细胞 RNA,实时荧光定量 PCR(RT-PCR)检测生物钟基因(Bmal1、Per2、Rev-erba)表达量变化,以确定1a,25(OH)2 D3最佳处理浓度。再将培养72 h 的原代心肌细胞分为3组,对照组:无血清培养基培养;血清休克组:含体积分数50%马血清的 DMEM培养2 h;1a,25(OH)2 D3处理组:最佳1a,25(OH)2 D3浓度培养2 h。分别于7个时间点(0 h、4 h、8 h、12 h、16 h、20 h、24 h)收集细胞,提取细胞总 RNA,再采用 RT-PCR 分析心肌细胞生物钟基因(Bmal1,Per2,Rev-erba)的转录水平。结果1a,25(OH)2 D3培养浓度为50 nmol/L 时, Bmal1 mRNA 表达水平最高,Per2和 Rev-erba mRNA 表达水平最低。与对照组相比,1a,25(OH)2 D3处理组和血清休克组均引起心肌细胞 Bmal1、Per2和 Rev-erba 基因呈日周期的节律振荡,且 Bmal1和 Per2的表达模式呈相反的时相表达,在12 h 时 Bmal1的表达量出现在波峰,而 Per2的表达量出现在波谷。Rev-erba 的表达变化趋势在8 h 开始上升,在12~16 h 出现最高表达量。结论1a,25(OH)2 D3可影响心肌细胞生物钟基因 Bmal1、Per2和 Rev-erba mRNA 表达。%Objective To explore the effect of 1a,25(OH)2 D3 on circadian clock gene expressions in cardiac myocytes.Methods Cultured cardiac myocytes isolated from 7 -day -old Sprague -Dawley(SD)rats were identified by immunofluorescence.The medium including 1a,25 (OH)2 D3 (final concentrations were 0 nmol/L,1 nmol/L, 10 nmol/L,50 nmol/L and 100 nmol/L)were added to primary myocardial cells to

  13. Alternative splicing in the differentiation of human embryonic stem cells into cardiac precursors.

    Directory of Open Access Journals (Sweden)

    Nathan Salomonis

    2009-11-01

    Full Text Available The role of alternative splicing in self-renewal, pluripotency and tissue lineage specification of human embryonic stem cells (hESCs is largely unknown. To better define these regulatory cues, we modified the H9 hESC line to allow selection of pluripotent hESCs by neomycin resistance and cardiac progenitors by puromycin resistance. Exon-level microarray expression data from undifferentiated hESCs and cardiac and neural precursors were used to identify splice isoforms with cardiac-restricted or common cardiac/neural differentiation expression patterns. Splice events for these groups corresponded to the pathways of cytoskeletal remodeling, RNA splicing, muscle specification, and cell cycle checkpoint control as well as genes with serine/threonine kinase and helicase activity. Using a new program named AltAnalyze (http://www.AltAnalyze.org, we identified novel changes in protein domain and microRNA binding site architecture that were predicted to affect protein function and expression. These included an enrichment of splice isoforms that oppose cell-cycle arrest in hESCs and that promote calcium signaling and cardiac development in cardiac precursors. By combining genome-wide predictions of alternative splicing with new functional annotations, our data suggest potential mechanisms that may influence lineage commitment and hESC maintenance at the level of specific splice isoforms and microRNA regulation.

  14. Impact of intra-aortic balloon counterpulsation with different balloon volumes on cardiac performance in humans.

    Science.gov (United States)

    Cohen, Marc; Fasseas, Panayotis; Singh, Varinder P; McBride, Ruth; Orford, James L; Kussmaul, William G

    2002-10-01

    Intra-aortic balloon (IAB) counterpulsation can augment the cardiac output. However, the effect of different IAB volumes on cardiac performance has not been adequately evaluated in humans. Eighty-two patients (52 males [63%]; mean age, 65 +/- 12 years; mean body surface area [BSA], 1.8 +/- 0.2 m(2)) had IAB counterpulsation for cardiogenic shock, refractory angina, and preoperatively for high-risk cardiac surgery. Cardiac hemodynamics were prospectively studied during IAB with inflation volumes of 32 vs. 40 cc. Hemodynamic data collected included aortic pressure, pulmonary artery pressure, systemic and mixed venous oxygen saturations, and cardiac output (by Fick). Transthoracic echocardiography (TTE) was used to obtain both velocity time integrals (VTIs) and the area of the left ventricular outflow tract (LVOT). Left ventricular stroke volume was then calculated as LVOT area x VTI. Cardiac output (CO) determined by the Fick method and VTI did not differ significantly (P = NS) between the two inflation volumes (y = 0.002 + 0.97x). In a subgroup of 33 patients with BSA

  15. High resolution 3-Dimensional imaging of the human cardiac conduction system from microanatomy to mathematical modeling.

    Science.gov (United States)

    Stephenson, Robert S; Atkinson, Andrew; Kottas, Petros; Perde, Filip; Jafarzadeh, Fatemeh; Bateman, Mike; Iaizzo, Paul A; Zhao, Jichao; Zhang, Henggui; Anderson, Robert H; Jarvis, Jonathan C; Dobrzynski, Halina

    2017-08-03

    Cardiac arrhythmias and conduction disturbances are accompanied by structural remodelling of the specialised cardiomyocytes known collectively as the cardiac conduction system. Here, using contrast enhanced micro-computed tomography, we present, in attitudinally appropriate fashion, the first 3-dimensional representations of the cardiac conduction system within the intact human heart. We show that cardiomyocyte orientation can be extracted from these datasets at spatial resolutions approaching the single cell. These data show that commonly accepted anatomical representations are oversimplified. We have incorporated the high-resolution anatomical data into mathematical simulations of cardiac electrical depolarisation. The data presented should have multidisciplinary impact. Since the rate of depolarisation is dictated by cardiac microstructure, and the precise orientation of the cardiomyocytes, our data should improve the fidelity of mathematical models. By showing the precise 3-dimensional relationships between the cardiac conduction system and surrounding structures, we provide new insights relevant to valvar replacement surgery and ablation therapies. We also offer a practical method for investigation of remodelling in disease, and thus, virtual pathology and archiving. Such data presented as 3D images or 3D printed models, will inform discussions between medical teams and their patients, and aid the education of medical and surgical trainees.

  16. Human paraoxonase gene cluster overexpression alleviates angiotensin II-induced cardiac hypertrophy in mice.

    Science.gov (United States)

    Pei, Jian-Fei; Yan, Yun-Fei; Tang, Xiaoqiang; Zhang, Yang; Cui, Shen-Shen; Zhang, Zhu-Qin; Chen, Hou-Zao; Liu, De-Pei

    2016-11-01

    Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase (PON) gene cluster (PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene (PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type (WT) control. The same protective tendency was also observed with an Apoe (-/-) background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family.

  17. Effect of Transmurally Heterogeneous Myocyte Excitation-Contraction Coupling on Left Ventricular Electromechanics

    OpenAIRE

    2009-01-01

    The excitation-contraction coupling properties of cardiac myocytes isolated from different regions of the mammalian left ventricular wall have been shown to vary considerably, with uncertain effects on ventricular function. We embedded a cell-level excitation-contraction coupling model with region-dependent parameters within a simple finite element model of left ventricular geometry to study effects of electromechanical heterogeneity on local myocardial mechanics and global hemodynamics. This...

  18. Cell origin of human mesenchymal stem cells determines a different healing performance in cardiac regeneration.

    Directory of Open Access Journals (Sweden)

    Ralf Gaebel

    Full Text Available The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC derived from umbilical cord blood (CB, adipose tissue (AT or bone marrow (BM for the treatment of myocardial infarction (MI remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105(+-CB treated groups compared to CB and nontreated MI group (MI-C. Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular acidification and apoptosis has been detected from CB-hMSC compared to BM and CD105 purified CB-derived hMSC. Our findings suggests that hMSC originating from different sources showed a different healing performance in cardiac regeneration and CD105(+ hMSC exhibited a favorable survival pattern in infarcted hearts, which translates into a more robust preservation of cardiac function.

  19. Pacemaker current inhibition in experimental human cardiac sympathetic activation: a double-blind, randomized, crossover study

    NARCIS (Netherlands)

    Schroeder, C.; Heusser, K.; Zoerner, A.A.; Grosshennig, A.; Wenzel, D.; May, M.; Sweep, F.C.; Mehling, H.; Luft, F.C.; Tank, J.; Jordan, J.

    2014-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated 4 (HCN4) channels comprise the final pathway for autonomic heart rate (HR) regulation. We hypothesized that HCN4 inhibition could reverse autonomic imbalance in a human model of cardiac sympathetic activation. Nineteen healthy men ingested oral me

  20. Fetal myocardium in the kidney capsule: an in vivo model of repopulation of myocytes by bone marrow cells.

    Directory of Open Access Journals (Sweden)

    Eric Y Zhang

    Full Text Available Debate surrounds the question of whether the heart is a post-mitotic organ in part due to the lack of an in vivo model in which myocytes are able to actively regenerate. The current study describes the first such mouse model--a fetal myocardial environment grafted into the adult kidney capsule. Here it is used to test whether cells descended from bone marrow can regenerate cardiac myocytes. One week after receiving the fetal heart grafts, recipients were lethally irradiated and transplanted with marrow from green fluorescent protein (GFP-expressing C57Bl/6J (B6 donors using normal B6 recipients and fetal donors. Levels of myocyte regeneration from GFP marrow within both fetal myocardium and adult hearts of recipients were evaluated histologically. Fetal myocardium transplants had rich neovascularization and beat regularly after 2 weeks, continuing at checkpoints of 1, 2, 4, 6, 8 and12 months after transplantation. At each time point, GFP-expressing rod-shaped myocytes were found in the fetal myocardium, but only a few were found in the adult hearts. The average count of repopulated myocardium with green rod-shaped myocytes was 996.8 cells per gram of fetal myocardial tissue, and 28.7 cells per adult heart tissue, representing a thirty-five fold increase in fetal myocardium compared to the adult heart at 12 months (when numbers of green rod-shaped myocytes were normalized to per gram of myocardial tissue. Thus, bone marrow cells can differentiate to myocytes in the fetal myocardial environment. The novel in vivo model of fetal myocardium in the kidney capsule appears to be valuable for testing repopulating abilities of potential cardiac progenitors.

  1. Myocyte repolarization modulates myocardial function in aging dogs.

    Science.gov (United States)

    Sorrentino, Andrea; Signore, Sergio; Qanud, Khaled; Borghetti, Giulia; Meo, Marianna; Cannata, Antonio; Zhou, Yu; Wybieralska, Ewa; Luciani, Marco; Kannappan, Ramaswamy; Zhang, Eric; Matsuda, Alex; Webster, Andrew; Cimini, Maria; Kertowidjojo, Elizabeth; D'Alessandro, David A; Wunimenghe, Oriyanhan; Michler, Robert E; Royer, Christopher; Goichberg, Polina; Leri, Annarosa; Barrett, Edward G; Anversa, Piero; Hintze, Thomas H; Rota, Marcello

    2016-04-01

    Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.

  2. Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms.

    Science.gov (United States)

    Mearini, Giulia; Gedicke, Christina; Schlossarek, Saskia; Witt, Christian C; Krämer, Elisabeth; Cao, Peirang; Gomes, Marcelo D; Lecker, Stewart H; Labeit, Siegfried; Willis, Monte S; Eschenhagen, Thomas; Carrier, Lucie

    2010-01-15

    Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

  3. Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+ handling.

    Directory of Open Access Journals (Sweden)

    Christopher J Traynham

    Full Text Available Nitric oxide (NO and superoxide (O(2 (- are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O(2 (.- must exist at defined levels. Unfortunately, the NO and O(2 (.- levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc. leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O(2 (.- while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2-2-3,4-dihydro-2H-pyrrole 1-oxide (EMEPO. We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6 and NOS1 knockout (NOS1(-/- mice, a known model of NO/O(2 (.- imbalance, and incubated with EMEPO. EMEPO significantly reduced O(2 (.- (lucigenin-enhanced chemiluminescence and elevated NO (DAF-FM diacetate levels in NOS1(-/- myocytes. Furthermore, EMEPO increased NOS1(-/- myocyte basal contraction (Ca(2+ transients, Fluo-4AM; shortening, video-edge detection, the force-frequency response and the contractile response to β-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca(2+ leak/load relationship and phospholamban Serine16 phosphorylation (Western blot. We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1(-/- myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O(2 (.- levels may have therapeutic potential in the treatment of various cardiomyopathies.

  4. Mouse atrial tumor myocytes AT-1 in cardiac electrophysiologic and pharmacologic studies%小鼠心房肌瘤AT-1细胞在心脏电生理学和药理学研究中的应用

    Institute of Scientific and Technical Information of China (English)

    杨韬

    2001-01-01

    Atrial tumor myocytes (AT-1 cells) were derived from tumor-bearing mice in which SV40 T large antigen drives atrial natriuretic promoter. AT-1 cells possess many features typically seen in cardiac cells from other species, such as spontaneous beating, multiple ion channels and responses to drugs, etc. Major ion channels are present in AT-1 cells: sodium, calcium and potassium channels. Sodium channel in AT-1 cells is highly sensitive to blocker tetrodotoxin. L- and T-type calcium channels respond to classical blockers. An important outward potassium current in AT-1 cells is the rapidly-activating delayed rectifier (IKr). IKr Is the sole time-dependently repolarizing potassium current in AT-1 cells. AT-1 cells are a very useful tool for studies in cardiac electrophysiology and pharmacology. As yet, a great number of cardiac and non-cardiac drugs have been tested for their IKr blocking actions in AT-1 cells. The polymorphic ventricular arrhythmia (torsade de pointes) is a severe side effect of IKr blockers.%小鼠心房肌瘤细胞(AT-1)系从转基因小鼠体内分化出来的一种心肌样细胞. AT-1细胞具有许多其他动物心脏细胞的典型特征,例如自发性搏动,多种离子通道和对许多药物起反应等. AT-1细胞上的主要离子通道包括钠,钙和钾通道. 钠通道对阻滞剂河豚毒高度敏感. L型和T型钙通道对典型的阻滞剂起反应. AT-1细胞上的一种重要外向钾电流是快速激活型迟缓整流外向钾电流(IKr). IKr是AT-1细胞上主要的时间依赖性复极化钾电流. 研究证明,AT-1细胞可作为心脏电生理学和药理学研究中的一种非常有用的工具. 迄今为止,在AT-1细胞上已经测试了许多心脏作用和非心脏作用药物的IKr阻滞作用. IKr阻滞药的一种严重副作用是可引起致死性的尖端扭转型室性心动过速.

  5. Negligible effect of eNOS palmitoylation on fatty acid regulation of contraction in ventricular myocytes from healthy and hypertensive rats.

    Science.gov (United States)

    Jin, Chun Li; Wu, Yu Na; Jang, Ji Hyun; Zhao, Zai Hao; Oh, Goo Taeg; Kim, Sung Joon; Zhang, Yin Hua

    2017-04-25

    S-palmitoylation is an important post-translational modification that affects the translocation and the activity of target proteins in a variety of cell types including cardiomyocytes. Since endothelial nitric oxide synthase (eNOS) is known to be palmitoylated and the activity of eNOS is essential in fatty acid-dependent β-oxidation in muscle, we aimed to test whether palmitoylation of eNOS is involved in palmitic acid (PA) regulation of left ventricular (LV) myocyte contraction from healthy (sham) and hypertensive (HTN) rats. Our results showed that PA, a predominant metabolic substrate for cardiac β-oxidation, significantly increased contraction and oxygen consumption rate (OCR) in LV myocytes from sham. Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) or eNOS gene deletion prevented PA regulation of the myocyte contraction or OCR, indicating the pivotal role of eNOS in mediating the effects of PA in cardiac myocytes. PA increased the palmitoylation of eNOS in LV myocytes and depalmitoylation with 2-bromopalmitate (2BP; 100 μM) abolished the increment. Furthermore, although PA did not increase eNOS-Ser(1177), 2BP reduced eNOS-Ser(1177) with and without PA. Intriguingly, PA-induced increases in contraction and OCR were unaffected by 2BP treatment. In HTN, PA did not affect eNOS palmitoylation, eNOS-Ser(1177), or myocyte contraction. However, 2BP diminished eNOS palmitoylation and eNOS-Ser(1177) in the presence and absence of PA but did not change myocyte contraction. Collectively, our results confirm eNOS palmitoylation in LV myocytes from sham and HTN rats and its upregulation by PA in sham. However, such post-transcriptional modification plays negligible role in PA regulation of myocyte contraction and mitochondrial activity in sham and HTN.

  6. Human cardiac-derived adherent proliferating cells reduce murine acute Coxsackievirus B3-induced myocarditis.

    Directory of Open Access Journals (Sweden)

    Kapka Miteva

    Full Text Available BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs. They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis.

  7. High Glucose Causes Human Cardiac Progenitor Cell Dysfunction by Promoting Mitochondrial Fission: Role of a GLUT1 Blocker.

    Science.gov (United States)

    Choi, He Yun; Park, Ji Hye; Jang, Woong Bi; Ji, Seung Taek; Jung, Seok Yun; Kim, Da Yeon; Kang, Songhwa; Kim, Yeon Ju; Yun, Jisoo; Kim, Jae Ho; Baek, Sang Hong; Kwon, Sang-Mo

    2016-07-01

    Cardiovascular disease is the most common cause of death in diabetic patients. Hyperglycemia is the primary characteristic of diabetes and is associated with many complications. The role of hyperglycemia in the dysfunction of human cardiac progenitor cells that can regenerate damaged cardiac tissue has been investigated, but the exact mechanism underlying this association is not clear. Thus, we examined whether hyperglycemia could regulate mitochondrial dynamics and lead to cardiac progenitor cell dysfunction, and whether blocking glucose uptake could rescue this dysfunction. High glucose in cardiac progenitor cells results in reduced cell viability and decreased expression of cell cycle-related molecules, including CDK2 and cyclin E. A tube formation assay revealed that hyperglycemia led to a significant decrease in the tube-forming ability of cardiac progenitor cells. Fluorescent labeling of cardiac progenitor cell mitochondria revealed that hyperglycemia alters mitochondrial dynamics and increases expression of fission-related proteins, including Fis1 and Drp1. Moreover, we showed that specific blockage of GLUT1 improved cell viability, tube formation, and regulation of mitochondrial dynamics in cardiac progenitor cells. To our knowledge, this study is the first to demonstrate that high glucose leads to cardiac progenitor cell dysfunction through an increase in mitochondrial fission, and that a GLUT1 blocker can rescue cardiac progenitor cell dysfunction and downregulation of mitochondrial fission. Combined therapy with cardiac progenitor cells and a GLUT1 blocker may provide a novel strategy for cardiac progenitor cell therapy in cardiovascular disease patients with diabetes.

  8. Denoising human cardiac diffusion tensor magnetic resonance images using sparse representation combined with segmentation

    Energy Technology Data Exchange (ETDEWEB)

    Bao, L J; Zhu, Y M; Liu, W Y; Pu, Z B; Magnin, I E [HIT-INSA Sino French Research Centre for Biomedical Imaging, Harbin Institute of Technology, Harbin (China); Croisille, P; Robini, M [CREATIS-LRMN, CNRS UMR 5220, Inserm U630, INSA of Lyon, University of Lyon 1, Villeurbanne (France)], E-mail: baolij@gmail.com

    2009-03-21

    Cardiac diffusion tensor magnetic resonance imaging (DT-MRI) is noise sensitive, and the noise can induce numerous systematic errors in subsequent parameter calculations. This paper proposes a sparse representation-based method for denoising cardiac DT-MRI images. The method first generates a dictionary of multiple bases according to the features of the observed image. A segmentation algorithm based on nonstationary degree detector is then introduced to make the selection of atoms in the dictionary adapted to the image's features. The denoising is achieved by gradually approximating the underlying image using the atoms selected from the generated dictionary. The results on both simulated image and real cardiac DT-MRI images from ex vivo human hearts show that the proposed denoising method performs better than conventional denoising techniques by preserving image contrast and fine structures.

  9. Human cardiac telocytes: 3D imaging by FIB-SEM tomography.

    Science.gov (United States)

    Cretoiu, D; Hummel, E; Zimmermann, H; Gherghiceanu, M; Popescu, L M

    2014-11-01

    Telocyte (TC) is a newly identified type of cell in the cardiac interstitium (www.telocytes.com). TCs are described by classical transmission electron microscopy as cells with very thin and long telopodes (Tps; cellular prolongations) having podoms (dilations) and podomers (very thin segments). TCs' three-dimensional (3D) morphology is still unknown. Cardiac TCs seem to be particularly involved in long and short distance intercellular signalling and, therefore, their 3D architecture is important for understanding their spatial connections. Using focused ion beam scanning electron microscopy (FIB-SEM) we show, for the first time, the whole ultrastructural anatomy of cardiac TCs. 3D reconstruction of cardiac TCs by FIB-SEM tomography confirms that they have long, narrow but flattened (ribbon-like) telopodes, with humps generated by the podoms. FIB-SEM tomography also confirms the network made by TCs in the cardiac interstitium through adherens junctions. This study provides the first FIB-SEM tomography of a human cell type.

  10. Towards a clinical use of human embryonic stem cell-derived cardiac progenitors: a translational experience.

    Science.gov (United States)

    Menasché, Philippe; Vanneaux, Valérie; Fabreguettes, Jean-Roch; Bel, Alain; Tosca, Lucie; Garcia, Sylvie; Bellamy, Valérie; Farouz, Yohan; Pouly, Julia; Damour, Odile; Périer, Marie-Cécile; Desnos, Michel; Hagège, Albert; Agbulut, Onnik; Bruneval, Patrick; Tachdjian, Gérard; Trouvin, Jean-Hugues; Larghero, Jérôme

    2015-03-21

    There is now compelling evidence that cells committed to a cardiac lineage are most effective for improving the function of infarcted hearts. This has been confirmed by our pre-clinical studies entailing transplantation of human embryonic stem cell (hESC)-derived cardiac progenitors in rat and non-human primate models of myocardial infarction. These data have paved the way for a translational programme aimed at a phase I clinical trial. The main steps of this programme have included (i) the expansion of a clone of pluripotent hESC to generate a master cell bank under good manufacturing practice conditions (GMP); (ii) a growth factor-induced cardiac specification; (iii) the purification of committed cells by immunomagnetic sorting to yield a stage-specific embryonic antigen (SSEA)-1-positive cell population strongly expressing the early cardiac transcription factor Isl-1; (iv) the incorporation of these cells into a fibrin scaffold; (v) a safety assessment focused on the loss of teratoma-forming cells by in vitro (transcriptomics) and in vivo (cell injections in immunodeficient mice) measurements; (vi) an extensive cytogenetic and viral testing; and (vii) the characterization of the final cell product and its release criteria. The data collected throughout this process have led to approval by the French regulatory authorities for a first-in-man clinical trial of transplantation of these SSEA-1(+) progenitors in patients with severely impaired cardiac function. Although several facets of this manufacturing process still need to be improved, these data may yet provide a useful platform for the production of hESC-derived cardiac progenitor cells under safe and cost-effective GMP conditions. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  11. Cardiac Dysfunction in HIV-1 Transgenic Mouse: Role of Stress and BAG3.

    Science.gov (United States)

    Cheung, Joseph Y; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Tilley, Douglas G; Gao, Erhe; Koch, Walter J; Rabinowitz, Joseph; Klotman, Paul E; Khalili, Kamel; Feldman, Arthur M

    2015-08-01

    Since highly active antiretroviral therapy improved long-term survival of acquired immunodeficiency syndrome (AIDS) patients, AIDS cardiomyopathy has become an increasingly relevant clinical problem. We used human immunodeficiency virus (HIV)-1 transgenic (Tg26) mouse to explore molecular mechanisms of AIDS cardiomyopathy. Tg26 mice had significantly lower left ventricular (LV) mass and smaller end-diastolic and end-systolic LV volumes. Under basal conditions, cardiac contractility and relaxation and single myocyte contraction dynamics were not different between wild-type (WT) and Tg26 mice. Ten days after open heart surgery, contractility and relaxation remained significantly depressed in Tg26 hearts, suggesting that Tg26 mice did not tolerate surgical stress well. To simulate heart failure in which expression of Bcl2-associated athanogene 3 (BAG3) is reduced, we down-regulated BAG3 by small hairpin ribonucleic acid in WT and Tg26 hearts. BAG3 down-regulation significantly reduced contractility in Tg26 hearts. BAG3 overexpression rescued contractile abnormalities in myocytes expressing the HIV-1 protein Tat. We conclude: (i) Tg26 mice exhibit normal contractile function at baseline; (ii) Tg26 mice do not tolerate surgical stress well; (iii) BAG3 down-regulation exacerbated cardiac dysfunction in Tg26 mice; (iv) BAG3 overexpression rescued contractile abnormalities in myocytes expressing HIV-1 protein Tat; and (v) BAG3 may occupy a role in pathogenesis of AIDS cardiomyopathy. © 2015 Wiley Periodicals, Inc.

  12. The oral iron chelator ICL670A (deferasirox) does not protect myocytes against doxorubicin.

    Science.gov (United States)

    Hasinoff, Brian B; Patel, Daywin; Wu, Xing

    2003-12-01

    The oral iron chelating agent ICL670A (deferasirox) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were compared for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle through iron-mediated oxygen radical damage. While dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, ICL670A, in contrast, depending upon the concentration, synergistically increased or did not affect the cytotoxicity of doxorubicin. This occurred in spite of the fact that ICL670A quickly and efficiently removed iron(III) from its complex with doxorubicin, and rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex. Continuous exposure of ICL670A to either myocytes or Chinese hamster ovary (CHO) cells resulted in cytotoxicity while treatment of CHO cells with the ferric complex of ICL670A did not. These results suggest that ICL670A was cytotoxic either by removing or withholding iron from critical iron-containing proteins. Electron paramagnetic resonance spectroscopy was used to show that neither ICL670A nor its ferric complex were able to generate free radicals in either oxidizing or reducing systems suggesting that its cytotoxicity is not due to radical generation.

  13. Mechanotransduction in cardiac hypertrophy and failure.

    Science.gov (United States)

    Lyon, Robert C; Zanella, Fabian; Omens, Jeffrey H; Sheikh, Farah

    2015-04-10

    Cardiac muscle cells have an intrinsic ability to sense and respond to mechanical load through a process known as mechanotransduction. In the heart, this process involves the conversion of mechanical stimuli into biochemical events that induce changes in myocardial structure and function. Mechanotransduction and its downstream effects function initially as adaptive responses that serve as compensatory mechanisms during adaptation to the initial load. However, under prolonged and abnormal loading conditions, the remodeling processes can become maladaptive, leading to altered physiological function and the development of pathological cardiac hypertrophy and heart failure. Although the mechanisms underlying mechanotransduction are far from being fully elucidated, human and mouse genetic studies have highlighted various cytoskeletal and sarcolemmal structures in cardiac myocytes as the likely candidates for load transducers, based on their link to signaling molecules and architectural components important in disease pathogenesis. In this review, we summarize recent developments that have uncovered specific protein complexes linked to mechanotransduction and mechanotransmission within the sarcomere, the intercalated disc, and at the sarcolemma. The protein structures acting as mechanotransducers are the first step in the process that drives physiological and pathological cardiac hypertrophy and remodeling, as well as the transition to heart failure, and may provide better insights into mechanisms driving mechanotransduction-based diseases. © 2015 American Heart Association, Inc.

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

    Science.gov (United States)

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

    2015-08-01

    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.

  15. A TASER conducted electrical weapon with cardiac biomonitoring capability: Proof of concept and initial human trial.

    Science.gov (United States)

    Stopyra, Jason P; Ritter, Samuel I; Beatty, Jennifer; Johnson, James C; Kleiner, Douglas M; Winslow, James E; Gardner, Alison R; Bozeman, William P

    2016-10-01

    Despite research demonstrating the overall safety of Conducted Electrical Weapons (CEWs), commonly known by the brand name TASER(®), concerns remain regarding cardiac safety. The addition of cardiac biomonitoring capability to a CEW could prove useful and even lifesaving in the rare event of a medical crisis by detecting and analyzing cardiac rhythms during the period immediately after CEW discharge. To combine an electrocardiogram (ECG) device with a CEW to detect and store ECG signals while still allowing the CEW to perform its primary function of delivering an incapacitating electrical discharge. This work was performed in three phases. In Phase 1 standard law enforcement issue CEW cartridges were modified to demonstrate transmission of ECG signals. In Phase 2, a miniaturized ECG recorder was combined with a standard issue CEW and tested. In Phase 3, a prototype CEW with on-board cardiac biomonitoring was tested on human volunteers to assess its ability to perform its primary function of electrical incapacitation. Bench testing demonstrated that slightly modified CEW cartridge wires transmitted simulated ECG signals produced by an ECG rhythm generator and from a human volunteer. Ultimately, a modified CEW incorporating ECG monitoring successfully delivered incapacitating current to human volunteers and successfully recorded ECG signals from subcutaneous CEW probes after firing. An ECG recording device was successfully incorporated into a standard issue CEW without impeding the functioning of the device. This serves as proof-of-concept that safety measures such as cardiac biomonitoring can be incorporated into CEWs and possibly other law enforcement devices. Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  16. A novel LQT3 mutation implicates the human cardiac sodium channel domain IVS6 in inactivation kinetics

    NARCIS (Netherlands)

    Groenewegen, WA; Bezzina, CR; van Tintelen, JP; Hoorntje, TM; Mannens, MMAM; Wilde, AAM; Jongsma, HJ; Rook, MB

    2003-01-01

    The Long QT3 syndrome is associated with mutations in the cardiac sodium channel gene SCN5A. Objective: The aim of the present study was the identification and functional characterization of a mutation in a family with the long QT3 syndrome. Methods: The human cardiac sodium channel gene SCN5A was s

  17. Novel Perspectives in Redox Biology and Pathophysiology of Failing Myocytes: Modulation of the Intramyocardial Redox Milieu for Therapeutic Interventions—A Review Article from the Working Group of Cardiac Cell Biology, Italian Society of Cardiology

    Science.gov (United States)

    Arcaro, Alessia; Pirozzi, Flora; Angelini, Annalisa; Chimenti, Cristina; Crotti, Lia; Giordano, Carla; Mancardi, Daniele; Torella, Daniele; Tocchetti, Carlo G.

    2016-01-01

    The prevalence of heart failure (HF) is still increasing worldwide, with enormous human, social, and economic costs, in spite of huge efforts in understanding pathogenetic mechanisms and in developing effective therapies that have transformed this syndrome into a chronic disease. Myocardial redox imbalance is a hallmark of this syndrome, since excessive reactive oxygen and nitrogen species can behave as signaling molecules in the pathogenesis of hypertrophy and heart failure, leading to dysregulation of cellular calcium handling, of the contractile machinery, of myocardial energetics and metabolism, and of extracellular matrix deposition. Recently, following new interesting advances in understanding myocardial ROS and RNS signaling pathways, new promising therapeutical approaches with antioxidant properties are being developed, keeping in mind that scavenging ROS and RNS tout court is detrimental as well, since these molecules also play a role in physiological myocardial homeostasis. PMID:26881035

  18. Novel Perspectives in Redox Biology and Pathophysiology of Failing Myocytes: Modulation of the Intramyocardial Redox Milieu for Therapeutic Interventions—A Review Article from the Working Group of Cardiac Cell Biology, Italian Society of Cardiology

    Directory of Open Access Journals (Sweden)

    Alessia Arcaro

    2016-01-01

    Full Text Available The prevalence of heart failure (HF is still increasing worldwide, with enormous human, social, and economic costs, in spite of huge efforts in understanding pathogenetic mechanisms and in developing effective therapies that have transformed this syndrome into a chronic disease. Myocardial redox imbalance is a hallmark of this syndrome, since excessive reactive oxygen and nitrogen species can behave as signaling molecules in the pathogenesis of hypertrophy and heart failure, leading to dysregulation of cellular calcium handling, of the contractile machinery, of myocardial energetics and metabolism, and of extracellular matrix deposition. Recently, following new interesting advances in understanding myocardial ROS and RNS signaling pathways, new promising therapeutical approaches with antioxidant properties are being developed, keeping in mind that scavenging ROS and RNS tout court is detrimental as well, since these molecules also play a role in physiological myocardial homeostasis.

  19. Human in vivo cardiac phosphorus NMR spectroscopy at 3.0 Tesla

    Science.gov (United States)

    Bruner, Angela Properzio

    One of the newest methods with great potential for use in clinical diagnosis of heart disease is human, cardiac, phosphorus NMR spectroscopy (cardiac p 31 MRS). Cardiac p31 MRS is able to provide quantitative, non-invasive, functional information about the myocardial energy metabolites such as pH, phosphocreatine (PCr), and adenosinetriphosphate (ATP). In addition to the use of cardiac p3l MRS for other types of cardiac problems, studies have shown that the ratio of PCr/ATP and pH are sensitive and specific markers of ischemia at the myocardial level. In human studies, typically performed at 1.5 Tesla, PCr/ATP has been relatively easy to measure but often requires long scan times to provide adequate signal-to-noise (SNR). In addition, pH which relies on identification of inorganic phosphate (Pi), has rarely been obtained. Significant improvement in the quality of cardiac p31 MRS was achieved through the use of the General Electric SIGNATM 3.0 Tesla whole body magnet, improved coil designs and optimized pulse sequences. Phantom and human studies performed on many types of imaging and spectroscopy sequences, identified breathhold gradient-echo imaging and oblique DRESS p31 spectroscopy as the best compromises between SNR, flexibility and quality localization. Both single-turn and quadrature 10-cm diameter, p31 radiofrequency coils, were tested with the quadrature coil providing greater SNR, but at a greater depth to avoid skeletal muscle contamination. Cardiac p31 MRS obtained in just 6 to 8 minutes, gated, showed both improved SNR and discernment of Pi allowing for pH measurement. A handgrip, in-magnet exerciser was designed, created and tested at 1.5 and 3.0 Tesla on volunteers and patients. In ischemic patients, this exercise was adequate to cause a repeated drop in PCr/ATP and pH with approximately eight minutes of isometric exercise at 30% maximum effort. As expected from literature, this exercise did not cause a drop in PCr/ATP for reference volunteers.

  20. Calcium handling by vascular myocytes in hypertension

    Directory of Open Access Journals (Sweden)

    R.C.A. Tostes

    1997-03-01

    Full Text Available Calcium ions (Ca2+ trigger the contraction of vascular myocytes and the level of free intracellular Ca2+ within the myocyte is precisely regulated by sequestration and extrusion mechanisms. Extensive evidence indicates that a defect in the regulation of intracellular Ca2+ plays a role in the augmented vascular reactivity characteristic of clinical and experimental hypertension. For example, arteries from spontaneously hypertensive rats (SHR have an increased contractile sensitivity to extracellular Ca2+ and intracellular Ca2+ levels are elevated in aortic smooth muscle cells of SHR. We hypothesize that these changes are due to an increase in membrane Ca2+ channel density and possibly function in vascular myocytes from hypertensive animals. Several observations using various experimental approaches support this hypothesis: 1 the contractile activity in response to depolarizing stimuli is increased in arteries from hypertensive animals demonstrating increased voltage-dependent Ca2+ channel activity in hypertension; 2 Ca2+ channel agonists such as Bay K 8644 produce contractions in isolated arterial segments from hypertensive rats and minimal contraction in those from normotensive rats; 3 intracellular Ca2+ concentration is abnormally increased in vascular myocytes from hypertensive animals following treatment with Ca2+ channel agonists and depolarizing interventions, and 4 using the voltage-clamp technique, the inward Ca2+ current in arterial myocytes from hypertensive rats is nearly twice as large as that from myocytes of normotensive rats. We suggest that an alteration in Ca2+ channel function and/or an increase in Ca2+ channel density, resulting from increased channel synthesis or reduced turnover, underlies the increased vascular reactivity characteristic of hypertension

  1. Cardiac sympathetic nerve terminal function in congestive heart failure

    Institute of Scientific and Technical Information of China (English)

    Chang-seng LIANG

    2007-01-01

    Increased cardiac release of norepinephrine (NE) and depleted cardiac stores of NE are two salient features of the human failing heart. Researches from my labo-ratory have shown that these changes are accompanied by a functional defect of NE uptake in the cardiac sympathetic nerve terminals. Our studies have shown that the decrease of NE uptake is caused by reduction of NE transporter density in the sympathetic nerve endings, and this change is responsible, at least in part, for the increased myocardial interstitial NE, decreased myocardial adrenoceptor density, and increased myocyte apoptosis in experimental cardiomyopathies. We have also provided evidence in both intact animals and cultured PC12 cells that the decrease of NE transporter is induced by the actions of oxidative metabolites of exogenous NE, involving endoplasmic reticulum stress and impaired N-glycosylation of the NE transporter. This change in the cardiac sympathetic NE uptake function, as demonstrated by [123I] metaiodobenzylguanidine in human studies, may not only serve as an important prognostic variable in patients with congestive heart failure, but also be used as a surrogate for the efficacies of various therapeutic interventions for heart failure. Finally, increasing evidence suggests and further studies are needed to show that the cardiac sympathetic nerve terminal function may be a direct target for pharmacologic treatment of congestive heart failure.

  2. Cardiac protein kinases: the cardiomyocyte kinome and differential kinase expression in human failing hearts

    OpenAIRE

    Fuller, Stephen J.; Osborne, Sally A.; Leonard, Sam J.; Hardyman, Michelle A.; Vaniotis, George; Allen, Bruce G.; Sugden, Peter H.; Clerk, Angela

    2015-01-01

    Aims. Protein kinases are potential therapeutic targets for heart failure, but most studies of cardiac protein kinases derive from other systems, an approach that fails to account for specific kinases expressed in the heart and the contractile cardiomyocytes. We aimed to define the cardiomyocyte kinome (i.e. the protein kinases expressed in cardiomyocytes) and identify kinases with altered expression in human failing hearts. Methods and Results. Expression profiling (Affymetrix microarrays) d...

  3. Human induced pluripotent stem cell-derived beating cardiac tissues on paper.

    Science.gov (United States)

    Wang, Li; Xu, Cong; Zhu, Yujuan; Yu, Yue; Sun, Ning; Zhang, Xiaoqing; Feng, Ke; Qin, Jianhua

    2015-11-21

    There is a growing interest in using paper as a biomaterial scaffold for cell-based applications. In this study, we made the first attempt to fabricate a paper-based array for the culture, proliferation, and direct differentiation of human induced pluripotent stem cells (hiPSCs) into functional beating cardiac tissues and create "a beating heart on paper." This array was simply constructed by binding a cured multi-well polydimethylsiloxane (PDMS) mold with common, commercially available paper substrates. Three types of paper material (print paper, chromatography paper and nitrocellulose membrane) were tested for adhesion, proliferation and differentiation of human-derived iPSCs. We found that hiPSCs grew well on these paper substrates, presenting a three-dimensional (3D)-like morphology with a pluripotent property. The direct differentiation of human iPSCs into functional cardiac tissues on paper was also achieved using our modified differentiation approach. The cardiac tissue retained its functional activities on the coated print paper and chromatography paper with a beating frequency of 40-70 beats per min for up to three months. Interestingly, human iPSCs could be differentiated into retinal pigment epithelium on nitrocellulose membrane under the conditions of cardiac-specific induction, indicating the potential roles of material properties and mechanical cues that are involved in regulating stem cell differentiation. Taken together, these results suggest that different grades of paper could offer great opportunities as bioactive, low-cost, and 3D in vitro platforms for stem cell-based high-throughput drug testing at the tissue/organ level and for tissue engineering applications.

  4. Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue

    National Research Council Canada - National Science Library

    Takeichiro Nakane; Hidetoshi Masumoto; Joseph P Tinney; Fangping Yuan; William J Kowalski; Fei Ye; Amanda J Leblanc; Ryuzo Sakata; Jun K Yamashita; Bradley B Keller

    2017-01-01

    ...) composed of human induced pluripotent stem cells (hiPSCs) derived multiple lineage cardiac cells with varied 3D geometries and cell densities developed towards the goal of scale-up for large animal pre-clinical studies...

  5. Sex-dependent alterations of Ca2+ cycling in human cardiac hypertrophy and heart failure.

    Science.gov (United States)

    Fischer, Thomas H; Herting, Jonas; Eiringhaus, Jörg; Pabel, Steffen; Hartmann, Nico H; Ellenberger, David; Friedrich, Martin; Renner, André; Gummert, Jan; Maier, Lars S; Zabel, Markus; Hasenfuss, Gerd; Sossalla, Samuel

    2016-09-01

    Clinical studies have shown differences in the propensity for malignant ventricular arrhythmias between women and men suffering from cardiomyopathies and heart failure (HF). This is clinically relevant as it impacts therapies like prophylactic implantable cardioverter-defibrillator implantation but the pathomechanisms are unknown. As an increased sarcoplasmic reticulum (SR) Ca(2+) leak is arrhythmogenic, it could represent a cellular basis for this paradox. We evaluated the SR Ca(2+) leak with respect to sex differences in (i) afterload-induced cardiac hypertrophy (Hy) with preserved left ventricular (LV) function and (ii) end-stage HF. Cardiac function did not differ between sexes in both cardiac pathologies. Human cardiomyocytes isolated from female patients with Hy showed a significantly lower Ca(2+) spark frequency (CaSpF, confocal microscopy, Fluo3-AM) compared with men (P cardiac impairment. Since the SR Ca(2+) leak triggers delayed afterdepolarizations, our findings may explain why women are less prone to ventricular arrhythmias and confirm the rationale of therapeutic measures reducing the SR Ca(2+) leak. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

  6. Angiogenesis and cardiac hypertrophy: maintenance of cardiac function and causative roles in heart failure.

    Science.gov (United States)

    Oka, Toru; Akazawa, Hiroshi; Naito, Atsuhiko T; Komuro, Issei

    2014-01-31

    Cardiac hypertrophy is an adaptive response to physiological and pathological overload. In response to the overload, individual cardiac myocytes become mechanically stretched and activate intracellular hypertrophic signaling pathways to re-use embryonic transcription factors and to increase the synthesis of various proteins, such as structural and contractile proteins. These hypertrophic responses increase oxygen demand and promote myocardial angiogenesis to dissolve the hypoxic situation and to maintain cardiac contractile function; thus, these responses suggest crosstalk between cardiac myocytes and microvasculature. However, sustained pathological overload induces maladaptation and cardiac remodeling, resulting in heart failure. In recent years, specific understanding has increased with regard to the molecular processes and cell-cell interactions that coordinate myocardial growth and angiogenesis. In this review, we summarize recent advances in understanding the regulatory mechanisms of coordinated myocardial growth and angiogenesis in the pathophysiology of cardiac hypertrophy and heart failure.

  7. Responses of hypertrophied myocytes to reactive species: implications for glycolysis and electrophile metabolism.

    Science.gov (United States)

    Sansbury, Brian E; Riggs, Daniel W; Brainard, Robert E; Salabei, Joshua K; Jones, Steven P; Hill, Bradford G

    2011-04-15

    During cardiac remodelling, the heart generates higher levels of reactive species; yet an intermediate 'compensatory' stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischaemia or in the failing heart. For this, we measured energetics in control and PE (phenylephrine)-treated NRCMs (neonatal rat cardiomyocytes) under basal conditions and when stressed with HNE (4-hydroxynonenal). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a 2-fold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homoeostasis under conditions of increased oxidative stress.

  8. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells

    Science.gov (United States)

    van Meer, Berend J.; Tertoolen, Leon G. J.

    2017-01-01

    ABSTRACT Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs. PMID:28279973

  9. Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells

    Directory of Open Access Journals (Sweden)

    Giacomo Palazzolo

    2016-01-01

    Full Text Available The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs. We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C, known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

  10. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.

    Directory of Open Access Journals (Sweden)

    Paul W Burridge

    Full Text Available BACKGROUND: The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC, including hiPSC generated from CD34(+ cord blood using non-viral, non-integrating methods. METHODOLOGY/PRINCIPAL FINDINGS: We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5% oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89% of cardiac troponin I(+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. CONCLUSION/SIGNIFICANCE: This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically

  11. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Armin Arshi, Yasuhiro Nakashima, Haruko Nakano, Sarayoot Eaimkhong, Denis Evseenko, Jason Reed, Adam Z Stieg, James K Gimzewski and Atsushi Nakano

    2013-01-01

    Full Text Available While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES cells providing an endless reservoir. In addition to secreted factors and cell–cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

  12. Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

    Science.gov (United States)

    Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z.; Gimzewski, James K.; Nakano, Atsushi

    2013-04-01

    While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

  13. Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish

    Science.gov (United States)

    Mishra, Shikha; Guan, Jian; Plovie, Eva; Seldin, David C.; Connors, Lawreen H.; Merlini, Giampaolo; Falk, Rodney H.; MacRae, Calum A.

    2013-01-01

    Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies. PMID:23624626

  14. Ultrasensitive cardiac troponin I antibody based nanohybrid sensor for rapid detection of human heart attack.

    Science.gov (United States)

    Bhatnagar, Deepika; Kaur, Inderpreet; Kumar, Ashok

    2017-02-01

    An ultrasensitive cardiac troponin I antibody conjugated with graphene quantum dots (GQD) and polyamidoamine (PAMAM) nanohybrid modified gold electrode based sensor was developed for the rapid detection of heart attack (myocardial infarction) in human. Screen printed gold (Au) electrode was decorated with 4-aminothiophenol for amine functionalization of the Au surface. These amino groups were further coupled with carboxyl functionalities of GQD with EDC-NHS reaction. In order to enhance the sensitivity of the sensor, PAMAM dendrimer was successively embedded on GQD through carbodiimide coupling to provide ultra-high surface area for antibody immobilization. The activated cardiac troponin I (cTnI) monoclonal antibody was immobilized on PAMAM to form nanoprobe for sensing specific heart attack marker cTnI. Various concentrations of cardiac marker, cTnI were electrochemically measured using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in human blood serum. The modifications on sensor surface were characterized by FTIR and AFM techniques. The sensor is highly specific to cTnI and showed negligible response to non-specific antigens. The sensitivity of the sensor was 109.23μAcm(-2)μg(-1) and lower limit of detection of cTnI was found 20fgmL(-1).

  15. Actin dynamics is rapidly regulated by the PTEN and PIP2 signaling pathways leading to myocyte hypertrophy.

    Science.gov (United States)

    Li, Jieli; Tanhehco, Elaine J; Russell, Brenda

    2014-12-01

    Mature cardiac myocytes are terminally differentiated, and the heart has limited capacity to replace lost myocytes. Thus adaptation of myocyte size plays an important role in the determination of cardiac function. The hypothesis tested is that regulation of the dynamic exchange of actin leads to cardiac hypertrophy. ANG II was used as a hypertrophic stimulant in mouse heart and neonatal rat ventricular myocytes (NRVMs) in culture for assessment of a mechanism for regulation of actin dynamics by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin dynamics in NRVMs rapidly increased in a PIP2-dependent manner, measured by imaging and fluorescence recovery after photobleaching (FRAP). A significant increase in PIP2 levels was found by immunoblotting in both adult mouse heart tissue and cultured NRVMs. Inhibition of phosphatase and tensin homolog (PTEN) in NRVMs markedly blunted ANG II-induced increases in actin dynamics, the PIP2 level, and cell size. Furthermore, PTEN activity was dramatically upregulated in ANG II-treated NRVMs but downregulated when PTEN inhibitors were used. The time course of the rise in the PIP2 level was inversely related to the fall in the PIP3 level, which was significant by 30 min in ANG II-treated NRVMs. However, significant translocation of PTEN to the plasma membrane occurred by 10 min, suggesting a crucial initial step for PTEN for the cellular responses to ANG II. In conclusion, PTEN and PIP2 signaling may play an important role in myocyte hypertrophy by the regulation of actin filament dynamics, which is induced by ANG II stimulation. Copyright © 2014 the American Physiological Society.

  16. Genetically engineered cardiac pacemaker: Stem cells transfected with HCN2 gene and myocytes—A model

    Science.gov (United States)

    Kanani, S.; Pumir, A.; Krinsky, V.

    2008-01-01

    One of the successfully tested methods to design genetically engineered cardiac pacemaker cells consists in transfecting a human mesenchymal stem cell (hMSC) with a HCN2 gene and connecting it to a myocyte. We develop and study a mathematical model, describing a myocyte connected to a hMSC transfected with a HCN2 gene. The cardiac action potential is described both with the simple Beeler Reuter model, as well as with the elaborate dynamic Luo Rudy model. The HCN2 channel is described by fitting electrophysiological records, in the spirit of Hodgkin Huxley. The model shows that oscillations can occur in a pair myocyte-stem cell, that was not observed in the experiments yet. The model predicted that: (1) HCN pacemaker channels can induce oscillations only if the number of expressed I channels is low enough. At too high an expression level of I channels, oscillations cannot be induced, no matter how many pacemaker channels are expressed. (2) At low expression levels of I channels, a large domain of values in the parameter space (n, N) exists, where oscillations should be observed. We denote N the number of expressed pacemaker channels in the stem cell, and n the number of gap junction channels coupling the stem cell and the myocyte. (3) The expression levels of I channels observed in ventricular myocytes, both in the Beeler Reuter and in the dynamic Luo Rudy models are too high to allow to observe oscillations. With expression levels below ˜1/4 of the original value, oscillations can be observed. The main consequence of this work is that in order to obtain oscillations in an experiment with a myocyte-stem cell pair, increasing the values of n, N is unlikely to be helpful, unless the expression level of I has been reduced enough. The model also allows us to explore levels of gene expression not yet achieved in experiments, and could be useful to plan new experiments, aimed at improving the robustness of the oscillations.

  17. Stimulation of isolated ventricular myocytes within an open architecture microarray.

    Science.gov (United States)

    Klauke, Norbert; Smith, Godfrey L; Cooper, Jonathan M

    2005-03-01

    prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca2+ transients and cell shortening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.

  18. ISL1 directly regulates FGF10 transcription during human cardiac outflow formation.

    Directory of Open Access Journals (Sweden)

    Christelle Golzio

    Full Text Available The LIM homeodomain gene Islet-1 (ISL1 encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations.

  19. Functional high-resolution time-course expression analysis of human embryonic stem cells undergoing cardiac induction

    Directory of Open Access Journals (Sweden)

    Ilaria Piccini

    2016-12-01

    Full Text Available Cardiac induction of human embryonic stem cells (hESCs is a process bearing increasing medical relevance, yet it is poorly understood from a developmental biology perspective. Anticipated technological progress in deriving stably expandable cardiac precursor cells or in advancing cardiac subtype specification protocols will likely require deeper insights into this fascinating system. Recent improvements in controlling hESC differentiation now enable a near-homogeneous induction of the cardiac lineage. This is based on an optimized initial stimulation of mesoderm-inducing signaling pathways such as Activin and/or FGF, BMP, and WNT, followed by WNT inhibition as a secondary requirement. Here, we describe a comprehensive data set based on varying hESC differentiation conditions in a systematic manner and recording high-resolution differentiation time-courses analyzed by genome-wide expression profiling (GEO accession number GSE67154. As a baseline, hESCs were differentiated into cardiomyocytes under optimal conditions. Moreover, in additional time-series, individual signaling factors were withdrawn from the initial stimulation cocktail to reveal their specific roles via comparison to the standard condition. Hence, this data set presents a rich resource for hypothesis generation in studying human cardiac induction, as we reveal numbers of known as well as uncharacterized genes prominently marking distinct intermediate stages in the process. These data will also be useful for identifying putative cardiac master regulators in the human system as well as for characterizing expandable cardiac stem cells.

  20. Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells

    DEFF Research Database (Denmark)

    Oberwallner, Barbara; Brodarac, Andreja; Anić, Petra;

    2015-01-01

    lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell......OBJECTIVES: Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation...... that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. METHODS: Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic...

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

    Science.gov (United States)

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

    2015-08-01

    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.

  2. Characteristics of the Nonselective Cation Current (NSCCs) in Rabbit Left Ventricular Epicardial, Midmyocardial and Endocardial Myocytes

    Institute of Scientific and Technical Information of China (English)

    Min Zong; Xinchun Yang; Xiulan Liu; Liang Shi; Taifeng Liu

    2007-01-01

    Recent studies have described regional differences in the electrophysiology and pharmacology of ventricular myocardium in canine,feline,rat,guinea pig,and human hearts.This has been shown to be due to a smaller IKs and a lager sodium-calcium exchange current (INa-Ca ) and late INa in M region (deep subepicardial to midmyocardial).Studies from our laboratory have found a new repolarization current-nonselective cation current (NSCCs) existing in rabbit right ventricular myocytes.Methods We examined the characteristics of NSCCs in epicardial,M region,and endocardial cells isolated from the rabbit left ventricle with standard microelectrode and whole-cell patch-clamp techniques.The permeability to Na+,K + ,Li + ,Cs + but not to Cl-indicating that it was a nonselective cation current.Gd3+ (0.1 mmol/1) and La3+ (0.1 mmol/1) can block the current markedly.Results Further characterization of NSCCs was significantly smaller in M cells than in epicardial and endocardial cells.NSCCs current density was significantly smaller in M cells than in epicardial and endocardial cells.With repolarization to-80 mV,INs current density was (-0.44 ±0.05) PA/PF in endocardial cells,(-0.12 ±0.05) PA/PF in M cells and (-0.28 ±0.07) PA/PF in epicardial cells;and with repolarization to + 30 mV,INs current density was ( 1.09 ± 0.29) PA/PF in endocardial cells,(0.38 ± 0.09) PA/PF in M cells and (0.91 ± 0.32) PA/PF in epicardial cells.Conclusions Transmural dispersion of repolarization was due to the heterogeneity of NSCCs in rabbit left ventricle epicardial,endocardial myocytes and M cells.These findings may advance our understanding of the ionic basis for our understanding of factors contributing to the development of cardiac arrhythmias.

  3. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    Science.gov (United States)

    Zhang, YouEn; Wang, JiaNing; Li, Hua; Yuan, LiangJun; Wang, Lei; Wu, Bing; Ge, JunBo

    2015-11-01

    In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

  4. Assembly of the cardiac intercalated disk during pre- and postnatal development of the human heart.

    Directory of Open Access Journals (Sweden)

    Arnold Vreeker

    Full Text Available BACKGROUND: In cardiac muscle, the intercalated disk (ID at the longitudinal cell-edges of cardiomyocytes provides as a macromolecular infrastructure that integrates mechanical and electrical coupling within the heart. Pathophysiological disturbance in composition of this complex is well known to trigger cardiac arrhythmias and pump failure. The mechanisms underlying assembly of this important cellular domain in human heart is currently unknown. METHODS: We collected 18 specimens from individuals that died from non-cardiovascular causes. Age of the specimens ranged from a gestational age of 15 weeks through 11 years postnatal. Immunohistochemical labeling was performed against proteins comprising desmosomes, adherens junctions, the cardiac sodium channel and gap junctions to visualize spatiotemporal alterations in subcellular location of the proteins. RESULTS: Changes in spatiotemporal localization of the adherens junction proteins (N-cadherin and ZO-1 and desmosomal proteins (plakoglobin, desmoplakin and plakophilin-2 were identical in all subsequent ages studied. After an initial period of diffuse and lateral labelling, all proteins were fully localized in the ID at approximately 1 year after birth. Nav1.5 that composes the cardiac sodium channel and the gap junction protein Cx43 follow a similar pattern but their arrival in the ID is detected at (much later stages (two years for Nav1.5 and seven years for Cx43, respectively. CONCLUSION: Our data on developmental maturation of the ID in human heart indicate that generation of the mechanical junctions at the ID precedes that of the electrical junctions with a significant difference in time. In addition arrival of the electrical junctions (Nav1.5 and Cx43 is not uniform since sodium channels localize much earlier than gap junction channels.

  5. RSK3 – A Regulator of Pathological Cardiac Remodeling

    Science.gov (United States)

    Martinez, Eliana C.; Passariello, Catherine L.; Li, Jinliang; Matheson, Christopher J.; Dodge-Kafka, Kimberly; Reigan, Philip; Kapiloff, Michael S.

    2015-01-01

    Summary The family of p90 ribosomal S6 kinases (RSK) are pleiotropic effectors for extracellular signal-regulated kinase (ERK) signaling pathways. Recently, RSK3 was shown to be important for pathological remodeling of the heart. While cardiac myocyte hypertrophy can be compensatory for increased wall stress, in chronic heart diseases this non-mitotic cell growth is usually associated with interstitial fibrosis, increased cell death, and decreased cardiac function. Although RSK3 is less abundant in the cardiac myocyte than other RSK family members, RSK3 appears to serve a unique role in cardiac myocyte stress responses. A potential mechanism conferring RSK3’s unique function in the heart is anchoring by the scaffold protein muscle A-kinase Anchoring Protein β (mAKAPβ). Recent findings suggest that RSK3 should be considered as a therapeutic target for the prevention of heart failure, a clinical syndrome of major public health significance. PMID:25988524

  6. Ionic mechanisms underlying cardiac toxicity of the organochloride solvent trichloromethane.

    Science.gov (United States)

    Zhou, Yuan; Wu, Hui-Jun; Zhang, Yan-Hui; Sun, Hai-Ying; Wong, Tak-Ming; Li, Gui-Rong

    2011-12-18

    Trichloromethane (chloroform) is widely used for industrial chemical synthesis and also as an organic solvent in laboratories or ingredient of pesticides. Sudden death resulted from cardiac arrhythmias has been reported in clinic with acute trichloromethane intoxication. The present study was designed to investigate ionic mechanisms underlying arrhythmogenic effect (cardiac toxicity) of trichloromethane in isolated rat hearts and ventricular myocytes and HEK 293 cells stably expressing human Nav1.5, HCN2, or hERG channel using conventional electrophysiological approaches. It was found that trichloromethane (5mM) induced bradycardia and atrial-ventricular conduction blockade or ventricular fibrillation, and inhibited cardiac contractile function in isolated rat hearts. It shortened action potential duration (APD) in isolated rat ventricular myocytes, and increased the threshold current for triggering action potential, but had no effect on the inward rectifier K(+) current I(K1). However, trichloromethane significantly inhibited the L-type calcium current I(Ca.L) and the transient outward potassium current I(to) in a concentration-dependent manner (IC(50)s: 1.01 and 2.4mM, respectively). In HEK 293 cells stably expressing cardiac ion channel genes, trichloromethane reduced hNav1.5, HCN2, and hERG currents with IC(50)s of 8.2, 3.3, and 4.0mM, respectively. These results demonstrate for the first time that trichloromethane can induce bradycardia or ventricular fibrillation, and the arrhythmogenic effect of trichloromethane is related to the inhibition of multiple ionic currents including I(Ca.L), I(to), I(Na), HCN2, and hERG channels.

  7. Human Mesenchymal Stromal Cells Improve Cardiac Perfusion in an Ovine Immunocompetent Animal Model.

    Science.gov (United States)

    Dayan, Victor; Sotelo, Veronica; Delfina, Valentina; Delgado, Natalia; Rodriguez, Carlos; Suanes, Carol; Langhain, María; Ferrando, Rodolfo; Keating, Armand; Benech, Alejandro; Touriño, Cristina

    2016-08-01

    Mesenchymal stromal cells (MSCs) hold considerable promise in the treatment of ischemic heart disease. Most preclinical studies of MSCs for acute myocardial infarction (AMI) have been performed either in syngeneic animal models or with human cells in xenogeneic immunodeficient animals. A preferable pre-clinical model, however, would involve human MSCs in an immunocompetent animal. AMI was generated in adult sheep by inducing ischemia reperfusion of the second diagonal branch. Sheep (n = 10) were randomized to receive an intravenous injection of human MSCs (1 × 10(6) cells/kg) or phosphate buffered saline. Cardiac function and remodeling were evaluated with echocardiography. Perfusion scintigraphy was used to identify sustained myocardial ischemia. Interaction between human MSCs and ovine lymphocytes was assessed by a mixed lymphocyte response (MLR). Sheep receiving human MSCs showed significant improvement in myocardial perfusion at 1 month compared with baseline measurements. There was no change in ventricular dimensions in either group after 1 month of AMI. No adverse events or symptoms were observed in the sheep receiving human MSCs. The MLR was negative. The immunocompetent ovine AMI model demonstrates the clinical safety and efficacy of human MSCs. The human cells do not appear to be immunogenic, further suggesting that immunocompetent sheep may serve as a suitable pre-clinical large animal model for testing human MSCs.

  8. Using a human cardiovascular-respiratory model to characterize cardiac tamponade and pulsus paradoxus

    Science.gov (United States)

    Ramachandran, Deepa; Luo, Chuan; Ma, Tony S; Clark, John W

    2009-01-01

    Background Cardiac tamponade is a condition whereby fluid accumulation in the pericardial sac surrounding the heart causes elevation and equilibration of pericardial and cardiac chamber pressures, reduced cardiac output, changes in hemodynamics, partial chamber collapse, pulsus paradoxus, and arterio-venous acid-base disparity. Our large-scale model of the human cardiovascular-respiratory system (H-CRS) is employed to study mechanisms underlying cardiac tamponade and pulsus paradoxus. The model integrates hemodynamics, whole-body gas exchange, and autonomic nervous system control to simulate pressure, volume, and blood flow. Methods We integrate a new pericardial model into our previously developed H-CRS model based on a fit to patient pressure data. Virtual experiments are designed to simulate pericardial effusion and study mechanisms of pulsus paradoxus, focusing particularly on the role of the interventricular septum. Model differential equations programmed in C are solved using a 5th-order Runge-Kutta numerical integration scheme. MATLAB is employed for waveform analysis. Results The H-CRS model simulates hemodynamic and respiratory changes associated with tamponade clinically. Our model predicts effects of effusion-generated pericardial constraint on chamber and septal mechanics, such as altered right atrial filling, delayed leftward septal motion, and prolonged left ventricular pre-ejection period, causing atrioventricular interaction and ventricular desynchronization. We demonstrate pericardial constraint to markedly accentuate normal ventricular interactions associated with respiratory effort, which we show to be the distinct mechanisms of pulsus paradoxus, namely, series and parallel ventricular interaction. Series ventricular interaction represents respiratory variation in right ventricular stroke volume carried over to the left ventricle via the pulmonary vasculature, whereas parallel interaction (via the septum and pericardium) is a result of

  9. Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

    Science.gov (United States)

    Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

    2016-02-01

    Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic

  10. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Christophe M Raynaud

    Full Text Available Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs derived in vitro from human embryonic stem cells (hESCs. Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5, MEF2C, HAND2 and MYOCD. Nevertheless, NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.

  11. Expression of Rho/Rho kinase of cardiac myocyte of heart failure model of pressure overload rats and intervention of fasudil%压力负荷心力衰竭大鼠心肌细胞内Rho/Rho激酶的表达及药物干预

    Institute of Scientific and Technical Information of China (English)

    张曼; 曾定尹

    2005-01-01

    目的探讨升主动脉缩窄压力超负荷心力衰竭大鼠心肌细胞内Rho/Rho激酶的表达及法舒地尔(fasudil)--Rho/Rho激酶拮抗剂对心力衰竭的影响.方法制备升主动脉缩窄大鼠心力衰竭及假手术组(S组)模型.将升主动脉缩窄术后Wistar雌性大鼠随机分为2组,一组为心力衰竭组(H组),给予生理盐水0.1ml,腹腔注射,每日2次;一组为fasudil治疗组(F组),治疗组给予fasudil 5 mg/Kg,腹腔注射,每日2次;治疗4周.H组、F组及S组每组各10只大鼠.观察各组大鼠各项指标变化.结果H组鼠心肌细胞RhoA,Rho激酶mRNA表达显著增高,F组心肌细胞RhoA mRNA,Rho激酶mRNA表达下降.结论心肌细胞RhoA、Rho激酶表达与充血性心力衰竭密切相关,法舒地尔可有效降低心肌细胞内RhoA mRNA,Rho激酶mRNA表达,缓解心力衰竭症状,可能为一种新的、有效的治疗心力衰竭的血管扩张剂.%Objective: To study the expression of Rho/Rho kinase of cardiac myocyte of heart failure pressure overload rat models subsequent to coarctation of ascending aorta and the effects of fasudil on heart failure. Methods:A model of heart failure induced by coarctation of ascending aorta was used in this study. 20 female wistar operation rats were divided randomly into two groups (n =10). Heart failure (Natrii chloridi, 0.1 ml), fasudil (5 mg/Kg), Bid i.p,20 weeks after coarctation of ascending aorta operation, 10 agematched sham operation group as control. Treatment time was 4 weeks. Hemodynamics, ratio of LV weight to body weight, expression of RhoA and Rho kinase mRNA were investigated in the two groups and sham operation group. Results: RhoA, Rho kinase mRNA level were higher in heart failure when compared with sham. Fasudil could change the expression of RhoA, Rho kinase mRNA level.Conclusion: These results indicate that heart failure is probably related to activating of RhoA, Rho kinase. Fasudil may contribute to the observed beneficial effects on heart

  12. Turbulent electrical activity at sharp-edged inexcitable obstacles in a model for human cardiac tissue.

    Science.gov (United States)

    Majumder, Rupamanjari; Pandit, Rahul; Panfilov, A V

    2014-10-01

    Wave propagation around various geometric expansions, structures, and obstacles in cardiac tissue may result in the formation of unidirectional block of wave propagation and the onset of reentrant arrhythmias in the heart. Therefore, we investigated the conditions under which reentrant spiral waves can be generated by high-frequency stimulation at sharp-edged obstacles in the ten Tusscher-Noble-Noble-Panfilov (TNNP) ionic model for human cardiac tissue. We show that, in a large range of parameters that account for the conductance of major inward and outward ionic currents of the model [fast inward Na(+) current (INa), L-type slow inward Ca(2+) current (ICaL), slow delayed-rectifier current (IKs), rapid delayed-rectifier current (IKr), inward rectifier K(+) current (IK1)], the critical period necessary for spiral formation is close to the period of a spiral wave rotating in the same tissue. We also show that there is a minimal size of the obstacle for which formation of spirals is possible; this size is ∼2.5 cm and decreases with a decrease in the excitability of cardiac tissue. We show that other factors, such as the obstacle thickness and direction of wave propagation in relation to the obstacle, are of secondary importance and affect the conditions for spiral wave initiation only slightly. We also perform studies for obstacle shapes derived from experimental measurements of infarction scars and show that the formation of spiral waves there is facilitated by tissue remodeling around it. Overall, we demonstrate that the formation of reentrant sources around inexcitable obstacles is a potential mechanism for the onset of cardiac arrhythmias in the presence of a fast heart rate.

  13. Improved Cardiac Contractility of Human Recombinant Growth Hormone on the Congestive Heart Failure of Pig

    Institute of Scientific and Technical Information of China (English)

    Yang Ping; He Yu-quan; Zeng Hong; Ni Jin-song; Yun Qing-jun; Huang Xiao-ping; Li Shu-mei

    2005-01-01

    The enhanced cardiac contractility effect of human recombinant growth hormone (hr-GH) on the congestive heart failure (CHF) was studied on the pig. To build a pig model of congestive heart failure, a temporary artificial cardiac pacemaker was implanted in the pig's body and paced at 220 beats to 240 beats per minute for 1 week. After the model of congestive heart failure was successfully set up, the frequency of the pacemaker was changed to 150 beats to 180 beats per minute to maintain the CHF model stable. Pigs were divided into three groups: The hr-GH group in which 0.5 mg/kg per day of hr-GH was administrated intramuscularly for 15 days, the injection control group in which an equal amount of physiological saline was injected intramuscularly, and a normal control group. The left ventricular diastolic end pressure was (10.60±2.41 ) mmHg in the hr-GH group, but (19.00±3.81) mmHg in the saline control group (P<0.01); Cardiac output was (1.86±0.13) L/min in the hr-GH group, but (1.56 ±0.18) L/min in the saline control group (P<0.05); Peripheral min) -1 in the saline control group (P<0.05); ± dp/dtmax was (2900 ±316.23) and (2280 ±286.36) in the hr-HG group and the saline control group respectively (P<0.05). The results show that hr-GH enhances myocardial contractility of CHF, and the CHF model built by a temporary artificial cardiac pacemaker at a high rate of stimulation is reasonable and applicable.

  14. SKF-96365 strongly inhibits voltage-gated sodium current in rat ventricular myocytes.

    Science.gov (United States)

    Chen, Kui-Hao; Liu, Hui; Yang, Lei; Jin, Man-Wen; Li, Gui-Rong

    2015-06-01

    SKF-96365 (1-(beta-[3-(4-methoxy-phenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride) is a general TRPC channel antagonist commonly used to characterize the potential functions of TRPC channels in cardiovascular system. Recent reports showed that SKF-96365 induced a reduction in cardiac conduction. The present study investigates whether the reduced cardiac conduction caused by SKF-96365 is related to the blockade of voltage-gated sodium current (I Na) in rat ventricular myocytes using the whole-cell patch voltage-clamp technique. It was found that SKF-96365 inhibited I Na in rat ventricular myocytes in a concentration-dependent manner. The compound (1 μM) negatively shifted the potential of I Na availability by 9.5 mV, increased the closed-state inactivation of I Na, and slowed the recovery of I Na from inactivation. The inhibition of cardiac I Na by SKF-96365 was use-dependent and frequency-dependent, and the IC₅₀ was decreased from 1.36 μM at 0.5 Hz to 1.03, 0.81, 0.61, 0.56 μM at 1, 2, 5, 10 Hz, respectively. However, the selective TRPC3 antagonist Pyr3 decreased cardiac I Na by 8.5% at 10 μM with a weak use and frequency dependence. These results demonstrate that the TRPC channel antagonist SKF-96365 strongly blocks cardiac I Na in use-dependent and frequency-dependent manners. Caution should be taken for interpreting the alteration of cardiac electrical activity when SKF-96365 is used in native cells as a TRPC antagonist.

  15. Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs.

    Science.gov (United States)

    Jost, Norbert; Virág, László; Comtois, Philippe; Ordög, Balázs; Szuts, Viktória; Seprényi, György; Bitay, Miklós; Kohajda, Zsófia; Koncz, István; Nagy, Norbert; Szél, Tamás; Magyar, János; Kovács, Mária; Puskás, László G; Lengyel, Csaba; Wettwer, Erich; Ravens, Ursula; Nánási, Péter P; Papp, Julius Gy; Varró, András; Nattel, Stanley

    2013-09-01

    The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50-100 nmol l(-1) dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l(-1) BaCl2) and IKs block (1 μmol l(-1) HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ~30% larger and ~29% smaller in human, and Na(+)-Ca(2+) exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for

  16. Genetic dissection of cardiac growth control pathways

    Science.gov (United States)

    MacLellan, W. R.; Schneider, M. D.

    2000-01-01

    Cardiac muscle cells exhibit two related but distinct modes of growth that are highly regulated during development and disease. Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle irreversibly soon after birth, following which the predominant form of growth shifts from hyperplastic to hypertrophic. Much research has focused on identifying the candidate mitogens, hypertrophic agonists, and signaling pathways that mediate these processes in isolated cells. What drives the proliferative growth of embryonic myocardium in vivo and the mechanisms by which adult cardiac myocytes hypertrophy in vivo are less clear. Efforts to answer these questions have benefited from rapid progress made in techniques to manipulate the murine genome. Complementary technologies for gain- and loss-of-function now permit a mutational analysis of these growth control pathways in vivo in the intact heart. These studies have confirmed the importance of suspected pathways, have implicated unexpected pathways as well, and have led to new paradigms for the control of cardiac growth.

  17. Development and application of human virtual excitable tissues and organs: from premature birth to sudden cardiac death.

    Science.gov (United States)

    Holden, Arun V

    2010-12-01

    The electrical activity of cardiac and uterine tissues has been reconstructed by detailed computer models in the form of virtual tissues. Virtual tissues are biophysically and anatomically detailed, and represent quantitatively predictive models of the physiological and pathophysiological behaviours of tissue within an isolated organ. The cell excitation properties are quantitatively reproduced by equations that describe the kinetics of a few dozen proteins. These equations are derived from experimental measurements of membrane potentials, ionic currents, fluxes, and concentrations. Some of the measurements were taken from human cells and human ion channel proteins expressed in non-human cells, but they were mostly taken from cells of other animal species. Data on tissue geometry and architecture are obtained from the diffusion tensor magnetic resonance imaging of ex vivo or post mortem tissue, and are used to compute the spread of current in the tissue. Cardiac virtual tissues are well established and reproduce normal and pathological patterns of cardiac excitation within the atria or ventricles of the human heart. They have been applied to increase the understanding of normal cardiac electrophysiology, to evaluate the candidate mechanisms for re-entrant arrhythmias that lead to sudden cardiac death, and to predict the tissue level effects of mutant or pharmacologically-modified ion channels. The human full-term virtual uterus is still in development. This virtual tissue reproduces the in vitro behaviour of uterine tissue biopsies, and provides possible mechanisms for premature labour.

  18. Hypertrophy of neurons within cardiac ganglia in human, canine, and rat heart failure: the potential role of nerve growth factor.

    Science.gov (United States)

    Singh, Sanjay; Sayers, Scott; Walter, James S; Thomas, Donald; Dieter, Robert S; Nee, Lisa M; Wurster, Robert D

    2013-08-19

    Autonomic imbalances including parasympathetic withdrawal and sympathetic overactivity are cardinal features of heart failure regardless of etiology; however, mechanisms underlying these imbalances remain unknown. Animal model studies of heart and visceral organ hypertrophy predict that nerve growth factor levels should be elevated in heart failure; whether this is so in human heart failure, though, remains unclear. We tested the hypotheses that neurons in cardiac ganglia are hypertrophied in human, canine, and rat heart failure and that nerve growth factor, which we hypothesize is elevated in the failing heart, contributes to this neuronal hypertrophy. Somal morphology of neurons from human (579.54±14.34 versus 327.45±9.17 μm(2); Phypertrophy of neurons in cardiac ganglia compared with controls. Western blot analysis shows that nerve growth factor levels in the explanted, failing human heart are 250% greater than levels in healthy donor hearts. Neurons from cardiac ganglia cultured with nerve growth factor are significantly larger and have greater dendritic arborization than neurons in control cultures. Hypertrophied neurons are significantly less excitable than smaller ones; thus, hypertrophy of vagal postganglionic neurons in cardiac ganglia would help to explain the parasympathetic withdrawal that accompanies heart failure. Furthermore, our observations suggest that nerve growth factor, which is elevated in the failing human heart, causes hypertrophy of neurons in cardiac ganglia.

  19. Wavelet transform analysis to assess oscillations in pial artery pulsation at the human cardiac frequency.

    Science.gov (United States)

    Winklewski, P J; Gruszecki, M; Wolf, J; Swierblewska, E; Kunicka, K; Wszedybyl-Winklewska, M; Guminski, W; Zabulewicz, J; Frydrychowski, A F; Bieniaszewski, L; Narkiewicz, K

    2015-05-01

    Pial artery adjustments to changes in blood pressure (BP) may last only seconds in humans. Using a novel method called near-infrared transillumination backscattering sounding (NIR-T/BSS) that allows for the non-invasive measurement of pial artery pulsation (cc-TQ) in humans, we aimed to assess the relationship between spontaneous oscillations in BP and cc-TQ at frequencies between 0.5 Hz and 5 Hz. We hypothesized that analysis of very short data segments would enable the estimation of changes in the cardiac contribution to the BP vs. cc-TQ relationship during very rapid pial artery adjustments to external stimuli. BP and pial artery oscillations during baseline (70s and 10s signals) and the response to maximal breath-hold apnea were studied in eighteen healthy subjects. The cc-TQ was measured using NIR-T/BSS; cerebral blood flow velocity, the pulsatility index and the resistive index were measured using Doppler ultrasound of the left internal carotid artery; heart rate and beat-to-beat systolic and diastolic blood pressure were recorded using a Finometer; end-tidal CO2 was measured using a medical gas analyzer. Wavelet transform analysis was used to assess the relationship between BP and cc-TQ oscillations. The recordings lasting 10s and representing 10 cycles with a frequency of ~1 Hz provided sufficient accuracy with respect to wavelet coherence and wavelet phase coherence values and yielded similar results to those obtained from approximately 70cycles (70s). A slight but significant decrease in wavelet coherence between augmented BP and cc-TQ oscillations was observed by the end of apnea. Wavelet transform analysis can be used to assess the relationship between BP and cc-TQ oscillations at cardiac frequency using signals intervals as short as 10s. Apnea slightly decreases the contribution of cardiac activity to BP and cc-TQ oscillations.

  20. Beta2-adrenergic signaling affects the phenotype of human cardiac progenitor cells through EMT modulation.

    Science.gov (United States)

    Pagano, Francesca; Angelini, Francesco; Siciliano, Camilla; Tasciotti, Julia; Mangino, Giorgio; De Falco, Elena; Carnevale, Roberto; Sciarretta, Sebastiano; Frati, Giacomo; Chimenti, Isotta

    2017-01-15

    Human cardiac progenitor cells (CPCs) offer great promises to cardiac cell therapy for heart failure. Many in vivo studies have shown their therapeutic benefits, paving the way for clinical translation. The 3D model of cardiospheres (CSs) represents a unique niche-like in vitro microenvironment, which includes CPCs and supporting cells. CSs have been shown to form through a process mediated by epithelial-to-mesenchymal transition (EMT). β2-Adrenergic signaling significantly affects stem/progenitor cells activation and mobilization in multiple tissues, and crosstalk between β2-adrenergic signaling and EMT processes has been reported. In the present study, we aimed at investigating the biological response of CSs to β2-adrenergic stimuli, focusing on EMT modulation in the 3D culture system of CSs. We treated human CSs and CS-derived cells (CDCs) with the β2-blocker butoxamine (BUT), using either untreated or β2 agonist (clenbuterol) treated CDCs as control. BUT-treated CS-forming cells displayed increased migration capacity and a significant increase in their CS-forming ability, consistently associated with increased expression of EMT-related genes, such as Snai1. Moreover, long-term BUT-treated CDCs contained a lower percentage of CD90+ cells, and this feature has been previously correlated with higher cardiogenic and therapeutic potential of the CDCs population. In addition, long-term BUT-treated CDCs had an increased ratio of collagen-III/collagen-I gene expression levels, and showed decreased release of inflammatory cytokines, overall supporting a less fibrosis-prone phenotype. In conclusion, β2 adrenergic receptor block positively affected the stemness vs commitment balance within CSs through the modulation of type1-EMT (so called "developmental"). These results further highlight type-1 EMT to be a key process affecting the features of resident cardiac progenitor cells, and mediating their response to the microenvironment.

  1. Oxidative stress decreases microtubule growth and stability in ventricular myocytes

    OpenAIRE

    Drum, BML; Yuan, C.; Li, L; Liu, Q.; Wordeman, L; Santana, LF

    2016-01-01

    © 2016 Elsevier Ltd.Microtubules (MTs) have many roles in ventricular myocytes, including structural stability, morphological integrity, and protein trafficking. However, despite their functional importance, dynamic MTs had never been visualized in living adult myocytes. Using adeno-associated viral vectors expressing the MT-associated protein plus end binding protein 3 (EB3) tagged with EGFP, we were able to perform live imaging and thus capture and quantify MT dynamics in ventricular myocyt...

  2. Vitamin D Levels Are Associated with Cardiac Autonomic Activity in Healthy Humans

    Directory of Open Access Journals (Sweden)

    Linda Ellis

    2013-06-01

    Full Text Available Vitamin D deficiency (≤50nmol/L 25-hydroxy vitamin D is a cardiovascular (CV risk factor that affects approximately one billion people worldwide, particularly those affected by chronic kidney disease (CKD. Individuals with CKD demonstrate abnormal cardiac autonomic nervous system activity, which has been linked to the significant rates of CV-related mortality in this population. Whether vitamin D deficiency has a direct association with regulation of cardiac autonomic activity has never been explored in humans. Methods: Thirty-four (34 healthy, normotensive subjects were studied and categorized based on 25-hydroxy vitamin D deficiency (deficient vs. non-deficient, n = 7 vs. 27, as well as 1,25-dihydroxy vitamin D levels (above vs. below 25th percentile, n = 8 vs. 26. Power spectral analysis of electrocardiogram recordings provided measures of cardiac autonomic activity across low frequency (LF and high frequency (HF, representative of vagal contribution bands, representative of the sympathetic and vagal limbs of the autonomic nervous system when transformed to normalized units (nu, respectively, as well as overall cardiosympathovagal balance (LF:HF during graded angiotensin II (AngII challenge (3 ng/kg/min × 30 min, 6 ng/kg/min × 30 min. Results: At baseline, significant suppression of sympathovagal balance was observed in the 25-hydroxy vitamin D-deficient participants (LF:HF, p = 0.02 vs. non-deficient, although no other differences were observed throughout AngII challenge. Participants in the lowest 1,25-dihydroxy VD quartile experienced significant withdrawal of inhibitory vagal control, as well as altered overall sympathovagal balance throughout AngII challenge (HF, mean difference = −6.98 ± 3 nu, p = 0.05; LF:HF, mean difference = 0.34 ± 0.1, p = 0.043 vs. above 25th percentile. Conclusions: Vitamin D deficiency is associated with suppression of resting cardiac autonomic activity, while low 1,25-dihydroxy vitamin D levels are

  3. Optimisation of recombinant production of active human cardiac SERCA2a ATPase.

    Science.gov (United States)

    Antaloae, Ana V; Montigny, Cédric; le Maire, Marc; Watson, Kimberly A; Sørensen, Thomas L-M

    2013-01-01

    Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.

  4. Reference values for total blood volume and cardiac output in humans

    Energy Technology Data Exchange (ETDEWEB)

    Williams, L.R. [Indiana Univ., South Bend, IN (United States). Division of Liberal Arts and Sciences

    1994-09-01

    Much research has been devoted to measurement of total blood volume (TBV) and cardiac output (CO) in humans but not enough effort has been devoted to collection and reduction of results for the purpose of deriving typical or {open_quotes}reference{close_quotes} values. Identification of normal values for TBV and CO is needed not only for clinical evaluations but also for the development of biokinetic models for ultra-short-lived radionuclides used in nuclear medicine (Leggett and Williams 1989). The purpose of this report is to offer reference values for TBV and CO, along with estimates of the associated uncertainties that arise from intra- and inter-subject variation, errors in measurement techniques, and other sources. Reference values are derived for basal supine CO and TBV in reference adult humans, and differences associated with age, sex, body size, body position, exercise, and other circumstances are discussed.

  5. Effects of acetylcholine and noradrenalin on action potentials of isolated rabbit sinoatrial and atrial myocytes

    Directory of Open Access Journals (Sweden)

    Arie O. Verkerk

    2012-05-01

    Full Text Available The autonomic nervous system controls heart rate and contractility through sympathetic and parasympathetic inputs to the cardiac tissue, with acetylcholine (ACh and noradrenalin (NA as the chemical transmitters. In recent years, it has become clear that specific Regulators of G protein Signalling proteins (RGS proteins suppress muscarinic sensitivity and parasympathetic tone, identifying RGS proteins as intriguing potential therapeutic targets. In the present study, we have identified the effects of 1 µM ACh and 1 µM NA on the intrinsic action potentials of sinotrial (SA nodal and atrial myocytes. Single cells were enzymatically isolated from the SA node or from the left atrium of rabbit hearts. Action potentials were recorded using the amphotericin-perforated patch-clamp technique in the absence and presence of ACh, NA or a combination of both. In SA nodal myocytes, ACh increased cycle length and decreased diastolic depolarization rate, whereas NA decreased cycle length and increased diastolic depolarization rate. Both ACh and NA increased maximum upstroke velocity. Furthermore, ACh hyperpolarized the maximum diastolic potential. In atrial myocytes stimulated at 2 Hz, both ACh and NA hyperpolarized the maximum diastolic potential, increased the action potential amplitude, and increased the maximum upstroke velocity. Action potential duration at 50 and 90% repolarization was decreased by ACh, but increased by NA. The effects of both ACh and NA on action potential duration showed a dose dependence in the range of 1–1,000 nM, while a clear-cut frequency dependence in the range of 1–4 Hz was absent. Intermediate results were obtained in the combined presence of ACh and NA in both SA nodal and atrial myocytes. Our data uncover the extent to which SA nodal and atrial action potentials are intrinsically dependent on ACh, NA or a combination of both and may thus guide further experiments with RGS proteins.

  6. Atrial myocyte function and Ca2+ handling is associated with inborn aerobic capacity.

    Directory of Open Access Journals (Sweden)

    Anne Berit Johnsen

    Full Text Available Although high aerobic capacity is associated with effective cardiac function, the effect of aerobic capacity on atrial function, especially in terms of cellular mechanisms, is not known. We aimed to investigate whether rats with low inborn maximal oxygen uptake (VO2 max had impaired atrial myocyte contractile function when compared to rats with high inborn VO2 max.Atrial myocyte function was depressed in Low Capacity Runners (LCR relative to High Capacity Runners (HCR which was associated with impaired Ca(2+ handling. Fractional shortening was 52% lower at 2 Hz and 60% lower at 5 Hz stimulation while time to 50% relengthening was 43% prolonged and 55% prolonged, respectively. Differences in Ca(2+ amplitude and diastolic Ca(2+ level were observed at 5 Hz stimulation where Ca(2+ amplitude was 70% lower and diastolic Ca(2+ level was 11% higher in LCR rats. Prolonged time to 50% Ca(2+ decay was associated with reduced sarcoplasmic reticulum (SR Ca(2+ ATPase function in LCR (39%. Na(+/Ca(2+ exchanger activity was comparable between the groups. Diastolic SR Ca(2+ leak was increased by 109%. This could be partly explained by increased ryanodine receptors phosphorylation at the Ca(2+-calmodulin-dependent protein kinase-II specific Ser-2814 site in LCR rats. T-tubules were present in 68% of HCR cells whereas only 33% LCR cells had these structures. In HCR, the significantly higher numbers of cells with T-tubules were combined with greater numbers of myocytes where Ca(2+ release in the cell occurred simultaneously in central and peripheral regions, giving rise to faster and more spatial homogenous Ca(2+-signal onset.This data demonstrates that contrasting for low or high aerobic capacity leads to diverse functional and structural remodelling of atrial myocytes, with impaired contractile function in LCR compared to HCR rats.

  7. Acute improvement of cardiac function with intravenous L-propionylcarnitine in humans.

    Science.gov (United States)

    Bartels, G L; Remme, W J; Pillay, M; Schönfeld, D H; Cox, P H; Kruijssen, H A; Knufman, N M

    1992-07-01

    As the myocardial carnitine content, a key control factor in myocardial oxidative metabolism and energy transfer, is reduced in heart failure, administration of L-propionylcarnitine (LPC), a potent analogue of L-carnitine, potentially may improve cardiac function, possibly through a positive inotropic effect. As its hemodynamic profile is unknown in humans, 32 fasting normotensive patients with coronary artery disease received either 15 mg/kg of LPC (n = 16) or vehicle (mannitol/acetate, n = 16) infused over 5 min. Hemodynamic, radionuclide [peak ejection and filling rates (PER and PFR, respectively)], and metabolic variables (myocardial O2, lactate, and carnitine uptake) were studied at baseline and 1, 3, 5, 10, 15, and 45 min postdrug. The baseline ejection fraction was depressed in LPC patients (40 +/- 3% vs. 48 +/- 4% in the vehicle group, p less than 0.05) as a result of a significant high incidence of previous infarctions. Immediately following LPC, the cardiac total carnitine uptake changed from 102 +/- 181 to 5,335 +/- 1,761 mumol/L (p less than 0.05). In both groups, left ventricular systolic and end-diastolic pressures increased significantly by 5 and 20%, respectively, during the first 5 min. In the vehicle group, contractility decreased by 5%, accompanied by a significant 11% fall in the stroke volume. In contrast, following LPC, isovolumetric contractility indices remained unaltered. Instead, both the PER and PFR improved by 16% at 45 min. Moreover, the cardiac output increased by 8%. LPC did not affect systemic or coronary hemodynamics. Lactate uptake increased by 42%, but myocardial O2 consumption did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts

    Directory of Open Access Journals (Sweden)

    F Di Meglio

    2009-08-01

    Full Text Available The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (a-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively inclines toward an increase in both a-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

  9. CD13 and ROR2 Permit Isolation of Highly Enriched Cardiac Mesoderm from Differentiating Human Embryonic Stem Cells.

    Science.gov (United States)

    Skelton, Rhys J P; Brady, Bevin; Khoja, Suhail; Sahoo, Debashis; Engel, James; Arasaratnam, Deevina; Saleh, Kholoud K; Abilez, Oscar J; Zhao, Peng; Stanley, Edouard G; Elefanty, Andrew G; Kwon, Murray; Elliott, David A; Ardehali, Reza

    2016-01-12

    The generation of tissue-specific cell types from human embryonic stem cells (hESCs) is critical for the development of future stem cell-based regenerative therapies. Here, we identify CD13 and ROR2 as cell-surface markers capable of selecting early cardiac mesoderm emerging during hESC differentiation. We demonstrate that the CD13+/ROR2+ population encompasses pre-cardiac mesoderm, which efficiently differentiates to all major cardiovascular lineages. We determined the engraftment potential of CD13+/ROR2+ in small (murine) and large (porcine) animal models, and demonstrated that CD13+/ROR2+ progenitors have the capacity to differentiate toward cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells in vivo. Collectively, our data show that CD13 and ROR2 identify a cardiac lineage precursor pool that is capable of successful engraftment into the porcine heart. These markers represent valuable tools for further dissection of early human cardiac differentiation, and will enable a detailed assessment of human pluripotent stem cell-derived cardiac lineage cells for potential clinical applications.

  10. CD13 and ROR2 Permit Isolation of Highly Enriched Cardiac Mesoderm from Differentiating Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Rhys J.P. Skelton

    2016-01-01

    Full Text Available The generation of tissue-specific cell types from human embryonic stem cells (hESCs is critical for the development of future stem cell-based regenerative therapies. Here, we identify CD13 and ROR2 as cell-surface markers capable of selecting early cardiac mesoderm emerging during hESC differentiation. We demonstrate that the CD13+/ROR2+ population encompasses pre-cardiac mesoderm, which efficiently differentiates to all major cardiovascular lineages. We determined the engraftment potential of CD13+/ROR2+ in small (murine and large (porcine animal models, and demonstrated that CD13+/ROR2+ progenitors have the capacity to differentiate toward cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells in vivo. Collectively, our data show that CD13 and ROR2 identify a cardiac lineage precursor pool that is capable of successful engraftment into the porcine heart. These markers represent valuable tools for further dissection of early human cardiac differentiation, and will enable a detailed assessment of human pluripotent stem cell-derived cardiac lineage cells for potential clinical applications.

  11. Sarcolemmal Ca(2+)-entry through L-type Ca(2+) channels controls the profile of Ca(2+)-activated Cl(-) current in canine ventricular myocytes.

    Science.gov (United States)

    Horváth, Balázs; Váczi, Krisztina; Hegyi, Bence; Gönczi, Mónika; Dienes, Beatrix; Kistamás, Kornél; Bányász, Tamás; Magyar, János; Baczkó, István; Varró, András; Seprényi, György; Csernoch, László; Nánási, Péter P; Szentandrássy, Norbert

    2016-08-01

    Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

  12. Analysis of Pregnancy-Associated Plasma Protein A Production in Human Adult Cardiac Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Piera D’Elia

    2013-01-01

    Full Text Available IGF-binding proteins (IGFBPs and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily, thus positively affecting IGF signalling. Therefore, our results strongly support the importance of PAPP-A for the IGFs/IGFBPs/PAPP-A axis in CPCs biology.

  13. Effects of mibefradil on intracellular Ca2+ release in cultured rat cardiac fibroblasts and human platelets.

    Science.gov (United States)

    Eberhard, M; Miyagawa, K; Hermsmeyer, K; Erne, P

    1995-12-01

    The Ca2+ antagonist mibefradil at supratherapeutic concentrations induced a sustained increase of cytosolic Ca2+ in cultured rat cardiac fibroblasts and human platelets which lack sensitivity to K+ depolarization and Ca2+ channel block by verapamil or other Ca2+ antagonists. At concentrations above 10 microM, mibefradil elevated substantially cytosolic [Ca2+] without affecting the peak level of agonist-induced Ca2+ transients. These Ca2+-mobilizing actions of 10 or 100 microM mibefradil stand in contrast to the Ca2+ antagonism and relaxation of vascular muscle at 1 microM concentrations. Since a substantial part of mibefradil-induced increase in cytosolic Ca2+ was independent of extracellular Ca2+, and in order to define better the mechanism of Ca2+ increase, we exposed permeabilized cultured rat cardiac fibroblasts and human platelets to mibefradil at concentrations sufficiently high to identify covert effects. In permeabilized fibroblasts or platelets mibefradil at concentrations above 10 microM activated dose-dependent Ca2+ release from intracellular Ca2+ stores. Verapamil had no effect at concentrations of up to 100 microM. Mibefradil-induced Ca2+ release was not affected by ryanodine, thapsigargin, removal of ATP or dithioerythreitol, indicating that neither Ca2+ - nor disulfide reagent-induced Ca2+ release were involved and that mibefradil did not release Ca2+ by inhibition of the Ca2+-ATPase pump of endoplasmic reticulum. The rate, but not the amplitude, of mibefradil-induced Ca2+ release is increased up to fourfold in the presence of pentosan polysulphate or heparin, two potent inhibitors of inositol 1,4,5-trisphosphate-induced Ca2+ release. Depletion of Ca2+ stores of permeabilized cells inositol 1,4,5-trisphosphate in the presence of thapsigargin completely blocked mibefradil-induced Ca2+ release, and depletion of Ca2+ stores by mibefradil prevented further Ca2+ release by inositol 1,4,5-trisphosphate. Mibefradil at supratherapeutic concentrations (> or

  14. Cardiac lipid levels show diurnal changes and long-term variations in healthy human subjects.

    Science.gov (United States)

    Ith, Michael; Stettler, Christoph; Xu, Jian; Boesch, Chris; Kreis, Roland

    2014-11-01

    (1) H-MRS is regularly applied to determine lipid content in ectopic tissue - mostly skeletal muscle and liver - to investigate physiological and/or pathologic conditions, e.g. insulin resistance. Technical developments also allow non-invasive in vivo assessment of cardiac lipids; however, basic data about methodological reliability (repeatability) and physiological variations are scarce. The aim of the presented work was to determine potential diurnal changes of cardiac lipid stores in humans, and to put the results in relation to methodological repeatability and normal physiological day-to-day variations. Optimized cardiac- and respiratory-gated (1) H-MRS was used for non-invasive quantification of intracardiomyocellular lipids (ICCL), creatine, trimethyl-ammonium compounds (TMA), and taurine in nine healthy young men at three time points per day on two days separated by one week. This design allowed determination of (a) diurnal changes, (b) physiological variation over one week and (c) methodological repeatability of the ICCL levels. Comparison of fasted morning to post-absorptive evening measurements revealed a significant 37 ± 19% decrease of ICCL during the day (p = 0.0001). There was a significant linear correlation between ICCL levels in the morning and their decrease during the day (p = 0.015). Methodological repeatability for the ICCL/creatine ratio was excellent, with a coefficient of variance of ~5%, whereas physiological variation was found to be considerably higher (22%) in spite of a standardized physiological preparation protocol. In contrast, TMA levels remained stable over this time period. The proposed (1) H-MRS technique provides a robust way to investigate relevant physiological changes in cardiac metabolites, in particular ICCL. The present results suggest that ICCL reveal a diurnal course, with higher levels in the morning as compared to evening. In addition, a considerable long-term variation of ICCL levels, in both the morning and evening

  15. Acute alteration of cardiac ECG, action potential, I{sub Kr} and the human ether-a-go-go-related gene (hERG) K{sup +} channel by PCB 126 and PCB 77

    Energy Technology Data Exchange (ETDEWEB)

    Park, Mi-Hyeong; Park, Won Sun; Jo, Su-Hyun, E-mail: suhyunjo@kangwon.ac.kr

    2012-07-01

    Polychlorinated biphenyls (PCBs) have been known as serious persistent organic pollutants (POPs), causing developmental delays and motor dysfunction. We have investigated the effects of two PCB congeners, 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) on ECG, action potential, and the rapidly activating delayed rectifier K{sup +} current (I{sub Kr}) of guinea pigs' hearts, and hERG K{sup +} current expressed in Xenopus oocytes. PCB 126 shortened the corrected QT interval (QTc) of ECG and decreased the action potential duration at 90% (APD{sub 90}), and 50% of repolarization (APD{sub 50}) (P < 0.05) without changing the action potential duration at 20% (APD{sub 20}). PCB 77 decreased APD{sub 20} (P < 0.05) without affecting QTc, APD{sub 90}, and APD{sub 50}. The PCB 126 increased the I{sub Kr} in guinea-pig ventricular myocytes held at 36 °C and hERG K{sup +} current amplitude at the end of the voltage steps in voltage-dependent mode (P < 0.05); however, PCB 77 did not change the hERG K{sup +} current amplitude. The PCB 77 increased the diastolic Ca{sup 2+} and decreased Ca{sup 2+} transient amplitude (P < 0.05), however PCB 126 did not change. The results suggest that PCB 126 shortened the QTc and decreased the APD{sub 90} possibly by increasing I{sub Kr}, while PCB 77 decreased the APD{sub 20} possibly by other modulation related with intracellular Ca{sup 2+}. The present data indicate that the environmental toxicants, PCBs, can acutely affect cardiac electrophysiology including ECG, action potential, intracellular Ca{sup 2+}, and channel activity, resulting in toxic effects on the cardiac function in view of the possible accumulation of the PCBs in human body. -- Highlights: ► PCBs are known as serious environmental pollutants and developmental disruptors. ► PCB 126 shortened QT interval of ECG and action potential duration. ► PCB 126 increased human ether-a-go-go-related K{sup +} current and I{sub Kr}.

  16. Modeling CICR in rat ventricular myocytes: voltage clamp studies

    Directory of Open Access Journals (Sweden)

    Palade Philip T

    2010-11-01

    Full Text Available Abstract Background The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR in cardiac myocytes, with voltage clamp (VC studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca2+ loading of the sarcoplasmic reticulum (SR, and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca2+ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca2+ concentration ([Ca2+]myo. Methods The cell model consists of an electrical-equivalent model for the cell membrane and a fluid-compartment model describing the flux of ionic species between the extracellular and several intracellular compartments (cell cytosol, SR and the dyadic coupling unit (DCU, in which resides the mechanistic basis of CICR. The DCU is described as a controller-actuator mechanism, internally stabilized by negative feedback control of the unit's two diametrically-opposed Ca2+ channels (trigger-channel and release-channel. It releases Ca2+ flux into the cyto-plasm and is in turn enclosed within a negative feedback loop involving the SERCA pump, regulating[Ca2+]myo. Results Our model reproduces measured VC data published by several laboratories, and generates graded Ca2+ release at high Ca2+ gain in a homeostatically-controlled environment where [Ca2+]myo is precisely regulated. We elucidate the importance of the DCU elements in this process, particularly the role of the ryanodine receptor in controlling SR Ca2+ release, its activation by trigger Ca2+, and its

  17. Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

    Science.gov (United States)

    Zhao, Weiwei; Fast, Vladimir G.; Ye, Tong; Ai, Xun

    2015-01-01

    Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development. PMID:25867896

  18. Tetrahydroacridine inhibits voltage-dependent Na+ current in guinea-pig ventricular myocytes

    Institute of Scientific and Technical Information of China (English)

    Wei WANG; Yi-ping WANG; Guo-yuan HU

    2004-01-01

    AIM: To study the effects of tetrahydroacridine (tacrine) on voltage-gated Na+ channels in cardiac tissues.METHODS: Single ventricular myocytes were enzymatically dissociated from adult guinea-pig heart. Voltagedependent Na+ current was recorded using whole cell voltage-clamp technique. RESULTS: (1) Tacrine reversibly inhibited Na+ current with an IC50 value of 120 μmol/L (95 % confidence range: 108-133 μmol/L). (2) The inhibitory effects of tacrine on Na+ current exhibited both a tonic nature and use-dependence. (3) Tacrine at 100 μmol/L caused a negative shift (about 10 mV) in the voltage-dependence of steady-state inactivation of Na+ current, and retarded its recovery from inactivation, but did not affect its activation curve. (4) Intracellular application of tacrine significantly inhibited Na+ current. CONCLUSION: In addition to blocking other voltage-gated ion channels,tacrine blocked Na+ channels in guinea-pig ventricular myocytes. Tactine acted as inactivation stabilizer of Na+channels in cardiac tissues.

  19. Production and Characterisation of Anti-Cardiac Troponin-I Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Kh. H. Haider

    1994-01-01

    Full Text Available Cardiac troponin-I (cTn-I was isolated from bovine left ventricular tissue and used as immunogen. Sixteen murine hybridoma lines were produced with two of them. I D 12 and 5F4, showing a high specificity for cTn-I; both of these monoclonal antibodies (McAbs were isotyped as IgG I with kappa - light chains. The specificity of the McAbs for cTn-1 was confirmed by ELISA, western blotting and by the ability of the antibodies to block actomyosin ATPase inhibition by cTn-I. The McAbs may be useful for human ill vivo imaging of myocardial infarcts and other pathological conditions related to cardiac myocyte damage.

  20. Fabrication of Anti-human Cardiac Troponin I Immunogold Nanorods for Sensing Acute Myocardial Damage

    Science.gov (United States)

    Guo, Z. R.; Gu, C. R.; Fan, X.; Bian, Z. P.; Wu, H. F.; Yang, D.; Gu, N.; Zhang, J. N.

    2009-12-01

    A facile, rapid, solution-phase method of detecting human cardiac troponin I for sensing myocardial damage has been described using gold nanorods-based biosensors. The sensing is demonstrated by the distinct change of the longitudinal surface plasmon resonance wavelength of the gold nanorods to specific antibody-antigen binding events. For a higher sensitivity, the aspect ratio of gold nanorods is increased up to ca 5.5 by simply adding small amount of HCl in seed-mediated growth solution. Experimental results show that the detecting limit of the present method is 10 ng/mL. Contrast tests reveal that these gold nanorods-based plasmonic biosensors hold much higher sensitivity than that of conventionally spherical gold nanoparticles.

  1. Fabrication of Anti-human Cardiac Troponin I Immunogold Nanorods for Sensing Acute Myocardial Damage

    Directory of Open Access Journals (Sweden)

    Fan X

    2009-01-01

    Full Text Available Abstract A facile, rapid, solution-phase method of detecting human cardiac troponin I for sensing myocardial damage has been described using gold nanorods-based biosensors. The sensing is demonstrated by the distinct change of the longitudinal surface plasmon resonance wavelength of the gold nanorods to specific antibody–antigen binding events. For a higher sensitivity, the aspect ratio of gold nanorods is increased up to ca 5.5 by simply adding small amount of HCl in seed-mediated growth solution. Experimental results show that the detecting limit of the present method is 10 ng/mL. Contrast tests reveal that these gold nanorods-based plasmonic biosensors hold much higher sensitivity than that of conventionally spherical gold nanoparticles.

  2. Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Jason; Hsueh, Carlin; Gimzewski, James K [Department of Chemistry and Biochemistry, UCLA, 607 Charles Young Drive East, Los Angeles, CA 90095 (United States); Mishra, Bud [Courant Institute of Mathematical Sciences, NYU, 251 Mercer Street, New York, NY 10012 (United States)], E-mail: jreed@chem.ucla.edu, E-mail: gim@chem.ucla.edu

    2008-09-24

    We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.

  3. Right and left ventricular cardiac function in a developed world population with human immunodeficiency virus studied with radionuclide ventriculography

    DEFF Research Database (Denmark)

    Lebech, Anne-Mette; Gerstoft, Jan; Hesse, Birger

    2004-01-01

    BACKGROUND: Cardiac dysfunction has been reported in a substantial part of patients infected with the human immunodeficiency virus (HIV). However, most studies are from a time before the introduction of highly active antiretroviral treatment (HAART), which has significantly reduced HIV-associated......BACKGROUND: Cardiac dysfunction has been reported in a substantial part of patients infected with the human immunodeficiency virus (HIV). However, most studies are from a time before the introduction of highly active antiretroviral treatment (HAART), which has significantly reduced HIV......-associated morbidity and mortality rates. Accordingly, the prevalence of HIV-associated cardiac dysfunction may also have changed. The aim of the study was to establish the prevalence of right- and left-sided cardiac dysfunction in a Danish HIV population, most of whom were undergoing HAART, with radionuclide...... ventricular ejection fraction and 6 (7%) had a reduced right ventricle ejection fraction (0.35-0.42) compared with reference values from the age- and sex-matched reference population. Patients with HIV and reduced cardiac function did not differ in the duration of HIV, CD4 count, CD4 nadir, or HIV RNA load...

  4. A new non-invasive statistical method to assess the spontaneous cardiac baroreflex in humans.

    Science.gov (United States)

    Ducher, M; Fauvel, J P; Gustin, M P; Cerutti, C; Najem, R; Cuisinaud, G; Laville, M; Pozet, N; Paultre, C Z

    1995-06-01

    1. A new method was developed to evaluate cardiac baroreflex sensitivity. The association of a high systolic blood pressure with a low heart rate or the converse is considered to be under the influence of cardiac baroreflex activity. This method is based on the determination of the statistical dependence between systolic blood pressure and heart rate values obtained non-invasively by a Finapres device. Our computerized analysis selects the associations with the highest statistical dependence. A 'Z-coefficient' quantifies the strength of the statistical dependence. The slope of the linear regression, computed on these selected associations, is used to estimate baroreflex sensitivity. 2. The present study was carried out in 11 healthy resting male subjects. The results obtained by the 'Z-coefficient' method were compared with those obtained by cross-spectrum analysis, which has already been validated in humans. Furthermore, the reproducibility of both methods was checked after 1 week. 3. The results obtained by the two methods were significantly correlated (r = 0.78 for the first and r = 0.76 for the second experiment, P < 0.01). When repeated after 1 week, the average results were not significantly different. Considering individual results, test-retest correlation coefficients were higher with the Z-analysis (r = 0.79, P < 0.01) than with the cross-spectrum analysis (r = 0.61, P < 0.05). 4. In conclusion, as the Z-method gives results similar to but more reproducible than the cross-spectrum method, it might be a powerful and reliable tool to assess baroreflex sensitivity in humans.

  5. Signaling Pathways Involved in Cardiac Hypertrophy

    Institute of Scientific and Technical Information of China (English)

    Tao Zewei; Li Longgui

    2006-01-01

    Cardiac hypertrophy is the heart's response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress.Traditionally, it has been considered a beneficial mechanism; however, sustained hypertrophy has been associated with a significant increase in the risk of cardiovascular disease and mortality. Delineating intracellular signaling pathways involved in the different aspects of cardiac hypertrophy will permit future improvements in potential targets for therapeutic intervention. Generally, there are two types of cardiac hypertrophies, adaptive hypertrophy, including eutrophy (normal growth) and physiological hypertrophy (growth induced by physical conditioning), and maladaptive hypertrophy, including pathologic or reactive hypertrophy (growth induced by pathologic stimuli) and hypertrophic growth caused by genetic mutations affecting sarcomeric or cytoskeletal proteins. Accumulating observations from animal models and human patients have identified a number of intracellular signaling pathways that characterized as important transducers of the hypertrophic response,including calcineurin/nuclear factor of activated Tcells, phosphoinositide 3-kinases/Akt (PI3Ks/Akt),G protein-coupled receptors, small G proteins,MAPK, PKCs, Gp130/STAT'3, Na+/H+ exchanger,peroxisome proliferator-activated receptors, myocyte enhancer factor 2/histone deacetylases, and many others. Furthermore, recent evidence suggests that adaptive cardiac hypertrophy is regulated in large part by the growth hormone/insulin-like growth factors axis via signaling through the PI3K/Akt pathway. In contrast, pathological or reactive hypertrophy is triggered by autocrine and paracrine neurohormonal factors released during biomechanical stress that signal through the Gq/phosphorlipase C pathway, leading to an increase in cytosolic calcium and activation of PKC.

  6. Effects of Angiotensin Ⅱ on Expression of the Gap Junction Channel Protein Connexin 43 in Neonatal Rat Ventricular Myocytes

    Institute of Scientific and Technical Information of China (English)

    Jun Yang; Wei Wu

    2007-01-01

    To study the effects of angiotensin Ⅱ,as a mediator of cardiac hypertrophy,on expression of connexin 43 (Cx43) in cultured neonatal rat ventricular myocytes and correlation of expression of Cx43 and cardiomyocyte hypertrophy.Methods Cardiomyocytes were isolated from newborn SD rats.Angiotensin Ⅱ was added into the media to induce myocyte hypertrophy.Cultures were exposed to 10 ~6 mol/L angiotensin Ⅱ for 72 h,Cx43 expression was characterized by RT-PCR and Immunofluorescence methods.Results Immunofluorescence analysis revealed decreased Cx43 immunoreactivity in cells treated for 72 h with angiotensin Ⅱ.RT-PCR analysis demonstrated there was an obvious decrease of Cx43 mRNA level in cells exposed to angiotensin Ⅱ for 72 h.The changes of expression of connexin 43 were related to its entrance into S phase of the cell cycle.Cultured neonatal rat cardiomyocytes were exposed for 72 h to increase concentrations of angiotensin Ⅱ ( 1.0 × 10-9 ~ 1.0 × 10-6mol/L),resulting in significantly decreased Cx43 expression.Conclusions Angiotensin Ⅱ leads to a concentration-dependent decrease in Cx43 protein in cultured neonatal rat ventricular myocytes by decreasing Cx43 mRNA synthesis.Signal transduction pathways activated by angiotensin Ⅱ under pathophysiologic conditions of cardiac hypertrophy could initiate remodeling of gap junctions.

  7. α-Catenin localization and sarcomere self-organization on N-cadherin adhesive patterns are myocyte contractility driven.

    Directory of Open Access Journals (Sweden)

    Anant Chopra

    Full Text Available The N-cadherin (N-cad complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. α-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that α-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that α-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.

  8. Block of Human Cardiac Sodium Channels by Lacosamide: Evidence for Slow Drug Binding along the Activation Pathway

    OpenAIRE

    Wang, Ging Kuo; Wang, Sho-Ya

    2014-01-01

    Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na+ channels for its therapeutic action. Cardiac Na+ channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na+ channels. Lacosamide showed little effect on hNav1.5 Na+ currents at 300 µM when cells were held at −140 mV. With 30-second condi...

  9. Immune modulation of cardiac repair and regeneration: the art of mending broken hearts

    Directory of Open Access Journals (Sweden)

    Ivana Zlatanova

    2016-10-01

    Full Text Available The accumulation of immune cells is amongst the earliest responses that manifest in the cardiac tissue after injury. Both innate and adaptive immunity coordinate distinct and mutually non-exclusive events governing cardiac repair including elimination of the cellular debris, compensatory growth of the remaining cardiac tissue, activation of resident or circulating precursor cells, quantitative and qualitative modifications of the vascular network and formation of a fibrotic scar. The present review summarizes the mounting evidence suggesting that the inflammatory response also guides the regenerative process following cardiac damage. In particular, recent literature has reinforced the central role of monocytes/macrophages in poising the refreshment of cardiomyocytes in myocardial infarction- or apical resection-induced cardiac insult. Macrophages dictate cardiac myocyte renewal through stimulation of pre-existing cardiomyocyte proliferation and/or neovascularization. Nevertheless, substantial efforts are required to identify the nature of these macrophage-derived factors as well as the molecular mechanisms engendered by the distinct subsets of macrophages pertaining in the cardiac tissue. Among the growing inflammatory intermediaries that have been recognized as essential player in heart regeneration, we will focus on the role of interleukin-6 and interleukin-13. Finally, it is likely that within the mayhem of the injured cardiac tissue, additional types of inflammatory cells, such as neutrophils, will enter the dance to ignite and refresh the broken heart. However, the protective and detrimental inflammatory pathways have been mainly deciphered in animal models. Future research should be focused on understanding the cellular effectors and molecular signals regulating inflammation in human heart to pave the way for the development of factual therapies targeting the inflammatory compartment in cardiac diseases.

  10. Anabolic steroids impair the exercise-induced growth of the cardiac capillary bed.

    Science.gov (United States)

    Tagarakis, C V; Bloch, W; Hartmann, G; Hollmann, W; Addicks, K

    2000-08-01

    Concomitant application of anabolic-androgenic steroids and physical exercise can induce cardiac hypertrophy. These experiments investigate the still unknown response of the cardiac myocytes and capillaries to the combined influence of various anabolic steroids and muscular exercise. Female SPF-NMRI mice were divided into the following groups: a) sedentary control, b) exercise (treadmill running); c) sedentary receiving Dianabol; d) exercise + Dianabol; e) exercise + Oral-Turinabol. After 3 and 6 weeks the left ventricular papillary muscles were studied morphometrically. Evaluated variables: minimal myocyte diameter, number of capillaries around a single myocyte, capillary density and intercapillary distance. Only the anabolic steroids + exercise groups showed a mild myocyte hypertrophy. In contrast, only exercise alone caused a significant increase of the capillary density after both experimental periods; e.g. capillary density after 6 weeks (capillaries/mm2, mean values +/- standard deviation, p turinabol + exercise (4,053 +/- 306). Moreover, unlike all other regimens, only exercise alone shortened the intercapillary distance. Finally, exercise without drugs induced the greatest increase in the number of capillaries around a single myocyte. Anabolic steroids combined with exercise: 1) induce mild hypertrophy of the cardiac myocytes, 2) impair the cardiac microvascular adaptation to physical conditioning. The microvascular impairment may cause a detrimental alteration of the myocardial oxygen supply, especially during muscular exercise.

  11. Dual-Energy Computed Tomography Gemstone Spectral Imaging: A Novel Technique to Determine Human Cardiac Calculus Composition.

    Science.gov (United States)

    Cheng, Ching-Li; Chang, Hsiao-Huang; Ko, Shih-Chi; Huang, Pei-Jung; Lin, Shan-Yang

    2016-01-01

    Understanding the chemical composition of any calculus in different human organs is essential for choosing the best treatment strategy for patients. The purpose of this study was to assess the capability of determining the chemical composition of a human cardiac calculus using gemstone spectral imaging (GSI) mode on a single-source dual-energy computed tomography (DECT) in vitro. The cardiac calculus was directly scanned on the Discovery CT750 HD FREEdom Edition using GSI mode, in vitro. A portable fiber-optic Raman spectroscopy was also applied to verify the quantitative accuracy of the DECT measurements. The results of spectral DECT measurements indicate that effective Z values in 3 designated positions located in this calculus were 15.02 to 15.47, which are close to values of 15.74 to 15.86, corresponding to the effective Z values of calcium apatite and hydroxyapatite. The Raman spectral data were also reflected by the predominant Raman peak at 960 cm for hydroxyapatite and the minor peak at 875 cm for calcium apatite. A potential single-source DECT with GSI mode was first used to examine the morphological characteristics and chemical compositions of a giant human cardiac calculus, in vitro. The CT results were consistent with the Raman spectral data, suggesting that spectral CT imaging techniques could be accurately used to diagnose and characterize the compositional materials in the cardiac calculus.

  12. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells.

    Science.gov (United States)

    Månsson-Broberg, Agneta; Rodin, Sergey; Bulatovic, Ivana; Ibarra, Cristián; Löfling, Marie; Genead, Rami; Wärdell, Eva; Felldin, Ulrika; Granath, Carl; Alici, Evren; Le Blanc, Katarina; Smith, C I Edvard; Salašová, Alena; Westgren, Magnus; Sundström, Erik; Uhlén, Per; Arenas, Ernest; Sylvén, Christer; Tryggvason, Karl; Corbascio, Matthias; Simonson, Oscar E; Österholm, Cecilia; Grinnemo, Karl-Henrik

    2016-04-12

    The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs) has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN)-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

  13. Wnt/β-Catenin Stimulation and Laminins Support Cardiovascular Cell Progenitor Expansion from Human Fetal Cardiac Mesenchymal Stromal Cells

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    Agneta Månsson-Broberg

    2016-04-01

    Full Text Available The intrinsic regenerative capacity of human fetal cardiac mesenchymal stromal cells (MSCs has not been fully characterized. Here we demonstrate that we can expand cells with characteristics of cardiovascular progenitor cells from the MSC population of human fetal hearts. Cells cultured on cardiac muscle laminin (LN-based substrata in combination with stimulation of the canonical Wnt/β-catenin pathway showed increased gene expression of ISL1, OCT4, KDR, and NKX2.5. The majority of cells stained positive for PDGFR-α, ISL1, and NKX2.5, and subpopulations also expressed the progenitor markers TBX18, KDR, c-KIT, and SSEA-1. Upon culture of the cardiac MSCs in differentiation media and on relevant LNs, portions of the cells differentiated into spontaneously beating cardiomyocytes, and endothelial and smooth muscle-like cells. Our protocol for large-scale culture of human fetal cardiac MSCs enables future exploration of the regenerative functions of these cells in the context of myocardial injury in vitro and in vivo.

  14. Expression of ATP7B in human gastric cardiac carcinomas in comparison with distal gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Da-Long Wu; Hui-Xing Yi; Feng-Ying Sui; Xiao-Hong Jiang; Xiao-Ming Jiang; Ying-Ying Zhao

    2006-01-01

    AIM: To analyze expression of ATP7B in gastric cardiac adenocarcinomas, its clinicopathologic significance, in comparison with distal gastric adenocarcinomas.METHODS: Immunohistochemical avidin-biotin peroxidase complex method was applied to detect the expression of ATP7B in 49 cases of cardiac carcinomas,the corresponding adjacent non-neoplastic epithelium and 55 cases of distal gastric carcinomas.RESULTS: The proportion of ATP7B positive samples in gastric cardiac carcinomas (51.0%, 25 of 49) was significantly higher than that in the corresponding adjacent non-neoplastic epithelium (22.4%, 11 of 49)(P = 0.003). ATP7B expression in poorly differentiated gastric cardiac carcinomas was significantly higher than that in well/moderately differentiated gastric cardiac carcinomas (P = 0.030). ATP7B expression in gastric cardiac carcinomas was independent of age, tumor size, nodal stage and metastasis status. ATP7B protein was detected in 30.9% (17/55 cases) of distal gastric carcinomas, markedly lower than that in gastric cardiac carcinomas (P = 0.037).CONCLUSION: ATP7B protein is frequently overexpressed in gastric cardiac carcinomas, and correlated with the differentiation of cardiac carcinoma. ATP7B expression in gastric cardiac carcinomas is significantly higher than that in distal gastric carcinomas, which might partially explain the difference of chemotherapy response and prognosis between these two gastric carcinomas.

  15. Correlation between endogenous polyamines in human cardiac tissues and clinical parameters in patients with heart failure.

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    Meana, Clara; Rubín, José Manuel; Bordallo, Carmen; Suárez, Lorena; Bordallo, Javier; Sánchez, Manuel

    2016-02-01

    Polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy in experimental animals. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with cyclic adenosine monophosphate (cAMP) increases. The aim of the study was to establish the role of these in the human heart in living patients. For this, polyamines (by high performance liquid chromatography) and the activity of ODC and N(1)-acetylpolyamine oxidases (APAO) were determined in the right atrial appendage of 17 patients undergoing extracorporeal circulation to correlate with clinical parameters. There existed enzymatic activity associated with the homeostasis of polyamines. Left atria size was positively associated with ODC (r = 0.661, P = 0.027) and negatively with APAO-N(1) -acetylspermine (r = -0.769, P = 0.026), suggesting that increased levels of polyamines are associated with left atrial hemodynamic overload. Left ventricular ejection fraction (LVEF) and heart rate were positively associated with spermidine (r = 0.690, P = 0.003; r = 0.590, P = 0.021) and negatively with N(1)-acetylspermidine (r = -0.554, P = 0.032; r = -0.644, P = 0.018). LVEF was negatively correlated with cAMP levels (r = -0.835, P = 0.001) and with cAMP/ODC (r = -0.794, P = 0.011), cAMP/spermidine (r = -0.813, P = 0.001) and cAMP/spermine (r = -0.747, P = 0.003) ratios. Abnormal LVEF patients showed decreased ODC activity and spermidine, and increased N(1) -acetylspermidine, and cAMP. Spermine decreased in congestive heart failure patients. The trace amine isoamylamine negatively correlated with septal wall thickness (r = -0.634, P = 0.008) and was increased in cardiac heart failure. The results indicated that modifications in polyamine homeostasis might be associated with cardiac function and remodelling. Increased cAMP might have a deleterious effect on function. Further studies should confirm these findings and the involvement of

  16. In Vitro Epigenetic Reprogramming of Human Cardiac Mesenchymal Stromal Cells into Functionally Competent Cardiovascular Precursors

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    Vecellio, Matteo; Meraviglia, Viviana; Nanni, Simona; Barbuti, Andrea; Scavone, Angela; DiFrancesco, Dario; Farsetti, Antonella; Pompilio, Giulio; Colombo, Gualtiero I.; Capogrossi, Maurizio C.

    2012-01-01

    Adult human cardiac mesenchymal-like stromal cells (CStC) represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS) in the presence of 5 µM all-trans Retinoic Acid (ATRA), 5 µM Phenyl Butyrate (PB), and 200 µM diethylenetriamine/nitric oxide (DETA/NO), to create a novel epigenetically active cocktail (EpiC). Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker If current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors. PMID:23284745

  17. In vitro epigenetic reprogramming of human cardiac mesenchymal stromal cells into functionally competent cardiovascular precursors.

    Directory of Open Access Journals (Sweden)

    Matteo Vecellio

    Full Text Available Adult human cardiac mesenchymal-like stromal cells (CStC represent a relatively accessible cell type useful for therapy. In this light, their conversion into cardiovascular precursors represents a potential successful strategy for cardiac repair. The aim of the present work was to reprogram CStC into functionally competent cardiovascular precursors using epigenetically active small molecules. CStC were exposed to low serum (5% FBS in the presence of 5 µM all-trans Retinoic Acid (ATRA, 5 µM Phenyl Butyrate (PB, and 200 µM diethylenetriamine/nitric oxide (DETA/NO, to create a novel epigenetically active cocktail (EpiC. Upon treatment the expression of markers typical of cardiac resident stem cells such as c-Kit and MDR-1 were up-regulated, together with the expression of a number of cardiovascular-associated genes including KDR, GATA6, Nkx2.5, GATA4, HCN4, NaV1.5, and α-MHC. In addition, profiling analysis revealed that a significant number of microRNA involved in cardiomyocyte biology and cell differentiation/proliferation, including miR 133a, 210 and 34a, were up-regulated. Remarkably, almost 45% of EpiC-treated cells exhibited a TTX-sensitive sodium current and, to a lower extent in a few cells, also the pacemaker I(f current. Mechanistically, the exposure to EpiC treatment introduced global histone modifications, characterized by increased levels of H3K4Me3 and H4K16Ac, as well as reduced H4K20Me3 and H3s10P, a pattern compatible with reduced proliferation and chromatin relaxation. Consistently, ChIP experiments performed with H3K4me3 or H3s10P histone modifications revealed the presence of a specific EpiC-dependent pattern in c-Kit, MDR-1, and Nkx2.5 promoter regions, possibly contributing to their modified expression. Taken together, these data indicate that CStC may be epigenetically reprogrammed to acquire molecular and biological properties associated with competent cardiovascular precursors.

  18. Cardiac disease modeling using induced pluripotent stemcell-derived human cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Patrizia Dell’Era; Patrizia Benzoni; Elisabetta Crescini; Matteo Valle; Er Xia; Antonella Consiglio; Maurizio Memo

    2015-01-01

    Causative mutations and variants associated with cardiacdiseases have been found in genes encoding cardiac ionchannels, accessory proteins, cytoskeletal components,junctional proteins, and signaling molecules. In mostcases the functional evaluation of the genetic alterationhas been carried out by expressing the mutated proteinsin in-vitro heterologous systems. While these studieshave provided a wealth of functional details that havegreatly enhanced the understanding of the pathologicalmechanisms, it has always been clear that heterologousexpression of the mutant protein bears the intrinsiclimitation of the lack of a proper intracellular environmentand the lack of pathological remodeling. The resultsobtained from the application of the next generationsequencing technique to patients suffering from cardiacdiseases have identified several loci, mostly in non-codingDNA regions, which still await functional analysis. Theisolation and culture of human embryonic stem cells hasinitially provided a constant source of cells from whichcardiomyocytes (CMs) can be obtained by differentiation.Furthermore, the possibility to reprogram cellular fateto a pluripotent state, has opened this process to thestudy of genetic diseases. Thus induced pluripotentstem cells (iPSCs) represent a completely new cellularmodel that overcomes the limitations of heterologousstudies. Importantly, due to the possibility to keepspontaneously beating CMs in culture for several months,during which they show a certain degree of maturation/aging, this approach will also provide a system in whichto address the effect of long-term expression of themutated proteins or any other DNA mutation, in termsof electrophysiological remodeling. Moreover, sinceiPSC preserve the entire patients' genetic context, thesystem will help the physicians in identifying the mostappropriate pharmacological intervention to correct thefunctional alteration. This article summarizes the currentknowledge of cardiac genetic

  19. Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate adenocarcinoma cells.

    Science.gov (United States)

    McConkey, D J; Lin, Y; Nutt, L K; Ozel, H Z; Newman, R A

    2000-07-15

    Cardiac glycosides are used clinically to increase contractile force in patients with cardiac disorders. Their mechanism of action is well established and involves inhibition of the plasma membrane Na+/K+-ATPase, leading to alterations in intracellular K+ and Ca(2+) levels. Here, we report that the cardiac glycosides oleandrin, ouabain, and digoxin induce apoptosis in androgen-independent human prostate cancer cell lines in vitro. Cell death was associated with early release of cytochrome c from mitochondria, followed by proteolytic processing of caspases 8 and 3. Oleandrin also promoted caspase activation, detected by cleavage poly(ADP-ribose) polymerase and hydrolysis of a peptide substrate (DEVD-pNA). Comparison of the rates of apoptosis in poorly metastatic PC3 M-Pro4 and highly metastatic PC3 M-LN4 subclones demonstrated that cell death was delayed in the latter because of a delay in mitochondrial cytochrome c release. Single-cell imaging of intracellular Ca(2+) fluxes demonstrated that the proapoptotic effects of the cardiac glycosides were linked to their abilities to induce sustained Ca(2+) increases in the cells. Our results define a novel activity for cardiac glycosides that could prove relevant to the treatment of metastatic prostate cancer.

  20. Three-Dimensional Human Cardiac Tissue Engineered by Centrifugation of Stacked Cell Sheets and Cross-Sectional Observation of Its Synchronous Beatings by Optical Coherence Tomography.

    Science.gov (United States)

    Haraguchi, Yuji; Hasegawa, Akiyuki; Matsuura, Katsuhisa; Kobayashi, Mari; Iwana, Shin-Ichi; Kabetani, Yasuhiro; Shimizu, Tatsuya

    2017-01-01

    Three-dimensional (3D) tissues are engineered by stacking cell sheets, and these tissues have been applied in clinical regenerative therapies. The optimal fabrication technique of 3D human tissues and the real-time observation system for these tissues are important in tissue engineering, regenerative medicine, cardiac physiology, and the safety testing of candidate chemicals. In this study, for aiming the clinical application, 3D human cardiac tissues were rapidly fabricated by human induced pluripotent stem (iPS) cell-derived cardiac cell sheets with centrifugation, and the structures and beatings in the cardiac tissues were observed cross-sectionally and noninvasively by two optical coherence tomography (OCT) systems. The fabrication time was reduced to approximately one-quarter by centrifugation. The cross-sectional observation showed that multilayered cardiac cell sheets adhered tightly just after centrifugation. Additionally, the cross-sectional transmissions of beatings within multilayered human cardiac tissues were clearly detected by OCT. The observation showed the synchronous beatings of the thicker 3D human cardiac tissues, which were fabricated rapidly by cell sheet technology and centrifugation. The rapid tissue-fabrication technique and OCT technology will show a powerful potential in cardiac tissue engineering, regenerative medicine, and drug discovery research.

  1. Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4

    Institute of Scientific and Technical Information of China (English)

    Azhar Maqbool; Emma J Spary; Iain W Manfield; Michaela Ruhmann; Lorena Zuliani-Alvarez; Filomena O Gamboa-Esteves; Karen E Porter; Mark J Drinkhill; Kim S Midwood; Neil A Turner

    2016-01-01

    AIM:To investigate the effect of Tenascin C(TNC)on the expression of pro-inflammatory cytokines and matrixmetalloproteinases in human cardiac myofibroblasts(CMF).METHODS:CMF were isolated and cultured from patients undergoing coronary artery bypass grafting.Cultured cells were treated with either TNC(0.1μmol/L,24 h)or a recombinant protein corresponding to different domains of the TNC protein;fibrinogen-like globe(FBG)and fibronectin typeⅢ-like repeats(TNⅢ5-7)(both 1μmol/L,24 h).The expression of the proinflammatory cytokines;interleukin(IL)-6,IL-1β,TNFαand the matrix metalloproteinases;MMPs(MMP1,2,3,9,10,MT1-MMP)was assessed using real time RT-PCR and western blot analysis.RESULTS:TNC increased both IL-6 and MMP3(P<0.01)mR NA levels in cultured human CMF but had no significant effect on the other markers studied.The increase in IL-6 mR NA expression was mirrored by an increase in protein secretion as assessed by enzymelinked immunosorbant assay(P<0.01).Treating CMF with the recombinant protein FBG increased IL-6mR NA and protein(P<0.01)whereas the recombinant protein TNⅢ5-7 had no effect.Neither FBG nor TNⅢ5-7 had any significant effect on MMP3 expression.The expression of toll-like receptor 4(TLR4)in human CMF was confirmed by real time RT-PCR,western blot and immunohistochemistry.Pre-incubation of cells with TLR4neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mR NA and protein expression.CONCLUSION:TNC up-regulates IL-6 expression in human CMF,an effect mediated through the FBG domain of TNC and via the TLR4 receptor.

  2. The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway.

    Science.gov (United States)

    Pan, Li; Zhang, Yuming; Zhao, Wanlu; Zhou, Xia; Wang, Chunxia; Deng, Fan

    2017-07-01

    Evidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer. However, the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown. The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells. The cytotoxicity and IC50 of five small molecule compounds (oleandrin, neriifolin, strophanthidin, gitoxigenin, and convallatoxin) in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays, respectively. Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide, followed by flow cytometry. Intracellular Ca(2+) was determined using Fluo-3 AM,glutathione (GSH) levels were measured using a GSH detection kit,and the activity of caspase-3, -9 was measured using a peptide substrate. BAX, pro-caspase-3, -9, cytochrome C and BCL-2 expression were determined by Western blotting. Oleandrin significantly decreased cell viabilities in SW480, HCT116 and RKO cells. The IC50 for SW480 cells was 0.02 µM, whereas for NCM460 cells 0.56 µM. More interestingly, the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells. Oleandrin downregulated protein expression of pro-caspase-3, -9, but enhanced caspase-3, -9 activities. These effects were accompanied by upregulation of protein expression of cytochrome C and BAX, and downregulation of BCL-2 protein expression in a concentration-dependent manner. Furthermore, oleandrin increased intracellular Ca(2+) concentration, but decreased GSH concentration in the cells. The present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway. Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.

  3. Comparative study of myocytes from normal and mdx mice iPS cells.

    Science.gov (United States)

    Chen, Fei; Cao, Jiqing; Liu, Qiang; Qin, Jie; Kong, Jie; Wang, Yanyun; Li, Yaqin; Geng, Jia; Li, Qiuling; Yang, Liqing; Xiang, Andy Peng; Zhang, Cheng

    2012-02-01

    Recently, induced pluripotent stem cells (iPS cells) have been derived from various techniques and show great potential for therapy of human diseases. Furthermore, the iPS technique can be used to provide cell models to explore pathological mechanisms of many human diseases in vitro, such as Duchenne muscular dystrophy (DMD), which is a severe recessive X-linked form of muscular dystrophy without effective treatment. In this study, we try to determine whether there are different characteristics of myocytes from mdx iPS cells and C57BL/10 iPS cells. Our results showed that both of mdx and C57BL/10 cells could be induced into iPS cells in vitro, whereas colony-forming ability of mdx iPS cells was much weaker than that of C57BL/10 iPS cells. Meanwhile, mdx iPS cells could be induced to differentiate into myocytes, whereas their differentiation efficiency was much lower than that of C57BL/10 iPS cells. And, the number of apoptotic cells in differentiated myocytes from mdx iPS cells was significantly higher than that from C57BL/10 iPS cells. More importantly, treatment of a pan-caspase inhibitor (Z-VAD) produced a significant decrease in apoptotic cells. This study might add some insight to the biology study of dystrophin gene.

  4. Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes

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    Higashi Richard M

    2008-10-01

    Full Text Available Abstract Background The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30 and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis. Results The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells. Conclusion The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells

  5. The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression.

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    Annina Kelloniemi

    Full Text Available The phosphatase and actin regulator 1 (PHACTR1 locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI. However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05 at 3 days but 40% lower (P<0.05 at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks' follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550. Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.

  6. Laminar arrangement of ventricular myocytes influences electrical behavior of the heart.

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    Hooks, Darren A; Trew, Mark L; Caldwell, Bryan J; Sands, Gregory B; LeGrice, Ian J; Smaill, Bruce H

    2007-11-01

    The response of the heart to electrical shock, electrical propagation in sinus rhythm, and the spatiotemporal dynamics of ventricular fibrillation all depend critically on the electrical anisotropy of cardiac tissue. A long-held view of cardiac electrical anisotropy is that electrical conductivity is greatest along the myocyte axis allowing most rapid propagation of electrical activation in this direction, and that conductivity is isotropic transverse to the myocyte axis supporting a slower uniform spread of activation in this plane. In this context, knowledge of conductivity in two directions, parallel and transverse to the myofiber axis, is sufficient to characterize the electrical action of the heart. Here we present new experimental data that challenge this view. We have used a novel combination of intramural electrical mapping, and experiment-specific computer modeling, to demonstrate that left ventricular myocardium has unique bulk conductivities associated with three microstructurally-defined axes. We show that voltage fields induced by intramural current injection are influenced by not only myofiber direction, but also the transmural arrangement of muscle layers or myolaminae. Computer models of these experiments, in which measured 3D tissue structure was reconstructed in-silico, best matched recorded voltages with conductivities in the myofiber direction, and parallel and normal to myolaminae, set in the ratio 4:2:1, respectively. These findings redefine cardiac tissue as an electrically orthotropic substrate and enhance our understanding of how external shocks may act to successfully reset the fibrillating heart into a uniform electrical state. More generally, the mechanisms governing the destabilization of coordinated electrical propagation into ventricular arrhythmia need to be evaluated in the light of this discovery.

  7. Longstanding hyperthyroidism is associated with normal or enhanced intrinsic cardiomyocyte function despite decline in global cardiac function.

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    Nathan Y Weltman

    Full Text Available Thyroid hormones (THs play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH. LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function.

  8. Longstanding Hyperthyroidism Is Associated with Normal or Enhanced Intrinsic Cardiomyocyte Function despite Decline in Global Cardiac Function

    Science.gov (United States)

    Redetzke, Rebecca A.; Gerdes, A. Martin

    2012-01-01

    Thyroid hormones (THs) play a pivotal role in cardiac homeostasis. TH imbalances alter cardiac performance and ultimately cause cardiac dysfunction. Although short-term hyperthyroidism typically leads to heightened left ventricular (LV) contractility and improved hemodynamic parameters, chronic hyperthyroidism is associated with deleterious cardiac consequences including increased risk of arrhythmia, impaired cardiac reserve and exercise capacity, myocardial remodeling, and occasionally heart failure. To evaluate the long-term consequences of chronic hyperthyroidism on LV remodeling and function, we examined LV isolated myocyte function, chamber function, and whole tissue remodeling in a hamster model. Three-month-old F1b hamsters were randomized to control or 10 months TH treatment (0.1% grade I desiccated TH). LV chamber remodeling and function was assessed by echocardiography at 1, 2, 4, 6, 8, and 10 months of treatment. After 10 months, terminal cardiac function was assessed by echocardiography and LV hemodynamics. Hyperthyroid hamsters exhibited significant cardiac hypertrophy and deleterious cardiac remodeling characterized by myocyte lengthening, chamber dilatation, decreased relative wall thickness, increased wall stress, and increased LV interstitial fibrotic deposition. Importantly, hyperthyroid hamsters demonstrated significant LV systolic and diastolic dysfunction. Despite the aforementioned remodeling and global cardiac decline, individual isolated cardiac myocytes from chronically hyperthyroid hamsters had enhanced function when compared with myocytes from untreated age-matched controls. Thus, it appears that long-term hyperthyroidism may impair global LV function, at least in part by increasing interstitial ventricular fibrosis, in spite of normal or enhanced intrinsic cardiomyocyte function. PMID:23056390

  9. Bisphenol A binds to the local anesthetic receptor site to block the human cardiac sodium channel.

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    Andrias O O'Reilly

    Full Text Available Bisphenol A (BPA has attracted considerable public attention as it leaches from plastic used in food containers, is detectable in human fluids and recent epidemiologic studies link BPA exposure with diseases including cardiovascular disorders. As heart-toxicity may derive from modified cardiac electrophysiology, we investigated the interaction between BPA and hNav1.5, the predominant voltage-gated sodium channel subtype expressed in the human heart. Electrophysiology studies of heterologously-expressed hNav1.5 determined that BPA blocks the channel with a K(d of 25.4±1.3 µM. By comparing the effects of BPA and the local anesthetic mexiletine on wild type hNav1.5 and the F1760A mutant, we demonstrate that both compounds share an overlapping binding site. With a key binding determinant thus identified, an homology model of hNav1.5 was generated based on the recently-reported crystal structure of the bacterial voltage-gated sodium channel NavAb. Docking predictions position both ligands in a cavity delimited by F1760 and contiguous with the DIII-IV pore fenestration. Steered molecular dynamics simulations used to assess routes of ligand ingress indicate that the DIII-IV pore fenestration is a viable access pathway. Therefore BPA block of the human heart sodium channel involves the local anesthetic receptor and both BPA and mexiletine may enter the closed-state pore via membrane-located side fenestrations.

  10. Influence of the cardiac myosin hinge region on contractile activity.

    Science.gov (United States)

    Margossian, S S; Krueger, J W; Sellers, J R; Cuda, G; Caulfield, J B; Norton, P; Slayter, H S

    1991-06-01

    The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

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

    Science.gov (United States)

    Vincent, N D; Cummins, P

    1985-04-01

    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

  12. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

    Science.gov (United States)

    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

  13. Human cardiac beta1- or beta2-adrenergic receptor stimulation and the negative chronotropic effect of low-dose pirenzepine.

    Science.gov (United States)

    Jakubetz, J; Schmuck, S; Wochatz, G; Ruhland, B; Poller, U; Radke, J; Brodde, O E

    2000-05-01

    The M1-muscarinic receptor antagonist pirenzepine in low doses (pirenzepine differ in volunteers with activated cardiac beta1-adrenergic receptors versus activated cardiac beta2-adrenergic receptors. In 17 male volunteers (25 +/- 1 years) we studied effects of pirenzepine infusion (0.5 mg intravenous bolus followed by continuous infusion of 0.15 microg/kg/min) on heart rate and heart rate-corrected duration of electromechanical systole (QS2c, as a measure of inotropism) that had been stimulated by activation of cardiac beta1-adrenergic receptors (bicycle exercise in the supine position for 60 minutes at 25 W) or cardiac beta2-adrenergic receptors (continuous intravenous infusion of 100 ng/kg/min terbutaline). Bicycle exercise and terbutaline infusion significantly increased heart rate and shortened QS2c. When pirenzepine was infused 20 minutes after the beginning of the exercise or terbutaline infusion, heart rate decreased in both settings by approximately the same extent (approximately -10 to -14 beats/min), although exercise and terbutaline infusion continued; however, QS2c was not affected. Pirenzepine (0.05 to 1 mg intravenous bolus)-induced decrease in heart rate was abolished after 6 days of transdermal scopolamine treatment of volunteers. Low-dose pirenzepine decreased heart rate by muscarinic receptor stimulation, because this was blocked by scopolamine. Moreover, low-dose pirenzepine did not differentiate between cardiac beta1- or beta2-adrenergic receptor stimulation; however, low-dose pirenzepine did not affect cardiac contractility as measured by QS2c. Low-dose pirenzepine therefore exerted a unique pattern of action in the human heart: it decreased heart rate (basal and beta1- and/or beta2-adrenergic receptor-stimulated) without affecting contractility.

  14. Transcription of the human beta enolase gene (ENO-3) is regulated by an intronic muscle-specific enhancer that binds myocyte-specific enhancer factor 2 proteins and ubiquitous G-rich-box binding factors.

    Science.gov (United States)

    Feo, S; Antona, V; Barbieri, G; Passantino, R; Calì, L; Giallongo, A

    1995-01-01

    To provide evidence for the cis-regulatory DNA sequences and trans-acting factors involved in the complex pattern of tissue- and stage-specific expression of the beta enolase gene, constructs containing fragments of the gene fused to the chloramphenicol acetyltransferase gene were used in transient-transfection assays of C2C12 myogenic cells. Deletion analysis revealed the presence of four major regions: two negative regions in the 5'-flanking sequence, a basal promoter region which directs expression at low levels in proliferating and differentiated muscle cells, and a positive region within the first intron that confers cell-type-specific and differentiation-induced expression. This positive regulatory element is located in the 3'-proximal portion of the first intron (nucleotides +504 to +637) and acts as an enhancer irrespective of orientation and position from the homologous beta enolase promoter or the heterologous thymidine kinase promoter, conferring in both cases muscle-specific expression to the linked reporter gene. Deletion of a putative myocyte-specific enhancer factor 1 (MEF-1) binding site, containing a canonical E-box motif, had no effects on muscle-specific transcription, indicating that this site is not required for the activity of the enhancer. Gel mobility shift assays, competition analysis, DNase I footprinting, and mutagenesis studies indicated that this element interacts through an A/T-rich box with a MEF-2 protein(s) and through a G-rich box with a novel ubiquitous factor(s). Mutation of either the G-rich box or the A/T-rich box resulted in a significantly reduced activity of the enhancer in transient-transfection assays. These data indicate that MEF-2 and G-rich-box binding factors are each necessary for tissue-specific expression of the beta enolase gene in skeletal muscle cells. PMID:7565752

  15. Biphasic effects of haloperidol on sodium currents in guinea pig ventricular myocytes

    Institute of Scientific and Technical Information of China (English)

    Lu-feng CHENG; Dong YAN; Subat TURDI; Parhat KERRAM

    2007-01-01

    Aim: To study the effects of haloperidol on sodium currents (INa) in guinea pig ventricular myocytes. Method: Whole-cell patch clamp technique was employed to evaluate the effects of haloperidol on INa, in individual ventricular myocytes.Results: Haloperidol (0.1-3 μmol/L) inhibited INa in a concentration-dependent manner with an IC50 of 0.253±0.015 μmol/L. The inhibition rate of haloperidol (0.3 μmol/L) on INa was 22.14%±0.02%, and the maximum conductance was reduced.Haloperidol significantly reduced the midpoints for the activation and inactiva-tion of INa by 2.09 and 4.09 mV, respectively. The time constant of recovery was increased. The increase in time intervals could only recover by 90.14%±1.4% (n=6); however, haloperidol at 0.03 μmol/L enhanced INa conductance. The mid-points for the activation and inactivation of INa were shifted by 1.38 and 5.69 mV,respectively, at this concentration of haloperidol.Conclusion: Haloperidol dis-played a biphasic effect on INa in guinea pig cardiac myocytes. High concentra-tions of haloperidol inhibited INa, while lower concentrations of haloperidol shifted the activation and inactivation curve to the left. Full recovery of recovery curve was not achieved after 0.3 μmol/L haloperidol administration, indicating that the drug affects the inactivated state of sodium channels.

  16. Human cardiac troponin I sensor based on silver nanoparticle doped microsphere resonator

    Science.gov (United States)

    Saliminasab, Maryam; Bahrampour, Alireza; Zandi, Mohammad Hossein

    2012-12-01

    Human cardiac troponin I (cTnI) is a specific biomarker for diagnosis of acute myocardial infarction (AMI). In this paper, a composite sensing system of an optical microsphere resonator and silver nanoparticles based on surface enhanced Raman scattering (SERS) and stimulated Raman scattering (SRS) techniques towards a point of care diagnostic system for AMI using the cTnI biomarker in HEPES buffered solution (HBS) is proposed. Pump and Raman signals enter the optical fiber coupling into the microsphere, and then SRS occurs in the microsphere. The presence of silver nanoparticles on the microsphere surface provides a tremendous enhancement of the resulting Raman signal through an electromagnetic enhancement of both the laser excitation and Stokes-shifted light of the order of 1010. This enhancement occurs in metals as surface plasmon resonance (SPR), which increases the Raman gain through the SERS effect. Our simulation results show that this sensor presents a linear response for cTnI detection. The calculated enhanced Raman signal can be employed to detect the cTnI molecules around the microsphere.

  17. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Vincent C. Chen

    2015-09-01

    Full Text Available To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2 × 109 CM/L at scales up to 1 L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  18. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

    Science.gov (United States)

    Chen, Vincent C; Ye, Jingjing; Shukla, Praveen; Hua, Giau; Chen, Danlin; Lin, Ziguang; Liu, Jian-chang; Chai, Jing; Gold, Joseph; Wu, Joseph; Hsu, David; Couture, Larry A

    2015-09-01

    To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  19. Ubiquitin-Specific Protease 4 Is an Endogenous Negative Regulator of Pathological Cardiac Hypertrophy.

    Science.gov (United States)

    He, Ben; Zhao, Yi-Chao; Gao, Ling-Chen; Ying, Xiao-Ying; Xu, Long-Wei; Su, Yuan-Yuan; Ji, Qing-Qi; Lin, Nan; Pu, Jun

    2016-06-01

    Dysregulation of the ubiquitin proteasome system components ubiquitin ligases and proteasome plays an important role in the pathogenesis of cardiac hypertrophy. However, little is known about the role of another ubiquitin proteasome system component, the deubiquitinating enzymes, in cardiac hypertrophy. Here, we revealed a crucial role of ubiquitin specific protease 4 (USP4), a deubiquitinating enzyme prominently expressed in the heart, in attenuating pathological cardiac hypertrophy and dysfunction. USP4 levels were consistently decreased in human failing hearts and in murine hypertrophied hearts. Adenovirus-mediated gain- and loss-of-function approaches indicated that deficiency of endogenous USP4 promoted myocyte hypertrophy induced by angiotensin II in vitro, whereas restoration of USP4 significantly attenuated the prohypertrophic effect of angiotensin II. To corroborate the role of USP4 in vivo, we generated USP4 global knockout mice and mice with cardiac-specific overexpression of USP4. Consistent with the in vitro study, USP4 depletion exacerbated the hypertrophic phenotype and cardiac dysfunction in mice subjected to pressure overload, whereas USP4 transgenic mice presented ameliorated pathological cardiac hypertrophy compared with their control littermates. Molecular analysis revealed that USP4 deficiency augmented the activation of the transforming growth factor β-activated kinase 1 (TAK1)-(JNK1/2)/P38 signaling in response to hypertrophic stress, and blockage of TAK1 activation abolished the pathological effects of USP4 deficiency in vivo. These findings provide the first evidence for the involvement of USP4 in cardiac hypertrophy, and shed light on the therapeutic potential of targeting USP4 in the treatment of cardiac hypertrophy.

  20. Effect of global cardiac ischemia on human ventricular fibrillation: insights from a multi-scale mechanistic model of the human heart.

    Science.gov (United States)

    Kazbanov, Ivan V; Clayton, Richard H; Nash, Martyn P; Bradley, Chris P; Paterson, David J; Hayward, Martin P; Taggart, Peter; Panfilov, Alexander V

    2014-11-01

    Acute regional ischemia in the heart can lead to cardiac arrhythmias such as ventricular fibrillation (VF), which in turn compromise cardiac output and result in secondary global cardiac ischemia. The secondary ischemia may influence the underlying arrhythmia mechanism. A recent clinical study documents the effect of global cardiac ischaemia on the mechanisms of VF. During 150 seconds of global ischemia the dominant frequency of activation decreased, while after reperfusion it increased rapidly. At the same time the complexity of epicardial excitation, measured as the number of epicardical phase singularity points, remained approximately constant during ischemia. Here we perform numerical studies based on these clinical data and propose explanations for the observed dynamics of the period and complexity of activation patterns. In particular, we study the effects on ischemia in pseudo-1D and 2D cardiac tissue models as well as in an anatomically accurate model of human heart ventricles. We demonstrate that the fall of dominant frequency in VF during secondary ischemia can be explained by an increase in extracellular potassium, while the increase during reperfusion is consistent with washout of potassium and continued activation of the ATP-dependent potassium channels. We also suggest that memory effects are responsible for the observed complexity dynamics. In addition, we present unpublished clinical results of individual patient recordings and propose a way of estimating extracellular potassium and activation of ATP-dependent potassium channels from these measurements.

  1. Effects of the venom of the spider Ornithoctonus hainana on neonatal rat ventricular myocytes cellular and ionic electrophysiology.

    Science.gov (United States)

    Zhang, Yiya; Liu, Jinyan; Liu, Zhonghua; Wang, Meichi; Wang, Jing; Lu, Shanshan; Zhu, Li; Zeng, Xiongzhi; Liang, Songping

    2014-09-01

    Cardiac ion channels are membrane-spanning proteins that allow the passive movement of ions across the cell membrane along its electrochemical gradient, which regulates the resting membrane potential as well as the shape and duration of the cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters, hormones and drugs of cardiac diseases. Spider venoms contain high abundant of toxins that target diverse ion channels and have been considered as a potential resource of new constituents with specific pharmacological properties. However, few peptides from spider venoms were detected as cardiac channel antagonists. In order to explore the effects of the venom of Ornithoctonus hainana on the action potential and ionic currents of neonatal rat ventricular myocytes (NRVMs), whole cell patch clamp technique was used to record action potential duration (APD), sodium current (INa), L calcium current (ICaL), rapidly activating and inactivating transient outward currents (Ito1), rapid (IKr) and slow (IKs) components of the delayed rectifier currents and the inward rectifier currents (IK1). Our results showed that 100 μg/mL venom obviously prolonged APDs. Significantly, the venom could inhibit INa and ICaL effectively, while no evident inhibitory effects on cardiac K(+) channels (Ito1, Iks, Ikr and Ik1) were observed, suggesting that the venom represented a multifaceted pharmacological profile. The effect of venom on Na(+) and Ca(2+) currents of ventricular myocytes revealed that the hainan venom as a rich resource of cardiac channel antagonists might be valuable tools for the investigation of both channels and drug development.

  2. Rational promoter selection for gene transfer into cardiac cells

    NARCIS (Netherlands)

    Maass, A; Langer, SJ; Oberdorf-Maass, S; Bauer, S; Neyses, L; Leinwand, LA

    2003-01-01

    Cardiomyocytes (CMCs) are extremely difficult to transfect with non-viral techniques, but they are efficiently infected by adenoviruses. The most commonly used promoters to drive protein expression in cardiac myocytes are of viral origin, since they are believed to be constitutively active and minim

  3. Quantification of a cardiac biomarker in human serum using extraordinary optical transmission (EOT.

    Directory of Open Access Journals (Sweden)

    Tao Ding

    Full Text Available Nanoimprinting lithography (NIL is a manufacturing process that can produce macroscale surface areas with nanoscale features. In this paper, this technique is used to solve three fundamental issues for the application of localized surface plasmonic resonance (LSPR in practical clinical measurements: assay sensitivity, chip-to-chip variance, and the ability to perform assays in human serum. Using NIL, arrays of 140 nm square features were fabricated on a sensing area of 1.5 mm x 1.5 mm with low cost. The high reproducibility of NIL allowed for the use of a one-chip, one-measurement approach with 12 individually manufactured surfaces with minimal chip-to-chip variations. To better approximate a real world setting, all chips were modified with a biocompatible, multi-component monolayer and inter-chip variability was assessed by measuring a bioanalyte standard (2.5-75 ng/ml in the presence of a complex biofluid, human serum. In this setting, nanoimprinted LSPR chips were able to provide sufficient characteristics for a 'low-tech' approach to laboratory-based bioanalyte measurement, including: 1 sufficient size to interface with a common laboratory light source and detector without the need for a microscope, 2 high sensitivity in serum with a cardiac troponin limit of detection of 0.55 ng/ml, and 3 very low variability in chip manufacturing to produce a figure of merit (FOM of 10.5. These findings drive LSPR closer to technical comparability with ELISA-based assays while preserving the unique particularities of a LSPR based sensor, suitability for multiplexing and miniaturization, and point-of-care detections.

  4. Quantification of a cardiac biomarker in human serum using extraordinary optical transmission (EOT).

    Science.gov (United States)

    Ding, Tao; Hong, Minghui; Richards, A Mark; Wong, Ten It; Zhou, Xiaodong; Drum, Chester Lee

    2015-01-01

    Nanoimprinting lithography (NIL) is a manufacturing process that can produce macroscale surface areas with nanoscale features. In this paper, this technique is used to solve three fundamental issues for the application of localized surface plasmonic resonance (LSPR) in practical clinical measurements: assay sensitivity, chip-to-chip variance, and the ability to perform assays in human serum. Using NIL, arrays of 140 nm square features were fabricated on a sensing area of 1.5 mm x 1.5 mm with low cost. The high reproducibility of NIL allowed for the use of a one-chip, one-measurement approach with 12 individually manufactured surfaces with minimal chip-to-chip variations. To better approximate a real world setting, all chips were modified with a biocompatible, multi-component monolayer and inter-chip variability was assessed by measuring a bioanalyte standard (2.5-75 ng/ml) in the presence of a complex biofluid, human serum. In this setting, nanoimprinted LSPR chips were able to provide sufficient characteristics for a 'low-tech' approach to laboratory-based bioanalyte measurement, including: 1) sufficient size to interface with a common laboratory light source and detector without the need for a microscope, 2) high sensitivity in serum with a cardiac troponin limit of detection of 0.55 ng/ml, and 3) very low variability in chip manufacturing to produce a figure of merit (FOM) of 10.5. These findings drive LSPR closer to technical comparability with ELISA-based assays while preserving the unique particularities of a LSPR based sensor, suitability for multiplexing and miniaturization, and point-of-care detections.

  5. Cardiac mitochondria exhibit dynamic functional clustering

    Directory of Open Access Journals (Sweden)

    Felix Tobias Kurz

    2014-09-01

    Full Text Available Multi-oscillatory behavior of mitochondrial inner membrane potential ΔΨm in self-organized cardiac mitochondrial networks can be triggered by metabolic or oxidative stress. Spatio-temporal analyses of cardiac mitochondrial networks have shown that mitochondria are heterogeneously organized in synchronously oscillating clusters in which the mean cluster frequency and size are inversely correlated, thus suggesting a modulation of cluster frequency through local inter-mitochondrial coupling. In this study, we propose a method to examine the mitochondrial network's topology through quantification of its dynamic local clustering coefficients. Individual mitochondrial ΔΨm oscillation signals were identified for each cardiac myocyte and cross-correlated with all network mitochondria using previously described methods (Kurz et al., 2010. Time-varying inter-mitochondrial connectivity, defined for mitochondria in the whole network whose signals are at least 90% correlated at any given time point, allowed considering functional local clustering coefficients. It is shown that mitochondrial clustering in isolated cardiac myocytes changes dynamically and is significantly higher than for random mitochondrial networks that are constructed using the Erdös-Rényi model based on the same sets of vertices. The network's time-averaged clustering coefficient for cardiac myocytes was found to be 0.500 ± 0.051 (N=9 versus 0.061 ± 0.020 for random networks, respectively. Our results demonstrate that cardiac mitochondria constitute a network with dynamically connected constituents whose topological organization is prone to clustering. Cluster partitioning in networks of coupled oscillators has been observed in scale-free and chaotic systems and is therefore in good agreement with previous models of cardiac mitochondrial networks (Aon et al., 2008.

  6. Effect of increases in cardiac contractility on cerebral blood flow in humans.

    Science.gov (United States)

    Ogoh, Shigehiko; Moralez, Gilbert; Washio, Takuro; Sarma, Satyam; Hieda, Michinari; Romero, Steven A; Cramer, Matthew N; Shibasaki, Manabu; Crandall, Craig G

    2017-09-15

    The effect of acute increases in cardiac contractility on cerebral blood flow (CBF) remains unknown. We hypothesized that the external carotid artery (ECA) downstream vasculature modifies the direct influence of acute increases in heart rate and cardiac function on CBF regulation. Twelve healthy subjects received two infusions of dobutamine (first a low dose; 5 μg/kg/min and then a high dose; 15 μg/kg/min) for 12 min each. Cardiac output, blood flow through the internal carotid artery (ICA) and ECA and echocardiographic measurements were performed during dobutamine infusions. Despite increases in cardiac contractility, cardiac output and arterial pressure with dobutamine, ICA blood flow and conductance slightly decreased from resting baseline during both low and high dose infusions. In contrast, ECA blood flow and conductance increased appreciably during both low and high dose infusions. Greater ECA vascular conductance and corresponding increases in blood flow may protect over-perfusion of intracranial cerebral arteries during enhanced cardiac contractility and associated increases in cardiac output and perfusion pressure. Importantly, these findings suggest that the acute increase of blood perfusion due to dobutamine administration does not cause cerebral over-perfusion or an associated risk of cerebral vascular damage. Copyright © 2017, American Journal of Physiology-Heart and Circulatory Physiology.

  7. TPEN prevents rapid pacing-induced calcium overload and nitration stress in HL-1 myocytes.

    Science.gov (United States)

    Yang, Shusen; Xu, Wenjing; Dong, Zengxiang; Zhou, Mo; Lin, Chaolan; Jin, Hongbo; Su, Yafen; Li, Qingyu; Wang, Xu; Chang, Huiying; Han, Wei

    2015-08-01

    Atrial fibrillation (AF) is the most common cardiac arrhythmia. However, the current drug interference of antiarrhythmia has limited efficacy and off-target effects. Accumulating evidence has implicated a potential role of nitration stress in the pathogenesis of AF. The aim of the study was to determine whether TPEN provided antinitration effects on atrial myocytes during AF, especially under circumstances of nitration stress. We utilized a rapid paced HL-1 cells model for AF. The changes of electrophysiological characteristics and structure of paced HL-1 cells were determined by a patch clamp and a TEM method. The effects of TPEN on pacing and ONOO(-) pretreated HL-1 cells were examined using MTT assay, TUNEL technique, confocal microscope experiment, and Western blot analysis. The results revealed that ONOO(-) reduced the viability of HL-1 cells in a dose-dependent manner, and 1 μmol/L TPEN sign