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1
Orita, Hiroyuki, Manabu Fukasawa, Hideaki Uchino, Kana Fukui, Minoru Kohi, and Masahiko Washio. "Modulation of the viability of immature cardiac myocytes by cardiac fibroblasts after hypothermic preservation—its values as a technique for evaluation of storage solutions." Cardiology in the Young 5, no. 2 (April 1995): 110–17. http://dx.doi.org/10.1017/s1047951100011665.
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AbstractWe evaluated the modulation of the viability of immature cardiac myocytes by cardiac fibroblasts after hypothermic preservation using three types of storage solutions—saline, University of Wisconsin solution, and MCDB 107 medium. Cardiac myocytes and fibroblasts were isolated from neonatal rat ventricles, and cultures of myocytes only or co-cultures with fibroblasts (myocyte: fibroblast 2:1) were established. On the fourth day of culture, the cultures were incubated at 4 °C for 6, 12, 18 and 24 hours in the different storage solutions. Enzymes were measured in the storage solutions immediately before and after hypothermic incubation. The cultures were then incubated for an additional 24 hours at 37 °C to evaluate the recovery of the myocyte beating rate. The myocyte beating rate in the co-culture groups showed significantly higher recovery ratios than the corresponding groups in which only myocytes were cultured. Complete recovery was observed in the group co-cultured in MCDB medium 24 hours after hypothermic incubation (83.4% of control—beating rate prior to hypothermic incubation) compared to the other co-cultured groups (15.4, 0%, respectively). Release of enzymes in the co-cultures was significantly suppressed compared to the cultured myocytes, and the greatest suppression was found after 24 hours of incubation in MCDB medium (CPK: 36.6 mIU/flask, LDH: 281.2 mIU/flask) compared to the other two co-cultured groups (CPK: 181.1, 281.1; LDH: 501.7, 773.2). Cardiac fibroblasts diminished myocytic injury after hypothermic preservation using various storage solutions, in which MCDB 107 medium showed the best overall protective effect. Thus, cardiac fibroblasts may play an important role in controlling myocytic viability under various hypothermic conditions.
2
Krishnan, Anirudh, Emily Chilton, Jaishankar Raman, Pankaj Saxena, Craig McFarlane, Alexandra F. Trollope, Robert Kinobe, and Lisa Chilton. "Are Interactions between Epicardial Adipose Tissue, Cardiac Fibroblasts and Cardiac Myocytes Instrumental in Atrial Fibrosis and Atrial Fibrillation?" Cells 10, no. 9 (September 21, 2021): 2501. http://dx.doi.org/10.3390/cells10092501.
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Atrial fibrillation is very common among the elderly and/or obese. While myocardial fibrosis is associated with atrial fibrillation, the exact mechanisms within atrial myocytes and surrounding non-myocytes are not fully understood. This review considers the potential roles of myocardial fibroblasts and myofibroblasts in fibrosis and modulating myocyte electrophysiology through electrotonic interactions. Coupling with (myo)fibroblasts in vitro and in silico prolonged myocyte action potential duration and caused resting depolarization; an optogenetic study has verified in vivo that fibroblasts depolarized when coupled myocytes produced action potentials. This review also introduces another non-myocyte which may modulate both myocardial (myo)fibroblasts and myocytes: epicardial adipose tissue. Epicardial adipocytes are in intimate contact with myocytes and (myo)fibroblasts and may infiltrate the myocardium. Adipocytes secrete numerous adipokines which modulate (myo)fibroblast and myocyte physiology. These adipokines are protective in healthy hearts, preventing inflammation and fibrosis. However, adipokines secreted from adipocytes may switch to pro-inflammatory and pro-fibrotic, associated with reactive oxygen species generation. Pro-fibrotic adipokines stimulate myofibroblast differentiation, causing pronounced fibrosis in the epicardial adipose tissue and the myocardium. Adipose tissue also influences myocyte electrophysiology, via the adipokines and/or through electrotonic interactions. Deeper understanding of the interactions between myocytes and non-myocytes is important to understand and manage atrial fibrillation.
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Jonker, Sonnet S., Lubo Zhang, Samantha Louey, George D. Giraud, Kent L. Thornburg, and J. Job Faber. "Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart." Journal of Applied Physiology 102, no. 3 (March 2007): 1130–42. http://dx.doi.org/10.1152/japplphysiol.00937.2006.
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The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from ∼115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after ∼115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.
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Kabaeva, Zhyldyz, Mei Zhao, and Daniel E. Michele. "Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression." American Journal of Physiology-Heart and Circulatory Physiology 294, no. 4 (April 2008): H1667—H1674. http://dx.doi.org/10.1152/ajpheart.01144.2007.
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The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.
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Evans, Heather J., Janea K. Sweet, Robert L. Price, Michael Yost, and Richard L. Goodwin. "Novel 3D culture system for study of cardiac myocyte development." American Journal of Physiology-Heart and Circulatory Physiology 285, no. 2 (August 2003): H570—H578. http://dx.doi.org/10.1152/ajpheart.01027.2002.
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Insufficient myocardial repair after pathological processes contributes to cardiovascular disease, which is a major health concern. Understanding the molecular mechanisms that regulate the proliferation and differentiation of cardiac myocytes will aid in designing therapies for myocardial repair. Models are needed to delineate these molecular mechanisms. Here we report the development of a model system that recapitulates many aspects of cardiac myocyte differentiation that occur during early cardiac development. A key component of this model is a novel three-dimensional tubular scaf-fold engineered from aligned type I collagen strands. In this model embryonic ventricular myocytes undergo a transition from a hyperplastic to a quiescent phenotype, display significant myofibrillogenesis, and form critical cell-cell connections. In addition, embryonic cardiac myocytes grown on the tubular substrate have an aligned phenotype that closely resembles in vivo neonatal ventricular myocytes. We propose that embryonic cardiac myocytes grown on the tube substrate develop into neonatal cardiac myocytes via normal in vivo mechanisms. This model will aid in the elucidation of the molecular mechanisms that regulate cardiac myocyte proliferation and differentiation, which will provide important insights into myocardial development.
6
Li, F., M. R. McNelis, K. Lustig, and A. M. Gerdes. "Hyperplasia and hypertrophy of chicken cardiac myocytes during posthatching development." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 273, no. 2 (August 1, 1997): R518—R526. http://dx.doi.org/10.1152/ajpregu.1997.273.2.r518.
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For characterization of the growth pattern of cardiac myocytes during posthatching development, cardiac myocytes were enzymatically isolated from the ventricles of 1-, 15-, 29-, and 42-day-old chickens for measurement of myocyte nucleation, length, width, volume, and number, and for immunolabeling of cytoskeletal proteins. Ventricular myocyte number increased 156% from day 1 to day 42. Average cell volume increased more than 400%, and myocytes lengthened 125%, but cell width only increased 53% during this period. All myocytes were mononucleated at day 1. At day 15, 18% of myocytes became binucleated with < 1% of myocytes containing more than two nuclei. Interestingly, binucleated myocytes were able to divide with two nuclei going through mitosis at the same time. As demonstrated by staining with tubulin and alpha-actinin antibodies, two mitotic spindles and two cleavage furrows were formed in dividing binucleated myocytes. At day 42, binucleated myocytes increased to 44% with 11% of myocytes containing more than two nuclei. Sarcomeric alpha-actinin was partially disassembled in prometaphase and was reorganized into regular Z lines of sarcomeres in telophase. Desmin was disassembled in prophase and was reassembled during late telophase. These results suggest that chicken myocytes undergo hypertrophy and continue to proliferate during posthatching maturation, although it is currently believed that myocytes of all vertebrates withdraw from the cell cycle shortly after birth. We provide direct evidence for the first time of in vivo myocyte division in 6-wk-old chicken hearts.
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Pinsky, David J., Walif Aji, Matthias Szabolcs, Eleni S. Athan, Youping Liu, Yi Ming Yang, Richard P. Kline, Kim E. Olson, and Paul J. Cannon. "Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture." American Journal of Physiology-Heart and Circulatory Physiology 277, no. 3 (September 1, 1999): H1189—H1199. http://dx.doi.org/10.1152/ajpheart.1999.277.3.h1189.
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Excessive nitric oxide (NO) production within the heart is implicated in the pathogenesis of myocyte death, but the mechanism whereby NO kills cardiac myocytes is not known. To determine whether NO may trigger programmed cell death (apoptosis) of adult rat ventricular myocytes in culture, the NO donor S-nitroso- N-acetylpenicillamine (SNAP) was shown to kill purified cardiac myocytes in a dose-dependent fashion. In situ analysis of ventricular myocytes plated on chamber slides using nick-end labeling of DNA demonstrated that SNAP induces cardiac myocyte apoptosis, which was confirmed by the identification of oligonucleosomal DNA fragmentation on agarose gel electrophoresis. Similarly, treatment of cardiac myocytes with cytokines that induce inducible NO synthase was shown to cause an NO-dependent induction of apoptosis. Addition of reduced hemoglobin to scavenge NO liberated by SNAP extinguished both the increase in percentage of apoptotic cells and the appearance of DNA ladders. Treatment with SNAP (but not with N-acetylpenicillamine or SNAP + hemoglobin) not only induced apoptosis but resulted in a marked increase in p53 expression in cardiac myocytes detected by Western blotting and immunohistochemistry. These data indicate that NO has the capacity to kill cardiac myocytes by triggering apoptosis and suggest the involvement of p53 in this process.
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Chen, Hua, Xueyin N. Huang, Alexandre F. R. Stewart, and Jorge L. Sepulveda. "Gene expression changes associated with fibronectin-induced cardiac myocyte hypertrophy." Physiological Genomics 18, no. 3 (August 11, 2004): 273–83. http://dx.doi.org/10.1152/physiolgenomics.00104.2004.
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Fibronectin (FN) is an extracellular matrix protein that binds to integrin receptors and couples cardiac myocytes to the basal lamina. Cardiac FN expression is elevated in models of pressure overload, and FN causes cultured cardiac myocytes to hypertrophy by a mechanism that has not been characterized in detail. In this study, we analyzed the gene expression changes induced by FN in purified rat neonatal ventricular myocytes using the Affymetrix RAE230A microarray, to understand how FN affects gene expression in cardiac myocytes and to separate the effects contributed by cardiac nonmyocytes in vivo. Pathway analysis using z-score statistics and comparison with a mouse model of cardiac hypertrophy revealed several pathways stimulated by FN in cardiac myocytes. In addition to the known cardiac myocyte hypertrophy markers, FN significantly induced metabolic pathways including virtually all of the enzymes of cholesterol biosynthesis, fatty acid biosynthesis, and the mitochondrial electron transport chain. FN also increased the expression of genes coding for ribosomal proteins, translation factors, and the ubiquitin-proteasome pathway. Interestingly, cardiac myocytes plated on FN showed elevated expression of the fibrosis-promoting peptides connective tissue growth factor (CTGF), WNT1 inducible signaling pathway protein 2 (WISP2), and secreted acidic cysteine-rich glycoprotein (SPARC). Our data complement in vivo studies and reveal several novel genes and pathways stimulated by FN, pointing to cardiac myocyte-specific mechanisms that lead to development of the hypertrophic phenotype.
9
Brady, A. J., J. B. Warren, P. A. Poole-Wilson, T. J. Williams, and S. E. Harding. "Nitric oxide attenuates cardiac myocyte contraction." American Journal of Physiology-Heart and Circulatory Physiology 265, no. 1 (July 1, 1993): H176—H182. http://dx.doi.org/10.1152/ajpheart.1993.265.1.h176.
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Cardiac muscle fibers have microvessels in close proximity, the distance from the nearest capillary being no greater than 8 microns. We performed experiments on isolated, electrically stimulated, contracting guinea pig cardiac myocytes to test whether NO from endothelium or nitrovasodilators or directly superfused in solution might affect myocyte contractility. In endothelium-myocyte coculture experiments, 10(-7) M bradykinin reduced myocyte shortening by 11 +/- 3.5%. This effect was abolished in the presence of NG-nitro-L-arginine methyl ester and was unaffected by indomethacin. Sodium nitroprusside, but not organic nitrovasodilators, reduced myocyte contraction amplitude by 23% at 3 x 10(-5) M. This effect was reversed by methylene blue. Superfusion with NO solution had an effect similar to sodium nitroprusside, as did exposure to 8-bromoguanosine 3',5'-cyclic monophosphate. Thus the present study shows that cardiac myocyte contraction is attenuated by NO, which appears to act via production of guanosine 3',5'-cyclic monophosphate within the myocytes. Because cardiac myocytes in vivo are in such close proximity to endothelium, the effects of endothelial products on cardiac myocyte contractility may be important in myocardial function.
10
Jiang, Jianming, Patrick G. Burgon, Hiroko Wakimoto, Kenji Onoue, Joshua M. Gorham, Caitlin C. O’Meara, Gregory Fomovsky, et al. "Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis." Proceedings of the National Academy of Sciences 112, no. 29 (July 7, 2015): 9046–51. http://dx.doi.org/10.1073/pnas.1511004112.
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Homozygous cardiac myosin binding protein C-deficient (Mybpct/t) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpct/t myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpct/t myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpct/t mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3+/− individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3−/− mice is primarily myocyte hyperplasia.
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Moore, R. L., T. I. Musch, R. V. Yelamarty, R. C. Scaduto, A. M. Semanchick, M. Elensky, and J. Y. Cheung. "Chronic exercise alters contractility and morphology of isolated rat cardiac myocytes." American Journal of Physiology-Cell Physiology 264, no. 5 (May 1, 1993): C1180—C1189. http://dx.doi.org/10.1152/ajpcell.1993.264.5.c1180.
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Chronic exercise training elicits positive adaptations in cardiac contractile function and ventricular dimension. The potential contribution of single myocyte morphological and functional adaptations to these global responses to training was determined in this study. Left ventricular cardiac myocytes were isolated from the hearts of sedentary control (Sed) or exercise-trained (TR) rats. Training elicited an approximately 5% increase in resting myocyte length (Sed, 121.0 +/- 2.0 vs. TR, 126.7 +/- 2.0 microns; P < 0.05), whereas resting sarcomere length and midpoint cell width were unaffected. These data suggest that longitudinal myocyte growth contributes to the training-induced increase in end-diastolic dimension. Single myocytes (28 degrees C) were stimulated at 0.067 and 0.2 Hz and shortening dynamics assessed at extracellular Ca2+ concentrations ([Ca2+]o) of 0.6, 1.1, and 2.0 mM. In both groups, maximal extent of myocyte shortening (ESmax) increased as [Ca2+]o increased and decreased as contraction frequency increased. TR myocytes were more strongly influenced by the effects of [Ca2+]o and frequency. At 0.067 Hz and 2.0 mM, ESmax was greater in TR than in Sed myocytes. The magnitude of this difference decreased as [Ca2+]o was reduced. At 0.2 Hz, ESmax was similar in Sed and TR myocytes at 2.0 mM [Ca2+]o. As [Ca2+]o was reduced, ESmax decreased more rapidly in TR than in Sed myocytes; at 0.6 mM, ESmax was greater in Sed than in TR myocytes. Our data indicate that chronic exercise influences cardiac contractile function at the single myocyte level. This study also provides evidence in support of the hypothesis that chronic exercise influences myocyte Ca2+ influx and efflux pathways.(ABSTRACT TRUNCATED AT 250 WORDS)
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Hershman, Kenneth M., and Edwin S. Levitan. "RPTPμ and protein tyrosine phosphorylation regulate K+channel mRNA expression in adult cardiac myocytes." American Journal of Physiology-Cell Physiology 278, no. 2 (February 1, 2000): C397—C403. http://dx.doi.org/10.1152/ajpcell.2000.278.2.c397.
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Previously, we reported that cell-cell contact regulates K+channel mRNA expression in cultured adult rat cardiac myocytes. Here we show that exposing cardiac myocytes to tyrosine kinase inhibitors (genistein, tyrphostin A25), but not inactive analogs, prevents downregulation of Kv1.5 mRNA and upregulation of Kv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells that endogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells) express the receptor-type protein tyrosine phosphatase μ (RPTPμ) display Kv1.5 mRNA levels paralleling that which was observed in myocytes cultured under high-density conditions and in intact tissue. In contrast, myocytes cocultured with control cells failed to produce this response. Finally, it is shown that Kv4.2 mRNA expression is unaffected by RPTPμ. These findings reveal that multiple tyrosine phosphorylation-dependent mechanisms control cardiac myocyte K+channel genes. Furthermore, we conclude that RPTPμ specifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus this receptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart.
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Ahuja, Preeti, Patima Sdek, and W. Robb MacLellan. "Cardiac Myocyte Cell Cycle Control in Development, Disease, and Regeneration." Physiological Reviews 87, no. 2 (April 2007): 521–44. http://dx.doi.org/10.1152/physrev.00032.2006.
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Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle soon after birth in mammals. Although the extent to which adult cardiac myocytes are capable of cell cycle reentry is controversial and species-specific differences may exist, it appears that for the vast majority of adult cardiac myocytes the predominant form of growth postnatally is an increase in cell size (hypertrophy) not number. Unfortunately, this limits the ability of the heart to restore function after any significant injury. Interest in novel regenerative therapies has led to the accumulation of much information on the mechanisms that regulate the rapid proliferation of cardiac myocytes in utero, their cell cycle exit in the perinatal period, and the permanent arrest (terminal differentiation) in adult myocytes. The recent identification of cardiac progenitor cells capable of giving rise to cardiac myocyte-like cells has challenged the dogma that the heart is a terminally differentiated organ and opened new prospects for cardiac regeneration. In this review, we summarize the current understanding of cardiomyocyte cell cycle control in normal development and disease. In addition, we also discuss the potential usefulness of cardiomyocyte self-renewal as well as feasibility of therapeutic manipulation of the cardiac myocyte cell cycle for cardiac regeneration.
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Burczynski, F. J., Z. S. Cai, J. B. Moran, T. Geisbuhler, and M. Rovetto. "Palmitate uptake by cardiac myocytes and endothelial cells." American Journal of Physiology-Heart and Circulatory Physiology 268, no. 4 (April 1, 1995): H1659—H1666. http://dx.doi.org/10.1152/ajpheart.1995.268.4.h1659.
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The mechanisms regulating the cellular uptake of long-chain fatty acids are poorly understood. Although there is evidence that hepatocytes facilitate the uptake of ligands from the protein-bound fraction, it is not known whether cardiac myocytes also facilitate the uptake process. The present studies were designed to address the role of albumin in the uptake of long-chain fatty acids by cardiac myocytes isolated from adult male rats. At low albumin concentrations (1 microM), the myocyte palmitate clearance rate did not exceed that predicted by the diffusion-reaction model. At high albumin concentrations (300 and 600 microM), the clearance ratio test was used to determine whether myocytes facilitate the uptake of palmitate. As with the low albumin concentrations, the diffusion-reaction model accounted for the overall clearance rates. Because endothelial cells might be involved in enhancing fatty acid transport into myocytes, we also determined the effects of endothelial cells on palmitate uptake by cardiac myocytes. Based on cell number, the palmitate clearance rate by endothelial cells in the presence of albumin was only 7% of the cardiac myocyte clearance rate. Combining the endothelial cells with the myocytes did not result in any synergistic effect on the palmitate clearance rate.
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Peng, Chang-Fu, Yi Wei, Jeffrey M. Levsky, Thomas V. McDonald, Geoffrey Childs, and Richard N. Kitsis. "Microarray analysis of global changes in gene expression during cardiac myocyte differentiation." Physiological Genomics 9, no. 3 (June 3, 2002): 145–55. http://dx.doi.org/10.1152/physiolgenomics.00027.2002.
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Significant progress has been made in defining pathways that mediate the formation of the mammalian heart. Little is known, however, about the genetic program that directs the differentiation of cardiac myocytes from their precursor cells. A major hindrance to this kind of investigation has been the absence of an appropriate cell culture model of cardiac myocyte differentiation. Recently, a subline of P19 cells (P19CL6) was derived that, following dimethyl sulfoxide (DMSO) treatment, differentiate efficiently over 10 days into spontaneously beating cardiac myocytes. We demonstrate that these cells are indeed cardiac myocytes as they express cell type-specific markers and exhibit electrophysiological properties indicative of cardiac myocytes. The requirement for DMSO stimulation in this paradigm was shown to be limited to the first 4 days, suggesting that critical events in the differentiation process occur over this interval. To uncover relationships among known genes and identify novel genes that mediate cardiac myocyte differentiation, a detailed time course of changes in global gene expression was carried out using cDNA microarrays. In addition to the activation of genes encoding cardiac transcription factors and structural proteins, increases were noted in the expression of multiple known genes and expressed sequence tags (ESTs). Analysis of the former suggested the involvement of a variety of signaling pathways in cardiac myocyte differentiation. The 16 ESTs whose expression was increased during the early, stimulus-dependent phase of cardiac myocyte differentiation may be novel regulators of this process. Thus this first report of large-scale changes in gene expression during cardiac myocyte differentiation has delineated relationships among the expression patterns of known genes and identified a number of novel genes that merit further study.
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Zhang, Xue-Qian, Anwer Qureshi, Jianliang Song, Lois L. Carl, Qiang Tian, Richard C. Stahl, David J. Carey, Lawrence I. Rothblum, and Joseph Y. Cheung. "Phospholemman modulates Na+/Ca2+exchange in adult rat cardiac myocytes." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 1 (January 1, 2003): H225—H233. http://dx.doi.org/10.1152/ajpheart.00698.2002.
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Previous studies have shown that overexpression of phospholemman (PLM) affected contractile function and Ca2+ homeostasis in adult rat myocytes. We tested the hypothesis that PLM modulated Na+/Ca2+exchanger (NCX1) activity. PLM was overexpressed in adult rat myocytes by adenovirus-mediated gene transfer. After 72 h, the half-time of relaxation from caffeine-induced contracture, an estimate of forward NCX1 activity, was prolonged 1.8-fold ( P < 0.003) in myocytes overexpressing PLM compared with control myocytes overexpressing green fluorescent protein alone. Reverse NCX1 current (3 Na+ out:1 Ca2+ in) was significantly ( P < 0.0001) lower in PLM myocytes, especially at more positive voltages. Immunofluorescence demonstrated colocalization of PLM and NCX1 to the plasma membrane and t-tubules. Resting membrane potential, action potential amplitude and duration, myocyte size, and NCX1 and calsequestrin protein levels were not affected by PLM overexpression. At 5 mM extracellular [Ca2+] ([Ca2+]o), the depressed contraction amplitudes in PLM myocytes were increased towards normal by cooverexpression with NCX1. At 0.6 mM [Ca2+]o, the supranormal contraction amplitudes in PLM myocytes were reduced by cooverexpression with NCX1. We conclude that PLM modulated myocyte contractility partly by inhibiting Na+/Ca2+ exchange.
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McArthur, Lisa, Lisa Chilton, Godfrey L. Smith, and Stuart A. Nicklin. "Electrical consequences of cardiac myocyte: fibroblast coupling." Biochemical Society Transactions 43, no. 3 (June 1, 2015): 513–18. http://dx.doi.org/10.1042/bst20150035.
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Gap junctions are channels which allow electrical signals to propagate through the heart from the sinoatrial node and through the atria, conduction system and onwards to the ventricles, and hence are essential for co-ordinated cardiac contraction. Twelve connexin (Cx) proteins make up one gap junction channel, of which there are three main subtypes in the heart; Cx40, Cx43 and Cx45. In the cardiac myocyte, gap junctions are present mainly at the intercalated discs between neighbouring myocytes, and assist in rapid electrical conduction throughout the ventricular myocardium. Fibroblasts provide the structural skeleton of the myocardium and fibroblast numbers significantly increase in heart disease. Fibroblasts also express connexins and this may facilitate heterocellular electrical coupling between myocytes and fibroblasts in the setting of cardiac disease. Interestingly, cardiac fibroblasts have been demonstrated to increase Cx43 expression in experimental models of myocardial infarction and functional gap junctions between myocytes and fibroblasts have been reported. Therefore, in the setting of heart disease enhanced cardiac myocyte: fibroblast coupling may influence the electrical activity of the myocyte and contribute to arrhythmias.
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Knight, Walter E., Si Chen, Yishuai Zhang, Masayoshi Oikawa, Meiping Wu, Qian Zhou, Clint L. Miller, et al. "PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction." Proceedings of the National Academy of Sciences 113, no. 45 (October 20, 2016): E7116—E7125. http://dx.doi.org/10.1073/pnas.1607728113.
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Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-β–stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance.
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Kim, Song-Jung, Kenji Iizuka, Ralph A. Kelly, Yong-Jian Geng, Sanford P. Bishop, Guiping Yang, Amelia Kudej, et al. "An α-cardiac myosin heavy chain gene mutation impairs contraction and relaxation function of cardiac myocytes." American Journal of Physiology-Heart and Circulatory Physiology 276, no. 5 (May 1, 1999): H1780—H1787. http://dx.doi.org/10.1152/ajpheart.1999.276.5.h1780.
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Left Ventricular (LV) myocytes were isolated from 15-wk-old male mice bearing the Arg403 → Gln α-cardiac myosin heavy chain missense mutation (α-MHC403/+), a model of familial hypertrophic cardiomyopathy. LV myocytes were classified morphologically: type I, rod shaped with parallel myofibrils; type II, irregularly shaped, shorter and wider than wild-type (WT) control cells, with parallel myofibrils; and type III, irregularly shaped with disoriented myofibrils. Compared with WT myocytes, α-MHC403/+ myocytes had fewer type I cells (WT = 74 ± 3%, α-MHC403/+ = 41 ± 4%, P < 0.01) and more type III cells (WT= 12 ± 3%, α-MHC403/+ = 49 ± 7%, P < 0.01). In situ histology also demonstrated marked myofibrillar disarray in the α-MHC403/+ hearts. With the use of video edge detection, myocytes were paced at 1 Hz (37°C) to determine the effects of the mutation on myocyte function. End-diastolic length was reduced in mutant myocytes, but fractional shortening (% contraction) and sarcomere length were not. Velocity of contraction (−d L/d t max) was depressed in mutant cells, but more in type II and III cells (−31%) than in type I cells (−18%). Velocity of relaxation (+d L/d t) was also depressed more in type II and III cells (−38%) than in type I cells (−16%). Using fura 2 dye with intracellular Ca2+ transients, we demonstrated that in α-MHC403/+ myocytes, the amplitude of the Ca2+ signal during contraction was unchanged but that the time required for decay of the signal to decrease 70% from its maximum was delayed significantly (WT = 159 ± 8 ms; α-MHC403/+ = 217 ± 14 ms, P < 0.01). Sarco(endo)plasmic reticulum Ca2+-ATPase mRNA levels in α-MHC403/+ and WT mice were similar. These data indicate that the altered cardiac dysfunction of α-MHC403/+ myocytes is directly due to defective myocyte function rather than to secondary changes in global cardiac function and/or loading conditions.
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Brady, A. J., P. A. Poole-Wilson, S. E. Harding, and J. B. Warren. "Nitric oxide production within cardiac myocytes reduces their contractility in endotoxemia." American Journal of Physiology-Heart and Circulatory Physiology 263, no. 6 (December 1, 1992): H1963—H1966. http://dx.doi.org/10.1152/ajpheart.1992.263.6.h1963.
Full textAbstract:
We investigated whether increased nitric oxide (NO) synthase activity within cardiac myocytes contributes to the depressed cardiac contractility observed in endotoxic shock. Isolated ventricular myocytes were studied to examine the effects of substrates and inhibitors of NO synthase on myocyte contractility. When stimulated electrically, the resting length of myocytes from control animals shortened by 5.3 +/- 0.3% (means +/- SE, n = 32). Baseline contraction of myocytes from endotoxin-treated animals was reduced to 3.0 +/- 0.3% (n = 17, P < 0.001). The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M) had no effect on myocytes from control animals, but it increased the contraction of myocytes from endotoxin-treated animals by 40% (fractional shortening increased to 4.3 +/- 0.4%, P < 0.01). Similar results were obtained with NG-methyl-L-arginine. The effect of L-NAME could be reversed by excess L-arginine, but not D-arginine. The effect of endotoxin was abolished by dexamethasone pretreatment. Methylene blue also reversed the effects of endotoxin but had toxic effects on myocytes. Agents that either prevent synthesis or the effects of NO reverse the depression of myocyte contraction seen following endotoxin treatment.
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Balse, Elise, David F. Steele, Hugues Abriel, Alain Coulombe, David Fedida, and Stéphane N. Hatem. "Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes." Physiological Reviews 92, no. 3 (July 2012): 1317–58. http://dx.doi.org/10.1152/physrev.00041.2011.
Full textAbstract:
Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.
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Metzger, J. M., W. I. Lin, and L. C. Samuelson. "Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells." Journal of Cell Biology 126, no. 3 (August 1, 1994): 701–11. http://dx.doi.org/10.1083/jcb.126.3.701.
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Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell-derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.
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Stewart, Michael J., Kathleen Smoak, Mary Ann Blum, and Barbara Sherry. "Basal and Reovirus-Induced Beta Interferon (IFN-β) and IFN-β-Stimulated Gene Expression Are Cell Type Specific in the Cardiac Protective Response." Journal of Virology 79, no. 5 (March 1, 2005): 2979–87. http://dx.doi.org/10.1128/jvi.79.5.2979-2987.2005.
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ABSTRACT Viral myocarditis is an important human disease, with a wide variety of viruses implicated. Cardiac myocytes are not replenished yet are critical for host survival and thus may have a unique response to infection. Previously, we determined that the extent of reovirus induction of beta interferon (IFN-β) and IFN-β-mediated protection in primary cardiac myocyte cultures was inversely correlated with the extent of reovirus-induced cardiac damage in a mouse model. Surprisingly, and in contrast, the IFN-β response did not determine reovirus replication in skeletal muscle cells. Here we compared the IFN-β response in cardiac myocytes to that in primary cardiac fibroblast cultures, a readily replenished cardiac cell type. We compared basal and reovirus-induced expression of IFN-β, IRF-7 (an interferon-stimulated gene [ISG] that further induces IFN-β), and another ISG (561) in the two cell types by using real-time reverse transcription-PCR. Basal IFN-β, IRF-7, and 561 expression was higher in cardiac myocytes than in cardiac fibroblasts. Reovirus T3D induced greater expression of IFN-β in cardiac myocytes than in cardiac fibroblasts but equivalent expression of IRF-7 and 561 in the two cell types (though fold induction for IRF-7 and 561 was higher in fibroblasts than in myocytes because of the differences in basal expression). Interestingly, while reovirus replicated to equivalent titers in cardiac myocytes and cardiac fibroblasts, removal of IFN-β resulted in 10-fold-greater reovirus replication in the fibroblasts than in the myocytes. Together the data suggest that the IFN-β response controls reovirus replication equivalently in the two cell types. In the absence of reovirus-induced IFN-β, however, reovirus replicates to higher titers in cardiac fibroblasts than in cardiac myocytes, suggesting that the higher basal IFN-β and ISG expression in myocytes may play an important protective role.
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Wang, Z., R. Mukherjee, C. F. Lam, and F. G. Spinale. "Spatial characterization of contracting cardiac myocytes by computer-assisted, video-based image processing." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 2 (February 1, 1996): H769—H779. http://dx.doi.org/10.1152/ajpheart.1996.270.2.h769.
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The goals of the present study were to develop and validate a computer-assisted, video-based image processing (CAVIP) system to measure time-dependent changes in isolated myocyte geometry during contraction and to use the CAVIP system to examine spatial characteristics of the myocyte during contraction in normal myocytes and in myocytes after development of dilated cardiomyopathy (DCM). Myocytes were isolated from the left ventricles of five control pigs and five pigs that developed chronic tachycardia (240 beats/min; 3 wk)-induced DCM. Isolated myocytes were stimulated and recorded using a high-speed camera interfaced with a standard video recording system. There was a significant linear relation between the indexes of time-dependent changes in myocyte length as measured by a conventional video edge-detector system and by the CAVIP system (r > 0.96; P < 0.01). After this validation procedure, dynamic changes in myocyte width and profile area with DCM were examined. Myocyte resting profile area was 33% larger in DCM myocytes compared with controls. However, there was no difference in the rate of area change with contraction between the two groups. Percent changes in myocyte width and profile area at peak contraction were significantly lower in the DCM group (43 and 46% respectively, P < 0.05). Therefore, the present study demonstrated that the CAVIP system provides unique information on time-dependent changes in myocyte geometry during contraction, particularly with the development of cardiomyopathic disease.
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Abi-Char, Joëlle, Saïd El-Haou, Elise Balse, Nathalie Neyroud, Roger Vranckx, Alain Coulombe, and Stéphane N. Hatem. "The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes." American Journal of Physiology-Heart and Circulatory Physiology 294, no. 4 (April 2008): H1851—H1861. http://dx.doi.org/10.1152/ajpheart.01045.2007.
Full textAbstract:
Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K+ current, IKur (5.64 ± 0.57 pA/pF in SAP97 myocytes vs. 3.23 ± 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K+ current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.
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Lefroy, D. C., T. Crake, F. Del Monte, G. Vescovo, L. Dalla Libera, S. Harding, and P. A. Poole-Wilson. "Angiotensin II and contraction of isolated myocytes from human, guinea pig, and infarcted rat hearts." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 6 (June 1, 1996): H2060—H2069. http://dx.doi.org/10.1152/ajpheart.1996.270.6.h2060.
Full textAbstract:
The effects of angiotensin II on myocardial contractility were assessed in isolated cardiac myocyte preparations, using video microscopy with a computerized edge-detection system. Angiotensin II (1 nM-10 microM) did not affect the contraction of rat (n = 10), guinea pig (n = 11), or human ventricular myocytes (n = 8) or of human atrial myocytes (n = 12). Isoproterenol or raised extracellular calcium increased the contraction amplitude of the cardiac myocytes to a maximum of between 150 and 560% above basal, and there were corresponding increases in the velocities of contraction and relaxation. In rat and guinea pig ventricular myocytes 1 microM angiotensin II did not affect the inotropic response to isoproterenol. Seven days after left coronary artery ligation in seven rats, the basal contraction amplitude was reduced in myocytes from the infarcted region (4.0 +/- 1.9%) compared with the noninfarcted region (5.0 +/- 2.8%, P = 0.03) and with myocytes from six sham-operated hearts (5.0 +/- 2.8%, P = 0.03). There was a switch in myosin isoform expression from the V1 to the V3 isoform in myocytes from both the infarcted and noninfarcted regions. Angiotensin II (1 nM-10 microM) had no significant effect on the contraction characteristics of myocytes from the infarcted rat hearts. In conclusion, angiotensin II had no significant inotropic effect on isolated cardiac myocyte preparations from guinea pig ventricle, normal and infarcted rat ventricle, human ventricle, and human atrium.
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Hershman, Kenneth M., and Edwin S. Levitan. "Cell-cell contact between adult rat cardiac myocytes regulates Kv1.5 and Kv4.2 K+channel mRNA expression." American Journal of Physiology-Cell Physiology 275, no. 6 (December 1, 1998): C1473—C1480. http://dx.doi.org/10.1152/ajpcell.1998.275.6.c1473.
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Regulation of voltage-gated K+channel genes represents an important mechanism for modulating cardiac excitability. Here we demonstrate that expression of two K+channel mRNAs is reciprocally controlled by cell-cell interactions between adult cardiac myocytes. It is shown that culturing acutely dissociated rat ventricular myocytes for 3 h results in a dramatic downregulation of Kv1.5 mRNA and a modest upregulation of Kv4.2 mRNA. These effects are specific, because similar changes are not detected with other channel mRNAs. Increasing myocyte density promotes maintenance of Kv1.5 gene expression, whereas Kv4.2 mRNA expression was found to be inversely proportional to cell density. Conditioned culture medium did not mimic the effects of high cell density. However, paraformaldehyde-fixed myocytes were comparable to live cells in their ability to influence K+channel message levels. Thus the reciprocal effects of cell density on the expression of Kv1.5 and Kv4.2 genes are mediated by direct contact between adult cardiac myocytes. These findings reveal for the first time that cardiac myocyte gene expression is influenced by signaling induced by cell-cell contact.
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Buerke, M., A. S. Weyrich, and A. M. Lefer. "Isolated cardiac myocytes are sensitized by hypoxia-reoxygenation to neutrophil-released mediators." American Journal of Physiology-Heart and Circulatory Physiology 266, no. 1 (January 1, 1994): H128—H136. http://dx.doi.org/10.1152/ajpheart.1994.266.1.h128.
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We exposed isolated rat cardiac myocytes to 20 min of hypoxia followed by 20 min of reoxygenation and observed the effect of supernatants of stimulated neutrophils [polymorphonuclear leukocytes (PMNs)] given at the beginning of reoxygenation. PMN supernatants induced cardiac myocyte injury, which was characterized by a significant (P < 0.01) reduction in cell viability to 53 +/- 3%, vs. 84 +/- 3% in rat myocytes subjected to hypoxia-reoxygenation (H/R) alone. The PMN supernatants also resulted in elevated creatine kinase (CK) activities in the myocyte medium. To examine specific PMN-released mediators that may contribute to this cell death, we studied the effects of hydrogen peroxide (H2O2), elastase, and platelet-activating factor on H/R cardiac myocytes. Incubation of myocytes after hypoxia with 10, 50, and 100 microM H2O2 decreased viability in a concentration-dependent manner (from 83 +/- 2 to 37 +/- 2%; P < 0.01). CK release of H/R myocytes was also significantly increased by 100 microM H2O2 (to 28 +/- 5 from 12 +/- 1% for H/R alone; P < 0.01). Similarly, elastase (5 micrograms/ml) given after hypoxia significantly reduced cardiac myocyte viability during reoxygenation (viability 58 +/- 1 vs. 85 +/- 1% H/R alone; P < 0.05) and increased CK release (to 29 +/- 3 from 11 +/- 1% for H/R alone; P < 0.01), an effect that was abolished by L-680,833, an elastase inhibitor. Unlike H2O2 and elastase, platelet-activating factor had no significant effect on myocyte viability or CK release after H/R.(ABSTRACT TRUNCATED AT 250 WORDS)
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Poon, Betty Y., Christopher A. Ward, Conan B. Cooper, Wayne R. Giles, Alan R. Burns, and Paul Kubes. "α4-Integrin Mediates Neutrophil-Induced Free Radical Injury to Cardiac Myocytes." Journal of Cell Biology 152, no. 5 (February 26, 2001): 857–66. http://dx.doi.org/10.1083/jcb.152.5.857.
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Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via β2-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the α4-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) β2-integrins (CD18) still signal oxidant production, or if this process is now coupled to the α4-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte–PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30–50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both α4-integrin antibody (Ab)-treated PMNs and NADPH oxidase–deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti–α4-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the α4-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed α4-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the α4-integrin–coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD.
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Shen, Jian-Bing, Robin Shutt, Mariela Agosto, Achilles Pappano, and Bruce T. Liang. "Reversal of cardiac myocyte dysfunction as a unique mechanism of rescue by P2X4 receptors in cardiomyopathy." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 4 (April 2009): H1089—H1095. http://dx.doi.org/10.1152/ajpheart.01316.2008.
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Binary cardiac transgenic (Tg) overexpression of P2X4 receptors (P2X4R) improved the survival of the cardiomyopathic calsequestrin (CSQ) mice. Here we studied the mechanism of rescue using binary P2X4R/CSQ Tg and CSQ Tg mice as models. Cellular and intact heart properties were determined by simultaneous sarcomere shortening (SS) and Ca2+ transients in vitro and echocardiography in vivo. Similar to a delay in death, binary mice exhibited a slowed heart failure progression with a greater left ventricular (LV) fractional shortening (FS) and thickness and a concomitant lesser degree of LV dilatation in both systole and diastole at 8 or 12 wk. By 16 wk, binary hearts showed similarly depressed FS and thinned out LV and equal enlargement of LV as did 12-wk-old CSQ hearts. Binary cardiac myocytes showed higher peak basal cell shortening (CS) and SS as well as greater basal rates of shortening and relaxation than did the CSQ myocytes at either 8 or 12 wk. Similar data were obtained in comparing the Ca2+ transient. At 16 wk, binary myocytes were like the 12-wk-old CSQ myocytes with equally depressed CS, SS, and Ca2+ transient. CSQ myocytes were longer than myocytes from wild-type and binary mice at 12 wk of age. At 16 wk, the binary myocyte length increased to that of the 12-wk-old CSQ myocyte, parallel to LV dilatation. The data suggest a unique mechanism, which involves a reversal of cardiac myocyte dysfunction and a delay in heart failure progression. It represents an example of targeting the abnormal failing myocyte in treating heart failure.
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Chen, Yue-Feng, Suleman Said, Scott E. Campbell, and A. Martin Gerdes. "A method to collect isolated myocytes and whole tissue from the same heart." American Journal of Physiology-Heart and Circulatory Physiology 293, no. 3 (September 2007): H2004—H2006. http://dx.doi.org/10.1152/ajpheart.00479.2007.
Full textAbstract:
A technique for isolation of cardiac myocytes and collection of whole heart tissue from individual hearts of adult rats is described in this study. After excision of the apical half of the left ventricle (LV) and cauterization of the cut edge, aortas were cannulated and high-quality isolated cardiac myocytes were collected after collagenase perfusion of the basal portion. Myocyte dimensions from the basal portion of cauterized and noncauterized hearts from matching rats were identical. Additionally, myocyte dimensions from the basal and apical halves of the LV were compared with the use of whole heart-isolated myocyte preps. No regional differences between basal and apical LV myocyte size were found. Therefore, this cauterization method can be used to collect isolated myocytes from the basal half and whole heart tissue from the apical half, with each half being representative of the other with respect to myocyte dimensions.
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Long, C. S., C. J. Henrich, and P. C. Simpson. "A growth factor for cardiac myocytes is produced by cardiac nonmyocytes." Cell Regulation 2, no. 12 (December 1991): 1081–95. http://dx.doi.org/10.1091/mbc.2.12.1081.
Full textAbstract:
Cardiac nonmyocytes, primarily fibroblasts, surround cardiac myocytes in vivo. We examined whether nonmyocytes could modulate myocyte growth by production of one or more growth factors. Cardiac myocyte hypertrophic growth was stimulated in cultures with increasing numbers of cardiac nonmyocytes. This effect of nonmyocytes on myocyte size was reproduced by serum-free medium conditioned by the cardiac nonmyocytes. The majority of the nonmyocyte-derived myocyte growth-promoting activity bound to heparin-Sepharose and was eluted with 0.75 M NaCl. Several known polypeptide growth factors found recently in cardiac tissue, namely acidic fibroblast growth factor (aFGF), basic FGF (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta 1 (TGF beta 1), also caused hypertrophy of cardiac myocytes in a dose-dependent manner. However, the nonmyocyte-derived growth factor (tentatively named NMDGF) could be distinguished from these other growth factors by different heparin-Sepharose binding profiles (TNF alpha, aFGF, bFGF, and TGF beta 1) by neutralizing growth factor-specific antisera (PDGF, TNF alpha, aFGF, bFGF, and TGF beta 1), by the failure of NMDGF to stimulate phosphatidylinositol hydrolysis (PDGF and TGF beta 1), and, finally, by the apparent molecular weight of NMDGF (45-50 kDa). This nonmyocyte-derived heparin-binding growth factor may represent a novel paracrine growth mechanism in myocardium.
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Heller, Lois Jane, David E. Mohrman, and Joseph R. Prohaska. "Decreased passive stiffness of cardiac myocytes and cardiac tissue from copper-deficient rat hearts." American Journal of Physiology-Heart and Circulatory Physiology 278, no. 6 (June 1, 2000): H1840—H1847. http://dx.doi.org/10.1152/ajpheart.2000.278.6.h1840.
Full textAbstract:
Passive stiffness characteristics of isolated cardiac myocytes, papillary muscles, and aortic strips from male Holtzman rats fed a copper-deficient diet for ∼5 wk were compared with those of rats fed a copper-adequate diet to determine whether alterations in these characteristics might accompany the well-documented cardiac hypertrophy and high incidence of ventricular rupture characteristic of copper deficiency. Stiffness of isolated cardiac myocytes was assessed from measurements of cellular dimensional changes to varied osmotic conditions. Stiffness of papillary muscles and aortic strips was determined from resting length-tension analyses and included steady-state characteristics, dynamic viscoelastic stiffness properties, and maximum tensile strength. The primary findings were that copper deficiency resulted in cardiac hypertrophy with increased cardiac myocyte size and fragility, decreased cardiac myocyte stiffness, and decreased papillary muscle passive stiffness, dynamic stiffness, and tensile strength and no alteration in aortic connective tissue passive stiffness or tensile strength. These findings suggest that a reduction of cardiac myocyte stiffness and increased cellular fragility could contribute to the reduced overall cardiac tissue stiffness and the high incidence of ventricular aneurysm observed in copper-deficient rats.
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L'Ecuyer, Thomas, Sanjeev Sanjeev, Ronald Thomas, Raymond Novak, Lauri Das, Wendy Campbell, and Richard Vander Heide. "DNA damage is an early event in doxorubicin-induced cardiac myocyte death." American Journal of Physiology-Heart and Circulatory Physiology 291, no. 3 (September 2006): H1273—H1280. http://dx.doi.org/10.1152/ajpheart.00738.2005.
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Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.
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Zhang, Hongyu, Xiongwen Chen, Erhe Gao, Scott M. MacDonnell, Wei Wang, Mikhail Kolpakov, Hiroyuki Nakayama, et al. "Increasing Cardiac Contractility After Myocardial Infarction Exacerbates Cardiac Injury and Pump Dysfunction." Circulation Research 107, no. 6 (September 17, 2010): 800–809. http://dx.doi.org/10.1161/circresaha.110.219220.
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Rationale: Myocardial infarction (MI) leads to heart failure (HF) and premature death. The respective roles of myocyte death and depressed myocyte contractility in the induction of HF after MI have not been clearly defined and are the focus of this study. Objectives: We developed a mouse model in which we could prevent depressed myocyte contractility after MI and used it to test the idea that preventing depression of myocyte Ca 2+ -handling defects could avert post-MI cardiac pump dysfunction. Methods and Results: MI was produced in mice with inducible, cardiac-specific expression of the β2a subunit of the L-type Ca 2+ channel. Myocyte and cardiac function were compared in control and β2a animals before and after MI. β2a myocytes had increased Ca 2+ current; sarcoplasmic reticulum Ca 2+ load, contraction and Ca 2+ transients (versus controls), and β2a hearts had increased performance before MI. After MI, cardiac function decreased. However, ventricular dilation, myocyte hypertrophy and death, and depressed cardiac pump function were greater in β2a versus control hearts after MI. β2a animals also had poorer survival after MI. Myocytes isolated from β2a hearts after MI did not develop depressed Ca 2+ handling, and Ca 2+ current, contractions, and Ca 2+ transients were still above control levels (before MI). Conclusions: Maintaining myocyte contractility after MI, by increasing Ca 2+ influx, depresses rather than improves cardiac pump function after MI by reducing myocyte number.
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Lee, Peiyee, Masataka Sata, David J. Lefer, Stephen M. Factor, Kenneth Walsh, and Richard N. Kitsis. "Fas pathway is a critical mediator of cardiac myocyte death and MI during ischemia-reperfusion in vivo." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 2 (February 1, 2003): H456—H463. http://dx.doi.org/10.1152/ajpheart.00777.2002.
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Fas is a widely expressed cell surface receptor that can initiate apoptosis when activated by its ligand (FasL). Whereas Fas abundance on cardiac myocytes increases in response to multiple pathological stimuli, direct evidence supporting its role in the pathogenesis of heart disease is lacking. Moreover, controversy exists even as to whether Fas activation induces apoptosis in cardiac myocytes. In this study, we show that adenoviral overexpression of FasL, but not β-galactosidase, results in marked apoptosis both in cultures of primary neonatal cardiac myocytes and in the myocardium of intact adult rats. Myocyte killing by FasL is a specific event, because it does not occur in lpr (lymphoproliferative) mice that lack functional Fas. To assess the contribution of the Fas pathway to myocardial infarction (MI) in vivo, lpr mice were subjected to 30 min of ischemia followed by 24 h of reperfusion. Compared with wild-type mice, lpr mice exhibited infarcts that were 62.3% smaller with 63.8% less myocyte apoptosis. These data provide direct evidence that activation of Fas can induce apoptosis in cardiac myocytes and that Fas is a critical mediator of MI due to ischemia-reperfusion in vivo.
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McDonald, Kerry S., Laurin M. Hanft, Timothy L. Domeier, and Craig A. Emter. "Length and PKA Dependence of Force Generation and Loaded Shortening in Porcine Cardiac Myocytes." Biochemistry Research International 2012 (2012): 1–12. http://dx.doi.org/10.1155/2012/371415.
Full textAbstract:
In healthy hearts, ventricular ejection is determined by three myofibrillar properties; force, force development rate, and rate of loaded shortening (i.e., power). The sarcomere length and PKA dependence of these mechanical properties were measured in porcine cardiac myocytes. Permeabilized myocytes were prepared from left ventricular free walls and myocyte preparations were calcium activated to yield ~50% maximal force after which isometric force was measured at varied sarcomere lengths. Porcine myocyte preparations exhibited two populations of length-tension relationships, one being shallower than the other. Moreover, myocytes with shallow length-tension relationships displayed steeper relationships following PKA. Sarcomere length-Ktrrelationships also were measured andKtrremained nearly constant over ~2.30 μm to ~1.90 μm and then increased at lengths below 1.90 μm. Loaded-shortening and peak-normalized power output was similar at ~2.30 μm and ~1.90 μm even during activations with the same [Ca2+], implicating a myofibrillar mechanism that sustains myocyte power at lower preloads. PKA increased myocyte power and yielded greater shortening-induced cooperative deactivation in myocytes, which likely provides a myofibrillar mechanism to assist ventricular relaxation. Overall, the bimodal distribution of myocyte length-tension relationships and the PKA-mediated changes in myocyte length-tension and power are likely important modulators of Frank-Starling relationships in mammalian hearts.
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Liang, B. T. "Direct preconditioning of cardiac ventricular myocytes via adenosine A1 receptor and KATP channel." American Journal of Physiology-Heart and Circulatory Physiology 271, no. 5 (November 1, 1996): H1769—H1777. http://dx.doi.org/10.1152/ajpheart.1996.271.5.h1769.
Full textAbstract:
Both adenosine receptor and ATP-sensitive K (KATP) channel mediate the protective effect of ischemic preconditioning in the intact heart. The objective of the present study was to determine the role of adenosine receptor and KATP channel as well as their interaction in simulating and mediating preconditioning of the cardiac myocyte. Cardiac ventricular myocytes cultured from chick embryos 14 days in ovo were developed as a myocyte model of preconditioning. Myocytes were preconditioned by exposing them to 5-min hypoxia, termed preconditioning hypoxia, before a second 90-min hypoxia. Preconditioning resulted in a 64 +/- 3% decrease in the amount of creatine kinase released and a 66 +/- 2% reduction in the percentage of myocytes (+/-SE, n = 11) killed. Glibenclamide or 5-hydroxydecanoic acid (5-HD), when present during the preconditioning hypoxia, blocked the preconditioning effect. Prior exposure of the myocytes to pinacidil also led to a decrease in the injury sustained during the 90-min hypoxia. The protective effect of pinacidil was blocked by glibenclamide or 5-HD, suggesting that KATP channel activation can mimic as well as mediate preconditioning. Adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT) blocked the protective effect of preconditioning hypoxia. Adenosine or the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) can replace preconditioning hypoxia and mimic preconditioning; this effect was fully antagonized by 8-SPT, glibenclamide, or 5-HD. Adenosine A1-receptor activation caused a glibenclamide-sensitive inhibition of the basal 45Ca influx and basal myocyte contractile amplitude, consistent with coupling of A1 receptor to stimulation of KATP channel in the myocytes. The data provide direct evidence that myocyte KATP channel is the effector downstream from adenosine A1 receptor in mediating the direct preconditioning of cardiac myocytes.
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Colligan, PB, HM Brown-Borg, J. Duan, BH Ren, and J. Ren. "Cardiac contractile function is enhanced in isolated ventricular myocytes from growth hormone transgenic mice." Journal of Endocrinology 173, no. 2 (May 1, 2002): 257–64. http://dx.doi.org/10.1677/joe.0.1730257.
Full textAbstract:
Growth hormone (GH) plays a key role in cardiac growth and function. However, excessive levels of GH often result in cardiac dysfunction, which is the major cause of death in acromegalic patients. Transgenic mice with GH over-expression serve as useful models for acromegaly and exhibit impaired cardiac functions using echocardiography, similar to those of human acromegaly. However, the mechanism underscoring the impaired ventricular function has not been well defined. This study was designed to evaluate the cardiac excitation-contraction coupling in GH over-expressing transgenic mice at the single ventricular myocyte level. Myocytes were isolated from GH and age-matched wild-type mouse hearts. Mechanical properties were evaluated using an IonOptix MyoCam system. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS) and time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ properties were evaluated by fura-2. GH transgenic mice exhibited significantly increased body weights and enlarged heart and myocyte size. Myocytes from GH transgenic mice displayed significantly enhanced PS and+/-dL/dt associated with similar TPS and TR(90) compared with the wild-type littermates. Myocytes from GH transgenic mice displayed a similar resting intracellular Ca2+ level and Ca2+ removal rate but exhibited an elevated peak intracellular Ca2+ level compared with the wild-type group. Myocytes from both groups were equally responsive to increases in extracellular Ca2+ concentration and stimulating frequency. These results suggest that GH over-expression is associated with enhanced contractile function in isolated myocytes and that the impaired cardiac function observed in whole hearts may not be due to defects at the myocyte level.
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Song, Jianliang, Xue-Qian Zhang, JuFang Wang, Ellina Cheskis, Tung O. Chan, Arthur M. Feldman, Amy L. Tucker, and Joseph Y. Cheung. "Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+-K+-ATPase." American Journal of Physiology-Heart and Circulatory Physiology 295, no. 4 (October 2008): H1615—H1625. http://dx.doi.org/10.1152/ajpheart.00287.2008.
Full textAbstract:
Phospholemman (PLM) regulates cardiac Na+/Ca2+ exchanger (NCX1) and Na+-K+-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser68, 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 Ca2+-ATPase levels, and retained near normal contractility, but α1-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 Ca2+ concentration ([Ca2+]i) transients reverted back to those observed in cultured WT myocytes. Both Na+-K+-ATPase current ( Ipump) and Na+/Ca2+ exchange current ( INaCa) 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 INaCa but had no effect on Ipump. Contractility, [Ca2+]i transient amplitudes, and sarcoplasmic reticulum Ca2+ 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 INaCa but decreased Ipump. Contractility, [Ca2+]i transient amplitudes, and sarcoplasmic reticulum Ca2+ 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 [Ca2+]i homeostasis primarily by its direct inhibitory effects on Na+/Ca2+ exchange.
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Heller, Lois Jane, David E. Mohrman, Juline A. Smith, and Kendall B. Wallace. "Multitrack system for superfusing isolated cardiac myocytes." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 5 (May 1, 2003): H1872—H1878. http://dx.doi.org/10.1152/ajpheart.00914.2002.
Full textAbstract:
A new system for studying mechanical activity of freshly isolated cardiac myocytes from up to four experimental groups simultaneously is described. Suspensions of cardiac myocytes isolated from adult rat hearts were drawn into microhematocrit capillary tubes, which were then mounted in parallel fashion between two four-channel tubing manifolds placed on the movable stage of an inverted microscope. Within a few minutes, cells settled and attached to the bottom of the tubes and then could be superfused with various test solutions. The system allowed for electrical field stimulation, rapid changes in bathing solutions, control of temperature, and simulation of ischemia and reperfusion with measurements of the effects of such interventions on both populations of cells (low power survey) and individual myocytes (high power). Myocyte responses to these various interventions are described. The primary advantage of this system is the ability to conduct experiments on cardiac myocytes isolated concurrently from multiple experimental groups at the same time and under identical conditions.
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Lew, W. Y., J. Ryan, and S. Yasuda. "Lipopolysaccharide induces cell shrinkage in rabbit ventricular cardiac myocytes." American Journal of Physiology-Heart and Circulatory Physiology 272, no. 6 (June 1, 1997): H2989—H2993. http://dx.doi.org/10.1152/ajpheart.1997.272.6.h2989.
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The effects of 10 ng/ml of lipopolysaccharide (LPS) on cell volume were examined in rabbit left ventricular myocytes. The myocytes were isolated with depyrogenated digestive enzymes (< 0.7 ng/ml of LPS) to minimize tolerance. Myocyte cross-sectional area (CSA) did not change after 1 h of LPS. However after 8 h, the CSA decreased to 0.93 +/- 0.01 (SE) of the baseline CSA (time = 0) in 19 LPS-exposed myocytes compared with 1.00 +/- 0.01 in 13 control myocytes (P = 0.0015). LPS-induced cell shrinkage was completely blocked by coincubation with 1 mM N-monomethyl-L-arginine, indicating a nitric oxide-mediated mechanism. Cardiac guanosine 3',5'-cyclic monophosphate (cGMP) did not change after 1 h but increased 6 h after LPS (548 +/- 31 vs. 312 +/- 20 fmol/mg protein in control cells; P < 0.05). After 8 h, bumetanide (10 microM for 30 min), a Na+/K+/2Cl- cotransport inhibitor, decreased the CSA in 15 control myocytes to 0.92 +/- 0.02 of the baseline CSA. However, in 19 myocytes with a CSA of 0.93 +/- 0.01 of baseline after 8 h of LPS, the addition of bumetanide caused no additional cell shrinkage. We conclude that low levels of LPS increase cardiac cGMP to inhibit Na+/K+/2Cl- cotransport, causing significant cell shrinkage in cardiac myocytes.
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O'Connell, T. D., J. E. Berry, A. K. Jarvis, M. J. Somerman, and R. U. Simpson. "1,25-Dihydroxyvitamin D3 regulation of cardiac myocyte proliferation and hypertrophy." American Journal of Physiology-Heart and Circulatory Physiology 272, no. 4 (April 1, 1997): H1751—H1758. http://dx.doi.org/10.1152/ajpheart.1997.272.4.h1751.
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We previously demonstrated that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits myocyte maturation (T. D. O'Connell, D. A. Giacherio, A. K. Jarvis, and R. U. Simpson. Endocrinology 136: 482-488, 1995). To define further the role of 1,25(OH)2D3 in regulating myocardial development, we examined the effects of 1,25(OH)2D3 on proliferation and growth of primary cultures of ventricular myocytes isolated from neonatal rat hearts. When neonatal myocytes were grown in a serum-supplemented medium, cell number approximately doubled, and treating these myocytes with 1,25(OH)2D3 inhibited their proliferation by 56.56% after 4 days. Flow cytometry revealed that 1,25(OH)2D3 reduced the percentage of cells in the S phase of the cell cycle by 31.39% after 4 days. We show for the first time that proliferating cell nuclear antigen protein levels were specifically reduced by 1,25(OH)2D3. Protooncogene c-myc protein levels were also reduced by this hormone. Interestingly, a phorbol ester had a similar effect on myocyte proliferation. Furthermore, 1,25(OH)2D3 increased myocyte protein levels and increased cell size, suggesting that it induces cardiac myocyte hypertrophy. Our findings indicate that 1,25(OH)2D3 and phorbol esters directly regulate myocyte proliferation and induce myocyte hypertrophy. Finally, the data demonstrate that the mechanism by which 1,25(OH)2D3 regulates myocyte proliferation involves blocking entry into the S phase of the cell cycle.
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Haddad, J., M. L. Decker, L. C. Hsieh, M. Lesch, A. M. Samarel, and R. S. Decker. "Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture." American Journal of Physiology-Cell Physiology 255, no. 1 (July 1, 1988): C19—C27. http://dx.doi.org/10.1152/ajpcell.1988.255.1.c19.
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The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk. Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum. The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days. During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study. Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM). This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium. These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function.
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Tang, Mingxin, Xiaoying Zhang, Yingxin Li, Yinzheng Guan, Xiaojie Ai, Christopher Szeto, Hiroyuki Nakayama, et al. "Enhanced basal contractility but reduced excitation-contraction coupling efficiency and β-adrenergic reserve of hearts with increased Cav1.2 activity." American Journal of Physiology-Heart and Circulatory Physiology 299, no. 2 (August 2010): H519—H528. http://dx.doi.org/10.1152/ajpheart.00265.2010.
Full textAbstract:
Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type Ca2+ channel (Cav1.2). However, the effects of enhanced Cav1.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the β-adrenergic system are not clear. To recapitulate the increased Cav1.2 activity, a double transgenic (DTG) mouse model overexpressing the Cavβ2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon β-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: 1) in DTG myocytes, L-type Ca2+ current ( ICa,L) density, myocyte fractional shortening (FS), peak Ca2+ transients, and sarcoplasmic reticulum (SR) Ca2+ content (caffeine-induced Ca2+ transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/d t and −dP/d t) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/d t, and −dP/d t)-enhancing effect of the β-adrenergic agonist isoproterenol (2 μg/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of β-adrenergic stimulation on contraction, Ca2+ transients, ICa,L, and SR Ca2+ content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cav1.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart.
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Cheedipudi, Sirisha M., Jinzhu Hu, Siyang Fan, Ping Yuan, Jennifer Karmouch, Grace Czernuszewicz, Matthew J. Robertson, et al. "Exercise restores dysregulated gene expression in a mouse model of arrhythmogenic cardiomyopathy." Cardiovascular Research 116, no. 6 (July 26, 2019): 1199–213. http://dx.doi.org/10.1093/cvr/cvz199.
Full textAbstract:
Abstract Aims Arrhythmogenic cardiomyopathy (ACM) is a myocardial disease caused mainly by mutations in genes encoding desmosome proteins ACM patients present with ventricular arrhythmias, cardiac dysfunction, sudden cardiac death, and a subset with fibro-fatty infiltration of the right ventricle predominantly. Endurance exercise is thought to exacerbate cardiac dysfunction and arrhythmias in ACM. The objective was to determine the effects of treadmill exercise on cardiac phenotype, including myocyte gene expression in myocyte-specific desmoplakin (Dsp) haplo-insufficient (Myh6-Cre:DspW/F) mice. Methods and results Three months old sex-matched wild-type (WT) and Myh6-Cre:DspW/F mice with normal cardiac function, as assessed by echocardiography, were randomized to regular activity or 60 min of daily treadmill exercise (5.5 kJ work per run). Cardiac myocyte gene expression, cardiac function, arrhythmias, and myocardial histology, including apoptosis, were analysed prior to and after 3 months of routine activity or treadmill exercise. Fifty-seven and 781 genes were differentially expressed in 3- and 6-month-old Myh6-Cre:DspW/F cardiac myocytes, compared to the corresponding WT myocytes, respectively. Genes encoding secreted proteins (secretome), including inhibitors of the canonical WNT pathway, were among the most up-regulated genes. The differentially expressed genes (DEGs) predicted activation of epithelial–mesenchymal transition (EMT) and inflammation, and suppression of oxidative phosphorylation pathways in the Myh6-Cre:DspW/F myocytes. Treadmill exercise restored transcript levels of two-third (492/781) of the DEGs and the corresponding dysregulated transcriptional and biological pathways, including EMT, inflammation, and secreted inhibitors of the canonical WNT. The changes were associated with reduced myocardial apoptosis and eccentric cardiac hypertrophy without changes in cardiac function. Conclusion Treadmill exercise restored transcript levels of the majority of dysregulated genes in cardiac myocytes, reduced myocardial apoptosis, and induced eccentric cardiac hypertrophy without affecting cardiac dysfunction in a mouse model of ACM. The findings suggest that treadmill exercise has potential beneficial effects in a subset of cardiac phenotypes in ACM.
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Noga, Anna A., Carrie-Lynn M. Soltys, Amy J. Barr, Suzanne Kovacic, Gary D. Lopaschuk, and Jason R. B. Dyck. "Expression of an active LKB1 complex in cardiac myocytes results in decreased protein synthesis associated with phenylephrine-induced hypertrophy." American Journal of Physiology-Heart and Circulatory Physiology 292, no. 3 (March 2007): H1460—H1469. http://dx.doi.org/10.1152/ajpheart.01133.2006.
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AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADα) and MO25α. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADα/MO25α expression, LKB1 activity, and AMPKα phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADα/MO25α complex in neonatal rat cardiac myocytes inhibited the increase in protein synthesis observed in cells treated with phenylephrine (measured via [3H]phenylalanine incorporation). This was associated with a decreased phosphorylation of p70S6 kinase and its substrate S6 ribosomal protein, key regulators of protein synthesis. In addition, we show that the pathological cardiac hypertrophy in transgenic mice with cardiac-specific expression of activated calcineurin is associated with a significant decrease in LKB1 expression. Together, our data show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1 activation may be a method for the prevention of pathological cardiac hypertrophy.
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Carlson, Deborah L., Ellis Lightfoot, Debora D. Bryant, Sandra B. Haudek, David Maass, Jureta Horton, and Brett P. Giroir. "Burn plasma mediates cardiac myocyte apoptosis via endotoxin." American Journal of Physiology-Heart and Circulatory Physiology 282, no. 5 (May 1, 2002): H1907—H1914. http://dx.doi.org/10.1152/ajpheart.00393.2001.
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Thermal trauma is associated with cardiac myocyte apoptosis in vivo. To determine whether cardiac myocyte apoptosis could be secondary to burn-induced cytokines or inflammatory mediators, we investigated the effects of tumor necrosis factor-α (TNF-α) and burn plasma on a murine cardiac myocyte cell line and primary culture myocytes. HL-1 cells were exposed to plasma isolated from burned or sham rats. Burn, but not sham plasma, induced significant increases in caspase-3 activity and DNA fragmentation. Similar results were obtained in primary culture rat myocytes. A dose-dependent increase in caspase-3 activity was observed when HL-1 cells were incubated with increasing concentrations of TNF-α. Even though TNF-α increased apoptosis, enzyme-linked immunosorbent assay detected no TNF-α in burn plasma. Burn plasma also failed to induce TNF-α mRNA, eliminating an autocrine mechanism of TNF-α secretion and binding. Also, treatment of burn plasma containing rhuTNFR:Fc failed to inhibit apoptosis. To examine the possibility that endotoxin within burn plasma might account for the apoptotic effect, burn plasma was preincubated with rBPI21. Caspase-3 activity was reduced to control levels. These data indicate that burn plasma induces apoptosis in cardiac myocytes via an endotoxin-dependent mechanism and suggest that systemic inhibition of endotoxin may provide a therapeutic approach for treatment of burn-associated cardiac dysfunction.
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Hebbar, Latha, Hugh B. Dorman, Mark J. Clair, Raymond C. Roy, and Francis G. Spinale. "Negative and Selective Effects of Propofol on Isolated Swine Myocyte Contractile Function in Pacing-induced Congestive Heart Failure." Anesthesiology 86, no. 3 (March 1, 1997): 649–59. http://dx.doi.org/10.1097/00000542-199703000-00018.
Full textAbstract:
Background Although propofol (2-6 di-isopropylphenol) is commonly used to induce and maintain anesthesia and sedation for surgery, systematic hypotension and reduced cardiac output can occur in patients with or without intrinsic cardiac disease. The effect of propofol on myocyte contractility after the development of congestive heart failure (CHF) remains unknown. This study tested the hypothesis that propofol would have direct effects on myocyte contractile function in both healthy and CHF cardiac myocyte preparations. Methods Isolated left ventricular (LV) myocyte contractile function (shortening velocity, micron/s) was examined in myocytes from five control pigs and in five pigs with pacing-induced CHF (240 beats/min, for 3 weeks) in the presence of propofol concentrations ranging from 1-6 micrograms/ml. In addition, myocyte contractility in response to beta-adrenergic receptor stimulation (isoproterenol, 10-50 nM) in the presence of propofol (3 micrograms/ml) was examined. Results Three weeks of pacing caused LV dysfunction consistent with CHF as evidenced by increased LV end-diastolic diameter (control 3.3 +/- 0.1 cm vs. CHF 5.6 +/- 0.2 cm; P < 0.05) and reduced LV fractional shortening (control 34 +/- 3% vs. CHF 12 +/- 2%, P < 0.05). Propofol (6 micrograms/ml) caused a concentration-dependent negative effect on velocity of shortening from baseline in both control (67 +/- 2 microns/s vs. 27 +/- 3 microns/s; P < 0.05) and CHF myocytes (29 +/- 1 microns/s vs. 15 +/- 1 microns/s; P < 0.05). Importantly, CHF myocytes were more sensitive than control myocytes to the negative effects of propofol on velocity of shortening at the lower concentration (1 microgram/ml). beta-adrenergic responsiveness was reduced by propofol (3 micrograms/ml) in control myocytes only. Conclusions Propofol has a direct and negative effect on basal myocyte contractile processes in the setting of CHF, which is more pronounced than that on healthy myocytes at reduced propofol concentrations.
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Rook, M. B., A. C. van Ginneken, B. de Jonge, A. el Aoumari, D. Gros, and H. J. Jongsma. "Differences in gap junction channels between cardiac myocytes, fibroblasts, and heterologous pairs." American Journal of Physiology-Cell Physiology 263, no. 5 (November 1, 1992): C959—C977. http://dx.doi.org/10.1152/ajpcell.1992.263.5.c959.
Full textAbstract:
Cultures of neonatal rat heart cells contain predominantly myocytes and fibroblastic cells. Most abundant are groups of synchronously contracting myocytes, which are electrically well coupled through large gap junctions. Cardiac fibroblasts may be electrically coupled to each other and to adjacent myocytes, be it with low intercellular conductances. Nevertheless, synchronously beating myocytes interconnected via a fibroblast were present, demonstrating that nonexcitable cardiac cells are capable of passive impulse conduction. In fibroblast pairs as well as in myocyte-fibroblast cell pairs, no sensitivity to junctional voltage could be detected when transjunctional conductance was > 1-2 nS. However, in pairs coupled by a conductance of < 1 nS, complex voltage-dependent gating was evident; gap junction channel open probability decreased with increasing junctional voltage but a nongated residual conductance remained at all voltages tested. Single gap junction channel conductance between fibroblasts was approximately 21 pS, very similar to an approximately 18-pS channel conductance that was found between myocytes next to the major conductance of 43 pS. Single-channel conductance in heterologous myocyte-fibroblast gap junctions was approximately 32 pS, which matches the theoretical value of 29 pS for gap junction channels composed of a fibroblast connexon and the major myocyte connexon. A site-directed antibody against rat heart gap junction protein connexin43 recognized gap junctions between neonatal cardiomyocytes, as demonstrated by immunocytochemical labeling. In contrast, junctions between fibroblasts showed no labeling, while in myocyte-fibroblast junctions labeling occasionally was present. Our results suggest the existence of two gap junction proteins between neonatal rat cardiocytes, connexin43 and another yet unidentified connexin. An alternative explanation (cell-specific regulation of the conductance of connexin43 channels) is discussed.