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Sample records for heart induces cardiomyocyte

  1. Derivation and cardiomyocyte differentiation of induced pluripotent stem cells from heart failure patients.

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    Zwi-Dantsis, Limor; Huber, Irit; Habib, Manhal; Winterstern, Aaron; Gepstein, Amira; Arbel, Gil; Gepstein, Lior

    2013-06-01

    Myocardial cell replacement therapies are hampered by a paucity of sources for human cardiomyocytes and by the expected immune rejection of allogeneic cell grafts. The ability to derive patient-specific human-induced pluripotent stem cells (hiPSCs) may provide a solution to these challenges. We aimed to derive hiPSCs from heart failure (HF) patients, to induce their cardiomyocyte differentiation, to characterize the generated hiPSC-derived cardiomyocytes (hiPSC-CMs), and to evaluate their ability to integrate with pre-existing cardiac tissue. Dermal fibroblasts from two HF patients were reprogrammed by retroviral delivery of Oct4, Sox2, and Klf4 or by using an excisable polycistronic lentiviral vector. The resulting HF-hiPSCs displayed adequate reprogramming properties and could be induced to differentiate into cardiomyocytes with the same efficiency as control hiPSCs (derived from human foreskin fibroblasts). Gene expression and immunostaining studies confirmed the cardiomyocyte phenotype of the differentiating HF-hiPSC-CMs. Multi-electrode array recordings revealed the development of a functional cardiac syncytium and adequate chronotropic responses to adrenergic and cholinergic stimulation. Next, functional integration and synchronized electrical activities were demonstrated between hiPSC-CMs and neonatal rat cardiomyocytes in co-culture studies. Finally, in vivo transplantation studies in the rat heart revealed the ability of the HF-hiPSC-CMs to engraft, survive, and structurally integrate with host cardiomyocytes. Human-induced pluripotent stem cells can be established from patients with advanced heart failure and coaxed to differentiate into cardiomyocytes, which can integrate with host cardiac tissue. This novel source for patient-specific heart cells may bring a unique value to the emerging field of cardiac regenerative medicine.

  2. Cited2 participates in cardiomyocyte apoptosis and maternal diabetes-induced congenital heart abnormality.

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    Su, Dongmei; Song, Jun-Xian; Gao, Qianqian; Guan, Lina; Li, Qian; Shi, Cuige; Ma, Xu

    2016-10-28

    Gestational diabetes mellitus is a risk factor for abnormal heart development, but the molecular basis remains obscure. To further analyze this, the hyperglycemia rat and cell model were established in this study. The results showed that hyperglycemic rats gained significantly less weight during gestation than controls. The number of embryos per litter was significantly reduced in diabetic mothers compared to controls. Ventricular wall thickness was often decreased in the diabetic offspring and cardiomyocyte apoptosis participated in ventricular wall thinness. Our results also indicated that Cited2 expression decreased in the heart tissues of diabetic-exposed embryos comparing with the control. The vitro results showed that down-regulation of Cited2 was associated with high glucose-induced apoptosis in cardiomyocytes in vitro. Over-expression of Cited2 gene restrained the cardiomyocyte apoptosis induced by high glucose. Furthermore, Cited2 S192G mutation partly inhibited the capacity of Cited2 to suppress apoptosis induced by high glucose in cardiomyocytes. This showed the critical role of Cited2 in high glucose-induced cardiomyocytes apoptosis. Data from this study found the association of Cited2 down regulation with cardiomyocytes apoptosis and maternal diabetes-induced ventricular wall thinness genesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Towards regenerating the mammalian heart: challenges in evaluating experimentally induced adult mammalian cardiomyocyte proliferation.

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    Zebrowski, David C; Becker, Robert; Engel, Felix B

    2016-05-01

    In recent years, there has been a dramatic increase in research aimed at regenerating the mammalian heart by promoting endogenous cardiomyocyte proliferation. Despite many encouraging successes, it remains unclear if we are any closer to achieving levels of mammalian cardiomyocyte proliferation for regeneration as seen during zebrafish regeneration. Furthermore, current cardiac regenerative approaches do not clarify whether the induced cardiomyocyte proliferation is an epiphenomena or responsible for the observed improvement in cardiac function. Moreover, due to the lack of standardized protocols to determine cardiomyocyte proliferation in vivo, it remains unclear if one mammalian regenerative factor is more effective than another. Here, we discuss current methods to identify and evaluate factors for the induction of cardiomyocyte proliferation and challenges therein. Addressing challenges in evaluating adult cardiomyocyte proliferation will assist in determining 1) which regenerative factors should be pursued in large animal studies; 2) if a particular level of cell cycle regulation presents a better therapeutic target than another (e.g., mitogenic receptors vs. cyclins); and 3) which combinatorial approaches offer the greatest likelihood of success. As more and more regenerative studies come to pass, progress will require a system that not only can evaluate efficacy in an objective manner but can also consolidate observations in a meaningful way. Copyright © 2016 the American Physiological Society.

  4. Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy.

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    Ji Zhang

    Full Text Available Fatty acid binding protein 4 (FABP4 is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG mice using α myosin-heavy chain (α-MHC promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway.

  5. Cancer induces cardiomyocyte remodeling and hypoinnervation in the left ventricle of the mouse heart.

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    Christian Mühlfeld

    Full Text Available Cancer is often associated with cachexia, cardiovascular symptoms and autonomic dysregulation. We tested whether extracardiac cancer directly affects the innervation of left ventricular myocardium. Mice injected with Lewis lung carcinoma cells (tumor group, TG or PBS (control group, CG were analyzed after 21 days. Cardiac function (echocardiography, serum levels of TNF-α and Il-6 (ELISA, structural alterations of cardiomyocytes and their innervation (design-based stereology and levels of innervation-related mRNA (quantitative RT-PCR were analysed. The groups did not differ in various functional parameters. Serum levels of TNF-α and Il-6 were elevated in TG. The total length of axons in the left ventricle was reduced. The number of dense core vesicles per axon profile was reduced. Decreased myofibrillar volume, increased sarcoplasmic volume and increased volume of lipid droplets were indicative of metabolic alterations of TG cardiomyocytes. In the heart, the mRNA level of nerve growth factor was reduced whereas that of β1-adrenergic receptor was unchanged in TG. In the stellate ganglion of TG, mRNA levels of nerve growth factor and neuropeptide Y were decreased and that of tyrosine hydroxylase was increased. In summary, cancer induces a systemic pro-inflammatory state, a significant reduction in myocardial innervation and a catabolic phenotype of cardiomyocytes in the mouse. Reduced expression of nerve growth factor may account for the reduced myocardial innervation.

  6. Diabetes-induced effects on cardiomyocytes in chick embryonic heart micromass and mouse embryonic D3 differentiated stem cells.

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    Mohammed, Omar J; Latif, Muhammad Liaque; Pratten, Margaret K

    2017-04-01

    Diabetes mellitus during pregnancy is a considerable medical challenge, since it is related to ‎augmented morbidity and mortality concerns for both the fetus ‎and the pregnant woman. Records show that the etiology of diabetic ‎embryopathy is complicated, as many teratological factors might be involved ‎in the mechanisms of diabetes mellitus-induced congenital malformation. ‎In this study, the potential cardiotoxic effect of hyperglycemia with hyperketonemia was investigated by using two in vitro models; primary chick embryonic cardiomyocytes and stem cell derived cardiomyocytes, where adverse effects were recorded in both systems. The cells were evaluated by changes in beating activity, cell activity, protein content, ROS production, DNA damage and differentiating stem cell migration. The diabetic formulae used produced an increase in DNA damage and a decline in cell migration in mouse embryonic stem cells. These results provide an additional insight into adverse effects during gestational diabetes mellitus and a recommendation for expectant mothers and maternity staff to monitor glycaemic levels months ahead of conception. This study also supports the recommendation of using antioxidants during pregnancy to prevent DNA damage by the production of ROS, which might result in heart defects as well as other developmental anomalies. Copyright © 2017. Published by Elsevier Inc.

  7. Effect of beta2-adrenergic agonist clenbuterol on ischemia/reperfusion injury in isolated rat hearts and cardiomyocyte apoptosis induced by hydrogen peroxide.

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    Liu, Ping; Xiang, Ji-zhou; Zhao, Lei; Yang, Lei; Hu, Ben-rong; Fu, Qin

    2008-06-01

    To observe the effect of beta2-adrenergic agonist clenbuterol on ischemia/reperfusion (I/R) injury in isolated rat hearts and hydrogen peroxide (H2O2)-induced cardiomyocyte apoptosis. Isolated rat hearts were subjected to 30 min global ischemia and 60 min reperfusion on a Langendorff apparatus. Cardiac function was evaluated by heart rate, left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure, maximal rise rate of left ventricular pressure [+dp/dt(max)], and the coronary effluent (CF). Lactate dehydrogenase (LDH) in the coronary effluent, malondialdehyde (MDA), superoxide dismutase (SOD), and Ca2+-ATPase activity in the cardiac tissue were measured using commercial kits. The apoptotic cardiomyocyte was detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay. Bax/Bcl-2 mRNA levels and the expression of caspase-3 were detected by RT-PCR and immunoblotting, respectively. Cultured newborn rat cardiomyocytes were preincubated with clenbuterol, and oxidative stress injury was induced by H2O2. Cell viability and cardiomyocyte apoptosis were evaluated by flow cytometry (FCM). In the isolated rat hearts after I/R injury, clenbuterol significantly improved diastolic function (LVEDP and CF) and Ca2+-ATPase activity. Treatment with clenbuterol increased SOD activity and decreased the MDA level and LDH release compared with the I/R group (Pclenbuterol decreased apoptosis, which was associated with a reduction in TUNEL-positive cells, Bax/Bcl-2 mRNA, and caspase-3 expression. In H2O2-induced cardiomyocyte injury, clenbuterol increased cell viability and attenuated cardiomyocyte apoptosis. Pretreatment with ICI118551 (selective beta2-adrenergic antagonist) decreased these effects compared with the clenbuterol-treated group (PClenbuterol ameliorated ventricular diastolic function by enhancing Ca2+-ATPase activity and reduced oxidative stress and cardiac myocyte apoptosis in an experimental rat model

  8. Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Encapsulating Bioactive Hydrogels Improve Rat Heart Function Post Myocardial Infarction.

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    Chow, Andre; Stuckey, Daniel J; Kidher, Emaddin; Rocco, Mark; Jabbour, Richard J; Mansfield, Catherine A; Darzi, Ara; Harding, Sian E; Stevens, Molly M; Athanasiou, Thanos

    2017-10-04

    Tissue engineering offers an exciting possibility for cardiac repair post myocardial infarction. We assessed the effects of combined polyethylene glycol hydrogel (PEG), human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), and erythropoietin (EPO) therapy in a rat model of myocardial infarction. PEG with/out iPSC-CMs and EPO; iPSC-CMs in saline; or saline alone was injected into infarcted hearts shortly after infarction. Injection of almost any combination of the therapeutics limited acute elevations in chamber volumes. After 10 weeks, attenuation of ventricular remodeling was identified in all groups that received PEG injections, while ejection fractions were significantly increased in the gel-EPO, cell, and gel-cell-EPO groups. In all treatment groups, infarct thickness was increased and regions of muscle were identified within the scar. However, no grafted cells were detected. Hence, iPSC-CM-encapsulating bioactive hydrogel therapy can improve cardiac function post myocardial infarction and increase infarct thickness and muscle content despite a lack of sustained donor-cell engraftment. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Heart failure induces significant changes in nuclear pore complex of human cardiomyocytes.

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    Estefanía Tarazón

    Full Text Available AIMS: The objectives of this study were to analyse the effect of heart failure (HF on several proteins of nuclear pore complex (NPC and their relationship with the human ventricular function. METHODS AND RESULTS: A total of 88 human heart samples from ischemic (ICM, n = 52 and dilated (DCM, n = 36 patients undergoing heart transplant and control donors (CNT, n = 9 were analyzed by Western blot. Subcellular distribution of nucleoporins was analysed by fluorescence and immunocytochemistry. When we compared protein levels according to etiology, ICM showed significant higher levels of NDC1 (65%, p<0.0001, Nup160 (88%, p<0.0001 and Nup153 (137%, p = 0.004 than those of the CNT levels. Furthermore, DCM group showed significant differences for NDC1 (41%, p<0.0001, Nup160 (65%, p<0.0001, Nup153 (155%, p = 0.006 and Nup93 (88%, p<0.0001 compared with CNT. However, Nup155 and translocated promoter region (TPR did not show significant differences in their levels in any etiology. Regarding the distribution of these proteins in cell nucleus, only NDC1 showed differences in HF. In addition, in the pathological group we obtained good relationship between the ventricular function parameters (LVEDD and LVESD and Nup160 (r = -0382, p = 0.004; r = -0.290, p = 0.033; respectively. CONCLUSIONS: This study shows alterations in specific proteins (NDC1, Nup160, Nup153 and Nup93 that compose NPC in ischaemic and dilated human heart. These changes, related to ventricular function, could be accompanied by alterations in the nucleocytoplasmic transport. Therefore, our findings may be the basis for a new approach to HF management.

  10. Analysis of cardiomyocyte movement in the developing murine heart

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    Hashimoto, Hisayuki [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Tabata, Hidenori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan); Nakajima, Kazunori [Department of Anatomy, Keio University School of Medicine, Tokyo (Japan); Sakaue-Sawano, Asako; Miyawaki, Atsushi [Life Function and Dynamics, ERATO, JST, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198 (Japan); Fukuda, Keiichi [Department of Cardiology, Keio University School of Medicine, Tokyo (Japan)

    2015-09-04

    The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.

  11. Necroptosis Induced by Ad-HGF Activates Endogenous C-Kit+ Cardiac Stem Cells and Promotes Cardiomyocyte Proliferation and Angiogenesis in the Infarcted Aged Heart

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    Jiabao Liu

    2016-12-01

    Full Text Available Background/Aims: The discovery of c-kit+ cardiac stem cells (CSCs provided us with new therapeutic targets to repair the damaged heart. However, the precise mechanisms regulating CSC proliferation and differentiation in the aged heart remained elusive. Necroptosis, a type of regulated cell death, has recently been shown to occur following myocardial infarction (MI; however, its effect on c-kit+ CSCs remains unknown. We investigated the effects of hepatocyte growth factor (HGF and necroptosis on the proliferation and differentiation of endogenous c-kit+ CSCs in aged rat hearts following MI. Methods: The c-kit+ CSCs and HGF/p-Met expression levels in neonatal, adult and aged rats were compared using immunofluorescence and Western blotting. Immediately after MI, adenovirus carrying the HGF gene (Ad-HGF was injected into the left ventricular wall surrounding the infarct areas of the aged rat heart. The proliferation and differentiation of the endogenous c-kit+ CSCs were studied using immunofluorescence. The signalling pathways were analysed via Western blotting and ELISA. Results: HGF/p-Met expression levels and c-kit+ CSC abundance gradually decreased with age. Ad-HGF promoted c-kit+ CSC differentiation into precursor cells of cardiomyocyte, endothelial and smooth muscle cell lineages and enhanced cardiomyocyte proliferation and angiogenesis in aged rats; these effects were reversed by the inhibition of necroptosis. Ad-HGF administration induced necroptosis by increasing the expression of receptor interacting protein kinase (RIP 1 and receptor interacting protein kinase (RIP 3 proteins in the infarcted heart. Moreover, Ad-HGF-induced necroptosis increased high-mobility group box 1 protein (HMGB1 levels and enhanced the abundance of c-kit+ cells in the bone marrow, which may partly account for the beneficial effect of necroptosis on the c-kit+ CSCs. Conclusion: Ad-HGF-induced necroptosis facilitated aged heart repair after MI by promoting c-kit+ CSC

  12. Essential role of Cdc42 in cardiomyocyte proliferation and cell-cell adhesion during heart development.

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    Li, Jieli; Liu, Yang; Jin, Yixin; Wang, Rui; Wang, Jian; Lu, Sarah; VanBuren, Vincent; Dostal, David E; Zhang, Shenyuan L; Peng, Xu

    2017-01-15

    Cdc42 is a member of the Rho GTPase family and functions as a molecular switch in regulating cell migration, proliferation, differentiation and survival. However, the role of Cdc42 in heart development remains largely unknown. To determine the function of Cdc42 in heart formation, we have generated a Cdc42 cardiomyocyte knockout (CCKO) mouse line by crossing Cdc42 flox mice with myosin light chain (MLC) 2a-Cre mice. The inactivation of Cdc42 in embryonic cardiomyocytes induced lethality after embryonic day 12.5. Histological analysis of CCKO embryos showed cardiac developmental defects that included thin ventricular walls and ventricular septum defects. Microarray and real-time PCR data also revealed that the expression level of p21 was significantly increased and cyclin B1 was dramatically decreased, suggesting that Cdc42 is required for cardiomyocyte proliferation. Phosphorylated Histone H3 staining confirmed that the inactivation of Cdc42 inhibited cardiomyocytes proliferation. In addition, transmission electron microscope studies showed disorganized sarcomere structure and disruption of cell-cell contact among cardiomyocytes in CCKO hearts. Accordingly, we found that the distribution of N-cadherin/β-Catenin in CCKO cardiomyocytes was impaired. Taken together, our data indicate that Cdc42 is essential for cardiomyocyte proliferation, sarcomere organization and cell-cell adhesion during heart development. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart.

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    Kawamura, Masashi; Miyagawa, Shigeru; Fukushima, Satsuki; Saito, Atsuhiro; Miki, Kenji; Ito, Emiko; Sougawa, Nagako; Kawamura, Takuji; Daimon, Takashi; Shimizu, Tatsuya; Okano, Teruo; Toda, Koichi; Sawa, Yoshiki

    2013-09-10

    Transplantation of cardiomyocytes that are derived from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) shows promise in generating new functional myocardium in situ, whereas the survival and functionality of the transplanted cells are critical in considering this therapeutic impact. Cell-sheet method has been used to transplant many functional cells; however, potential ischemia might limit cell survival. The omentum, which is known to have rich vasculature, is expected to be a source of blood supply. We hypothesized that transplantation of hiPS-CM cell sheets combined with an omentum flap may deliver a large number of functional hiPS-CMs with enhanced blood supply. Retrovirally established human iPS cells were treated with Wnt signaling molecules to induce cardiomyogenic differentiation, followed by superparamagnetic iron oxide labeling. Cell sheets were created from the magnetically labeled hiPS-CMs using temperature-responsive dishes and transplanted to porcine hearts with or without the omentum flap (n=8 each). Two months after transplantation, the survival of superparamagnetic iron oxide-labeled hiPS-CMs, assessed by MRI, was significantly greater in mini-pigs with the omentum than in those without it; histologically, vascular density in the transplanted area was significantly greater in mini-pigs with the omentum than in those without it. The transplanted tissues contained abundant cardiac troponin T-positive cells surrounded by vascular-rich structures. The omentum flap enhanced the survival of hiPS-CMs after transplantation via increased angiogenesis, suggesting that this strategy is useful in clinical settings. The combination of hiPS-CMs and the omentum flap may be a promising technique for the development of tissue-engineered vascular-rich new myocardium in vivo.

  14. The Adipokine Chemerin Induces Apoptosis in Cardiomyocytes

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    Diego Rodríguez-Penas

    2015-08-01

    Full Text Available Background: The adipokine chemerin has been associated with cardiovascular disease. We investigated the effects of chemerin on viability and intracellular signalling in murine cardiomyocytes, and the effects of insulin and TNF-α on cardiomyocyte chemerin production. Methods: Hoechst dye vital staining and cell cycle analysis were used to analyse the viability of murine cardiac cells in culture. Western blot was used to explore the phosphorylation of AKT and caspase-9 activity in neonatal rat cardiomyocytes and HL-1 cells. Finally, RT-qPCR, ELISA and western blot were performed to examine chemerin and CMKLR1 expression after insulin and TNF-α treatment in cardiac cells. Results: Chemerin treatment increased apoptosis, reduced phosphorylation of AKT at Thr308 and increased caspase-9 activity in murine cardiomyocytes. Insulin treatment lowered chemerin and CMKLR1 mRNA and protein levels, and the amount of chemerin in the cell media, while TNF-α treatment increased chemerin mRNA and protein levels but decreased expression of the CMKLR1 gene. Conclusion: Chemerin induces apoptosis, reduces AKT phosphorylation and increases the cleavage of caspase-9 in murine cardiomyocytes. The expression of chemerin is regulated by important metabolic (insulin and inflammatory (TNF-α mediators at cardiac level. Our results suggest that chemerin could play a role in the physiopathology of cardiac diseases.

  15. NOX2 Antisense Attenuates Hypoxia-Induced Oxidative Stress and Apoptosis in Cardiomyocyte.

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    Yu, Bo; Meng, Fanbo; Yang, Yushuang; Liu, Dongna; Shi, Kaiyao

    2016-01-01

    Heart ischemia is a hypoxia related disease. NOX2 and HIF-1α proteins were increased in cardiomyocytes after acute myocardial infarction. However, the relationship of the hypoxia-induced HIF-1α. NOX2-derived oxidative stress and apoptosis in cardiomyocyte remains unclear. In the current study, we use NOX2 antisense strategy to investigate the role of NOX2 in hypoxia-induced oxidative stress and apoptosis in rat cardiomyocytes. Here, we show that transduction of ADV-NOX2-AS induces potent silencing of NOX2 in cardiomyocytes, and resulting in attenuation of hypoxia-induced oxidative stress and apoptosis. This study indicates the potential of antisense-based therapies and validates NOX2 as a potent therapeutic candidate for heart ischemia.

  16. Immunological Properties of Murine Parthenogenetic Stem Cell-Derived Cardiomyocytes and Engineered Heart Muscle

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    Michael Didié

    2017-08-01

    Full Text Available Pluripotent parthenogenetic stem cells (pSCs can be derived by pharmacological activation of unfertilized oocytes. Homozygosity of the major histocompatibility complex (MHC in pSCs makes them an attractive cell source for applications in allogeneic tissue repair. This was recently demonstrated for pSC-based tissue-engineered heart repair. A detailed analysis of immunological properties of pSC-derived cardiomyocytes and engineered heart muscle (EHM thereof is, however, lacking. The aim of this study was to determine baseline and cytokine-inducible MHC class I and MHC class II as well as programmed death ligand-1 (PDL-1 and co-stimulatory protein (CD40, CD80, CD86 expression in pSC-derived cardiomyocytes and pSC-EHM in vitro and in vivo. Cardiomyocytes from an MHC-homologous (H2d/d pSC-line were enriched to ~90% by making use of a recently developed cardiomyocyte-specific genetic selection protocol. MHC class I and MHC class II expression in cardiomyocytes could only be observed after stimulation with interferon gamma (IFN-γ. PDL-1 was markedly upregulated under IFN-γ. CD40, CD80, and CD86 were expressed at low levels and not upregulated by IFN-γ. EHM constructed from H2d/d cardiomyocytes expressed similarly low levels of MHC class I, MHC class II, and costimulatory molecules under basal conditions. However, in EHM only MHC class I, but not MHC class II, molecules were upregulated after IFN-γ-stimulation. We next employed a cocultivation system with MHC-matched and MHC-mismatched splenocytes and T-cells to analyze the immune stimulatory properties of EHMs. Despite MHC-mismatched conditions, EHM did not induce splenocyte or T-cell proliferation in vitro. To evaluate the immunogenicity of pSC-derived cardiomyocytes in vivo, we implanted pSC-derived embryoid bodies after elimination of non-cardiomyocytes (cardiac bodies under the kidney capsules of MHC-matched and -mismatched mice. Spontaneous beating of cardiac bodies could be observed for 28

  17. Immunological Properties of Murine Parthenogenetic Stem Cell-Derived Cardiomyocytes and Engineered Heart Muscle

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    Didié, Michael; Galla, Satish; Muppala, Vijayakumar; Dressel, Ralf; Zimmermann, Wolfram-Hubertus

    2017-01-01

    Pluripotent parthenogenetic stem cells (pSCs) can be derived by pharmacological activation of unfertilized oocytes. Homozygosity of the major histocompatibility complex (MHC) in pSCs makes them an attractive cell source for applications in allogeneic tissue repair. This was recently demonstrated for pSC-based tissue-engineered heart repair. A detailed analysis of immunological properties of pSC-derived cardiomyocytes and engineered heart muscle (EHM) thereof is, however, lacking. The aim of this study was to determine baseline and cytokine-inducible MHC class I and MHC class II as well as programmed death ligand-1 (PDL-1) and co-stimulatory protein (CD40, CD80, CD86) expression in pSC-derived cardiomyocytes and pSC-EHM in vitro and in vivo. Cardiomyocytes from an MHC-homologous (H2d/d) pSC-line were enriched to ~90% by making use of a recently developed cardiomyocyte-specific genetic selection protocol. MHC class I and MHC class II expression in cardiomyocytes could only be observed after stimulation with interferon gamma (IFN-γ). PDL-1 was markedly upregulated under IFN-γ. CD40, CD80, and CD86 were expressed at low levels and not upregulated by IFN-γ. EHM constructed from H2d/d cardiomyocytes expressed similarly low levels of MHC class I, MHC class II, and costimulatory molecules under basal conditions. However, in EHM only MHC class I, but not MHC class II, molecules were upregulated after IFN-γ-stimulation. We next employed a cocultivation system with MHC-matched and MHC-mismatched splenocytes and T-cells to analyze the immune stimulatory properties of EHMs. Despite MHC-mismatched conditions, EHM did not induce splenocyte or T-cell proliferation in vitro. To evaluate the immunogenicity of pSC-derived cardiomyocytes in vivo, we implanted pSC-derived embryoid bodies after elimination of non-cardiomyocytes (cardiac bodies) under the kidney capsules of MHC-matched and -mismatched mice. Spontaneous beating of cardiac bodies could be observed for 28 days in the

  18. Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

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    Wu Yihe

    2012-04-01

    Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

  19. Testosterone Antagonizes Doxorubicin-Induced Senescence of Cardiomyocytes.

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    Altieri, Paola; Barisione, Chiara; Lazzarini, Edoardo; Garuti, Anna; Bezante, Gian Paolo; Canepa, Marco; Spallarossa, Paolo; Tocchetti, Carlo Gabriele; Bollini, Sveva; Brunelli, Claudio; Ameri, Pietro

    2016-01-08

    Chronic cardiotoxicity is less common in male than in female patients receiving doxorubicin and other anthracyclines at puberty and adolescence. We hypothesized that this sex difference might be secondary to distinct activities of sex hormones on cardiomyocyte senescence, which is thought to be central to the development of long-term anthracycline cardiomyopathy. H9c2 cells and neonatal mouse cardiomyocytes were exposed to doxorubicin with or without prior incubation with testosterone or 17β-estradiol, the main androgen and estrogen, respectively. Testosterone, but not 17β-estradiol, counteracted doxorubicin-elicited senescence. Downregulation of telomere binding factor 2, which has been pinpointed previously as being pivotal to doxorubicin-induced senescence, was also prevented by testosterone, as were p53 phosphorylation and accumulation. Pretreatment with the androgen receptor antagonist flutamide, the phosphatidylinositol 3 kinase inhibitor LY294002, and the nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester abrogated the reduction in senescence and the normalization of telomere binding factor 2 levels attained by testosterone. Consistently, testosterone enhanced the phosphorylation of AKT and nitric oxide synthase 3. In H9c2 cells, doxorubicin-stimulated senescence was still observed up to 21 days after treatment and increased further when cells were rechallenged with doxorubicin 14 days after the first exposure to mimic the schedule of anthracycline-containing chemotherapy. Remarkably, these effects were also inhibited by testosterone. Testosterone protects cardiomyocytes against senescence caused by doxorubicin at least in part by modulating telomere binding factor 2 via a pathway involving the androgen receptor, phosphatidylinositol 3 kinase, AKT, and nitric oxide synthase 3. This is a potential mechanism by which pubescent and adolescent boys are less prone to chronic anthracycline cardiotoxicity than girls. © 2016 The Authors. Published on

  20. Targeting Cardiomyocyte Ca2+ Homeostasis in Heart Failure

    Science.gov (United States)

    Røe, Åsmund T.; Frisk, Michael; Louch, William E.

    2015-01-01

    Improved treatments for heart failure patients will require the development of novel therapeutic strategies that target basal disease mechanisms. Disrupted cardiomyocyte Ca2+ homeostasis is recognized as a major contributor to the heart failure phenotype, as it plays a key role in systolic and diastolic dysfunction, arrhythmogenesis, and hypertrophy and apoptosis signaling. In this review, we outline existing knowledge of the involvement of Ca2+ homeostasis in these deficits, and identify four promising targets for therapeutic intervention: the sarcoplasmic reticulum Ca2+ ATPase, the Na+-Ca2+ exchanger, the ryanodine receptor, and t-tubule structure. We discuss experimental data indicating the applicability of these targets that has led to recent and ongoing clinical trials, and suggest future therapeutic approaches. PMID:25483944

  1. Functional Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells

    National Research Council Canada - National Science Library

    Zhang, Jianhua; Wilson, Gisela F; Soerens, Andrew G; Koonce, Chad H; Yu, Junying; Palecek, Sean P; Thomson, James A; Kamp, Timothy J

    2009-01-01

    Human induced pluripotent stem (iPS) cells hold great promise for cardiovascular research and therapeutic applications, but the ability of human iPS cells to differentiate into functional cardiomyocytes has not yet been demonstrated...

  2. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    Energy Technology Data Exchange (ETDEWEB)

    Aguilar, David; Strom, Joshua; Chen, Qin M., E-mail: qchen@email.arizona.edu

    2014-04-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA.

  3. Nerve Growth Factor Stimulates Cardiac Regeneration via Cardiomyocyte Proliferation in Experimental Heart Failure

    Science.gov (United States)

    Lam, Nicholas T.; Currie, Peter D.; Lieschke, Graham J.; Rosenthal, Nadia A.; Kaye, David M.

    2012-01-01

    Although the adult heart likely retains some regenerative capacity, heart failure (HF) typically remains a progressive disorder. We hypothesise that alterations in the local environment contribute to the failure of regeneration in HF. Previously we showed that nerve growth factor (NGF) is deficient in the failing heart and here we hypothesise that diminished NGF limits the cardiac regenerative response in HF. The capacity of NGF to augment cardiac regeneration was tested in a zebrafish model of HF. Cardiac injury with a HF phenotype was induced in zebrafish larvae at 72 hours post fertilization (hpf) by exposure to aristolochic acid (AA, 2.5 µM, 72–75 hpf). By 168 hpf, AA induced HF and death in 37.5% and 20.8% of larvae respectively (pheart by 4.8 fold (pheart, mediated by stimulation of cardiomyocyte proliferation. PMID:23300892

  4. MicroRNAs Inducing Proliferation of Quiescent Adult Cardiomyocytes.

    Science.gov (United States)

    Pandey, Raghav; Ahmed, Rafeeq P H

    In the United States, each year over 700,000 people suffer from a heart attack and over 25% of deaths are related to heart disease, making it the leading cause of death. Following ischemic injury a part of the heart muscle is replaced by a scar tissue, reducing its functioning capacity. Recent advancements in surgical intervention and pharmacotherapy only provide symptomatic relief and do not address the root cause of the problem which is the massive loss of cardiomyocytes (CM). Therefore, the development of novel therapeutic intervention for the repair and regeneration of ischemic myocardium remains an area of intense research. While existing CM in zebra fish and neonatal mice are known to proliferate and replenish the infarcted heart, it has been shown that adult mammalian CM lose this ability, thus preventing regeneration of the scar tissue. There have been many attempts to facilitate regeneration of ischemic heart but have met with limited success. Micro-RNAs (miRNAs) are one of the promising candidates towards this goal as they are known to play important regulatory roles during differentiation and tissue regeneration, and regulate genetic information by post-transcriptional modification as well as regulation of other miRNAs. While previous work by Eulalio et al., showed miRNAs inducing proliferation in neonatal CM (NCM), we here identify miRNAs inducing proliferation of rat adult-CM (ACM). This commentary while analyses recent work by Eulalio et al[1] also shows some new data with microRNAs in rat adult-CMs. Further work into the mechanism of these miRNAs can determine their therapeutic potential towards regenerating cardiac tissue post ischemic injury.

  5. Stress-induced cell-cycle activation in Tip60 haploinsufficient adult cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Joseph B Fisher

    Full Text Available BACKGROUND: Tat-interactive protein 60 (Tip60 is a member of the MYST family of histone acetyltransferases. Studies using cultured cells have shown that Tip60 has various functions including DNA repair, apoptosis and cell-cycle regulation. We globally ablated the Tip60 gene (Htatip, observing that Tip60-null embryos die at the blastocyst stage (Hu et al. Dev.Dyn.238:2912;2009. Although adult heterozygous (Tip60(+/- mice reproduce normally without a haploinsufficient phenotype, stress caused by Myc over-expression induced B-cell lymphoma in Tip60(+/- adults, suggesting that Tip60 is a tumor suppressor (Gorrini et al. Nature 448:1063;2007. These findings prompted assessment of whether Tip60, alternative splicing of which generates two predominant isoforms termed Tip60α and Tip60β, functions to suppress the cell-cycle in adult cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting revealed that Tip60α is the predominant Tip60 isoprotein in the embryonic heart, transitioning at neonatal stages to Tip60β, which is the only isoprotein in the adult heart wherein it is highly enriched. Over-expression of Tip60β, but not Tip60α, inhibited cell proliferation in NIH3T3 cells; and, Tip60-haploinsufficient cultured neonatal cardiomyocytes exhibited increased cell-cycle activity. To address whether Tip60β suppresses the cardiomyocyte cell-cycle in the adult heart, hypertrophic stress was induced in Tip60(+/+ and Tip(+/- littermates via two methods, Myc over-expression and aortic banding. Based on immunostaining cell-cycle markers and western blotting cyclin D, stress increased cardiomyocyte cell-cycle mobilization in Tip60(+/- hearts, in comparison with Tip60(+/+ littermates. Aortic-banded Tip60(+/- hearts also exhibited significantly decreased apoptosis. CONCLUSIONS/SIGNIFICANCE: These findings provide evidence that Tip60 may function in a tumor suppressor pathway(s to maintain adult cardiomyocytes in replicative senescence.

  6. Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

    Directory of Open Access Journals (Sweden)

    Nicolas Christoforou

    Full Text Available The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS cells, which once differentiated allow for the enrichment of Nkx2-5(+ cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+ cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological

  7. Hypoxia changes the expression of the epidermal growth factor (EGF) system in human hearts and cultured cardiomyocytes

    DEFF Research Database (Denmark)

    Munk, Mathias; Memon, Ashfaque Ahmed; Goetze, Jens Peter

    2012-01-01

    the mRNA expression by real time PCR of the 4 receptors and 12 ligands from the EGF-system in paired normoxic and hypoxic biopsies isolated from human hearts during coronary artery bypass operation. Compared to normoxic biopsies, hypoxic samples showed down-regulation of HER2 (P = 0.0005) and NRG1 (both......2 down-regulation seen in the human heart in cultured cardiomyocytes inhibited their proliferation under hypoxic conditions. Interestingly, HB-EGF is induced in the hypoxic human hearts, and rescues hypoxic cardiomyocytes from the effect of HER2 inhibition in the in vitro model. The results have...... revealed nuclear translocation of activated MAPK and the activity of this downstream signaling molecule was decreased by HER2 inhibition (20 nM trastuzumab), and re-established by HB-EGF (10 nM). CONCLUSIONS/SIGNIFICANCE: Hypoxia in the human heart alters the expression of the EGF system. Mimicking the HER...

  8. Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications.

    Directory of Open Access Journals (Sweden)

    Maresha S Gay

    Full Text Available The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1, 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.

  9. Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications

    Science.gov (United States)

    Gay, Maresha S.; Li, Yong; Xiong, Fuxia; Lin, Thant; Zhang, Lubo

    2015-01-01

    The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner. PMID:25923220

  10. A p53-based genetic tracing system to follow postnatal cardiomyocyte expansion in heart regeneration.

    Science.gov (United States)

    Xiao, Qi; Zhang, Guoxin; Wang, Huijuan; Chen, Lai; Lu, Shuangshuang; Pan, Dejing; Liu, Geng; Yang, Zhongzhou

    2017-02-15

    In the field of heart regeneration, the proliferative potential of cardiomyocytes in postnatal mice is under intense investigation. However, solely relying on immunostaining of proliferation markers, the long-term proliferation dynamics and potential of the cardiomyocytes cannot be readily addressed. Previously, we found that a p53 promoter-driving reporter predominantly marked the proliferating lineages in mice. Here, we established a p53-based genetic tracing system to investigate postnatal cardiomyocyte proliferation and heart regeneration. By selectively tracing proliferative cardiomyocytes, a differential pattern of clonal expansion in p53(+) cardiac myocytes was revealed in neonatal, adolescent and adult stages. In addition, the percentage of p53(+) lineage cardiomyocytes increased continuously in the first month. Furthermore, these cells rapidly responded to heart injury and greatly contributed to the replenished myocardium. Therefore, this study reveals complex proliferating dynamics in postnatal cardiomyocytes and heart repair, and provides a novel genetic tracing strategy for studying postnatal cardiac turnover and regeneration. © 2017. Published by The Company of Biologists Ltd.

  11. Cardiomyocyte-specific ablation of CD36 accelerates the progression from compensated cardiac hypertrophy to heart failure.

    Science.gov (United States)

    Sung, Miranda M; Byrne, Nikole J; Kim, Ty T; Levasseur, Jody; Masson, Grant; Boisvenue, Jamie J; Febbraio, Maria; Dyck, Jason R B

    2017-03-01

    Previous studies have shown that loss of CD36 protects the heart from dysfunction induced by pressure overload in the presence of diet-induced insulin resistance and/or obesity. The beneficial effects of CD36 ablation in this context are mediated by preventing excessive cardiac fatty acid (FA) entry and reducing lipotoxic injury. However, whether or not the loss of CD36 can prevent pressure overload-induced cardiac dysfunction in the absence of chronic exposure to high circulating FAs is presently unknown. To address this, we utilized a tamoxifen-inducible cardiomyocyte-specific CD36 knockout (icCD36KO) mouse and genetically deleted CD36 in adulthood. Control mice (CD36 floxed/floxed mice) and icCD36KO mice were treated with tamoxifen and subsequently subjected to transverse aortic constriction (TAC) surgery to generate pressure overload-induced cardiac hypertrophy. Consistent with CD36 mediating a significant proportion of FA entry into the cardiomyocyte and subsequent FA utilization for ATP production, hearts from icCD36KO mice were metabolically inefficient and displayed signs of energetic stress, including activation of the energetic stress kinase, AMPK. In addition, impaired energetics in icCD36KO mice contributed to a rapid progression from compensated hypertrophy to heart failure. However, icCD36KO mice fed a medium-chain FA diet, whereby medium-chain FAs can enter into the cardiomyocyte independent from CD36, were protected from TAC-induced heart failure. Together these data suggest that limiting FA uptake and partial inhibition of FA oxidation in the heart via CD36 ablation may be detrimental for the compensated hypertrophic heart in the absence of sufficiently elevated circulating FAs to provide an adequate energy source. NEW & NOTEWORTHY Limiting CD36-mediated fatty acid uptake in the setting of obesity and/or insulin resistance protects the heart from cardiac hypertrophy and dysfunction. However, cardiomyocyte-specific CD36 ablation in the absence of

  12. Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block.

    Science.gov (United States)

    Salomonsson, Stina; Sonesson, Sven-Erik; Ottosson, Lars; Muhallab, Saad; Olsson, Tomas; Sunnerhagen, Maria; Kuchroo, Vijay K; Thorén, Peter; Herlenius, Eric; Wahren-Herlenius, Marie

    2005-01-03

    Congenital heart block develops in fetuses after placental transfer of Ro/SSA autoantibodies from rheumatic mothers. The condition is often fatal and the majority of live-born children require a pacemaker at an early age. The specific antibody that induces the heart block and the mechanism by which it mediates the pathogenic effect have not been elucidated. In this study, we define the cellular mechanism leading to the disease and show that maternal autoantibodies directed to a specific epitope within the leucine zipper amino acid sequence 200-239 (p200) of the Ro52 protein correlate with prolongation of fetal atrioventricular (AV) time and heart block. This finding was further confirmed experimentally in that pups born to rats immunized with p200 peptide developed AV block. p200-specific autoantibodies cloned from patients bound cultured cardiomyocytes and severely affected Ca2+ oscillations, leading to accumulating levels and overload of intracellular Ca2+ levels with subsequent loss of contractility and ultimately apoptosis. These findings suggest that passive transfer of maternal p200 autoantibodies causes congenital heart block by dysregulating Ca2+ homeostasis and inducing death in affected cells.

  13. Transcription factor GATA4 inhibits doxorubicin-induced autophagy and cardiomyocyte death.

    Science.gov (United States)

    Kobayashi, Satoru; Volden, Paul; Timm, Derek; Mao, Kai; Xu, Xianmin; Liang, Qiangrong

    2010-01-01

    Doxorubicin (DOX) is a potent anti-tumor drug known to cause heart failure. The transcription factor GATA4 antagonizes DOX-induced cardiotoxicity. However, the protective mechanism remains obscure. Autophagy is the primary cellular pathway for lysosomal degradation of long-lived proteins and organelles, and its activation could be either protective or detrimental depending on specific pathophysiological conditions. Here we investigated the ability of GATA4 to inhibit autophagy as a potential mechanism underlying its protection against DOX toxicity in cultured neonatal rat cardiomyocytes. DOX markedly increased autophagic flux in cardiomyocytes as indicated by the difference in protein levels of LC3-II (microtubule-associated protein light chain 3 form 2) or numbers of autophagic vacuoles in the absence and presence of the lysosomal inhibitor bafilomycin A1. DOX-induced cardiomyocyte death determined by multiple assays was aggravated by a drug or genetic approach that activates autophagy, but it was attenuated by manipulations that inhibit autophagy, suggesting that autophagy contributes to DOX cardiotoxicity. DOX treatment depleted GATA4 protein levels, which predisposed cardiomyocytes to DOX toxicity. Indeed, GATA4 gene silencing triggered autophagy that rendered DOX more toxic, whereas GATA4 overexpression inhibited DOX-induced autophagy, reducing cardiomyocyte death. Mechanistically, GATA4 up-regulated gene expression of the survival factor Bcl2 and suppressed DOX-induced activation of autophagy-related genes, which may likely be responsible for the anti-apoptotic and anti-autophagic effects of GATA4. Together, these findings suggest that activation of autophagy mediates DOX cardiotoxicity, and preservation of GATA4 attenuates DOX cardiotoxicity by inhibiting autophagy through modulation of the expression of Bcl2 and autophagy-related genes.

  14. CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart

    Science.gov (United States)

    Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F.; Roy, Anna R.; Galjart, Niels; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis

    2017-01-01

    Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development. PMID:28846746

  15. CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

    Science.gov (United States)

    Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Tena, Juan J; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F; Roy, Anna R; Galjart, Niels; Delgado-Olguin, Paul; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis; Manzanares, Miguel

    2017-08-01

    Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

  16. Talin Is Required Continuously for Cardiomyocyte Remodeling during Heart Growth in Drosophila.

    Directory of Open Access Journals (Sweden)

    Simina Bogatan

    Full Text Available Mechanotransduction of tension can govern the remodeling of cardiomyocytes during growth or cardiomyopathy. Tension is signaled through the integrin adhesion complexes found at muscle insertions and costameres but the relative importance of signalling during cardiomyocyte growth versus remodelling has not been assessed. Employing the Drosophila cardiomyocyte as a genetically amenable model, we depleted the levels of Talin, a central component of the integrin adhesion complex, at different stages of heart growth and remodeling. We demonstrate a continuous requirement for Talin during heart growth to maintain the one-to-one apposition of myofibril ends between cardiomyocytes. Retracted myofibrils cannot regenerate appositions to adjacent cells after restoration of normal Talin expression, and the resulting deficit reduces heart contraction and lifespan. Reduction of Talin during heart remodeling after hatching or during metamorphosis results in pervasive degeneration of cell contacts, myofibril length and number, for which restored Talin expression is insufficient for regeneration. Resultant dilated cardiomyopathy results in a fibrillating heart with poor rhythmicity. Cardiomyocytes have poor capacity to regenerate deficits in myofibril orientation and insertion, despite an ongoing capacity to remodel integrin based adhesions.

  17. Regression of Copper-Deficient Heart Hypertrophy: Reduction in the Size of Hypertrophic Cardiomyocytes

    Science.gov (United States)

    Dietary copper deficiency causes cardiac hypertrophy and its transition to heart failure in a mouse model. Copper repletion results in a rapid regression of cardiac hypertrophy and prevention of heart failure. The present study was undertaken to understand dynamic changes of cardiomyocytes in the hy...

  18. Finding the rhythm of sudden cardiac death: new opportunities using induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Sallam, Karim; Li, Yingxin; Sager, Philip T; Houser, Steven R; Wu, Joseph C

    2015-06-05

    Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death. © 2015 American Heart Association, Inc.

  19. Overexpression of Cardiomyocyte Alpha1A-Adrenergic Receptors Attenuates Post-Infarct Remodeling by Inducing Angiogenesis Through Heterocellular Signaling

    Science.gov (United States)

    Zhao, Xin; Balaji, Poornima; Pachon, Ronald; Beniamen, Daniella M.; Vatner, Dorothy E.; Graham, Robert M.; Vatner, Stephen F.

    2015-01-01

    Objective We tested the hypothesis that simulation of cardiac α1A-adrenergic receptors (α1A-AR) protects against the development of heart failure through induction of angiogenesis. Approach and Results 4–6 weeks after permanent coronary artery occlusion (CAO), transgenic (TG) rats with cardiomyocyte-specific α1A-AR overexpression had less remodeling than their non-transgenic littermates (NTLs), with less fibrosis, hypertrophy and lung wt, and preserved left ventricular ejection fraction and wall stress, all p<0.05. Coronary blood flow, measured with microspheres, increased in the infarct zone in TG compared to NTLs (1.4±0.2 vs. 0.5±0.08ml/min/g) (p<0.05), which is consistent with angiogenesis, as reflected by a 20% increase in capillary density in the zone adjacent to the infarct. The question arose, how does TG overexpression of a gene in cardiomyocytes induce angiogenesis? We identified a paracrine mechanism, whereby VEGF-A mRNA and protein were increased in isolated TG cardiomyocytes, and also by NTL cardiomyocytes treated with an α1A-agonist, resulting in angiogenesis. Conditioned medium from cultured TG cardiomyocytes enhanced human umbilical vein endothelial cell (HUVEC) tubule formation, which was blocked by an anti-VEGF-A antibody. Moreover, improved cardiac function, blood flow and increased capillary density after chronic CAO in TG rats were blocked by either a MEK or a VEGF-A inhibitor. Conclusions Cardiomyocyte-specific overexpression of the α1A-AR resulted in enhanced MEK-dependent cardiomyocyte VEGF-A expression, which stimulates angiogenesis via a paracrine mechanism involving heterocellular cardiomyocyte/endothelial cell signalling, protecting against remodeling and heart failure following chronic CAO. PMID:26338300

  20. MiR-21 Protected Cardiomyocytes against Doxorubicin-Induced Apoptosis by Targeting BTG2

    Directory of Open Access Journals (Sweden)

    Zhongyi Tong

    2015-06-01

    Full Text Available Doxorubicin (DOX is an anthracycline drug with a wide spectrum of antineoplastic activities. However, it causes cardiac cytotoxicity, and this limits its clinical applications. MicroRNA-21 (miR-21 plays a vital role in regulating cell proliferation and apoptosis. While miR-21 is preferentially expressed in adult cardiomyocytes and involved in cardiac development and heart disease, little is known regarding its biological functions in responding to DOX-induced cardiac cytotoxicity. In this study, the effects of DOX on mouse cardiac function and the expression of miR-21 were examined in both mouse heart tissues and rat H9C2 cardiomyocytes. The results showed that the cardiac functions were more aggravated in chronic DOX injury mice compared with acute DOX-injury mice; DOX treatment significantly increased miR-21 expression in both mouse heart tissue and H9C2 cells. Over-expression of miR-21 attenuated DOX-induced apoptosis in cardiamyocytes whereas knocking down its expression increased DOX-induced apoptosis. These gain- and loss- of function experiments showed that B cell translocation gene 2 (BTG2 was a target of miR-21. The expression of BTG2 was significantly decreased both in myocardium and H9C2 cells treated with DOX. The present study has revealed that miR-21 protects mouse myocardium and H9C2 cells against DOX-induced cardiotoxicity probably by targeting BTG2.

  1. Insulin/NFκB protects against ischemia-induced necrotic cardiomyocyte death.

    Science.gov (United States)

    Díaz, Ariel; Humeres, Claudio; González, Verónica; Gómez, María Teresa; Montt, Natalia; Sanchez, Gina; Chiong, Mario; García, Lorena

    2015-11-13

    In the heart, insulin controls key functions such as metabolism, muscle contraction and cell death. However, all studies have been focused on insulin action during reperfusion. Here we explore the cardioprotective action of this hormone during ischemia. Rat hearts were perfused ex vivo with an ischemia/reperfusion Langendorff model in absence or presence of insulin. Additionally, cultured rat cardiomyocytes were exposed to simulated ischemia in the absence or presence of insulin. Cytoprotective effects were measured by myocardial infarct size, trypan blue exclusion, released LDH and DNA fragmentation by flow cytometry. We found that insulin protected against cardiac ischemia ex vivo and in vitro. Moreover, insulin protected cardiomyocytes from simulated ischemia by reducing necrotic cell death. Protective effects of insulin were dependent of Akt and NFκB. These novel results show that insulin reduces ischemia-induced cardiomyocyte necrosis through an Akt/NF-κB dependent mechanism. These novel findings clarify the role of insulin during ischemia and further support its use in early GIK perfusion to treat myocardial infarction. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. NF-κB (p65) negatively regulates myocardin-induced cardiomyocyte hypertrophy through multiple mechanisms.

    Science.gov (United States)

    Liao, Xing-Hua; Wang, Nan; Zhao, Dong-Wei; Zheng, De-Liang; Zheng, Li; Xing, Wen-Jing; Zhou, Hao; Cao, Dong-Sun; Zhang, Tong-Cun

    2014-12-01

    Myocardin is well known to play a key role in the development of cardiomyocyte hypertrophy. But the exact molecular mechanism regulating myocardin stability and transactivity to affect cardiomyocyte hypertrophy has not been studied clearly. We now report that NF-κB (p65) can inhibit myocardin-induced cardiomyocyte hypertrophy. Then we explore the molecular mechanism of this response. First, we show that p65 can functionally repress myocardin transcriptional activity and also reduce the protein expression of myocardin. Second, the function of myocardin can be regulated by epigenetic modifications. Myocardin sumoylation is known to transactivate cardiac genes, but whether p65 can inhibit SUMO modification of myocardin is still not clear. Our data show that p65 weakens myocardin transcriptional activity through attenuating SUMO modification of myocardin by SUMO1/PIAS1, thereby impairing myocardin-mediated cardiomyocyte hypertrophy. Furthermore, the expression of myocardin can be regulated by several microRNAs, which play important roles in the development and function of the heart and muscle. We next investigated potential role of miR-1 in cardiac hypotrophy. Our results show that p65 can upregulate the level of miR-1 and miR-1 can decrease protein expression of myocardin in cardiac myocytes. Notably, miR-1 expression is also controlled by myocardin, leading to a feedback loop. These data thus provide important and novel insights into the function that p65 inhibits myocardin-mediated cardiomyocyte hypertrophy by downregulating the expression and SUMO modification of myocardin and enhancing the expression of miR-1. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Growth hormone secretagogues preserve the electrophysiological properties of mouse cardiomyocytes isolated from in vitro ischemia/reperfusion heart.

    Science.gov (United States)

    Ma, Yi; Zhang, Lin; Launikonis, Bradley S; Chen, Chen

    2012-11-01

    Ischemic heart diseases often induce cardiac arrhythmia with irregular cardiac action potential (AP). This study aims to demonstrate that GH secretagogues (GHS) ghrelin and its synthetic analog hexarelin can preserve the electrophysiological properties of cardiomyocytes experiencing ischemia/reperfusion (I/R). Isolated hearts from adult male mice underwent 20 min global ischemia followed by 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nM) or hexarelin (1 nM) was administered in the perfusion solution either 10 min before or after ischemia, termed pre- or posttreatments. Cardiomyocytes isolated from these hearts were used for whole-cell patch clamping to measure AP, voltage-gated L-type calcium current (I(CaL)), transient outward potassium current (I(to)), and sodium current (I(Na)). AP amplitude and duration were significantly decreased by I/R, but GHS treatments maintained their normality. GHS treatments prevented the decrease in I(CaL) and I(Na) after I/R, thereby maintaining AP amplitude. Although the significant increase in I(to) after I/R partially explained the shortened AP duration, the normalization of it by GHS treatments might contribute to the preservation of AP duration. Phosphorylated p38 and c-Jun NH(2)-terminal kinase and the downstream active caspase-9 in the cellular apoptosis pathway were significantly increased after I/R but not when GHS treatments were included, whereas phosphorylation of ERK1/2 associated with cell survival showed increase after I/R and a further increase after GHS treatments by binding to its receptor GHS receptor type 1a. These results suggest GHS can not only preserve the electrophysiological properties of cardiomyocytes after I/R but also inhibit cardiomyocyte apoptosis and promote cell survival by modification of MAPK pathways through activating GHS receptor type 1a.

  4. Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations

    Science.gov (United States)

    Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H.; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns

    2013-01-01

    With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47–50 or 48–50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart. PMID:23829870

  5. Liensinine- and Neferine-Induced Cardiotoxicity in Primary Neonatal Rat Cardiomyocytes and Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Yangyang Yu

    2016-01-01

    Full Text Available Due to drug-induced potential congestive heart failure and irreversible dilated cardiomyopathies, preclinical evaluation of cardiac dysfunction is important to assess the safety of traditional or novel treatments. The embryos of Nelumbo nucifera Gaertner seeds are a homology of traditional Chinese medicine and food. In this study, we applied the real time cellular analysis (RTCA Cardio system, which can real-time monitor the contractility of cardiomyocytes (CMs, to evaluate drug safety in rat neonatal CMs and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs. This study showed detailed biomechanical CM contractility in vitro, and provided insights into the cardiac dysfunctions associated with liensinine and neferine treatment. These effects exhibited dose and time-dependent recovery. Neferine showed stronger blocking effect in rat neonatal CMs than liensinine. In addition, the effects of liensinine and neferine were further evaluated on hiPS-CMs. Our study also indicated that both liensinine and neferine can cause disruption of calcium homeostasis. For the first time, we demonstrated the potential cardiac side effects of liensinine or neferine. While the same inhibition was observed on hiPS-CMs, more importantly, this study introduced an efficient and effective approach to evaluate the cardiotoxicity of the existing and novel drug candidates.

  6. DHEA prevents mineralo- and glucocorticoid receptor-induced chronotropic and hypertrophic actions in isolated rat cardiomyocytes.

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    Mannic, Tiphaine; Mouffok, Mounira; Python, Magaly; Yoshida, Takehisa; Maturana, Andres D; Vuilleumier, Nicolas; Rossier, Michel F

    2013-03-01

    Corticosteroids have been involved in the genesis of ventricular arrhythmias associated with pathological heart hypertrophy, although molecular mechanisms responsible for these effects have not been completely explained. Because mineralocorticoid receptor (MR) antagonists have been demonstrated to be beneficial on the cardiac function, much attention has been given to the action of aldosterone on the heart. However, we have previously shown that both aldosterone and corticosterone in vitro induce a marked acceleration of the spontaneous contractions, as well as a significant cell hypertrophy in isolated neonate rat ventricular cardiomyocytes. Moreover, a beneficial role of the steroid hormone dehydroepiandrosterone (DHEA) has been also proposed, but the mechanism of its putative cardioprotective function is not known. We found that DHEA reduces both the chronotropic and the hypertrophic responses of cardiomyocytes upon stimulation of MR and glucocorticoid receptor (GR) in vitro. DHEA inhibitory effects were accompanied by a decrease of T-type calcium channel expression and activity, as assessed by quantitative PCR and the patch-clamp technique. Prevention of cell hypertrophy by DHEA was also revealed by measuring the expression of A-type natriuretic peptide and BNP. The kinetics of the negative chronotropic effect of DHEA, and its sensitivity to actinomycin D, pointed out the presence of both genomic and nongenomic mechanisms of action. Although the genomic action of DHEA was effective mostly upon MR activation, its rapid, nongenomic response appeared related to DHEA antioxidant properties. On the whole, these results suggest new mechanisms for a putative cardioprotective role of DHEA in corticosteroid-associated heart diseases.

  7. Substance P Receptor Signaling Mediates Doxorubicin-Induced Cardiomyocyte Apoptosis and Triple-Negative Breast Cancer Chemoresistance.

    Science.gov (United States)

    Robinson, Prema; Kasembeli, Moses; Bharadwaj, Uddalak; Engineer, Nikita; Eckols, Kris T; Tweardy, David J

    2016-01-01

    Doxorubicin (DOX), an anthracycline, is broadly considered the most active single agent available for treating breast cancer but has been known to induce cardiotoxicity. Although DOX is highly effective in treating triple-negative breast cancer (TNBC), DOX can have poor outcomes owing to induction of chemoresistance. There is an urgent need to develop new therapies for TNBC aimed at improving DOX outcome and DOX-induced cardiotoxicity. Substance P (SP), a neuropeptide involved in pain transmission is known to stimulate production of reactive oxygen species (ROS). Elevated cardiac ROS is linked with heart injury and failure. We investigated the role of SP in chemotherapy-associated death of cardiomyocytes and chemoresistance. We showed that pretreating a cardiomyocyte cell line (H9C2) and a TNBC cell line (MDA-MB 231) with aprepitant, a SP receptor antagonist that is routinely used to treat chemotherapy-associated associated nausea, decreased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in cardiomyocytes and increased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in TNBC cells compared with cells treated with DOX alone. Our findings demonstrate the ability of aprepitant to decrease DOX-induced killing of cardiomyocytes and to increase cancer cell sensitivity to DOX, which has tremendous clinical significance.

  8. Lactation Is a Risk Factor of Postpartum Heart Failure in Mice with Cardiomyocyte-specific Apelin Receptor (APJ) Overexpression.

    Science.gov (United States)

    Murata, Kazuya; Ishida, Junji; Ishimaru, Tomohiro; Mizukami, Hayase; Hamada, Juri; Saito, Chiaki; Fukamizu, Akiyoshi

    2016-05-20

    The G protein-coupled receptor APJ and its ligand apelin are highly expressed in cardiovascular tissues and are associated with the regulation of blood pressure and cardiac function. Although accumulating evidence suggests that APJ plays a crucial role in the heart, it remains unclear whether up-regulation of APJ affects cardiac function. Here we generated cardiomyocyte-specific APJ-overexpressing (APJ-TG) mice and investigated the cardiac phenotype in APJ-TG mice. Male and non-pregnant APJ-TG mice showed cardiac hypertrophy, contractile dysfunction, and elevation of B-type natriuretic peptide gene expression in the heart but not cardiac fibrosis and symptoms of heart failure, including breathing abnormality and pleural effusion. We further examined the influence of APJ overexpression in response to physiological stress induced by pregnancy and lactation in the heart. Interestingly, repeating pregnancy and lactation (pregnancy-lactation cycle) exacerbated cardiac hypertrophy and systolic dysfunction and induced cardiac fibrosis, lung congestion, pleural effusion, and abnormal breathing in APJ-TG mice. These data indicate that female APJ-TG mice develop postpartum cardiomyopathy. We showed that lactation, but not parturition, was critical for the onset of postpartum cardiomyopathy in APJ-TG mice. Furthermore, we found that lactating APJ-TG mice showed impaired myocardial angiogenesis and imbalance of pro- and antiangiogenic gene expression in the heart. These results demonstrate that overexpression of APJ in cardiomyocytes has adverse effects on cardiac function in male and non-pregnant mice and that lactation contributes to the development of postpartum cardiomyopathy in the heart with APJ overexpression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. The role of oxidative stress in high glucose-induced apoptosis in neonatal rat cardiomyocytes.

    Science.gov (United States)

    Zhou, Xiang; Lu, Xiang

    2013-08-01

    Accumulating evidence has demonstrated that apoptosis plays a critical role in the pathogenesis of diabetic cardiomyopathy. However, the exact molecular mechanisms by which hyperglycaemia induces cardiomyocyte apoptosis are not fully understood. The present study was designed to investigate the role of oxidative stress in high glucose-induced apoptosis in cultured neonatal rat cardiomyocytes. The MTT assay was used to detect the viability of cardiomyocytes exposed to different concentrations of glucose. Oxidative stress was evaluated by measuring intracellular reactive oxygen species with 2',7'-dichlorofluoresce diacetate staining and by detecting malondialdehyde and superoxide dismutase in the supernatant of culture media. Cardiomyocyte apoptosis was determined by flow cytometry and confocal laser scanning microscopy with Annexin V/PI staining. Our results showed that high glucose can induce oxidative stress and promote apoptosis in neonatal rat cardiomyocytes and the antioxidant can protect against high glucose-induced apoptosis, which suggests that oxidative stress is involved in high glucose-induced cardiomyocyte apoptosis. Furthermore, caspase-3 was found to be activated in the process of high glucose-induced oxidative stress, which subsequently contributes to increased apoptosis in neonatal rat cardiomyocytes. In conclusion, our study demonstrates that oxidative stress is involved in high glucose-induced cardiomyocyte apoptosis via activation of caspase-3.

  10. SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ashok, Ajay [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288 (United States); Kanwar, Jagat Rakesh [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Krishnan, Uma Maheswari [Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), School of Chemical & Biotechnology (SCBT), SASTRA University, Thanjavur 613401 (India); Kanwar, Rupinder Kaur, E-mail: rupinder.kanwar@deakin.edu.au [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia)

    2017-01-01

    Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxic microenvironment in vitro, well characterized HCM were treated with 100 µm (48 h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1 µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic injury

  11. Modeling Catecholaminergic Polymorphic Ventricular Tachycardia using Induced Pluripotent Stem Cell-derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Atara Novak

    2012-07-01

    Full Text Available Catecholaminergic polymorphic ventricular tachycardia (CPVT is an inherited arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias induced by physical or emotional stress, in the absence structural heart abnormalities. The arrhythmias may cause syncope or degenerate into cardiac arrest and sudden death which usually occurs during childhood. Recent studies have shown that CPVT is caused by mutations in the cardiac ryanodine receptor type 2 (RyR2 or calsequestrin 2 (CASQ2 genes. Both proteins are key contributors to the intracellular Ca2+ handling process and play a pivotal role in Ca2+ release from the sarcoplasmic reticulum to the cytosol during systole. Although the molecular pathogenesis of CPVT is not entirely clear, it was suggested that the CPVT mutations promote excessive sarcoplasmic reticulum Ca2+ leak, which initiates delayed afterdepolarizations (DADs and triggered arrhythmias in cardiac myocytes. The recent breakthrough discovery of induced pluripotent stem cells (iPSC generated from somatic cells (e.g. fibroblasts, keratinocytes now enables researches to investigate mutated cardiomyocytes generated from the patient’s iPSC. To this end, in the present article we review recent studies on CPVT iPSC-derived cardiomyocytes, thus demonstrating in the mutated cells catecholamine-induced DADs and triggered arrhythmias.

  12. Calcium handling in human induced pluripotent stem cell derived cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Ilanit Itzhaki

    Full Text Available BACKGROUND: The ability to establish human induced pluripotent stem cells (hiPSCs by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca(2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs. METHODOLOGY/PRINCIPAL FINDINGS: RT-PCR and immunocytochemistry experiments identified the expression of key Ca(2+-handling proteins. Detailed laser confocal Ca(2+ imaging demonstrated spontaneous whole-cell [Ca(2+](i transients. These transients required Ca(2+ influx via L-type Ca(2+ channels, as demonstrated by their elimination in the absence of extracellular Ca(2+ or by administration of the L-type Ca(2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR-mediated sarcoplasmic reticulum (SR Ca(2+ store, contributing to [Ca(2+](i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca(2+ and ryanodine (decreasing [Ca(2+](i. Similarly, the importance of Ca(2+ reuptake into the SR via the SR Ca(2+ ATPase (SERCA pump was demonstrated by the inhibiting effect of its blocker (thapsigargin, which led to [Ca(2+](i transients elimination. Finally, the presence of an IP3-releasable Ca(2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca(2+](i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB and the phospholipase C inhibitor U73122. CONCLUSIONS/SIGNIFICANCE: Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca(2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca(2+](i transients in hiPSC-CMs on both sarcolemmal Ca(2+ entry via L-type Ca(2+ channels and intracellular store Ca(2+ release.

  13. Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model.

    Science.gov (United States)

    Sugiura, Tadahisa; Hibino, Narutoshi; Breuer, Christopher K; Shinoka, Toshiharu

    2016-12-01

    Thousands of babies are born with congenital heart defects that require surgical repair involving a prosthetic implant. Lack of growth in prosthetic grafts is especially detrimental in pediatric surgery. Cell seeded biodegradable tissue engineered grafts are a novel solution to this problem. The purpose of the present study is to evaluate the feasibility of seeding human induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) onto a biodegradable cardiac patch. The hiPS-CMs were cultured on a biodegradable patch composed of a polyglycolic acid (PGA) and a 50:50 poly (l-lactic-co-ε-caprolactone) copolymer (PLCL) for 1 week. Male athymic rats were randomly divided into 2 groups of 10 animals each: 1. hiPS-CM seeded group, and 2. Unseeded group. After culture, the cardiac patch was implanted to repair a defect with a diameter of 2 mm created in the right ventricular outflow tract (RVOT) wall. Hearts were explanted at 4 (n = 2), 8 (n = 2), and 16 (n = 6) weeks after patch implantation. Explanted patches were assessed immunohistochemically. Seeded patch explants did not stain positive for α-actinin (marker of cardiomyocytes) at the 4 week time point, suggesting that the cultured hiPS-CMs evacuated the patch in the early phase of tissue remodeling. However, after 16 weeks implantation, the area fraction of positively stained α-actinin cells was significantly higher in the seeded group than in the unseeded group (Seeded group: 6.1 ± 2.8% vs. Unseeded group: 0.95 ± 0.50%, p = 0.004), suggesting cell seeding promoted regenerative proliferation of host cardiomyocytes. Seeded hiPS-CMs were not present in the patch after 4 weeks. However, we surmise that they influenced the regeneration of host cardiomyocytes via a paracrine mechanism. Tissue-engineered hiPS-CMs seeded cardiac patches warrant further investigation for use in the repair of congenital heart diseases.

  14. Propofol ameliorates doxorubicin-induced oxidative stress and cellular apoptosis in rat cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lai, H.C. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yeh, Y.C. [Graduate Institute of Natural Healing Sciences, Nanhua University, Chiayi, Taiwan (China); Wang, L.C. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Ting, C.T.; Lee, W.L. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Lee, H.W. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, K.Y. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine, Chung-Shan Medical University, Taichung, Taiwan (China); Wu, A. [College of Biological Science, University of California, Davis (United States); Su, C.S. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Liu, T.J., E-mail: trliu@vghtc.gov.tw [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China)

    2011-12-15

    Background: Propofol is an anesthetic with pluripotent cytoprotective properties against various extrinsic insults. This study was designed to examine whether this agent could also ameliorate the infamous toxicity of doxorubicin, a widely-used chemotherapeutic agent against a variety of cancer diseases, on myocardial cells. Methods: Cultured neonatal rat cardiomyocytes were administrated with vehicle, doxorubicin (1 {mu}M), propofol (1 {mu}M), or propofol plus doxorubicin (given 1 h post propofol). After 24 h, cells were harvested and specific analyses regarding oxidative/nitrative stress and cellular apoptosis were conducted. Results: Trypan blue exclusion and MTT assays disclosed that viability of cardiomyocytes was significantly reduced by doxorubicin. Contents of reactive oxygen and nitrogen species were increased and antioxidant enzymes SOD1, SOD2, and GPx were decreased in these doxorubicin-treated cells. Mitochondrial dehydrogenase activity and membrane potential were also depressed, along with activation of key effectors downstream of mitochondrion-dependent apoptotic signaling. Besides, abundance of p53 was elevated and cleavage of PKC-{delta} was induced in these myocardial cells. In contrast, all of the above oxidative, nitrative and pro-apoptotic events could be suppressed by propofol pretreatment. Conclusions: Propofol could extensively counteract oxidative/nitrative and multiple apoptotic effects of doxorubicin in the heart; hence, this anesthetic may serve as an adjuvant agent to assuage the untoward cardiac effects of doxorubicin in clinical application. -- Highlights: Black-Right-Pointing-Pointer We evaluate how propofol prevents doxorubicin-induced toxicity in cardiomyocytes. Black-Right-Pointing-Pointer Propofol reduces doxorubicin-imposed nitrative and oxidative stress. Black-Right-Pointing-Pointer Propofol suppresses mitochondrion-, p53- and PKC-related apoptotic signaling. Black-Right-Pointing-Pointer Propofol ameliorates apoptosis and

  15. Sulforaphane protects H9c2 cardiomyocytes from angiotensin II-induced hypertrophy.

    Science.gov (United States)

    Wu, Q-Q; Zong, J; Gao, L; Dai, J; Yang, Z; Xu, M; Fang, Y; Ma, Z-G; Tang, Q-Z

    2014-05-01

    Cardiac hypertrophy is an adaptive process of the heart in response to various stimuli, but sustained cardiac hypertrophy will finally lead to heart failure. Sulforaphane-extracted from cruciferous vegetables of the genus Brassica such as broccoli, brussels sprouts, and cabbage-has been evaluated for its anticarcinogenic and antioxidant effects. To investigate the effect of sulforaphane on angiotensin II (Ang II)-induced cardiac hypertrophy in vitro. Embryonic rat heart-derived H9c2 cells were co-incubated with sulforaphane and Ang II. The cell surface area and mRNA levels of hypertrophic markers were measured to clarify the effect of sulforaphane on cardiac hypertrophy. The underlying mechanism was further investigated by detecting the activation of Akt and NF-κB signaling pathways. We found that H9c2 cells pretreated with sulforaphane were protected from Ang II-induced hypertrophy. The increasing mRNA levels of ANP, BNP, and β-MHC in Ang II-stimulated cells were also down-regulated after sulforaphane treatment. Moreover, sulforaphane repressed the Ang II-induced phosphorylation of Akt, GSK3β, mTOR, eIF4e, as well as of IκBα and NF-κB. Based on our results, sulforaphane attenuates Ang II-induced hypertrophy of H9c2 cardiomyocytes mediated by the inhibition of intracellular signaling pathways including Akt and NF-κB.

  16. E2F6 protein levels modulate drug induced apoptosis in cardiomyocytes.

    Science.gov (United States)

    Major, Jennifer L; Salih, Maysoon; Tuana, Balwant S

    2017-12-01

    The E2F/Rb pathway regulates cell growth, differentiation, and death. In particular, E2F1 promotes apoptosis in all cells including those of the heart. E2F6, which represses E2F activity, was found to induce dilated cardiomyopathy in the absence of apoptosis in murine post-natal heart. Here we evaluate the anti-apoptotic potential of E2F6 in neonatal cardiomyocytes (NCM) from E2F6-Tg hearts which showed significantly less caspase-3 cleavage, a lower Bax/Bcl2 ratio, and improved cell viability in response to CoCl2 exposure. This correlated with a decrease in the pro-apoptotic E2F3 protein levels. In contrast, no difference in apoptotic markers or cell viability was observed in response to Doxorubicin (Dox) treatment between Wt and Tg-NCM. Dox caused a rapid and dramatic loss of the E2F6 protein in Tg-NCM within 6h and was undetectable after 12h. The level of e2f6 transcript was unchanged in Wt NCM, but was dramatically decreased in Tg cells in response to both Dox and CoCl2. This was related to an impact of the drugs on the α-myosin heavy chain promoter used to drive the E2F6 transgene. By comparison in HeLa, Dox induced apoptosis through upregulation of endogenous E2F1 involving post-transcriptional mechanisms, while E2F6 was down regulated with induction of the Checkpoint kinase-1 and proteasome degradation. These data imply that E2F6 serves to modulate E2F activity and protect cells including cardiomyocytes from apoptosis and improve survival. Strategies to modulate E2F6 levels may be therapeutically useful to mitigate cell death associated disorders. Copyright © 2017. Published by Elsevier Inc.

  17. EMF protects cardiomyocytes against hypoxia-induced injury via heat shock protein 70 activation.

    Science.gov (United States)

    Wei, Jinhong; Tong, Jie; Yu, Liying; Zhang, Jianbao

    2016-03-25

    Intracellular calcium (Ca(2+)i) overload induced by chronic hypoxia alters Ca(2+)i homeostasis, whereas ameliorating calcium homeostasis is believed to be responsible for cardioprotection. We hypothesize that cardioprotection by electromagnetic fields (EMF) exposure may restore Ca(2+)i homeostasis altered by hypoxia insults. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to chronic hypoxia (1% O2, 5% CO2, 37 °C). We observed that cardiomyocytes injury and hypertrophy were alleviated in hypoxic cardiomyocytes exposed with EMF preconditioning. Compared with hypoxic cardiomyocytes, the diastolic [Ca(2+)]i was decreased, the amplitude of Ca(2+)i oscillations was recovered when cardiomyocytes exposed with EMF. In addition, we also found that EMF exposure significantly increased heat shock protein 70 (HSP70) mRNA expression in hypoxic cardiomyocytes. However, treatment with HSP70 blocker KNK437, almost completely inhibited the EMF induced-cardioprotection and the beneficial effects of Ca(2+) oscillation in hypoxic cardiomyocytes. These results suggest that EMF preconditioning ameliorates Ca(2+)i homeostasis through activating HSP70, thereby producing the cardioprotective effect and reduction in hypoxic cardiomyocytes damage. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Thymosin β4 Prevents Angiotensin II-Induced Cardiomyocyte Growth by Regulating Wnt/WISP Signaling.

    Science.gov (United States)

    Li, Li; Guleria, Rakeshwar S; Thakur, Suresh; Zhang, Cheng-Lin; Pan, Jing; Baker, Kenneth M; Gupta, Sudhiranjan

    2016-08-01

    Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tβ4 in cardiomyocyte hypertrophy is currently unknown. The purpose of this study was to determine the cardio-protective effect of Tβ4 in angiotensin II (Ang II)-induced cardiomyocyte growth. Neonatal rat ventricular cardiomyocytes (NRVM) were pretreated with Tβ4 followed by Ang II stimulation. Cell size, hypertrophy marker gene expression and Wnt signaling components, β-catenin, and Wnt-induced secreted protein-1 (WISP-1) were evaluated by quantitative real-time PCR, Western blotting and fluorescent microscopy. Pre-treatment of Tβ4 resulted in reduction of cell size, hypertrophy marker genes and Wnt-associated gene expression, and protein levels; induced by Ang II in cardiomyocyte. WISP-1 was overexpressed in NRVM and, the effect of Tβ4 in Ang II-induced cardiomyocyte growth was evaluated. WISP-1 overexpression promoted cardiomyocytes growth and was reversed by pretreatment with Tβ4. This is the first report which demonstrates that Tβ4 targets Wnt/WISP-1 to protect Ang II-induced cardiomyocyte growth. J. Cell. Physiol. 231: 1737-1744, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  19. Coexpression of VEGF and angiopoietin-1 promotes angiogenesis and cardiomyocyte proliferation reduces apoptosis in porcine myocardial infarction (MI) heart.

    Science.gov (United States)

    Tao, Zhengxian; Chen, Bo; Tan, Xiao; Zhao, Yingming; Wang, Liansheng; Zhu, Tiebing; Cao, Kejiang; Yang, Zhijian; Kan, Yuet Wai; Su, Hua

    2011-02-01

    VEGF and angiopoietin-1 (Ang1) are two major angiogenic factors being investigated for the treatment of myocardial infarction (MI). Targeting VEGF and Ang1 expression in the ischemic myocardium can increase their local therapeutic effects and reduce possible adverse effects. Adeno-associated viral vectors (AAVs) expressing cardiac-specific and hypoxia-inducible VEGF [AAV-myosin light chain-2v (MLC)VEGF] and Ang1 (AAV-MLCAng1) were coinjected (VEGF/Ang1 group) into six different sites of the porcine myocardium at the peri-infarct zone immediately after ligating the left descending coronary artery. An identical dose of AAV-Cytomegalovirus (CMV)LacZ or saline was injected into control animals. AAV genomes were detected in the liver in addition to the heart. RT-PCR, Western blotting, and ELISA analyses showed that VEGF and Ang1 were predominantly expressed in the myocardium in the infarct core and border of the infarct heart. Gated single-photon emission computed tomography analyses showed that the VEGF/Ang1 group had better cardiac function and myocardial perfusion at 8 wk than at 2 wk after vector injection. Compared with the saline and LacZ controls, the VEGF/Ang1 group expressed higher phosphorylated Akt and Bcl-xL, less Caspase-3 and Bad, and had higher vascular density, more proliferating cardiomyocytes, and less apoptotic cells in the infarct and peri-infarct zones. Thus, cardiac-specific and hypoxia-induced coexpression of VEGF and Ang1 improves the perfusion and function of porcine MI heart through the induction of angiogenesis and cardiomyocyte proliferation, activation of prosurvival pathways, and reduction of cell apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Yi Ting

    2012-11-01

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

  1. MicroRNA-145 suppresses ROS-induced Ca{sup 2+} overload of cardiomyocytes by targeting CaMKIIδ

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Min-Ji [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jang, Jin-Kyung [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Lee, Chang Yeon; Park, Jun-Hee [Department of Integrated Omics for Biomedical Sciences, Graduate School, Yonsei University, 50 Yonsei-ro, Seodamun-gu, Seoul 120-759 (Korea, Republic of); Lee, Jiyun; Seo, Hyang-Hee [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Choi, Eunhyun [Severance Integrative Research Institute for Cerebral and Cardiovascular Disease, Yonsei University Health System, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jeon, Woo-min [Department of Animal Resource, Sahmyook University, Seoul 139-742 (Korea, Republic of); Hwang, Hye Jin [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Shin, Hyun-Taek [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); and others

    2013-06-14

    Highlights: •CaMKIIδ mediates H{sub 2}O{sub 2}-induced Ca{sup 2+} overload in cardiomyocytes. •miR-145 can inhibit Ca{sup 2+} overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca{sup 2+}) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca{sup 2+} signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca{sup 2+}-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H{sub 2}O{sub 2}-mediated Ca{sup 2+} overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca{sup 2+} overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca{sup 2+}-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca{sup 2+} overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

  2. Hybrid mathematical model of cardiomyocyte turnover in the adult human heart.

    Directory of Open Access Journals (Sweden)

    Jeremy A Elser

    Full Text Available The capacity for cardiomyocyte regeneration in the healthy adult human heart is fundamentally relevant for both myocardial homeostasis and cardiomyopathy therapeutics. However, estimates of cardiomyocyte turnover rates conflict greatly, with a study employing C14 pulse-chase methodology concluding 1% annual turnover in youth declining to 0.5% with aging and another using cell population dynamics indicating substantial, age-increasing turnover (4% increasing to 20%.Create a hybrid mathematical model to critically examine rates of cardiomyocyte turnover derived from alternative methodologies.Examined in isolation, the cell population analysis exhibited severe sensitivity to a stem cell expansion exponent (20% variation causing 2-fold turnover change and apoptosis rate. Similarly, the pulse-chase model was acutely sensitive to assumptions of instantaneous incorporation of atmospheric C14 into the body (4-fold impact on turnover in young subjects while numerical restrictions precluded otherwise viable solutions. Incorporating considerations of primary variable sensitivity and controversial model assumptions, an unbiased numerical solver identified a scenario of significant, age-increasing turnover (4-6% increasing to 15-22% with age that was compatible with data from both studies, provided that successive generations of cardiomyocytes experienced higher attrition rates than predecessors.Assignment of histologically-observed stem/progenitor cells into discrete regenerative phenotypes in the cell population model strongly influenced turnover dynamics without being directly testable. Alternatively, C14 trafficking assumptions and restrictive models in the pulse-chase model artificially eliminated high-turnover solutions. Nevertheless, discrepancies among recent cell turnover estimates can be explained and reconciled. The hybrid mathematical model provided herein permits further examination of these and forthcoming datasets.

  3. IL-33 attenuates anoxia/reoxygenation-induced cardiomyocyte apoptosis by inhibition of PKCβ/JNK pathway.

    Directory of Open Access Journals (Sweden)

    Tao Rui

    Full Text Available Interleukin-33 (IL-33 is a new member of the IL-1 cytokine family. The objectives of present study are to assess whether IL-33 can protect cardiomyocytes from anoxia/reoxygenation (A/R-induced injury and the mechanism involved in the protection.Cardiomyocytes derived from either wild type or JNK1(-/- mice were challenged with an A/R with or without IL-33. Myocyte apoptosis was assessed by measuring caspase 3 activity, fragmented DNA and TUNEL staining. In addition, cardiomyocyte oxidative stress was assessed by measuring DHR123 oxidation; PKCβII and JNK phosphorylation were assessed with Western blot.Challenge of cardiomyocytes with an A/R resulted in cardiomyocyte oxidative stress, PKCβII and JNK phosphorylation, and myocyte apoptosis. Treatment of the cardiomyocytes with IL-33 attenuated the A/R-induced myocyte oxidative stress, prevented PKCβII and JNK phosphorylation and attenuated the A/R-induced myocyte apoptosis. The protective effect of the IL-33 did not show in cardiac myocytes with siRNA specific to PKCβII or myocytes deficient in JNK1. Inhibition of PKCβII prevented the A/R-induced JNK phosphorylation, but inhibition of JNK1 showed no effect on A/R-induced PKCβII phosphorylation.Our results indicate that IL-33 prevents the A/R-induced myocyte apoptosis through inhibition of PKCβ/JNK pathway.

  4. Resveratrol protects the loss of connexin 43 induced by ethanol exposure in neonatal mouse cardiomyocytes.

    Science.gov (United States)

    Tu, Su; Cao, Fu-Tao; Fan, Xiao-Chun; Yang, Cheng-Jian

    2017-06-01

    Excessive alcohol consumption provides risk to cardiomyopathy with unknown mechanisms. Resveratrol, a plant polyphenol, is widely reported for its cardiovascular benefits, while its effect on alcohol-induced impairments in cardiomyocytes largely remains unknown. Effects of resveratrol on the cardiomyocytes under ethanol insult were studied in vitro. Ethanol exposure in mouse neonatal cardiomyocytes increased cell death and induced a specific loss of tight junction protein, connexin 43. In spite of adverse effects at higher concentrations, resveratrol at 10 μM improved cell viability of cardiomyocytes in the presence of a deleterious dose of ethanol. Importantly, the co-treatment of resveratrol with ethanol exhibited the restoration of connexin 43 protein. Further assays showed that these effects were likely associated with the antioxidative actions of resveratrol, and correlated with the alleviation of MAP kinase activation in cultured cardiomyocytes in response to ethanol. Our data suggests a novel mechanism of cardiomyocyte cell loss under ethanol exposure and provides new evidence of protective effects of resveratrol in the cardiomyocytes.

  5. Anti-addiction Drug Ibogaine Prolongs the Action Potential in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Science.gov (United States)

    Rubi, Lena; Eckert, Daniel; Boehm, Stefan; Hilber, Karlheinz; Koenig, Xaver

    2017-04-01

    Ibogaine is a plant alkaloid used as anti-addiction drug in dozens of alternative medicine clinics worldwide. Recently, alarming reports of life-threatening cardiac arrhythmias and cases of sudden death associated with the ingestion of ibogaine have accumulated. Using whole-cell patch clamp recordings, we assessed the effects of ibogaine and its main metabolite noribogaine on action potentials in human ventricular-like cardiomyocytes derived from induced pluripotent stem cells. Therapeutic concentrations of ibogaine and its long-lived active metabolite noribogaine significantly retarded action potential repolarization in human cardiomyocytes. These findings represent the first experimental proof that ibogaine application entails a cardiac arrhythmia risk for humans. In addition, they explain the clinically observed delayed incidence of cardiac adverse events several days after ibogaine intake. We conclude that therapeutic concentrations of ibogaine retard action potential repolarization in the human heart. This may give rise to a prolongation of the QT interval in the electrocardiogram and cardiac arrhythmias.

  6. Resveratrol Attenuated Low Ambient Temperature-Induced Myocardial Hypertrophy via Inhibiting Cardiomyocyte Apoptosis

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    Kun Yin

    2015-04-01

    Full Text Available Background/Aims: Low ambient temperature is an important risk factor for cardiovascular diseases, and has been shown to lead to cardiac hypertrophy. In this study, we aim to investigate if Resveratrol may inhibit cold exposure-induced cardiac hypertrophy in mice, and if so to clarify its molecular mechanism. Methods: Adult male mice were randomly assigned to Control group (kept at room temperature, Cold group (kept at low air temperature range from 3°C to 5°C and Resveratrol treatment group (100mg/kg/day for eight weeks. HE staining, Masson staining and Transmission electron microscopy were employed to detect cardiac structure, fibrosis and myocardial ultrastructure, respectively. Echocardiogram was used to measure myocardial functions. Western blot was used to detect the expression of MAPK pathway and apoptotic proteins. TUENL assay was performed to evaluate cardiomyocyte apoptosis. qRT-PCR was employed to measure the mRNA level. Results: Cold-treated mice showed a higher heart/body weight ratio and heart weight/tibia length ratio compared with control mice, and Resveratrol treatment may suppress these changes in cold-treated mice. Myocardial cross-section area and cardiac collagen volume were larger in cold group than control group, which also can be attenuated by Resveratrol treatment. Also, Resveratrol improved the ultrastructure damage of myocardium such as myofibril disarray in cold group. Echocardiogram measurement showed that EF and FS values in cold group declined apparently as compared to control group, and Resveratrol may improve the reduction of heart functions. The expression of p-JNK, p-p38 and p-ERK relative to total JNK, p38 and ERK in cold group was not altered in cold group and Resveratrol group as compared to control group. Cold-treated mouse hearts also showed the upregulation of hypertrophy-related miRNA-miR-328 but not miR-23a, and Resveratrol treatment can inhibit the increase of miR-328. Finally, Resveratrol treatment

  7. Role of Nodal-PITX2C signaling pathway in glucose-induced cardiomyocyte hypertrophy.

    Science.gov (United States)

    Su, Dongmei; Jing, Sun; Guan, Lina; Li, Qian; Zhang, Huiling; Gao, Xiaobo; Ma, Xu

    2014-06-01

    Pathological cardiac hypertrophy is a major cause of morbidity and mortality in cardiovascular disease. Recent studies have shown that cardiomyocytes, in response to high glucose (HG) stimuli, undergo hypertrophic growth. While much work still needs to be done to elucidate this important mechanism of hypertrophy, previous works have showed that some pathways or genes play important roles in hypertrophy. In this study, we showed that sublethal concentrations of glucose (25 mmol/L) could induce cardiomyocyte hypertrophy with an increase in the cellular surface area and the upregulation of the atrial natriuretic peptide (ANP) gene, a hypertrophic marker. High glucose (HG) treatments resulted in the upregulation of the Nodal gene, which is under-expressed in cardiomyocytes. We also determined that the knockdown of the Nodal gene resisted HG-induced cardiomyocyte hypertrophy. The overexpression of Nodal was able to induce hypertrophy in cardiomyocytes, which was associated with the upregulation of the PITX2C gene. We also showed that increases in the PITX2C expression, in response to Nodal, were mediated by the Smad4 signaling pathway. This study is highly relevant to the understanding of the effects of the Nodal-PITX2C pathway on HG-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms.

  8. Antioxidant treatment attenuates hyperglycemia-induced cardiomyocyte death in rats.

    Science.gov (United States)

    Fiordaliso, Fabio; Bianchi, Roberto; Staszewsky, Lidia; Cuccovillo, Ivan; Doni, Mirko; Laragione, Teresa; Salio, Monica; Savino, Costanza; Melucci, Silvia; Santangelo, Francesco; Scanziani, Eugenio; Masson, Serge; Ghezzi, Pietro; Latini, Roberto

    2004-11-01

    Diabetes and oxidative stress concur to cardiac myocyte death in various experimental settings. We assessed whether N-acetyl-L-cysteine (NAC), an antioxidant and glutathione precursor, has a protective role in a rat model of streptozotocin (STZ)-induced diabetes and in isolated myocytes exposed to high glucose (HG). Diabetic rats were treated with NAC (0.5 g/kg per day) or vehicle for 3 months. At sacrifice left ventricle (LV) myocyte number and size, collagen deposition and reactive oxygen species (ROS) were measured by quantitative histological methods. Diabetes reduced LV myocyte number by 29% and increased myocyte volume by 20% compared to non-diabetic controls. NAC protected from myocyte loss (+25% vs. untreated diabetics, P < 0.05) and reduced reactive hypertrophy (-16% vs. untreated diabetics, P < 0.05). Perivascular fibrosis was high in diabetic rats (+88% vs. control, P < 0.001) but prevented by NAC. ROS production and fraction of ROS-positive cardiomyocyte nuclei were drastically raised in diabetic rats (2.4- and 5.1-fold vs. control, P < 0.001) and normalized by NAC. In separate experiments, isolated adult rat ventricular myocytes were incubated in a medium containing high concentrations of glucose (HG, 25 mM) +/- 0.01 mM NAC; myocyte survival (Trypan blue exclusion and apoptosis by TUNEL) and glutathione content were evaluated. The number of dead and apoptotic myocytes increased five and 6.7-fold in HG and glutathione decreased by 48% (P < 0.05). NAC normalized cell death and apoptosis and prevented glutathione loss. NAC effectively protects from hyperglycemia-induced myocyte cell death and compensatory hypertrophy through direct scavenging of ROS and replenishment of the intracellular glutathione content.

  9. Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Shan, Tizhong; Liang, Xinrong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding, Ministry of Agriculture, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2014-09-12

    Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.

  10. Role of Oxidative Stress in Thyroid Hormone-Induced Cardiomyocyte Hypertrophy and Associated Cardiac Dysfunction: An Undisclosed Story

    Directory of Open Access Journals (Sweden)

    Mohammad T. Elnakish

    2015-01-01

    Full Text Available Cardiac hypertrophy is the most documented cardiomyopathy following hyperthyroidism in experimental animals. Thyroid hormone-induced cardiac hypertrophy is described as a relative ventricular hypertrophy that encompasses the whole heart and is linked with contractile abnormalities in both right and left ventricles. The increase in oxidative stress that takes place in experimental hyperthyroidism proposes that reactive oxygen species are key players in the cardiomyopathy frequently reported in this endocrine disorder. The goal of this review is to shed light on the effects of thyroid hormones on the development of oxidative stress in the heart along with the subsequent cellular and molecular changes. In particular, we will review the role of thyroid hormone-induced oxidative stress in the development of cardiomyocyte hypertrophy and associated cardiac dysfunction, as well as the potential effectiveness of antioxidant treatments in attenuating these hyperthyroidism-induced abnormalities in experimental animal models.

  11. Role of Oxidative Stress in Thyroid Hormone-Induced Cardiomyocyte Hypertrophy and Associated Cardiac Dysfunction: An Undisclosed Story

    Science.gov (United States)

    Elnakish, Mohammad T.; Ahmed, Amany A. E.; Mohler, Peter J.; Janssen, Paul M. L.

    2015-01-01

    Cardiac hypertrophy is the most documented cardiomyopathy following hyperthyroidism in experimental animals. Thyroid hormone-induced cardiac hypertrophy is described as a relative ventricular hypertrophy that encompasses the whole heart and is linked with contractile abnormalities in both right and left ventricles. The increase in oxidative stress that takes place in experimental hyperthyroidism proposes that reactive oxygen species are key players in the cardiomyopathy frequently reported in this endocrine disorder. The goal of this review is to shed light on the effects of thyroid hormones on the development of oxidative stress in the heart along with the subsequent cellular and molecular changes. In particular, we will review the role of thyroid hormone-induced oxidative stress in the development of cardiomyocyte hypertrophy and associated cardiac dysfunction, as well as the potential effectiveness of antioxidant treatments in attenuating these hyperthyroidism-induced abnormalities in experimental animal models. PMID:26146529

  12. Embryonic template-based generation and purification of pluripotent stem cell-derived cardiomyocytes for heart repair

    NARCIS (Netherlands)

    Dierickx, P.; Doevendans, P.A.; Geijsen, N.; van Laake, L.W.

    2012-01-01

    Cardiovascular disease remains a leading cause of death in Western countries. Many types of cardiovascular diseases are due to a loss of functional cardiomyocytes, which can result in irreversible cardiac failure. Since the adult human heart has limited regenerative potential, cardiac

  13. ERK-MAPK Signaling Opposes Rho-Kinase to Reduce Cardiomyocyte Apoptosis in Heart Ischemic Preconditioning

    Science.gov (United States)

    Juan-Zhang; Bian, Hong-Jun; Li, Xiao-Xing; Liu, Xiao-Bo; Sun, Jun-Ping; Na-Li; Yun-Zhang; Ji, Xiao-Ping

    2010-01-01

    We and others have reported that Rho-kinase plays an important role in the pathogenesis of heart ischemia/reperfusion (I/R) injury. Studies have also demonstrated that the activation of Rho-kinase is reversed in ischemic preconditioning (IPC). However, the mechanisms by which Rho-kinase is increased in I/R and reversed in IPC are not thoroughly understood. In female Wistar rats, we created I/R by ligating the left anterior–descending branch of the coronary artery (LAD) for 30 min and releasing the ligature for 180 min. IPC rats underwent IPC (two cycles of 5-min ligation of the LAD and 5-min reflow) before I/R. IPC caused a significant increase in extracellular signal–regulated kinase (ERK)1/2 activity and reduced Rho-kinase activity and cardiomyocyte apoptosis (P IPC). Western-blot analysis showed that administration of PD98059 increased Rho-kinase activity. Treatment with fasudil, an inhibitor of Rho-kinase, reversed cell apoptosis caused by treatment with PD98059 in IPC. In addition, ROCK1 (Rho-kinase 1) may be the major Rho-kinase isoform that is opposed by ERK-MAPK signaling in IPC. These results indicate that ERK-MAPK signaling is required in IPC to oppose Rho-kinase activity in cardiomyocyte apoptosis in vivo. PMID:20383434

  14. Cardiomyocyte­-specific expression of the nuclear matrix protein, CIZ1, stimulates production of mono-nucleated cells with an extended window of proliferation in the postnatal mouse heart

    Directory of Open Access Journals (Sweden)

    Sumia A. Bageghni

    2017-01-01

    Full Text Available Myocardial injury in mammals leads to heart failure through pathological cardiac remodelling that includes hypertrophy, fibrosis and ventricular dilatation. Central to this is inability of the mammalian cardiomyocyte to self-renew due to entering a quiescent state after birth. Modulation of the cardiomyocyte cell-cycle after injury is therefore a target mechanism to limit damage and potentiate repair and regeneration. Here, we show that cardiomyocyte-specific over-expression of the nuclear-matrix­-associated DNA replication protein, CIZ1, extends their window of proliferation during cardiac development, delaying onset of terminal differentiation without compromising function. CIZ1-expressing hearts are enlarged, but the cardiomyocytes are smaller with an overall increase in number, correlating with increased DNA replication after birth and retention of an increased proportion of mono-nucleated cardiomyocytes into adulthood. Furthermore, these CIZ1 induced changes in the heart reduce the impact of myocardial injury, identifying CIZ1 as a putative therapeutic target for cardiac repair.

  15. Vitamin E Reversed Apoptosis of Cardiomyocytes Induced by Exposure to High Dose Formaldehyde During Mice Pregnancy.

    Science.gov (United States)

    Wu, Dongyuan; Jiang, Zhirong; Gong, Bing; Dou, Yue; Song, Mingxuan; Song, Xiaoxia; Tian, Yu

    2017-10-21

    In this study, we investigated the protection effect of Vitamin E (Vit E) on formaldehyde (FA) exposure during pregnancy induced apoptosis of cardiomyocytes, and used an HL-1 cell line to confirmed the findings in vivo.Pregnant mice received different doses of FA (0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 0.1 μg Vit E, or 1.5 mg/kg + 0.1 μg Vit E). TUNEL staining was used to reveal the apoptosis in cardiomyocytes, and SOD, MDA, GSH, Livin, and Caspase-3 in cardiomyocytes were detected by ELISA, RT-PCR, and Western blot. For in vitro study, HL-1 cells were treated with vehicle, 5 μmol/L FA, 25 μmol/L FA, 50 μmol/L FA, 10 mg/L Vit. E, and 50 μmol/L FA+ 10 mg/L Vit E, respectively. CCK-8 assay and flow cytometry were used to evaluate cell vitality and apoptosis. A high dose of FA exposure led to cytotoxicity in both pregnant mice and offspring, as TUNEL staining revealed a significant apoptosis of cardiomyocytes, and the alternation in SOD, GSH, MDA, Livin, and Caspase-3 was found in cardiomyocytes. 0.1 μg Vit. E could reverse high doses of FA exposure induced apoptosis of cardiomyocytes in both pregnant mice and offspring. The in vitro study revealed that FA exposure induced a decrease of cell viability and increased cell apoptosis, as well as oxidative stress in HL-1 cells with alternation in SOD, GSH, MDA, Livin, and Caspase-3.This study revealed a high dose of FA induced oxidative stress and apoptosis of cardiomyocytes in both pregnant mice and offspring, and Vit E supplement during pregnancy reversed the systemic and myocardial toxicity of FA.

  16. Mechanosensitive Kinases Regulate Stiffness-Induced Cardiomyocyte Maturation

    Science.gov (United States)

    Young, Jennifer L.; Kretchmer, Kyle; Ondeck, Matthew G.; Zambon, Alexander C.; Engler, Adam J.

    2014-01-01

    Cells secrete and assemble extracellular matrix throughout development, giving rise to time-dependent, tissue-specific stiffness. Mimicking myocardial matrix stiffening, i.e. ~10-fold increase over 1 week, with a hydrogel system enhances myofibrillar organization of embryonic cardiomyocytes compared to static hydrogels, and thus we sought to identify specific mechanosensitive proteins involved. Expression and/or phosphorylation state of 309 unique protein kinases were examined in embryonic cardiomyocytes plated on either dynamically stiffening or static mature myocardial stiffness hydrogels. Gene ontology analysis of these kinases identified cardiogenic pathways that exhibited time-dependent up-regulation on dynamic versus static matrices, including PI3K/AKT and p38 MAPK, while GSK3β, a known antagonist of cardiomyocyte maturation, was down-regulated. Additionally, inhibiting GSK3β on static matrices improved spontaneous contraction and myofibril organization, while inhibiting agonist AKT on dynamic matrices reduced myofibril organization and spontaneous contraction, confirming its role in mechanically-driven maturation. Together, these data indicate that mechanically-driven maturation is at least partially achieved via active mechanosensing at focal adhesions, affecting expression and phosphorylation of a variety of protein kinases important to cardiomyogenesis. PMID:25236849

  17. Probenecid Improves Cardiac Function in Patients With Heart Failure With Reduced Ejection Fraction In Vivo and Cardiomyocyte Calcium Sensitivity In Vitro.

    Science.gov (United States)

    Robbins, Nathan; Gilbert, Mark; Kumar, Mohit; McNamara, James W; Daly, Patrick; Koch, Sheryl E; Conway, Ginger; Effat, Mohamed; Woo, Jessica G; Sadayappan, Sakthivel; Rubinstein, Jack

    2018-01-13

    Transient receptor potential vanilloid 2 is a calcium channel activated by probenecid. Probenecid is a Food and Drug Administration-approved uricosuric drug that has recently been shown to induce positive lusitropic and inotropic effects in animal models through cardiomyocyte transient receptor potential vanilloid 2 activation. The aim of this study was to test the hypothesis that oral probenecid can improve cardiac function and symptomatology in patients with heart failure with reduced ejection fraction and to further elucidate its calcium-dependent effects on myocyte contractility. The clinical trial recruited stable outpatients with heart failure with reduced ejection fraction randomized in a single-center, double-blind, crossover design. Clinical data were collected including a dyspnea assessment, physical examination, ECG, echocardiogram to assess systolic and diastolic function, a 6-minute walk test, and laboratory studies. In vitro force generation studies were performed on cardiomyocytes isolated from murine tissue exposed to probenecid or control treatments. The clinical trial recruited 20 subjects (mean age 57 years, mean baseline fractional shortening of 13.6±1.0%). Probenecid therapy increased fractional shortening by 2.1±1.0% compared with placebo -1.7±1.0% ( P =0.007). Additionally, probenecid improved diastolic function compared with placebo by decreasing the E/E' by -2.95±1.21 versus 1.32±1.21 in comparison to placebo ( P =0.03). In vitro probenecid increased myofilament force generation (92.36 versus 80.82 mN/mm 2 , P Probenecid improves cardiac function with minimal effects on symptomatology and no significant adverse effects after 1 week in patients with heart failure with reduced ejection fraction and increases force development and calcium sensitivity at the cardiomyocyte level. URL: https://www.clinicaltrials.gov. Unique identifier: NCT01814319. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  18. The case for induced pluripotent stem cell-derived cardiomyocytes in pharmacological screening

    Science.gov (United States)

    Khan, Jaffar M; Lyon, Alexander R; Harding, Sian E

    2013-01-01

    The current drug screening models are deficient, particularly in detecting cardiac side effects. Human stem cell-derived cardiomyocytes could aid both early cardiotoxicity detection and novel drug discovery. Work over the last decade has generated human embryonic stem cells as potentially accurate sources of human cardiomyocytes, but ethical constraints and poor efficacy in establishing cell lines limit their use. Induced pluripotent stem cells do not require the use of human embryos and have the added advantage of producing patient-specific cardiomyocytes, allowing both generic and disease- and patient-specific pharmacological screening, as well as drug development through disease modelling. A critical question is whether sufficient standards have been achieved in the reliable and reproducible generation of ‘adult-like’ cardiomyocytes from human fibroblast tissue to progress from validation to safe use in practice and drug discovery. This review will highlight the need for a new experimental system, assess the validity of human induced pluripotent stem cell-derived cardiomyocytes and explore what the future may hold for their use in pharmacology. LINKED ARTICLES This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-2 PMID:22845396

  19. Moderate ethanol administration accentuates cardiomyocyte contractile dysfunction and mitochondrial injury in high fat diet-induced obesity.

    Science.gov (United States)

    Yuan, Fang; Lei, Yonghong; Wang, Qiurong; Esberg, Lucy B; Huang, Zaixing; Scott, Glenda I; Li, Xue; Ren, Jun

    2015-03-18

    Light to moderate drinking confers cardioprotection although it remains unclear with regards to the role of moderate drinking on cardiac function in obesity. This study was designed to examine the impact of moderate ethanol intake on myocardial function in high fat diet intake-induced obesity and the mechanism(s) involved with a focus on mitochondrial integrity. C57BL/6 mice were fed low or high fat diet for 16 weeks prior to ethanol challenge (1g/kg/d for 3 days). Cardiac contractile function, intracellular Ca(2+) homeostasis, myocardial histology, and mitochondrial integrity [aconitase activity and the mitochondrial proteins SOD1, UCP-2 and PPARγ coactivator 1α (PGC-1α)] were assessed 24h after the final ethanol challenge. Fat diet intake compromised cardiomyocyte contractile and intracellular Ca(2+) properties (depressed peak shortening and maximal velocities of shortening/relengthening, prolonged duration of relengthening, dampened intracellular Ca(2+) rise and clearance without affecting duration of shortening). Although moderate ethanol challenge failed to alter cardiomyocyte mechanical property under low fat diet intake, it accentuated high fat diet intake-induced changes in cardiomyocyte contractile function and intracellular Ca(2+) handling. Moderate ethanol challenge failed to affect fat diet intake-induced cardiac hypertrophy as evidenced by H&E staining. High fat diet intake reduced myocardial aconitase activity, downregulated levels of mitochondrial protein UCP-2, PGC-1α, SOD1 and interrupted intracellular Ca(2+) regulatory proteins, the effect of which was augmented by moderate ethanol challenge. Neither high fat diet intake nor moderate ethanol challenge affected protein or mRNA levels as well as phosphorylation of Akt and GSK3β in mouse hearts. Taken together, our data revealed that moderate ethanol challenge accentuated high fat diet-induced cardiac contractile and intracellular Ca(2+) anomalies as well as mitochondrial injury. Copyright

  20. Creating frog heart as an organ: in vitro-induced heart functions as a circulatory organ in vivo.

    Science.gov (United States)

    Kinoshita, Masayoshi; Ariizumi, Takashi; Yuasa, Shinsuke; Miyoshi, Shunichirou; Komazaki, Shinji; Fukuda, Keiichi; Asashima, Makoto

    2010-01-01

    Cardiomyocytes have been induced from various pluripotent cells, such as embryonic stem cells and myeloid stem cells; however, the generation of cardiac tissues beyond two-dimensional cell-sheets has not been reported. Creating higher order, three-dimensional structures that are unique to heart is the long-awaited next step in realizing cardiac regenerative medicine. We have previously shown that cardiomyocytes can be induced in vitro from undifferentiated cells (animal caps) excised from Xenopus embryos. Cardiomyocytes were induced by first dissociating the animal caps and then reaggregating them following treatment with activin. Here, we describe an interesting method for creating a complete ectopic heart in vivo, involving the introduction of in vitro-created tissue during early embryogenesis. Thus, animal cap reaggregates were transplanted into the abdomen of late-neurula-stage embryos, resulting in two-chambered hearts being formed. The dual-heart larvae matured into adult animals with transplanted hearts intact. Involvement of transplanted hearts in systemic circulation was demonstrated. Moreover, the ectopic hearts possessed higher order structures such as atrium and ventricle, and were morphologically, histologically, and electrophysiologically identical to original hearts. This system should facilitate the study of heart organogenesis and may promote a shift from tissue to organ engineering for clinical applications.

  1. Excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kane, Christopher; Couch, Liam; Terracciano, Cesare M N

    2015-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation-contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca(2+) handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.

  2. Excitation–contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes

    Science.gov (United States)

    Kane, Christopher; Couch, Liam; Terracciano, Cesare M. N.

    2015-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation–contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca2+ handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells. PMID:26484342

  3. Xenotransplantation of Human Cardiomyocyte Progenitor Cells Does Not Improve Cardiac Function in a Porcine Model of Chronic Ischemic Heart Failure. Results from a Randomized, Blinded, Placebo Controlled Trial

    NARCIS (Netherlands)

    Jansen of Lorkeers, SJ; Gho, Johannes M I H; Koudstaal, Stefan; van Hout, Geert; Zwetsloot, Peter Paul M; van Oorschot, Joep W M; van Eeuwijk, Esther C M; Leiner, Tim; Höfer, Imo E.; Goumans, Marie-José; Doevendans, Pieter A; Sluijter, Joost P G; Chamuleau, Steven A J

    2015-01-01

    BACKGROUND: Recently cardiomyocyte progenitor cells (CMPCs) were successfully isolated from fetal and adult human hearts. Direct intramyocardial injection of human CMPCs (hCMPCs) in experimental mouse models of acute myocardial infarction significantly improved cardiac function compared to controls.

  4. Cardiomyocyte specific expression of Acyl-coA thioesterase 1 attenuates sepsis induced cardiac dysfunction and mortality

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Congying [Departments of Internal Medicine and Institute of Hypertension, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan (China); Dong, Ruolan [Department of Geriatric Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030 (China); Chen, Chen [Departments of Internal Medicine and Institute of Hypertension, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan (China); Wang, Hong, E-mail: hong.wang1988@yahoo.com [Departments of Internal Medicine and Institute of Hypertension, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan (China); Wang, Dao Wen, E-mail: dwwang@tjh.tjmu.edu.cn [Departments of Internal Medicine and Institute of Hypertension, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan (China)

    2015-12-25

    Compromised cardiac fatty acid oxidation (FAO) induced energy deprivation is a critical cause of cardiac dysfunction in sepsis. Acyl-CoA thioesterase 1 (ACOT1) is involved in regulating cardiac energy production via altering substrate metabolism. This study aims to clarify whether ACOT1 has a potency to ameliorate septic myocardial dysfunction via enhancing cardiac FAO. Transgenic mice with cardiomyocyte specific expression of ACOT1 (αMHC-ACOT1) and their wild type (WT) littermates were challenged with Escherichia coli lipopolysaccharide (LPS; 5 mg/kg i.p.) and myocardial function was assessed 6 h later using echocardiography and hemodynamics. Deteriorated cardiac function evidenced by reduction of the percentage of left ventricular ejection fraction and fractional shortening after LPS administration was significantly attenuated by cardiomyocyte specific expression of ACOT1. αMHC-ACOT1 mice exhibited a markedly increase in glucose utilization and cardiac FAO compared with LPS-treated WT mice. Suppression of cardiac peroxisome proliferator activated receptor alpha (PPARa) and PPARγ-coactivator-1α (PGC1a) signaling observed in LPS-challenged WT mice was activated by the presence of ACOT1. These results suggest that ACOT1 has potential therapeutic values to protect heart from sepsis mediated dysfunction, possibly through activating PPARa/PGC1a signaling. - Highlights: • ACOT1 has potential therapeutic values to protect heart from sepsis mediated dysfunction. • ACOT1 can regulate PPARa/PGC1a signaling pathway. • We first generate the transgenic mice with cardiomyocyte specific expression of ACOT1.

  5. Br-DIF-1 accelerates dimethyl sulphoxide-induced differentiation of P19CL6 embryonic carcinoma cells into cardiomyocytes.

    Science.gov (United States)

    Seya, K; Kanemaru, K; Matsuki, M; Hongo, K; Kitahara, H; Kikuchi, H; Oshima, Y; Kubohara, Y; Okumura, K; Motomura, S; Furukawa, K-I

    2012-02-01

    Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells. P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca(2+) channel blocker. Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3-3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca(2+) channel blocker; 100 nM) and a small interfering RNA for the T-type Ca(2+) channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca(2+) channel gene. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  6. Nanofibrous clinical-grade collagen scaffolds seeded with human cardiomyocytes induces cardiac remodeling in dilated cardiomyopathy.

    Science.gov (United States)

    Joanne, Pierre; Kitsara, Maria; Boitard, Solène-Emmanuelle; Naemetalla, Hany; Vanneaux, Valérie; Pernot, Mathieu; Larghero, Jérôme; Forest, Patricia; Chen, Yong; Menasché, Philippe; Agbulut, Onnik

    2016-02-01

    Limited data are available on the effects of stem cells in non-ischemic dilated cardiomyopathy (DCM). Since the diffuse nature of the disease calls for a broad distribution of cells, this study investigated the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) in a mouse model of DCM. Nanofibrous scaffolds were produced using a clinical grade atelocollagen which was electrospun and cross-linked under different conditions. As assessed by scanning electron microscopy and shearwave elastography, the optimum crosslinking conditions for hiPS-CM colonization proved to be a 10% concentration of citric acid crosslinking agent and 150 min of post-electrospinning baking. Acellular collagen scaffolds were first implanted in both healthy mice and those with induced DCM by a cardiac-specific invalidation of serum response factor (SRF). Seven and fourteen days after implantation, the safety of the scaffold was demonstrated by echocardiography and histological assessments. The subsequent step of implantation of the scaffolds seeded with hiPS-CM in DCM induced mice, using cell-free scaffolds as controls, revealed that after fourteen days heart function decreased in controls while it remained stable in the treated mice. This pattern was associated with an increased number of endothelial cells, in line with the greater vascularity of the scaffold. Moreover, a lesser degree of fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling was observed. These results support the interest of the proposed hiPS-CM seeded electrospun scaffold for the stabilization of the DCM outcome with potential for its clinical use in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Bile acid-induced arrhythmia is mediated by muscarinic M2 receptors in neonatal rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Siti H Sheikh Abdul Kadir

    Full Text Available BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC, which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM. Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart. METHODS AND RESULTS: Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M(2 receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M(2 receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters. CONCLUSION: We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M(2 receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.

  8. Identification and characterization of calcium sparks in cardiomyocytes derived from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Guang Qin Zhang

    Full Text Available INTRODUCTION: Ca2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs-derived cardiomyocytes (hiPSC-CMs may display unique structural and functional properties than mature adult cardiomyocytes. METHODS AND RESULTS: Ca2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM and full duration at half maximum (FDHM of spontaneous Ca2+ sparks. CONCLUSIONS: For the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.

  9. Ursodeoxycholic acid protects cardiomyocytes against cobalt chloride induced hypoxia by regulating transcriptional mediator of cells stress hypoxia inducible factor 1α and p53 protein.

    Science.gov (United States)

    Mohamed, Anis Syamimi; Hanafi, Noorul Izzati; Sheikh Abdul Kadir, Siti Hamimah; Md Noor, Julina; Abdul Hamid Hasani, Narimah; Ab Rahim, Sharaniza; Siran, Rosfaiizah

    2017-10-01

    In hepatocytes, ursodeoxycholic acid (UDCA) activates cell signalling pathways such as p53, intracellular calcium ([Ca2+ ]i ), and sphingosine-1-phosphate (S1P)-receptor via Gαi -coupled-receptor. Recently, UDCA has been shown to protect the heart against hypoxia-reoxygenation injury. However, it is not clear whether UDCA cardioprotection against hypoxia acts through a transcriptional mediator of cells stress, HIF-1α and p53. Therefore, in here, we aimed to investigate whether UDCA could protect cardiomyocytes (CMs) against hypoxia by regulating expression of HIF-1α, p53, [Ca2+ ]i , and S1P-Gαi -coupled-receptor. Cardiomyocytes were isolated from newborn rats (0-2 days), and hypoxia was induced by using cobalt chloride (CoCl2 ). Cardiomyocytes were treated with UDCA and cotreated with either FTY720 (S1P-receptor agonist) or pertussis toxin (PTX; Gαi inhibitor). Cells were subjected for proliferation assay, beating frequency, QuantiGene Plex assay, western blot, immunofluorescence, and calcium imaging. Our findings showed that UDCA counteracted the effects of CoCl2 on cell viability, beating frequency, HIF-1α, and p53 protein expression. We found that these cardioprotection effects of UDCA were similar to FTY720, S1P agonist. Furthermore, we observed that UDCA protects CMs against CoCl2 -induced [Ca2+ ]i dynamic alteration. Pharmacological inhibition of the Gαi -sensitive receptor did not abolish the cardioprotection of UDCA against CoCl2 detrimental effects, except for cell viability and [Ca2+ ]i . Pertussis toxin is partially effective in inhibiting UDCA protection against CoCl2 effects on CM cell viability. Interestingly, PTX fully inhibits UDCA cardioprotection on CoCl2 -induced [Ca2+ ]i dynamic changes. We conclude that UDCA cardioprotection against CoCl2 -induced hypoxia is similar to FTY720, and its actions are not fully mediated by the Gαi -coupled protein sensitive pathways. Ursodeoxycholic acid is the most hydrophilic bile acid and is currently

  10. Tanshinone IIA Prevents Leu27IGF-II-Induced Cardiomyocyte Hypertrophy Mediated by Estrogen Receptor and Subsequent Akt Activation.

    Science.gov (United States)

    Weng, Yueh-Shan; Wang, Hsueh-Fang; Pai, Pei-Ying; Jong, Gwo-Ping; Lai, Chao-Hung; Chung, Li-Chin; Hsieh, Dennis Jine-Yuan; HsuanDay, Cecilia; Kuo, Wei-Wen; Huang, Chih-Yang

    2015-01-01

    IGF-IIR plays important roles as a key regulator in myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound in Danshen and is structurally similar to 17[Formula: see text]-estradiol (E[Formula: see text]. However, whether tanshinone IIA improves cardiomyocyte survival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Akt phosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequent NFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced the cell size and suppressed ANP and BNP, inhibiting antihypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed by ICI. Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3K inhibitor), and PI3K siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activating the PI3K/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATC3. Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective ER modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy.

  11. How does the heart (not) die? The role of autophagy in cardiomyocyte homeostasis and cell death.

    Science.gov (United States)

    Dhesi, Pavittarpaul; Tehrani, Faramarz; Fuess, Justin; Schwarz, Ernst R

    2010-01-01

    Autophagy plays a critical and seemingly dual-purposed role in cardiomyocytes, being implicated as a mechanism of both cellular survival, for example, during ischemia/reperfusion injury and a mechanism of cell death at stages in which progressive myocyte alterations are beyond repair. This review aims to highlight the current literature as it relates to autophagy in cardiomyocytes. It provides background into the mechanisms of cell death, discusses the details that are known about the ubiquitin proteasome system and autophagy, delves into the pathways that are known to initiate and inhibit autophagy, and comments on the role of autophagy in cardiomyocyte homeostasis and cell death.

  12. Sympathetic Innervation Induced in Engrafted Engineered Cardiomyocyte Sheets by Glial Cell Line Derived Neurotrophic Factor In Vivo

    Directory of Open Access Journals (Sweden)

    Xian-ming Fu

    2013-01-01

    Full Text Available The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF. GFP-transgene (control group or GDNF overexpressing (GDNF group engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF.

  13. Atomic force microscopy observation of lipopolysaccharide-induced cardiomyocyte cytoskeleton reorganization.

    Science.gov (United States)

    Wang, Liqun; Chen, Tangting; Zhou, Xiang; Huang, Qiaobing; Jin, Chunhua

    2013-08-01

    We applied atomic force microscopy (AFM) to observe lipopolysaccharide (LPS)-induced intracellular cytoskeleton reorganization in primary cardiomyocytes from neonatal mouse. The nonionic detergent Triton X-100 was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized by AFM. Using three-dimensional technique of AFM, we were able to quantify the changes of cytoskeleton by the "density" and total "volume" of the cytoskeleton fibers. Compared to the control group, the density of cytoskeleton was remarkably decreased and the volume of cytoskeleton was significantly increased after LPS treatment, which suggests that LPS may induce the cytoskeleton reorganization and change the cardiomyocyte morphology. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation

    DEFF Research Database (Denmark)

    Zebrowski, David C.; Jensen, Charlotte H.; Becker, Robert

    2017-01-01

    tested the hypothesis that apical resection either inhibits, delays, or reverses cardiomyocyte centrosome disassembly and binucleation. Our data show that apical resection rather transiently accelerates centrosome disassembly as well as the rate of binucleation. Consistent with the nearly 2-fold...

  15. [Influence of Fatty Acids on Oxygen Consumption in Isolated Cardiomyocytes of Rats with Ischemic or Diabetic Heart Disease].

    Science.gov (United States)

    Afanasiev, S A; Egorova, M V; Kutsykova, T V; Popov, S V

    2016-01-01

    one of the reasons of violation of the functional viability of the myocardium is considered to be the oxygen deprivation and lack of energy. The reason is the inhibitory effect of fatty acids on glucose oxidation. Recently, however, new data have been published proving the need for fatty acids and their importance in the maintenance and regulation of the functional activity of the myocardium in chronic pathology. to investigate the influence of free polyunsaturated and saturated fatty acids (FA) on the oxygen uptake of isolated cardiomyocytes in intact rats and animals with ischemic or diabetic heart disease. the executed non-randomized controlled study. It includied 3 groups of male rats of Wistar line (weight 250-300g) with 10 animals in each group. Myocardial infarction ("heart attack" group) was caused by ligation of the left coronary artery, diabetes ("diabetes" group)--by intraperitoneal injection of streptozotocin, and "control" group (intact animals). Myocardial infarction caused by ligation of the left coronary artery, and diabetes by intraperitoneal injection of streptozotocin. Isolated cardiac myocytes were obtained by the enzymatic method. Oxygen consumption was assessed polarographically at different saturation incubation medium with oxygen ([O₂] ≤ 8 mg/l and ([O₂] ≥ 16 mg/l). Arachidonic and palmitic acids were applied as fatty acids. It is established that the introduction of the incubation medium 20 µm arachidonic or palmitic fatty acid significantly increased the oxygen consumption of intact cardiomyocytes of rats. Both at the ischemic and at the diabetic injury to the heart the opposite result was obtained. The most pronounced decrease in oxygen consumption was indicated in the group with diabetes mellitus. The inhibitory effect of LCD on the rate of oxygen consumption may be associated with the influence of the ischemic or diabetic injury to the heart on the barrierfunction ofmitochondrial membranes of cardiomyocytes, the activity of

  16. Production of Single Contracting Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Matrigel Mattress Technique.

    Science.gov (United States)

    Cadar, Adrian G; Feaster, Tromondae K; Durbin, Matthew D; Hong, Charles C

    2017-08-14

    This unit describes the published Matrigel mattress method. Briefly, we describe the preparation of the mattress, replating of the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) on the Matrigel mattress, and hiPSC-CM mattress maintenance. Adherence to this protocol will yield individual, robustly shortening hiPSC-CMs, which can be used for downstream applications. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  17. Induced Pluripotent Stem Cell–Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function

    Science.gov (United States)

    Chauveau, Samuel; Anyukhovsky, Evgeny P.; Ben-Ari, Meital; Naor, Shulamit; Jiang, Ya-Ping; Danilo, Peter; Rahim, Tania; Burke, Stephanie; Qiu, Xiaoliang; Potapova, Irina A.; Doronin, Sergey V.; Brink, Peter R.; Binah, Ofer

    2017-01-01

    Background— Although multiple approaches have been used to create biological pacemakers in animal models, induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) have not been investigated for this purpose. We now report pacemaker function of iPSC-CMs in a canine model. Methods and Results— Embryoid bodies were derived from human keratinocytes, their action potential characteristics determined, and their gene expression profiles and markers of differentiation identified. Atrioventricular blocked dogs were immunosuppressed, instrumented with VVI pacemakers, and injected subepicardially into the anterobasal left ventricle with 40 to 75 rhythmically contracting embryoid bodies (totaling 1.3–2×106 cells). ECG and 24-hour Holter monitoring were performed biweekly. After 4 to 13 weeks, epinephrine (1 μg kg−1 min−1) was infused, and the heart removed for histological or electrophysiological study. iPSC-CMs largely lost the markers of pluripotency, became positive for cardiac-specific markers. and manifested If-dependent automaticity. Epicardial pacing of the injection site identified matching beats arising from that site by week 1 after implantation. By week 4, 20% of beats were electronically paced, 60% to 80% of beats were matching, and mean and maximal biological pacemaker rates were 45 and 75 beats per minute. Maximum night and day rates of matching beats were 53±6.9 and 69±10.4 beats per minute, respectively, at 4 weeks. Epinephrine increased rate of matching beats from 35±4.3 to 65±4.0 beats per minute. Incubation of embryoid bodies with the vital dye, Dil, revealed the persistence of injected cells at the site of administration. Conclusions— iPSC-CMs can integrate into host myocardium and create a biological pacemaker. Although this is a promising development, rate and rhythm of the iPSC-CMs pacemakers remain to be optimized. PMID:28500172

  18. Xenoreactive natural antibodies and induced antibodies--their effects of beating cardiomyocytes as a model of a xenograft.

    Science.gov (United States)

    Müller-Werdan, U; Koidl, B; Autenrieth, A; Klein, D; Werdan, K; Hammer, C

    1996-01-01

    Xenotransplantation has been complicated by hyperacute rejection reactions, which are supposedly triggered by preformed natural antibodies (PNAb) of the recipient organism, whereas the role of antibodies specifically induced by previous antigen contact (IAb) is less clear. Primary cultures of spontaneously beating neonatal rat cardiomyocytes were used as a model of the heart to elaborate the effects of both PNAb and IAb from xenogeneic species and to investigate into their mechanisms of action. An experimental setup allowing for rapid medium exchange under continuous observation was employed. Sera containing PNAb reproducibly bring about a stereotype pattern of altered contractility including an initial increase in beating frequency followed by a temporary cessation of beating within the first minutes after administration. After recovery of spontaneous contractions, the cells within the monolayer exhibited a dissociation of the synchronicity of the beating persisting for several hours. The temporary pause in beating was prevented by a very high extracellular calcium concentration, but not by extracellular electrical stimulation sufficient to trigger contractions in control cells. Electrophysiological measurements carried out in adult ventricular guinea pig heart muscle cells under the same experimental conditions revealed an increase of the excitation threshold of the cells after application of sera containing PNAb due to an enhanced input resistance. These results indicate that the effect of PNAb is the consequence rather of a generally reduced excitability of the cell than of the inhibition of a singular ionic conductance. After specific absorption of PNAb directed against rat antigens beating of neonatal rate cardiomyocytes ensued without interruption. Sera specimens devoid of complement produced similar effects on contractility, although the duration of the standstill period was significantly shorter. The increase in input resistance visualized in guinea pig

  19. Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

    Science.gov (United States)

    Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

    2016-12-01

    Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na(+ )current (INa), nifedipine, a blocker of L-type Ca(2+ )current (ICaL), and E4031, a blocker of the rapid component of delayed rectifier K(+ )current (IKr). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K(+ )current (IKs). In hiPSC-derived cardiomyocytes of cardiac origin, INa, ICaL, IKr, and IKs were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic cell origins

  20. The Role of Sulfur Dioxide in the Regulation of Mitochondrion-Related Cardiomyocyte Apoptosis in Rats with Isopropylarterenol-Induced Myocardial Injury

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    Junbao Du

    2013-05-01

    Full Text Available The authors investigated the regulatory effects of sulfur dioxide (SO2 on myocardial injury induced by isopropylarterenol (ISO hydrochloride and its mechanisms. Wistar rats were divided into four groups: control group, ISO group, ISO plus SO2 group, and SO2 only group. Cardiac function was measured and cardiomyocyte apoptosis was detected. Bcl-2, bax and cytochrome c (cytc expressions, and caspase-9 and caspase-3 activities in the left ventricular tissues were examined in the rats. The opening status of myocardial mitochondrial permeability transition pore (MPTP and membrane potential were analyzed. The results showed that ISO-treated rats developed heart dysfunction and cardiac injury. Furthermore, cardiomyocyte apoptosis in the left ventricular tissues was augmented, left ventricular tissue bcl-2 expression was down-regulated, bax expression was up-regulated, mitochondrial membrane potential was significantly reduced, MPTP opened, cytc release from mitochondrion into cytoplasm was significantly increased, and both caspase-9 and caspase-3 activities were increased. Administration of an SO2 donor, however, markedly improved heart function and relieved myocardial injury of the ISO-treated rats; it lessened cardiomyocyte apoptosis, up-regulated myocardial bcl-2, down-regulated bax expression, stimulated mitochondrial membrane potential, closed MPTP, and reduced cytc release as well as caspase-9 and caspase-3 activities in the left ventricular tissue. Hence, SO2 attenuated myocardial injury in association with the inhibition of apoptosis in myocardial tissues, and the bcl-2/cytc/caspase-9/caspase-3 pathway was possibly involved in this process.

  1. Protective effect of pomegranate seed oil against H2O2 -induced oxidative stress in cardiomyocytes

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    Mehdi Bihamta

    2017-01-01

    Full Text Available Objective: It has been well documented that oxidative stress is involved in the pathogenesis of cardiac diseases. Previous studies have shown that pomegranate seed oil (PSO has antioxidant properties. This study was designed to investigate probable protective effects of PSO against hydrogen peroxide (H2O2-induced damage in H9c2 cardiomyocytes.Materials and Methods: The cells were pretreated 24 hr with PSO 1 hr before exposure to 200 µM H2O2. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium (MTT assay. The level of reactive oxygen species (ROS and lipid peroxidation were measured by fluorimetric methods.Results: H2O2 significantly decreased cell viability which was accompanied by an increase in ROS production and lipid peroxidation and a decline in superoxide dismutase activity. Pretreatment with PSO increased viability of cardiomyocytes and decrease the elevated ROS production and lipid peroxidation. Also, PSO was able to restore superoxide dismutase activity.Conclusion: PSO has protective effect against oxidative stress-induced damage in cardiomyocytes and can be considered as a natural cardioprotective agent to prevent cardiovascular diseases.

  2. The Correlation of PPARα Activity and Cardiomyocyte Metabolism and Structure in Idiopathic Dilated Cardiomyopathy during Heart Failure Progression

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    E. Czarnowska

    2016-01-01

    Full Text Available This study aimed to define relationship between PPARα expression and metabolic-structural characteristics during HF progression in hearts with DCM phenotype. Tissue endomyocardial biopsy samples divided into three groups according to LVEF ((I 45–50%, n=10; (II 30–40%, n=15; (III 60%, n=6 were investigated. The PPARα mRNA expression in the failing hearts was low in Group (I, high in Group (II, and comparable to that of the control in Group (III. There were analogous changes in the expression of FAT/CD36 and CPT-1 mRNA in contrast to continuous overexpression of GLUT-4 mRNA and significant increase of PDK-4 mRNA in Group (II. In addition, significant structural changes of cardiomyocytes with glycogen accumulation were accompanied by increased expression of PPARα. For the entire study population with HF levels of FAT/CD36 mRNA showed a strong tendency of negative correlation with LVEF. In conclusion, PPARα elevated levels may be a direct cause of adverse remodeling, both metabolic and structural. Thus, there is limited time window for therapy modulating cardiac metabolism and protecting cardiomyocyte structure in failing heart.

  3. Combinatorial polymer matrices enhance in vitro maturation of human induced pluripotent stem cell-derived cardiomyocytes.

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    Chun, Young Wook; Balikov, Daniel A; Feaster, Tromondae K; Williams, Charles H; Sheng, Calvin C; Lee, Jung-Bok; Boire, Timothy C; Neely, M Diana; Bellan, Leon M; Ess, Kevin C; Bowman, Aaron B; Sung, Hak-Joon; Hong, Charles C

    2015-10-01

    Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions. Published by Elsevier Ltd.

  4. Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

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    Khan, Mahmood; Xu, Yanyi; Hua, Serena; Johnson, Jed; Belevych, Andriy; Janssen, Paul M. L.; Gyorke, Sandor; Guan, Jianjun; Angelos, Mark G.

    2015-01-01

    Introduction Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. Methods hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. Results SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Conclusions Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes

  5. Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch.

    Science.gov (United States)

    Khan, Mahmood; Xu, Yanyi; Hua, Serena; Johnson, Jed; Belevych, Andriy; Janssen, Paul M L; Gyorke, Sandor; Guan, Jianjun; Angelos, Mark G

    2015-01-01

    Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment

  6. Bradykinin inhibits oxidative stress-induced cardiomyocytes senescence via regulating redox state.

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    Ruolan Dong

    Full Text Available BACKGROUND: Cell senescence is central to a large body of age related pathology, and accordingly, cardiomyocytes senescence is involved in many age related cardiovascular diseases. In consideration of that, delaying cardiomyocytes senescence is of great importance to control clinical cardiovascular diseases. Previous study indicated that bradykinin (BK protected endothelial cells from senescence induced by oxidative stress. However, the effects of bradykinin on cardiomyocytes senescence remain to be elucidated. In this study, we investigated the effect of bradykinin on H2O2-induced H9C2 cells senescence. METHODS AND RESULTS: Bradykinin pretreatment decreased the senescence induced by H2O2 in cultured H9C2 cells in a dose dependent manner. Interestingly, 1 nmol/L of BK almost completely inhibited the increase in senescent cell number and p21 expression induced by H2O2. Since H2O2 induces senescence through superoxide-induced DNA damage, we also observed the DNA damage by comet assay, and BK markedly reduced DNA damage induced by H2O2, and moreover, BK treatment significantly prevented reactive oxygen species (ROS production in H9C2 cells treated with H2O2. Importantly, when co-incubated with bradykinin B2 receptor antagonist HOE-140 or eNOS inhibitor N-methyl-L-arginine acetate salt (L-NAME, the protective effects of bradykinin on H9C2 senescence were totally blocked. Furthermore, BK administration significantly prevented the increase in nicotinamide adenine dinucleotide phosphate (NADPH oxidase activity characterized by increased ROS generation and gp91 expression and increased translocation of p47 and p67 to the membrane and the decrease in superoxide dismutase (SOD activity and expression induced by H2O2 in H9C2 cells, which was dependent on BK B2 receptor mediated nitric oxide (NO release. CONCLUSIONS: Bradykinin, acting through BK B2 receptor induced NO release, upregulated antioxidant Cu/Zn-SOD and Mn-SOD activity and expression while

  7. The impact of left ventricular stretching in model cultivations with neonatal cardiomyocytes in a whole-heart bioreactor.

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    Hülsmann, Jörn; Aubin, Hug; Wehrmann, Alexander; Lichtenberg, Artur; Akhyari, Payam

    2017-05-01

    Here, we investigate the impact of integrated three-dimensional (3D) left ventricular (LV) stretching on myocardial maturation in a whole-heart bioreactor setting. Therefore, decellularized rat hearts were selectively repopulated with rodent neonatal cardiomyocytes (5 · 10(6) cells per heart) and cultured over 5 days. Continuous medium perfusion was maintained through the coronary artery system in a customized whole-heart bioreactor system with or without integrated biomechanical stimulation of LV. 3D repopulation effectiveness and cellular vitality were evaluated by repetitive metabolic WST-1 assays and 3D confocal microscopy analysis through fluorescent staining, also assessing cellular organization. Moreover, specific myocardial vitality was verified by detecting spontaneous electrophysiological activity using a multielectrode assay. Western blot analysis of cardiac myosin heavychain (MHC) and quantitative RT-PCR for Connexin 43 was used to analyze cardiomyocyte maturation. Decellularized whole-heart constructs repopulated with neonatal cardiomyocytes (repopWHC) showed vital 3D cell populations throughout the repopulation sites within the LV with a significant increase in metabolic activity (326 ± 113% for stimulated constructs vs. 162 ± 32% for non-stimulated controls after 96 h of continuous cultivation as compared to their state 24 h after injection, directly prior to bioreactor cultivation). Further, bioreactor cultivation under integrated mechanical LV stimulation not only led to a higher degree of cellular organization and an increased MHC content, but also to a significant increase of Cx43 gene expression resulting in a regain of 60 ± 19% of native neonatal hearts expression level in contrast to 20 ± 9% for non-stimulated controls (P = 0.03). Therefore, our study suggests that the integration of LV stretching into whole-heart bioreactor cultivation may enhance cardiac maturation not only by promoting cellular organization

  8. Overexpression of KCNJ2 in induced pluripotent stem cell-derived cardiomyocytes for the assessment of QT-prolonging drugs

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    Min Li

    2017-06-01

    Full Text Available Human induced pluripotent stem cell (hiPSC-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (IK1 channel is a determinant of spontaneous activity. To understand impact of the low IK1 expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the IK1 channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 μM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.

  9. Exercise during pregnancy decreases doxorubicin-induced cardiotoxic effects on neonatal hearts.

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    Brito, Verônica B; Nascimento, Leopoldo V M; Nunes, Ramiro B; Moura, Dinara J; Lago, Pedro Dal; Saffi, Jenifer

    2016-08-10

    Cancer treatment with Doxorubicin (DOX) is limited due its dose-dependent cardiotoxicity, mainly related to the oxidative stress production. In experimental models of DOX treatment exercise can be used as a beneficial adjuvant therapy. This work aimed to investigate the effects of exercise during pregnancy on DOX-induced cardiotoxicity in cardiomyocytes of progeny, examining the possible intergenerational cardioprotective effects of maternal exercise. For this purpose pregnant rats were divided in control and exercise groups and pre-treated during gestational days. Hearts of newborns were used to obtain a culture of cardiomyocytes to be treated with DOX for analyses of cell viability, apoptosis and necrosis; ROS production; DNA damage; SOD and CAT activities; and Sirt6 protein expression. The results showed that exercise during pregnancy induced an increase in the viability of neonatal cardiomyocytes and a decrease in DOX-induced apoptotic and necrotic death which were correlated to the decrease in ROS production and an increase in antioxidant defenses. Exercise also protected neonatal cardiomyocytes from DOX-induced DNA damage, demonstrating a reduction in the oxidative DNA breaks. Likewise, exercise induced an increase in expression of Sirt6 in neonatal cardiomyocytes. Therefore, these results demonstrate for the first time that exercise performed by mothers protects the neonatal heart against DOX-induced toxicity. Our data demonstrate the intergenerational effect of exercise in cardiomyocytes of progeny, where the modulation of oxidative stress through antioxidant enzymes, and DNA integrity via Sirt6, were induced due to exercise in mothers, increasing the resistance of the neonatal heart against DOX toxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Effects of cardioactive drugs on human induced pluripotent stem cell derived long QT syndrome cardiomyocytes.

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    Kuusela, Jukka; Kujala, Ville J; Kiviaho, Anna; Ojala, Marisa; Swan, Heikki; Kontula, Kimmo; Aalto-Setälä, Katriina

    2016-01-01

    Human induced pluripotent stem cells (hiPSC) have enabled a major step forward in pathophysiologic studies of inherited diseases and may also prove to be valuable in in vitro drug testing. Long QT syndrome (LQTS), characterized by prolonged cardiac repolarization and risk of sudden death, may be inherited or result from adverse drug effects. Using a microelectrode array platform, we investigated the effects of six different drugs on the electrophysiological characteristics of human embryonic stem cell-derived cardiomyocytes as well as hiPSC-derived cardiomyocytes from control subjects and from patients with type 1 (LQT1) and type 2 (LQT2) of LQTS. At baseline the repolarization time was significantly longer in LQTS cells compared to controls. Isoprenaline increased the beating rate of all cell lines by 10-73 % but did not show any arrhythmic effects in any cell type. Different QT-interval prolonging drugs caused prolongation of cardiac repolarization by 3-13 % (cisapride), 10-20 % (erythromycin), 8-23 % (sotalol), 16-42 % (quinidine) and 12-27 % (E-4031), but we did not find any systematic differences in sensitivity between the control, LQT1 and LQT2 cell lines. Sotalol, quinidine and E-4031 also caused arrhythmic beats and beating arrests in some cases. In summary, the drug effects on these patient-specific cardiomyocytes appear to recapitulate clinical observations and provide further evidence that these cells can be applied for in vitro drug testing to probe their vulnerability to arrhythmia.

  11. Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway.

    Science.gov (United States)

    Altamirano, Francisco; Oyarce, César; Silva, Patricio; Toyos, Marcela; Wilson, Carlos; Lavandero, Sergio; Uhlén, Per; Estrada, Manuel

    2009-08-01

    Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of beta-myosin heavy chain, alpha-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP(3)/Ca(2+) and MEK/ERK1/2 to induce cardiomyocyte hypertrophy.

  12. Activation of β-Adrenoceptors by Dobutamine May Induce a Higher Expression of Peroxisome Proliferator-Activated Receptors δ (PPARδ in Neonatal Rat Cardiomyocytes

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    Ming-Ting Chou

    2012-01-01

    Full Text Available Recent evidence showed the role of peroxisome proliferator-activated receptors (PPARs in cardiac function. Cardiac contraction induced by various agents is critical in restoring the activity of peroxisome proliferator-activated receptors δ (PPARδ in cardiac myopathy. Because dobutamine is an agent widely used to treat heart failure in emergency setting, this study is aimed to investigate the change of PPARδ in response to dobutamine. Neonatal rat cardiomyocytes were used to examine the effects of dobutamine on PPARδ expression levels and cardiac troponin I (cTnI phosphorylation via Western blotting analysis. We show that treatment with dobutamine increased PPARδ expression and cTnI phosphorylation in a time- and dose-dependent manner in neonatal rat cardiomyocytes. These increases were blocked by the antagonist of β1-adrenoceptors. Also, the action of dobutamine was related to the increase of calcium ions and diminished by chelating intracellular calcium. Additionally, dobutamine-induced action was reduced by the inhibition of downstream messengers involved in this calcium-related pathway. Moreover, deletion of PPARδ using siRNA generated the reduction of cTnI phosphorylation in cardiomyocytes treated with dobutamine. Thus, we concluded that PPARδ is increased by dobutamine in cardiac cells.

  13. Protective effect of pioglitazone on cardiomyocyte apoptosis in low-dose streptozotocin & high-fat diet-induced type-2 diabetes in rats

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    Uma Bhandari

    2015-01-01

    Full Text Available Background & objectives: Cardiomyocyte apoptosis is one of the pathologic phenomena associated with diabetes and related conditions including obesity, insulin resistance and hyperlipidaemia. In the present study, the protective effects of pioglitazone on cardiomyocyte apoptosis was evaluated in experimental diabetes induced by low dose of streptozoticin (STZ combined with high fat diet (HFD in rats. Methods: Male Wistar rats (150-200 g were injected with low-dose STZ (45 mg/kg, i.v., single dose and orally fed with a HFD (20 g/day/rat for a period of 28 days and simultaneously treated with pioglitazone (20 mg/kg/p.o. for a period of 21 days (from 8 th day to 28 th day. On 29 th day blood was collected, serum separated and used for biochemical parameters. Heart tissue was used for cardiomyocyte apoptosis measurement and also for histopathological examination. Results: Pioglitazone treatment resulted in a decrease in cardiomyocyte apoptosis as revealed by a decrease in cardiac caspase-3, lactate dehydrogenase (LDH levels and DNA fragmentation, and an increase in Na+K+ATPase levels in diabetic rats. Cardiac histology of diabetic control rats showed dense focal fatty infiltration in the myocardial cells whereas normal architecture with regular morphology and well preserved cytoplasm was observed with pioglitazone treatment. Pioglitazone treatment significantly reduced the heart rate, mean arterial blood pressure, body mass index (BMI and levels of serum glucose, leptin, insulin, HOMA-IR, total cholesterol (TC and triglycerides (TGs, apoliproprotein-B glycosylated haemoglobin (HbA1c levels and atherogenic index, and increased the levels of serum high density lipoprotein cholesterol (HDL-C and cardiac antioxidant enzymes. Interpretation & conclusions: The present study results suggest that pioglitazone possesses cardiac anti-apoptotic potential in diabetic rat model and can be further explored for its use for treatment of diabetic cardiomyopathy.

  14. Characterization of a Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for the Study of Variant Pathogenicity: Validation of a KCNJ2 Mutation.

    Science.gov (United States)

    Gélinas, Roselle; El Khoury, Nabil; Chaix, Marie-A; Beauchamp, Claudine; Alikashani, Azadeh; Ethier, Nathalie; Boucher, Gabrielle; Villeneuve, Louis; Robb, Laura; Latour, Frédéric; Mondesert, Blandine; Rivard, Lena; Goyette, Philippe; Talajic, Mario; Fiset, Céline; Rioux, John David

    2017-10-01

    Long-QT syndrome is a potentially fatal condition for which 30% of patients are without a genetically confirmed diagnosis. Rapid identification of causal mutations is thus a priority to avoid at-risk situations that can lead to fatal cardiac events. Massively parallel sequencing technologies are useful for the identification of sequence variants; however, electrophysiological testing of newly identified variants is crucial to demonstrate causality. Long-QT syndrome could, therefore, benefit from having a standardized platform for functional characterization of candidate variants in the physiological context of human cardiomyocytes. Using a variant in Kir2.1 (Gly52Val) revealed by whole-exome sequencing in a patient presenting with symptoms of long-QT syndrome as a proof of principle, we demonstrated that commercially available human induced pluripotent stem cell-derived cardiomyocytes are a powerful model for screening variants involved in genetic cardiac diseases. Immunohistochemistry experiments and whole-cell current recordings in human embryonic kidney cells expressing the wild-type or the mutant Kir2.1 demonstrated that Kir2.1-52V alters channel cellular trafficking and fails to form a functional channel. Using human induced pluripotent stem cell-derived cardiomyocytes, we not only confirmed these results but also further demonstrated that Kir2.1-52V is associated with a dramatic prolongation of action potential duration with evidence of arrhythmic activity, parameters which could not have been studied using human embryonic kidney cells. Our study confirms the pathogenicity of Kir2.1-52V in 1 patient with long-QT syndrome and also supports the use of isogenic human induced pluripotent stem cell-derived cardiomyocytes as a physiologically relevant model for the screening of variants of unknown function. © 2017 American Heart Association, Inc.

  15. Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes

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    Streckfuss-Boemeke, K.; Tiburcy, M.; Fomin, A.; Luo, X; Li, W; Fischer, C; Oezcelik, C; Perrot, A; Sossalla, S; Haas, J; VIDAL, R. O.; Rebs, S.; Khadjeh, S.; Meder, B; Bonn?, S.

    2017-01-01

    The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric {a...

  16. Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Lee, Sujeong; Lee, Hyang-Ae; Choi, Sung Woo; Kim, Sung Joon; Kim, Ki-Suk

    2016-04-01

    The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effects of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD90) and 50% (APD50) repolarization, reduced the maximum upstroke velocity (dV/dtmax) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including IKr, IKs, INa, and ICa. Among them, IKr and INa showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K(+) channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease

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    Yalin Liao

    2016-08-01

    Full Text Available RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism.

  18. miR-138 protects cardiomyocytes from hypoxia-induced apoptosis via MLK3/JNK/c-jun pathway

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    He, Siyi; Liu, Peng; Jian, Zhao; Li, Jingwei; Zhu, Yun; Feng, Zezhou; Xiao, Yingbin, E-mail: xiaoyb@vip.sina.com

    2013-11-29

    Highlights: •First time to find miR-138 is up-regulated in hypoxic cardiomyocytes. •First time to find miR-138 targets MLK3 and regulates JNK/c-jun pathway. •Rare myocardial biopsy of patients with CHD were collected. •Both silence and overexpression of miR-138 were implemented. •Various methods were used to detect cell function. -- Abstract: Cardiomyocytes experience a series of complex endogenous regulatory mechanisms against apoptosis induced by chronic hypoxia. MicroRNAs are a class of endogenous small non-coding RNAs that regulate cellular pathophysiological processes. Recently, microRNA-138 (miR-138) has been found related to hypoxia, and beneficial for cell proliferation. Therefore, we intend to study the role of miR-138 in hypoxic cardiomyocytes and the main mechanism. Myocardial samples of patients with congenital heart disease (CHD) were collected to test miR-138 expression. Agomir or antagomir of miR-138 was transfected into H9C2 cells to investigate its effect on cell apoptosis. Higher miR-138 expression was observed in patients with cyanotic CHD, and its expression gradually increased with prolonged hypoxia time in H9C2 cells. Using MTT and LDH assays, cell growth was significantly greater in the agomir group than in the negative control (NC) group, while antagomir decreased cell survival. Dual luciferase reporter gene and Western-blot results confirmed MLK3 was a direct target of miR-138. It was found that miR-138 attenuated hypoxia-induced apoptosis using TUNEL, Hoechst staining and Annexin V-PE/7-AAD flow cytometry analysis. We further detected expression of apoptosis-related proteins. In the agomir group, the level of pro-apoptotic proteins such as cleaved-caspase-3, cleaved-PARP and Bad significantly reduced, while Bcl-2 and Bcl-2/Bax ratio increased. Opposite changes were observed in the antagomir group. Downstream targets of MLK3, JNK and c-jun, were also suppressed by miR-138. Our study demonstrates that up-regulation of miR-138 plays

  19. Damage of guinea pig heart and arteries by a trioleate-enriched diet and of cultured cardiomyocytes by oleic acid.

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    Josef Krieglstein

    2010-03-01

    Full Text Available Mono-unsaturated fatty acids (MUFAs like oleic acid have been shown to cause apoptosis of cultured endothelial cells by activating protein phosphatase type 2C alpha and beta (PP2C. The question arises whether damage of endothelial or other cells could be observed in intact animals fed with a trioleate-enriched diet.Dunkin-Hartley guinea pigs were fed with a trioleate-enriched diet for 5 months. Advanced atherosclerotic changes of the aorta and the coronary arteries could not be seen but the arteries appeared in a pre-atherosclerotic stage of vascular remodelling. However, the weight and size of the hearts were lower than in controls and the number of apoptotic myocytes increased in the hearts of trioleate-fed animals. To confirm the idea that oleic acid may have caused this apoptosis by activation of PP2C, cultured cardiomyocytes from guinea pigs and mice were treated with various lipids. It was demonstrable that oleic acid dose-dependently caused apoptosis of cardiomyocytes from both species, yet, similar to previous experiments with cultured neurons and endothelial cells, stearic acid, elaidic acid and oleic acid methylester did not. The apoptotic effect caused by oleic acid was diminished when PP2C alpha and beta were downregulated by siRNA showing that PP2C was causally involved in apoptosis caused by oleic acid.The glycerol trioleate diet given to guinea pigs for 5 months did not cause marked atherosclerosis but clearly damaged the hearts by activating PP2C alpha and beta. The diet used with 24% (wt/wt glycerol trioleate is not comparable to human diets. The detrimental role of MUFAs for guinea pig heart tissue in vivo is shown for the first time. Whether it is true for humans remains to be shown.

  20. Selenium deficiency aggravates T-2 toxin-induced injury of primary neonatal rat cardiomyocytes through ER stress.

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    Xu, Jing; Pan, Shengchi; Gan, Fang; Hao, Shu; Liu, Dandan; Xu, Haibin; Huang, Kehe

    2018-02-16

    Keshan disease is a potentially fatal cardiomyopathy in humans. Selenium deficiency, T-2 toxin, and myocarditis virus are thought to be the major factors contributing to Keshan disease. But the relationship among these three factors is poorly described. This study aims to explore whether selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury and its underlying mechanism. Cardiomyocytes were isolated from neonatal rat and cultured at the physiological (2.0 μM) or lower concentrations of selenium with different concentrations of T-2 toxin. Our results showed that selenium deficiencies aggravated T-2 toxin-induced cardiomyocyte injury in a concentration-dependent manner as demonstrated by MTT bioassay, LDH activity, reactive oxygen species levels and caspase 3 protein expressions. T-2 toxin treatment significantly increased mRNA expressions for stress proteins GRP78 and CHOP in cardiomyocytes compared with the control. Selenium deficiencies further promoted GRP78, CHOP and p-eIF2α expressions. Knockdown of CHOP by the specific small interfering RNA eliminated the effect of selenium deficiencies on T-2 toxin-induced injury. It could be concluded that selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury through initiating more aggressive endoplasmic reticulum stress. Copyright © 2018. Published by Elsevier B.V.

  1. Bioinformatics method identifies potential biomarkers of dilated cardiomyopathy in a human induced pluripotent stem cell-derived cardiomyocyte model.

    Science.gov (United States)

    Zhuang, Yu; Gong, Yu-Jia; Zhong, Bei-Fen; Zhou, Yi; Gong, Li

    2017-10-01

    Dilated cardiomyopathy (DCM) is the most common type of cardiomyopathy that account for the majority of heart failure cases. The present study aimed to reveal the underlying molecular mechanisms of DCM and provide potential biomarkers for detection of this condition. The public dataset of GSE35108 was downloaded, and 4 normal induced pluripotent stem cell (iPSC)-derived cardiomyocytes (N samples) and 4 DCM iPSC-derived cardiomyocytes (DCM samples) were utilized. Raw data were preprocessed, followed by identification of differentially expressed genes (DEGs) between N and DCM samples. Crucial functions and pathway enrichment analysis of DEGs were investigated, and protein-protein interaction (PPI) network analysis was conducted. Furthermore, a module network was extracted from the PPI network, followed by enrichment analysis. A set of 363 DEGs were identified, including 253 upregulated and 110 downregulated genes. Several biological processes (BPs), such as blood vessel development and vasculature development (FLT1 and MMP2), cell adhesion (CDH1, ITGB6, COL6A3, COL6A1 and LAMC2) and extracellular matrix (ECM)-receptor interaction pathway (CDH1, ITGB6, COL6A3, COL6A1 and LAMC2), were significantly enriched by these DEGs. Among them, MMP2, CDH1 and FLT1 were hub nodes in the PPI network, while COL6A3, COL6A1, LAMC2 and ITGB6 were highlighted in module 3 network. In addition, PENK and APLNR were two crucial nodes in module 2, which were linked to each other. In conclusion, several potential biomarkers for DCM were identified, such as MMP2, FLT1, CDH1, ITGB6, COL6A3, COL6A1, LAMC2, PENK and APLNR. These genes may serve significant roles in DCM via involvement of various BPs, such as blood vessel and vasculature development and cell adhesion, and the ECM-receptor interaction pathway.

  2. Experimental research on recombinant human endostatin-induced cardiomyocyte apoptosis in rats

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    Jing QIN

    2014-03-01

    Full Text Available Objective To explore the recombinant human endostatin (rh-ES-induced cardiotoxicity in rats and its mechanism. Methods Twenty four female Wistar rats were randomly divided into four groups (6 each. Rats in low, moderate and high dose group received rh-ES with a dosage of 3, 6 and 12mg/(kg·d, respectively, by intraperitoneal injection, and rats in control group received the same amount of normal saline alone. Half of rats in each group were sacrificed by spinal dislocation after 4 weeks and 8 weeks of the treatment. Pathomorphologic and ultrastructural changes in rat's myocardial tissue were evaluated by light microscopy and transmission electron microscopy. Cardiomyocyte apoptosis was detected with TdT-mediated dUTP nick end labeling (TUNEL assay. Microvessel density (MVD in myocardial tissue was measured by immunohistochemically marking endothelial cell with CD34. Results No pathomorphologic and ultrastrucural changes were found under light microscope and transmission electron microscope in the low dose and moderate dose groups, but cardiomyocyte damage were found in the high dose group. TUNEL assay revealed more apoptotic cells in high and moderate (only 8 weeks dose groups than in control group (P=0.033, P=0.000, and the apoptosis index was highest in the high dose group at 8 weeks. In addition, compared with the control group, MVD significantly increased in high dose groups at 4 weeks and 8 weeks (P<0.05. Conclusions rh-ES induces the cardiotoxicity in rats, and cardiomyocyte apoptosis is involved in the pathological course of cardiac toxicity. DOI: 10.11855/j.issn.0577-7402.2014.01.02

  3. N-glycans: phenotypic homology and structural differences between myocardial cells and induced pluripotent stem cell-derived cardiomyocytes.

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    Takuji Kawamura

    Full Text Available Cell surface glycans vary widely, depending on cell properties. We hypothesized that glycan expression on induced pluripotent stem cells (iPSCs might change during cardiomyogenic differentiation toward the myocardial phenotype. N-glycans were isolated from iPSCs, iPSC-derived cardiomyocytes (iPSC-CM, and original C57BL/6 mouse myocardium (Heart. Their structures were analyzed by a mapping technique based on HPLC elution times and MALDI-TOF/MS spectra. Sixty-eight different N-glycans were isolated; the structures of 60 of these N-glycans were identified. The quantity of high-mannose type (immature N-glycans on the iPSCs decreased with cardiomyogenic differentiation, but did not reach the low levels observed in the heart. We observed a similar reduction in neutral N-glycans and an increase in fucosylated or sialyl N-glycans. Some structural differences were detected between iPSC-CM and Heart. No N-glycolyl neuraminic acid (NeuGc structures were detected in iPSC-CM, whereas the heart contained numerous NeuGc structures, corresponding to the expression of cytidine monophosphate-N-acetylneuraminic acid hydroxylase. Furthermore, several glycans containing Galα1-6 Gal, rarely identified in the other cells, were detected in the iPSC-CM. The expression of N-glycan on murine iPSCs changed toward the myocardial phenotype during cardiomyogenic differentiation, leaving the structural differences of NeuGc content or Galα1-6 Gal structures. Further studies will be warranted to reveal the meaning of the difference of N-glycans between the iPSC-CM and the myocardium.

  4. Ca(2+)-Currents in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Effects of Two Different Culture Conditions.

    Science.gov (United States)

    Uzun, Ahmet U; Mannhardt, Ingra; Breckwoldt, Kaja; Horváth, András; Johannsen, Silke S; Hansen, Arne; Eschenhagen, Thomas; Christ, Torsten

    2016-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM). Here we investigated whether a 3D engineered heart tissue (EHT) culture format favors maturation and addressed the L-type Ca(2+)-current (ICa,L) as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML) and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were two- to three-fold smaller than adult CM, independently of culture format [capacitance ML 45 ± 1 pF (n = 289), EHT 45 ± 1 pF (n = 460), atrial CM 87 ± 3 pF (n = 196), ventricular CM 126 ± 8 pF (n = 50)]. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10 ± 1 pA/pF (n = 207) for ML and 12 ± 1 pA/pF (n = 361) for EHT and was larger than in adult CM [7 ± 1 pA/pF (p < 0.05, n = 196) for atrial CM and 6 ± 1 pA/pF (p < 0.05, n = 47) for ventricular CM]. However, ML and EHT showed robust T-type Ca(2+)-currents (ICa,T). While (-)-Bay K 8644, that activates ICa,L directly, increased ICa,Lto the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (-)-Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE) and epinephrine (Epi), respectively), but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi). Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation.

  5. Ca2+-currents in human induced pluripotent stem cell-derived cardiomyocytes - effects of two different culture conditions

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    Ahmet Umur Uzun

    2016-09-01

    Full Text Available Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM. Here we investigated whether a 3D engineered heart tissue (EHT culture format favors maturation and addressed the L-type Ca2+-current (ICa,L as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were 2-3 fold smaller than adult CM, independently of culture format (capacitance ML 45±1 pF (n=289, EHT 45±1 pF (n=460, atrial CM 87±3 pF (n=196, ventricular CM 126±8 pF (n=50. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10±1 pA/pF (n=207 for ML and 12±1 pA/pF (n=361 for EHT and was larger than in adult CM (7±1 pA/pF (p<0.05, n=196 for atrial CM and 6±1 pA/pF (p<0.05, n=47 for ventricular CM. However, ML and EHT showed robust T-type Ca2+-currents (ICa,T. While (--Bay K 8644, that activates ICa,L directly, increased ICa,L to the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (--Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE and epinephrine (Epi, respectively, but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi. Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation.

  6. Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration.

    Science.gov (United States)

    Chen, William C W; Wang, Zhouguang; Missinato, Maria Azzurra; Park, Dae Woo; Long, Daniel Ward; Liu, Heng-Jui; Zeng, Xuemei; Yates, Nathan A; Kim, Kang; Wang, Yadong

    2016-11-01

    Heart attack is a global health problem that leads to significant morbidity, mortality, and health care burden. Adult human hearts have very limited regenerative capability after injury. However, evolutionarily primitive species generally have higher regenerative capacity than mammals. The extracellular matrix (ECM) may contribute to this difference. Mammalian cardiac ECM may not be optimally inductive for cardiac regeneration because of the fibrotic, instead of regenerative, responses in injured adult mammalian hearts. Given the high regenerative capacity of adult zebrafish hearts, we hypothesize that decellularized zebrafish cardiac ECM (zECM) made from normal or healing hearts can induce mammalian heart regeneration. Using zebrafish and mice as representative species of lower vertebrates and mammals, we show that a single administration of zECM, particularly the healing variety, enables cardiac functional recovery and regeneration of adult mouse heart tissues after acute myocardial infarction. zECM-treated groups exhibit proliferation of the remaining cardiomyocytes and multiple cardiac precursor cell populations and reactivation of ErbB2 expression in cardiomyocytes. Furthermore, zECM exhibits pro-proliferative and chemotactic effects on human cardiac precursor cell populations in vitro. These contribute to the structural preservation and correlate with significantly higher cardiac contractile function, notably less left ventricular dilatation, and substantially more elastic myocardium in zECM-treated hearts than control animals treated with saline or decellularized adult mouse cardiac ECM. Inhibition of ErbB2 activity abrogates beneficial effects of zECM administration, indicating the possible involvement of ErbB2 signaling in zECM-mediated regeneration. This study departs from conventional focuses on mammalian ECM and introduces a new approach for cardiac tissue regeneration.

  7. ALDH2 Inhibition Potentiates High Glucose Stress-Induced Injury in Cultured Cardiomyocytes

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    Guodong Pan

    2016-01-01

    Full Text Available Aldehyde dehydrogenase (ALDH gene superfamily consists of 19 isozymes. They are present in various organs and involved in metabolizing aldehydes that are biologically generated. For instance, ALDH2, a cardiac mitochondrial ALDH isozyme, is known to detoxify 4-hydroxy-2-nonenal, a reactive aldehyde produced upon lipid peroxidation in diabetic conditions. We hypothesized that inhibition of ALDH leads to the accumulation of unmetabolized 4HNE and consequently exacerbates injury in cells subjected to high glucose stress. H9C2 cardiomyocyte cell lines were pretreated with 10 μM disulfiram (DSF, an inhibitor of ALDH2 or vehicle (DMSO for 2 hours, and then subjected to high glucose stress {33 mM D-glucose (HG or 33 mM D-mannitol as an osmotic control (Ctrl} for 24 hrs. The decrease in ALDH2 activity with DSF pretreatment was higher in HG group when compared to Ctrl group. Increased 4HNE adduct formation with DSF pretreatment was higher in HG group compared to Ctrl group. Pretreatment with DSF leads to potentiated HG-induced cell death in cultured H9C2 cardiomyocytes by lowering mitochondrial membrane potential. Our results indicate that ALDH2 activity is important in preventing high glucose induced cellular dysfunction.

  8. Urocortin-induced cardiomyocytes hypertrophy is associated with regulation of the GSK-3β pathway.

    Science.gov (United States)

    Gruson, Damien; Ginion, Audrey; Decroly, Noémie; Lause, Pascale; Vanoverschelde, Jean-Louis; Ketelslegers, Jean-Marie; Bertrand, Luc; Thissen, Jean-Paul

    2012-03-01

    Urocortin-1 (UCN), a member of the corticotropin-releasing factor, is a cardioprotective peptide, and is also involved in cardiac hypertrophy. The involvement of GSK-3β, a pivotal kinase in cardiac hypertrophy, in response to UCN is not yet documented. Cardiomyocytes from adult rats were stimulated for 48 h with UCN. Cell size, protein, and DNA contents were determined. Phosphorylated and total forms GSK-3β and the total amount of β-catenin were quantified by Western immunoblots. The effects of astressin, a UCN competitive receptor antagonist, were also evaluated. UCN increased cell size and the protein-to-DNA ratio, in accordance with a hypertrophic response. This effect was associated with increased phosphorylation of GSK-3β and marked accumulation of β-catenin, a downstream element to GSK-3β. All these effects were prevented by astressin and LY294002, an inhibitor of the phosphatidyl-inositol-3-kinase. UCN-induced cardiomyocytes hypertrophy is associated with regulation of GSK-3β, a pivotal kinase involved in cardiac hypertrophy, in a PI3K-dependent manner. Furthermore, the pharmacological blockade of UCN receptors was able to prevent UCN-induced hypertrophy, which leads to inhibition of the Akt/GSK-3β pathway.

  9. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

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    Daniel R. Bayzigitov

    2016-01-01

    Full Text Available Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

  10. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    Science.gov (United States)

    Bayzigitov, Daniel R.; Medvedev, Sergey P.; Dementyeva, Elena V.; Bayramova, Sevda A.; Pokushalov, Evgeny A.; Karaskov, Alexander M.; Zakian, Suren M.

    2016-01-01

    Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes. PMID:27110425

  11. CRISPR correction of the PRKAG2 gene mutation in a patient's induced pluripotent stem cell-derived cardiomyocytes eliminates electrophysiological and structural abnormalities.

    Science.gov (United States)

    Ben Jehuda, Ronen; Eisen, Binyamin; Shemer, Yuval; Mekies, Lucy N; Szantai, Agnes; Reiter, Irina; Cui, Huanhuan; Guan, Kaomei; Haron-Khun, Shiraz; Freimark, Dov; Sperling, Silke R; Gherghiceanu, Mihaela; Arad, Michael; Binah, Ofer

    2017-09-14

    Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial Wolff-Parkinson-White (WPW) syndrome. Patients carrying the R302Q mutation in PRKAG2 present with sinus bradycardia, escape rhythms, ventricular preexcitation, supraventricular tachycardia, and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW syndrome, HCM, and arrhythmias remains unknown. The purpose of this study was to investigate the pathological changes caused by the PRKAG2 mutation. We tested the hypothesis that a patient's induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease. Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we corrected the mutation and then generated isogenic iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. Using a microelectrode array setup, we recorded electrograms from iPSC-CM clusters. Transmission electron microscopy was used to detect ultrastructural abnormalities in the mutated iPSC-CMs. PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations, triggered arrhythmias, and augmented beat rate variability. Importantly, CRISPR correction eliminated the electrophysiological abnormalities, the augmented glycogen, storage, and cardiomyocyte hypertrophy. PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using CRISPR technology. Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  12. Polyamine Depletion Attenuates Isoproterenol-Induced Hypertrophy and Endoplasmic Reticulum Stress in Cardiomyocytes

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    Yan Lin

    2014-10-01

    Full Text Available Background/Aim: Polyamines (putrescine, spermidine and spermine play an essential role in cell growth, differentiation and apoptosis. Hypertrophy is accompanied by an increase in polyamine synthesis and endoplasmic reticulum stress (ERS in cardiomyocytes. The present study was undertaken to elucidate the molecular interactions between polyamines, ERS and cardiac hypertrophy. Methods: Myocardial hypertrophy was simulated by incubating cultured neonatal rat cardiomyocytes in 100 nM isoproterenol (ISO. Polyamine deletion was achieved using 0.5 mM difluoromethylornithine (DFMO. Hypertrophy was estimated using cell surface area measurements, total protein concentrations and atrial natriuretic peptide (ANP gene expression. Apoptosis was measured using flow cytometry and transmission electron microscopy. Expression of ornithine decarboxylase (ODC and spermidine/spermine N1-acetyltransferase (SSAT were analyzed via real-time PCR and Western blotting. Protein expression of ERS and apoptosis factors were analyzed using Western blotting. Results: DFMO (0.5 mM and 2 mM treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO also decreased lactate dehydrogenase (LDH and malondialdehyde (MDA level in the culture medium. In addition, DFMO (0.5 mM down regulated the expression of ODC, glucose-regulated protein 78 (GRP78, C/EBP homologous protein (CHOP, cleaved caspase-12, and Bax and up regulated the expression of SSAT and Bcl-2. Finally, these changes were partly reversed by the addition of exogenous putrescine (0.5 mM. Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

  13. Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells

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    Hongran Wang

    2016-03-01

    Full Text Available Induced pluripotent stem (iPS cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc, without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs, iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin, cardiac alpha myosin heavy chain (α-MHC, cardiac troponin T (cTnT, and connexin 43 (CX43, as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5 and gata binding protein 4 (gata4. The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI in the future.

  14. Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation

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    Hinits, Yaniv; Pan, Luyuan; Walker, Charline; Dowd, John; Moens, Cecilia B.; Hughes, Simon M.

    2013-01-01

    Summary Mef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart. PMID:22750409

  15. Basal and β-Adrenergic Cardiomyocytes Contractility Dysfunction Induced by Dietary Protein Restriction is Associated with Downregulation of SERCA2a Expression and Disturbance of Endoplasmic Reticulum Ca2+ Regulation in Rats

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    Arlete R. Penitente

    2014-07-01

    Full Text Available Background: The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. Methods: After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20 and a protein-restricted group (PRG, n = 20, receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca2+sparks analysis. Results: PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca2+sparks were observed in PRG cardiomyocytes. Conclusion: The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca2+ intracellular kinetics.

  16. Acetylcholine Attenuates Hydrogen Peroxide-Induced Intracellular Calcium Dyshomeostasis Through Both Muscarinic and Nicotinic Receptors in Cardiomyocytes

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    Siripong Palee

    2016-06-01

    Full Text Available Background/Aims: Oxidative stress induced intracellular Ca2+ overload plays an important role in the pathophysiology of several heart diseases. Acetylcholine (ACh has been shown to suppress reactive oxygen species generation during oxidative stress. However, there is little information regarding the effects of ACh on the intracellular Ca2+ regulation in the presence of oxidative stress. Therefore, we investigated the effects of ACh applied before or after hydrogen peroxide (H2O2 treatment on the intracellular Ca2+ regulation in isolated cardiomyocytes. Methods: Single ventricular myocytes were isolated from the male Wistar rats for the intracellular Ca2+ transient study by a fluorimetric ratio technique. Results: H2O2 significantly decreased both of intracellular Ca2+ transient amplitude and decay rate. ACh applied before, but not after, H2O2 treatment attenuated the reduction of intracellular Ca2+ transient amplitude and decay rate. Both atropine (a muscarinic acetylcholine receptor blocker and mecamylamine (a nicotinic acetylcholine receptor blocker significantly decreased the protective effects of acetylcholine on the intracellular Ca2+ regulation. Moreover, the combination of atropine and mecamylamine completely abolished the protective effects of acetylcholine on intracellular Ca2+ transient amplitude and decay rate. Conclusion: ACh pretreatment attenuates H2O2-induced intracellular Ca2+ dyshomeostasis through both muscarinic and nicotinic receptors.

  17. Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy.

    Science.gov (United States)

    Tigchelaar, Wardit; Yu, Hongjuan; de Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Silljé, Herman H W

    2015-01-15

    Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy. Copyright © 2015 the American Physiological Society.

  18. Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

    Science.gov (United States)

    Jung, Gwanghyun; Fajardo, Giovanni; Ribeiro, Alexandre J S; Kooiker, Kristina Bezold; Coronado, Michael; Zhao, Mingming; Hu, Dong-Qing; Reddy, Sushma; Kodo, Kazuki; Sriram, Krishna; Insel, Paul A; Wu, Joseph C; Pruitt, Beth L; Bernstein, Daniel

    2016-04-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used β-adrenergic receptor (β-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), β2-ARs are a primary source of cAMP/PKA signaling. With longer culture, β1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic β-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30vs 55% at d 90. Moreover, β-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of β-AR functionalvs remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functionalvs remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation. © FASEB.

  19. Spermine inhibits Endoplasmic Reticulum Stress - induced Apoptosis: a New Strategy to Prevent Cardiomyocyte Apoptosis

    Directory of Open Access Journals (Sweden)

    Can Wei

    2016-02-01

    Full Text Available Background/Aims: Endoplasmic reticulum stress (ERS plays an important role in the progression of acute myocardial infarction (AMI, in part by mediating apoptosis. Polyamines, including putrescine, spermidine, and spermine, are polycations with anti-oxidative, anti-aging, and cell growth-promoting activities. This study aimed to determine the mechanisms by which spermine protects against ERS-induced apoptosis in rats following AMI. Methods and Results: AMI was established by ligation of the left anterior descending coronary artery (LAD in rats, and exogenous spermine was administered by intraperitoneal injection (2.5 mg/ml daily for 7 days pre-AMI. Spermine treatment limited infarct size, attenuated cardiac troponin I and creatinine kinase-MB release, improved cardiac function, and decreased ERS and apoptosis related protein expression. Isolated cardiomyocytes subjected to hypoxia showed significant increase in reactive oxygen species (ROS and the expression of apoptosis and ERS related proteins; these effects occurred through PERK and eIF2α phosphorylation. The addition of spermine attenuated cardiomyocyte apoptosis, suppressed the production of ROS, and inhibited ERS related pathways. Conclusions: Spermine was an effective pre-treatment strategy to attenuate cardiac ERS injury in rats, and the cardioprotective mechanism occurring through inhibition of ROS production and down regulation of the PERK-eIF2α pathway. These findings provide a novel target for the prevention of apoptosis in the setting of AMI.

  20. Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Josè Manuel Pioner

    2016-06-01

    Full Text Available Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs. Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G. These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations.

  1. Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy

    Science.gov (United States)

    Putinski, Charis; Abdul-Ghani, Mohammad; Stiles, Rebecca; Brunette, Steve; Dick, Sarah A.; Fernando, Pasan; Megeney, Lynn A.

    2013-01-01

    Cardiomyocyte hypertrophy is the cellular response that mediates pathologic enlargement of the heart. This maladaptation is also characterized by cell behaviors that are typically associated with apoptosis, including cytoskeletal reorganization and disassembly, altered nuclear morphology, and enhanced protein synthesis/translation. Here, we investigated the requirement of apoptotic caspase pathways in mediating cardiomyocyte hypertrophy. Cardiomyocytes treated with hypertrophy agonists displayed rapid and transient activation of the intrinsic-mediated cell death pathway, characterized by elevated levels of caspase 9, followed by caspase 3 protease activity. Disruption of the intrinsic cell death pathway at multiple junctures led to a significant inhibition of cardiomyocyte hypertrophy during agonist stimulation, with a corresponding reduction in the expression of known hypertrophic markers (atrial natriuretic peptide) and transcription factor activity [myocyte enhancer factor-2, nuclear factor kappa B (NF-κB)]. Similarly, in vivo attenuation of caspase activity via adenoviral expression of the biologic effector caspase inhibitor p35 blunted cardiomyocyte hypertrophy in response to agonist stimulation. Treatment of cardiomyocytes with procaspase 3 activating compound 1, a small-molecule activator of caspase 3, resulted in a robust induction of the hypertrophy response in the absence of any agonist stimulation. These results suggest that caspase-dependent signaling is necessary and sufficient to promote cardiomyocyte hypertrophy. These results also confirm that cell death signal pathways behave as active remodeling agents in cardiomyocytes, independent of inducing an apoptosis response. PMID:24101493

  2. Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

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    Xu, Aibin; Liu, Jingyi [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Institute of Cardiovascular Disease, General Hospital of Beijing Command, PLA, Beijing (China); Liu, Peilin; Jia, Min; Wang, Han [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Tao, Ling, E-mail: lingtao2006@gmail.com [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China)

    2014-04-18

    Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H{sub 2}O{sub 2} led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H{sub 2}O{sub 2} and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the

  3. Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

    Science.gov (United States)

    Xu, Jiqian; Hu, Houxiang; Chen, Bin; Yue, Rongchuan; Zhou, Zhou; Liu, Yin; Zhang, Shuang; Xu, Lei; Wang, Huan; Yu, Zhengping

    2015-01-01

    Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes. PMID:26291709

  4. Drug-mediated shortening of action potentials in LQTS2 human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Duncan, Gary; Firth, Karl; George, Vinoj; Hoang, Minh Duc; Staniforth, Andrew; Smith, Godfrey; Denning, Chris

    2017-10-09

    Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are now a well-established modality for modelling genetic disorders of the heart. This is especially so for long QT syndrome (LQTS), which is caused by perturbation of ion channel function, and can lead to fainting, malignant arrhythmias and sudden cardiac death. LQTS2 is caused by mutations in KCNH2, a gene whose protein product contributes to IKr (also known as HERG), which is the predominant repolarising potassium current in CMs. b-blockers are the mainstay treatment for patients with LQTS, functioning by reducing heart rate and arrhythmogenesis. However, they are not effective in around a quarter of LQTS2 patients, in part because they do not correct the defining feature of the condition, which is excessively prolonged QT interval. Since new therapeutics are needed, in this report, we biopsied skin fibroblasts from a patient who was both genetically and clinically diagnosed with LQTS2. By producing LQTS-hiPSC-CMs, we assessed the impact of different drugs on action potential duration (APD), which is used as an in vitro surrogate for QT interval. Not surprisingly, the patient's own b-blocker medication, propranolol, had a marginal effect on APD in the LQTS-hiPSC-CMs. However, APD could be significantly reduced by up to 19% with compounds that enhanced the IKr current by direct channel binding or by indirect mediation via the PPARd/protein 14-3-3 epsilon/HERG pathway. Drug-induced enhancement of an alternative potassium current, IKATP, also reduced APD by up to 21%. This study demonstrates the utility of LQTS-hiPSC-CMs is evaluating whether drugs can shorten APD and, importantly, shows that PPARd agonists may form a new class of therapeutics for this condition.

  5. Ghrelin inhibits apoptosis induced by high glucose and sodium palmitate in adult rat cardiomyocytes through the PI3K-Akt signaling pathway.

    Science.gov (United States)

    Kui, Liu; Weiwei, Zhang; Ling, Liu; Daikun, He; Guoming, Zhou; Linuo, Zhou; Renming, Hu

    2009-06-05

    Ghrelin is a gastric acyl-peptide that has been identified as an endogenous ligand for the growth hormone secretagogue receptor. It has been reported to have cardioprotective activities independent of growth hormone release. We investigated the effect of ghrelin on apoptosis induced by high glucose and sodium palmitate and the mechanisms underlying the cardioprotective activities of ghrelin. Cardiomyocytes were isolated from hearts of adult rats and cultured in serum-free MEM. High glucose (30 mM) or sodium palmitate (0.5 mM) were used to induce apoptosis. Apoptosis was detected using an annexin V-FITC/PI binding assay and a caspase 3 activity assay. Reactive oxygen species were detected using a DCFH-DA fluorescent probe. Phospho-Akt, phospho-ERK, and NF kappaB levels were determined using ELISA. The transcription of genes was analyzed using real-time PCR. Ghrelin can inhibit apoptosis induced by oxidative stress in cardiomyocytes from adult rats through the activation of the PI3K-Akt signaling pathway. In addition, ghrelin does not decrease intracellular oxidative stress. Activation of the MEK-ERK1/2 signaling pathway has no influence on the inhibition of apoptosis. Finally, ghrelin activates NF kappaB and subsequently increases the transcription of survival genes such as Bcl-2, Bcl-xL, c-iap, and c-fos. Our research provides evidence that ghrelin may act as a survival factor under oxidative stress in cardiomyocytes. This may provide a clue for therapy for myocardial disease in diabetes mellitus.

  6. Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Chaudhari, Umesh; Nemade, Harshal; Sureshkumar, Poornima; Vinken, Mathieu; Ates, Gamze; Rogiers, Vera; Hescheler, Jürgen; Hengstler, Jan Georg; Sachinidis, Agapios

    2017-09-22

    There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

  7. PEP-1-CAT protects hypoxia/reoxygenation-induced cardiomyocyte apoptosis through multiple sigaling pathways

    Science.gov (United States)

    2013-01-01

    Background Catalase (CAT) breaks down H2O2 into H2O and O2 to protects cells from oxidative damage. However, its translational potential is limited because exogenous CAT cannot enter living cells automatically. This study is aimed to investigate if PEP-1-CAT fusion protein can effectively protect cardiomyocytes from oxidative stress due to hypoxia/reoxygenation (H/R)-induced injury. Methods H9c2 cardomyocytes were pretreated with catalase (CAT) or PEP-1-CAT fusion protein followed by culturing in a hypoxia and re-oxygenation condition. Cell apoptosis were measured by Annexin V and PI double staining and Flow cytometry. Intracellular superoxide anion level was determined, and mitochondrial membrane potential was measured. Expression of apoptosis-related proteins including Bcl-2, Bax, Caspase-3, PARP, p38 and phospho-p38 was analyzed by western blotting. Results PEP-1-CAT protected H9c2 from H/R-induced morphological alteration and reduced the release of lactate dehydrogenase (LDH) and malondialdehyde content. Superoxide anion production was also decreased. In addition, PEP-1-CAT inhibited H9c2 apoptosis and blocked the expression of apoptosis stimulator Bax while increased the expression of Bcl-2, leading to an increased mitochondrial membrane potential. Mechanistically, PEP-1-CAT inhibited p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways, resulting in blockade of Bcl2/Bax/mitochondrial apoptotic pathway. Conclusion Our study has revealed a novel mechanism by which PEP-1-CAT protects cardiomyocyte from H/R-induced injury. PEP-1-CAT blocks Bcl2/Bax/mitochondrial apoptotic pathway by inhibiting p38 MAPK while activating PI3K/Akt and Erk1/2 signaling pathways. PMID:23642335

  8. Development of correction formula for field potential duration of human induced pluripotent stem cell-derived cardiomyocytes sheets

    Directory of Open Access Journals (Sweden)

    Hiroko Izumi-Nakaseko

    2017-09-01

    Full Text Available Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs have been used in many studies to assess proarrhythmic risks of chemical compounds. In those studies, field potential durations (FPD of hiPSC-CMs have been corrected by clinically used Fridericia's and/or Bazett's formulae, however, the rationale for the use of these formulae has not been well established. In the present study, we developed a correction formula for experiments using hiPSC-CMs. First, we analyzed the effect of beating rate on FPD in the hiPSC-CMs sheets with electrical stimuli and a HCN channel inhibitor zatebradine. Next, we examined the relationship between the electrophysiological properties and the expression levels of ion channel genes in the cell sheets. Zatebradine slowed the beating rate and allowed to analyze FPD changes at various pacing cycle lengths. Rate-dependent change in the repolarization period was smaller in the cell sheets than that reported on the human hearts, which can be partly explained by lower gene expression level of hKCNJ2 and hKCNE1. Thus, non-linear equation for correcting FPD in the cell sheet; FPDc = FPD/RR0.22 with RR given in second was obtained, which may make it feasible to assess net repolarization delay by various chemical compounds with a chronotropic action.

  9. Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.

    Science.gov (United States)

    Ong, Chin Siang; Fukunishi, Takuma; Zhang, Huaitao; Huang, Chen Yu; Nashed, Andrew; Blazeski, Adriana; DiSilvestre, Deborah; Vricella, Luca; Conte, John; Tung, Leslie; Tomaselli, Gordon F; Hibino, Narutoshi

    2017-07-04

    We have developed a novel method to deliver stem cells using 3D bioprinted cardiac patches, free of biomaterials. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblasts (FB) and endothelial cells (EC) were aggregated to create mixed cell spheroids. Cardiac patches were created from spheroids (CM:FB:EC = 70:15:15, 70:0:30, 45:40:15) using a 3D bioprinter. Cardiac patches were analyzed with light and video microscopy, immunohistochemistry, immunofluorescence, cell viability assays and optical electrical mapping. Cardiac tissue patches of all cell ratios beat spontaneously after 3D bioprinting. Patches exhibited ventricular-like action potential waveforms and uniform electrical conduction throughout the patch. Conduction velocities were higher and action potential durations were significantly longer in patches containing a lower percentage of FBs. Immunohistochemistry revealed staining for CM, FB and EC markers, with rudimentary CD31+ blood vessel formation. Immunofluorescence revealed the presence of Cx43, the main cardiac gap junction protein, localized to cell-cell borders. In vivo implantation suggests vascularization of 3D bioprinted cardiac patches with engraftment into native rat myocardium. This constitutes a significant step towards a new generation of stem cell-based treatment for heart failure.

  10. Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes.

    Science.gov (United States)

    Tohyama, Shugo; Fujita, Jun; Fujita, Chihana; Yamaguchi, Miho; Kanaami, Sayaka; Ohno, Rei; Sakamoto, Kazuho; Kodama, Masami; Kurokawa, Junko; Kanazawa, Hideaki; Seki, Tomohisa; Kishino, Yoshikazu; Okada, Marina; Nakajima, Kazuaki; Tanosaki, Sho; Someya, Shota; Hirano, Akinori; Kawaguchi, Shinji; Kobayashi, Eiji; Fukuda, Keiichi

    2017-10-04

    Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 10(6) hiPSCs per layer yielded 7.2 × 10(8) hiPSCs in 4-layer CPs and 1.7 × 10(9) hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%-87%. Approximately 6.2-7.0 × 10(8) cells (4-layer) and 1.5-2.8 × 10(9) cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Development of correction formula for field potential duration of human induced pluripotent stem cell-derived cardiomyocytes sheets.

    Science.gov (United States)

    Izumi-Nakaseko, Hiroko; Kanda, Yasunari; Nakamura, Yuji; Hagiwara-Nagasawa, Mihoko; Wada, Takeshi; Ando, Kentaro; Naito, Atsuhiko T; Sekino, Yuko; Sugiyama, Atsushi

    2017-09-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used in many studies to assess proarrhythmic risks of chemical compounds. In those studies, field potential durations (FPD) of hiPSC-CMs have been corrected by clinically used Fridericia's and/or Bazett's formulae, however, the rationale for the use of these formulae has not been well established. In the present study, we developed a correction formula for experiments using hiPSC-CMs. First, we analyzed the effect of beating rate on FPD in the hiPSC-CMs sheets with electrical stimuli and a HCN channel inhibitor zatebradine. Next, we examined the relationship between the electrophysiological properties and the expression levels of ion channel genes in the cell sheets. Zatebradine slowed the beating rate and allowed to analyze FPD changes at various pacing cycle lengths. Rate-dependent change in the repolarization period was smaller in the cell sheets than that reported on the human hearts, which can be partly explained by lower gene expression level of hKCNJ2 and hKCNE1. Thus, non-linear equation for correcting FPD in the cell sheet; FPDc = FPD/RR(0.22) with RR given in second was obtained, which may make it feasible to assess net repolarization delay by various chemical compounds with a chronotropic action. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  12. Expression of Foxm1 transcription factor in cardiomyocytes is required for myocardial development.

    Directory of Open Access Journals (Sweden)

    Craig Bolte

    Full Text Available Forkhead Box M1 (Foxm1 is a transcription factor essential for organ morphogenesis and development of various cancers. Although complete deletion of Foxm1 in Foxm1(-/- mice caused embryonic lethality due to severe abnormalities in multiple organ systems, requirements for Foxm1 in cardiomyocytes remain to be determined. This study was designed to elucidate the cardiomyocyte-autonomous role of Foxm1 signaling in heart development. We generated a new mouse model in which Foxm1 was specifically deleted from cardiomyocytes (Nkx2.5-Cre/Foxm1(fl/f mice. Deletion of Foxm1 from cardiomyocytes was sufficient to disrupt heart morphogenesis and induce embryonic lethality in late gestation. Nkx2.5-Cre/Foxm1(fl/fl hearts were dilated with thinning of the ventricular walls and interventricular septum, as well as disorganization of the myocardium which culminated in cardiac fibrosis and decreased capillary density. Cardiomyocyte proliferation was diminished in Nkx2.5-Cre/Foxm1(fl/fl hearts owing to altered expression of multiple cell cycle regulatory genes, such as Cdc25B, Cyclin B(1, Plk-1, nMyc and p21(cip1. In addition, Foxm1 deficient hearts displayed reduced expression of CaMKIIδ, Hey2 and myocardin, which are critical mediators of cardiac function and myocardial growth. Our results indicate that Foxm1 expression in cardiomyocytes is critical for proper heart development and required for cardiomyocyte proliferation and myocardial growth.

  13. DPP4 deficiency exerts protective effect against H2O2 induced oxidative stress in isolated cardiomyocytes.

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    Hui-Chun Ku

    Full Text Available Apart from the antihyperglycemic effects, DPP4 inhibitors and GLP-1 molecules are involved in the preservation of cardiac functions. We have demonstrated that DPP4-deficient rats possess resistance to endotoxemia and ischemia/reperfusion stress. However, whether the decrease of DPP4 activity simply augmented the GLP-1 signaling or that such decrease resulted in a change of cellular function remain unclear. Accordingly, we investigated the responses of H(2O(2-induced oxidative stress in adult wild-type and DPP4-deficient rats isolated cardiomyocytes. The coadministration of GLP-1 or DPP4 inhibitor was also performed to define the mechanisms. Cell viability, ROS concentration, catalase activity, glucose uptake, prosurvival, proapoptotic signaling, and contractile function were examined after cells exposed to H(2O(2. DPP4-deficient cardiomyocytes were found to be resistant to H(2O(2-induced cell death via activating AKT signaling, enhancing glucose uptake, preserving catalase activity, diminishing ROS level and proapoptotic signaling. GLP-1 concentration-dependently improved cell viability in wild-type cardiomyocyte against ROS stress, and the ceiling response concentration (200 nM was chosen for studies. GLP-1 was shown to decrease H(2O(2-induced cell death by its receptor-dependent AKT pathway in wild-type cardiomyocytes, but failed to cause further activation of AKT in DPP4-deficient cardiomyocytes. Acute treatment of DPP4 inhibitor only augmented the protective effect of low dose GLP-1, but failed to alter fuel utilization or ameliorate cell viability in wild-type cardiomyocytes after H(2O(2 exposure. The improvement of cell viability after H(2O(2 exposure was correlated with the alleviation of cellular contractile dysfunction in both DPP4-deficient and GLP-1 treated wild-type cardiomyocytes. These findings demonstrated that GLP-1 receptor-dependent pathway is important and exert protective effect in wild-type cardiomyocyte. Long term loss of

  14. Autoantibodies in dilated cardiomyopathy induce vascular endothelial growth factor expression in cardiomyocytes

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    Saygili, Erol, E-mail: erol.saygili@med.uni-duesseldorf.de [Division of Cardiology, Pulmonology, and Vascular Medicine, University Hospital Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf (Germany); Noor-Ebad, Fawad; Schröder, Jörg W.; Mischke, Karl [Department of Cardiology, University RWTH Aachen, Pauwelsstr. 30, D-52074 Aachen (Germany); Saygili, Esra [Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, D-40225 Düsseldorf (Germany); Rackauskas, Gediminas [Department of Cardiovascular Medicine, Vilnius University Hospital Santariskiu Klinikos, Vilnius University (Lithuania); Marx, Nikolaus [Department of Cardiology, University RWTH Aachen, Pauwelsstr. 30, D-52074 Aachen (Germany); Kelm, Malte; Rana, Obaida R. [Division of Cardiology, Pulmonology, and Vascular Medicine, University Hospital Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf (Germany)

    2015-09-11

    Background: Autoantibodies have been identified as major predisposing factors for dilated cardiomyopathy (DCM). Patients with DCM show elevated serum levels of vascular endothelial growth factor (VEGF) whose source is unknown. Besides its well-investigated effects on angiogenesis, evidence is present that VEGF signaling is additionally involved in fibroblast proliferation and cardiomyocyte hypertrophy, hence in cardiac remodeling. Whether autoimmune effects in DCM impact cardiac VEGF signaling needs to be elucidated. Methods: Five DCM patients were treated by the immunoadsorption (IA) therapy on five consecutive days. The eluents from the IA columns were collected and prepared for cell culture. Cardiomyocytes from neonatal rats (NRCM) were incubated with increasing DCM-immunoglobulin-G (IgG) concentrations for 48 h. Polyclonal IgG (Venimmun N), which was used to restore IgG plasma levels in DCM patients after the IA therapy was additionally used for control cell culture purposes. Results: Elevated serum levels of VEGF decreased significantly after IA (Serum VEGF (ng/ml); DCM pre-IA: 45 ± 9.1 vs. DCM post–IA: 29 ± 6.7; P < 0.05). In cell culture, pretreatment of NRCM by DCM-IgG induced VEGF expression in a time and dose dependent manner. Biologically active VEGF that was secreted by NRCM significantly increased BNP mRNA levels in control cardiomyocytes and induced cell-proliferation of cultured cardiac fibroblast (Fibroblast proliferation; NRCM medium/HC-IgG: 1 ± 0.0 vs. NRCM medium/DCM-IgG 100 ng/ml: 5.6 ± 0.9; P < 0.05). Conclusion: The present study extends the knowledge about the possible link between autoimmune signaling in DCM and VEGF induction. Whether this observation plays a considerable role in cardiac remodeling during DCM development needs to be further elucidated. - Highlights: • Mechanisms of remodeling in dilated cardiomyopathy (DCM) are not fully understood. • Autoantibodies have been identified as major predisposing factors

  15. Modeling Inherited Arrhythmia Disorders Using Induced Pluripotent Stem Cell-Derived Cardiomyocytes

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    Bezzerides, Vassilios J.; Zhang, Donghui; Pu, William T.

    2017-01-01

    Inherited arrhythmia disorders are a group of potentially lethal diseases that remain diagnostic and management challenges. While the genetic basis for many of these disorders is well known, the pathogenicity of individual mutations and the resulting clinical outcomes are difficult to predict. Treatment options remain imperfect, and optimizing therapy for individual patients can be difficult. Recent advances in the derivation of induced pluripotent stem cells (iPSCs) from patients and creation of genetically engineered human models using CRISPR/Cas9 has the potential to dramatically advance translational arrhythmia research. In this review, we discuss the current state of modeling inherited arrhythmia disorders using human iPSC-derived cardiomyocytes. We also discuss current limitations and areas for further study. PMID:27916777

  16. Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.

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    Correia, Cláudia; Serra, Margarida; Espinha, Nuno; Sousa, Marcos; Brito, Catarina; Burkert, Karsten; Zheng, Yunjie; Hescheler, Jürgen; Carrondo, Manuel J T; Sarić, Tomo; Alves, Paula M

    2014-12-01

    Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

  17. Thyroid and Glucocorticoid Hormones Promote Functional T-tubule Development in Human-Induced Pluripotent Stem Cell Derived Cardiomyocytes.

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    Parikh, Shan S; Blackwell, Daniel J; Gomez-Hurtado, Nieves; Frisk, Michael; Wang, Lili; Kim, Kyungsoo; Dahl, Christen P; Fiane, Arnt E; Tønnessen, Theis; Kryshtal, Dmytro O; Louch, William E; Knollmann, Bjorn C

    2017-10-02

    Rationale: Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are increasingly being used for modeling heart disease and are under development for regeneration of the injured heart. However, incomplete structural and functional maturation of hiPSC-CM including lack of t-tubules, immature excitation-contraction (EC) coupling, and inefficient Ca-induced Ca release (CICR) remain major limitations. Objective: Thyroid and glucocorticoid hormones are critical for heart maturation. We hypothesized that their addition to standard protocols would promote t-tubule development and mature EC coupling of hiPSC-CM when cultured on extracellular matrix with physiological stiffness (Matrigel mattress). Methods and Results: HiPSC-CM were generated using a standard chemical differentiation method supplemented with triiodo-L-thyronine (T3) and/or dexamethasone (Dex) during days 16-30 followed by single-cell culture for 5 days on Matrigel mattress. HiPSC-CM treated with T3+Dex, but not with either T3 or Dex alone, developed an extensive t-tubule network. Notably, Matrigel mattress was necessary for t-tubule formation. Compared to adult human ventricular CM, t-tubules in T3+Dex-treated hiPSC-CM were less organized and had more longitudinal elements. Confocal line scans demonstrated spatially and temporally uniform Ca release that is characteristic of EC coupling in the heart ventricle. T3+Dex enhanced elementary Ca release measured by Ca sparks as well as promoted ryanodine receptor (RyR2) structural organization. Simultaneous measurements of L-type Ca current and intracellular Ca release confirmed enhanced functional coupling between L-type Ca channels and RyR2 in T3+Dex cells. Conclusions: Our results suggest a permissive role of combined thyroid and glucocorticoid hormones during the cardiac differentiation process which, when coupled with further maturation on Matrigel mattress, is sufficient for t-tubule development, enhanced CICR, and more ventricular-like EC

  18. Microcurrent stimulation promotes reverse remodelling in cardiomyocytes.

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    Kapeller, Barbara; Mueller, Johannes; Losert, Udo; Podesser, Bruno K; Macfelda, Karin

    2016-06-01

    It has been shown that electrical stimulation can improve tissue repair in patients. Imbalances in the extracellular matrix composition induce manifestation of heart failure. Here we investigated the application of microcurrent (MC) to modulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in cardiomyocytes in vitro and in vivo to reverse remodelling in the heart in spontaneous hypertensive rats (SHR). Cardiomyocytes from young SHR (7 months) and old SHR (14 months) were stimulated in vitro and in vivo with MC. MMP and TIMP expression were analysed by qPCR and immunofluorescence to evaluate the modulation of MC treatment. Modulation of cardiomyocytes with MC enhances proliferation with no morphological changes in vitro. By electrical stimulation dual effects, increase and decrease, on MMP-2, MMP-9, TIMP-3, and TIMP-4 mRNA as well as protein expression were observed, depending on the age of the cardiomyocytes. In our in vivo study, MC down-regulated MMP-2, MMP-9, and TIMP-4 and increased TIMP-3 in young SHR. In old SHR MMP-2, MMP-9, and TIMP-4 were up-regulated, whereas TIMP-3 was unaffected. Our data indicate that treatment of MC can modulate the expression of MMPs and TIMPs in vitro and in vivo in SHR. Based on these results new treatments for heart failure could be developed.

  19. Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

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    Chen, Ai-Lan [Department of Cardiology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Ou, Cai-Wen [The Fourth Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); He, Zhao-Chu [Department of Cardiology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Liu, Qi-Cai [Experimental Medical Research Center, Guangzhou Medical University, Guangzhou (China); Dong, Qi [Department of Physiology, Guangzhou Medical University, Guangzhou (China); Chen, Min-Sheng [Guangzhou Key Laboratory of Cardiovascular Disease, Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China)

    2012-10-15

    Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [{sup 3}H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy.

  20. MiR-195 enhances cardiomyocyte apoptosis induced by hypoxia/reoxygenation injury via downregulating c-myb.

    Science.gov (United States)

    Chen, C; Jia, K-Y; Zhang, H-L; Fu, J

    2016-08-01

    In this study, we explored the regulative effect of miR-195 on c-myb expression and also investigated the role of miR-195 and c-myb in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) injury. QRT-PCR analysis was performed to measure mature miR-195 expression. H9c2 cells were transfected for miR-195 overexpression or knockdown or c-myb overexpression using Lipofectamine 2000. The cells were subjected to H/R treatment and following flow cytometric analysis of active caspase-3 or florescent study of reactive oxygen species (ROS) generation. The binding sites between miR-195 and 3'UTR of MYB mRNA were predicted using TargetScan 7.0. The binding sites were verified using dual luciferase assay and Western blot analysis. MiR-195 is significantly upregulated after H/R treatment in H9c2 cells. H/R injury induced active caspase-3 expression. However, the cells with miR-195 suppression had substantially lower ratio of cells with active caspase-3. MiR-195 can decrease c-myb protein expression. Dual luciferase assay verified two binding sites between miR-195 and 3'UTR of MYB mRNA. C-myb overexpression can suppress mitochondrial superoxide generation and cardiomyocyte apoptosis after H/R. MiR-195 is significantly increased due to H/R and can enhance cardiomyocyte apoptosis. MYB is a target gene of miR-195 in cardiomyocytes. The miR-195-MYB axis is involved in regulation of cardiomyocyte apoptosis induced by H/R.

  1. Clematichinenoside (AR Attenuates Hypoxia/Reoxygenation-Induced H9c2 Cardiomyocyte Apoptosis via a Mitochondria-Mediated Signaling Pathway

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    Haiyan Ding

    2016-05-01

    Full Text Available Mitochondria-mediated cardiomyocyte apoptosis is involved in myocardial ischemia/reperfusion (MI/R injury. Clematichinenoside (AR is a triterpenoid saponin isolated from the roots of Clematis chinensis with antioxidant and anti-inflammatory cardioprotection effects against MI/R injury, yet the anti-apoptotic effect and underlying mechanisms of AR in MI/R injury remain unclear. We hypothesize that AR may improve mitochondrial function to inhibit MI/R-induced cardiomyocyte apoptosis. In this study, we replicated an in vitro H9c2 cardiomyocyte MI/R model by hypoxia/reoxygenation (H/R treatment. The viability of H9c2 cardiomyocytes was determined by MTT assay; apoptosis was evaluated by flow cytometry and TUNEL experiments; mitochondrial permeability transition pore (mPTP opening was analyzed by a calcein-cobalt quenching method; and mitochondrial membrane potential (ΔΨm was detected by JC-1. Moreover, we used western blots to determine the mitochondrial cytochrome c translocation to cytosolic and the expression of caspase-3, Bcl-2, and Bax proteins. These results showed that the application of AR decreased the ratio of apoptosis and the extent of mPTP opening, but increased ΔΨm. AR also inhibited H/R-induced release of mitochondrial cytochrome c and decreased the expression of the caspase-3, Bax proteins. Conversely, it remarkably increased the expression of Bcl-2 protein. Taken together, these results revealed that AR protects H9c2 cardiomyocytes against H/R-induced apoptosis through mitochondrial-mediated apoptotic signaling pathway.

  2. Induced pluripotent stem cell derived cardiomyocytes as models for cardiac arrhythmias

    Directory of Open Access Journals (Sweden)

    Maaike eHoekstra

    2012-08-01

    Full Text Available Cardiac arrhythmias are a major cause of morbidity and mortality. In younger patients, the majority of sudden cardiac deaths have an underlying Mendelian genetic cause. Over the last 15 years, enormous progress has been made in identifying the distinct clinical phenotypes and in studying the basic cellular and genetic mechanisms associated with the primary Mendelian (monogenic arrhythmia syndromes. Investigation of the electrophysiological consequences of an ion channel mutation is ideally done in the native cardiomyocyte environment. However, the majority of such studies so far have relied on heterologous expression systems in which single ion channel genes are expressed in non-cardiac cells. In some cases, transgenic mouse models haven been generated, but these also have significant shortcomings, primarily related to species differences.The discovery that somatic cells can be reprogrammed to pluripotency as induced pluripotent stem cells (iPSC has generated much interest since it presents an opportunity to generate patient- and disease-specific cell lines from which normal and diseased human cardiomyocytes can be obtained These genetically diverse human model systems can be studied in vitro and used to decipher mechanisms of disease and identify strategies and reagents for new therapies. Here we review the present state of the art with respect to cardiac disease models already generated using IPSC technology and which have been (partially characterized.Human iPSC (hiPSC models have been described for the cardiac arrhythmia syndromes, including LQT1, LQT2, LQT3-Brugada Syndrome, LQT8/Timothy syndrome and catecholaminergic polymorphic ventricular tachycardia. In most cases, the hiPSC-derived cardiomyoctes recapitulate the disease phenotype and have already provided opportunities for novel insight into cardiac pathophysiology. It is expected that the lines will be useful in the development of pharmacological agents for the management of these

  3. Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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    Liu, Qiang-Ni; Trudeau, Matthew C

    2015-01-01

    Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (IKr) current in the heart. The N-terminal 'eag' domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.

  4. Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Qiang-Ni Liu

    Full Text Available Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (IKr current in the heart. The N-terminal 'eag' domain, which is composed of a Per-Arnt-Sim (PAS domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET between i-eag domains tagged with Cyan fluorescent protein (CFP and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine, indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.

  5. Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure.

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    Mani Sadredini

    Full Text Available Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial

  6. Ectopic expression of Cdk8 induces eccentric hypertrophy and heart failure.

    Science.gov (United States)

    Hall, Duane D; Ponce, Jessica M; Chen, Biyi; Spitler, Kathryn M; Alexia, Adrianne; Oudit, Gavin Y; Song, Long-Sheng; Grueter, Chad E

    2017-08-03

    Widespread changes in cardiac gene expression occur during heart failure, yet the mechanisms responsible for coordinating these changes remain poorly understood. The Mediator complex represents a nodal point for modulating transcription by bridging chromatin-bound transcription factors with RNA polymerase II activity; it is reversibly regulated by its cyclin-dependent kinase 8 (Cdk8) kinase submodule. Here, we identified increased Cdk8 protein expression in human failing heart explants and determined the consequence of this increase in cardiac-specific Cdk8-expressing mice. Transgenic Cdk8 overexpression resulted in progressive dilated cardiomyopathy, heart failure, and premature lethality. Prior to functional decline, left ventricular cardiomyocytes were dramatically elongated, with disorganized transverse tubules and dysfunctional calcium handling. RNA sequencing results showed that myofilament gene isoforms not typically expressed in adult cardiomyocytes were enriched, while oxidative phosphorylation and fatty acid biosynthesis genes were downregulated. Interestingly, candidate upstream transcription factor expression levels and MAPK signaling pathways thought to determine cardiomyocyte size remained relatively unaffected, suggesting that Cdk8 functions within a novel growth regulatory pathway. Our findings show that manipulating cardiac gene expression through increased Cdk8 levels is detrimental to the heart by establishing a transcriptional program that induces pathological remodeling and eccentric hypertrophy culminating in heart failure.

  7. The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Munoz, Juan Pablo; Collao, Andres; Chiong, Mario; Maldonado, Carola; Adasme, Tatiana; Carrasco, Loreto; Ocaranza, Paula; Bravo, Roberto; Gonzalez, Leticia; Diaz-Araya, Guillermo [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Hidalgo, Cecilia [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Lavandero, Sergio, E-mail: slavander@uchile.cl [Centro FONDAP Estudios Moleculares de la Celula, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Facultad de Ciencias Quimicas y Farmaceuticas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile); Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago 8380492 (Chile)

    2009-10-09

    Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-induced MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal {alpha}-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.

  8. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

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    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

  9. Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, Tbx5

    Science.gov (United States)

    Chen, J.X.; Krane, M.; Deutsch, M. A.; Wang, L.; Rav-Acha, M.; Gregoire, S.; Engels, M. C.; Rajarajan, K.; Karra, R.; Abel, E. D.; Wu, J. C.; Milan, D.; Wu, S. M.

    2012-01-01

    Rationale Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. Objective To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). Methods and Results We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes. Conclusions Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy. PMID:22581928

  10. Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers

    Science.gov (United States)

    Hashemi, S; Salma, J; Wales, S; McDermott, JC

    2015-01-01

    β1-Adrenergic receptor (β1-AR) stimulation increases apoptosis in cardiomyocytes through activation of cAMP/protein kinase A (PKA) signaling. The myocyte enhancer factor 2 (MEF2) proteins function as important regulators of myocardial gene expression. Previously, we reported that PKA signaling directly represses MEF2 activity. We determined whether (a) MEF2 has a pro-survival function in cardiomyocytes, and (b) whether β-adrenergic/PKA signaling modulates MEF2 function in cardiomyocytes. Initially, we observed that siRNA-mediated gene silencing of MEF2 induces cardiomyocyte apoptosis as indicated by flow cytometry. β1-AR activation by isoproterenol represses MEF2 activity and promotes apoptosis in cultured neonatal cardiomyocytes. Importantly, β1-AR mediated apoptosis was abrogated in cardiomyocytes expressing a PKA-resistant form of MEF2D (S121/190A). We also observed that a β1-blocker, Atenolol, antagonizes isoproterenol-induced apoptosis while concomitantly enhancing MEF2 transcriptional activity. β-AR stimulation modulated MEF2 cellular localization in cardiomyocytes and this effect was reversed by β-blocker treatment. Furthermore, Kruppel-like factor 6, a MEF2 target gene in the heart, functions as a downstream pro-survival factor in cardiomyocytes. Collectively, these data indicate that (a) MEF2 has an important pro-survival role in cardiomyocytes, and (b) β-adrenergic signaling antagonizes the pro-survival function of MEF2 in cardiomyocytes and β-blockers promote it. These observations have important clinical implications that may contribute to novel strategies for preventing cardiomyocyte apoptosis associated with heart pathology. PMID:27551452

  11. Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kopljar, Ivan; De Bondt, An; Vinken, Petra; Teisman, Ard; Damiano, Bruce; Goeminne, Nick; Van den Wyngaert, Ilse; Gallacher, David J; Lu, Hua Rong

    2017-11-01

    In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity. Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment. Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression. We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery. This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc. © 2017 The British Pharmacological Society.

  12. Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling.

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    Ting-Ting Xiao

    Full Text Available Accumulating evidence indicates that oxymatrine (OMT possesses variously pharmacological properties, especially on the cardiovascular system. We previously demonstrated that activated calpain/apoptosis-inducing factor (AIF-mediated pathway was the key molecular mechanism in aldosterone (ALD induces cardiomyocytes apoptosis. In the present study, we extended the experimentation by investigating the effect of OMT on cardiomyocytes exposed to ALD, as compared to spironolactone (Spiro, a classical ALD receptor antagonist. Cardiomyocytes were pre-incubated with OMT, Spiro or vehicle for 1 h, and then, cardiomyocytes were exposed to ALD 24 h. The cell injury was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and lactate dehydrogenase (LDH leakage ratio. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, annexin V/PI staining, and relative caspase-3 activity assay. Furthermore, expression of pro-apoptotic proteins including truncated Bid (tBid, calpain and AIF were evaluated by western blot analysis. ALD stimulation increased cardiomyocytes apoptosis, caspase-3 activity and protein expression of calpain, tBid and AIF in the cytosol (p<0.05. Pre-incubated with cardiomyocytes injury and increased caspase-3 activity were significantly attenuated (p<0.05. Furthermore, OMT suppressed ALD-induced high expression of calpain and AIF. And these effects of OMT could be comparable to Spiro. These findings indicated that OMT might be a potential cardioprotective-agent against excessive ALD-induced cardiotoxicity, at least in part, mediated through inhibition of calpain/AIF signaling.

  13. Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart

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    Nikolay Naumenko

    2017-05-01

    Full Text Available Vascular endothelial growth factor B (VEGF-B is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60–70. These changes were partly caused by downregulation of sodium current (INa due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K+ currents, namely decreased density of transient outward current (Ito and total K+ current (Ipeak. At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2, a known modulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.

  14. CCND2 Overexpression Enhances the Regenerative Potency of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Remuscularization of Injured Ventricle.

    Science.gov (United States)

    Zhu, Wuqiang; Zhao, Meng; Mattapally, Saidulu; Chen, Sifeng; Zhang, Jianyi

    2017-10-10

    Rationale: The effectiveness of transplanted, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for treatment of ischemic myocardial injury is limited by the exceptionally low engraftment rate. Objective: To determine whether overexpression of the cell-cycle activator CCND2 in hiPSC-CMs can increase the graft size and improve myocardial recovery in a mouse model of myocardial infarction (MI) by increasing the proliferation of grafted cells. Methods and Results: Human CCND2 was delivered to hiPSCs via lenti-viral mediated gene transfection. In cultured cells, markers for cell-cycle activation and proliferation were ~3-7 folds higher in CCND2-overexpressing hiPSC-CMs (hiPSC-CCND2(OE)CMs) than in hiPSC-CMs with normal levels of CCND2 (hiPSC-CCND2(WT)CMs) (p<0.01). The pluripotent genes (Oct 4, Sox2, Nanog) decrease to minimal- and undetectable -levels at day 1 and 10 after differentiate to CMs. In the mouse MI model, cardiac function, infarct size, and the number of engrafted cells were similar at week 1 after treatment with hiPSC-CCND2(OE)CMs or hiPSC-CCND2(WT)CMs but was about tripled in hiPSC-CCND2(OE)CM-treated than in hiPSC-CCND2(WT)CM-treated animals at week 4 (p<0.01). The cardiac function and infarct size were significantly better in both cell-treatment groups hearts than in control hearts, which was most prominent in hiPSC-CCND2(OE)CM-treated animals (p<0.05, each). No tumor formation was observed in any hearts. Conclusions: CCND2 overexpression activates cell-cycle progression in hiPSC-CMs that results in a significant enhanced potency for myocardial repair as evidenced by remuscularization of injured myocardium. This LV muscle regeneration and increased angiogenesis in border zone are accompanied by a significant improvement of LV chamber function.

  15. A Systemized Approach to Investigate Ca(2+) Synchronization in Clusters of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes.

    Science.gov (United States)

    Jones, Aled R; Edwards, David H; Cummins, Michael J; Williams, Alan J; George, Christopher H

    2015-01-01

    Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM) are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB) vs. "on plate" culture on spontaneous activity and regional Ca(2+) synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca(2+) spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca(2+) synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (>2 weeks). The maintenance of all spontaneously active IPS-CM clusters under "on plate" culture conditions promoted the progressive reduction in regional Ca(2+) synchronization and the loss of spontaneous Ca(2+) spiking. Raising the extracellular [Ca(2+)] surrounding these quiescent IPS-CM clusters from ~0.4 to 1.8 mM unmasked discrete behaviors typified by either (a) long-lasting Ca(2+) elevation that returned to baseline or (b) persistent, large-amplitude Ca(2+) oscillations around an increased cytoplasmic [Ca(2+)]. The different responses of IPS-CM to elevated extracellular [Ca(2+)] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.

  16. A systemized approach to investigate Ca2+ synchronization in clusters of human induced pluripotent stem-cell derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Aled R Jones

    2016-01-01

    Full Text Available Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB versus ‘on plate’ culture on spontaneous activity and regional Ca2+ synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca2+ spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca2+ synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (> 2 weeks. The maintenance of all spontaneously active IPS-CM clusters under ‘on plate’ culture conditions promoted the progressive reduction in regional Ca2+ synchronization and the loss of spontaneous Ca2+ spiking. Raising the extracellular [Ca2+] surrounding these quiescent IPS-CM clusters from approximately 0.4 to 1.8 mM unmasked discrete behaviours typified by either a long-lasting Ca2+ elevation that returned to baseline or b persistent, large-amplitude Ca2+ oscillations around an increased cytoplasmic [Ca2+]. The different responses of IPS-CM to elevated extracellular [Ca2+] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.

  17. Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene

    National Research Council Canada - National Science Library

    Gay, Maresha S; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela; Zhang, Lubo

    2016-01-01

    .... Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone...

  18. INTERRELATION BETWEEN PERSISTENT NECROSIS OF CARDIOMYOCYTES AND PROGNOSIS IN PATIENTS WITH CHRONIC HEART FAILURE

    OpenAIRE

    E. N. Golovenko; D. A. Napalkov; V. A. Sulimov

    2010-01-01

    Background. Chronic heart failure (CHF) progression is accompanied by remodeling of muscular, collagen and vascular elements of myocardium. This can lead to increase in serum concentrations of myocardial lesion markers (cardiac troponin I (TrI) and myoglobin) which seem to correlate with poor prognosis in patients with CHF.Aim. To estimate correlations between cardiac TrI, myoglobin, creatine phosphokinase MB-fraction (MB-CPK) serum concentrations and disease severity and prognosis in CHF pat...

  19. Curcumin attenuates cardiomyocyte hypertrophy induced by high glucose and insulin via the PPARγ/Akt/NO signaling pathway.

    Science.gov (United States)

    Chen, Rongchun; Peng, Xiaofeng; Du, Weimin; Wu, Yang; Huang, Bo; Xue, Lai; Wu, Qin; Qiu, Hongmei; Jiang, Qingsong

    2015-05-01

    To investigate the potential effect of curcumin on cardiomyocyte hypertrophy and a possible mechanism involving the PPARγ/Akt/NO signaling pathway in diabetes. The cardiomyocyte hypertrophy induced by high glucose (25.5mmol/L) and insulin (0.1μmol/L) (HGI) and the antihypertrophic effect of curcumin were evaluated in primary culture by measuring the cell surface area, protein content and atrial natriuretic factor (ANF) mRNA expression. The mRNA and protein expressions were assayed by reverse transcription PCR and Western blotting, whereas the NO concentration and endothelial NO synthase (eNOS) activity were determined using nitrate reduction and ELISA methods, respectively. The cardiomyocyte hypertrophy induced by HGI was characterized by increasing ANF mRNA expression, total protein content, and cell surface area, with downregulated mRNA and protein expressions of both PPARγ and Akt, which paralleled the declining eNOS mRNA expression, eNOS content, and NO concentration. The effects of HGI were inhibited by curcumin (1, 3, 10μmol/L) in a concentration-dependent manner. GW9662 (10μmol/L), a selective PPARγ antagonist, could abolish the effects of curcumin. LY294002 (20μmol/L), an Akt blocker, and N(G)-nitro-l-arginine-methyl ester (100μmol/L), a NOS inhibitor, could also diminish the effects of curcumin. The results suggested that curcumin supplementation can improve HGI-induced cardiomyocytes hypertrophy in vitro through the activation of PPARγ/Akt/NO signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Aberrant α-Adrenergic Hypertrophic Response in Cardiomyocytes from Human Induced Pluripotent Cells

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    Gabor Földes

    2014-11-01

    Full Text Available Cardiomyocytes from human embryonic stem cells (hESC-CMs and induced pluripotent stem cells (hiPSC-CMs represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR agonist phenylephrine (PE compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.

  1. In vitro model to study the effects of matrix stiffening on Ca(2+) handling and myofilament function in isolated adult rat cardiomyocytes.

    Science.gov (United States)

    van Deel, Elza D; Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão-Pires, Inês; van der Velden, Jolanda

    2017-07-15

    This paper describes a novel model that allows exploration of matrix-induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function. Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca(2+) handling and myofilament function. Cell shortening and Ca(2+) handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix. Matrix stiffness-impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness. Matrix stiffness-induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte-matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix-induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca(2+) handling but does not alter myofilament-generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness-induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness-induced changes in cardiomyocyte

  2. Functional Effects of a Tissue-Engineered Cardiac Patch From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Rat Infarct Model.

    Science.gov (United States)

    Wendel, Jacqueline S; Ye, Lei; Tao, Ran; Zhang, Jianyi; Zhang, Jianhua; Kamp, Timothy J; Tranquillo, Robert T

    2015-11-01

    A tissue-engineered cardiac patch provides a method to deliver cardiomyoctes to the injured myocardium with high cell retention and large, controlled infarct coverage, enhancing the ability of cells to limit remodeling after infarction. The patch environment can also yield increased survival. In the present study, we sought to assess the efficacy of a cardiac patch made from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to engraft and limit left ventricular (LV) remodeling acutely after infarction. Cardiac patches were created from hiPSC-CMs and human pericytes (PCs) entrapped in a fibrin gel and implanted acutely onto athymic rat hearts. hiPSC-CMs not only remained viable after in vivo culture, but also increased in number by as much as twofold, consistent with colocalization of human nuclear antigen, cardiac troponin T, and Ki-67 staining. CM+PC patches led to reduced infarct sizes compared with myocardial infarction-only controls at week 4, and CM+PC patch recipient hearts exhibited greater fractional shortening over all groups at both 1 and 4 weeks after transplantation. However, a decline occurred in fractional shortening for all groups over 4 weeks, and LV thinning was not mitigated. CM+PC patches became vascularized in vivo, and microvessels were more abundant in the host myocardium border zone, suggesting a paracrine mechanism for the improved cardiac function. PCs in a PC-only control patch did not survive 4 weeks in vivo. Our results indicate that cardiac patches containing hiPSC-CMs engraft onto acute infarcts, and the hiPSC-CMs survive, proliferate, and contribute to a reduction in infarct size and improvements in cardiac function. In the present study, a cardiac patch was created from human induced pluripotent stem cell-derived cardiomyocytes and human pericytes entrapped in a fibrin gel, and it was transplanted onto infarcted rat myocardium. It was found that a patch that contained both cardiomyocytes and pericytes survived

  3. Characterization of the mechanodynamic response of cardiomyocytes with atomic force microscopy.

    Science.gov (United States)

    Chang, Wei-Tien; Yu, David; Lai, Yu-Cheng; Lin, Kuen-You; Liau, Ian

    2013-02-05

    Coordinated and synchronous contraction of cardiomyocytes ensures a normal cardiac function while deranged contraction of cardiomyocytes can lead to heart failure and circulatory dysfunction. Detailed assessment of the contractile property of cardiomyocytes not only helps elucidate the pathophysiology of heart failure but also facilitates development of novel therapies. Herein, we report application of atomic force microscopy to determine essential mechanodynamic characteristics of self-beating cardiomyocytes including the contractile amplitude, force, and frequency. The contraction was continuously measured on the same point of the cell surface; the result assessed postintervention was then compared with the baseline, and the fractional change was obtained. We employed short-time Fourier transform to analyze the time-varying contractile properties and calculate the spectrogram, based on which subtle dynamic changes in the contractile rhythmicity were delicately illustrated. To demonstrate potential applications of this approach, we examined the inotropic and chronotropic responses of cardiomyocyte contraction induced by various pharmacological interventions. The administration of epinephrine significantly increased the contractile amplitude, force, and frequency whereas esmolol markedly decreased these contractile properties. As uniquely illustrated in the spectrogram, doxorubicin not only impaired the contractility of cardiomyocytes but also drastically compromised the rhythmicity. We envision that our approach should be useful in research fields that require detailed evaluation of the mechanodynamic response of cardiomyocytes, for example, to screen drugs that possess cardiac activity or cardiotoxicity, or to assess chemicals that could direct differentiation of stem cells into functioning cardiomyocytes.

  4. Insulin-like growth factor-1 overexpression in cardiomyocytes diminishes ex vivo heart functional recovery after acute ischemia.

    Science.gov (United States)

    Prêle, Cecilia M; Reichelt, Melissa E; Mutsaers, Steven E; Davies, Marilyn; Delbridge, Lea M; Headrick, John P; Rosenthal, Nadia; Bogoyevitch, Marie A; Grounds, Miranda D

    2012-01-01

    Acute insulin-like growth factor-1 administration has been shown to have beneficial effects in cardiac pathological conditions. The aim of the present study was to assess the structural and ex vivo functional impacts of long-term cardiomyocyte-specific insulin-like growth factor-1 overexpression in hearts of transgenic αMHC-IGF-1 Ea mice. Performance of isolated transgenic αMHC-IGF-1 Ea and littermate wild-type control hearts was compared under baseline conditions and in response to 20-min ischemic insult. Cardiac desmin and laminin expression patterns were determined histologically, and myocardial hydroxyproline was measured to assess collagen content. Overexpression of insulin-like growth factor-1 did not modify expression patterns of desmin or laminin but was associated with a pronounced increase (∼30%) in cardiac collagen content (from ∼3.7 to 4.8 μg/mg). Baseline myocardial contractile function and coronary flow were unaltered by insulin-like growth factor-1 overexpression. In contrast to prior evidence of acute cardiac protection, insulin-like growth factor-1 overexpression was associated with significant impairment of acute functional response to ischemia-reperfusion. Insulin-like growth factor-1 overexpression did not modify ischemic contracture development, but postischemic diastolic dysfunction was aggravated (51±5 vs. 22±6 mmHg in nontransgenic littermates). Compared with wild-type control, recovery of pressure development and relaxation indices relative to baseline performance were significantly reduced in transgenic αMHC-IGF-1 Ea after 60-min reperfusion (34±7% vs. 62±7% recovery of +dP/dt; 35±11% vs. 57±8% recovery of -dP/dt). Chronic insulin-like growth factor-1 overexpression is associated with reduced functional recovery after acute ischemic insult. Collagen deposition is elevated in transgenic αMHC-IGF-1 Ea hearts, but there is no change in expression of the myocardial structural proteins desmin and laminin. These findings suggest

  5. Angiotensin-(1-7-Mediated Signaling in Cardiomyocytes

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    Enéas R. M. Gomes

    2012-01-01

    Full Text Available The Renin-Angiotensin System (RAS acts at multiple targets and has its synthesis machinery present in different tissues, including the heart. Actually, it is well known that besides Ang II, the RAS has other active peptides. Of particular interest is the heptapeptide Ang-(1-7 that has been shown to exert cardioprotective effects. In this way, great compilations about Ang-(1-7 actions in the heart have been presented in the literature. However, much less information is available concerning the Ang-(1-7 actions directly in cardiomyocytes. In this paper, we show the actual knowledge about Ang-(1-7-mediated signaling in cardiac cells more specifically we provide a brief overview of ACE2/Ang-(1-7/Mas axis; and highlight the discoveries made in cardiomyocyte physiology through the use of genetic approaches. Finally, we discuss the protective signaling induced by Ang-(1-7 in cardiomyocytes and point molecular determinants of these effects.

  6. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    Energy Technology Data Exchange (ETDEWEB)

    Zaja, Ivan [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bai, Xiaowen, E-mail: xibai@mcw.edu [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bosnjak, Zeljko J. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States)

    2014-10-31

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  7. Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology.

    Science.gov (United States)

    Yechikov, Sergey; Copaciu, Raul; Gluck, Jessica M; Deng, Wenbin; Chiamvimonvat, Nipavan; Chan, James W; Lieu, Deborah K

    2016-11-01

    Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016;34:2670-2680. © 2016 AlphaMed Press.

  8. Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology

    Science.gov (United States)

    Yechikov, Sergey; Copaciu, Raul; Gluck, Jessica M.; Deng, Wenbin; Chiamvimonvat, Nipavan; Chan, James W.; Lieu, Deborah K.

    2018-01-01

    Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers—hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1—at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. PMID:27434649

  9. Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongyan; Li, Yongqiang; Yan, Lijie; Yang, Haitao; Wu, Jintao; Qian, Peng; Li, Bing; Wang, Shanling, E-mail: shanglingwang@126.com

    2015-07-01

    Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H{sub 2}O{sub 2} production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling.

  10. INTERRELATION BETWEEN PERSISTENT NECROSIS OF CARDIOMYOCYTES AND PROGNOSIS IN PATIENTS WITH CHRONIC HEART FAILURE

    Directory of Open Access Journals (Sweden)

    E. N. Golovenko

    2010-01-01

    Full Text Available Background. Chronic heart failure (CHF progression is accompanied by remodeling of muscular, collagen and vascular elements of myocardium. This can lead to increase in serum concentrations of myocardial lesion markers (cardiac troponin I (TrI and myoglobin which seem to correlate with poor prognosis in patients with CHF.Aim. To estimate correlations between cardiac TrI, myoglobin, creatine phosphokinase MB-fraction (MB-CPK serum concentrations and disease severity and prognosis in CHF patients.Material and methods. Fifty eight patients with CHF of different etiology were included into the study. Physical examination, clinical and biochemical blood assays, chest X-ray study, echocardiography and ECG daily monitoring (initially and at the end of the study were carried out. TrI and myoglobin levels were estimated by immunoenzymometric assay. The follow-up period was 6 months. The following end points were used: CHF worsening caused hospital admission, acute myocardial infarction, and lethal outcome.Results. Mean TrI and myoglobin concentrations equaled 0.04 [0.02; 0.06] ng/ml and 62.95 [35.86; 77.28] μg/l, respectively. Demographic characteristics of patients (gender, age did not influence these markers levels. TrI concentration correlated with CHF severity: it was significantly higher in patients with CHF of functional class (FC III-IV than in those with CHF of FC I-II (p<0.001. Negative correlation was revealed between TrI level and ejection fraction (p<0.001. TrI concentration was significantly higher in patients with life-threatening ventricular arrhythmia than in patients without it (p=0.001. Besides, TrI level was significantly higher in patients with registered end points than in those with stable CHF course (p=0.001.Conclusion. TrI correlates with CHF severity and probably has prognostic value in these patients.

  11. MicroRNA-15a inhibition protects against hypoxia/reoxygenation-induced apoptosis of cardiomyocytes by targeting mothers against decapentaplegic homolog 7.

    Science.gov (United States)

    Yang, Yang; Ding, Shiao; Xu, Gaojun; Chen, Fei; Ding, Fangbao

    2017-06-01

    Myocardial ischemia/reperfusion (I/R) injury is a major pathological process in coronary heart disease and cardiac surgery, and is associated with aberrant microRNA (miR) expression. Previous studies have demonstrated that inhibition of miR-15a expression may ameliorate I/R‑induced myocardial injury. In the present study, the potential role and underlying mechanism of miR‑15a in hypoxia/reoxygenation‑induced apoptosis of cardiomyocytes was investigated. Myocardial I/R was simulated in cultured H9c2 cells by 24 h hypoxia followed by 24 h reoxygenation. Using recombinant lentivirus vectors, the inhibition of miR‑15a was indicated to significantly reduce cardiomyocyte apoptosis and release of lactate dehydrogenase and malondialdehyde. Conversely, upregulated miR‑15a expression was pro‑apoptotic. Mothers against decapentaplegic homolog 7 (SMAD7) was identified by bioinformatics analysis as a potential target of miR‑15a. Luciferase reporter assays and western blotting for endogenous SMAD7 protein indicated that miR‑15a inhibited SMAD7 expression via its 3'‑untranslated region. Nuclear levels of nuclear factor‑κB (NF‑κB) p65 were increased by miR‑15a expression and decreased by miR‑15a inhibition, which is consistent with the possibility that the inhibition of SMAD7 by miR-15a results in NF‑κB activation. These findings suggested that the therapeutic effects of miR‑15a inhibition on I/R injury may potentially be explained by its ability to release SMAD‑7‑dependent NF‑κB inhibition. This may provide evidence for miR‑15a as a potential therapeutic target for the treatment of cardiac I/R injury.

  12. Induced pluripotent stem cell derived cardiomyocytes as models for cardiac arrhythmias

    Science.gov (United States)

    Hoekstra, Maaike; Mummery, Christine L.; Wilde, Arthur A. M.; Bezzina, Connie R.; Verkerk, Arie O.

    2012-01-01

    Cardiac arrhythmias are a major cause of morbidity and mortality. In younger patients, the majority of sudden cardiac deaths have an underlying Mendelian genetic cause. Over the last 15 years, enormous progress has been made in identifying the distinct clinical phenotypes and in studying the basic cellular and genetic mechanisms associated with the primary Mendelian (monogenic) arrhythmia syndromes. Investigation of the electrophysiological consequences of an ion channel mutation is ideally done in the native cardiomyocyte (CM) environment. However, the majority of such studies so far have relied on heterologous expression systems in which single ion channel genes are expressed in non-cardiac cells. In some cases, transgenic mouse models have been generated, but these also have significant shortcomings, primarily related to species differences. The discovery that somatic cells can be reprogrammed to pluripotency as induced pluripotent stem cells (iPSC) has generated much interest since it presents an opportunity to generate patient- and disease-specific cell lines from which normal and diseased human CMs can be obtained These genetically diverse human model systems can be studied in vitro and used to decipher mechanisms of disease and identify strategies and reagents for new therapies. Here, we review the present state of the art with respect to cardiac disease models already generated using IPSC technology and which have been (partially) characterized. Human iPSC (hiPSC) models have been described for the cardiac arrhythmia syndromes, including LQT1, LQT2, LQT3-Brugada Syndrome, LQT8/Timothy syndrome and catecholaminergic polymorphic ventricular tachycardia (CPVT). In most cases, the hiPSC-derived cardiomyoctes recapitulate the disease phenotype and have already provided opportunities for novel insight into cardiac pathophysiology. It is expected that the lines will be useful in the development of pharmacological agents for the management of these disorders. PMID

  13. Inhibiting microRNA-144 abates oxidative stress and reduces apoptosis in hearts of streptozotocin-induced diabetic mice.

    Science.gov (United States)

    Yu, Manli; Liu, Yu; Zhang, Bili; Shi, Yicheng; Cui, Ling; Zhao, Xianxian

    2015-01-01

    Hyperglycemia-induced reactive oxygen species (ROS) generation contributes to the development of diabetic cardiomyopathy. However, little is known about the role of microRNAs in the regulation of ROS formation and myocardial apoptosis in streptozotocin (STZ)-induced diabetic mice. It was observed that microRNA-144 (miR-144) level was lower in heart tissues of STZ-induced diabetic mice. High glucose exposure also reduced miR-144 levels in cultured cardiomyocytes. Moreover, miR-144 modulated high glucose-induced oxidative stress in cultured cardiomyocytes by directly targeting nuclear factor-erythroid 2-related factor 2 (Nrf2), which was a central regulator of cellular response to oxidative stress. The miR-144 mimics aggravated high glucose-induced ROS formation and apoptosis in cardiomyocytes, which could be attenuated by treatment with Dh404, an activator of Nrf2. Meanwhile, inhibition of miR-144 suppressed ROS formation and apoptosis induced by high glucose in cultured cardiomyocytes. What was more important is that reduced myocardial oxidative stress and apoptosis and improved cardiac function were identified in STZ-induced diabetic mice when treated with miR-144 antagomir. Although miR-144 cannot explain the increased oxidative stress in STZ, therapeutic interventions directed at decreasing miR-144 may help to decrease oxidative stress in these hearts. Inhibition of miR-144 might have clinical potential to abate oxidative stress as well as to reduce cardiomyocyte apoptosis and improve cardiac function in diabetic cardiomyopathy. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47(phox).

    NARCIS (Netherlands)

    Sipkens, J.A.; Krijnen, P.A.; Hahn, N.E.; Wassink, M.; Meischl, C.; Smith, D.E.; Musters, R.J.; Stehouwer, C.D.A.; Rauwerda, J.A.; Hinsbergh, V.W.H. van; Niessen, H.W.M.

    2011-01-01

    We previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is

  15. From beat rate variability in induced pluripotent stem cell-derived pacemaker cells to heart rate variability in human subjects.

    Science.gov (United States)

    Ben-Ari, Meital; Schick, Revital; Barad, Lili; Novak, Atara; Ben-Ari, Erez; Lorber, Avraham; Itskovitz-Eldor, Joseph; Rosen, Michael R; Weissman, Amir; Binah, Ofer

    2014-10-01

    We previously reported that induced pluripotent stem cell-derived cardiomyocytes manifest beat rate variability (BRV) resembling heart rate variability (HRV) in the human sinoatrial node. We now hypothesized the BRV-HRV continuum originates in pacemaker cells. To investigate whether cellular BRV is a source of HRV dynamics, we hypothesized 3 levels of interaction among different cardiomyocyte entities: (1) single pacemaker cells, (2) networks of electrically coupled pacemaker cells, and (3) the in situ sinoatrial node. We measured BRV/HRV properties in single pacemaker cells, induced pluripotent stem cell-derived contracting embryoid bodies (EBs), and electrocardiograms from the same individual. Pronounced BRV/HRV was present at all 3 levels. The coefficient of variance of interbeat intervals and Poincaré plot indices SD1 and SD2 for single cells were 20 times greater than those for EBs (P heart (the latter two were similar; P > .05). We also compared BRV magnitude among single cells, small EBs (~5-10 cells), and larger EBs (>10 cells): BRV indices progressively increased with the decrease in the cell number (P heart rhythm. The decreased BRV magnitude in transitioning from the single cell to the EB suggests that the HRV of in situ hearts originates from the summation and integration of multiple cell-based oscillators. Hence, complex interactions among multiple pacemaker cells and intracellular Ca(2+) handling determine HRV in humans and cardiomyocyte networks. Copyright © 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  16. High-fat diet induces cardiomyocyte apoptosis via the inhibition of autophagy.

    Science.gov (United States)

    Hsu, Hsiu-Ching; Chen, Ching-Yi; Lee, Bai-Chin; Chen, Ming-Fong

    2016-10-01

    Excessive fat intake induces obesity and causes cardiac injury. Intracellular degradation process involving destruction of long-lived proteins and organelles maintains homeostasis for cells under stress. The purpose of this study was to explore the relation of high-fat diet (HFD)-induced cardiac injury and intracellular degradation process with regard to autophagy and ER stress. HFD feeding for 24 weeks induced hyperglycemia, hyperlipidemia, and cardiac hypertrophy in adult male C57BL/6 mice. In the heart, PARP cleavage, an indicator of apoptosis, levels of LC3-II and p62, indicators of autophagy, and CHOP, indicator of ER stress, were increased. A palmitate-treated cardiomyoblast (H9C2) cell culture was examined to explore how HFD induced myocardial injury. Excessive palmitate (400 μM) treatment induced apoptosis and increased the number of autophagosomes and acid vacuoles of H9C2 cells. Besides, it elevated the expression of LC3-II, p62, and PARP cleavage. Induction of autophagy by rapamycin ameliorated palmitate-induced apoptosis, while inhibition of autophagy by 3-methyladenine or LC3 siRNA exacerbated palmitate-induced apoptosis. Palmitate treatment also induced CHOP expression which is associated with ER stress. HFD can cause cardiac injury by induction of apoptosis which is associated with autophagy dysregulation and ER stress. In addition, autophagy deficiency augments cardiac apoptosis, suggesting that autophagy serves as a pro-survival role in lipotoxic condition.

  17. Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

    Science.gov (United States)

    Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

    2014-11-01

    Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

  18. In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes

    Science.gov (United States)

    Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão‐Pires, Inês; van der Velden, Jolanda

    2017-01-01

    Key points This paper describes a novel model that allows exploration of matrix‐induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function.Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca2+ handling and myofilament function.Cell shortening and Ca2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix.Matrix stiffness‐impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness.Matrix stiffness‐induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Abstract Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte–matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix‐induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca2+ handling but does not alter myofilament‐generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness‐induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness‐induced

  19. Assessment of acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Louisse, Jochem; Wüst, Rob C I; Pistollato, Francesca; Palosaari, Taina; Barilari, Manuela; Macko, Peter; Bremer, Susanne; Prieto, Pilar

    2017-08-01

    The present study assesses acute and chronic toxicity of doxorubicin in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), with the aim to obtain in vitro biomarkers that can be used as readouts to predict in vivo cardiotoxicity. Possible acute toxicity was investigated by assessing effects on the beating rate and the field potential duration (FPD) of doxorubicin-exposed cardiomyocytes by measuring electrical activity using multi-electrode array (MEA) analyses. No effects on the beating rate and FPD were found at concentrations up to 6μM, whereas at 12μM no electrical activity was recorded, indicating that the cardiomyocytes stopped beating. Acute and chronic effects of doxorubicin on mitochondria, which have been reported to be affected in doxorubicin-induced cardiotoxicity, were assessed using high content imaging techniques. To this end hiPSC-CMs were exposed to 150 or 300nM doxorubicin using both single dosing (3h and 2days) and repetitive dosing (3 times, of 2days each), including washout studies to assess delayed effects (assessment at day 14) and effects on cell number, mitochondrial density, mitochondrial membrane potential, mitochondrial superoxide levels and mitochondrial calcium levels were assessed. No effects of doxorubicin were found on mitochondrial density and mitochondrial superoxide levels, whereas doxorubicin reduced cell survival and slightly altered mitochondrial membrane potential and mitochondrial calcium levels, which was most profound in the washout studies. Altogether, the results of the present study show that concentrations of doxorubicin in the micromolar range were required to affect electrical activity of hiPSC-CMs, whereas nanomolar concentrations already affected cell viability and caused mitochondrial disturbances. Integration of these data with other in vitro data may enable the selection of a series of in vitro biomarkers that can be used as readouts to screen chemicals for possible cardiotoxicity

  20. Fluorescent gene reporters in human pluripotent stem cells : as model for studying human heart development and cardiomyocyte differentiation

    NARCIS (Netherlands)

    Hartogh, den S.C.

    2016-01-01

    It is critical to gain knowledge in the underlying mechanisms that control human cardiovascular developm ent, which helps us to understand the onset of congenital cardiovascular diseases, and to develop optimal culture methods for efficient in vitro cardiomyocyte differentiation from hPSCs, which

  1. Patient Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Development and Screening In Catecholaminergic Polymorphic Ventricular Tachycardia.

    Science.gov (United States)

    Ronen, Ben Jehuda; Lili, Barad

    2016-01-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmia often leading to sudden cardiac death in children and young adults, is characterized by polymorphic/bidirectional ventricular tachycardia induced by adrenergic stimulation associated with emotionally stress or physical exercise. There are two forms of CPVT: 1. CPVT1 is caused by mutations in the RYR2 gene, encoding for ryanodine receptor type 2. CPVT1 is the most common form of CPVT in the population, and is inherited by a dominant mechanism. 2. CPVT2 is caused by mutations in the CASQ2 gene, encoding for cardiac calsequestrin 2 and is inherited by recessive mechanism. Patient-specific induced Pluripotent Stem Cells (iPSC) have the ability to differentiate into cardiomyocytes carrying the patient's genome including CPVT-linked mutations and expressing the disease phenotype in vitro at the cellular level. The potency for in vitro modeling using iPSC-derived cardiomyocytes (iPSC-CMs) has been exploited to investigate a variety of inherited diseases including cardiac arrhythmias such as CPVT. In this review we attempted to cover the majority of CPVT patient specific iPSC research studies previously published. CPVT patient-specific iPSC model enables the in vitro investigation of the molecular and cellular disease-mechanisms by the means of electrophysiologycal and Ca(+2) imaging methodologies. Furthermore, this in vitro model allows the screening of various antiarrhythmic drugs, specifically for each patient, also known as "personalized medicine".

  2. Fractalkine depresses cardiomyocyte contractility.

    Directory of Open Access Journals (Sweden)

    David Taube

    Full Text Available Our laboratory reported that male mice with cardiomyocyte-selective knockout of the prostaglandin E2 EP4 receptor sub-type (EP4 KO exhibit reduced cardiac function. Gene array on left ventricles (LV showed increased fractalkine, a chemokine implicated in heart failure. We therefore hypothesized that fractalkine is regulated by PGE2 and contributes to depressed contractility via alterations in intracellular calcium.Fractalkine was measured in LV of 28-32 week old male EP4 KO and wild type controls (WT by ELISA and the effect of PGE2 on fractalkine secretion was measured in cultured neonatal cardiomyocytes and fibroblasts. The effect of fractalkine on contractility and intracellular calcium was determined in Fura-2 AM-loaded, electrical field-paced cardiomyocytes. Cardiomyocytes (AVM from male C57Bl/6 mice were treated with fractalkine and responses measured under basal conditions and after isoproterenol (Iso stimulation.LV fractalkine was increased in EP4 KO mice but surprisingly, PGE2 regulated fractalkine secretion only in fibroblasts. Fractalkine treatment of AVM decreased both the speed of contraction and relaxation under basal conditions and after Iso stimulation. Despite reducing contractility after Iso stimulation, fractalkine increased the Ca(2+ transient amplitude but decreased phosphorylation of cardiac troponin I, suggesting direct effects on the contractile machinery.Fractalkine depresses myocyte contractility by mechanisms downstream of intracellular calcium.

  3. Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient.

    Science.gov (United States)

    Selga, Elisabet; Sendfeld, Franziska; Martinez-Moreno, Rebecca; Medine, Claire N; Tura-Ceide, Olga; Wilmut, Sir Ian; Pérez, Guillermo J; Scornik, Fabiana S; Brugada, Ramon; Mills, Nicholas L

    2017-10-09

    Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems. Copyright © 2017. Published by Elsevier Ltd.

  4. Paradoxically, iron overload does not potentiate doxorubicin-induced cardiotoxicity in vitro in cardiomyocytes and in vivo in mice

    Energy Technology Data Exchange (ETDEWEB)

    Guenancia, Charles [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cardiology Department, University Hospital, Dijon (France); Li, Na [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Hachet, Olivier [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cardiology Department, University Hospital, Dijon (France); Rigal, Eve [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cottin, Yves [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cardiology Department, University Hospital, Dijon (France); Dutartre, Patrick; Rochette, Luc [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Vergely, Catherine, E-mail: cvergely@u-bourgogne.fr [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France)

    2015-04-15

    Doxorubicin (DOX) is known to induce serious cardiotoxicity, which is believed to be mediated by oxidative stress and complex interactions with iron. However, the relationship between iron and DOX-induced cardiotoxicity remains controversial and the role of iron chelation therapy to prevent cardiotoxicity is called into question. Firstly, we evaluated in vitro the effects of DOX in combination with dextran–iron on cell viability in cultured H9c2 cardiomyocytes and EMT-6 cancer cells. Secondly, we used an in vivo murine model of iron overloading (IO) in which male C57BL/6 mice received a daily intra-peritoneal injection of dextran–iron (15 mg/kg) for 3 weeks (D0–D20) and then (D21) a single sub-lethal intra-peritoneal injection of 6 mg/kg of DOX. While DOX significantly decreased cell viability in EMT-6 and H9c2, pretreatment with dextran–iron (125–1000 μg/mL) in combination with DOX, paradoxically limited cytotoxicity in H9c2 and increased it in EMT-6. In mice, IO alone resulted in cardiac hypertrophy (+ 22%) and up-regulation of brain natriuretic peptide and β-myosin heavy-chain (β-MHC) expression, as well as an increase in cardiac nitro-oxidative stress revealed by electron spin resonance spectroscopy. In DOX-treated mice, there was a significant decrease in left-ventricular ejection fraction (LVEF) and an up-regulation of cardiac β-MHC and atrial natriuretic peptide (ANP) expression. However, prior IO did not exacerbate the DOX-induced fall in LVEF and there was no increase in ANP expression. IO did not impair the capacity of DOX to decrease cancer cell viability and could even prevent some aspects of DOX cardiotoxicity in cardiomyocytes and in mice. - Highlights: • The effects of iron on cardiomyocytes were opposite to those on cancer cell lines. • In our model, iron overload did not potentiate anthracycline cardiotoxicity. • Chronic oxidative stress induced by iron could mitigate doxorubicin cardiotoxicity. • The role of iron in

  5. Metabolomic Profiling of Pompe Disease-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals That Oxidative Stress Is Associated With Cardiac and Skeletal Muscle Pathology.

    Science.gov (United States)

    Sato, Yohei; Kobayashi, Hiroshi; Higuchi, Takashi; Shimada, Yohta; Ida, Hiroyuki; Ohashi, Toya

    2016-08-18

    : Pompe disease (PD) is a lysosomal storage disease that is caused by a deficiency of the acid α-glucosidase, which results in glycogen accumulation in the lysosome. The major clinical symptoms of PD include skeletal muscle weakness, respiratory failure, and cardiac hypertrophy. Based on its severity and symptom onset, PD is classified into infantile and late-onset forms. Lysosomal accumulation of glycogen can promote many types of cellular dysfunction, such as autophagic dysfunction, endoplasmic reticulum stress, and abnormal calcium signaling within skeletal muscle. However, the disease mechanism underlying PD cardiomyopathy is not fully understood. Several researchers have shown that PD induced pluripotent stem cell (iPSC)-derived cardiomyocytes successfully replicate the disease phenotype and are useful disease models. We have analyzed the metabolomic profile of late-onset PD iPSC-derived cardiomyocytes and found that oxidative stress and mitochondrial dysfunction are likely associated with cardiac complications. Furthermore, we have validated that these disease-specific changes were also observed in the cardiomyocytes and skeletal muscle of a genetically engineered murine PD model. Oxidative stress may contribute to skeletal muscle and cardiomyocyte dysfunction in PD mice; however, NF-E2-related factor 2 was downregulated in cardiomyocytes and skeletal muscle, despite evidence of oxidative stress. We hypothesized that oxidative stress and an impaired antioxidative stress response mechanism may underlie the molecular pathology of late-onset PD. Pompe disease (PD) is a lysosomal storage disease that is caused by a deficiency of the acid α-glucosidase, which results in glycogen accumulation in the lysosome. An analysis of the metabolomic profile of late-onset PD induced pluripotent stem cell-derived cardiomyocytes found that oxidative stress and mitochondrial dysfunction are likely associated with cardiac complications. Furthermore, these disease

  6. β-Arrestin2 Improves Post-Myocardial Infarction Heart Failure via Sarco(endo)plasmic Reticulum Ca2+-ATPase-Dependent Positive Inotropy in Cardiomyocytes.

    Science.gov (United States)

    McCrink, Katie A; Maning, Jennifer; Vu, Angela; Jafferjee, Malika; Marrero, Christine; Brill, Ava; Bathgate-Siryk, Ashley; Dabul, Samalia; Koch, Walter J; Lymperopoulos, Anastasios

    2017-11-01

    Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein-coupled receptor) adapter proteins βarr (β-arrestin)-1 and βarr-2 are functionally distinct in the heart. βarr1 is cardiotoxic, decreasing contractility by opposing β 1 AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post-myocardial infarction (MI). Conversely, βarr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether βarr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that βarr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca 2+ -ATPase) in vivo and in vitro in a β 1 AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. βarr1 lacks this effect. Moreover, βarr2 does not desensitize β 1 AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to βarr1. In vivo, post-MI heart failure mice overexpressing cardiac βarr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, βarr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac βAR number or cAMP levels in vivo. In conclusion, enhancement of cardiac βarr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac βarr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure. © 2017 American Heart Association, Inc.

  7. Inhibiting Na+/K+ ATPase can impair mitochondrial energetics and induce abnormal Ca2+ cycling and automaticity in guinea pig cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Qince Li

    Full Text Available Cardiac glycosides have been used for the treatment of heart failure because of their capabilities of inhibiting Na+/K+ ATPase (NKA, which raises [Na+]i and attenuates Ca2+ extrusion via the Na+/Ca2+ exchanger (NCX, causing [Ca2+]i elevation. The resulting [Ca2+]i accumulation further enhances Ca2+-induced Ca2+ release, generating the positive inotropic effect. However, cardiac glycosides have some toxic and side effects such as arrhythmogenesis, confining their extensive clinical applications. The mechanisms underlying the proarrhythmic effect of glycosides are not fully understood. Here we investigated the mechanisms by which glycosides could cause cardiac arrhythmias via impairing mitochondrial energetics using an integrative computational cardiomyocyte model. In the simulations, the effect of glycosides was mimicked by blocking NKA activity. Results showed that inhibiting NKA not only impaired mitochondrial Ca2+ retention (thus suppressed reactive oxygen species (ROS scavenging but also enhanced oxidative phosphorylation (thus increased ROS production during the transition of increasing workload, causing oxidative stress. Moreover, concurrent blocking of mitochondrial Na+/Ca2+ exchanger, but not enhancing of Ca2+ uniporter, alleviated the adverse effects of NKA inhibition. Intriguingly, NKA inhibition elicited Ca2+ transient and action potential alternans under more stressed conditions such as severe ATP depletion, augmenting its proarrhythmic effect. This computational study provides new insights into the mechanisms underlying cardiac glycoside-induced arrhythmogenesis. The findings suggest that targeting both ion handling and mitochondria could be a very promising strategy to develop new glycoside-based therapies in the treatment of heart failure.

  8. The volatile oil of Nardostachyos Radix et Rhizoma inhibits the oxidative stress-induced cell injury via reactive oxygen species scavenging and Akt activation in H9c2 cardiomyocyte.

    Science.gov (United States)

    Maiwulanjiang, Maitinuer; Chen, Jianping; Xin, Guizhong; Gong, Amy G W; Miernisha, Abudureyimu; Du, Crystal Y Q; Lau, Kei M; Lee, Pinky S C; Chen, Jihang; Dong, Tina T X; Aisa, Haji A; Tsim, Karl W K

    2014-04-28

    Nardostachyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a well-known medicinal herb widely used in Chinese, Uyghur and Ayurvedic medicines for the treatment of cardiovascular disorders. The oxidative stress-induced cardiomyocyte loss is the major pathogenesis of heart disorders. Here, the total volatile oil of NRR was isolated, and its function in preventing the cell death of cardiomyocyte was demonstrated. The cyto-protective effect of volatile oil of NRR against tBHP-induced H9c2 cardiomyocyte injury was measured by MTT assay. A promoter-report construct (pARE-Luc) containing four repeats of antioxidant response element (ARE) was applied to study the transcriptional activation of ARE. The amounts of phase ΙΙ antioxidant enzymes were analyzed by quantitative real-time polymer chain reaction (qPCR) upon the volatile oil treatment at 30 μg/mL for 24 h. The activation of Akt pathway was analyzed by western blot. In cultured H9c2 cardiomyocytes, application of NRR volatile oil exhibited strong potency in preventing tBHP-induced cell death and accumulation of intracellular reactive oxygen species (ROS) in a concentration-dependent manner. In addition, the application of NRR volatile oil in cultures stimulated the gene expressions of self-defense antioxidant enzymes, which was mediated by the transcriptional activation of antioxidant response element (ARE). The induced genes were glutathione S-transferase, NAD(P)H quinone oxidoreductase, glutamate-cysteine ligase catalytic and modulatory subunits. In addition, the volatile oil of NRR activated the phosphorylation of Akt in cultured H9c2 cells. The treatment of LY294002, an Akt inhibitor, significantly inhibited the volatile oil-mediated ARE transcriptional activity, as well as the cell protective effect of NRR oil. These results demonstrated that NRR volatile oil prevented the oxidative stress-induced cell death in H9c2 cells by (i) reducing intracellular ROS production, (ii

  9. Supervised Machine Learning for Classification of the Electrophysiological Effects of Chronotropic Drugs on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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    Christopher Heylman

    Full Text Available Supervised machine learning can be used to predict which drugs human cardiomyocytes have been exposed to. Using electrophysiological data collected from human cardiomyocytes with known exposure to different drugs, a supervised machine learning algorithm can be trained to recognize and classify cells that have been exposed to an unknown drug. Furthermore, the learning algorithm provides information on the relative contribution of each data parameter to the overall classification. Probabilities and confidence in the accuracy of each classification may also be determined by the algorithm. In this study, the electrophysiological effects of β-adrenergic drugs, propranolol and isoproterenol, on cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CM were assessed. The electrophysiological data were collected using high temporal resolution 2-photon microscopy of voltage sensitive dyes as a reporter of membrane voltage. The results demonstrate the ability of our algorithm to accurately assess, classify, and predict hiPS-CM membrane depolarization following exposure to chronotropic drugs.

  10. Supervised Machine Learning for Classification of the Electrophysiological Effects of Chronotropic Drugs on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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    Heylman, Christopher; Datta, Rupsa; Sobrino, Agua; George, Steven; Gratton, Enrico

    2015-01-01

    Supervised machine learning can be used to predict which drugs human cardiomyocytes have been exposed to. Using electrophysiological data collected from human cardiomyocytes with known exposure to different drugs, a supervised machine learning algorithm can be trained to recognize and classify cells that have been exposed to an unknown drug. Furthermore, the learning algorithm provides information on the relative contribution of each data parameter to the overall classification. Probabilities and confidence in the accuracy of each classification may also be determined by the algorithm. In this study, the electrophysiological effects of β-adrenergic drugs, propranolol and isoproterenol, on cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CM) were assessed. The electrophysiological data were collected using high temporal resolution 2-photon microscopy of voltage sensitive dyes as a reporter of membrane voltage. The results demonstrate the ability of our algorithm to accurately assess, classify, and predict hiPS-CM membrane depolarization following exposure to chronotropic drugs.

  11. AMP-Activated Protein Kinase Activation during Cardioplegia-Induced Hypoxia/Reoxygenation Injury Attenuates Cardiomyocytic Apoptosis via Reduction of Endoplasmic Reticulum Stress

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    Chi-Hsiao Yeh

    2010-01-01

    Our results revealed that AMPK activation during cardioplegia-induced H/R injury attenuates cardiomyocytic apoptosis, via enhancement of antiapoptotic and reduction of proapoptotic responses, resulting from lessening ER stress and the UPR. AMPK activation may serve as a future pharmacological target to reduce H/R injury in the clinical setting.

  12. Paradoxically, iron overload does not potentiate doxorubicin-induced cardiotoxicity in vitro in cardiomyocytes and in vivo in mice.

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    Guenancia, Charles; Li, Na; Hachet, Olivier; Rigal, Eve; Cottin, Yves; Dutartre, Patrick; Rochette, Luc; Vergely, Catherine

    2015-04-15

    Doxorubicin (DOX) is known to induce serious cardiotoxicity, which is believed to be mediated by oxidative stress and complex interactions with iron. However, the relationship between iron and DOX-induced cardiotoxicity remains controversial and the role of iron chelation therapy to prevent cardiotoxicity is called into question. Firstly, we evaluated in vitro the effects of DOX in combination with dextran-iron on cell viability in cultured H9c2 cardiomyocytes and EMT-6 cancer cells. Secondly, we used an in vivo murine model of iron overloading (IO) in which male C57BL/6 mice received a daily intra-peritoneal injection of dextran-iron (15mg/kg) for 3weeks (D0-D20) and then (D21) a single sub-lethal intra-peritoneal injection of 6mg/kg of DOX. While DOX significantly decreased cell viability in EMT-6 and H9c2, pretreatment with dextran-iron (125-1000μg/mL) in combination with DOX, paradoxically limited cytotoxicity in H9c2 and increased it in EMT-6. In mice, IO alone resulted in cardiac hypertrophy (+22%) and up-regulation of brain natriuretic peptide and β-myosin heavy-chain (β-MHC) expression, as well as an increase in cardiac nitro-oxidative stress revealed by electron spin resonance spectroscopy. In DOX-treated mice, there was a significant decrease in left-ventricular ejection fraction (LVEF) and an up-regulation of cardiac β-MHC and atrial natriuretic peptide (ANP) expression. However, prior IO did not exacerbate the DOX-induced fall in LVEF and there was no increase in ANP expression. IO did not impair the capacity of DOX to decrease cancer cell viability and could even prevent some aspects of DOX cardiotoxicity in cardiomyocytes and in mice. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes

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    Tadahiro Shinozawa

    2017-02-01

    Full Text Available To predict drug-induced serious adverse events (SAE in clinical trials, a model using a panel of cells derived from human induced pluripotent stem cells (hiPSCs of individuals with different susceptibilities could facilitate major advancements in translational research in terms of safety and pharmaco-economics. However, it is unclear whether hiPSC-derived cells can recapitulate interindividual differences in drug-induced SAE susceptibility in populations not having genetic disorders such as healthy subjects. Here, we evaluated individual differences in SAE susceptibility based on an in vitro model using hiPSC-derived cardiomyocytes (hiPSC-CMs as a pilot study. hiPSCs were generated from blood samples of ten healthy volunteers with different susceptibilities to moxifloxacin (Mox-induced QT prolongation. Different Mox-induced field potential duration (FPD prolongation values were observed in the hiPSC-CMs from each individual. Interestingly, the QT interval was significantly positively correlated with FPD at clinically relevant concentrations (r > 0.66 in multiple analyses including concentration-QT analysis. Genomic analysis showed no interindividual significant differences in known target-binding sites for Mox and other drugs such as the hERG channel subunit, and baseline QT ranges were normal. The results suggest that hiPSC-CMs from healthy subjects recapitulate susceptibility to Mox-induced QT prolongation and provide proof of concept for in vitro preclinical trials.

  14. Trastuzumab Alters the Expression of Genes Essential for Cardiac Function and Induces Ultrastructural Changes of Cardiomyocytes in Mice

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    ElZarrad, M. Khair; Mukhopadhyay, Partha; Mohan, Nishant; Hao, Enkui; Dokmanovic, Milos; Hirsch, Dianne S.; Shen, Yi; Pacher, Pal; Wu, Wen Jin

    2013-01-01

    Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity. PMID:24255707

  15. Trastuzumab alters the expression of genes essential for cardiac function and induces ultrastructural changes of cardiomyocytes in mice.

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    M Khair ElZarrad

    Full Text Available Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2, very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI and cardiac myosin light chain-1 (cMLC1. The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity.

  16. Growth factor PDGF-BB stimulates cultured cardiomyocytes to synthesize the extracellular matrix component hyaluronan.

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    Urban Hellman

    Full Text Available BACKGROUND: Hyaluronan (HA is a glycosaminoglycan located in the interstitial space which is essential for both structural and cell regulatory functions in connective tissue. We have previously shown that HA synthesis is up-regulated in a rat model of experimental cardiac hypertrophy and that cardiac tissue utilizes two different HA synthases in the hypertrophic process. Cardiomyocytes and fibroblasts are two major cell types in heart tissue. The fibroblasts are known to produce HA, but it has been unclear if cardiomyocytes share the same feature, and whether or not the different HA synthases are activated in the different cell types. METHODOLOGY/PRINCIPAL FINDINGS: This study shows, for the first time that cardiomyocytes can produce HA. Cardiomyocytes (HL-1 and fibroblasts (NIH 3T3 were cultivated in absence or presence of the growth factors FGF2, PDGF-BB and TGFB2. HA concentration was quantified by ELISA, and the size of HA was estimated using dynamic light scattering. Cardiomyocytes synthesized HA but only when stimulated by PDGF-BB, whereas fibroblasts synthesized HA without addition of growth factors as well as when stimulated by any of the three growth factors. When fibroblasts were stimulated by the growth factors, reverse dose dependence was observed, where the highest dose induced the least amount of HA. With the exception of TGFB2, a trend of reverse dose dependence of HA size was also observed. CONCLUSIONS/SIGNIFICANCE: Co-cultivation of cardiomyocytes and fibroblasts (80%/20% increased HA concentration far more that can be explained by HA synthesis by the two cell types separately, revealing a crosstalk between cardiomyocytes and fibroblasts that induces HA synthesis. We conclude that dynamic changes of the myocardium, such as in cardiac hypertrophy, do not depend on the cardiomyocyte alone, but are achieved when both cardiomyocytes and fibroblasts are present.

  17. Evaluation of the cardiotoxicity of mitragynine and its analogues using human induced pluripotent stem cell-derived cardiomyocytes.

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    Jun Lu

    Full Text Available Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa, a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs.The rapid delayed rectifier potassium current (IKr, L-type Ca2+ current (ICa,L and action potential duration (APD were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM suppressed IKr in hiPSC-CMs by 67%∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90 (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression, and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine.Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes.

  18. Evaluation of the Cardiotoxicity of Mitragynine and Its Analogues Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    Science.gov (United States)

    Wu, Jianjun; Jamil, Mohd Fadzly Amar; Tan, Mei Lan; Adenan, Mohd Ilham; Wong, Philip; Shim, Winston

    2014-01-01

    Introduction Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and Results The rapid delayed rectifier potassium current (IKr), L-type Ca2+ current (ICa,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed IKr in hiPSC-CMs by 67% ∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression,and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine. Conclusions Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes. PMID:25535742

  19. Blueberry polyphenols prevent cardiomyocyte death by preventing calpain activation and oxidative stress.

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    Louis, Xavier Lieben; Thandapilly, Sijo Joseph; Kalt, Wilhelmina; Vinqvist-Tymchuk, Melinda; Aloud, Basma Milad; Raj, Pema; Yu, Liping; Le, Hoa; Netticadan, Thomas

    2014-08-01

    The purpose of this study was to examine the efficacy of an aqueous wild blueberry extract and five wild blueberry polyphenol fractions on an in vitro model of heart disease. Adult rat cardiomyocytes were pretreated with extract and fractions, and then exposed to norepinephrine (NE). Cardiomyocyte hypertrophy, cell death, oxidative stress, apoptosis and cardiomyocyte contractile function as well as the activities of calpain, superoxide dismutase (SOD) and catalase (CAT) were measured in cardiomyocytes treated with and without NE and blueberry fraction (BF). Four of five blueberry fractions prevented cell death and cardiomyocyte hypertrophy induced by NE. Total phenolic fraction was used for all further analysis. The NE-induced increase in oxidative stress, nuclear condensation, calpain activity and lowering of SOD and CAT activities were prevented upon pretreatment with BF. Reduced contractile function was also significantly improved with BF pretreatment. Blueberry polyphenols prevent NE-induced adult cardiomyocyte hypertrophy and cell death. The protective effects of BF may be in part attributed to a reduction in calpain activity and oxidative stress.

  20. Evidence for Cardiomyocyte Renewal in Humans

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    Bergmann, O; Bhardwaj, R D; Bernard, S; Zdunek, S; Barnabe-Heider, F; Walsh, S; Zupicich, J; Alkass, K; Buchholz, B A; Druid, H; Jovinge, S; Frisen, J

    2008-10-14

    It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of {sup 14}C, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 20 to 0.3% at the age of 75. Less than 50% of cardiomyocytes are exchanged during a normal lifespan. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work towards the development of therapeutic strategies aiming to stimulate this process in cardiac pathologies.

  1. Cox-2 Inhibition Protects against Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Akt-Dependent Enhancement of iNOS Expression

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    Lei Pang

    2016-01-01

    Full Text Available The present study explored the potential causal link between ischemia-driven cyclooxygenase-2 (COX-2 expression and enhanced apoptosis during myocardial ischemia/reperfusion (I/R by using H9C2 cardiomyocytes and primary rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R. The results showed that H/R resulted in higher COX-2 expression than that of controls, which was prevented by pretreatment with Helenalin (NFκB specific inhibitor. Furthermore, pretreatment with NS398 (COX-2 specific inhibitor significantly attenuated H/R-induced cell injury [lower lactate dehydrogenase (LDH leakage and enhanced cell viability] and apoptosis (higher Bcl2 expression and lower level of cleaved caspases-3 and TUNEL-positive cells in cardiomyocytes. The amelioration of posthypoxic apoptotic cell death was paralleled by significant attenuation of H/R-induced increases in proinflammatory cytokines [interleukin 6 (IL6 and tumor necrosis factor (TNFα] and reactive oxygen species (ROS production and by higher protein expression of phosphorylated Akt and inducible nitric oxide synthase (iNOS and enhanced nitric oxide production. Moreover, the application of LY294002 (Akt-specific inhibitor or 1400W (iNOS-selective inhibitor cancelled the cellular protective effects of NS398. Findings from the current study suggest that activation of NFκB during cardiomyocyte H/R induces the expression of COX-2 and that higher COX-2 expression during H/R exacerbates cardiomyocyte H/R injury via mechanisms that involve cross talks among inflammation, ROS, and Akt/iNOS/NO signaling.

  2. Heart failure induced by perinatal ablation of cardiac myosin light chain kinase

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    Yasmin F. K. Islam

    2016-10-01

    Full Text Available Background: Germline knockout mice are invaluable in understanding the function of the targeted genes. Sometimes, however, unexpected phenotypes are encountered, due in part to the activation of compensatory mechanisms. Germline ablation of cardiac myosin light chain kinase (cMLCK causes mild cardiac dysfunction with cardiomyocyte hypertrophy, whereas ablation in adult hearts results in acute heart failure with cardiomyocyte atrophy. We hypothesized that compensation after ablation of cMLCK is dependent on developmental staging and perinatal-onset of cMLCK ablation will result in more evident heart failure than germline ablation, but less profound when compared to adult-onset ablation.Methods and Results: The floxed-Mylk3 gene was ablated at the beginning of the perinatal stage using a single intra-peritoneal tamoxifen injection of 50 mg/kg into pregnant mice on the 19th day of gestation, this being the final day of gestation. The level of cMLCK protein level could no longer be detected 3 days after the injection, with these mice hereafter denoted as the perinatal Mylk3-KO. At postnatal day 19, shortly before weaning age, these mice showed reduced cardiac contractility with a fractional shortening 22.8 ± 1.0% (n = 7 as opposed to 31.4 ± 1.0% (n = 11 in controls. The ratio of the heart weight relative to body weight was significantly increased at 6.68 ± 0.28 mg/g (n = 12 relative to the two control groups, 5.90 ± 0.16 (flox/flox, n = 11 and 5.81 ± 0.33 (wild/wild/Cre, n = 5, accompanied by reduced body weight. Furthermore, their cardiomyocytes were elongated without thickening, with a long-axis of 101.8 ± 2.4 μm (n = 320 as opposed to 87.1 ± 1.6 μm (n = 360 in the controls. Conclusion: Perinatal ablation of cMLCK produces an increase of heart weight/body weight ratio, a reduction of contractility, and an increase in the expression of fetal genes. The perinatal Mylk3-KO cardiomyocytes were elongated in the absence of thickening, differing

  3. The protective effect of lycopene on hypoxia/reoxygenation-induced endoplasmic reticulum stress in H9C2 cardiomyocytes.

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    Gao, Yang; Jia, Pengyu; Shu, WenQi; Jia, Dalin

    2016-03-05

    Nowadays, drugs protecting ischemia/reperfusion (I/R) myocardium become more suitable for clinic. It has been confirmed lycopene has various protections, but lacking the observation of its effect on endoplasmic reticulum stress (ERS)-mediated apoptosis caused by hypoxia/reoxygenation (H/R). This study aims to clarify the protective effect of lycopene on ERS induced by H/R in H9C2 cardiomyocytes. Detect the survival rate, lactic dehydrogenase (LDH) activity, apoptosis ratio, glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP), c-Jun-N-terminal protein Kinase (JNK) and Caspase-12 mRNA and protein expression and phosphorylation of JNK (p-JNK) protein expression. LDH activity, apoptosis ratio and GRP78 protein expression increase in the H/R group, reduced by lycopene. The survival rate reduces in the H/R and thapsigargin (TG) groups; lycopene and 4-phenyl butyric acid (4-PBA) can improve it caused by H/R, lycopene also can improve it caused by TG. The apoptosis ratio, the expression of GRP78, CHOP and Caspase-12 mRNA and protein and p-JNK protein increase in the H/R and TG groups, weaken in the lycopene+H/R, 4-PBA+H/R and lycopene+TG groups. There is no obvious change in the expression of JNK mRNA or protein. Hence, our results provide the evidence that 10 μM lycopene plays an obviously protective effect on H/R H9C2 cardiomyocytes, realized through reducing ERS and apoptosis. The possible mechanism may be related to CHOP, p-JNK and Caspase-12 pathways. Copyright © 2016. Published by Elsevier B.V.

  4. Cardiac fibroblast–derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy

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    Bang, Claudia; Batkai, Sandor; Dangwal, Seema; Gupta, Shashi Kumar; Foinquinos, Ariana; Holzmann, Angelika; Just, Annette; Remke, Janet; Zimmer, Karina; Zeug, Andre; Ponimaskin, Evgeni; Schmiedl, Andreas; Yin, Xiaoke; Mayr, Manuel; Halder, Rashi; Fischer, Andre; Engelhardt, Stefan; Wei, Yuanyuan; Schober, Andreas; Fiedler, Jan; Thum, Thomas

    2014-01-01

    In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast–derived exosomes revealed a relatively high abundance of many miRNA passenger strands (“star” miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal–derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II–induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA–enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target. PMID:24743145

  5. RAGE modulates hypoxia/reoxygenation injury in adult murine cardiomyocytes via JNK and GSK-3beta signaling pathways.

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    Linshan Shang

    2010-04-01

    cardiomyocyte injury induced by hypoxia/reoxygenation and indicate that the effects of RAGE are mediated by JNK activation and dephosphorylation of GSK-3beta. The outcome in this study lends further support to the potential use of RAGE blockade as an adjunctive therapy for protection of the ischemic heart.

  6. A Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte (hiPSC-CM) Multielectrode Array Assay for Preclinical Cardiac Electrophysiology Safety Screening.

    Science.gov (United States)

    Harris, Kate

    2015-12-08

    Cardiotoxicity is a leading cause of compound attrition during drug development. Preclinical models used to assess the risk for compound-induced effects on cardiac electrophysiology largely rely on animals that can differ in terms of sensitivity and specificity to the targeted clinical response. There is currently no in vitro human cardiomyocyte model for routine preclinical compound screening, as adult human cardiac tissue is unsuitable for such screening. The commercial availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) makes possible the development of assays for assessing compound-induced effects on cardiac function in a human cardiomyocyte-like model. Using multielectrode array (MEA) technology with hiPSC-CMs provides a facile screen for compound-induced effects on cardiac electrophysiology. The MEA data generated from hiPSC-CMs correlate well with the results of conventional preclinical assays and clinical findings. Described in this unit is a technique for measuring extracellular field potentials from hiPSC-CMs using MEA technology to screen for compound-induced effects on cardiac electrophysiology. Copyright © 2015 John Wiley & Sons, Inc.

  7. Stimulation of cardiomyocyte regeneration in neonatal mice and in human myocardium with neuregulin reveals a therapeutic window

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    Polizzotti, Brian D.; Ganapathy, Balakrishnan; Walsh, Stuart; Choudhury, Sangita; Ammanamanchi, Niyatie; Bennett, David G.; dos Remedios, Cristobal G.; Haubner, Bernhard J.; Penninger, Josef M.; Kuhn, Bernhard

    2015-01-01

    Background Pediatric patients with heart failure are treated with medical therapies that were developed for adult patients. These therapies have been shown to be ineffective in pediatric trials, leading to the recognition that new pediatric-specific therapies must be developed. We have previously shown that administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates heart muscle cell (cardiomyocyte) regeneration in adult mice. We hypothesized that rNRG1 administration may be more effective in the neonatal period, which could provide a new therapeutic paradigm for treating heart failure in pediatric patients. Methods We used a cryoinjury model to induce myocardial dysfunction and scar formation for evaluating the effectiveness of rNRG1-administration in neonatal mice. We evaluated the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. Results After cryoinjury in neonatal mice, early administration of rNRG1 from birth for 34 days improved myocardial function and reduced the prevalence of transmural scars. In contrast, late administration of rNRG1 from 4 to 34 days after cryoinjury transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease less than 6 months of age. Conclusion Our results identify a more effective time period within which to execute future clinical trials of rNRG1 for stimulating cardiomyocyte regeneration. PMID:25834111

  8. Heme oxygenase-1 expression protects the heart from acute injury caused by inducible Cre recombinase.

    Science.gov (United States)

    Hull, Travis D; Bolisetty, Subhashini; DeAlmeida, Angela C; Litovsky, Silvio H; Prabhu, Sumanth D; Agarwal, Anupam; George, James F

    2013-08-01

    The protective effect of heme oxygenase-1 (HO-1) expression in cardiovascular disease has been previously demonstrated using transgenic animal models in which HO-1 is constitutively overexpressed in the heart. However, the temporal requirements for protection by HO-1 induction relative to injury have not been investigated, but are essential to employ HO-1 as a therapeutic strategy in human cardiovascular disease states. Therefore, we generated mice with cardiac-specific, tamoxifen (TAM)-inducible overexpression of a human HO-1 (hHO-1) transgene (myosin heavy chain (MHC)-HO-1 mice) by breeding mice with cardiac-specific expression of a TAM-inducible Cre recombinase (MHC-Cre mice), with mice containing an hHO-1 transgene preceded by a floxed-stop signal. MHC-HO-1 mice overexpress HO-1 mRNA and the enzymatically active protein following TAM administration (40 mg/kg body weight on 2 consecutive days). In MHC-Cre controls, TAM administration leads to severe, acute cardiac toxicity, cardiomyocyte necrosis, and 80% mortality by day 3. This cardiac toxicity is accompanied by a significant increase in inflammatory cells in the heart that are predominantly neutrophils. In MHC-HO-1 mice, HO-1 overexpression ameliorates the depression of cardiac function and high mortality rate observed in MHC-Cre mice following TAM administration and attenuates cardiomyocyte necrosis and neutrophil infiltration. These results highlight that HO-1 induction is sufficient to prevent the depression of cardiac function observed in mice with TAM-inducible Cre recombinase expression by protecting the heart from necrosis and neutrophil infiltration. These findings are important because MHC-Cre mice are widely used in cardiovascular research despite the limitations imposed by Cre-induced cardiac toxicity, and also because inflammation is an important pathological component of many human cardiovascular diseases.

  9. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation.

    Science.gov (United States)

    Nozaki, Yumiko; Honda, Yayoi; Tsujimoto, Shinji; Watanabe, Hitoshi; Kunimatsu, Takeshi; Funabashi, Hitoshi

    2014-07-01

    Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K(+) channel and Ca(2+) channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2min every 10min for 30min after drug exposure for the vehicle and each drug concentration. IKr and IKs blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca(2+) channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the IKr blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Up-regulation of alpha-smooth muscle actin in cardiomyocytes from non-hypertrophic and non-failing transgenic mouse hearts expressing N-terminal truncated cardiac troponin I

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    Stephanie Kern

    2014-01-01

    Full Text Available We previously reported that a restrictive N-terminal truncation of cardiac troponin I (cTnI-ND is up-regulated in the heart in adaptation to hemodynamic stresses. Over-expression of cTnI-ND in the hearts of transgenic mice revealed functional benefits such as increased relaxation and myocardial compliance. In the present study, we investigated the subsequent effect on myocardial remodeling. The alpha-smooth muscle actin (α-SMA isoform is normally expressed in differentiating cardiomyocytes and is a marker for myocardial hypertrophy in adult hearts. Our results show that in cTnI-ND transgenic mice of between 2 and 3 months of age (young adults, a significant level of α-SMA is expressed in the heart as compared with wild-type animals. Although blood vessel density was increased in the cTnI-ND heart, the mass of smooth muscle tissue did not correlate with the increased level of α-SMA. Instead, immunocytochemical staining and Western blotting of protein extracts from isolated cardiomyocytes identified cardiomyocytes as the source of increased α-SMA in cTnI-ND hearts. We further found that while a portion of the up-regulated α-SMA protein was incorporated into the sarcomeric thin filaments, the majority of SMA protein was found outside of myofibrils. This distribution pattern suggests dual functions for the up-regulated α-SMA as both a contractile component to affect contractility and as possible effector of early remodeling in non-hypertrophic, non-failing cTnI-ND hearts.

  11. Influence of field potential duration on spontaneous beating rate of human induced pluripotent stem cell-derived cardiomyocytes: Implications for data analysis and test system selection.

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    Rast, Georg; Kraushaar, Udo; Buckenmaier, Sandra; Ittrich, Carina; Guth, Brian D

    Field potential duration in human pluripotent stem cell (hiPSC)-derived cardiomyocytes is discussed as parameter for the assessment of drug-induced delayed repolarization. In spontaneously beating hiPSC-derived cardiomyocytes field potential duration varies depending on beating rate but beating rate can also be influenced by field potential duration. This interdependence is not fully understood and therefore mandates careful data analysis and cautious interpretation of the results. We analysed data from several types of hiPSC-derived cardiomyocytes and, for comparison, primary embryonic chick cardiomyocytes using reference compounds to study the relationship between spontaneous rate and field potential duration. Based on such data we developed a method based on a regression model of drug-induced changes in the inter-beat interval versus changes in the field potential duration to distinguish primary rate from repolarisation effects. We demonstrate the application of this approach with reference and research compounds. Cells from different sources differed with regard to the direct or indirect effects of reference compounds on spontaneous beating. All cell types showed an adaptation of field potential duration upon rate changes induced by reference compounds, however, the adaptation of the spontaneous rate after compound-induced changes in field potential duration varied considerably between cell types. As shown by comparison with data from guinea pig papillary muscle, an ex vivo model with a fixed stimulation rate, this approach is more appropriate than the application of correction algorithms routinely used for in vivo data since such algorithms do not account for a dependence of rate on field potential duration. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Iron overload and apoptosis of HL-1 cardiomyocytes: effects of calcium channel blockade.

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    Mei-pian Chen

    Full Text Available Iron overload cardiomyopathy that prevails in some forms of hemosiderosis is caused by excessive deposition of iron into the heart tissue and ensuing damage caused by a raise in labile cell iron. The underlying mechanisms of iron uptake into cardiomyocytes in iron overload condition are still under investigation. Both L-type calcium channels (LTCC and T-type calcium channels (TTCC have been proposed to be the main portals of non-transferrinic iron into heart cells, but controversies remain. Here, we investigated the roles of LTCC and TTCC as mediators of cardiac iron overload and cellular damage by using specific Calcium channel blockers as potential suppressors of labile Fe(II and Fe(III ingress in cultured cardiomyocytes and ensuing apoptosis.Fe(II and Fe(III uptake was assessed by exposing HL-1 cardiomyocytes to iron sources and quantitative real-time fluorescence imaging of cytosolic labile iron with the fluorescent iron sensor calcein while iron-induced apoptosis was quantitatively measured by flow cytometry analysis with Annexin V. The role of calcium channels as routes of iron uptake was assessed by cell pretreatment with specific blockers of LTCC and TTCC.Iron entered HL-1 cardiomyocytes in a time- and dose-dependent manner and induced cardiac apoptosis via mitochondria-mediated caspase-3 dependent pathways. Blockade of LTCC but not of TTCC demonstrably inhibited the uptake of ferric but not of ferrous iron. However, neither channel blocker conferred cardiomyocytes with protection from iron-induced apoptosis.Our study implicates LTCC as major mediators of Fe(III uptake into cardiomyocytes exposed to ferric salts but not necessarily as contributors to ensuing apoptosis. Thus, to the extent that apoptosis can be considered a biological indicator of damage, the etiopathology of cardiosiderotic damage that accompanies some forms of hemosiderosis would seem to be unrelated to LTCC or TTCC, but rather to other routes of iron ingress present in

  13. Comparison of Non-Coding RNAs in Exosomes and Functional Efficacy of Human Embryonic Stem Cell- versus Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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    Lee, Won Hee; Chen, Wen-Yi; Shao, Ning-Yi; Xiao, Dan; Qin, Xulei; Baker, Natalie; Bae, Hye Ryeong; Wei, Tzu-Tang; Wang, Yongjun; Shukla, Praveen; Wu, Haodi; Kodo, Kazuki; Ong, Sang-Ging; Wu, Joseph C

    2017-10-01

    Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived CMs (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here, we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome. Human ESCs and iPSCs were differentiated into CMs using small molecule inhibitors. Fluorescence-activated cell sorting analysis confirmed ∼85% and ∼83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable with the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective. We demonstrate that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because, unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. Stem Cells 2017;35:2138-2149. © 2017 AlphaMed Press.

  14. Human adipose tissue-derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes

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    Metzele, Roxana; Alt, Christopher; Bai, Xiaowen; Yan, Yasheng; Zhang, Zhi; Pan, Zhizhong; Coleman, Michael; Vykoukal, Jody; Song, Yao-Hua; Alt, Eckhard

    2011-01-01

    that fusion, even if artificially induced in our study, could indeed be a mechanism for cardiomyocyte renewal in the heart.—Metzele, R., Alt, C., Bai, X., Yan, Y., Zhang, Z., Pan, Z., Coleman, M., Vykoukal, J., Song, Y.-H., Alt, E. Human adipose tissue-derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes. PMID:21059751

  15. Myofibrillogenesis regulator-1 attenuated hypoxia/reoxygenation-induced apoptosis by inhibiting the PERK/Nrf2 pathway in neonatal rat cardiomyocytes.

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    Tao, Tian-Qi; Wang, Xiao-Reng; Liu, Mi; Xu, Fei-Fei; Liu, Xiu-Hua

    2015-03-01

    The purpose of this study was to investigate the role of myofibrillogenesis regulator-1 (MR-1) in cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R), through protein kinase R-like ER kinase (PERK)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. To address this aim, an H/R model of neonatal rat cardiomyocytes was used. MR-1 was overexpressed using an adenoviral vector system and knocked down using MR-1 specific siRNA. Apoptosis was assessed by using Annexin V/PI double staining, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay, and the Bcl-2/Bax ratio. Western blotting was used to detect the protein levels of MR-1, glucose-regulated protein 78 (GRP78), total and phosphorylated PERK, Nrf2, activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), Bcl-2 and Bax. Immunofluorescence staining was used to assess the subcellular location of Nrf2. We found that H/R induced significant apoptosis in neonatal rat cardiomyocytes. MR-1 overexpression attenuated H/R-induced apoptosis, decreased GRP78 (P apoptosis, increased expression of GRP78 and CHOP (P apoptosis through inhibition of the PERK/Nrf2 pathway.

  16. Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

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    Rebecca Josowitz

    Full Text Available The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN. When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

  17. Methanolic extract of onion (Allium cepa) attenuates ischemia/hypoxia-induced apoptosis in cardiomyocytes via antioxidant effect.

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    Park, Sok; Kim, Mi-Young; Lee, Dong Ha; Lee, Soo Hwan; Baik, Eun Joo; Moon, Chang-Hyun; Park, Se Won; Ko, Eun Young; Oh, Sei-Ryang; Jung, Yi-Sook

    2009-06-01

    Although there is growing awareness of the beneficial potential of onion intake to lower the risk of cardiovascular disease, there is little information about the effect of onion on ischemic heart injury, one of the most common cardiovascular diseases. This study investigates the effect of the methanol-soluble extract of onion on ischemic injury in heart-derived H9c2 cells in vitro and in rat hearts in vivo. The underlying mechanism is also investigated. To evaluate the effect of onion on ischemia-induced cell death, LDH release and TUNEL-positivity were assessed in H9c2 cells, and the infarct size was measured in a myocardial infarct model. To investigate the mechanism of the cardioprotection by onion, the reactive oxygen species (ROS) level and the mitochondrial membrane potential (DeltaPsi(m)) were measured using an imaging technique; the caspase-3 activity was assayed, and Western blotting was performed to examine cytochrome c release in H9c2 cells. The methanolic extract of onion had a preventive effect on ischemia/hypoxia-induced apoptotic death in H9c2 cells in vitro and in rat heart in vivo. The onion extract (0.05 g/ml) inhibited the elevation of the ROS, mitochondrial membrane depolarization, cytochrome c release and caspase-3 activation during hypoxia in H9c2 cells. In the in vivo rat myocardial infarction model, onion extract (10 g/kg) significantly reduced the infarct size, the apoptotic cell death of the heart and the plasma MDA level. In conclusion, the results of this study suggest that the methanolic extract of onion attenuates ischemia/hypoxia-induced apoptosis in heart-derived H9c2 cells in vitro and in rat hearts in vivo, through, at least in part, an antioxidant effect.

  18. Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction

    NARCIS (Netherlands)

    van Laake, Linda W.; Passier, Robert; Monshouwer-Kloots, Jantine; Verkleij, Arie J.; Lips, Daniel J.; Freund, Christian; den Ouden, Krista; Ward-van Oostwaard, Dorien; Korving, Jeroen; Tertoolen, Leon G.; van Echteld, Cees J.; Doevendans, Pieter A.; Mummery, Christine L.

    2007-01-01

    Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we

  19. Knockdown of CkrL by shRNA deteriorates hypoxia/reoxygenation-induced H9C2 cardiomyocyte apoptosis and survival inhibition Via Bax and downregulation of P-Erk1/2.

    Science.gov (United States)

    Zhang, Zhi-Sheng; Yang, Dong-Yan; Fu, Yan-Bo; Zhang, Lei; Zhao, Qian-Ping; Li, Gang

    2015-03-01

    Integrin β1 subunit and its downstream molecule integrin-linked kinase and focal adhesion kinase have been confirmed to be essential to cell survival and inhibition of apoptosis and hypoxia/reoxygenation (H/R)-induced injuries in cardiomyocytes. However, it is still unclear whether CrkL [v-crk avian sarcoma virus CT-10 oncogene homolog (Crk)-like], which acts also as a component of the integrin pathway, could also affect H/R-induced injuries in the cardiomyocytes. The rat-derived H9C2 cardiomyocytes were infected with a CrkL small hairpin RNA interference recombinant lentivirus, which knockdowns the endogenous CrkL expression in the cardiomyocytes. Apoptosis, cell proliferation and survival were examined in the H9C2 cardiomyocytes treated with either H/R or not. Results showed that knockdown of CrkL could significantly increase apoptosis and inhibition of the cell proliferation and survival and deteriorate the previously mentioned injuries induced by H/R. In contrast, overexpression of human CrkL could relieve the exacerbation of the previously mentioned injuries induced by CrkL knockdown in the H9C2 cardiomyocytes via regulation of Bax and extracellular signal-regulated kinase1/2 (p-ERK1/2). In conclusion, these results confirmed that knockdown of CrkL could deteriorate H/R-induced apoptosis and cell survival inhibition in rat-derived H9C2 cardiomyocytes via Bax and downregulation of p-ERK1/2. It implies that CrkL could mitigate H/R-induced injuries in the cardiomyocytes. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Ion channelopathies in human induced pluripotent stem cell derived cardiomyocytes: a dynamic clamp study with virtual IK1

    Science.gov (United States)

    Meijer van Putten, Rosalie M. E.; Mengarelli, Isabella; Guan, Kaomei; Zegers, Jan G.; van Ginneken, Antoni C. G.; Verkerk, Arie O.; Wilders, Ronald

    2015-01-01

    Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs—and consequently the profile of individual membrane currents active during that action potential—differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to “normalize” the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4–6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near −80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen–Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac

  1. Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr.

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    Michael Xavier Doss

    Full Text Available Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP from spontaneously beating clusters (BC micro-dissected from the EBs (n = 103; 37°C and to examine the response to 5 µM E-4031 (n = 21 or BaCl(2 (n = 22. Patch-clamp techniques were used to record I(Kr and I(K1 from cells enzymatically dissociated from BC (n = 49; 36°C. Spontaneous cycle length (CL and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21 increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001 and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl(2, at concentrations that selectively block I(K1 (50-100 µM, failed to depolarize the majority of clusters (13/22. Patch-clamp experiments revealed very low or negligible I(K1 in 53% (20/38 of the cells studied, but presence of I(Kr in all (11/11. Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I(K1 in the majority of cells, but robust expression of I(Kr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd. The vast majority have a maximum diastolic potential that depends critically on I(Kr due to the absence of

  2. The influence of physiological matrix conditions on permanent culture of induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Heras-Bautista, Carlos O; Katsen-Globa, Alisa; Schloerer, Nils E; Dieluweit, Sabine; Abd El Aziz, Osama M; Peinkofer, Gabriel; Attia, Wael A; Khalil, Markus; Brockmeier, Konrad; Hescheler, Jürgen; Pfannkuche, Kurt

    2014-08-01

    Cardiomyocytes (CMs) from induced pluripotent stem (iPS) cells mark an important achievement in the development of in vitro pharmacological, toxicological and developmental assays and in the establishment of protocols for cardiac cell replacement therapy. Using CMs generated from murine embryonic stem cells and iPS cells we found increased cell-matrix interaction and more matured embryoid body (EB) structures in iPS cell-derived EBs. However, neither suspension-culture in form of purified cardiac clusters nor adherence-culture on traditional cell culture plastic allowed for extended culture of CMs. CMs grown for five weeks on polystyrene exhibit signs of massive mechanical stress as indicated by α-smooth muscle actin expression and loss of sarcomere integrity. Hydrogels from polyacrylamide allow adapting of the matrix stiffness to that of cardiac tissue. We were able to eliminate the bottleneck of low cell adhesion using 2,5-Dioxopyrrolidin-1-yl-6-acrylamidohexanoate as a crosslinker to immobilize matrix proteins on the gels surface. Finally we present an easy method to generate polyacrylamide gels with a physiological Young's modulus of 55 kPa and defined surface ligand, facilitating the culture of murine and human iPS-CMs, removing excess mechanical stresses and reducing the risk of tissue culture artifacts exerted by stiff substrates. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Dedifferentiation and proliferation of mammalian cardiomyocytes.

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    Yiqiang Zhang

    2010-09-01

    Full Text Available It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1 cardiomyocyte purification from rat hearts, and 2 genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs, while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit(+.Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness

  4. Xenotransplantation of Human Cardiomyocyte Progenitor Cells Does Not Improve Cardiac Function in a Porcine Model of Chronic Ischemic Heart Failure. Results from a Randomized, Blinded, Placebo Controlled Trial.

    Science.gov (United States)

    Jansen of Lorkeers, Sanne J; Gho, Johannes M I H; Koudstaal, Stefan; van Hout, Gerardus P J; Zwetsloot, Peter Paul M; van Oorschot, Joep W M; van Eeuwijk, Esther C M; Leiner, Tim; Hoefer, Imo E; Goumans, Marie-José; Doevendans, Pieter A; Sluijter, Joost P G; Chamuleau, Steven A J

    2015-01-01

    Recently cardiomyocyte progenitor cells (CMPCs) were successfully isolated from fetal and adult human hearts. Direct intramyocardial injection of human CMPCs (hCMPCs) in experimental mouse models of acute myocardial infarction significantly improved cardiac function compared to controls. Here, our aim was to investigate whether xenotransplantation via intracoronary infusion of fetal hCMPCs in a pig model of chronic myocardial infarction is safe and efficacious, in view of translation purposes. We performed a randomized, blinded, placebo controlled trial. Four weeks after ischemia/reperfusion injury by 90 minutes of percutaneous left anterior descending artery occlusion, pigs (n = 16, 68.5 ± 5.4 kg) received intracoronary infusion of 10 million fetal hCMPCs or placebo. All animals were immunosuppressed by cyclosporin (CsA). Four weeks after infusion, endpoint analysis by MRI displayed no difference in left ventricular ejection fraction, left ventricular end diastolic and left ventricular end systolic volumes between both groups. Serial pressure volume (PV-)loop and echocardiography showed no differences in functional parameters between groups at any timepoint. Infarct size at follow-up, measured by late gadolinium enhancement MRI showed no difference between groups. Intracoronary pressure and flow measurements showed no signs of coronary obstruction 30 minutes after cell infusion. No premature death occurred in cell treated animals. Xenotransplantation via intracoronary infusion of hCMPCs is feasible and safe, but not associated with improved left ventricular performance and infarct size compared to placebo in a porcine model of chronic myocardial infarction.

  5. Rapid negative inotropic effect induced by TNF-α in rat heart perfused related to PKC activation.

    Science.gov (United States)

    Jude, B; Vetel, S; Giroux-Metges, M A; Pennec, J P

    2017-11-29

    Myocardial depression, frequently observed in septic shock, is mediated by circulating molecules such as cytokines. TNF-α appears to be the most important pro-inflammatory cytokine released during the early phase of a septic shock. It was previously shown that TNF-α had a negative inotropic effect on myocardium. Now, the aim of this study was to investigate the effects of the activation of PKC by TNF-α on heart function, and to determine if this cytokine could induce a decrease of membrane excitability. Isolated rat hearts (n = 6) were perfused with Tyrode solution containing TNF-α at 20 ng/ml during 30 min by using a Langendorff technique. Expressions of PKC-α and PKC-ε were analysed by western blot on membrane and cytosol proteins extracted from ventricular myocardium. Patch clamp was performed on freshly isolated cardiomyocytes (n = 8). Compared to control situation, 30 min of TNF-α perfusion led to cardiac dysfunction with a decrease of the heart rate (-83%), the force (-20%) and speed of relaxation (-18%) and the coronary flow (-25%). This is associated with an activation and a membrane targeting of both PKC-α and PKC-ε isoforms in ventricle with respectively +123% and +54% compared to control hearts. Nevertheless, TNF-α had no significant effect on voltage-gated sodium current (109.0%+/- 12.5) after addition of the cytokine when compared to control. These results showed that TNF-α had a negative inotropic effect on the isolated rat heart and can induce PKC activation leading to an impaired contractility of the heart. However the early heart dysfunction induced by the cytokine was not associated to a decrease of cardiomyocytes membrane excitability as it has been evidenced in skeletal muscle fibres. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

    Science.gov (United States)

    Guo, Liang; Eldridge, Sandy; Furniss, Mike; Mussio, Jodie; Davis, Myrtle

    2015-09-01

    There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. Copyright © 2015 John Wiley & Sons, Inc.

  7. Growth hormone-releasing hormone attenuates cardiac hypertrophy and improves heart function in pressure overload-induced heart failure.

    Science.gov (United States)

    Gesmundo, Iacopo; Miragoli, Michele; Carullo, Pierluigi; Trovato, Letizia; Larcher, Veronica; Di Pasquale, Elisa; Brancaccio, Mara; Mazzola, Marta; Villanova, Tania; Sorge, Matteo; Taliano, Marina; Gallo, Maria Pia; Alloatti, Giuseppe; Penna, Claudia; Hare, Joshua M; Ghigo, Ezio; Schally, Andrew V; Condorelli, Gianluigi; Granata, Riccarda

    2017-11-07

    It has been shown that growth hormone-releasing hormone (GHRH) reduces cardiomyocyte (CM) apoptosis, prevents ischemia/reperfusion injury, and improves cardiac function in ischemic rat hearts. However, it is still not known whether GHRH would be beneficial for life-threatening pathological conditions, like cardiac hypertrophy and heart failure (HF). Thus, we tested the myocardial therapeutic potential of GHRH stimulation in vitro and in vivo, using GHRH or its agonistic analog MR-409. We show that in vitro, GHRH(1-44)NH 2 attenuates phenylephrine-induced hypertrophy in H9c2 cardiac cells, adult rat ventricular myocytes, and human induced pluripotent stem cell-derived CMs, decreasing expression of hypertrophic genes and regulating hypertrophic pathways. Underlying mechanisms included blockade of Gq signaling and its downstream components phospholipase Cβ, protein kinase Cε, calcineurin, and phospholamban. The receptor-dependent effects of GHRH also involved activation of Gα s and cAMP/PKA, and inhibition of increase in exchange protein directly activated by cAMP1 (Epac1). In vivo, MR-409 mitigated cardiac hypertrophy in mice subjected to transverse aortic constriction and improved cardiac function. Moreover, CMs isolated from transverse aortic constriction mice treated with MR-409 showed improved contractility and reversal of sarcolemmal structure. Overall, these results identify GHRH as an antihypertrophic regulator, underlying its therapeutic potential for HF, and suggest possible beneficial use of its analogs for treatment of pathological cardiac hypertrophy. Copyright © 2017 the Author(s). Published by PNAS.

  8. Medicinal effect and its JP2/RyR2-based mechanism of Smilax glabra flavonoids on angiotensin II-induced hypertrophy model of cardiomyocytes.

    Science.gov (United States)

    Cai, Yueqin; Tu, Jue; Pan, Shuizhen; Jiang, Jianping; Shou, Qiyang; Ling, Yun; Chen, Yunxiang; Wang, Dejun; Yang, Weiji; Shan, Letian; Chen, Minli

    2015-07-01

    Rhizome and root of Smilax glabra Roxb (Liliaceae family) is a widely used traditional Chinese medicine (TCM) named Tu-fu-ling (TFL) for cardiac disease therapy. The TFL flavonoids (TFLF) has been extracted and proven to possess the anti-cardiac hypertrophy effect in our previous reports. Such effect could be mediated by the modulation of intracellular Ca(2+) flux in myocardial cells, in which junctophilin-2 (JP2) and ryanodine receptor 2 (RyR2) play an important role. However, its mechanism of the anti-cardiac hypertrophy effect remains unclarified. 2μmol/L Ang II was applied to induce hypertrophy model of rat primary cardiomyocytes. After treatment of TFLF at 0.25, 0.5 and 1.0mg/ml, the cell size was microscopic measured, and the protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were estimated by immunofluorescence imaging, ELISA and real-time PCR assay. Obvious hypertrophy of cardiomyocytes was induced by Ang II but reversed by TFLF from 0.5 to 1.0mg/ml. The protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were also inhibited by Ang II but restored by TFLF at its dose range. Such effect of TFLF was exerted at a dose dependent manner, which was even better than that of verapamil. Our findings may evidence the correlation between JP2/RyR2 and myocardiac hypertrophy, and indicate the JP2/RyR2-mediated anti-hypertrophy mechanism of TFLF for the first time. It deserves to be developed as a promising TCM candidate of new drug for myocardial hypertrophy treatment. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. The cardiomyocyte molecular clock regulates the circadian expression of Kcnh2 and contributes to ventricular repolarization.

    Science.gov (United States)

    Schroder, Elizabeth A; Burgess, Don E; Zhang, Xiping; Lefta, Mellani; Smith, Jennifer L; Patwardhan, Abhijit; Bartos, Daniel C; Elayi, Claude S; Esser, Karyn A; Delisle, Brian P

    2015-06-01

    Sudden cardiac death (SCD) follows a diurnal variation. Data suggest the timing of SCD is influenced by circadian (~24-hour) changes in neurohumoral and cardiomyocyte-specific regulation of the heart's electrical properties. The basic helix-loop-helix transcription factors brain muscle arnt-like1 (BMAL1) and circadian locomotor output control kaput (CLOCK) coordinate the circadian expression of select genes. We sought to test whether Bmal1 expression in cardiomyocytes contributes to K(+) channel expression and diurnal changes in ventricular repolarization. We used transgenic mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1(-/-)). We used quantitative polymerase chain reaction, voltage clamping, promoter-reporter bioluminescence assays, and electrocardiographic telemetry. Although several K(+) channel gene transcripts were downregulated in iCSΔBmal1(-/-)mouse hearts, only Kcnh2 exhibited a robust circadian pattern of expression that was disrupted in iCSΔBmal1(-/-) hearts. Kcnh2 underlies the rapidly activating delayed-rectifier K(+) current, and the rapidly activating delayed-rectifier K(+) current recorded from iCSΔBmal1(-/-) ventricular cardiomyocytes was ~50% smaller than control ventricular myocytes. Promoter-reporter assays demonstrated that the human Kcnh2 promoter is transactivated by the coexpression of BMAL1 and CLOCK. Electrocardiographic analysis showed that iCSΔBmal1(-/-) mice developed a prolongation in the heart rate-corrected QT interval during the light (resting) phase. This was secondary to an augmented circadian rhythm in the uncorrected QT interval without a corresponding change in the RR interval. The molecular clock in the heart regulates the circadian expression of Kcnh2, modifies K(+) channel gene expression, and is important for normal ventricular repolarization. Disruption of the cardiomyocyte circadian clock mechanism likely unmasks diurnal changes in ventricular repolarization that could contribute

  10. p21WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Ming-Yong Cao

    2011-10-01

    Full Text Available We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21WAF1 (p21 in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21WAF1 and a plasmid expressing small hairpin RNA (shRNA targeting p21WAF1 were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO2, balancing the incubator chamber with N2 for 6 h (hypoxia 6 h, then 21% oxygen for 8 h (reoxygenation 8 h. Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16INK4a, and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16INK4a expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451

  11. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation

    Energy Technology Data Exchange (ETDEWEB)

    Nozaki, Yumiko, E-mail: yumiko-nozaki@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Honda, Yayoi, E-mail: yayoi-honda@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Tsujimoto, Shinji, E-mail: shinji-tsujimoto@ds-pharma.co.jp [Regenerative and Cellular Medicine Office, Dainippon Sumitomo Pharma. Co., Ltd., Chuo-ku, Tokyo 104-0031 (Japan); Watanabe, Hitoshi, E-mail: hitoshi-1-watanabe@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Kunimatsu, Takeshi, E-mail: takeshi-kunimatsu@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Funabashi, Hitoshi, E-mail: hitoshi-funabashi@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan)

    2014-07-01

    Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K{sup +} channel and Ca{sup 2+} channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2 min every 10 min for 30 min after drug exposure for the vehicle and each drug concentration. I{sub Kr} and I{sub Ks} blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca{sup 2+} channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the I{sub Kr} blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. - Highlights: • We focused on hiPS-CMs to replace in vitro assays in preclinical screening studies. • hiPS-CMs FPD is useful as an indicator to predict drug potential for QT prolongation. • MEA assay can help detect EAD for drugs with TdP potentials. • MEA assay in hiPS-CMs is useful for accurately predicting drug TdP risk in humans.

  12. Pompe Disease Results in a Golgi-based Glycosylation Deficit in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes*

    Science.gov (United States)

    Raval, Kunil K.; Tao, Ran; White, Brent E.; De Lange, Willem J.; Koonce, Chad H.; Yu, Junying; Kishnani, Priya S.; Thomson, James A.; Mosher, Deane F.; Ralphe, John C.; Kamp, Timothy J.

    2015-01-01

    Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation. PMID:25488666

  13. Pompe disease results in a Golgi-based glycosylation deficit in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Raval, Kunil K; Tao, Ran; White, Brent E; De Lange, Willem J; Koonce, Chad H; Yu, Junying; Kishnani, Priya S; Thomson, James A; Mosher, Deane F; Ralphe, John C; Kamp, Timothy J

    2015-01-30

    Infantile-onset Pompe disease is an autosomal recessive disorder caused by the complete loss of lysosomal glycogen-hydrolyzing enzyme acid α-glucosidase (GAA) activity, which results in lysosomal glycogen accumulation and prominent cardiac and skeletal muscle pathology. The mechanism by which loss of GAA activity causes cardiomyopathy is poorly understood. We reprogrammed fibroblasts from patients with infantile-onset Pompe disease to generate induced pluripotent stem (iPS) cells that were differentiated to cardiomyocytes (iPSC-CM). Pompe iPSC-CMs had undetectable GAA activity and pathognomonic glycogen-filled lysosomes. Nonetheless, Pompe and control iPSC-CMs exhibited comparable contractile properties in engineered cardiac tissue. Impaired autophagy has been implicated in Pompe skeletal muscle; however, control and Pompe iPSC-CMs had comparable clearance rates of LC3-II-detected autophagosomes. Unexpectedly, the lysosome-associated membrane proteins, LAMP1 and LAMP2, from Pompe iPSC-CMs demonstrated higher electrophoretic mobility compared with control iPSC-CMs. Brefeldin A induced disruption of the Golgi in control iPSC-CMs reproduced the higher mobility forms of the LAMPs, suggesting that Pompe iPSC-CMs produce LAMPs lacking appropriate glycosylation. Isoelectric focusing studies revealed that LAMP2 has a more alkaline pI in Pompe compared with control iPSC-CMs due largely to hyposialylation. MALDI-TOF-MS analysis of N-linked glycans demonstrated reduced diversity of multiantennary structures and the major presence of a trimannose complex glycan precursor in Pompe iPSC-CMs. These data suggest that Pompe cardiomyopathy has a glycan processing abnormality and thus shares features with hypertrophic cardiomyopathies observed in the congenital disorders of glycosylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Severe DCM phenotype of patient harboring RBM20 mutation S635A can be modeled by patient-specific induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Streckfuss-Bömeke, Katrin; Tiburcy, Malte; Fomin, Andrey; Luo, Xiaojing; Li, Wener; Fischer, Claudia; Özcelik, Cemil; Perrot, Andreas; Sossalla, Samuel; Haas, Jan; Vidal, Ramon Oliveira; Rebs, Sabine; Khadjeh, Sara; Meder, Benjamin; Bonn, Stefan; Linke, Wolfgang A; Zimmermann, Wolfram-Hubertus; Hasenfuss, Gerd; Guan, Kaomei

    2017-09-21

    The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  15. Protective effect of guggulsterone against cardiomyocyte injury induced by doxorubicin in vitro

    Directory of Open Access Journals (Sweden)

    Wang Wen-Ching

    2012-08-01

    Full Text Available Abstract Background Doxorubicin (DOX is an effective antineoplastic drug; however, clinical use of DOX is limited by its dose-dependent cardiotoxicity. It is well known that reactive oxygen species (ROS play a vital role in the pathological process of DOX-induced cardiotoxicity. For this study, we evaluated the protective effects of guggulsterone (GS, a steroid obtained from myrrh, to determine its preliminary mechanisms in defending against DOX-induced cytotoxicity in H9C2 cells. Methods In this study, we used a 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl-2H-tetrazolium bromide (MTT assay, lactate dehydrogenase (LDH release measurements, and Hoechst 33258 staining to evaluate the protective effect of GS against DOX-induced cytotoxicity in H9C2 cells. In addition, we observed the immunofluorescence of intracellular ROS and measured lipid peroxidation, caspase-3 activity, and apoptosis-related proteins by using Western blotting. Results The MTT assay and LDH release showed that treatment using GS (1–30 μM did not cause cytotoxicity. Furthermore, GS inhibited DOX (1 μM-induced cytotoxicity in a concentration-dependent manner. Hoechst 33258 staining showed that GS significantly reduced DOX-induced apoptosis and cell death. Using GS at a dose of 10–30 μM significantly reduced intracellular ROS and the formation of MDA in the supernatant of DOX-treated H9C2 cells and suppressed caspase-3 activity to reference levels. In immunoblot analysis, pretreatment using GS significantly reversed DOX-induced decrease of PARP, caspase-3 and bcl-2, and increase of bax, cytochrome C release, cleaved-PARP and cleaved-caspase-3. In addition, the properties of DOX-induced cancer cell (DLD-1 cells death did not interfere when combined GS and DOX. Conclusion These data provide considerable evidence that GS could serve as a novel cardioprotective agent against DOX-induced cardiotoxicity.

  16. Adult Human Primary Cardiomyocyte-Based Model for the Simultaneous Prediction of Drug-Induced Inotropic and Pro-arrhythmia Risk

    Directory of Open Access Journals (Sweden)

    Nathalie Nguyen

    2017-12-01

    Full Text Available Cardiac safety remains the leading cause of drug development discontinuation. We developed a human cardiomyocyte-based model that has the potential to provide a predictive preclinical approach for simultaneously predicting drug-induced inotropic and pro-arrhythmia risk.Methods: Adult human primary cardiomyocytes from ethically consented organ donors were used to measure contractility transients. We used measures of changes in contractility parameters as markers to infer both drug-induced inotropic effect (sarcomere shortening and pro-arrhythmia (aftercontraction, AC; contractility escape (CE; time to 90% relaxation (TR90. We addressed the clinical relevance of this approach by evaluating the effects of 23 torsadogenic and 10 non-torsadogenic drugs. Each drug was tested separately at four multiples of the free effective therapeutic plasma concentration (fETPC.Results: Human cardiomyocyte-based model differentiated between torsadogenic and non-torsadogenic drugs. For example, dofetilide, a torsadogenic drug, caused ACs and increased TR90 starting at 10-fold the fETPC, while CE events were observed at the highest multiple of fETPC (100-fold. Verapamil, a non-torsadogenic drug, did not change TR90 and induced no AC or CE up to the highest multiple of fETPCs tested in this study (222-fold. When drug pro-arrhythmic activity was evaluated at 10-fold of the fETPC, AC parameter had excellent assay sensitivity and specificity values of 96 and 100%, respectively. This high predictivity supports the translational safety potential of this preparation and of the selected marker. The data demonstrate that human cardiomyocytes could also identify drugs associated with inotropic effects. hERG channel blockers, like dofetilide, had no effects on sarcomere shortening, while multi-ion channel blockers, like verapamil, inhibited sarcomere shortening.Conclusions: Isolated adult human primary cardiomyocytes can simultaneously predict risks associated with inotropic

  17. Role of microRNA-195 in cardiomyocyte apoptosis induced by ...

    Indian Academy of Sciences (India)

    VAV2, and CDC42. Hepatology 58, 642–653. Wu S. H., Hang L. W., Yang J. S., Chen H. Y., Lin H. Y., Chiang. J. H. et al. 2010 Curcumin induces apoptosis in human non-small cell lung cancer NCI-H460 cells through ER stress and caspase cascade- and mitochondria-dependent pathways. Anticancer Res. 30, 2125–2133.

  18. p21WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Dan Wang

    2011-10-01

    Full Text Available We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatalrat cardiomyocytes. In this research, we investigated the effects of p21WAF1 (p21 in hypoxia/reoxygenation-inducedsenescence, using H9c2 cells. A plasmid overexpressing wild type p21WAF1 and a plasmid expressing smallhairpin RNA (shRNA targeting p21WAF1 were constructed, and transfected into H9c2 cells to control the p21expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO2, balancing the incubator chamber withN2 for 6 h (hypoxia 6 h, then 21% oxygen for 8 h (reoxygenation 8 h. Cell cycle was examined using flowcytometry. Senescence was assessed using b-galactosidase staining. The expression of p53, p21, p16INK4a, andcyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution,stronger b-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the oppositeresults and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silencedcells had a smaller percentage of G1 cells, weaker b-galactosidase activity and lower 16INK4a expression, andhigher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data impliesthat p21WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescenceprocess. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451

  19. Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp.

    Science.gov (United States)

    Verkerk, Arie O; Veerman, Christiaan C; Zegers, Jan G; Mengarelli, Isabella; Bezzina, Connie R; Wilders, Ronald

    2017-08-30

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in "ventricular-like" and "atrial-like" hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs.

  20. Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

    Science.gov (United States)

    Veerman, Christiaan C.; Zegers, Jan G.; Mengarelli, Isabella; Bezzina, Connie R.

    2017-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs. PMID:28867785

  1. Propofol attenuates H2O2-induced oxidative stress and apoptosis via the mitochondria- and ER-medicated pathways in neonatal rat cardiomyocytes.

    Science.gov (United States)

    Liu, Xue-Ru; Cao, Lu; Li, Tao; Chen, Lin-Lin; Yu, Yi-Yan; Huang, Wen-Jun; Liu, Li; Tan, Xiao-Qiu

    2017-05-01

    Previous studies have shown that propofol, an intravenous anesthetic commonly used in clinical practice, protects the myocardium from injury. Mitochondria- and endoplasmic reticulum (ER)-mediated oxidative stress and apoptosis are two important signaling pathways involved in myocardial injury and protection. The present study aimed to test the hypothesis that propofol could exert a cardio-protective effect via the above two pathways. Cultured neonatal rat cardiomyocytes were treated with culture medium (control group), H2O2 at 500 μM (H2O2 group), propofol at 50 μM (propofol group), and H2O2 plus propofol (H2O2 + propofol group), respectively. The oxidative stress, mitochondrial membrane potential (ΔΨm) and apoptosis of the cardiomyocytes were evaluated by a series of assays including ELISA, flow cytometry, immunofluorescence microscopy and Western blotting. Propofol significantly suppressed the H2O2-induced elevations in the activities of caspases 3, 8, 9 and 12, the ratio of Bax/Bcl-2, and cell apoptosis. Propofol also inhibited the H2O2-induced reactive oxygen species (ROS) generation, lactic dehydrogenase (LDH) release and mitochondrial transmembrane potential (ΔΨm) depolarization, and restored the H2O2-induced reductions of glutathione (GSH) and superoxide dismutase (SOD). In addition, propofol decreased the expressions of glucose-regulated protein 78 kDa (Grp78) and inositol-requiring enzyme 1α (IRE1α), two important signaling molecules in the ER-mediated apoptosis pathway. Propofol protects cardiomyocytes from H2O2-induced injury by inhibiting the mitochondria- and ER-mediated apoptosis signaling pathways.

  2. Xenotransplantation of Human Cardiomyocyte Progenitor Cells Does Not Improve Cardiac Function in a Porcine Model of Chronic Ischemic Heart Failure. Results from a Randomized, Blinded, Placebo Controlled Trial.

    Directory of Open Access Journals (Sweden)

    Sanne J Jansen of Lorkeers

    Full Text Available Recently cardiomyocyte progenitor cells (CMPCs were successfully isolated from fetal and adult human hearts. Direct intramyocardial injection of human CMPCs (hCMPCs in experimental mouse models of acute myocardial infarction significantly improved cardiac function compared to controls.Here, our aim was to investigate whether xenotransplantation via intracoronary infusion of fetal hCMPCs in a pig model of chronic myocardial infarction is safe and efficacious, in view of translation purposes.We performed a randomized, blinded, placebo controlled trial. Four weeks after ischemia/reperfusion injury by 90 minutes of percutaneous left anterior descending artery occlusion, pigs (n = 16, 68.5 ± 5.4 kg received intracoronary infusion of 10 million fetal hCMPCs or placebo. All animals were immunosuppressed by cyclosporin (CsA. Four weeks after infusion, endpoint analysis by MRI displayed no difference in left ventricular ejection fraction, left ventricular end diastolic and left ventricular end systolic volumes between both groups. Serial pressure volume (PV-loop and echocardiography showed no differences in functional parameters between groups at any timepoint. Infarct size at follow-up, measured by late gadolinium enhancement MRI showed no difference between groups. Intracoronary pressure and flow measurements showed no signs of coronary obstruction 30 minutes after cell infusion. No premature death occurred in cell treated animals.Xenotransplantation via intracoronary infusion of hCMPCs is feasible and safe, but not associated with improved left ventricular performance and infarct size compared to placebo in a porcine model of chronic myocardial infarction.

  3. Resveratrol Attenuated Low Ambient Temperature-Induced Myocardial Hypertrophy via Inhibiting Cardiomyocyte Apoptosis

    OpenAIRE

    Kun Yin; Liang Zhao; Dan Feng; Wenya Ma; Yu Liu; Yang Wang; Jing Liang; Fan Yang; Chongwei Bi; Hongyang Chen; Xingda Li; Yanjie Lu; Benzhi Cai

    2015-01-01

    Background/Aims: Low ambient temperature is an important risk factor for cardiovascular diseases, and has been shown to lead to cardiac hypertrophy. In this study, we aim to investigate if Resveratrol may inhibit cold exposure-induced cardiac hypertrophy in mice, and if so to clarify its molecular mechanism. Methods: Adult male mice were randomly assigned to Control group (kept at room temperature), Cold group (kept at low air temperature range from 3°C to 5°C) and Resveratrol treatment group...

  4. High-calcium exposure to frog heart: a simple model representing hypercalcemia-induced ECG abnormalities.

    Science.gov (United States)

    Kazama, Itsuro

    2017-01-20

    By simply adding a high concentration of calcium solution to the surface of the bullfrog heart, we reproduced electrocardiogram (ECG) abnormalities representing those observed in hypercalcemia, such as Osborn waves and shortening of the QT interval. The rise in extracellular calcium concentration may have activated the outward potassium currents during phase 3 of the action potential, and thus decreased its duration. In addition to the known decrease in the duration of phase 2, such changes in phase 3 were also likely to contribute to the shortening of the QT interval. The dual recordings of the action potential in cardiomyocytes and the ECG waves enabled us to demonstrate the mechanisms of ECG abnormalities induced by hypercalcemia.

  5. Heart Failure as an Aging-Related Phenotype.

    Science.gov (United States)

    Morita, Hiroyuki; Komuro, Issei

    2018-01-27

    The molecular pathophysiology of heart failure, which is one of the leading causes of mortality, is not yet fully understood. Heart failure can be regarded as a systemic syndrome of aging-related phenotypes. Wnt/β-catenin signaling and the p53 pathway, both of which are key regulators of aging, have been demonstrated to play a critical role in the pathogenesis of heart failure. Circulating C1q was identified as a novel activator of Wnt/β-catenin signaling, promoting systemic aging-related phenotypes including sarcopenia and heart failure. On the other hand, p53 induces the apoptosis of cardiomyocytes in the failing heart. In these molecular mechanisms, the cross-talk between cardiomyocytes and non-cardiomyocytes (e,g,. endothelial cells, fibroblasts, smooth muscle cells, macrophages) deserves mentioning. In this review, we summarize recent advances in the understanding of the molecular pathophysiology underlying heart failure, focusing on Wnt/β-catenin signaling and the p53 pathway.

  6. Desmoglein-2 interaction is crucial for cardiomyocyte cohesion and function.

    Science.gov (United States)

    Schlipp, Angela; Schinner, Camilla; Spindler, Volker; Vielmuth, Franziska; Gehmlich, Katja; Syrris, Petros; Mckenna, William J; Dendorfer, Andreas; Hartlieb, Eva; Waschke, Jens

    2014-11-01

    We determined the contribution of the desmosomal cadherin desmoglein-2 to cell-cell cohesion in cardiomyocytes. In the intercalated disc, providing mechanical strength and electrical communication between adjacent cardiomyocytes, desmoglein-2 is closely associated with N-cadherin and gap junctions. We studied intercalated discs of HL-1 cardiomyocytes by immunostaining of desmoglein-2 and N-cadherin. Cohesion was measured using a liberase-based dissociation-assay and compared with cell-free single-molecule atomic force microscopy measurements. L-tryptophan caused irregular desmoglein-2 condensation, weakened cell-cell cohesion and impaired both homophilic desmoglein-2 and N-cadherin trans-interaction, whereas l-phenylalanine had no effect. L-tryptophan did not affect N-cadherin localization and its inhibitory effect on cell-cohesion and desmoglein-2 binding, but not on N-cadherin interaction, was blocked by a desmoglein-specific tandem peptide. Moreover, Ca(2+)-depletion, desmoglein-2 knockdown, a desmoglein-specific single peptide and certain desmoglein-2 mutations associated with arrhythmogenic cardiomyopathy reduced cell-cell cohesion, whereas cell adhesion was strengthened by desmoglein-2 overexpression. Since single peptide did not interfere with N-cadherin trans-interaction, these data indicate that (i) desmoglein-2 binding is crucial for cardiomyocyte cohesion and (ii) L-tryptophan reduced both desmoglein-2 and N-cadherin binding, whereas single and tandem peptide can be used to specifically target desmoglein-2-mediated adhesion. L-tryptophan and single peptide also induced ultrastructural alterations of areae compositae. Functional analyses at the organ level revealed reduced cardiomyocyte function and inefficient response to adrenergic stimulation in both L-tryptophan- and single peptide-challenged murine Langendorff hearts paralleled by redistribution of connexin 43 in L-tryptophan-treated heart slices. Our data demonstrate that desmoglein-2 plays a

  7. Sodium accumulation in SERCA knockout-induced heart failure.

    Science.gov (United States)

    Li, Liren; Louch, William E; Niederer, Steven A; Aronsen, Jan M; Christensen, Geir; Sejersted, Ole M; Smith, Nicolas P

    2012-05-02

    In cardiomyocytes, a major decrease in the level of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) can severely impair systolic and diastolic functions. In mice with cardiomyocyte-specific conditional excision of the Serca2 gene (SERCA2 KO), end-stage heart failure developed between four and seven weeks after gene deletion combined with [Na(+)](i) elevation and intracellular acidosis. In this study, to investigate the underpinning changes in Ca(2+) dynamics and metabolic homeostasis, we developed data-driven mathematical models of Ca(2+) dynamics in the ventricular myocytes of the control, four-week, and seven-week SERCA2 knockout (KO) mice. The seven-week KO model showed that elevated [Na(+)](i) was due to increased Na(+) influxes through the Na(+)/Ca(2+) exchanger (NCX) and the Na(+)/H(+) exchanger, with the latter exacerbated by intracellular acidosis. Furthermore, NCX upregulation in the seven-week KO model resulted in increased ATP consumption for ion transport. Na(+) accumulation in the SERCA KO due to NCX upregulation and intracellular acidosis potentially play a role in the development of heart failure, by initiating a reinforcing cycle involving: a mismatch between ATP demand and supply; an increasingly compromised metabolism; a decreased pH(i); and, finally, an even greater [Na(+)](i) elevation. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Glyceraldehyde-3-phosphate dehydrogenase interacts with proapoptotic kinase mst1 to promote cardiomyocyte apoptosis.

    Directory of Open Access Journals (Sweden)

    Bei You

    Full Text Available Mammalian sterile 20-like kinase 1 (Mst1 is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in the heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart remains unknown. In a yeast two-hybrid screen of a human heart cDNA library with Mst1 as bait, glyceraldehyde-3-phosphate dehydrogenase (GAPDH was identified as an Mst1-interacting protein. The interaction of GAPDH with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and mouse heart homogenates, in which GAPDH interacted with the kinase domain of Mst1, whereas the C-terminal catalytic domain of GAPDH mediated its interaction with Mst1. Moreover, interaction of Mst1 with GAPDH caused a robust phosphorylation of GAPDH and markedly increased the Mst1 activity in cells. Chelerythrine, a potent inducer of apoptosis, substantially increased the nuclear translocation and interaction of GAPDH and Mst1 in cardiomyocytes. Overexpression of GAPDH significantly augmented the Mst1 mediated apoptosis, whereas knockdown of GAPDH markedly attenuated the Mst1 activation and cardiomyocyte apoptosis in response to either chelerythrine or hypoxia/reoxygenation. These findings reveal a novel function of GAPDH in Mst1 activation and cardiomyocyte apoptosis and suggest that disruption of GAPDH interaction with Mst1 may prevent apoptosis related heart diseases such as heart failure and ischemic heart disease.

  9. Functional Differences in Engineered Myocardium from Embryonic Stem Cell-Derived versus Neonatal Cardiomyocytes

    NARCIS (Netherlands)

    Feinberg, Adam W.; Ripplinger, Crystal M.; van der Meer, Peter; Sheehy, Sean P.; Domian, Ibrahim; Chien, Kenneth R.; Parker, Kevin Kit

    2013-01-01

    Stem cell-derived cardiomyocytes represent unique tools for cell-and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated.

  10. Peroxisomes in cardiomyocytes and the peroxisome / peroxisome proliferator-activated receptor-loop.

    Science.gov (United States)

    Colasante, Claudia; Chen, Jiangping; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

    2015-03-01

    It is well established that the heart is strongly dependent on fatty acid metabolism. In cardiomyocytes there are two distinct sites for the β-oxidisation of fatty acids: the mitochondrion and the peroxisome. Although the metabolism of these two organelles is believed to be tightly coupled, the nature of this relationship has not been fully investigated. Recent research has established the significant contribution of mitochondrial function to cardiac ATP production under normal and pathological conditions. In contrast, limited information is available on peroxisomal function in the heart. This is despite these organelles harbouring metabolic pathways that are potentially cardio-protective, and findings that patients with peroxisomal diseases, such as adult Refsum´s disease, can develop heart failure. In this article, we provide a comprehensive overview on the current knowledge of peroxisomes and the regulation of lipid metabolism by PPARs in cardiomyocytes. We also present new experimental evidence on the differential expression of peroxisome-related genes in the heart chambers and demonstrate that even a mild peroxisomal biogenesis defect (Pex11α-/-) can induce profound alterations in the cardiomyocyte´s peroxisomal compartment and related gene expression, including the concomitant deregulation of specific PPARs. The possible impact of peroxisomal dysfunction in the heart is discussed and a model for the modulation of myocardial metabolism via a peroxisome/PPAR-loop is proposed.

  11. Endoplasmic reticulum stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) protects against pressure overload-induced heart failure and lung remodeling.

    Science.gov (United States)

    Liu, Xiaoyu; Kwak, Dongmin; Lu, Zhongbing; Xu, Xin; Fassett, John; Wang, Huan; Wei, Yidong; Cavener, Douglas R; Hu, Xinli; Hall, Jennifer; Bache, Robert J; Chen, Yingjie

    2014-10-01

    Studies have reported that development of congestive heart failure is associated with increased endoplasmic reticulum stress. Double stranded RNA-activated protein kinase R-like endoplasmic reticulum kinase (PERK) is a major transducer of the endoplasmic reticulum stress response and directly phosphorylates eukaryotic initiation factor 2α, resulting in translational attenuation. However, the physiological effect of PERK on congestive heart failure development is unknown. To study the effect of PERK on ventricular structure and function, we generated inducible cardiac-specific PERK knockout mice. Under unstressed conditions, cardiac PERK knockout had no effect on left ventricular mass, or its ratio to body weight, cardiomyocyte size, fibrosis, or left ventricular function. However, in response to chronic transverse aortic constriction, PERK knockout mice exhibited decreased ejection fraction, increased left ventricular fibrosis, enhanced cardiomyocyte apoptosis, and exacerbated lung remodeling in comparison with wild-type mice. PERK knockout also dramatically attenuated cardiac sarcoplasmic reticulum Ca(2+)-ATPase expression in response to aortic constriction. Our findings suggest that PERK is required to protect the heart from pressure overload-induced congestive heart failure. © 2014 American Heart Association, Inc.

  12. MicroRNA-297 promotes cardiomyocyte hypertrophy via targeting sigma-1 receptor.

    Science.gov (United States)

    Bao, Qinxue; Zhao, Mingyue; Chen, Li; Wang, Yu; Wu, Siyuan; Wu, Wenchao; Liu, Xiaojing

    2017-04-15

    Sigma-1 receptor (Sig-1R) is a ligand-regulated endoplasmic reticulum (ER) chaperone involved in cardiac hypertrophy, but it is not known whether Sig-1R is regulated by microRNAs (miRNAs). According to bioinformatic analysis, miR-297 was suggested as a potential target miRNA for Sig-1R. Therefore, we verified whether miR-297 could target Sig-1R and investigated the possible mechanisms underlying the role of miR-297 in cardiac hypertrophy. Bioinformatic analysis combined with laboratory experiments, including quantitative RT-PCR, Western blotting, and luciferase assay, were performed to identify the target miRNA of Sig-1R. Transverse aortic constriction (TAC) model and neonatal rat cardiomyocytes (NCMs) stimulated with angiotensin II (AngII) were used to explore the relationship between miR-297 and Sig-1R. Additionally, the function of miR-297 in cardiomyocyte hypertrophy and ER stress/unfolded protein response (UPR) signaling pathway was investigated by transfecting miR-297 mimics/inhibitor. miR-297 levels were increased in both TAC-induced hypertrophic heart tissue and AngII-induced cardiomyocyte hypertrophy. Up-regulation of miR-297 by specific mimics exacerbated AngII-induced cardiomyocyte hypertrophy, whereas inhibition of miR-297 suppressed the process. During cardiomyocyte hypertrophy, Sig-1R expression, which was negatively regulated by miR-297 by directly targeting its 3'untranslated region (UTR), was decreased. Furthermore, attenuation of miR-297 inhibited the activation of X-box binding protein 1 (Xbp1) and activating transcriptional factor 4 (ATF4) signaling pathways in NCMs. Our data demonstrate that miR-297 promotes cardiomyocyte hypertrophy by inhibiting the expression of Sig-1R and activation of ER stress signaling, which provides a novel interpretation for cardiac hypertrophy. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Chronic hypoxemia in late gestation decreases cardiomyocyte number but does not change expression of hypoxia-responsive genes.

    Science.gov (United States)

    Botting, Kimberley J; McMillen, I Caroline; Forbes, Heather; Nyengaard, Jens R; Morrison, Janna L

    2014-07-28

    Placental insufficiency is the leading cause of intrauterine growth restriction in the developed world and results in chronic hypoxemia in the fetus. Oxygen is essential for fetal heart development, but a hypoxemic environment in utero can permanently alter development of cardiomyocytes. The present study aimed to investigate the effect of placental restriction and chronic hypoxemia on total number of cardiomyocytes, cardiomyocyte apoptosis, total length of coronary capillaries, and expression of genes regulated by hypoxia. We induced experimental placental restriction from conception, which resulted in fetal growth restriction and chronic hypoxemia. Fetal hearts in the placental restriction group had fewer cardiomyocytes, but interestingly, there was no difference in the percentage of apoptotic cardiomyocytes; the abundance of the transcription factor that mediates hypoxia-induced apoptosis, p53; or expression of apoptotic genes Bax and Bcl2. Likewise, there was no difference in the abundance of autophagy regulator beclin 1 or expression of autophagic genes BECN1, BNIP3, LAMP1, and MAP1LC3B. Furthermore, fetuses exposed to normoxemia (control) or chronic hypoxemia (placental restriction) had similar mRNA expression of a suite of hypoxia-inducible factor target genes, which are essential for angiogenesis (VEGF, Flt1, Ang1, Ang2, and Tie2), vasodilation (iNOS and Adm), and glycolysis (GLUT1 and GLUT3). In addition, there was no change in the expression of PKC-ε, a cardioprotective gene with transcription regulated by hypoxia in a manner independent of hypoxia-inducible factors. There was an increased capillary length density but no difference in the total length of capillaries in the hearts of the chronically hypoxemic fetuses. The lack of upregulation of hypoxia target genes in response to chronic hypoxemia in the fetal heart in late gestation may be due to a decrease in the number of cardiomyocytes (decreased oxygen demand) and the maintenance of the total length

  14. Hyperkalemia-induced complete heart block

    Directory of Open Access Journals (Sweden)

    Alireza Baratloo

    2015-05-01

    Full Text Available Background: Potassium, as an extracellular ion, plays an important role in the electrophysiologic function of the myocardium and any change in extracellular concentration of this ion might have a marked impression upon myocyte electrophysiologic gain. High serum potassium levels are thought to impair pulse conduction in Purkinje fibers and ventricles more than that in the Atrioventricular (AV node. Therefore, although complete AV block can occur, it is a rare initial presentation. Case Report: We describe a 62-year-old man with a history of diabetes mellitus, ischemic heart disease and previous Coronary Artery Bypass Graft (CABG, who came to our emergency department due to generalized weakness starting 2 days before admission. The patient also had decreased force in lower limbs, exacerbating from the morning, and was finally diagnosed as a hyperkalemia-induced Complete Heart Block (CHB. It should also be noted that the patient responded dramatically to the administration of 10 mL of 10% calcium gluconate along with external pacing until potassium level correction became effective. Conclusion: In spite of the fact that Hyperkalemia can be associated with frequent Electrocardiogram (ECG abnormality, advanced heart blocks (second- and third-degree AV blocks are usually found only in patients with pre-existing heart failure, conduction abnormalities, or other cardiac diseases. Institution of effective treatment rapidly and forgiveness of traditional non-effective, time consumptive and sometimes risking full-adjustment modalities, such as sodium bicarbonate infusion or exchange resins that prevent their use in the emergent phase, can help minimize patient morbidity and mortality.

  15. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.

    Science.gov (United States)

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.

  16. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    Science.gov (United States)

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. PMID:25834424

  17. Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair.

    Science.gov (United States)

    Tiburcy, Malte; Hudson, James E; Balfanz, Paul; Schlick, Susanne; Meyer, Tim; Chang Liao, Mei-Ling; Levent, Elif; Raad, Farah; Zeidler, Sebastian; Wingender, Edgar; Riegler, Johannes; Wang, Mouer; Gold, Joseph D; Kehat, Izhak; Wettwer, Erich; Ravens, Ursula; Dierickx, Pieterjan; van Laake, Linda W; Goumans, Marie Jose; Khadjeh, Sara; Toischer, Karl; Hasenfuss, Gerd; Couture, Larry A; Unger, Andreas; Linke, Wolfgang A; Araki, Toshiyuki; Neel, Benjamin; Keller, Gordon; Gepstein, Lior; Wu, Joseph C; Zimmermann, Wolfram-Hubertus

    2017-05-09

    Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions. © 2017 American Heart Association, Inc.

  18. Micropost arrays for measuring stem cell-derived cardiomyocyte contractility.

    Science.gov (United States)

    Beussman, Kevin M; Rodriguez, Marita L; Leonard, Andrea; Taparia, Nikita; Thompson, Curtis R; Sniadecki, Nathan J

    2016-02-01

    Stem cell-derived cardiomyocytes have the potential to be used to study heart disease and maturation, screen drug treatments, and restore heart function. Here, we discuss the procedures involved in using micropost arrays to measure the contractile forces generated by stem cell-derived cardiomyocytes. Cardiomyocyte contractility is needed for the heart to pump blood, so measuring the contractile forces of cardiomyocytes is a straightforward way to assess their function. Microfabrication and soft lithography techniques are utilized to create identical arrays of flexible, silicone microposts from a common master. Micropost arrays are functionalized with extracellular matrix protein to allow cardiomyocytes to adhere to the tips of the microposts. Live imaging is used to capture videos of the deflection of microposts caused by the contraction of the cardiomyocytes. Image analysis code provides an accurate means to quantify these deflections. The contractile forces produced by a beating cardiomyocyte are calculated by modeling the microposts as cantilever beams. We have used this assay to assess techniques for improving the maturation and contractile function of stem cell-derived cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Different roles of β-arrestin and the PKA pathway in mitochondrial ROS production induced by acute β-adrenergic receptor stimulation in neonatal mouse cardiomyocytes.

    Science.gov (United States)

    Zhang, Jianshu; Xiao, Han; Shen, Jing; Wang, Nanping; Zhang, Youyi

    2017-08-05

    Reactive oxygen species (ROS) play a crucial role in various physiological and pathological processes mediated by β-adrenergic receptors (β-ARs) in cardiomyocytes. However, the sources and signaling pathways involved in ROS production induced by acute β-AR activation have not yet been fully defined. In primary neonatal mouse cardiomyocytes (NMCMs), the β-AR agonist isoproterenol (ISO) induced a rapid increase in mitochondrial ROS and total ROS production. Both the expression and activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2/4 (NOX 2/4) remained unchanged after 2 h of ISO treatment, suggesting that acute ISO stimulation mainly induces mitochondrial ROS production in NMCMs. Knockdown of β-arrestin1, but not β-arrestin2, inhibited ISO-induced mitochondrial ROS production within 1-2 h after ISO treatment. Moreover, forskolin, an adenylyl cyclase (AC) activator, rapidly increased mitochondrial ROS as early as 15 min after ISO treatment. Inhibition of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway abolished the mitochondrial ROS production within 15-60 min after ISO treatment. In conclusion, mitochondria are the major source of ROS production upon acute ISO stimulation. β-arrestin1, but not β-arrestin2, is involved in ISO-induced mitochondrial ROS production. Upon acute β-AR stimulation in NMCMs, the classical cAMP/PKA pathway is responsible for faster mitochondrial ROS production, whereas β-arrestin1 signaling is responsible for slower mitochondrial ROS production. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Chronic Hypoxemia in Late Gestation Decreases Cardiomyocyte Number but Does Not Change Expression of Hypoxia‐Responsive Genes

    Science.gov (United States)

    Botting, Kimberley J.; McMillen, I. Caroline; Forbes, Heather; Nyengaard, Jens R.; Morrison, Janna L.

    2014-01-01

    Background Placental insufficiency is the leading cause of intrauterine growth restriction in the developed world and results in chronic hypoxemia in the fetus. Oxygen is essential for fetal heart development, but a hypoxemic environment in utero can permanently alter development of cardiomyocytes. The present study aimed to investigate the effect of placental restriction and chronic hypoxemia on total number of cardiomyocytes, cardiomyocyte apoptosis, total length of coronary capillaries, and expression of genes regulated by hypoxia. Methods and Results We induced experimental placental restriction from conception, which resulted in fetal growth restriction and chronic hypoxemia. Fetal hearts in the placental restriction group had fewer cardiomyocytes, but interestingly, there was no difference in the percentage of apoptotic cardiomyocytes; the abundance of the transcription factor that mediates hypoxia‐induced apoptosis, p53; or expression of apoptotic genes Bax and Bcl2. Likewise, there was no difference in the abundance of autophagy regulator beclin 1 or expression of autophagic genes BECN1, BNIP3, LAMP1, and MAP1LC3B. Furthermore, fetuses exposed to normoxemia (control) or chronic hypoxemia (placental restriction) had similar mRNA expression of a suite of hypoxia‐inducible factor target genes, which are essential for angiogenesis (VEGF, Flt1, Ang1, Ang2, and Tie2), vasodilation (iNOS and Adm), and glycolysis (GLUT1 and GLUT3). In addition, there was no change in the expression of PKC‐ε, a cardioprotective gene with transcription regulated by hypoxia in a manner independent of hypoxia‐inducible factors. There was an increased capillary length density but no difference in the total length of capillaries in the hearts of the chronically hypoxemic fetuses. Conclusion The lack of upregulation of hypoxia target genes in response to chronic hypoxemia in the fetal heart in late gestation may be due to a decrease in the number of cardiomyocytes (decreased

  1. Functional innervation of human induced pluripotent stem cell-derived cardiomyocytes by co-culture with sympathetic neurons developed using a microtunnel technique.

    Science.gov (United States)

    Sakai, Koji; Shimba, Kenta; Ishizuka, Kazuma; Yang, Zhuonan; Oiwa, Kosuke; Takeuchi, Akimasa; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2017-10-14

    Microelectrode array (MEA) based-drug screening with human induced pluripotent stem cell-derived cardiomyocytes (hiPSCM) is a potent pre-clinical assay for efficiently assessing proarrhythmic risks in new candidates. Furthermore, predicting sympathetic modulation of the proarrhythmic side-effects is an important issue. Although we have previously developed a MEA-based co-culture system of rat primary cardiomyocyte and sympathetic neurons (rSNs), it is unclear if this co-culture approach is applicable to develop and investigate sympathetic innervation of hiPSCMs. In this study, we developed a co-culture of rSNs and hiPSCMs on MEA substrate, and assessed functional connections. The inter-beat interval of hiPSCM was significantly shortened by stimulation in SNs depending on frequency and pulse number, indicating functional connections between rSNs and hiPSCM and the dependency of chronotropic effects on rSN activity pattern. These results suggest that our co-culture approach can evaluate sympathetic effects on hiPSCMs and would be a useful tool for assessing sympathetic modulated-cardiotoxicity in human cardiac tissue. Copyright © 2017. Published by Elsevier Inc.

  2. Propofol Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor-Alpha Expression and Myocardial Depression through Decreasing the Generation of Superoxide Anion in Cardiomyocytes

    Science.gov (United States)

    Tang, Jing; Hu, Ji-Jie; Lu, Chun-Hua; Liang, Jia-Ni; Xiao, Jin-Fang; Liu, You-Tan; Lin, Chun-Shui; Qin, Zai-Sheng

    2014-01-01

    TNF-α has been shown to be a major factor responsible for myocardial depression in sepsis. The aim of this study was to investigate the effect of an anesthetic, propofol, on TNF-α expression in cardiomyocytes treated with LPS both in vivo and in vitro. In cultured cardiomyocytes, compared with control group, propofol significantly reduced protein expression of gp91phox and phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2) and p38 MAPK, which associates with reduced TNF-α production. In in vivo mice studies, propofol significantly improved myocardial depression and increased survival rate of mice after LPS treatment or during endotoxemia, which associates with reduced myocardial TNF-α production, gp91phox, ERK1/2, and p38 MAPK. It is concluded that propofol abrogates LPS-induced TNF-α production and alleviates cardiac depression through gp91phox/ERK1/2 or p38 MAPK signal pathway. These findings have great clinical importance in the application of propofol for patients enduring sepsis. PMID:25180066

  3. Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis.

    Science.gov (United States)

    Huang, Zhan-Peng; Kataoka, Masaharu; Chen, Jinghai; Wu, Gengze; Ding, Jian; Nie, Mao; Lin, Zhiqiang; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Zhou, Bin; Wakimoto, Hiroko; Zeng, Chunyu; Kyselovic, Jan; Deng, Zhong-Liang; Seidman, Christine E; Seidman, J G; Pu, William T; Wang, Da-Zhi

    2015-11-02

    Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression.

  4. Functional and Transcriptional Characterization of Histone Deacetylase Inhibitor-Mediated Cardiac Adverse Effects in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Science.gov (United States)

    Kopljar, Ivan; Gallacher, David J; De Bondt, An; Cougnaud, Laure; Vlaminckx, Eddy; Van den Wyngaert, Ilse; Lu, Hua Rong

    2016-05-01

    Histone deacetylase (HDAC) inhibitors possess therapeutic potential to reverse aberrant epigenetic changes associated with cancers, neurological diseases, and immune disorders. Unfortunately, clinical studies with some HDAC inhibitors displayed delayed cardiac adverse effects, such as atrial fibrillation and ventricular tachycardia. However, the underlying molecular mechanism(s) of HDAC inhibitor-mediated cardiotoxicity remains poorly understood and is difficult to detect in the early stages of preclinical drug development because of a delayed onset of effects. In the present study, we show for the first time in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) that HDAC inhibitors (dacinostat, panobinostat, vorinostat, entinostat, and tubastatin-a) induce delayed dose-related cardiac dysfunction at therapeutic concentrations associated with cardiac adverse effects in humans. HDAC inhibitor-mediated delayed effects on the beating properties of hiPS-CMs developed after 12 hours by decreasing the beat rate, shortening the field potential duration, and inducing arrhythmic behavior under form of sustained contractions and fibrillation-like patterns. Transcriptional changes that are common between the cardiotoxic HDAC inhibitors but different from noncardiotoxic treatments identified cardiac-specific genes and pathways related to structural and functional changes in cardiomyocytes. Combining the functional data with epigenetic changes in hiPS-CMs allowed us to identify molecular targets that might explain HDAC inhibitor-mediated cardiac adverse effects in humans. Therefore, hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and support identification of better HDAC inhibitors with an improved benefit-risk profile. Histone deacetylase (HDAC) inhibitors are a promising class of drugs to treat certain cancers, autoimmune, and neurodegenerative diseases. However, treated patients can experience various

  5. Effects of hawthorn (Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells.

    Science.gov (United States)

    Pahlavan, Sara; Tousi, Marziyeh Shalchi; Ayyari, Mahdi; Alirezalu, Abolfazl; Ansari, Hassan; Saric, Tomo; Baharvand, Hossein

    2017-11-13

    Cardiac arrhythmias are major life-threatening conditions. The landmark discovery of induced pluripotent stem cells has provided a promising in vitro system for modeling hereditary cardiac arrhythmias as well as drug development and toxicity testing. Nowadays, nutraceuticals are frequently used as supplements for cardiovascular therapy. Here we studied the cardiac effects of hawthorn (Crataegus pentagyna) leaf extract using cardiomyocytes (CMs) differentiated from healthy human embryonic stem cells, long QT syndrome type 2 (LQTS2), and catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) patient-specific induced pluripotent stem cells. The hydroalcoholic extract resulted in a dose-dependent negative chronotropic effect in all CM preparations leading to a significant reduction at 1000 µg/ml. This was accompanied by prolongation of field potential durations, although with different magnitudes in CMs from different human embryonic stem cell and iPSC lines. Hawthorn further prolonged field potential durations in LQTS2 CMs but reduced the beating frequencies and occurrence of immature field potentials triggered by β1-adrenergic stimulation in CPVT1 CMs at 300 and 1000 µg/ml. Furthermore, isoquercetin and vitexin flavonoids significantly slowed down isoproterenol (5 µM)-induced beating frequencies at 3 and 10 µg/ml. Therefore, C. pentagyna leaf extract and its isoquercetin and vitexin flavonoids may be introduced as a novel nutraceutical with antiarrhythmic potential for CPVT1 patients.-Pahlavan, S., Tousi, M. S., Ayyari, M., Alirezalu, A., Ansari, H., Saric, T., Baharvand, H. Effects of hawthorn (Crataegus pentagyna) leaf extract on electrophysiologic properties of cardiomyocytes derived from human cardiac arrhythmia-specific induced pluripotent stem cells. © FASEB.

  6. Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

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    Birkedal Rikke

    2009-12-01

    Full Text Available Abstract Background Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss, which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine. Results Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. Conclusions The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that

  7. Ablation of matrix metalloproteinase-9 prevents cardiomyocytes contractile dysfunction in diabetics

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    Priyanka ePrathipati

    2016-03-01

    Full Text Available Elevated expression and activity of matrix metalloproteinase-9 (MMP9 and decreased contractility of cardiomyocytes are documented in diabetic hearts. However, it is unclear whether MMP is involved in the regulation of contractility of cardiomyocytes in diabetic hearts. In the present study, we tested the hypothesis that MMP9 regulates contractility of cardiomyocytes in diabetic hearts, and ablation of matrix metalloproteinase-9 (MMP9 prevents impaired contractility of cardiomyocytes in diabetic hearts. To determine the specific role of MMP9 in cardiomyocyte contractility, we used twelve - fourteen week male WT (normoglycemic sibling of Akita, Akita, and Ins2+/- /MMP9-/- (DKO mice. DKO mice were generated by cross-breeding male Ins2+/- Akita (T1D with female MMP9 knockout (MMP9-/- mice. We isolated cardiomyocytes from the heart of the above three groups of mice and measured their contractility and calcium transients. Moreover, we determined mRNA and protein levels of sarco-endoplasmic reticulum calcium ATPase-2a (SERCA-2a, which is involved in calcium handling during contractility of cardiomyocytes, in WT, Akita, and DKO hearts using qPCR, Western blotting and immunoprecipitation, respectively. Our results revealed that in Akita hearts where increased expression and activity of MMP9 is reported, the rates of shortening and re-lengthening (± dL/dt of cardiomyocytes were decreased, time to 90% peak height and baseline during contractility was increased, rate of calcium decay was increased, and calcium transient was decreased as compared to WT cardiomyocytes. However, these changes in Akita were blunted in DKO cardiomyocytes. The molecular analyses of SERCA-2a in the hearts showed that it was downregulated in Akita as compared to WT but was comparatively upregulated in DKO. These results suggest that abrogation of MMP9 gene prevents contractility of cardiomyocytes, possibly by increasing SERCA-2a and calcium transients. We conclude that MMP9 plays

  8. Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

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    Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

    2017-10-02

    Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional

  9. The Role of Reactive Oxygen Species in β-Adrenergic Signaling in Cardiomyocytes from Mice with the Metabolic Syndrome.

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    Monica Llano-Diez

    Full Text Available The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS. Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice. We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.

  10. Rac1-PAK2 pathway is essential for zebrafish heart regeneration

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    Peng, Xiangwen [State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life Sciences, Fudan University, Shanghai 201203 (China); He, Quanze [Center for Reproduction and Genetics, Suzhou Municipal Hospital, Jiangsu 215002 (China); Li, Guobao; Ma, Jinmin [State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life Sciences, Fudan University, Shanghai 201203 (China); Zhong, Tao P., E-mail: taozhongfudan@yahoo.com [State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life Sciences, Fudan University, Shanghai 201203 (China); Department of Medicine, Vanderbilt University School of Medicine, TN 37232 (United States)

    2016-04-15

    P-21 activated kinases, or PAKs, are serine–threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.

  11. Integrative Analysis of the Developing Postnatal Mouse Heart Transcriptome.

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    Jingyi Gan

    Full Text Available In mammals, cardiomyocytes rapidly proliferate in the fetus and continue to do so for a few more days after birth. These cardiomyocytes then enter into growth arrest but the detailed molecular mechanisms involved have not been fully elucidated. We have addressed this issue by comparing the transcriptomes of 2-day-old (containing dividing cardiomyocytes with 13-day-old (containing growth arrested cardiomyocytes postnatal mouse hearts. We performed comparative microarray analysis on the heart tissues and then conducted Functional annotation, Gene ontology, KEGG pathway and Gene Set enrichment analyses on the differentially expressed genes. The bioinformatics analysis revealed that gene ontology categories associated with the "cell cycle", "DNA replication", "chromosome segregation" and "microtubule cytoskeleton" were down-regulated. Inversely, "immune response", "extracellular matrix", "cell differentiation" and "cell membrane" were up-regulated. Ingenuity Pathways Analysis (IPA has revealed that GATA4, MYH7 and IGF1R were the key drivers of the gene interaction networks. In addition, Regulator Effects network analysis suggested that TASP1, TOB1, C1orf61, AIF1, ROCK1, TFF2 and miR503-5p may be acting on the cardiomyocytes in 13-day-old mouse hearts to inhibit cardiomyocyte proliferation and G1/S phase transition. RT-qPCR was used to validate genes which were differentially expressed and genes that play a prominent role in the pathways and interaction networks that we identified. In sum, our integrative analysis has provided more insights into the transcriptional regulation of cardiomyocyte exit from the cell cycle during postnatal heart development. The results also pinpoint potential regulators that could be used to induce growth arrested cardiomyocytes to proliferate in the infarcted heart.

  12. Advanced Ring-Shaped Microelectrode Assay Combined with Small Rectangular Electrode for Quasi-In vivo Measurement of Cell-to-Cell Conductance in Cardiomyocyte Network

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    Nomura, Fumimasa; Kaneko, Tomoyuki; Hamada, Tomoyo; Hattori, Akihiro; Yasuda, Kenji

    2013-06-01

    To predict the risk of fatal arrhythmia induced by cardiotoxicity in the highly complex human heart system, we have developed a novel quasi-in vivo electrophysiological measurement assay, which combines a ring-shaped human cardiomyocyte network and a set of two electrodes that form a large single ring-shaped electrode for the direct measurement of irregular cell-to-cell conductance occurrence in a cardiomyocyte network, and a small rectangular microelectrode for forced pacing of cardiomyocyte beating and for acquiring the field potential waveforms of cardiomyocytes. The advantages of this assay are as follows. The electrophysiological signals of cardiomyocytes in the ring-shaped network are superimposed directly on a single loop-shaped electrode, in which the information of asynchronous behavior of cell-to-cell conductance are included, without requiring a set of huge numbers of microelectrode arrays, a set of fast data conversion circuits, or a complex analysis in a computer. Another advantage is that the small rectangular electrode can control the position and timing of forced beating in a ring-shaped human induced pluripotent stem cell (hiPS)-derived cardiomyocyte network and can also acquire the field potentials of cardiomyocytes. First, we constructed the human iPS-derived cardiomyocyte ring-shaped network on the set of two electrodes, and acquired the field potential signals of particular cardiomyocytes in the ring-shaped cardiomyocyte network during simultaneous acquisition of the superimposed signals of whole-cardiomyocyte networks representing cell-to-cell conduction. Using the small rectangular electrode, we have also evaluated the response of the cell network to electrical stimulation. The mean and SD of the minimum stimulation voltage required for pacing (VMin) at the small rectangular electrode was 166+/-74 mV, which is the same as the magnitude of amplitude for the pacing using the ring-shaped electrode (179+/-33 mV). The results showed that the

  13. Nitroxyl (HNO stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation.

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    Eliane Q Lin

    Full Text Available New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP however has not been investigated.Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II in the presence and absence of the HNO donor Angeli's salt (sodium trioxodinitrate or B-type natriuretic peptide, BNP (all 1 µmol/L. Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.We now demonstrate that Angeli's salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli's salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation, as well as p38 mitogen-activated protein kinase (p38MAPK. The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli's salt were mimicked by BNP. We also demonstrate that the effects of Angeli's salt are specifically mediated by HNO (with no role for NO• or nitrite, with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP.Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.

  14. Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload.

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    Anna N Panek

    Full Text Available Connective tissue growth factor (CTGF is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG. To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca(2+ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF's profibrotic function in the heart.

  15. Src is required for mechanical stretch-induced cardiomyocyte hypertrophy through angiotensin II type 1 receptor-dependent β-arrestin2 pathways.

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    Shijun Wang

    Full Text Available Angiotensin II (AngII type 1 receptor (AT1-R can be activated by mechanical stress (MS without the involvement of AngII during the development of cardiomyocyte hypertrophy, in which G protein-independent pathways are critically involved. Although β-arrestin2-biased signaling has been speculated, little is known about how AT1-R/β-arrestin2 leads to ERK1/2 activation. Here, we present a novel mechanism by which Src kinase mediates AT1-R/β-arrestin2-dependent ERK1/2 phosphorylation in response to MS. Differing from stimulation by AngII, MS-triggered ERK1/2 phosphorylation is neither suppressed by overexpression of RGS4 (the negative regulator of the G-protein coupling signal nor by inhibition of Gαq downstream protein kinase C (PKC with GF109203X. The release of inositol 1,4,5-triphosphate (IP3 is increased by AngII but not by MS. These results collectively suggest that MS-induced ERK1/2 activation through AT1-R might be independent of G-protein coupling. Moreover, either knockdown of β-arrestin2 or overexpression of a dominant negative mutant of β-arrestin2 prevents MS-induced activation of ERK1/2. We further identifies a relationship between Src, a non-receptor tyrosine kinase and β-arrestin2 using analyses of co-immunoprecipitation and immunofluorescence after MS stimulation. Furthermore, MS-, but not AngII-induced ERK1/2 phosphorylation is attenuated by Src inhibition, which also significantly improves pressure overload-induced cardiac hypertrophy and dysfunction in mice lacking AngII. Finally, MS-induced Src activation and hypertrophic response are abolished by candesartan but not by valsartan whereas AngII-induced responses can be abrogated by both blockers. Our results suggest that Src plays a critical role in MS-induced cardiomyocyte hypertrophy through β-arrestin2-associated angiotensin II type 1 receptor signaling.

  16. Hippo pathway deficiency reverses systolic heart failure after infarction.

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    Leach, John P; Heallen, Todd; Zhang, Min; Rahmani, Mahdis; Morikawa, Yuka; Hill, Matthew C; Segura, Ana; Willerson, James T; Martin, James F

    2017-10-12

    Mammalian organs vary widely in regenerative capacity. Poorly regenerative organs, such as the heart are particularly vulnerable to organ failure. Once established, heart failure commonly results in mortality. The Hippo pathway, a kinase cascade that prevents adult cardiomyocyte proliferation and regeneration, is upregulated in human heart failure. Here we show that deletion of the Hippo pathway component Salvador (Salv) in mouse hearts with established ischaemic heart failure after myocardial infarction induces a reparative genetic program with increased scar border vascularity, reduced fibrosis, and recovery of pumping function compared with controls. Using translating ribosomal affinity purification, we isolate cardiomyocyte-specific translating messenger RNA. Hippo-deficient cardiomyocytes have increased expression of proliferative genes and stress response genes, such as the mitochondrial quality control gene, Park2. Genetic studies indicate that Park2 is essential for heart repair, suggesting a requirement for mitochondrial quality control in regenerating myocardium. Gene therapy with a virus encoding Salv short hairpin RNA improves heart function when delivered at the time of infarct or after ischaemic heart failure following myocardial infarction was established. Our findings indicate that the failing heart has a previously unrecognized reparative capacity involving more than cardiomyocyte renewal.

  17. Testosterone suppresses oxidative stress via androgen receptor-independent pathway in murine cardiomyocytes.

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    Zhang, Li; Wu, Saizhu; Ruan, Yunjun; Hong, Lei; Xing, Xiaowen; Lai, Wenyan

    2011-01-01

    Evidence supports that oxidative stress exerts significant effects on the pathogenesis of heart dysfunction. On the other hand, the presence of specific androgen receptor (AR) in mammalian cardiomyocytes implies that androgen plays a physiological role in cardiac function, myocardial injury and the regulation of the redox state in the heart. This study used the testicular feminized (Tfm) and castrated male mice to investigate the effects of testosterone deficiency, physiological testosterone therapy and AR on oxidative stress in cardiomyocytes. Tfm mice have a non-functional AR and reduced circulating testosterone levels. Male littermates and Tfm mice were separated into 5 experimental groups: non-castrated littermate controls, castrated littermates, sham-operated Tfm, testosterone-treated castrated littermates and testosterone-treated sham-operated Tfm mice. Cardiomyocytes that were isolated from the left ventricle were used for determination of superoxide dismutase (SOD), glutathione peroxidase (GSH‑Px) enzyme activities, and malondialdehyde (MDA) levels. Additionally, mitochondrial DNA (mtDNA) deletion mutations were detected by nested PCR. The SOD and GSH-Px enzyme activities of cardiomyocytes were decreased, and the MDA levels and the proportion of mtDNA mutations were increased in castrated and sham-operated Tfm mice compared to control mice. However, an increase was observed in the activities of SOD and GSH-Px enzyme as well as a decrease in MDA levels and the proportion of mtDNA mutations in the mice that had received testosterone therapy. These changes were statistically similar in castrated and sham-operated Tfm mice after testosterone therapy. In conclusion, it is testosterone deficiency that induces oxidative stress in cardiomyocytes. Physiological testosterone therapy is able to suppress oxidative stress mediated via the AR-independent pathway.

  18. Urotensin II induction of adult cardiomyocytes hypertrophy involves the Akt/GSK-3beta signaling pathway.

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    Gruson, D; Ginion, A; Decroly, N; Lause, P; Vanoverschelde, J L; Ketelslegers, J M; Bertrand, L; Thissen, J P

    2010-07-01

    Urotensin II (UII) a potent vasoactive peptide is upregulated in the failing heart and promotes cardiomyocytes hypertrophy, in particular through mitogen-activated protein kinases. However, the regulation by UII of GSK-3beta, a recognized pivotal signaling element of cardiac hypertrophy has not yet been documented. We therefore investigated in adult cardiomyocytes, if UII phosphorylates GSK-3beta and Akt, one of its upstream regulators and stabilizes beta-catenin, a GSK-3beta dependent nuclear transcriptional co-activator. Primary cultures of adult rat cardiomyocytes were stimulated for 48h with UII. Cell size and protein/DNA contents were determined. Phosphorylated and total forms of Akt, GSK-3beta and the total amount of beta-catenin were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the chemical phosphatidyl-inositol-3-kinase inhibitor, LY294002, and urantide, a competitive UII receptor antagonist. UII increased cell size and the protein/DNA ratio, consistent with a hypertrophic response. UII also increased phosphorylation of Akt and its downstream target GSK-3beta. beta-Catenin protein levels were increased. All of these effects of UII were prevented by LY294002, and urantide. The UII-induced adult cardiomyocytes hypertrophy involves the Akt/GSK-3beta signaling pathways and is accompanied by the stabilization of the beta-catenin. All these effects are abolished by competitive inhibition of the UII receptor, consistent with new therapeutic perspectives for heart failure treatment. Copyright 2010 Elsevier Inc. All rights reserved.

  19. PTRF/Cavin-1 Deficiency Causes Cardiac Dysfunction Accompanied by Cardiomyocyte Hypertrophy and Cardiac Fibrosis.

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    Takuya Taniguchi

    Full Text Available Mutations in the PTRF/Cavin-1 gene cause congenital generalized lipodystrophy type 4 (CGL4 associated with myopathy. Additionally, long-QT syndrome and fatal cardiac arrhythmia are observed in patients with CGL4 who have homozygous PTRF/Cavin-1 mutations. PTRF/Cavin-1 deficiency shows reductions of caveolae and caveolin-3 (Cav3 protein expression in skeletal muscle, and Cav3 deficiency in the heart causes cardiac hypertrophy with loss of caveolae. However, it remains unknown how loss of PTRF/Cavin-1 affects cardiac morphology and function. Here, we present a characterization of the hearts of PTRF/Cavin-1-null (PTRF-/- mice. Electron microscopy revealed the reduction of caveolae in cardiomyocytes of PTRF-/- mice. PTRF-/- mice at 16 weeks of age developed a progressive cardiomyopathic phenotype with wall thickening of left ventricles and reduced fractional shortening evaluated by echocardiography. Electrocardiography revealed that PTRF-/- mice at 24 weeks of age had low voltages and wide QRS complexes in limb leads. Histological analysis showed cardiomyocyte hypertrophy accompanied by progressive interstitial/perivascular fibrosis. Hypertrophy-related fetal gene expression was also induced in PTRF-/- hearts. Western blotting analysis and quantitative RT-PCR revealed that Cav3 expression was suppressed in PTRF-/- hearts compared with that in wild-type (WT ones. ERK1/2 was activated in PTRF-/- hearts compared with that in WT ones. These results suggest that loss of PTRF/Cavin-1 protein expression is sufficient to induce a molecular program leading to cardiomyocyte hypertrophy and cardiomyopathy, which is partly attributable to Cav3 reduction in the heart.

  20. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

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    Tahereh Talaei-Khozani

    2014-03-01

    Full Text Available Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA and 5-Aza-2-Deoxycytidine (5-aza-dC. The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function.

  1. The cardiac maladaptive ATF3-dependent cross-talk between cardiomyocytes and macrophages is mediated by the IFNγ-CXCL10-CXCR3 axis.

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    Koren, L; Barash, U; Zohar, Y; Karin, N; Aronheim, A

    2017-02-01

    Pressure overload induces adaptive and maladaptive cardiac remodeling processes in the heart. Part of the maladaptive process is the cross-talk between cardiomyocytes and macrophages which is dependent on the function of the Activating Transcription Factor 3, ATF3. Yet, the molecular mechanism involved in cardiomyocytes-macrophages communication leading to macrophages recruitment to the heart and cardiac maladaptive remodeling is currently unknown. Isolated peritoneal macrophages from either wild type or ATF3-KO mice were cultured in serum free medium to collect conditioned medium (CM). CM was used to probe an antibody cytokine/chemokine array. The interferon γ induced protein 10kDa, CXCL10, was found to be enriched in wild type macrophages CM. Wild type cardiomyocytes treated with CXCL10 in vitro, resulted in significant increase in cell volume as compared to ATF3-KO cardiomyocytes. In vivo, pressure overload was induced by phenylephrine (PE) infusion using micro-osmotic pumps. Consistently, CXCL11 (CXCL10 competitive agonist) and CXCL10 receptor antagonist (AMG487) attenuated PE-dependent maladaptive cardiac remodeling. Significantly, we show that the expression of the CXCL10 receptor, CXCR3, is suppressed in cardiomyocytes and macrophages derived from ATF3-KO mice. CXCR3 is positively regulated by ATF3 through an ATF3 transcription response element found in its proximal promoter. Finally, mice lacking CXCR3 display a significant reduction of cardiac remodeling processes following PE infusion. Chronic PE infusion results in a unique cardiomyocytes-macrophages cross-talk that is mediated by IFNγ. Subsequently, macrophages that are recruited to the heart secrete CXCL10 resulting in maladaptive cardiac remodeling mediated by the CXCR3 receptor. ATF3-KO mice escape from PE-dependent maladaptive cardiac remodeling by suppressing the IFNγ-CXCL10-CXCR3 axis at multiple levels. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

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    Stephanie Friedrichs

    2015-01-01

    Full Text Available Disease-specific induced pluripotent stem (iPS cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

  3. Increased expression of microRNA-378a-5p in acute ethanol exposure of rat cardiomyocytes.

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    Wang, Zhongkai; Song, Jingwen; Zhang, Liang; Huang, Songqun; Bao, Lizhi; Chen, Feng; Zhao, Xianxian

    2017-03-01

    Alcohol abuse is a risk factor for a distinct form of congestive heart failure, known as alcoholic cardiomyopathy (ACM). Here, we investigate how microRNAs may participate in the induction of cardiomyocyte apoptosis associated with ethanol exposure in vitro. Increasing the concentrations of ethanol to primary rat cardiomyocytes resulted in elevated apoptosis assessed by annexin V and propidium iodide staining, and reduced expression of an enzyme for alcohol detoxification aldehyde dehydrogenase 2 (ALDH2). These ethanol effects were accompanied by a substantial elevation of miR-378a-5p. Driving miR-378a-5p overexpression in cardiomyocytes decreased ALDH2. The specific interaction of miR-378a-5p with the 3'UTR of ALDH2 was examined by luciferase reporter assays, and we found that miR-378a-5p activity depends on a complementary base pairing at the 3'-UTR region of ALDH2 mRNA. Finally, ethanol-induced apoptosis in cardiomyocytes was attenuated in the presence of anti-miR378a-5p. Collectively, these data implicate a likely involvement of miR-378a-5p in the stimulation of cardiomyocyte apoptosis through ALDH2 gene suppression, which might play a potential role in the pathogenesis of ACM.

  4. Use of Adeno-Associated Virus to Enrich Cardiomyocytes Derived from Human Stem Cells.

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    Guan, Xuan; Wang, Zejing; Czerniecki, Stefan; Mack, David; François, Virginie; Blouin, Veronique; Moullier, Philippe; Childers, Martin K

    2015-09-01

    Cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) show great promise as autologous donor cells to treat heart disease. A major technical obstacle to this approach is that available induction methods often produce heterogeneous cell population with low percentage of cardiomyocytes. Here we describe a cardiac enrichment approach using nonintegrating adeno-associated virus (AAV). We first examined several AAV serotypes for their ability to selectively transduce iPSC-derived cardiomyocytes. Results showed that AAV1 demonstrated the highest in vitro transduction efficiency among seven widely used serotypes. Next, differentiated iPSC derivatives were transduced with drug-selectable AAV1 expressing neomycin resistance gene. Selection with G418 enriched the cardiac cell fraction from 27% to 57% in 2 weeks. Compared with other enrichment strategies such as integrative genetic selection, mitochondria labeling, or surface marker cell sorting, this simple AAV method described herein bypasses antibody or dye labeling. These findings provide proof of concept for large-scale cardiomyocyte enrichment by exploiting AAV's intrinsic tissue tropism.

  5. ERK/PP1a/PLB/SERCA2a and JNK pathways are involved in luteolin-mediated protection of rat hearts and cardiomyocytes following ischemia/reperfusion.

    Directory of Open Access Journals (Sweden)

    Xin Wu

    Full Text Available Luteolin has long been used in traditional Chinese medicine for treatment of various diseases. Recent studies have suggested that administration of luteolin yields cardioprotective effects during ischemia/reperfusion (I/R in rats. However, the precise mechanisms of this action remain unclear. The aim of this study is to confirm that luteolin-mediated extracellular signal regulated kinase (ERK1/2 and c-Jun N-terminal kinase (JNK pathways are responsible for their cardioprotective effects during I/R. Wistar rats were divided into the following groups: (i DMSO group (DMSO; (ii I/R group (I/R; (iii luteolin+I/R group (Lut+I/R; (iv ERK1/2 inhibitor PD98059+I/R group (PD+I/R; (v PD98059+luteolin+I/R group (PD+Lut+I/R; and (vi JNK inhibitor SP600125+I/R group (SP+I/R. The following properties were measured: contractile function of isolated heart and cardiomyocytes; infarct size; the release of lactate dehydrogenase (LDH; the percentage of apoptotic cells; the expression levels of Bcl-2 and Bax; and phosphorylation status of ERK1/2, JNK, type 1 protein phosphatase (PP1a, phospholamban (PLB and sarcoplasmic reticulum Ca(2+-ATPase (SERCA2a. Our data showed that pretreatment with luteolin or SP600125 significantly improved the contraction of the isolated heart and cardiomyocytes, reduced infarct size and LDH activity, decreased the rate of apoptosis and increased the Bcl-2/Bax ratio. However, pretreatment with PD98059 alone before I/R had no effect on the above indexes. Further, these consequences of luteolin pretreatment were abrogated by co-administration of PD98059. We also found that pretreatment with PD98059 caused a significant increase in JNK expression, and SP600125 could cause ERK1/2 activation during I/R. In addition, we are the first to demonstrate that luteolin affects PP1a expression, which results in the up-regulation of the PLB, thereby relieving its inhibition of SERCA2a. These results showed that luteolin improves cardiomyocyte contractile

  6. HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer

    CSIR Research Space (South Africa)

    Rangel Lopes de Campos, W

    2014-10-01

    Full Text Available the direct and indirect effects of HIV-1 subtype C infection of cultured human cardiomyocytes and the mechanisms leading to cardiomyocytes damage; as well as a way to mitigate the damage. We evaluated a novel approach to mitigate HIVCM using a previously...

  7. Case of congestive heart failure induced by therapeutic irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kushigami, Motohiko; Suruda, Hidetoshi; Mizukoshi, Masato; Umemoto, Masaaki; Fujiwara, Setsuko; Yamamoto, Katsuhiro; Ueno, Yuji; Nishio, Ichiro; Masuyama, Yoshiaki

    1985-02-01

    Valvular insufficiency in radiation-induced heart disease is very rare. We described a patient, 53 years old woman, who developed congestive heart failure 2.5 years later following radiotherapy for esophageal carcinoma. The findings on examinations including cardiac catheterization revealed pericarditis with effusion, mitral and tricuspid valve insufficiency and pulmonary infarction. (author).

  8. The role of sex differences in autophagy in the heart during coxsackievirus B3-induced myocarditis.

    Science.gov (United States)

    Koenig, Andreas; Sateriale, Adam; Budd, Ralph C; Huber, Sally A; Buskiewicz, Iwona A

    2014-03-01

    Under normal conditions, autophagy maintains cardiomyocyte health and integrity through turnover of organelles. During stress, oxygen and nutrient deprivation, or microbial infection, autophagy prolongs cardiomyocyte survival. Sex differences in induction of cell death may to some extent explain the disparity between the sexes in many human diseases. However, sex differences in gene expression, which regulate cell death and autophagy, were so far not taken in consideration to explain the sex bias of viral myocarditis. Coxsackievirus B3 (CVB3)-induced myocarditis is a sex-biased disease, with females being substantially less susceptible than males and sex hormones largely determine this bias. CVB3 was shown to induce and subvert the autophagosome for its optimal viral RNA replication. Gene expression analysis on mouse and human, healthy and CVB3-infected, cardiac samples of both sexes, suggests sex differences in autophagy-related gene expression. This review discusses the aspects of sex bias in autophagy induction in cardiomyocytes.

  9. Inhibition of NAPDH Oxidase 2 (NOX2 Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Leroy C Joseph

    Full Text Available Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.

  10. Heart Failure-Induced Diaphragm Myopathy

    Directory of Open Access Journals (Sweden)

    Aline Regina Ruiz Lima

    2014-07-01

    Full Text Available Background: Intracellular signaling pathways involved in skeletal myosin heavy chain (MyHC isoform alterations during heart failure (HF are not completely understood. We tested the hypothesis that diaphragm expression of mitogen-activated protein kinases (MAPK and myogenic regulatory factors is changed in rats with myocardial infarction (MI induced HF. Methods: Six months after MI rats were subjected to transthoracic echocardiography. After euthanasia, infarcted rats were subdivided in MI/HF- group (with no HF evidence; n=10, and MI/HF+ (with right ventricular hypertrophy and lung congestion; n=10. Sham-operated rats were used as controls (n=10. MyHC isoforms were analyzed by electrophoresis. Statistical analysis: ANOVA and Pearson correlation. Results: MI/HF- had left cardiac chambers dilation with systolic and diastolic left ventricular dysfunction. Cardiac injury was more intense in MI/HF+ than MI/HF-. MyHC I isoform percentage was higher in MI/HF+ than MI/HF-, and IIb isoform lower in MI/HF+ than Sham. Left atrial diameter-to-body weight ratio positively correlated with MyHC I (p=0.005 and negatively correlated with MyHC IIb (p=0.02. TNF-a serum concentration positively correlated with MyHC I isoform. Total and phosphorylated ERK was lower in MI/HF- and MI/HF+ than Sham. Phosphorylated JNK was lower in MI/HF- than Sham. JNK and p38 did not differ between groups. Expression of NF-κB and the myogenic regulatory factors MyoD, myogenin, and MRF4 was similar between groups. Conclusion: Diaphragm MyHC fast-to-slow shift is related to cardiac dysfunction severity and TNF-a serum levels in infarcted rats. Reduced ERK expression seems to participate in MyHC isoform changes. Myogenic regulatory factors and NF-κB do not modulate diaphragm MyHC distribution during chronic HF.

  11. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells.

    Science.gov (United States)

    Giacomelli, Elisa; Bellin, Milena; Sala, Luca; van Meer, Berend J; Tertoolen, Leon G J; Orlova, Valeria V; Mummery, Christine L

    2017-03-15

    Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs. © 2017. Published by The Company of Biologists Ltd.

  12. A promising culture model for analyzing the interaction between adipose tissue and cardiomyocytes.

    Science.gov (United States)

    Anan, Mayumi; Uchihashi, Kazuyoshi; Aoki, Shigehisa; Matsunobu, Aki; Ootani, Akifumi; Node, Koichi; Toda, Shuji

    2011-04-01

    The heart has epicardial adipose tissue that produces adipokines and mesenchymal stem cells. Systemic adipose tissue is involved in the pathophysiology of obesity-related heart diseases. However, the method for analyzing the direct interaction between adipose tissue and cardiomyocytes has not been established. Here we show the novel model, using collagen gel coculture of adipose tissue fragments (ATFs) and HL-1 cardiomyocytes, and electron microscopy, immunohistochemistry, real-time RT-PCR, and ELISA. HL-1 cells formed a stratified layer on ATF-nonembedded gel, whereas they formed almost a monolayer on ATF-embedded gel. ATFs promoted the apoptosis, lipid accumulation, and fatty acid transport protein (FATP) expression of FATP4 and CD36 in HL-1 cells, whereas ATFs inhibited the growth and mRNA expression of myosin, troponin T, and atrial natriuretic peptide. Treatment of leptin (100 ng/ml) and adiponectin (10 μg/ml) neither replicated nor abolished the ATF-induced morphology of HL-1 cells, whereas that of FATP4 and CD36 antibodies (25 μg/ml) never abolished it. HL-1 cells prohibited the development of CD44+/CD105+ mesenchymal stem cell-like cells and lipid-laden preadipocytes from ATFs. HL-1 cells increased the production of adiponectin in ATFs, whereas they decreased that of leptin. The data indicate that our model actively creates adipose tissue-HL-1 cardiomyocyte interaction, suggesting first that ATFs may be related to the lipotoxiciy of HL-1 cells via unknown factors plus FATP4 and CD36 and second that HL-1 cells may help to retain the static state of ATFs, affecting adipokine secretion. Our model will serve to study adipose tissue-cardiomyocyte interaction and mechanisms of obesity-related lipotoxicity and heart diseases.

  13. Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Shan-Li [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Sun, Ming-Rui [Department of Pharmacology, Qiqihaer Medical College, Qiqihaer 160001 (China); Li, Ting-Ting; Yin, Xin [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Xu, Chang-Qing [Department of Pathophysiology, Harbin Medical University, Harbin 150086 (China); Sun, Yi-Hua, E-mail: syh200415@126.com [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China)

    2011-03-11

    Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3

  14. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

    Science.gov (United States)

    Mohamed, Tamer M. A.; Abou-Leisa, Riham; Stafford, Nicholas; Maqsood, Arfa; Zi, Min; Prehar, Sukhpal; Baudoin-Stanley, Florence; Wang, Xin; Neyses, Ludwig; Cartwright, Elizabeth J.; Oceandy, Delvac

    2016-01-01

    The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition. PMID:27020607

  15. A near-infrared fluorescent voltage-sensitive dye allows for moderate-throughput electrophysiological analyses of human induced pluripotent stem cell-derived cardiomyocytes

    Science.gov (United States)

    Lopez-Izquierdo, Angelica; Warren, Mark; Riedel, Michael; Cho, Scott; Lai, Shuping; Lux, Robert L.; Spitzer, Kenneth W.; Benjamin, Ivor J.; Jou, Chuanchau J.

    2014-01-01

    Human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM)-based assays are emerging as a promising tool for the in vitro preclinical screening of QT interval-prolonging side effects of drugs in development. A major impediment to the widespread use of human iPSC-CM assays is the low throughput of the currently available electrophysiological tools. To test the precision and applicability of the near-infrared fluorescent voltage-sensitive dye 1-(4-sulfanatobutyl)-4-{β[2-(di-n-butylamino)-6-naphthyl]butadienyl}quinolinium betaine (di-4-ANBDQBS) for moderate-throughput electrophysiological analyses, we compared simultaneous transmembrane voltage and optical action potential (AP) recordings in human iPSC-CM loaded with di-4-ANBDQBS. Optical AP recordings tracked transmembrane voltage with high precision, generating nearly identical values for AP duration (AP durations at 10%, 50%, and 90% repolarization). Human iPSC-CMs tolerated repeated laser exposure, with stable optical AP parameters recorded over a 30-min study period. Optical AP recordings appropriately tracked changes in repolarization induced by pharmacological manipulation. Finally, di-4-ANBDQBS allowed for moderate-throughput analyses, increasing throughput >10-fold over the traditional patch-clamp technique. We conclude that the voltage-sensitive dye di-4-ANBDQBS allows for high-precision optical AP measurements that markedly increase the throughput for electrophysiological characterization of human iPSC-CMs. PMID:25172899

  16. Low extracellular potassium prolongs repolarization and evokes early afterdepolarization in human induced pluripotent stem cell-derived cardiomyocytes

    Science.gov (United States)

    Kuusela, Jukka; Larsson, Kim; Shah, Disheet; Prajapati, Chandra; Aalto-Setälä, Katriina

    2017-01-01

    ABSTRACT Long QT syndrome (LQTS) is characterized by a prolonged QT-interval on electrocardiogram and by increased risk of sudden death. One of the most common and potentially life-threatening electrolyte disturbances is hypokalemia, characterized by low concentrations of K+. Using a multielectrode array platform and current clamp technique, we investigated the effect of low extracellular K+ concentration ([K+]Ex) on the electrophysiological properties of hiPSC-derived cardiomyocytes (CMs) generated from a healthy control subject (WT) and from two symptomatic patients with type 1 of LQTS carrying G589D (LQT1A) or IVS7-2A>G mutation (LQT1B) in KCNQ1. The baseline prolongations of field potential durations (FPDs) and action potential durations (APDs) were longer in LQT1-CMs than in WT-CMs. Exposure to low [K+]Ex prolonged FPDs and APDs in a concentration-dependent fashion. LQT1-CMs were found to be more sensitive to low [K+]Ex compared to WT-CMs. At baseline, LQT1A-CMs had more prolonged APDs than LQT1B-CMs, but low [K+]Ex caused more pronounced APD prolongation in LQT1B-CMs. Early afterdepolarizations in the action potentials were observed in a subset of LQT1A-CMs with further prolonged baseline APDs and triangular phase 2 profiles. This work demonstrates that the hiPSC-derived CMs are sensitive to low [K+]Ex and provide a platform to study acquired LQTS. PMID:28619993

  17. Low extracellular potassium prolongs repolarization and evokes early afterdepolarization in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kuusela, Jukka; Larsson, Kim; Shah, Disheet; Prajapati, Chandra; Aalto-Setälä, Katriina

    2017-06-15

    Long QT syndrome (LQTS) is characterized by a prolonged QT-interval on electrocardiogram and by increased risk of sudden death. One of the most common and potentially life-threatening electrolyte disturbances is hypokalemia, characterized by low concentrations of K(+) Using a multielectrode array platform and current clamp technique, we investigated the effect of low extracellular K(+) concentration ([K(+)]Ex) on the electrophysiological properties of hiPSC-derived cardiomyocytes (CMs) generated from a healthy control subject (WT) and from two symptomatic patients with type 1 of LQTS carrying G589D (LQT1A) or IVS7-2A>G mutation (LQT1B) in KCNQ1 The baseline prolongations of field potential durations (FPDs) and action potential durations (APDs) were longer in LQT1-CMs than in WT-CMs. Exposure to low [K(+)]Ex prolonged FPDs and APDs in a concentration-dependent fashion. LQT1-CMs were found to be more sensitive to low [K(+)]Ex compared to WT-CMs. At baseline, LQT1A-CMs had more prolonged APDs than LQT1B-CMs, but low [K(+)]Ex caused more pronounced APD prolongation in LQT1B-CMs. Early afterdepolarizations in the action potentials were observed in a subset of LQT1A-CMs with further prolonged baseline APDs and triangular phase 2 profiles. This work demonstrates that the hiPSC-derived CMs are sensitive to low [K(+)]Ex and provide a platform to study acquired LQTS. © 2017. Published by The Company of Biologists Ltd.

  18. Low extracellular potassium prolongs repolarization and evokes early afterdepolarization in human induced pluripotent stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jukka Kuusela

    2017-06-01

    Full Text Available Long QT syndrome (LQTS is characterized by a prolonged QT-interval on electrocardiogram and by increased risk of sudden death. One of the most common and potentially life-threatening electrolyte disturbances is hypokalemia, characterized by low concentrations of K+. Using a multielectrode array platform and current clamp technique, we investigated the effect of low extracellular K+ concentration ([K+]Ex on the electrophysiological properties of hiPSC-derived cardiomyocytes (CMs generated from a healthy control subject (WT and from two symptomatic patients with type 1 of LQTS carrying G589D (LQT1A or IVS7-2A>G mutation (LQT1B in KCNQ1. The baseline prolongations of field potential durations (FPDs and action potential durations (APDs were longer in LQT1-CMs than in WT-CMs. Exposure to low [K+]Ex prolonged FPDs and APDs in a concentration-dependent fashion. LQT1-CMs were found to be more sensitive to low [K+]Ex compared to WT-CMs. At baseline, LQT1A-CMs had more prolonged APDs than LQT1B-CMs, but low [K+]Ex caused more pronounced APD prolongation in LQT1B-CMs. Early afterdepolarizations in the action potentials were observed in a subset of LQT1A-CMs with further prolonged baseline APDs and triangular phase 2 profiles. This work demonstrates that the hiPSC-derived CMs are sensitive to low [K+]Ex and provide a platform to study acquired LQTS.

  19. ZYZ-772 Prevents Cardiomyocyte Injury by Suppressing Nox4-Derived ROS Production and Apoptosis

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    Ying Wang

    2017-02-01

    Full Text Available Nox-dependent signaling plays critical roles in the development of heart failure, cardiac hypertrophy, and myocardial infarction. NADPH oxidase 4 (Nox4 as a major source of oxidative stress in the heart offers a new therapeutic target in cardiovascular disease. In the present work, a novel flavonoid was isolated from Zanthoxylum bungeanum. Its structure was elucidated as Quercetin-3-O-(6′′-O-α-l-rhamnopyransoyl-β-d-glucopyranoside-7-O-β-d-glucopyranoside (ZYZ-772 for the first time. ZYZ-772 exhibited significant cardio-protective property against CoCl2 induced H9c2 cardiomyocyte cells injury. In CoCl2 stimulated cardiomyocyte injury, ZYZ-772 inhibited expression of Nox4, and alleviated ROS overproduction. Importantly, ROS triggered MAPKs phosphorylation and P53 signaling mediated apoptosis were restored by ZYZ-772. Our findings present the first piece of evidence for the therapeutic properties of ZYZ-772 in preventing cardiomyocyte injury, which could be attributed to the suppression of Nox4/MAPKs/P53 axis. This will offer a novel therapeutic strategy for the treatment of cardiac ischemia disease.

  20. Heme oxygenase suppresses markers of heart failure and ameliorates cardiomyopathy in L-NAME-induced hypertension.

    Science.gov (United States)

    Ndisang, Joseph Fomusi; Chibbar, Rajni; Lane, Nina

    2014-07-05

    Heart failure and related cardiac complications remains a great health challenge. We investigated the effects of upregulating heme-oxygenase (HO) on myocardial histo-pathological lesions, proinflammatory cytokines/chemokines, oxidative mediators and important markers of heart failure such as osteopontin and osteoprotergerin in N(ω)-nitro-l-arginine methyl ester (L-NAME)-induced hypertension. Treatment with the HO-inducer, heme-arginate improved myocardial morphology in L-NAME hypertensive rats by attenuating subendocardial injury, interstitial fibrosis, mononuclear-cell infiltration and cardiomyocyte hypertrophy. These were associated with the reduction of several inflammatory/oxidative mediators including chemokines/cytokines such as macrophage inflammatory protein-1 alpha (MIP-1α), macrophage chemoattractant protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, endothelin-1, 8-isoprostane, nitrotyrosine, and aldosterone. Similarly, heme-arginate abated the elevated levels of extracellular matrix/remodeling proteins including transforming-growth factor beta (TGF-β1) and collagen-IV in the myocardium. These were accompanied by significant reduction of proteins of heart failure such as osteopontin and osteoprotegerin. Interestingly, the cardio-protective effects of heme-arginate were associated with the potentiation of adiponectin, atrial-natriuretic peptide (ANP), HO-1, HO-activity, cyclic gnanosine monophosphate (cGMP) and the total-anti-oxidant capacity, whereas the HO-inhibitor, chromium-mesoporphyrin nullified the effects of heme-arginate, exacerbating inflammatory injury and oxidative insults. We conclude that heme-arginate therapy protects myocardial damage by potentiating the HO-adiponectin-ANP axis, which in turn suppressed the elevated levels of aldosterone, pro-inflammatory chemokines/cytokines, mononuclear-cell infiltration and oxidative stress, with concomitant reduction of extracellular matrix/remodeling proteins and

  1. Beat Rate Variability in Murine Embryonic Stem Cell-Derived Cardiomyocytes: Effect of Antiarrhythmic Drugs.

    Science.gov (United States)

    Niehoff, Julius; Matzkies, Matthias; Nguemo, Filomain; Hescheler, Jürgen; Reppel, Michael

    2016-01-01

    Heart rate variability (HRV) refers to the fluctuation of the time interval between consecutive heartbeats in humans. It has recently been discovered that cardiomyocytes derived from human embryonic and induced pluripotent stem cells show beat rate variability (BRV) that is similar to the HRV in humans. In the present study, clinical aspects of HRV were transferred to an in vitro model. The aims of the study were to explore the BRV in murine embryonic stem cell (mESC)-derived cardiomyocytes and to demonstrate the influence of antiarrhythmic drugs on BRV as has been shown in clinical trials previously. The Microelectrode Array (MEA) technique was used to perform short-term recordings of extracellular field potentials (FPs) of spontaneously beating cardiomyocytes derived from mESCs (D3 cell line, αPig-44). Offline analysis was focused on time domain and nonlinear methods. The Poincaré-Plot analysis of measurements without pharmacological intervention revealed that three different shapes of scatter plots occurred most frequently. Comparable shapes have been described in clinical studies before. The antiarrhythmic drugs Ivabradine, Verapamil and Sotalol augmented BRV, whereas Flecainide decreased BRV parameters at low concentrations (SDSD 79.0 ± 8.7% of control at 10(-9) M, p < 0.05) and increased variability measures at higher concentrations (SDNN 258.8 ± 42.7% of control at 10(-5) M, p < 0.05). Amiodarone and Metoprolol did not alter BRV significantly. Spontaneously beating cardiomyocytes derived from mESCs showed BRV that appears to be similar to the HRV known from humans. Antiarrhythmic drugs affected BRV parameters similar to clinical observations. Therefore, our study demonstrates that this in vitro model can contribute to a better understanding of electrophysiological properties of mESC-derived cardiomyocytes and might serve as a valuable tool for drug safety screening. © 2016 The Author(s) Published by S. Karger AG, Basel.

  2. Beat Rate Variability in Murine Embryonic Stem Cell-Derived Cardiomyocytes: Effect of Antiarrhythmic Drugs

    Directory of Open Access Journals (Sweden)

    Julius Niehoff

    2016-02-01

    Full Text Available Background/Aims: Heart rate variability (HRV refers to the fluctuation of the time interval between consecutive heartbeats in humans. It has recently been discovered that cardiomyocytes derived from human embryonic and induced pluripotent stem cells show beat rate variability (BRV that is similar to the HRV in humans. In the present study, clinical aspects of HRV were transferred to an in vitro model. The aims of the study were to explore the BRV in murine embryonic stem cell (mESC-derived cardiomyocytes and to demonstrate the influence of antiarrhythmic drugs on BRV as has been shown in clinical trials previously. Methods: The Microelectrode Array (MEA technique was used to perform short-term recordings of extracellular field potentials (FPs of spontaneously beating cardiomyocytes derived from mESCs (D3 cell line, αPig-44. Offline analysis was focused on time domain and nonlinear methods. Results: The Poincaré-Plot analysis of measurements without pharmacological intervention revealed that three different shapes of scatter plots occurred most frequently. Comparable shapes have been described in clinical studies before. The antiarrhythmic drugs Ivabradine, Verapamil and Sotalol augmented BRV, whereas Flecainide decreased BRV parameters at low concentrations (SDSD 79.0 ± 8.7% of control at 10-9 M, p -5 M, p Conclusions: Spontaneously beating cardiomyocytes derived from mESCs showed BRV that appears to be similar to the HRV known from humans. Antiarrhythmic drugs affected BRV parameters similar to clinical observations. Therefore, our study demonstrates that this in vitro model can contribute to a better understanding of electrophysiological properties of mESC-derived cardiomyocytes and might serve as a valuable tool for drug safety screening.

  3. In vitro cardiotoxicity screening of silver and metal oxide nanoparticles using human induced pluripotent stem cell-derived cardiomyocytes

    Science.gov (United States)

    Exposure risk to silver and metal oxide nanoparticles (NPs) continues to increase due to their widespread use in products and applications. In vivo studies have shown Ag, TiO2 and CeO2 NPs translocate to the heart following various routes of exposure. Thus, it is critical to asse...

  4. Inducible NO synthase is constitutively expressed in porcine myocardium and its level decreases along with tachycardia-induced heart failure.

    Science.gov (United States)

    Paslawska, Urszula; Kiczak, Liliana; Bania, Jacek; Paslawski, Robert; Janiszewski, Adrian; Dzięgiel, Piotr; Zacharski, Maciej; Tomaszek, Alicja; Michlik, Katarzyna

    2016-01-01

    The adverse effects of oxidative stress and the presence of proinflammatory factors in the heart have been widely demonstrated mainly on rodent models. However, larger clinical trials focusing on inflammation or oxidative stress in heart failure (HF) have not been carried out. This may be due to differences in the anatomy and physiology of the cardiovascular system between small rodents and large mammals. Thus, we investigated myocardial inflammatory factors, such as inducible NO synthase (iNOS) and oxidative stress indices in female pigs with chronic tachycardia-induced cardiomyopathy. Homogenous female siblings of Large White breed swine (n=15) underwent continuous right ventricular (RV) pacing at 170bpm, whereas five sham-operated subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were euthanized at subsequent stages of the disease: mild, moderate and severe HF. Left ventricle (LV) sections were examined with electron microscopy. The relative expression of iNOS in LV was determined by quantitative PCR. The protein level of iNOS was determined by Western blotting and immunohistochemistry. The level of the S-nitrosylated (S-NO) protein in LV was determined after S-NO moieties were substituted by biotin, followed by a colorimetrical detection with streptavidin. Malondialdehyde (MDA), a marker of lipid peroxidation, was evaluated in the LV and serum using thiobarbituric acid. The aconitase activity (based on measurement of the concomitant formation of NADPH from NADP(+)), a marker of oxidative stress, was analyzed in mitochondrial and cytosolic LV fractions. The concentration of interleukin-1β (IL-1β) was measured in LV homogenates using enzyme-linked immunosorbent assay. RV pacing resulted in an impairment of LV systolic function, LV dilatation and neurohormonal activation. The electron microscopy revealed abnormalities within the cardiomyocytes of failing hearts, i.e. swollen mitochondria and myofibril

  5. High-Throughput Phenotyping of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons Using Electric Field Stimulation and High-Speed Fluorescence Imaging.

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    Daily, Neil J; Du, Zhong-Wei; Wakatsuki, Tetsuro

    Electrophysiology of excitable cells, including muscle cells and neurons, has been measured by making direct contact with a single cell using a micropipette electrode. To increase the assay throughput, optical devices such as microscopes and microplate readers have been used to analyze electrophysiology of multiple cells. We have established a high-throughput (HTP) analysis of action potentials (APs) in highly enriched motor neurons and cardiomyocytes (CMs) that are differentiated from human induced pluripotent stem cells (iPSCs). A multichannel electric field stimulation (EFS) device enabled the ability to electrically stimulate cells and measure dynamic changes in APs of excitable cells ultra-rapidly (>100 data points per second) by imaging entire 96-well plates. We found that the activities of both neurons and CMs and their response to EFS and chemicals are readily discerned by our fluorescence imaging-based HTP phenotyping assay. The latest generation of calcium (Ca(2+)) indicator dyes, FLIPR Calcium 6 and Cal-520, with the HTP device enables physiological analysis of human iPSC-derived samples highlighting its potential application for understanding disease mechanisms and discovering new therapeutic treatments.

  6. Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP-2/MLCK-mediated cardiomyocyte protection from hypoxic injury.

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    Bil-Lula, Iwona; Lin, Han-Bin; Biały, Dariusz; Wawrzyńska, Magdalena; Diebel, Lucas; Sawicka, Jolanta; Woźniak, Mieczyslaw; Sawicki, Grzegorz

    2016-06-01

    Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase-2 (MMP-2). It has been shown that simultaneous inhibition of MMP-2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML-7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co-administration of nitric oxide synthase (NOS) inhibitor (1400W or L-NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia-reoxygenation (H-R)-induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP-2 by Doxy (25-100 μM), MLCK by ML-7 (0.5-5 μM) and NOS by L-NAME (25-100 μM) or 1400W (25-100 μM) protected myocyte contractility after H-R in a concentration-dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H-R at the level of highest single-drug concentration. The combination of subthreshold concentrations of NOS, MMP-2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP-2. The observed protection with addition of L-NAME or 1400W was better than previously reported combination of ML-7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  7. Effects of Polyphenols on Oxidative Stress-Mediated Injury in Cardiomyocytes

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    Mattera, Rosanna; Benvenuto, Monica; Giganti, Maria Gabriella; Tresoldi, Ilaria; Pluchinotta, Francesca Romana; Bergante, Sonia; Tettamanti, Guido; Masuelli, Laura; Manzari, Vittorio; Modesti, Andrea; Bei, Roberto

    2017-01-01

    Cardiovascular diseases are the main cause of mortality and morbidity in the world. Hypertension, ischemia/reperfusion, diabetes and anti-cancer drugs contribute to heart failure through oxidative and nitrosative stresses which cause cardiomyocytes nuclear and mitochondrial DNA damage, denaturation of intracellular proteins, lipid peroxidation and inflammation. Oxidative or nitrosative stress-mediated injury lead to cardiomyocytes apoptosis or necrosis. The reactive oxygen (ROS) and nitrogen species (RNS) concentration is dependent on their production and on the expression and activity of anti-oxidant enzymes. Polyphenols are a large group of natural compounds ubiquitously expressed in plants, and epidemiological studies have shown associations between a diet rich in polyphenols and the prevention of various ROS-mediated human diseases. Polyphenols reduce cardiomyocytes damage, necrosis, apoptosis, infarct size and improve cardiac function by decreasing oxidative stress-induced production of ROS or RNS. These effects are achieved by the ability of polyphenols to modulate the expression and activity of anti-oxidant enzymes and several signaling pathways involved in cells survival. This report reviews current knowledge on the potential anti-oxidative effects of polyphenols to control the cardiotoxicity induced by ROS and RNS stress. PMID:28531112

  8. Seeding bioreactor-produced embryonic stem cell-derived cardiomyocytes on different porous, degradable, polyurethane scaffolds reveals the effect of scaffold architecture on cell morphology.

    Science.gov (United States)

    Fromstein, Joanna D; Zandstra, Peter W; Alperin, Cecilia; Rockwood, Danielle; Rabolt, John F; Woodhouse, Kimberly A

    2008-03-01

    A successful regenerative therapy to treat damage incurred after an ischemic event in the heart will require an integrated approach including methods for appropriate revascularization of the infarct site, mechanical recovery of damaged tissue, and electrophysiological coupling with native cells. Cardiomyocytes are the ideal cell type for heart regeneration because of their inherent electrical and physiological properties, and cardiomyocytes derived from embryonic stem cells (ESCs) represent an attractive option for tissue-engineering therapies. An important step in developing tissue engineering-based approaches to cardiac cell therapy is understanding how scaffold architecture affects cell behavior. In this work, we generated large numbers of ESC-derived cardiomyocytes in bioreactors and seeded them on porous, 3-dimensional scaffolds prepared using 2 different techniques: electrospinning and thermally induced phase separation (TIPS). The effect of material macro-architecture on the adhesion, viability, and morphology of the seeded cells was determined. On the electrospun scaffolds, cells were elongated in shape, a morphology typical of cultured ESC-derived cardiomyocytes, whereas on scaffolds fabricated using TIPS, the cells retained a rounded morphology. Despite these gross phenotypic and physiological differences, sarcomeric myosin and connexin 43 expression was evident, and contracting cells were observed on both scaffold types, suggesting that morphological changes induced by material macrostructure do not directly correlate to functional differences.

  9. Inducible Nitric Oxide Synthase in Heart Tissue and Nitric Oxide in Serum of Trypanosoma cruzi-Infected Rhesus Monkeys: Association with Heart Injury

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    Carvalho, Cristiano Marcelo Espinola; Silverio, Jaline Coutinho; da Silva, Andrea Alice; Pereira, Isabela Resende; Coelho, Janice Mery Chicarino; Britto, Constança Carvalho; Moreira, Otacílio Cruz; Marchevsky, Renato Sergio; Xavier, Sergio Salles; Gazzinelli, Ricardo Tostes; da Glória Bonecini-Almeida, Maria; Lannes-Vieira, Joseli

    2012-01-01

    Background The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2 −/−) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. Methodology Rhesus monkeys and C57BL/6 and Nos2 −/− mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2+ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue. Results Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2+ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2 −/− mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue. Conclusion T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC

  10. Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury.

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    Cristiano Marcelo Espinola Carvalho

    Full Text Available BACKGROUND: The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2 is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2(-/- mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. METHODOLOGY: Rhesus monkeys and C57BL/6 and Nos2(-/- mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2(+ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG, echocardiogram (ECHO, creatine kinase heart isoenzyme (CK-MB activity levels in serum and connexin 43 (Cx43 expression in the cardiac tissue. RESULTS: Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC. Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2(+ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2(-/- mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue. CONCLUSION: T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute

  11. Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes

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    Azra Fatima

    2016-09-01

    Full Text Available We report here a transgenic murine induced pluripotent stem cell (iPSC line expressing puromycin N-acetyltransferase (PAC and enhanced green fluorescent protein (EGFP under the control of α-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols.

  12. Electrical Properties of Isolated Cardiomyocytes in a Rat Model of Thiamine Deficiency

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    Artur Santos-Miranda

    2015-03-01

    Full Text Available In modern society, thiamine deficiency (TD remains an important medical condition linked to altered cardiac function. There have been contradictory reports about the impact of TD on heart physiology, especially in the context of cardiac excitability. In order to address this particular question, we used a TD rat model and patch-clamp technique to investigate the electrical properties of isolated cardiomyocytes from epicardium and endocardium. Neither cell type showed substantial differences on the action potential waveform and transient outward potassium current. Based on our results we can conclude that TD does not induce major electrical remodeling in isolated cardiac myocytes in either endocardium or epicardium cells.

  13. Structural and functional screening in human induced-pluripotent stem cell-derived cardiomyocytes accurately identifies cardiotoxicity of multiple drug types.

    Science.gov (United States)

    Doherty, Kimberly R; Talbert, Dominique R; Trusk, Patricia B; Moran, Diarmuid M; Shell, Scott A; Bacus, Sarah

    2015-05-15

    Safety pharmacology studies that evaluate new drug entities for potential cardiac liability remain a critical component of drug development. Current studies have shown that in vitro tests utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) may be beneficial for preclinical risk evaluation. We recently demonstrated that an in vitro multi-parameter test panel assessing overall cardiac health and function could accurately reflect the associated clinical cardiotoxicity of 4 FDA-approved targeted oncology agents using hiPS-CM. The present studies expand upon this initial observation to assess whether this in vitro screen could detect cardiotoxicity across multiple drug classes with known clinical cardiac risks. Thus, 24 drugs were examined for their effect on both structural (viability, reactive oxygen species generation, lipid formation, troponin secretion) and functional (beating activity) endpoints in hiPS-CM. Using this screen, the cardiac-safe drugs showed no effects on any of the tests in our panel. However, 16 of 18 compounds with known clinical cardiac risk showed drug-induced changes in hiPS-CM by at least one method. Moreover, when taking into account the Cmax values, these 16 compounds could be further classified depending on whether the effects were structural, functional, or both. Overall, the most sensitive test assessed cardiac beating using the xCELLigence platform (88.9%) while the structural endpoints provided additional insight into the mechanism of cardiotoxicity for several drugs. These studies show that a multi-parameter approach examining both cardiac cell health and function in hiPS-CM provides a comprehensive and robust assessment that can aid in the determination of potential cardiac liability. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Structural and functional screening in human induced-pluripotent stem cell-derived cardiomyocytes accurately identifies cardiotoxicity of multiple drug types

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    Doherty, Kimberly R., E-mail: kimberly.doherty@quintiles.com; Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M.; Shell, Scott A.; Bacus, Sarah

    2015-05-15

    Safety pharmacology studies that evaluate new drug entities for potential cardiac liability remain a critical component of drug development. Current studies have shown that in vitro tests utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) may be beneficial for preclinical risk evaluation. We recently demonstrated that an in vitro multi-parameter test panel assessing overall cardiac health and function could accurately reflect the associated clinical cardiotoxicity of 4 FDA-approved targeted oncology agents using hiPS-CM. The present studies expand upon this initial observation to assess whether this in vitro screen could detect cardiotoxicity across multiple drug classes with known clinical cardiac risks. Thus, 24 drugs were examined for their effect on both structural (viability, reactive oxygen species generation, lipid formation, troponin secretion) and functional (beating activity) endpoints in hiPS-CM. Using this screen, the cardiac-safe drugs showed no effects on any of the tests in our panel. However, 16 of 18 compounds with known clinical cardiac risk showed drug-induced changes in hiPS-CM by at least one method. Moreover, when taking into account the Cmax values, these 16 compounds could be further classified depending on whether the effects were structural, functional, or both. Overall, the most sensitive test assessed cardiac beating using the xCELLigence platform (88.9%) while the structural endpoints provided additional insight into the mechanism of cardiotoxicity for several drugs. These studies show that a multi-parameter approach examining both cardiac cell health and function in hiPS-CM provides a comprehensive and robust assessment that can aid in the determination of potential cardiac liability. - Highlights: • 24 drugs were tested for cardiac liability using an in vitro multi-parameter screen. • Changes in beating activity were the most sensitive in predicting cardiac risk. • Structural effects add in

  15. Novel distribution of calreticulin to cardiomyocyte mitochondria and its increase in a rat model of dilated cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ming [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Department of Respiratory Medicine, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Wei, Jin, E-mail: weijindr@163.com [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Li, Yali [Department of Respiratory Medicine, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Shan, Hu; Yan, Rui; Lin, Lin [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Zhang, Qiuhong [Department of Respiratory Medicine, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China); Xue, Jiahong [Department of Cardiology, Second Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi (China)

    2014-06-20

    Highlights: • Calreticulin can also be found in cardiomyocyte mitochondria. • The mitochondrial content of calreticulin is increased in DCM hearts. • Increased expression of mitochondrial CRT may induce mitochondrial damage. • Mitochondrial CRT may inhibit the phosphorylation of mitochondrial STAT3. - Abstract: Background: Calreticulin (CRT), a Ca{sup 2+}-binding chaperone of the endoplasmic reticulum, can also be found in several other locations including the cytosol, nucleus, secretory granules, the outer side of the plasma membrane, and the extracellular matrix. Whether CRT is localized at mitochondria of cardiomyocytes and whether such localization is affected under DCM are still unclear. Methods and results: The DCM model was generated in rats by the daily oral administration of furazolidone for thirty weeks. Echocardiographic and hemodynamic studies demonstrated enlarged left ventricular dimensions and reduced systolic and diastolic function in DCM rats. Immuno-electron microscopy and Western blot showed that CRT was present in cardiomyocyte mitochondria and the mitochondrial content of CRT was increased in DCM hearts (P < 0.05). Morphometric analysis showed notable myocardial apoptosis and mitochondrial swelling with fractured or dissolved cristae in the DCM hearts. Compared with the control group, the mitochondrial membrane potential level of the freshly isolated cardiac mitochondria and the enzyme activities of cytochrome c oxidase and succinate dehydrogenase in the model group were significantly decreased (P < 0.05), and the myocardial apoptosis index and the caspase activities of caspase-9 and caspase-3 were significantly increased (P < 0.05). Pearson linear correlation analysis showed that the mitochondrial content of CRT had negative correlations with the mitochondrial function, and a positive correlation with myocardial apoptosis index (P < 0.001). The protein expression level of cytochrome c and the phosphorylation activity of STAT3 in the

  16. Intra-myocardial injection of both growth factors and heart derived Sca-1+/CD31- cells attenuates post-MI LV remodeling more than does cell transplantation alone: neither intervention enhances functionally significant cardiomyocyte regeneration.

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    Xiaohong Wang

    Full Text Available Insulin-like growth factor 1 (IGF-1 and hepatocyte growth factor (HGF are two potent cell survival and regenerative factors in response to myocardial injury (MI. We hypothesized that simultaneous delivery of IGF+HGF combined with Sca-1+/CD31- cells would improve the outcome of transplantation therapy in response to the altered hostile microenvironment post MI. One million adenovirus nuclear LacZ-labeled Sca-1+/CD31- cells were injected into the peri-infarction area after left anterior descending coronary artery (LAD ligation in mice. Recombinant mouse IGF-1+HGF was added to the cell suspension prior to the injection. The left ventricular (LV function was assessed by echocardiography 4 weeks after the transplantation. The cell engraftment, differentiation and cardiomyocyte regeneration were evaluated by histological analysis. Sca-1+/CD31- cells formed viable grafts and improved LV ejection fraction (EF (Control, 54.5+/-2.4; MI, 17.6+/-3.1; Cell, 28.2+/-4.2, n = 9, P<0.01. IGF+HGF significantly enhanced the benefits of cell transplantation as evidenced by increased EF (38.8+/-2.2; n = 9, P<0.01 and attenuated adverse structural remodeling. Furthermore, IGF+HGF supplementation increased the cell engraftment rate, promoted the transplanted cell survival, enhanced angiogenesis, and minimally stimulated endogenous cardiomyocyte regeneration in vivo. The in vitro experiments showed that IGF+HGF treatment stimulated Sca-1+/CD31- cell proliferation and inhibited serum free medium induced apoptosis. Supperarray profiling of Sca-1+/CD31- cells revealed that Sca-1+/CD31- cells highly expressed various trophic factor mRNAs and IGF+HGF treatment altered the mRNAs expression patterns of these cells. These data indicate that IGF-1+HGF could serve as an adjuvant to cell transplantation for myocardial repair by stimulating donor cell and endogenous cardiac stem cell survival, regeneration and promoting angiogenesis.

  17. Epigenetic revival of a dead cardiomyocyte through mitochondrial interventions.

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    Kunkel, George H; Chaturvedi, Pankaj; Tyagi, Suresh C

    2015-08-01

    Mitochondrial dysfunction has been reported to underline heart failure, and our earlier report suggests that mitochondrial fusion and fission contributes significantly to volume overload heart failure. Although ample studies highlight mitochondrial dysfunction to be a major cause, studies are lacking to uncover the role of mitochondrial epigenetics, i.e. epigenetic modifications of mtDNA in cardiomyocyte function. Additionally, mitochondrial proteases like calpain and Lon proteases are underexplored. Cardiomyopathies are correlated to mitochondrial damage via increased reactive oxygen species production and free calcium within cardiomyocytes. These abnormalities drive increased proteolytic activity from matrix metalloproteinases and calpains, respectively. These proteases degrade the cytoskeleton of the cardiomyocyte and lead to myocyte death. mtDNA methylation is another factor that can lead to myocyte death by silencing several genes of mitochondria or upregulating the expression of mitochondrial proteases by hypomethylation. Cardiomyocyte resuscitation can occur through mitochondrial interventions by decreasing the proteolytic activity and reverting back the epigenetic changes in the mtDNA which lead to myocyte dysfunction. Epigenetic changes in the mtDNA are triggered by environmental factors like pollution and eating habits with cigarette smoking. An analysis of mitochondrial epigenetics in cigarette-smoking mothers will reveal an underlying novel mechanism leading to mitochondrial dysfunction and eventually heart failure. This review is focused on the mitochondrial dysfunction mechanisms that can be reverted back to resuscitate cardiomyocytes.

  18. Macrophages Facilitate Electrical Conduction in the Heart.

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    Hulsmans, Maarten; Clauss, Sebastian; Xiao, Ling; Aguirre, Aaron D; King, Kevin R; Hanley, Alan; Hucker, William J; Wülfers, Eike M; Seemann, Gunnar; Courties, Gabriel; Iwamoto, Yoshiko; Sun, Yuan; Savol, Andrej J; Sager, Hendrik B; Lavine, Kory J; Fishbein, Gregory A; Capen, Diane E; Da Silva, Nicolas; Miquerol, Lucile; Wakimoto, Hiroko; Seidman, Christine E; Seidman, Jonathan G; Sadreyev, Ruslan I; Naxerova, Kamila; Mitchell, Richard N; Brown, Dennis; Libby, Peter; Weissleder, Ralph; Swirski, Filip K; Kohl, Peter; Vinegoni, Claudio; Milan, David J; Ellinor, Patrick T; Nahrendorf, Matthias

    2017-04-20

    Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11b(DTR) mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Myricitrin Attenuates High Glucose-Induced Apoptosis through Activating Akt-Nrf2 Signaling in H9c2 Cardiomyocytes

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    Bin Zhang

    2016-07-01

    Full Text Available Hyperglycemia, as well as diabetes mellitus, has been shown to trigger cardiac cell apoptosis. We have previously demonstrated that myricitrin prevents endothelial cell apoptosis. However, whether myricitrin can attenuate H9c2 cell apoptosis remains unknown. In this study, we established an experiment model in H9c2 cells exposed to high glucose. We tested the hypothesis that myricitrin may inhibit high glucose (HG-induced cardiac cell apoptosis as determined by TUNEL staining. Furthermore, myricitrin promoted antioxidative enzyme production, suppressed high glucose-induced reactive oxygen species (ROS production and decreased mitochondrial membrane potential (MMP in H9c2 cells. This agent significantly inhibited apoptotic protein expression, activated Akt and facilitated the transcription of NF-E2-related factor 2 (Nrf2-mediated protein (heme oxygenase-1 (HO-1 and quinone oxidoreductase 1 (NQO-1 expression as determined by Western blotting. Significantly, an Akt inhibitor (LY294002 or HO-1 inhibitor (ZnPP not only inhibited myricitrin-induced HO-1/NQO-1 upregulation but also alleviated its anti-apoptotic effects. In summary, these observations demonstrate that myricitrin activates Nrf2-mediated anti-oxidant signaling and attenuates H9c2 cell apoptosis induced by high glucose via activation of Akt signaling.

  20. The heart's content-renewable resources.

    Science.gov (United States)

    Faucherre, Adèle; Jopling, Chris

    2013-08-20

    Heart regeneration is a huge, complex area involving numerous lines of research ranging from the stem cell therapy to xenografts and bioengineering. This review will focus on two avenues of regenerative research, cardiac progenitor cells and adult cardiomyocyte proliferation, both of which offer great promise for the field of heart regeneration. However, the principles behind how this could be achieved by either technique are very different. Cardiac progenitor cells represent a population of somatic stem cells which reside within the adult heart. These cells appear to have the capacity to proliferate and differentiate into the different cell types found within the adult heart and thus have the potential, if the correct stimuli can be found, to effectively regenerate a heart damaged by ischemia/infarction. Inducing adult cardiomyocytes to proliferate offers a different approach to achieving the same goal. In this case, the cardiomyocytes that remain after the damage has occurred would need to be stimulated into effecting a regenerative response. In this review, we will discuss the current understanding of how heart regeneration could be achieved by either of these very different approaches. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  1. CSAHi study: Detection of drug-induced ion channel/receptor responses, QT prolongation, and arrhythmia using multi-electrode arrays in combination with human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kitaguchi, Takashi; Moriyama, Yuta; Taniguchi, Tomohiko; Maeda, Sanae; Ando, Hiroyuki; Uda, Takaaki; Otabe, Koji; Oguchi, Masao; Shimizu, Shigekazu; Saito, Hiroyuki; Toratani, Atsushi; Asayama, Mahoko; Yamamoto, Wataru; Matsumoto, Emi; Saji, Daisuke; Ohnaka, Hiroki; Miyamoto, Norimasa

    The use of multi-electrode arrays (MEA) in combination with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides a promising method to predict comprehensive cardiotoxicity, including drug-induced QT prolongation and arrhythmia. We previously demonstrated that MEA in combination with hiPSC-CMs could provide a generalizable platform by using 7 reference drugs at 10 testing facilities. Using this approach, we evaluated responses to reference drugs that modulate a range of cardiac ion currents and have a range of arrhythmogenic effects. We used the MEA system (MED64) and commercially available hiPSC-CMs (iCell cardiomyocytes) to evaluate drug effects on the beat rate, field potential duration (FPD), FPD corrected by Fridericia's formula (FPDc), and the incidence of arrhythmia-like waveforms. This assay detected the repolarization effects of Bay K8644, mibefradil, NS1643, levcromakalim, and ouabain; and the chronotropic effects of isoproterenol, ZD7288, and BaCl2. Chronotropy was also affected by K(+) and Ca(2+) current modulation. This system detected repolarization delays and the arrhythmogenic effects of quinidine, cisapride, thioridazine, astemizole, bepridil, and pimozide more sensitively than the established guinea pig papillary muscle action potential assay. It also predicted clinical QT prolongation by drugs with multiple ion channel effects (fluoxetine, amiodarone, tolterodine, vanoxerine, alfuzosin, and ranolazine). MEA in combination with hiPSC-CMs may provide a powerful method to detect various cardiac electrophysiological effects, QT prolongation, and arrhythmia during drug discovery. However, the data require careful interpretation to predict chronotropic effects and arrhythmogenic effects of candidate drugs with multiple ion channel effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Erythropoietin enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia through Akt/eNOS signalling pathway.

    Science.gov (United States)

    Qin, Chuan; Zhou, Shengkai; Xiao, Yingbin; Chen, Lin

    2014-03-01

    Adaptation of cardiomyocytes to chronic hypoxia in cyanotic patients remains unclear. Mitochondrial biogenesis is enhanced in myocardium from cyanotic patients, which is possibly an adaptive response. Erythropoietin (EPO) in blood and its receptor (EPOR) on cardiomyocytes are upregulated by chronic hypoxia, suggesting that EPO-EPOR interaction is increased, which is inferred to positively regulate mitochondrial biogenesis through protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signalling pathway. H9c2 cardiomyocytes were exposed to hypoxia (1% O(2)) for 1 week and treated with different doses of recombinant human erythropoietin (rhEPO). Mitochondrial number, mitochondrial DNA (mtDNA) copy number and peroxisome proliferator activated receptor gamma coactivator alpha (PGC-1α) mRNA expression increased in a dose-dependent manner induced by rhEPO. Akt and eNOS were significantly phosphorylated by rhEPO. Both blocking Akt with Wortmannin and silencing eNOS expression with shRNA plasmid decreased the mtDNA copy number and PGC-1α mRNA expression induced by rhEPO. Blocking Akt was associated with the decreased phosphorylation of Akt and eNOS. RNA interference led to a reduction in the total and phosphorylated proteins of eNOS. Thus EPO enhances mitochondrial biogenesis in cardiomyocytes exposed to chronic hypoxia, at least partly through Akt/eNOS signalling, which might be an adaptive mechanism of cardiomyocytes associated with the increased EPO-EPOR interaction in patients with cyanotic congenital heart disease (CCHD). © 2013 International Federation for Cell Biology.

  3. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Christophe M Raynaud

    Full Text Available Mesenchymal progenitors or stromal cells have shown promise as a therapeutic strategy for a range of diseases including heart failure. In this context, we explored the growth and differentiation potential of mesenchymal progenitors (MPs derived in vitro from human embryonic stem cells (hESCs. Similar to MPs isolated from bone marrow, hESC derived MPs (hESC-MPs efficiently differentiated into archetypical mesenchymal derivatives such as chondrocytes and adipocytes. Upon treatment with 5-Azacytidine or TGF-β1, hESC-MPs modified their morphology and up-regulated expression of key cardiac transcription factors such as NKX2-5, MEF2C, HAND2 and MYOCD. Nevertheless, NKX2-5+ hESC-MP derivatives did not form contractile cardiomyocytes, raising questions concerning the suitability of these cells as a platform for cardiomyocyte replacement therapy. Gene profiling experiments revealed that, although hESC-MP derived cells expressed a suite of cardiac related genes, they lacked the complete repertoire of genes associated with bona fide cardiomyocytes. Our results suggest that whilst agents such as TGF-β1 and 5-Azacytidine can induce expression of cardiac related genes, but treated cells retain a mesenchymal like phenotype.

  4. The role of hypoxia-inducible factor-1α and vascular endothelial growth factor in late-phase preconditioning with xenon, isoflurane and levosimendan in rat cardiomyocytes.

    Science.gov (United States)

    Goetzenich, Andreas; Hatam, Nima; Preuss, Stephanie; Moza, Ajay; Bleilevens, Christian; Roehl, Anna B; Autschbach, Rüdiger; Bernhagen, Jürgen; Stoppe, Christian

    2014-03-01

    The protective effects of late-phase preconditioning can be triggered by several stimuli. Unfortunately, the transfer from bench to bedside still represents a challenge, as concomitant medication or diseases influence the complex signalling pathways involved. In an established model of primary neonatal rat cardiomyocytes, we analysed the cardioprotective effects of three different stimulating pharmaceuticals of clinical relevance. The effect of additional β-blocker treatment was studied as these were previously shown to negatively influence preconditioning. Twenty-four hours prior to hypoxia, cells pre-treated with or without metoprolol (0.55 µg/ml) were preconditioned with isoflurane, levosimendan or xenon. The influences of these stimuli on hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) as well as inducible and endothelial nitric synthase (iNOS/eNOS) and cyclooxygenase-2 (COX-2) were analysed by polymerase chain reaction and western blotting. The preconditioning was proved by trypan blue cell counts following 5 h of hypoxia and confirmed by fluorescence staining. Five hours of hypoxia reduced cell survival in unpreconditioned control cells to 44 ± 4%. Surviving cell count was significantly higher in cells preconditioned either by 2 × 15 min isoflurane (70 ± 16%; P = 0.005) or by xenon (59 ± 8%; P = 0.049). Xenon-preconditioned cells showed a significantly elevated content of VEGF (0.025 ± 0.010 IDV [integrated density values when compared with GAPDH] vs 0.003 ± 0.006 IDV in controls; P = 0.0003). The protein expression of HIF-1α was increased both by levosimendan (0.563 ± 0.175 IDV vs 0.142 ± 0.042 IDV in controls; P = 0.0289) and by xenon (0.868 ± 0.222 IDV; P xenon but not by the other chosen stimuli. eNOS mRNA expression was found to be suppressed by β-blocker treatment for all stimuli. In our model, independently of the chosen stimulus, β-blocker treatment had no significant effect on cell survival. We found

  5. CaMKIIδB mediates aberrant NCX1 expression and the imbalance of NCX1/SERCA in transverse aortic constriction-induced failing heart.

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    Ying-Mei Lu

    Full Text Available Ca²⁺/calmodulin-dependent protein kinase II δB (CaMKIIδB is one of the predominant isoforms of CaMKII in the heart. The precise role of CaMKIIδB in the transcriptional cross-talk of Ca²⁺-handling proteins during heart failure remains unclear. In this work, we aim to determine the mechanism of CaMKIIδB in modulating the expression of sarcolemmal Na⁺-Ca²⁺ exchange (NCX1. We also aim to address the potential effects of calmodulin antagonism on the imbalance of NCX1 and sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA during heart failure. Eight weeks after transverse aortic constriction (TAC-induced heart failure in mice, we found that the heart weight/tibia length (HW/TL ratio and the lung weight/body weight (LW/BW ratio increased by 59% and 133%, respectively. We further found that the left ventricle-shortening fraction decreased by 40% compared with the sham-operated controls. Immunoblotting revealed that the phosphorylation of CaMKIIδB significantly increased 8 weeks after TAC-induced heart failure. NCX1 protein levels were also elevated, whereas SERCA2 protein levels decreased in the same animal model. Moreover, transfection of active CaMKIIδB significantly increased NCX1 protein levels in adult mouse cardiomyocytes via class IIa histone deacetylase (HDAC/myocyte enhancer factor-2 (MEF2-dependent signaling. In addition, pharmacological inhibition of calmodulin/CaMKIIδB activity improved cardiac function in TAC mice, which partially normalized the imbalance between NCX1 and SERCA2. These data identify NCX1 as a cellular target for CaMKIIδB. We also suggest that the CaMKIIδB-induced imbalance between NCX1 and SERCA2 is partially responsible for the disturbance of intracellular Ca²⁺ homeostasis and the pathological process of heart failure.

  6. The ATP-binding cassette transporter ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

    Science.gov (United States)

    Higashikuni, Yasutomi; Sainz, Julie; Nakamura, Kazuto; Takaoka, Minoru; Enomoto, Soichiro; Iwata, Hiroshi; Tanaka, Kimie; Sahara, Makoto; Hirata, Yasunobu; Nagai, Ryozo; Sata, Masataka

    2012-03-01

    ATP-binding cassette transporter subfamily G member 2 (ABCG2), expressed in microvascular endothelial cells in the heart, has been suggested to regulate several tissue defense mechanisms. This study was performed to elucidate its role in pressure overload-induced cardiac hypertrophy. Pressure overload was induced in 8- to 12-week-old wild-type and Abcg2-/- mice by transverse aortic constriction (TAC). Abcg2-/- mice showed exaggerated cardiac hypertrophy and ventricular remodeling after TAC compared with wild-type mice. In the early phase after TAC, functional impairment in angiogenesis and antioxidant response in myocardium was found in Abcg2-/- mice. In vitro experiments demonstrated that ABCG2 regulates transport of glutathione, an important endogenous antioxidant, from microvascular endothelial cells. Besides, glutathione transported from microvascular endothelial cells in ABCG2-dependent manner ameliorated oxidative stress-induced cardiomyocyte hypertrophy. In vivo, glutathione levels in plasma and the heart were increased in wild-type mice but not in Abcg2-/- mice after TAC. Treatment with the superoxide dismutase mimetic ameliorated cardiac hypertrophy in Abcg2-/- mice after TAC to the same extent as that in wild-type mice, although cardiac dysfunction with impaired angiogenesis was observed in Abcg2-/- mice. ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

  7. Sirtuin-3 (SIRT3) protein attenuates doxorubicin-induced oxidative stress and improves mitochondrial respiration in H9c2 cardiomyocytes

    Science.gov (United States)

    Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electro...

  8. Pregnancy-induced remodeling of heart valves.

    Science.gov (United States)

    Pierlot, Caitlin M; Moeller, Andrew D; Lee, J Michael; Wells, Sarah M

    2015-11-01

    Recent studies have demonstrated remodeling of aortic and mitral valves leaflets under the volume loading and cardiac expansion of pregnancy. Those valves' leaflets enlarge with altered collagen fiber architecture, content, and cross-linking and biphasic changes (decreases, then increases) in extensibility during gestation. This study extends our analyses to right-sided valves, with additional compositional measurements for all valves. Valve leaflets were harvested from nonpregnant heifers and pregnant cows. Leaflet structure was characterized by leaflet dimensions, and ECM composition was determined using standard biochemical assays. Histological studies assessed changes in cellular and ECM components. Leaflet mechanical properties were assessed using equibiaxial mechanical testing. Collagen thermal stability and cross-linking were assessed using denaturation and hydrothermal isometric tension tests. Pulmonary and tricuspid leaflet areas increased during pregnancy by 35 and 55%, respectively. Leaflet thickness increased by 20% only in the pulmonary valve and largely in the fibrosa (30% thickening). Collagen crimp length was reduced in both the tricuspid (61%) and pulmonary (42%) valves, with loss of crimped area in the pulmonary valve. Thermomechanics showed decreased collagen thermal stability with surprisingly maintained cross-link maturity. The pulmonary leaflet exhibited the biphasic change in extensibility seen in left side valves, whereas the tricuspid leaflet mechanics remained largely unchanged throughout pregnancy. The tricuspid valve exhibits a remodeling response during pregnancy that is significantly diminished from the other three valves. All valves of the heart remodel in pregnancy in a manner distinct from cardiac pathology, with much similarity valve to valve, but with interesting valve-specific responses in the aortic and tricuspid valves. Copyright © 2015 the American Physiological Society.

  9. Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics.

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    Yu-Fen Chang

    Full Text Available Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact. This combination can be applied to single rodent primary adult cardiomyocytes, and human stem cell derived cardiomyocytes, enabling contactless small molecule evaluation for inhibitors of sodium, potassium and calcium channels suggesting it may be useful for early toxicity work. In pancreatic beta-cells it reveals the effects of glucose and the KATP inhibitor gliclazide.

  10. SIRT1 Suppresses Doxorubicin-Induced Cardiotoxicity by Regulating the Oxidative Stress and p38MAPK Pathways

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    Yang Ruan

    2015-02-01

    Full Text Available Background: SIRT1, which belongs to the Sirtuin family of NAD-dependent enzymes, plays diverse roles in aging, metabolism, and disease biology. It could regulate cell survival and has been shown to be a protective factor in heart function. Hence, we verified the mechanism by which SIRT1 regulates doxorubicin induced cardiomyocyte injury in vivo and in vitro. Methods: We analyzed SIRT1 expression in doxorubicin-induced neonatal rat cardiomyocyte injury model and adult mouse heart failure model. SIRT1 was over-expressed in cultured neonatal rat cardiomyocyte by adenovirus mediated gene transfer. SIRT1 agonist resveratrol was used to treat the doxorubicin-induced heart failure mouse model. Echocardiography, reactive oxygen species (ROS production, TUNEL, qRT-PCR, and Western blotting were performed to analyze cell survival, oxidative stress, and inflammatory signal pathways in cardiomyocytes. Results: SIRT1 expression was down-regulated in doxorubicin induced cardiomocyte injury, accompanied by elevated oxidative stress and cell apoptosis. SIRT1 over-expression reduced doxorubicin induced cardiomyocyte apoptosis with the attenuated ROS production. SIRT1 also reduced cell apoptosis by inhibition of p38MAPK phosphorylation and caspase-3 activation. The SIRT1 agonist resveratrol was able to prevent doxorubicin-induced heart function loss. Moreover, the SIRT1 inhibitor niacinamide could reverse SIRT1's protective effect in cultured neonatal rat cardiomyocytes. Conclusions: These results support the role of SIRT1 as an important regulator of cardiomyocyte apoptosis during doxorubicin-induced heart injury, which may represent a potential therapeutic target for doxorubicin-induced cardiomyopathy.

  11. Metabolic changes in cardiomyocytes during sepsis

    OpenAIRE

    Douglas, James J.; Walley, Keith R.

    2013-01-01

    Different types of shock induce distinct metabolic changes. The myocardium at rest utilizes free fatty acids as its primary energy source, a mechanism that changes to aerobic glycolysis during sepsis and is in contrast to hemorrhagic shock. The immune system also uses this mechanism, changing its substrate utilization to activate innate and adaptive cells. Cardiomyocytes share a number of features similar to antigen-presenting cells and may use this mechanism to augment the immune response at...

  12. Metabolic changes in cardiomyocytes during sepsis.

    Science.gov (United States)

    Douglas, James J; Walley, Keith R

    2013-09-20

    Different types of shock induce distinct metabolic changes. The myocardium at rest utilizes free fatty acids as its primary energy source, a mechanism that changes to aerobic glycolysis during sepsis and is in contrast to hemorrhagic shock. The immune system also uses this mechanism, changing its substrate utilization to activate innate and adaptive cells. Cardiomyocytes share a number of features similar to antigen-presenting cells and may use this mechanism to augment the immune response at the reversible expense of cardiac function.

  13. Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers

    Science.gov (United States)

    Tang, Yadong; Liu, Li; Li, Junjun; Yu, Leqian; Wang, Li; Shi, Jian; Chen, Yong

    2016-07-01

    Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape of the hiPSC colony could be controlled from a flat layer to a hemisphere. Afterwards, the induction and differentiation of hiPSCs were achieved on the same patch, leading to a uniform cardiac layer with homogeneous contraction. This cardiac layer could then be used for extracellular recording with a commercial multi-electrode array, showing representative field potential waveforms of matured cardiac tissues with appropriate drug responses.Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell-medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape

  14. The Use of Ratiometric Fluorescence Measurements of the Voltage Sensitive Dye Di-4-ANEPPS to Examine Action Potential Characteristics and Drug Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Science.gov (United States)

    Hortigon-Vinagre, M P; Zamora, V; Burton, F L; Green, J; Gintant, G A; Smith, G L

    2016-12-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and higher throughput platforms have emerged as potential tools to advance cardiac drug safety screening. This study evaluated the use of high bandwidth photometry applied to voltage-sensitive fluorescent dyes (VSDs) to assess drug-induced changes in action potential characteristics of spontaneously active hiPSC-CM. Human iPSC-CM from 2 commercial sources (Cor.4U and iCell Cardiomyocytes) were stained with the VSD di-4-ANEPPS and placed in a specialized photometry system that simultaneously monitors 2 wavebands of emitted fluorescence, allowing ratiometric measurement of membrane voltage. Signals were acquired at 10 kHz and analyzed using custom software. Action potential duration (APD) values were normally distributed in cardiomyocytes (CMC) from both sources though the mean and variance differed significantly (APD 90 : 229 ± 15 ms vs 427 ± 49 ms [mean ± SD, P < 0.01]; average spontaneous cycle length: 0.99 ± 0.02 s vs 1.47 ± 0.35 s [mean ± SD, P < 0.01], Cor.4U vs iCell CMC, respectively). The 10-90% rise time of the AP (T rise ) was ∼6 ms and was normally distributed when expressed as 1/[Formula: see text] in both cell preparations. Both cell types showed a rate dependence analogous to that of adult human cardiac cells. Furthermore, nifedipine, ranolazine, and E4031 had similar effects on cardiomyocyte electrophysiology in both cell types. However, ranolazine and E4031 induced early after depolarization-like events and high intrinsic firing rates at lower concentrations in iCell CMC. These data show that VSDs provide a minimally invasive, quantitative, and accurate method to assess hiPSC-CM electrophysiology and detect subtle drug-induced effects for drug safety screening while highlighting a need to standardize experimental protocols across preparations. © The Author 2016. Published by Oxford University Press on behalf of the Society of

  15. Switch From Fetal to Adult SCN5A Isoform in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Unmasks the Cellular Phenotype of a Conduction Disease-Causing Mutation.

    Science.gov (United States)

    Veerman, Christiaan C; Mengarelli, Isabella; Lodder, Elisabeth M; Kosmidis, Georgios; Bellin, Milena; Zhang, Miao; Dittmann, Sven; Guan, Kaomei; Wilde, Arthur A M; Schulze-Bahr, Eric; Greber, Boris; Bezzina, Connie R; Verkerk, Arie O

    2017-07-24

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can recapitulate features of ion channel mutations causing inherited rhythm disease. However, the lack of maturity of these cells is considered a significant limitation of the model. Prolonged culture of hiPSC-CMs promotes maturation of these cells. We studied the electrophysiological effects of the I230T mutation in the sodium channel gene SCN5A in hiPSC-CMs generated from a homozygous (I230T(homo)) and a heterozygous (I230T(het)) individual from a family with recessive cardiac conduction disease. Since the I230T mutation occurs in the developmentally regulated "adult" isoform of SCN5A, we investigated the relationship between the expression fraction of the adult SCN5A isoform and the electrophysiological phenotype at different time points in culture. After a culture period of 20 days, sodium current (INa) was mildly reduced in I230T(homo) hiPSC-CMs compared with control hiPSC-CMs, while I230T(het) hiPSC-CMs displayed no reduction in INa. This coincided with a relatively high expression fraction of the "fetal" SCN5A isoform compared with the adult isoform as measured by quantitative polymerase chain reaction. Following prolonged culture to 66 days, the fraction of adult SCN5A isoform increased; this was paralleled by a marked decrease in INa in I230T(homo) hiPSC-CMs, in line with the severe clinical phenotype in homozygous patients. At this time in culture, I230T(het) hiPSC-CMs displayed an intermediate loss of INa, compatible with a gene dosage effect. Prolonged culture of hiPSC-CMs leads to an increased expression fraction of the adult sodium channel isoform. This new aspect of electrophysiological immaturity should be taken into account in studies that focus on the effects of SCN5A mutations in hiPSC-CMs. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  16. Congenital heart malformations induced by hemodynamic altering surgical interventions

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    Madeline eMidgett

    2014-08-01

    Full Text Available Embryonic heart formation results from a dynamic interplay between genetic and environmental factors. Blood flow during early embryonic stages plays a critical role in heart development, as interactions between flow and cardiac tissues generate biomechanical forces that modulate cardiac growth and remodeling. Normal hemodynamic conditions are essential for proper cardiac development, while altered blood flow induced by surgical manipulations in animal models result in heart defects similar to those seen in humans with congenital heart disease. This review compares the altered hemodynamics, changes in tissue properties, and cardiac defects reported after common surgical interventions that alter hemodynamics in the early chick embryo, and shows that interventions produce a wide spectrum of cardiac defects. Vitelline vein ligation and left atrial ligation decrease blood pressure and flow; and outflow tract banding increases blood pressure and flow velocities. These three surgical interventions result in many of the same cardiac defects, which indicate that the altered hemodynamics interfere with common looping, septation and valve formation processes that occur after intervention and that shape the four-chambered heart. While many similar defects develop after the interventions, the varying degrees of hemodynamic load alteration among the three interventions also result in varying incidence and severity of cardiac defects, indicating that the hemodynamic modulation of cardiac developmental processes is strongly dependent on hemodynamic load.

  17. Opposing Roles of FoxP1 and Nfat3 in Transcriptional Control of Cardiomyocyte Hypertrophy▿

    Science.gov (United States)

    Bai, Shoumei; Kerppola, Tom K.

    2011-01-01

    Cardiac homeostasis is maintained by a balance of growth-promoting and growth-modulating factors. Sustained elevation of calcium signaling can induce cardiac hypertrophy through activation of Nfat family transcription factors. FoxP family transcription factors are known to interact with Nfat proteins and to modulate their transcriptional activities in lymphocytes. We investigated FoxP1 interaction with Nfat3 (Nfatc4) and their effects on transcription of hypertrophy-associated genes in neonatal rat cardiomyocytes. FoxP1-Nfat3 complexes were visualized using bimolecular fluorescence complementation (BiFC) analysis. Calcineurin activation induced FoxP1-Nfat3 BiFC complex formation. Amino acid substitutions in the predicted interaction interface inhibited it. FoxP1 repressed hypertrophy-associated genes (Myh7, Rcan1, Cx43, Anf, and Bnp) and counteracted their activation by constitutively nuclear Nfat3 (cnNfat3). In contrast, FoxP1 activated genes that maintain normal heart functions (Myh6 and p57Kip2) and cnNfat3 counteracted their activation by FoxP1. Amino acid substitutions in FoxP1 or cnNfat3 that inhibited their interaction abrogated the activation of hypertrophy-associated gene transcription by cnNfat3 and the repression of these genes by FoxP1. FoxP1 and Nfat3 co-occupied the promoter regions of hypertrophy-associated genes in neonatal and adult heart tissue. FoxP1 counteracted hypertrophic cardiomyocyte growth, and connexin 43 mislocalization caused by cnNfat3 expression. These data suggest that the opposing transcriptional activities of FoxP1 and Nfat3 maintain cardiomyocyte homeostasis. PMID:21606195

  18. Locally expressed IGF1 propeptide improves mouse heart function in induced dilated cardiomyopathy by blocking myocardial fibrosis and SRF-dependent CTGF induction

    Directory of Open Access Journals (Sweden)

    Melissa Touvron

    2012-07-01

    Cardiac fibrosis is critically involved in the adverse remodeling accompanying dilated cardiomyopathies (DCMs, which leads to cardiac dysfunction and heart failure (HF. Connective tissue growth factor (CTGF, a profibrotic cytokine, plays a key role in this deleterious process. Some beneficial effects of IGF1 on cardiomyopathy have been described, but its potential role in improving DCM is less well characterized. We investigated the consequences of expressing a cardiac-specific transgene encoding locally acting IGF1 propeptide (muscle-produced IGF1; mIGF1 on disease progression in a mouse model of DCM [cardiac-specific and inducible serum response factor (SRF gene disruption] that mimics some forms of human DCM. Cardiac-specific mIGF1 expression substantially extended the lifespan of SRF mutant mice, markedly improved cardiac functions, and delayed both DCM and HF. These protective effects were accompanied by an overall improvement in cardiomyocyte architecture and a massive reduction of myocardial fibrosis with a concomitant amelioration of inflammation. At least some of the beneficial effects of mIGF1 transgene expression were due to mIGF1 counteracting the strong increase in CTGF expression within cardiomyocytes caused by SRF deficiency, resulting in the blockade of fibroblast proliferation and related myocardial fibrosis. These findings demonstrate that SRF plays a key role in the modulation of cardiac fibrosis through repression of cardiomyocyte CTGF expression in a paracrine fashion. They also explain how impaired SRF function observed in human HF promotes fibrosis and adverse cardiac remodeling. Locally acting mIGF1 efficiently protects the myocardium from these adverse processes, and might thus represent a therapeutic avenue to counter DCM.

  19. Phenotypic variability in LQT3 human induced pluripotent stem cell-derived cardiomyocytes and their response to antiarrhythmic pharmacologic therapy: An in silico approach.

    Science.gov (United States)

    Paci, Michelangelo; Passini, Elisa; Severi, Stefano; Hyttinen, Jari; Rodriguez, Blanca

    2017-07-27

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are in vitro models with the clear advantages of their human origin and suitability for human disease investigations. However, limitations include their incomplete characterization and variability reported in different cell lines and laboratories. The purpose of this study was to investigate in silico ionic mechanisms potentially explaining the phenotypic variability of hiPSC-CMs in long QT syndrome type 3 (LQT3) and their response to antiarrhythmic drugs. Populations of in silico hiPSC-CM models were constructed and calibrated for control (n = 1,463 models) and LQT3 caused by INaL channelopathy (n = 1,401 models), using experimental recordings for late sodium current (INaL) and action potentials (APs). Antiarrhythmic drug therapy was evaluated by simulating mexiletine and ranolazine multichannel effects. As in experiments, LQT3 hiPSC-CMs yield prolonged action potential duration at 90% repolarization (APD90) (+34.3% than controls) and large electrophysiological variability. LQT3 hiPSC-CMs with symptomatic APs showed overexpression of ICaL, IK1, and INaL, underexpression of IKr, and increased sensitivity to both drugs compared to asymptomatic LQT3 models. Simulations showed that both mexiletine and ranolazine corrected APD prolongation in the LQT3 population but also highlighted differences in drug response. Mexiletine stops spontaneous APs in more LQT3 hiPSC-CMs models than ranolazine (784/1,401 vs 53/1,401) due to its stronger action on INa. In silico simulations demonstrate our ability to recapitulate variability in LQT3 and control hiPSC-CM phenotypes, and the ability of mexiletine and ranolazine to reduce APD prolongation, in agreement with experiments. The in silico models also identify potential ionic mechanisms of phenotypic variability in LQT3 hiPSC-CMs, explaining APD prolongation in symptomatic vs asymptomatic LQT3 hiPSC-CMs. Copyright © 2017 The Authors. Published by Elsevier Inc

  20. Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes

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    Ziqing Liu

    2016-03-01

    Full Text Available Direct conversion of fibroblasts into induced cardiomyocytes (iCMs offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial−mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome

  1. Transformation of jaw muscle satellite cells to cardiomyocytes.

    Science.gov (United States)

    Daughters, Randall S; Keirstead, Susan A; Slack, Jonathan M W

    In the embryo a population of progenitor cells known as the second heart field forms not just parts of the heart but also the jaw muscles of the head. Here we show that it is possible to take skeletal muscle satellite cells from jaw muscles of the adult mouse and to direct their differentiation to become heart muscle cells (cardiomyocytes). This is done by exposing the cells to extracellular factors similar to those which heart progenitors would experience during normal embryonic development. By contrast, cardiac differentiation does not occur at all from satellite cells isolated from trunk and limb muscles, which originate from the somites of the embryo. The cardiomyocytes arising from jaw muscle satellite cells express a range of specific marker proteins, beat spontaneously, display long action potentials with appropriate responses to nifedipine, norepinephrine and carbachol, and show synchronized calcium transients. Our results show the existence of a persistent cardiac developmental competence in satellite cells of the adult jaw muscles, associated with their origin from the second heart field of the embryo, and suggest a possible method of obtaining cardiomyocytes from individual patients without the need for a heart biopsy. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  2. Predictive lethal proarrhythmic risk evaluation using a closed-loop-circuit cell network with human induced pluripotent stem cells derived cardiomyocytes

    Science.gov (United States)

    Nomura, Fumimasa; Hattori, Akihiro; Terazono, Hideyuki; Kim, Hyonchol; Odaka, Masao; Sugio, Yoshihiro; Yasuda, Kenji

    2016-06-01

    For the prediction of lethal arrhythmia occurrence caused by abnormality of cell-to-cell conduction, we have developed a next-generation in vitro cell-to-cell conduction assay, i.e., a quasi in vivo assay, in which the change in spatial cell-to-cell conduction is quantitatively evaluated from the change in waveforms of the convoluted electrophysiological signals from lined-up cardiomyocytes on a single closed loop of a microelectrode of 1 mm diameter and 20 µm width in a cultivation chip. To evaluate the importance of the closed-loop arrangement of cardiomyocytes for prediction, we compared the change in waveforms of convoluted signals of the responses in the closed-loop circuit arrangement with that of the response of cardiomyocyte clusters using a typical human ether a go-go related gene (hERG) ion channel blocker, E-4031. The results showed that (1) waveform prolongation and fluctuation both in the closed loops and clusters increased depending on the E-4031 concentration increase. However, (2) only the waveform signals in closed loops showed an apparent temporal change in waveforms from ventricular tachycardia (VT) to ventricular fibrillation (VF), which is similar to the most typical cell-to-cell conductance abnormality. The results indicated the usefulness of convoluted waveform signals of a closed-loop cell network for acquiring reproducible results acquisition and more detailed temporal information on cell-to-cell conduction.

  3. Radiation-induced heart disease in lung cancer radiotherapy

    Science.gov (United States)

    Ming, Xin; Feng, Yuanming; Yang, Chengwen; Wang, Wei; Wang, Ping; Deng, Jun

    2016-01-01

    Abstract Background: Radiation-induced heart disease (RIHD), which affects the patients’ prognosis with both acute and late side effects, has been published extensively in the radiotherapy of breast cancer, lymphoma and other benign diseases. Studies on RIHD in lung cancer radiotherapy, however, are less extensive and clear even though the patients with lung cancer are delivered with higher doses to the heart during radiation treatment. Methods: In this article, after extensive literature search and analysis, we reviewed the current evidence on RIHD in lung cancer patients after their radiation treatments and investigated the potential risk factors for RIHD as compared to other types of cancers. Result: Cardiac toxicity has been found highly relevant in lung cancer radiotherapy. So far, the crude incidence of cardiac complications in the lung cancer patients after radiotherapy has been up to 33%. Conclusion: The dose to the heart, the lobar location of tumor, the treatment modality, the history of heart and pulmonary disease and smoking were considered as potential risk factors for RIHD in lung cancer radiotherapy. As treatment techniques improve over the time with better prognosis for lung cancer survivors, an improved prediction model can be established to further reduce the cardiac toxicity in lung cancer radiotherapy. PMID:27741117

  4. Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscuta chinensis Lam on cardiomyocytes in aging rats.

    Science.gov (United States)

    Sun, Shou-Li; Guo, Li; Ren, Ya-Chao; Wang, Bing; Li, Rong-Hui; Qi, Yu-Shan; Yu, Hui; Chang, Nai-Dan; Li, Ming-Hui; Peng, Hai-Sheng

    2014-09-01

    To investigate the mechanism of apoptosis in myocardial cells of aging rats induced by D-galactose and to study the effect of the Polysaccharide isolated from the seeds of Cuscuta chinensis Lam (PCCL) on apoptosis of cardiomyocytes and its corresponding machinasim in aging rat model. Fifty male SD rats were randomly divided into 5 groups. Normal control group (NC). D-galactose (100 mg · kg(-1)d(-1) for 56 day) indued aging group (MC), D-galactose plus 100 mg kg(-1) d(-1) PCCL group (ML), D-galactose plus 200 mg kg(-1) d(-1) PCCL group (MM), and D-galactose plus 400 mg kg(-1) d(-1) PCCL group (MH). Same volume of solution (water, or PCCL aqueous solution) was given by gavage for 56 days. Then the hearts were collected and apoptosis parameters were evaluated. Caspase-3 and Cyt c were determined by fluorescence spectrometer, the apoptosis rate was assessed by AnnexinV-FITC method by Flow-Cytometry, [Ca(2+)]i and [Ca(2+)]i overloaded by KCL were observed by laser scanning confocal microscopy (LSCM); Bcl-2 and Bax were examined by immunohistochemistry. The content of Cyt C, [Ca(2+)]i of cardiomyocytes, the activity of Caspase-3, Bax expression level in D-galactose induced aging group were higher than NC (p aging group compared to NC. On the other hand, the content of Cyt C, [Ca(2+)]i of cardiomyocytes, the activity of Caspase-3 and apoptosis rate, as well as Bax expression level in all three PCCL groups were decreased compared to galactose induced group (p aging group. PCCL could decrease the apoptosis of cardiomyocytes by the mitochondria apoptosis pathway.

  5. [Effects of BmKIM on sodium current of isolated cardiomyocytes, transmembrane action potential and aconitine induced arrhythmia in vivo in rabbits].

    Science.gov (United States)

    Wang, Teng; Huang, Cong-Xin; Jiang, Hong; Tang, Qi-Zhu; Yang, Bo; Li, Geng-Shan

    2009-02-01

    To investigate the effects of recombinant BmKIM (poly-peptide derived from Asian Scorpion Buthus martensi Karsch) on the sodium current (I(Na)) of isolated ventricular myocytes, transmembrane action potential and aconitine induced arrhythmia in vivo in rabbits. Ventricular myocytes were enzymatically dissociated from adult rabbits. Whole-cell patch-clamp technique was used to record voltage-dependent I(Na). Standard transmembrane action potentials in rabbit hearts in vivo were recorded by using floating glass microelectrodes. Incidence of arrhythmias, the early after depolarization (EAD) and/or delay after depolarization (DAD) were measured in vivo in rabbits post aconitine (100 microg/kg, iv) in the absence or presence of BmKIM (50 microg/kg iv). (1) BmKIM significantly inhibited I(Na) in a voltage-dependent manner and significantly shifted the I-V curves of I(Na) upward. BmKIM left shifted the inactivation curve of I(Na) and voltages at 50% inactivation of I(Na) were changed from (-70.8 +/- 2.6) mV to (-84.8 +/- 3.5) mV (P action potential duration (APD(50) and APD(90)), and reduced action potential amplitude (APA), declined maximum up stroke velocity of action potential (V(max)) in vivo. The Q-T duration was shortened and heart rate significantly increased post BmKIM injection. (3) Incidence of aconitine induced ventricular arrhythmias (77.8%) was significantly reduced by BmKIM (22.2%, P action potential duration and reduce action potential amplitude and reduce the incidence of aconitine induced arrhythmias.

  6. Gene expression analysis of zebrafish heart regeneration.

    Directory of Open Access Journals (Sweden)

    Ching-Ling Lien

    2006-08-01

    Full Text Available Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required.

  7. Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kim, Yoon Young; Ku, Seung-Yup; Huh, Yul; Liu, Hung-Ching; Kim, Seok Hyun; Choi, Young Min; Moon, Shin Yong

    2013-10-01

    Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 μM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

  8. Single cardiomyocyte nuclear transcriptomes reveal a lincRNA-regulated de-differentiation and cell cycle stress-response in vivo.

    Science.gov (United States)

    See, Kelvin; Tan, Wilson L W; Lim, Eng How; Tiang, Zenia; Lee, Li Ting; Li, Peter Y Q; Luu, Tuan D A; Ackers-Johnson, Matthew; Foo, Roger S

    2017-08-09

    Cardiac regeneration may revolutionize treatment for heart failure but endogenous progenitor-derived cardiomyocytes in the adult mammalian heart are few and pre-existing adult cardiomyocytes divide only at very low rates. Although candidate genes that control cardiomyocyte cell cycle re-entry have been implicated, expression heterogeneity in the cardiomyocyte stress-response has never been explored. Here, we show by single nuclear RNA-sequencing of cardiomyocytes from both mouse and human failing, and non-failing adult hearts that sub-populations of cardiomyocytes upregulate cell cycle activators and inhibitors consequent to the stress-response in vivo. We characterize these subgroups by weighted gene co-expression network analysis and discover long intergenic non-coding RNAs (lincRNA) as key nodal regulators. KD of nodal lincRNAs affects expression levels of genes related to dedifferentiation and cell cycle, within the same gene regulatory network. Our study reveals that sub-populations of adult cardiomyocytes may have a unique endogenous potential for cardiac regeneration in vivo.Adult mammalian cardiomyocytes are predominantly binucleated and unable to divide. Using single nuclear RNA-sequencing of cardiomyocytes from mouse and human failing and non-failing adult hearts, See et al. show that some cardiomyocytes respond to stress by dedifferentiation and cell cycle re-entry regulated by lncRNAs.

  9. Spatially Resolved Genome-wide Transcriptional Profiling Identifies BMP Signaling as Essential Regulator of Zebrafish Cardiomyocyte Regeneration

    NARCIS (Netherlands)

    Wu, Chi-Chung; Kruse, Fabian; Vasudevarao, Mohankrishna Dalvoy; Junker, Jan Philipp; Zebrowski, David C; Fischer, Kristin; Noël, Emily S; Grün, Dominic; Berezikov, Eugene; Engel, Felix B; van Oudenaarden, Alexander; Weidinger, Gilbert; Bakkers, Jeroen

    2016-01-01

    In contrast to mammals, zebrafish regenerate heart injuries via proliferation of cardiomyocytes located near the wound border. To identify regulators of cardiomyocyte proliferation, we used spatially resolved RNA sequencing (tomo-seq) and generated a high-resolution genome-wide atlas of gene

  10. Human-induced pluripotent stem cell approaches to model inborn and acquired metabolic heart diseases.

    Science.gov (United States)

    Chanana, Anita M; Rhee, June-Wha; Wu, Joseph C

    2016-05-01

    The article provides an overview of advances in the induced pluripotent stem cell field to model cardiomyopathies of inherited inborn errors of metabolism and acquired metabolic syndromes in vitro. Several inborn errors of metabolism have been studied using 'disease in a dish' models, including Pompe disease, Danon disease, Fabry disease, and Barth syndrome. Disease phenotypes of complex metabolic syndromes, such as diabetes mellitus and aldehyde dehydrogenase 2 deficiency, have also been observed. Differentiation of patient and disease-specific induced pluripotent stem cell-derived cardiomyocytes has provided the capacity to model deleterious cardiometabolic diseases to understand molecular mechanisms, perform drug screens, and identify novel drug targets.

  11. The zebrafish heart regenerates after cryoinjury-induced myocardial infarction

    Directory of Open Access Journals (Sweden)

    Rainer Gregor

    2011-04-01

    Full Text Available Abstract Background In humans, myocardial infarction is characterized by irreversible loss of heart tissue, which becomes replaced with a fibrous scar. By contrast, teleost fish and urodele amphibians are capable of heart regeneration after a partial amputation. However, due to the lack of a suitable infarct model, it is not known how these animals respond to myocardial infarction. Results Here, we have established a heart infarct model in zebrafish using cryoinjury. In contrast to the common method of partial resection, cryoinjury results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts. As in mammals, the initial stages of the injury response include thrombosis, accumulation of fibroblasts and collagen deposition. However, at later stages, cardiac cells can enter the cell cycle and invade the infarct area in zebrafish. In the subsequent two months, fibrotic scar tissue is progressively eliminated by cell apoptosis and becomes replaced with a new myocardium, resulting in scarless regeneration. We show that tissue remodeling at the myocardial-infarct border zone is associated with accumulation of Vimentin-positive fibroblasts and with expression of an extracellular matrix protein Tenascin-C. Electrocardiogram analysis demonstrated that the reconstitution of the cardiac muscle leads to the restoration of the heart function. Conclusions We developed a new cryoinjury model to induce myocardial infarction in zebrafish. Although the initial stages following cryoinjury resemble typical healing in mammals, the zebrafish heart is capable of structural and functional regeneration. Understanding the key healing processes after myocardial infarction in zebrafish may result in identification of the barriers to efficient cardiac regeneration in mammals.

  12. Impact of leucine supplementation on exercise training induced anti-cardiac remodeling effect in heart failure mice.

    Science.gov (United States)

    de Moraes, Wilson Max Almeida Monteiro; Melara, Thaís Plasti; de Souza, Pamella Ramona Moraes; Guimarães, Fabiana de Salvi; Bozi, Luiz Henrique Marchesi; Brum, Patricia Chakur; Medeiros, Alessandra

    2015-05-15

    Leucine supplementation potentiates the effects of aerobic exercise training (AET) on skeletal muscle; however, its potential effects associated with AET on cardiac muscle have not been clarified yet. We tested whether leucine supplementation would potentiate the anti-cardiac remodeling effect of AET in a genetic model of sympathetic hyperactivity-induced heart failure in mice (α2A/α2CARKO). Mice were assigned to five groups: wild type mice treated with placebo and sedentary (WT, n = 11), α2A/α2CARKO treated with placebo and sedentary (KO, n = 9), α2A/α2CARKO treated with leucine and sedentary (KOL, n = 11), α2A/α2CARKO treated with placebo and AET (KOT, n = 12) or α2A/α2CARKO treated with leucine and AET (KOLT, n = 12). AET consisted of four weeks on a treadmill with 60 min sessions (six days/week, 60% of maximal speed) and administration by gavage of leucine (1.35 g/kg/day) or placebo (distilled water). The AET significantly improved exercise capacity, fractional shortening and re-established cardiomyocytes' diameter and collagen fraction in KOT. Additionally, AET significantly prevented the proteasome hyperactivity, increased misfolded proteins and HSP27 expression. Isolated leucine supplementation displayed no effect on cardiac function and structure (KOL), however, when associated with AET (KOLT), it increased exercise tolerance to a higher degree than isolated AET (KOT) despite no additional effects on AET induced anti-cardiac remodeling. Our results provide evidence for the modest impact of leucine supplementation on cardiac structure and function in exercised heart failure mice. Leucine supplementation potentiated AET effects on exercise tolerance, which might be related to its recognized impact on skeletal muscle.

  13. The diagnosis of anthracycline-induced cardiac damage and heart failure

    Directory of Open Access Journals (Sweden)

    Jarosław Dudka

    2009-05-01

    Full Text Available Routine examinations during chemotherapy containing anthracyclines evaluate heart function before treatment and monitor cardiotoxic effects during and after therapy. A number of methods are useful in cardiac assessment, including electrocardiography, radiology techniques (RTG, CT, MRI, PET-CT, PET-MRI, echocardiography, radioisotope imaging techniques (scyntygraphy, MUGA, PET, and ultra-structure evaluation in biopsy samples. Nevertheless, there is a continuous need for new methods to predict future damage at the initial stages of cardiac changes. In recent years the therapeutic usefulness of biochemical blood parameters in anthracycline-treated patients has been assessed. The levels of cardiac troponines (cTnI, cTnT, natriuretic peptides (ANP, BNP, and endothelin 1 have been included in the studies. Heart-type fatty acid binding protein (H-FABP is another promising factor showing cardiomyocytic impairment. However, the clinical use of biochemical parameters in diagnosing anthracycline-related cardiotoxicity is still a controversial issue.

  14. Rac1 modulates cardiomyocyte adhesion during mouse embryonic development

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    Abu-Issa, Radwan, E-mail: rabuissa@umich.edu

    2015-01-24

    Highlights: • Conditional knockout of Rac1 using Nkx2.5 Cre line is lethal at E13.5. • The myocardium of the mutant is thin and disorganized. • The phenotype is not due to cardiomyocyte low proliferation or apoptosis. • The phenotype is due to specific defect in cardiomyocyte adhesion. - Abstract: Rac1, a member of the Rho subfamily of small GTPases, is involved in morphogenesis and differentiation of many cell types. Here we define a role of Rac1 in cardiac development by specifically deleting Rac1 in the pre-cardiac mesoderm using the Nkx2.5-Cre transgenic driver line. Rac1-conditional knockout embryos initiate heart development normally until embryonic day 11.5 (E11.5); their cardiac mesoderm is specified, and the heart tube is formed and looped. However, by E12.5-E13.5 the mutant hearts start failing and embryos develop edema and hemorrhage which is probably the cause for the lethality observed soon after. The hearts of Rac1-cKO embryos exhibit disorganized and thin myocardial walls and defects in outflow tract alignment. No significant differences of cardiomyocyte death or proliferation were found between developing control and mutant embryos. To uncover the role of Rac1 in the heart, E11.5 primary heart cells were cultured and analyzed in vitro. Rac1-deficient cardiomyocytes were less spread, round and loosely attached to the substrate and to each other implying that Rac1-mediated signaling is required for appropriate cell–cell and/or cellmatrix adhesion during cardiac development.

  15. From teeth, skin, blood to heart : induced pluripotent stem cells as an in vitro model for cardiac disease

    NARCIS (Netherlands)

    Dambrot, Cheryl Susan

    2014-01-01

    Since the first reports of human induced pluripotent stem cells (hiPSC), the field of pluripotent stem cell (PSC) research has grown in leap and bounds, particularly in the area of (cardiac) disease modeling. This is in part because it is fairly easy to produce cardiomyocytes from hPSC and also

  16. Pathological alterations in liver injury following congestive heart failure induced by volume overload in rats

    OpenAIRE

    Shaqura, Mohammed; Mohamed, Doaa M.; Aboryag, Noureddin B.; Bedewi, Lama; Dehe, Lukas; Treskatsch, Sascha; Shakibaei, Mehdi; Schaefer, Michael; Mousa, Shaaban A

    2017-01-01

    Heart failure has emerged as a disease with significant public health implications. Following progression of heart failure, heart and liver dysfunction are frequently combined in hospitalized patients leading to increased morbidity and mortality. Here, we investigated the underlying pathological alterations in liver injury following heart failure. Heart failure was induced using a modified infrarenal aortocaval fistula (ACF) in male Wistar rats. Sham operated and ACF rats were compared for th...

  17. Assessment of extracellular field potential and Ca2+ transient signals for early QT/pro-arrhythmia detection using human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Abi-Gerges, Najah; Pointon, Amy; Oldman, Karen L; Brown, Martin R; Pilling, Mark A; Sefton, Clare E; Garside, Helen; Pollard, Christopher E

    Cardiovascular toxicity is a prominent reason for failures in drug development, resulting in the demand for assays that can predict this liability in early drug discovery. We investigated whether iCell® cardiomyocytes have utility as an early QT/TdP screen. Thirty clinical drugs with known QT/TdP outcomes were evaluated blind using label-free microelectrode array (parameters measured were beating period (BP), field potential duration (FPD), fast Na+ amplitude and slope) and live cell, fast kinetic fluorescent Ca2+ transient FLIPR® Tetra (parameters measured were peak count, width, amplitude) systems. Many FPD-altering drugs also altered BP. Correction for BP, using a Log-Log (LL) model, was required to appropriately interpret direct drug effects on FPD. In comparison with human QT effects and when drug activity was to be predicted at top test concentration (TTC), LL-corrected FPD and peak count had poor assay sensitivity and specificity values: 13%/64% and 65%/11%, respectively. If effective free therapeutic plasma concentration (EFTPC) was used instead of TTC, the values were 0%/100% and 6%/100%, respectively. When compared to LL-corrected FPD and peak count, predictive values of uncorrected FPD, BP, width and amplitude were not much different. If pro-arrhythmic risk was to be predicted using Ca2+ transient data, the values were 67%/100% and 78%/53% at EFTPC and TTC, respectively. Thus, iCell® cardiomyocytes have limited value as an integrated QT/TdP assay, highlighting the urgent need for improved experimental alternatives that may offer an accurate integrated cardiomyocyte safety model for supporting the development of new drugs without QT/TdP effects. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Role of endogenous PDGF-BB in cultured cardiomyocytes exposed to hypoxia.

    Science.gov (United States)

    Rong, Rong; Wang, You-Cui; Hu, Li-qun; He, Qin-qin; Zhou, Xin-Fu; Wang, Ting-hua; Bu, Pei-li

    2015-04-01

    Platelet-derived growth factor-BB (PDGF-BB) plays a critical role in cell proliferation, angiogenesis and fibrosis. However, its exact role in cardiomyocytes exposed to hypoxia is not well known. This study was therefore designed to detect whether PDGF-BB expression was changed in a hypoxic condition, then the possible role of endogenous PDGF-BB in cardiomyocytes was explored, with interference RNA in a lentiviral vector ex vivo. The results showed that cultured cardiomyocytes exhibited an optimal proliferation from 3 to 10 days. However, LDH level was significantly increased but the heart rhythm was not altered in cardiomyocytes exposed to hypoxia for 24 hours. PDGF-BB expression was substantially upregulated in hypoxic cardiomyocytes. In order to know the role of PDGF-BB, we performed PDGF-BB knockdown in cultured cardiomyocytes. The number of apoptotic cells and the level of LDH were significantly increased but the beat rhythm was reduced in cardiomyocytes with PDGF-BB knockdown. These findings suggest that endogenous PDGF-BB exerts a crucial protective effect to cultured cardiomyocytes exposed to hypoxia. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes.

    Science.gov (United States)

    Wu, Dan; Hu, Qingxun; Liu, Xinhua; Pan, Lilong; Xiong, Qinghui; Zhu, Yi Zhun

    2015-04-30

    Oxidative stress plays a great role in the pathogenesis of heart failure (HF). Oxidative stress results in apoptosis, which can cause the damage of cardiomyocytes. Hydrogen sulfide (H2S), the third gasotransmitter, is a good reactive oxygen species (ROS) scavenger, which has protective effect against HF. Sirtuin-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that plays a critical role in promoting cell survival under oxidative stress. The purpose of this article is to investigate the interaction between H2S and SIRT1 under oxidative stress in H9c2 cardiomyocytes. Oxidative stress was induced by hydrogen peroxide (H2O2). Treatment with NaSH (25-100 µmol/L) dose-dependently increased the cell viability and improved the cell apoptosis induced by H2O2 in H9c2 cardiomyocytes. The protective effect of NaSH against the apoptosis could be attenuated by SIRT1 inhibitor Ex 527 (10 µmol/L). Treatment with NaSH (100 µmol/L) could increase the expression of SIRT1 in time dependent manner, which decreased by different concentration of H2O2. NaSH (100 µmol/L) increased the cellular ATP level and the expression of ATPase. These effects were attenuated by Ex 527 (10 µmol/L). After NaSH (100 µmol/L) treatment, the decrease in ROS production and the enhancement in SOD, GPx and GST expression were observed. Ex 527 (10 µmol/L) reversed these effects. In conclusion, for the first time, this article can identify antioxidative effects of H2S under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Short term exercise induces PGC-1α, ameliorates inflammation and increases mitochondrial membrane proteins but fails to increase respiratory enzymes in aging diabetic hearts.

    Directory of Open Access Journals (Sweden)

    Amy Botta

    Full Text Available PGC-1α, a transcriptional coactivator, controls inflammation and mitochondrial gene expression in insulin-sensitive tissues following exercise intervention. However, attributing such effects to PGC-1α is counfounded by exercise-induced fluctuations in blood glucose, insulin or bodyweight in diabetic patients. The goal of this study was to investigate the role of PGC-1α on inflammation and mitochondrial protein expressions in aging db/db mice hearts, independent of changes in glycemic parameters. In 8-month-old db/db mice hearts with diabetes lasting over 22 weeks, short-term, moderate-intensity exercise upregulated PGC-1α without altering body weight or glycemic parameters. Nonetheless, such a regimen lowered both cardiac (macrophage infiltration, iNOS and TNFα and systemic (circulating chemokines and cytokines inflammation. Curiously, such an anti-inflammatory effect was also linked to attenuated expression of downstream transcription factors of PGC-1α such as NRF-1 and several respiratory genes. Such mismatch between PGC-1α and its downstream targets was associated with elevated mitochondrial membrane proteins like Tom70 but a concurrent reduction in oxidative phosphorylation protein expressions in exercised db/db hearts. As mitochondrial oxidative stress was predominant in these hearts, in support of our in vivo data, increasing concentrations of H2O2 dose-dependently increased PGC-1α expression while inhibiting expression of inflammatory genes and downstream transcription factors in H9c2 cardiomyocytes in vitro. We conclude that short-term exercise-induced oxidative stress may be key in attenuating cardiac inflammatory genes and impairing PGC-1α mediated gene transcription of downstream transcription factors in type 2 diabetic hearts at an advanced age.

  1. Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induces Post-Translational Modifications of AKAP121, DRP1 and OPA1 That Promote Mitochondrial Fission.

    Science.gov (United States)

    Tsushima, Kensuke; Bugger, Heiko; Wende, Adam R; Soto, Jamie; Jenson, Gregory A; Tor, Austin R; McGlauflin, Rose; Kenny, Helena C; Zhang, Yuan; Souvenir, Rhonda; Hu, Xiao X; Black, Crystal L; Pereira, Renata O; Lira, Vitor A; Spitzer, Kenneth; Sharp, Terry L; Shoghi, Kooresh I; Sparagna, Genevieve C; Rog-Zielinska, Eva A; Kohl, Peter; Khalimonchuk, Oleh; Schaffer, Jean E; Abel, E Dale

    2017-11-01

    Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes (NRVCs). Palmitate exposure to NRVCs initially activates mitochondrial respiration, coupled with increased mitochondrial membrane potential and adenosine triphosphate (ATP) synthesis. However, long-term exposure to palmitate (> 8h) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of A-kinase anchor protein (AKAP121) leading to reduced phosphorylation of DRP1 at Ser637 and altered proteolytic processing of OPA1. Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel

  2. Assessment of contractility in intact ventricular cardiomyocytes using the dimensionless 'Frank-Starling Gain' index.

    Science.gov (United States)

    Bollensdorff, Christian; Lookin, Oleg; Kohl, Peter

    2011-07-01

    This paper briefly recapitulates the Frank-Starling law of the heart, reviews approaches to establishing diastolic and systolic force-length behaviour in intact isolated cardiomyocytes, and introduces a dimensionless index called 'Frank-Starling Gain', calculated as the ratio of slopes of end-systolic and end-diastolic force-length relations. The benefits and limitations of this index are illustrated on the example of regional differences in Guinea pig intact ventricular cardiomyocyte mechanics. Potential applicability of the Frank-Starling Gain for the comparison of cell contractility changes upon stretch will be discussed in the context of intra- and inter-individual variability of cardiomyocyte properties.

  3. Higenamine Combined with [6]-Gingerol Suppresses Doxorubicin-Triggered Oxidative Stress and Apoptosis in Cardiomyocytes via Upregulation of PI3K/Akt Pathway

    Directory of Open Access Journals (Sweden)

    Yan-Ling Chen

    2013-01-01

    Full Text Available Sini decoction is a well-known formula of traditional Chinese medicine, which has been used to treat cardiovascular disease for many years. Previously, we demonstrated that Sini decoction prevented doxorubicin-induced heart failure in vivo. However, its active components are still unclear. Thus, we investigated the active components of Sini decoction and their cardioprotective mechanisms in the in vitro neonatal rat cardiomyocytes and H9c2 cell line models of doxorubicin-induced cytotoxicity. Our results demonstrated that treatment with higenamine or [6]-gingerol increased viability of doxorubicine-injured cardiomyocytes. Moreover, combined use of higenamine and [6]-gingerol exerted more profound protective effects than either drug as a single agent, with effects similar to those of dexrazoxane, a clinically approved cardiac protective agent. In addition, we found that treatment with doxorubicin reduced SOD activity, increased ROS generation, enhanced MDA formation, induced release of LDH, and triggered the intrinsic mitochondria-dependent apoptotic pathway in cardiomyocytes, which was inhibited by cotreatment of higenamine and [6]-gingerol. Most importantly, the cytoprotection of higenamine plus [6]-gingerol could be abrogated by LY294002, a PI3K inhibitor. In conclusion, combination of higenamine and [6]-gingerol exerts cardioprotective effect against doxorubicin-induced cardiotoxicity through activating the PI3K/Akt signaling pathway. Higenamine and [6]-gingerol may be the active components of Sini decoction.

  4. Simple Monolayer Differentiation of Murine Cardiomyocytes via Nutrient Deprivation-Mediated Activation of β-Catenin.

    Science.gov (United States)

    Hofbauer, Pablo; Jung, Jangwook P; McArdle, Tanner J; Ogle, Brenda M

    2016-12-01

    Methods to generate murine cardiomyocytes from pluripotent stem cells (PSCs) in vitro are resource and time intensive. All current protocols require exogenously provided soluble factors and almost all utilize embryoid body formation to modulate pathways associated with mesoderm specification and cardiomyocyte differentiation. Here, we developed a simple protocol without EBs and without exogenous soluble factors that enabled cardiomyocyte differentiation of a murine induced PSC line based on controlled nutrient deprivation in 2D monolayer cultures. We showed that this protocol reproducibly imposed metabolic stress and consequently modulated active β-catenin levels to yield functional cardiomyocytes. The yield of cardiomyocytes and calcium handling kinetics were comparable to existing approaches. However, this approach did not produce consistent results between murine PSC lines suggesting signaling pathways linking nutrient deprivation to β-catenin activation are not universally conserved and may be a remnant of the parent population from which the induced PSCs were derived.

  5. Inhibition of Cardiomyocytes Hypertrophy by Resveratrol Is Associated with Amelioration of Endoplasmic Reticulum Stress

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    Yan Lin

    2016-07-01

    Full Text Available Background/Aims: Resveratrol (Res, a polyphenol antioxidant found in red wine, has been shown to play a cardioprotective role. This study was undertaken to investigate whether Res can protect the heart suffering from hypertrophy injuries induced by isoproterenol (ISO, and whether the protective effect is mediated by endoplasmic reticulum (ER stress. Methods: Cardiomyocytes were randomly assigned to the control group, ISO group (100 nM ISO for 48 h, Res + ISO group (50 μM Res and 100 nM ISO for 48 h and Res group (50 μM Res for 48h only. Hypertrophy was estimated by measuring the cell surface area and the atrial natriuretic peptide (ANP gene expression. Apoptosis was measured using Hoechst 33258 staining and transmission electron microscopy. Protein expression of ER stress and apoptosis factors was analyzed using Western Blot analysis. Results: Res effectively suppress the cardiomyocytes hypertrophy and apoptosis induced by ISO, characterized by the reduction of the myocardial cell surface area, the ANP gene expression, the LDH and MDA leakage amount and the rate of cell apoptosis, while decrease of the protein expression of GRP78, GRP94 and CHOP, and reverse the expression of Bcl-2 and Bax. Conclusion: In summary, Res treatment effectively suppressed myocardial hypertrophy and apoptosis at least partially via inhibiting ER stress.

  6. A piezoelectric electrospun platform for in situ cardiomyocyte contraction analysis

    Science.gov (United States)

    Beringer, Laura Toth

    hyperpolarized state, proving their potential use as contractile analysis microdevices. The third and final aim of this dissertation was to be able to measure contraction events from both cultured cardiomyocytes and whole tissues in situ. Rat neonatal cardiomyocytes grew on the prepared collagen/PVDF-TrFe nanogenerators and yielded a distinct signal after 8 days of growth. These contractions were verified with live cell imaging and video recording. In addition, cardiomyocyte exposure to the drug isoproterenol increased contraction strength and frequency, which was reflected in the nanogenerator recordings. Frog whole heart and heart tissue slices also were interfaced with the fabricated nanogenerators and signals were recorded. The same held true for heart slices from male Sprague-Dawley rats. These signals were determined to be statistically different compared to the control baseline nanogenerator recordings in media in the absence of cell culture. Overall the fabricated nanogenerators have demonstrated their potential to be used as in situ analysis tools for contractile events and have potential in the field of personalized medicine and drug diagnostic assays. The facile fabrication and ease of setup to obtain the electrical voltage signal corresponding to the contractile events are what sets the nanogenerator apart from any polymer based sensor available today.

  7. A protective anti-arrhythmic role of ursodeoxycholic acid in an in-vitro rat model of the cholestatic fetal heart

    Science.gov (United States)

    Miragoli, Michele; Sheikh Abdul Kadir, Siti H; Sheppard, Mary N.; Salvarani, Nicolό; Virta, Matilda; Wells, Sarah; Lab, Max J.; Nikolaev, Viacheslav O.; Moshkov, Alexey; Hague, William M; Rohr, Stephan; Williamson, Catherine; Gorelik, Julia

    2016-01-01

    Intrahepatic cholestasis of pregnancy may be complicated by fetal arrhythmia, fetal hypoxia, preterm labour and, in severe cases, intrauterine death. The precise aetiology of the fetal death is not known. However, taurocholate has been demonstrated to cause arrhythmia and abnormal calcium dynamics in cardiomyocytes. To identify the underlying reason for increased susceptibility of fetal cardiomyocytes to arrhythmia we studied myofibroblasts, which appear during structural remodelling of the adult diseased heart. In-vitro, they depolarize rat cardiomyocytes via heterocellular gap junctional coupling. Recently, it has been hypothesized that ventricular myofibroblasts might appear in the developing human heart triggered by physiological fetal hypoxia. However, their presence in the fetal heart and their pro-arrhythmogenic effects have not been systematically characterized. Immunohistochemistry demonstrated that ventricular myofibroblasts transiently appear in the human fetal heart during gestation. We established two in-vitro models of the maternal and fetal heart both exposed to increasing doses of taurocholate. The maternal heart model consisted of confluent strands of rat cardiomyocytes, while for the fetal heart model we added cardiac myofibroblasts on top of cardiomyocytes. Taurocholate in the fetal heart model, but not in the maternal heart model, slowed the conduction velocity from 19 cm/s to 9 cm/s, induced early afterdepolarizations and resulted in sustained re-entrant arrhythmias. These arrhythmic events were prevented by ursodeoxycholic acid, which hyperpolarized the myofibroblast membrane potential by modulating potassium conductance. Conclusion These results illustrate that the appearance of myofibroblasts in the fetal heart may contribute to arrhythmias. The described mechanism represents a new therapeutic approach for cardiac arrhythmias at the level of myofibroblast. PMID:21809354

  8. CstF-64 is necessary for endoderm differentiation resulting in cardiomyocyte defects

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    Bradford A. Youngblood

    2014-11-01

    Full Text Available Although adult cardiomyocytes have the capacity for cellular regeneration, they are unable to fully repair severely injured hearts. The use of embryonic stem cell (ESC-derived cardiomyocytes as transplantable heart muscle cells has been proposed as a solution, but is limited by the lack of understanding of the developmental pathways leading to specification of cardiac progenitors. Identification of these pathways will enhance the ability to differentiate cardiomyocytes into a clinical source of transplantable cells. Here, we show that the mRNA 3′ end processing protein, CstF-64, is essential for cardiomyocyte differentiation in mouse ESCs. Loss of CstF-64 in mouse ESCs results in loss of differentiation potential toward the endodermal lineage. However, CstF-64 knockout (Cstf2E6 cells were able to differentiate into neuronal progenitors, demonstrating that some differentiation pathways were still intact. Markers for mesodermal differentiation were also present, although Cstf2E6 cells were defective in forming beating cardiomyocytes and expressing cardiac specific markers. Since the extraembryonic endoderm is needed for cardiomyocyte differentiation and endodermal markers were decreased, we hypothesized that endodermal factors were required for efficient cardiomyocyte formation in the Cstf2E6 cells. Using conditioned medium from the extraembryonic endodermal (XEN stem cell line we were able to restore cardiomyocyte differentiation in Cstf2E6 cells, suggesting that CstF-64 has a role in regulating endoderm differentiation that is necessary for cardiac specification and that extraembryonic endoderm signaling is essential for cardiomyocyte development.

  9. Potential role of microRNA-10b down-regulation in cardiomyocyte apoptosis in aortic stenosis patients.

    Science.gov (United States)

    Gallego, Idoia; Beaumont, Javier; López, Begoña; Ravassa, Susana; Gómez-Doblas, Juan José; Moreno, María Ujué; Valencia, Félix; de Teresa, Eduardo; Díez, Javier; González, Arantxa

    2016-12-01

    MicroRNAs have been associated with cardiomyocyte apoptosis, a process involved in myocardial remodelling in aortic valve (Av) stenosis (AS). Our aim was to analyse whether the dysregulation of myocardial microRNAs was related to cardiomyocyte apoptosis in AS patients. Endomyocardial biopsies were obtained from 28 patients with severe AS (based on pressure gradients and Av area) referred for Av replacement and from necropsies of 10 cardiovascular disease-free control subjects. AS patients showed an increased (P 0.08%; n=12). Group 2 patients presented lower cardiomyocyte density (P<0.001) and ejection fraction (P<0.05), and higher troponin T levels (P<0.05), prevalence of heart failure (HF; P<0.05) and NT-proBNP levels (P<0.05) than those from group 1. miRNA expression profile analysed in 5 patients randomly selected from each group showed 64 microRNAs down-regulated and 6 up-regulated (P<0.05) in group 2 compared with group 1. Those microRNAs with the highest fold-change were validated in the full two groups corroborating that miR-10b, miR-125b-2* and miR-338-3p were down-regulated (P<0.05) in group 2 compared with group 1 and control subjects. These three microRNAs were inversely correlated (P<0.05) with the CMAI. Inhibition of miR-10b induced an increase (P<0.05) of apoptosis and increased expression (P<0.05) of apoptosis protease-activating factor-1 (Apaf-1) in HL-1 cardiomyocytes. In conclusion, myocardial down-regulation of miR-10b may be involved in increased cardiomyocyte apoptosis in AS patients, probably through Apaf-1 up-regulation, contributing to cardiomyocyte damage and to the development of HF. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  10. Anti-Apoptotic and Pro-Survival Effect of Alpinate Oxyphyllae Fructus (AOF) in a d-Galactose-Induced Aging Heart.

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    Chang, Yung-Ming; Chang, Hen-Hong; Kuo, Wei-Wen; Lin, Hung-Jen; Yeh, Yu-Lan; Padma Viswanadha, Vijaya; Tsai, Chin-Chuan; Chen, Ray-Jade; Chang, Hsin-Nung; Huang, Chih-Yang

    2016-03-29

    Aging, a natural biological/physiological phenomenon, is accelerated by reactive oxygen species (ROS) accumulation and identified by a progressive decrease in physiological function. Several studies have shown a positive relationship between aging and chronic heart failure (HF). Cardiac apoptosis was found in age-related diseases. We used a traditional Chinese medicine, Alpinate Oxyphyllae Fructus (AOF), to evaluate its effect on cardiac anti-apoptosis and pro-survival. Male eight-week-old Sprague-Dawley (SD) rats were segregated into five groups: normal control group (NC), d-Galactose-Induced aging group (Aging), and AOF of 50 (AL (AOF low)), 100 (AM (AOF medium)), 150 (AH (AOF high)) mg/kg/day. After eight weeks, hearts were measured by an Hematoxylin-Eosin (H&E) stain, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assays and Western blotting. The experimental results show that the cardiomyocyte apoptotic pathway protein expression increased in the d-Galactose-Induced aging groups, with dose-dependent inhibition in the AOF treatment group (AL, AM, and AH). Moreover, the expression of the pro-survival p-Akt (protein kinase B (Akt)), Bcl-2 (B-cell lymphoma 2), anti-apoptotic protein (Bcl-xL) protein decreased significantly in the d-Galactose-induced aging group, with increased performance in the AOF treatment group with levels of p-IGFIR and p-PI3K (Phosphatidylinositol-3' kinase (PI3K)) to increase by dosage and compensatory performance. On the other hand, the protein of the Sirtuin 1 (SIRT1) pathway expression decreased in the aging groups and showed improvement in the AOF treatment group. Our results suggest that AOF strongly works against ROS-induced aging heart problems.

  11. Visualizing sodium dynamics in isolated cardiomyocytes using fluorescent nanosensors

    OpenAIRE

    Dubach, J. Matthew; Das, Saumya; Rosenzweig, Anthony; Clark, Heather A.

    2009-01-01

    Regulation of sodium flux across the cell membrane plays a vital role in the generation of action potentials and regulation of membrane excitability in cells such as cardiomyocytes and neurons. Alteration of sodium channel function has been implicated in diseases such as epilepsy, long QT syndrome, and heart failure. However, single cell imaging of sodium dynamics has been limited due to the narrow selection of fluorescent sodium indicators available to researchers. Here we report, the detect...

  12. miR-34a Modulates Angiotensin II-Induced Myocardial Hypertrophy by Direct Inhibition of ATG9A Expression and Autophagic Activity

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    Huang, He; Ye, Jing; Pan, Wei; Zhong, Yun; Cheng, Chuanfang; You, Xiangyu; Liu, Benrong; Xiong, Longgen; Liu, Shiming

    2014-01-01

    Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3′-UTR, but not to the mutated 3′-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity. PMID:24728149

  13. Burn-induced subepicardial injury in frog heart: a simple model mimicking ST segment changes in ischemic heart disease.

    Science.gov (United States)

    Kazama, Itsuro

    2016-02-01

    To mimic ischemic heart disease in humans, several animal models have been created, mainly in rodents by surgically ligating their coronary arteries. In the present study, by simply inducing burn injuries on the bullfrog heart, we reproduced abnormal ST segment changes in the electrocardiogram (ECG), mimicking those observed in ischemic heart disease, such as acute myocardial infarction and angina pectoris. The "currents of injury" created by a voltage gradient between the intact and damaged areas of the myocardium, negatively deflected the ECG vector during the diastolic phase, making the ST segment appear elevated during the systolic phase. This frog model of heart injury would be suitable to explain the mechanisms of ST segment changes observed in ischemic heart disease.

  14. Nicotine plus a high-fat diet triggers cardiomyocyte apoptosis.

    Science.gov (United States)

    Sinha-Hikim, Indrani; Friedman, Theodore C; Falz, Mark; Chalfant, Victor; Hasan, Mohammad Kamrul; Espinoza-Derout, Jorge; Lee, Desean L; Sims, Carl; Tran, Peter; Mahata, Sushil K; Sinha-Hikim, Amiya P

    2017-04-01

    Cigarette smoking is an important risk factor for diabetes, cardiovascular disease and non-alcoholic fatty liver disease. The health risk associated with smoking can be aggravated by obesity. Smoking might also trigger cardiomyocyte (CM) apoptosis. Given that CM apoptosis has been implicated as a potential mechanism in the development of cardiomyopathy and heart failure, we characterize the key signaling pathways in nicotine plus high-fat diet (HFD)-induced CM apoptosis. Adult C57BL6 male mice were fed a normal diet (ND) or HFD and received twice-daily intraperitoneal (IP) injections of nicotine (0.75 mg/kg body weight [BW]) or saline for 16 weeks. An additional group of nicotine-treated mice on HFD received twice-daily IP injections of mecamylamine (1 mg/kg BW), a non-selective nicotinic acetylcholine receptor antagonist, for 16 weeks. Nicotine when combined with HFD led to a massive increase in CM apoptosis that was fully prevented by mecamylamine treatment. Induction of CM apoptosis was associated with increased oxidative stress and activation of caspase-2-mediated intrinsic pathway signaling coupled with inactivation of AMP-activated protein kinase (AMPK). Furthermore, nicotine treatment significantly (P nicotine, when combined with HFD, triggers CM apoptosis through the generation of oxidative stress and inactivation of AMPK together with the activation of caspase-2-mediated intrinsic apoptotic signaling independently of FGF21 and SIRT1.

  15. Visualizing sodium dynamics in isolated cardiomyocytes using fluorescent nanosensors

    Science.gov (United States)

    Dubach, J. Matthew; Das, Saumya; Rosenzweig, Anthony; Clark, Heather A.

    2009-01-01

    Regulation of sodium flux across the cell membrane plays a vital role in the generation of action potentials and regulation of membrane excitability in cells such as cardiomyocytes and neurons. Alteration of sodium channel function has been implicated in diseases such as epilepsy, long QT syndrome, and heart failure. However, single cell imaging of sodium dynamics has been limited due to the narrow selection of fluorescent sodium indicators available to researchers. Here we report, the detection of spatially defined sodium activity during action potentials. Fluorescent nanosensors that measure sodium in real-time, are reversible and are completely selective over other cations such as potassium that were used to image sodium. The use of the nanosensors in vitro was validated by determining drug-induced activation in heterologous cells transfected with the voltage-gated sodium channel NaV1.7. Spatial information of sodium concentrations during action potentials will provide insight at the cellular level on the role of sodium and how slight changes in sodium channel function can affect the entirety of an action potential. PMID:19805271

  16. Radiation-Induced Heart Disease: Pathologic Abnormalities and Putative Mechanisms

    Directory of Open Access Journals (Sweden)

    Neil K Taunk

    2015-02-01

    Full Text Available Breast cancer is a common diagnosis in women. Breast radiation has become a critical in managing patients who receive breast conserving surgery, or have certain high-risk features after mastectomy. Most patients have an excellent prognosis, therefore understanding the late effects of radiation to the chest is important. Radiation induced heart disease (RIHD comprises a spectrum of cardiac pathology including myocardial fibrosis and cardiomyopathy, coronary artery disease, valvular disease, pericardial disease, and arrhythmias. Tissue fibrosis is a common mediator in RIHD. Multiple pathways converge with both acute and chronic cellular, molecular, and genetic changes to result in fibrosis. In this article, we review the pathophysiology of cardiac disease related to radiation therapy to the chest. Our understanding of these mechanisms has improved substantially, but much work remains to further refine radiation delivery techniques and develop therapeutics to battle late effects of radiation.

  17. Enterovirus-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis.

    Science.gov (United States)

    Ventéo, Lydie; Bourlet, Thomas; Renois, Fanny; Douche-Aourik, Fatima; Mosnier, Jean-François; Maison, Geoffroy Lorain De la Grand; Pluot, Michel; Pozzetto, Bruno; Andreoletti, Laurent

    2010-03-01

    We examined the impact of enterovirus (EV) cardiac replication activity on the endomyocardial mitochondrial pathway in patients with acute myocarditis. Levels of apoptotic cardiomyocytes were determined by TUNEL and ligation-mediated polymerase chain reaction (PCR) assays and EV replication activity was assessed by immunostaining of EV VP1 capsid protein in ventricular myocytes of patients with acute myocarditis (n = 25), and healthy heart controls (n = 15). Ratio of cytosolic/mitochondrial cytochrome c concentrations was determined by ELISA assay, levels of active caspase-9 were determined by western blot analysis and Bax/Bcl2 mRNA ratio was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in the same cardiac tissues. Patients with EV-associated acute myocarditis (n = 15) exhibited a significantly higher number of apoptotic cardiomyocytes than those with non-EV-associated acute myocarditis (n = 10) and controls (n = 15) (P < 0.001). Endomyocardial ratio of cytosolic/mitochondrial cytochrome c concentrations and levels of active caspase-9 protein were significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). Moreover, Bax/Bcl2 mRNA ratio was significantly increased in EV than in non-EV-related myocarditis patients (P < 0.001). Our findings evidence an EV-related activation of the cardiomyocyte mitochondrial apoptotic pathway in patients with acute myocarditis. Moreover, our results indicate that this EV-induced pro-apoptotic mechanism could be partly related to an up-regulation of Bax expression, and suggest that inhibition of this cell death process may constitute the basis for novel therapies.

  18. HDL protects against doxorubicin induced cardiotoxicity in a scavenger receptor class B type 1, PI3K, and AKT dependent manner.

    Science.gov (United States)

    Durham, Kristina; Chathely, Kevin; Mak, Kei Cheng; Momen, Abdul; Thomas, Cyrus T; Zhao, Yuan-Yuan; MacDonald, Melissa E; Curtis, Jonathan M; Husain, Mansoor; Trigatti, Bernardo L

    2017-10-06

    Doxorubicin is a widely used chemotherapeutic with deleterious cardiotoxic side effects. HDL has been shown to protect cardiomyocytes in vitro against doxorubicin-induced apoptosis. The scavenger receptor class B type I (SR-B1), a high affinity HDL receptor, mediates cytoprotective signaling by HDL through AKT. Here we assess whether increased HDL levels protects against doxorubicin-induced cardiotoxicity in vivo, and in cardiomyocytes in culture, and explore the intracellular signaling mechanisms involved, in particular the role of SR-B1. Mice with increased HDL levels, through overexpression of human ApoA1 (ApoA1(Tg/Tg)) and wild type mice (ApoA1(+/+)) with normal HDL levels were treated repeatedly with doxorubicin. Following treatment, ApoA1(+/+) mice displayed cardiac dysfunction as evidenced by reduced LVESP and +dP/dt, and histological analysis revealed cardiomyocyte atrophy and increased cardiomyocyte apoptosis following doxorubicin treatment. In contrast, ApoA1(Tg/Tg) mice were protected against doxorubicin-induced cardiac dysfunction and cardiomyocyte atrophy and apoptosis. When SR-B1 was knocked out, however, overexpression of ApoA1 overexpression did not protect against doxorubicin-induced cardiotoxicity. Using primary neonatal mouse cardiomyocytes and human immortalized ventricular cardiomyocytes in combination with genetic knockout, inhibitors, or siRNA mediated knockdown, we demonstrate that SR-B1 is required for HDL mediated protection of cardiomyocytes against doxorubicin-induced apoptosis in vitro, via a pathway involving PI3K and AKT1/2. Our findings provide proof of concept that raising ApoA1 to supra-physiological levels can dramatically protect against doxorubicin-induced cardiotoxicity via a pathway that is mediated by SR-B1 and involves AKT1/2 activation in cardiomyocytes. Copyright © 2016, American Journal of Physiology-Heart and Circulatory Physiology.

  19. Microparticles from apoptotic RAW 264.7 macrophage cells carry tumour necrosis factor-α functionally active on cardiomyocytes from adult mice

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    Edward Milbank

    2015-10-01

    Full Text Available After ischaemic injury and in patients with atherosclerosis, the pool of inflammatory macrophages is enlarged in the heart and in atherosclerotic plaques. Monocyte/macrophage-derived microparticles (MPs are part of the pathological process of unstable atherosclerotic plaques. The present study focused on effects of MPs, produced by apoptotic murine RAW 264.7 macrophage cell line, in adult murine cardiomyocytes. Flow cytometry and western blot analysis showed that these MPs contained the soluble form of tumour necrosis factor alpha (TNF-α. Cardiomyocyte sarcomere shortening amplitudes and kinetics were reduced within 5 min of exposure to these MPs. Conversely, Ca2+ transient amplitude and kinetics were not modified. The contractile effects of MPs were completely prevented after pretreatment with nitric oxide synthase, guanylate cyclase or TNF-α inhibitors as well as blocking TNF-α receptor 1 with neutralizing antibody. Microscopy showed that, after 1 h, MPs were clearly surrounding rod-shaped cardiomyocytes, and after 2 h they were internalized into cardiomyocytes undergoing apoptosis. After 4 h of treatment with MPs, cardiomyocytes expressed increased caspase-3, caspase-8, Bax and cytochrome C. Thus, MPs from apoptotic macrophages induced a negative inotropic effect and slowing of both contraction and relaxation, similar to that observed in the presence of TNF-α. The use of specific inhibitors strongly suggests that TNF-α receptors and the guanylate cyclase/cGMP/PKG pathway were involved in the functional responses to these MPs and that the mitochondrial intrinsic pathway was implicated in their proapoptotic effects. These data suggest that MPs issued from activated macrophages carrying TNF-α could contribute to propagation of inflammatory signals leading to myocardial infarction.

  20. Heart rate variability in porcine progressive peritonitis-induced sepsis

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    Dagmar eJarkovska

    2016-01-01

    Full Text Available Accumulating evidence suggests that heart rate variability (HRV alterations could serve as an indicator of sepsis progression and outcome, however, the relationships of HRV and major pathophysiological processes of sepsis remain unclear. Therefore, in this experimental study HRV was investigated in a clinically relevant long-term porcine model of severe sepsis/septic shock. HRV was analyzed by several methods and the parameters were correlated with pathophysiological processes of sepsis.In 16 anesthetized, mechanically ventilated and instrumented domestic pigs of either gender, sepsis was induced by fecal peritonitis. Experimental subjects were screened up to the refractory shock development or death. ECG was continuously recorded throughout the experiment, afterwards RR intervals were detected and HRV parameters computed automatically using custom made measurement and analysis MATLAB routines. In all septic animals, progressive hyperdynamic septic shock developed. The statistical measures of HRV, geometrical measures of HRV and Poincaré plot analysis revealed a pronounced reduction of HRV that developed quickly upon the onset of sepsis and was maintained throughout the experiment. The frequency domain analysis demonstrated a decrease in the high frequency component and increase in the low frequency component together with an increase of the low/high frequency component ratio. The reduction of HRV parameters preceded sepsis-associated hemodynamic changes including heart rate increase or shock progression.In a clinically relevant porcine model of peritonitis-induced progressive septic shock, reduction of HRV parameters heralded sepsis development. HRV reduction was associated with a pronounced parasympathetic inhibition and a shift of sympathovagal balance. Early reduction of HRV may serve as a non-invasive and sensitive marker of systemic inflammatory syndrome, thereby widening the therapeutic window for early interventions.

  1. An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.

    Science.gov (United States)

    Rast, Georg; Weber, Jürgen; Disch, Christoph; Schuck, Elmar; Ittrich, Carina; Guth, Brian D

    2015-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes are available from various sources and they are being evaluated for safety testing. Several platforms are available offering different assay principles and read-out parameters: patch-clamp and field potential recording, imaging or photometry, impedance measurement, and recording of contractile force. Routine use will establish which assay principle and which parameters best serve the intended purpose. We introduce a combination of field potential recording and calcium ratiometry from spontaneously beating cardiomyocytes as a novel assay providing a complementary read-out parameter set. Field potential recording is performed using a commercial multi-well multi-electrode array platform. Calcium ratiometry is performed using a fiber optic illumination and silicon avalanche photodetectors. Data condensation and statistical analysis are designed to enable statistical inference of differences and equivalence with regard to a solvent control. Simultaneous recording of field potentials and calcium transients from spontaneously beating monolayers was done in a nine-well format. Calcium channel blockers (e.g. nifedipine) and a blocker of calcium store release (ryanodine) can be recognized and discriminated based on the calcium transient signal. An agonist of L-type calcium channels, FPL 64176, increased and prolonged the calcium transient, whereas BAY K 8644, another L-type calcium channel agonist, had no effect. Both FPL 64176 and various calcium channel antagonists have chronotropic effects, which can be discriminated from typical "chronotropic" compounds, like (±)isoprenaline (positive) and arecaidine propargyl ester (negative), based on their effects on the calcium transient. Despite technical limitations in temporal resolution and exact matching of composite calcium transient with the field potential of a subset of cells, the combined recording platform enables a refined interpretation of the field potential

  2. Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

    Science.gov (United States)

    Hamada, Tomoyo; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

    2012-06-01

    We have developed a three-dimensionally controlled in vitro human cardiomyocyte network assay for the measurements of drug-induced conductivity changes and the appearance of fatal arrhythmia such as ventricular tachycardia/fibrillation for more precise in vitro predictive cardiotoxicity. To construct an artificial conductance propagation model of a human cardiomyocyte network, first, we examined the cell concentration dependence of the cell network heights and found the existence of a height limit of cell networks, which was double-layer height, whereas the cardiomyocytes were effectively and homogeneously cultivated within the microchamber maintaining their spatial distribution constant and their electrophysiological conductance and propagation were successfully recorded using a microelectrode array set on the bottom of the microchamber. The pacing ability of a cardiomyocyte's electrophysiological response has been evaluated using microelectrode extracellular stimulation, and the stimulation for pacing also successfully regulated the beating frequencies of two-layered cardiomyocyte networks, whereas monolayered cardiomyocyte networks were hardly stimulated by the external electrodes using the two-layered cardiomyocyte stimulation condition. The stability of the lined-up shape of human cardiomyocytes within the rectangularly arranged agarose microchambers was limited for a two-layered cardiomyocyte network because their stronger force genera