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Sample records for hl-1 mouse cardiomyocytes

  1. Impact of mitochondria on nitrite metabolism in HL-1 cardiomyocytes

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    Peter eDungel

    2013-05-01

    Full Text Available Apart from ATP synthesis mitochondria have many other functions, one being nitrite reductase activity. NO released from nitrite has been shown to protect the heart from ischemia/reperfusion injury in a cGMP-dependent manner. However, the exact impact of mitochondria on the release of NO from nitrite in cardiomyocytes is not completely understood. Besides mitochondria, a number of non-mitochondrial metalloproteins have been suggested to facilitate this process. The aim of this study was to investigate the impact of mitochondria on the bioactivation of nitrite in HL-1 cardiomyocytes.The levels of nitrosyl complexes of hemoglobin (NO-Hb and cGMP levels were measured by electron spin resonance spectroscopy and enzyme immunoassay. In addition the formation of free NO was determined by confocal microscopy as well as intracellular nitrite and S-nitrosothiols by chemoluminescence analysis. NO was released from nitrite in cell culture in an oxygen dependent manner. Application of specific inhibitors of the respiratory chain, p450, NO synthases and xanthine oxidoreductase showed that all four enzymatic systems are involved in the release of NO, but more than 50% of NO is released via the mitochondrial pathway. Only NO released by mitochondria activated cGMP synthesis. Cardiomyocytes co-cultured with red blood cells (RBC competed with RBC for nitrite, but free NO was detected only in HL-1 cells suggesting that RBC are not a source of NO in this model. Apart from activation of cGMP synthesis, NO formed in HL-1 cells diffused out of the cells and formed NO-Hb complexes. In addition nitrite was converted by HL-1 cells to S-nitrosyl complexes. In HL-1 cardiomyocytes, several enzymatic systems are involved in nitrite reduction to NO but only the mitochondrial pathway of NO release activates cGMP synthesis. Our data suggest that this pathway may be a key regulator of myocardial contractility especially under hypoxic conditions.

  2. 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

  3. Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

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    Stimers, Joseph R; Song, Li; Rusch, Nancy J; Rhee, Sung W

    2015-01-01

    Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

  4. Doxazosin stimulates galectin-3 expression and collagen synthesis in HL-1 cardiomyocytes independent of protein kinase C pathway

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    Xiaoqian Qian

    2016-12-01

    Full Text Available Doxazosin, a drug commonly prescribed for hypertension and prostate disease, increases heart failure risk. However, the underlying mechanism remains unclear. Galectin-3 is an important mediator that plays a pathogenic role in cardiac hypertrophy and heart failure. In the present study, we investigated whether doxazosin could stimulate galectin-3 expression and collagen synthesis in cultured HL-1 cardiomyocytes. We found that doxazosin dose-dependently induced galectin-3 protein expression, with a statistically significant increase in expression with a dose as low as 0.01 μM. Doxazosin upregulated collagen I and α-smooth muscle actin (α-SMA protein levels and also induced apoptotic protein caspase-3 in HL-1 cardiomyocytes. Although we previously reported that activation of protein kinase C (PKC stimulates galectin-3 expression, blocking the PKC pathway with the PKC inhibitor chelerythrine did not prevent doxazosin-induced galectin-3 and collagen expression. Consistently, doxazosin treatment did not alter total and phosphorylated PKC. These results suggest that doxazosin-stimulated galectin-3 is independent of PKC pathway. To determine if the α1-adrenergic pathway is involved, we pretreated the cells with the irreversible α-adrenergic receptor blocker phenoxybenzamine and found that doxazosin-stimulated galectin-3 and collagen expression was similar to controls, suggesting that doxazosin acts independently of α1-adrenergic receptor blockade. Collectively, we show a novel effect of doxazosin on cardiomycytes by stimulating heart fibrosis factor galectin-3 expression. The mechanism of action of doxazosin is not mediated through either activation of the PKC pathway or antagonism of α1-adrenergic receptors.

  5. Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

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    Graves Bridget M

    2012-06-01

    Full Text Available Abstract The phosphoinositide 3-kinases (PI3K/Akt dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20 μM, a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM; β (TGX-221; 100 nM and γ (AS-252424; 100 nM, to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20 μM, which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.

  6. Direct Exposure to Ethanol Disrupts Junctional Cell-Cell Contact and Hippo-YAP Signaling in HL-1 Murine Atrial Cardiomyocytes.

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    Kanako Noritake

    Full Text Available Direct exposure of cardiomyocytes to ethanol causes cardiac damage such as cardiac arrythmias and apoptotic cell death. Cardiomyocytes are connected to each other through intercalated disks (ID, which are composed of a gap junction (GJ, adherens junction, and desmosome. Changes in the content as well as the subcellular localization of connexin43 (Cx43, the main component of the cardiac GJ, are reportedly involved in cardiac arrythmias and subsequent damage. Recently, the hippo-YAP signaling pathway, which links cellular physical status to cell proliferation, differentiation, and apoptosis, has been implicated in cardiac homeostasis under physiological as well as pathological conditions. This study was conducted to explore the possible involvement of junctional intercellular communication, mechanotransduction through cytoskeletal organization, and the hippo-YAP pathway in cardiac damage caused by direct exposure to ethanol. HL-1 murine atrial cardiac cells were used since these cells retain cardiac phenotypes through ID formation and subsequent synchronous contraction. Cells were exposed to 0.5-2% ethanol; significant apoptotic cell death was observed after exposure to 2% ethanol for 48 hours. A decrease in Cx43 levels was already observed after 3 hours exposure to 2% ethanol, suggesting a rapid degradation of this protein. Upon exposure to ethanol, Cx43 translocated into lysosomes. Cellular cytoskeletal organization was also dysregulated by ethanol, as demonstrated by the disruption of myofibrils and intermediate filaments. Coinciding with the loss of cell-cell adherence, decreased phosphorylation of YAP, a hippo pathway effector, was also observed in ethanol-treated cells. Taken together, the results provide evidence that cells exposed directly to ethanol show 1 impaired cell-cell adherence/communication, 2 decreased cellular mechanotransduction by the cytoskeleton, and 3 a suppressed hippo-YAP pathway. Suppression of hippo-YAP pathway

  7. MitoNEET Protects HL-1 Cardiomyocytes from Oxidative Stress Mediated Apoptosis in an In Vitro Model of Hypoxia and Reoxygenation.

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    Anika Habener

    Full Text Available The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an in vitro model of hypoxia and reoxygenation (H/R. Our results show that siRNA knockdown (KD of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE, indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA, known to effectively inhibit glutathione reductase (GSR and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach

  8. MitoNEET Protects HL-1 Cardiomyocytes from Oxidative Stress Mediated Apoptosis in an In Vitro Model of Hypoxia and Reoxygenation.

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    Habener, Anika; Chowdhury, Arpita; Echtermeyer, Frank; Lichtinghagen, Ralf; Theilmeier, Gregor; Herzog, Christine

    2016-01-01

    The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an in vitro model of hypoxia and reoxygenation (H/R). Our results show that siRNA knockdown (KD) of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE), indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), known to effectively inhibit glutathione reductase (GSR) and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach for the

  9. 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.

  10. Genetic enrichment of cardiomyocytes derived from mouse ...

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    The transgenic ESC line, designated MN6 retained the undifferentiated state and the potential of cardiogenic differentiation. After G418 selection, more than 99% of cells expressed α-sarcomeric actin. Immunocytological and ultrastructural analysis demonstrated that, the selected cardiomyocytes were highly differentiated.

  11. Hypoxia Enhances Direct Reprogramming of Mouse Fibroblasts to Cardiomyocyte-Like Cells.

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    Wang, Yanyan; Shi, Shujun; Liu, Huiwen; Meng, Li

    2016-02-01

    Recent work has shown that mouse and human fibroblasts can be reprogrammed to cardiomyocyte-like cells with a combination of transcription factors. Current research has focused on improving the efficiency and mechanisms for fibroblast reprogramming. Previously, it has been reported that hypoxia enhances fibroblast cell reprogramming to pluripotent stem cells. In this study, we observed that 6 h of hypoxic conditions (2% oxygen) on newborn mouse dermal fibroblasts can improve the efficiency of reprogramming to cardiomyocyte-like cells. Expression of cardiac-related genes and proteins increased at 4 weeks after transfer of three transcription factors (Gata4/Mef2c/Tbx5 [GMT]). However, beating cardiomyocyte cells were not detected. The epigenetic mechanism of hypoxia-induced fibroblast reprogramming to cardiomyocyte cells requires further study.

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

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    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.

  13. Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H{sub 2}O{sub 2} in HL-1 mouse cardiac muscle cells

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    Rao, F. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Deng, C.Y. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Zhang, Q.H.; Xue, Y.M. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Xiao, D.Z.; Kuang, S.J.; Lin, Q.X.; Shan, Z.X.; Liu, X.Y.; Zhu, J.N. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Yu, X.Y. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Wu, S.L. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China)

    2013-09-06

    Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H{sub 2}O{sub 2}), but not angiotensin II, stimulated MIF expression in HL-1 cells. H{sub 2}O{sub 2}-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H{sub 2}O{sub 2}-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

  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. Rapid genetic algorithm optimization of a mouse computational model: benefits for anthropomorphization of neonatal mouse cardiomyocytes.

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    Bot, Corina T; Kherlopian, Armen R; Ortega, Francis A; Christini, David J; Krogh-Madsen, Trine

    2012-01-01

    While the mouse presents an invaluable experimental model organism in biology, its usefulness in cardiac arrhythmia research is limited in some aspects due to major electrophysiological differences between murine and human action potentials (APs). As previously described, these species-specific traits can be partly overcome by application of a cell-type transforming clamp (CTC) to anthropomorphize the murine cardiac AP. CTC is a hybrid experimental-computational dynamic clamp technique, in which a computationally calculated time-dependent current is inserted into a cell in real-time, to compensate for the differences between sarcolemmal currents of that cell (e.g., murine) and the desired species (e.g., human). For effective CTC performance, mismatch between the measured cell and a mathematical model used to mimic the measured AP must be minimal. We have developed a genetic algorithm (GA) approach that rapidly tunes a mathematical model to reproduce the AP of the murine cardiac myocyte under study. Compared to a prior implementation that used a template-based model selection approach, we show that GA optimization to a cell-specific model results in a much better recapitulation of the desired AP morphology with CTC. This improvement was more pronounced when anthropomorphizing neonatal mouse cardiomyocytes to human-like APs than to guinea pig APs. CTC may be useful for a wide range of applications, from screening effects of pharmaceutical compounds on ion channel activity, to exploring variations in the mouse or human genome. Rapid GA optimization of a cell-specific mathematical model improves CTC performance and may therefore expand the applicability and usage of the CTC technique.

  16. Genetic selection system allowing monitoring of myofibrillogenesis in living cardiomyocytes derived from mouse embryonic stem cells

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    R Bugorsky

    2009-08-01

    Full Text Available Embryonic stem (ES cell-derived cardiomyocytes recapitulate cardiomyogenesis in vitro and are a potential source of cells for cardiac repair. However, this requires enrichment of mixed populations of differentiating ES cells into cardiomyocytes. Toward this goal, we have generated bicistronic vectors that express both the blasticidin S deaminase (bsd gene and a fusion protein consisting of either myosin light chain (MLC-3f or human a-actinin 2A and enhanced green fluorescent protein (EGFP under the transcriptional control of the a-cardiac myosin heavy chain (a-MHC promoter. Insertion of the DNase I-hypersensitive site (HS-2 element from the b-globin locus control region, which has been shown to reduce transgene silencing in other cell systems, upstream of the transgene promoter enhanced MLC3f-EGFP gene expression levels in mouse ES cell lines. The a-MHC-a- actinin-EGFP, but not the a-MHC-MLC3f-EGFP, construct resulted in the correct incorporation of the newly synthesized fusion protein at the Z-band of the sarcomeres in ES cellderived cardiomyocytes. Exposure of embryoid bodies to blasticidin S selected for a relatively pure population of cardiomyocytes within 3 days. Myofibrillogenesis could be monitored by fluorescence microscopy in living cells due to sarcomeric epitope tagging. Therefore, this genetic system permits the rapid selection of a relatively pure population of developing cardiomyocytes from a heterogeneous population of differentiating ES cells, simultaneously allowing monitoring of early myofibrillogenesis in the selected myocytes.

  17. 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.

  18. 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.

  19. Regulatory Volume Increase and Regulatory Volume Decrease Responses in HL-1 Atrial Myocytes

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    Veronica I. Cacace

    2014-05-01

    Full Text Available Background/Aims: we have investigated whether cultured cardiomyocytes of the cell line HL-1 have the ability to perform regulatory volume responses both in hypotonic and hypertonic conditions. Furthermore, we characterized those regulatory responses and studied the effects of bumetanide and DIDS in volume regulation of HL-1 cells. Methods: we used a light scattering system to measure the transient volume changes of HL-1 cells when subjected to osmotic challenge. Results: We found that HL-1 cells correct for their volume excess by undergoing regulatory volume decrease (RVD, and also respond to hypertonic stress with a regulatory volume increase (RVI. Rate of RVD was 0.08 ± 0.04 intensity/min, and rate of RVI was 0.09 ± 0.01 intensity/min. Volume recovery was 83.68 ± 5.73 % for RVD and 92.3 ± 2.3 % for RVI. Bumetanide 50 µM inhibited volume recovery, from 92.3 ± 2.3 % (control to 24.6 ± 8.8 % and reduced the rate of RVI from 0.070 ± 0.020 intensity/min (control to 0.010 ± 0.005 intensity/min. 50 µM DIDS reduced volume recovery to 42.93 ± 7.7 % and rate of RVI, to 0.03 ± 0.01 intensity/min. Conclusions: these results suggest that bumetanide- and DIDS-sensitive mechanisms are involved in the RVI of HL-1 cells, which points to the involvement of the Na+/K+/2Cl- cotransporter and Cl-/bicarbonate exchanger in RVI, respectively.

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

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    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.

  1. 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

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

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    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

  3. Combined effects of bone morphogenetic protein 10 and crossveinless-2 on cardiomyocyte differentiation in mouse adipocyte-derived stem cells.

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    Jumabay, Medet; Zhumabai, Jiayinaguli; Mansurov, Nurlan; Niklason, Katharine C; Guihard, Pierre J; Cubberly, Mark R; Fogelman, Alan M; Iruela-Arispe, Luisa; Yao, Yucheng; Saparov, Arman; Boström, Kristina I

    2018-03-01

    Bone morphogenetic protein (BMP) 10, a cardiac-restricted BMP family member, is essential in cardiomyogenesis, especially during trabeculation. Crossveinless-2 (CV2, also known as BMP endothelial cell precursor derived regulator [BMPER]) is a BMP-binding protein that modulates the activity of several BMPs. The objective of this study was to examine the combined effects of BMP10 and CV2 on cardiomyocyte differentiation using mouse dedifferentiated fat (mDFAT) cells, which spontaneously differentiate into cardiomyocyte-like cells, as a model. Our results revealed that CV2 binds directly to BMP10, as determined by co-immunoprecipitation, and inhibits BMP10 from initiating SMAD signaling, as determined by luciferase reporter gene assays. BMP10 treatment induced mDFAT cell proliferation, whereas CV2 modulated the BMP10-induced proliferation. Differentiation of cardiomyocyte-like cells proceeded in a reproducible fashion in mDFAT cells, starting with small round Nkx2.5-positive progenitor cells that progressively formed myotubes of increasing length that assembled into beating colonies and stained strongly for Troponin I and sarcomeric alpha-actinin. BMP10 enhanced proliferation of the small progenitor cells, thereby securing sufficient numbers to support formation of myotubes. CV2, on the other hand, enhanced formation and maturation of large myotubes and myotube-colonies and was expressed by endothelial-like cells in the mDFAT cultures. Thus BMP10 and CV2 have important roles in coordinating cardiomyogenesis in progenitor cells. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

  5. miR-27b and miR-23b Modulate Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells

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    José Manuel Vilches

    2014-03-01

    Full Text Available Diverse types of stem cells represent a potentially attractive source of cardiac cells for the treatment of cardiovascular diseases. However, most of the functional benefits reported for stem cell have been modest and mainly due to paracrine effects rather than differentiation into cardiomyocytes of the applied cells. Therefore, new tools need to be developed in order to improve the efficiency of stem cell differentiation towards specific cardiovascular lineages. Here we show that microRNAs that display early differential expression during ventricular maturation, such as miR-27b, inhibits cardiac differentiation from mouse embryonic stem cells whereas miRNAs that display late differential expression, such as miR-23b, regulates the beating phenotype during in vitro cardiac differentiation from Embryonic Stem Cells (ESCs. This study could have an impact on regenerative medicine since we showed that miR-27b and miR-23b overexpression differentially modify the ESC cell fate towards the cardiac lineage.

  6. A promising culture model for analyzing the interaction between adipose tissue and cardiomyocytes.

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    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.

  7. Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

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    Zhenghang Zhao

    Full Text Available Recent studies have suggested that mitochondria may play important roles in the Ca(2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+ flux can regulate the generation of Ca(2+ waves (CaWs and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+ (Cai (2+ was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR Ca(2+ release and CaWs were induced in the presence of high (4 mM external Ca(2+ (Cao (2+. The protonophore carbonyl cyanide p-(trifluoromethoxyphenylhydrazone (FCCP reversibly raised basal Cai (2+ levels even after depletion of SR Ca(2+ in the absence of Cao (2+ , suggesting Ca(2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m or Ru360 (a mitochondrial Ca(2+ uniporter inhibitor, but not by oligomycin (an ATP synthase inhibitor or iodoacetic acid (a glycolytic inhibitor, excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+ release and uptake exquisitely control the local Ca(2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.

  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. 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.

  10. 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.

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

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    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.

  12. Comparative cardiac toxicity of anthracyclines in vitro and in vivo in the mouse.

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    Stefano Toldo

    Full Text Available PURPOSE: The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo. METHODS: The cardiomyocytes were incubated with the three anthracyclines (1 µM to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF, heart rate (HR and cardiac output (CO both prior to and 10 days after drug treatment. RESULTS: In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS, as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin. CONCLUSION: This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.

  13. 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

  14. 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.

  15. ADAM10 modulates calcitriol-regulated RAGE in cardiomyocytes.

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    Lee, Ting-Wei; Kao, Yu-Hsun; Lee, Ting-I; Chen, Yi-Jen

    2017-09-01

    Receptor for advanced glycation end products (RAGE) signalling plays a critical role in the pathogenesis of cardiovascular disease. Calcitriol modulates cardiac RAGE expression. This study explored the mechanisms underlying the effect of calcitriol on RAGE and soluble RAGE (sRAGE) expression in cardiomyocytes. Western blot, ELISA, fluorometric assay and PCR analyses were used to evaluate the RAGE, sRAGE, endogenous secretory RAGE (esRAGE), Jun N-terminal kinase (JNK), and a disintegrin and metalloprotease 10 (ADAM10) expression and enzyme activity in HL-1 atrial myocytes without and with calcitriol (10 and 100 nM), nuclear factor-κB (NF-κB) inhibitor (50 μg/mL), or ADAM10 inhibitor (5 μM) incubation for 48 h. Calcitriol (10 nM) significantly reduced RAGE protein expression and increased sRAGE concentrations in HL-1 cardiomyocytes compared with control cells. These changes were associated with increased protein expression and enzyme activity of ADAM10 and higher mRNA expression of esRAGE. In the presence of ADAM10 inhibitor, however, the suppressive effect of calcitriol on RAGE was diminished. Methylglyoxal (500 μM for 10 min)-mediated JNK phosphorylation was attenuated in the presence of calcitriol (10 nM). Moreover, control and NF-κB inhibitor-treated HL-1 cells had similar RAGE and sRAGE expression, suggesting that calcitriol-mediated RAGE modulation was independent of NF-κB signalling. We showed that RAGE downregulation and increased sRAGE production by calcitriol were mediated through ADAM10 activation in cardiomyocytes. The results suggest that calcitriol has therapeutic potential in treating RAGE-mediated cardiovascular complications. © 2017 Stichting European Society for Clinical Investigation Journal Foundation.

  16. [Study on attenuate and synergistic mechanism between aconiti lateralis praeparata radix and glycyrrhizae radix for toxicity reduction based on metabonomic of MI-RI mouse cardiomyocytes].

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    Li, Ying; Fu, Chao-Mei; Ren, Bo; Liu, Yuan; Gao, Fei; Yang, Huan; Peng, Wei; Gan, Yan-Xiong

    2014-08-01

    Sini decoction (SND), a classical traditional Chinese medicine emergency formula recorded in Shanghan Lun (Treatise on Febrile Diseases), which is composed of Aconiti Lateralis Preparata Radix, Glycyrrhizae Preparata Radix and Zingiberis Rhizoma. Modern clinical and pharmacological researches have shown that SND can protect the myocardium effectively during myocardial ischemia reperfusion injury (MI-RI). A myocardial ischemia reperfusion injury model of H9c2 cardiomyocytes in vitro has been established. Four groups, control group, MI-RI Model group, SND group and SND without Glycyrrhizae Radix group, were arranged. The livability, the level of LDH and CK activity in H9c2 cardiomyocytes in different groups were tested. By combining with principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares projection of latent structures-discriminant analysis (OPLS-DA), 17 biomarkers in extracellular fluid were identified and 15 of them were related to the pathway of biological processes. The results showed that the attenuate and synergistic mechanism between Aconiti Lateralis Praeparata Radix and Glycyrrhizae Radix for toxicity reduction was related with the glycolysis, lipid metabolism, citrate cycle and nitrogen metabolism of amino acids metabolism. The study proved the effect on H9c2 cardiomyocyte treated by MI-RI injury both SND group and SND without Glycyrrhizae Radix group, and compared with the SND without Glycyrrhizae Radix, the protective effect of myocardial ischemia reperfusion injury model of H9c2 cardiomyocyte from SND was stronger.

  17. 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

  18. Group B streptococcal beta-hemolysin/cytolysin directly impairs cardiomyocyte viability and function.

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    Mary E Hensler

    Full Text Available BACKGROUND: Group B Streptococcus (GBS is a leading cause of neonatal sepsis where myocardial dysfunction is an important contributor to poor outcome. Here we study the effects of the GBS pore-forming beta-hemolysin/cytolysin (Bh/c exotoxin on cardiomyocyte viability, contractility, and calcium transients. METHODOLOGY/PRINCIPAL FINDINGS: HL-1 cardiomyocytes exposed to intact wild-type (WT or isogenic Deltabeta h/c mutant GBS, or to cell-free extracts from either strain, were assessed for viability by trypan blue exclusion and for apoptosis by TUNEL staining. Functionality of exposed cardiomyocytes was analyzed by visual quantitation of the rate and extent of contractility. Mitochondrial membrane polarization was measured in TMRE-loaded cells exposed to GBS beta h/c. Effects of GBS beta h/c on calcium transients were studied in fura-2AM-loaded primary rat ventricular cardiomyocytes. Exposure of HL-1 cardiomyocytes to either WT GBS or beta h/c extracts significantly reduced both rate and extent of contractility and later induced necrotic and apoptotic cell death. No effects on cardiomyocyte viability or function were observed after treatment with Deltabeta h/c mutant bacteria or extracts. The beta h/c toxin was associated with complete and rapid loss of detectable calcium transients in primary neonatal rat ventricular cardiomyocytes and induced a loss of mitochondrial membrane polarization. These effects on viability and function were abrogated by the beta h/c inhibitor, dipalmitoyl phosphatidylcholine (DPPC. CONCLUSIONS/SIGNIFICANCE: Our data show a rapid loss of cardiomyocyte viability and function induced by GBS beta h/c, and these deleterious effects are inhibited by DPPC, a normal constituent of human pulmonary surfactant.. These findings have clinical implications for the cardiac dysfunction observed in neonatal GBS infections.

  19. 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.

  20. A Perilipin Gene from Clonostachys rosea f. Catenulata HL-1-1 Is Related to Sclerotial Parasitism

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    Zhan-Bin Sun

    2015-03-01

    Full Text Available Clonostachys rosea f. catenulata is a promising biocontrol agent against many fungal plant pathogens. To identify mycoparasitism-related genes from C. rosea f. catenulata, a suppression subtractive hybridization (SSH cDNA library of C. rosea f. catenulata HL-1-1 that parasitizes the sclerotia of S. sclerotiorum was constructed. 502 clones were sequenced randomly, and thereby 472 expressed sequence tags (ESTs were identified. Forty-three unigenes were annotated and exhibited similarity to a wide diversity of genes. Quantitative real -time PCR showed that a perilipin-like protein encoding gene, Per3, was up-regulated by 6.6-fold over the control at 96 h under the induction of sclerotia. The full-length sequence of Per3 was obtained via 5' and 3' rapid identification of cDNA ends. Overexpression of Per3 in HL-1-1 significantly enhanced the parasitic ability on sclerotia. The results indicated that Per3 might be involved in the mycoparasitism of C. rosea f. catenulata HL-1-1. This is the first report of a perilipin as a potential biocontrol gene in mycoparasites. The study provides usefu l insights into the interaction between C. rosea f. catenulata and fungal plant pathogens.

  1. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    Science.gov (United States)

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  2. Prevention of export of anoxia/reoxygenation injury from ischemic to nonischemic cardiomyocytes via inhibition of endocytosis.

    Science.gov (United States)

    Khaidakov, Magomed; Mercanti, Federico; Wang, Xianwei; Ding, Zufeng; Dai, Yao; Romeo, Francesco; Sawamura, Tatsuya; Mehta, Jawahar L

    2014-06-15

    Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caused by export of injury signals from the infarct zone. The countermeasures to limit infarct size, therefore, should be aimed at nonselective blockade of most, if not all, injury signals from entering nonischemic cells. To test whether inhibition of endocytosis might limit infarct size, HL-1 cardiomyocytes were subjected to anoxia (6 h) and reoxygenation (1 h). Anoxic and reoxygenated cells showed a multifold increase in mitochondrial ROS production accompanied with upregulation of scavenger receptors lectin-like oxidized low-density lipoprotein receptor-1 and CD36 and stimulation of stress signals, including NADPH oxidase subunit p22(phox), SOD2, and beclin-1. Incubation of healthy cardiomyocytes in media from anoxic and reoxygenated cells (conditioned media) resulted in qualitatively similar responses, including increase in the generation of mitochondrial ROS, p22(phox), SOD2, and beclin-1. Anoxia and reoxygenation caused collapse of clathrin-mediated endocytosis and stimulation of macropinocytosis, whereas in cultures exposed to conditioned media, the activity of endocytosis was uniformly higher. Conditioned media also significantly aggravated cytotoxic effects of TNF-α and angiotensin II, and suppression of endocytosis reversed these trends, resulting in an overall increase of metabolic activity. Moreover, inhibition of endocytosis prevented binding of oxidized cellular fragments with greater efficiency than targeted neutralization of the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1. Many of the observations in HL-1 cardiomyocytes were confirmed in primary cardiomyocyte cultures. Our data suggest that endocytosis is upregulated in border zone cardiomyocytes, and inhibition of endocytosis may be an effective approach to prevent export of injury signals from the infarct zone. Copyright © 2014 the American Physiological Society.

  3. Fractalkine depresses cardiomyocyte contractility.

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    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.

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

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    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

  5. Two inhibitory systems and CKIs regulate cell cycle exit of mammalian cardiomyocytes after birth

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    Tane, Shoji; Okayama, Hitomi; Ikenishi, Aiko; Amemiya, Yuki [School of Life Sciences, Faculty of Medicine, Tottori University, Yonago 683-8503 (Japan); Nakayama, Keiichi I. [Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 (Japan); Takeuchi, Takashi, E-mail: takeuchi@med.tottori-u.ac.jp [School of Life Sciences, Faculty of Medicine, Tottori University, Yonago 683-8503 (Japan)

    2015-10-16

    Mammalian cardiomyocytes actively proliferate during embryonic stages, following which they exit their cell cycle after birth, and the exit is maintained. Previously, we showed that two inhibitory systems (the G1-phase inhibitory system: repression of cyclin D1 expression; the M-phase inhibitory system: inhibition of CDK1 activation) maintain the cell cycle exit of mouse adult cardiomyocytes. We also showed that two CDK inhibitors (CKIs), p21{sup Cip1} and p27{sup Kip1}, regulate the cell cycle exit in a portion of postnatal cardiomyocytes. It remains unknown whether the two inhibitory systems are involved in the cell cycle exit of postnatal cardiomyocytes and whether p21{sup Cip1} and p27{sup Kip1} also inhibit entry to M-phase. Here, we showed that more than 40% of cardiomyocytes entered an additional cell cycle by induction of cyclin D1 expression at postnatal stages, but M-phase entry was inhibited in the majority of cardiomyocytes. Marked cell cycle progression and endoreplication were observed in cardiomyocytes of p21{sup Cip1} knockout mice at 4 weeks of age. In addition, tri- and tetranucleated cardiomyocytes increased significantly in p21{sup Cip1} knockout mice. These data showed that the G1-phase inhibitory system and two CKIs (p21{sup Cip1} and p27{sup Kip1}) inhibit entry to an additional cell cycle in postnatal cardiomyocytes, and that the M-phase inhibitory system and p21{sup Cip1} inhibit M-phase entry of cardiomyocytes which have entered the additional cell cycle. - Highlights: • Many postnatal cardiomyocytes entered an additional cell cycle by cyclin D1 induction. • The majority of cardiomyocytes could not enter M-phase after cyclin D1 induction. • Cell cycle progressed markedly in p21{sup Cip1} knockout mice after postnatal day 14. • Tri- and tetranucleated cardiomyocytes increased in p21{sup Cip1} knockout mice.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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

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    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.

  8. Chymase mediates injury and mitochondrial damage in cardiomyocytes during acute ischemia/reperfusion in the dog.

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    Junying Zheng

    Full Text Available Cardiac ischemia and reperfusion (I/R injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI. 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1 and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore

  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. 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.

  11. 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.

  12. 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.

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

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    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.

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

    Science.gov (United States)

    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.

  15. Attenuation of microRNA-16 derepresses the cyclins D1, D2 and E1 to provoke cardiomyocyte hypertrophy

    Science.gov (United States)

    Huang, Shuai; Zou, Xiao; Zhu, Jie-Ning; Fu, Yong-Heng; Lin, Qiu-Xiong; Liang, Ye-You; Deng, Chun-Yu; Kuang, Su-Juan; Zhang, Meng-Zhen; Liao, Yu-Lin; Zheng, Xi-Long; Yu, Xi-Yong; Shan, Zhi-Xin

    2015-01-01

    Cyclins/retinoblastoma protein (pRb) pathway participates in cardiomyocyte hypertrophy. MicroRNAs (miRNAs), the endogenous small non-coding RNAs, were recognized to play significant roles in cardiac hypertrophy. But, it remains unknown whether cyclin/Rb pathway is modulated by miRNAs during cardiac hypertrophy. This study investigates the potential role of microRNA-16 (miR-16) in modulating cyclin/Rb pathway during cardiomyocyte hypertrophy. An animal model of hypertrophy was established in a rat with abdominal aortic constriction (AAC), and in a mouse with transverse aortic constriction (TAC) and in a mouse with subcutaneous injection of phenylephrine (PE) respectively. In addition, a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocyte and based on Ang-II-induced neonatal mouse ventricular cardiomyocyte respectively. We demonstrated that miR-16 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes in rats and mice. Overexpression of miR-16 suppressed rat cardiac hypertrophy and hypertrophic phenotype of cultured cardiomyocytes, and inhibition of miR-16 induced a hypertrophic phenotype in cardiomyocytes. Expressions of cyclins D1, D2 and E1, and the phosphorylated pRb were increased in hypertrophic myocardium and hypertrophic cardiomyocytes, but could be reversed by enforced expression of miR-16. Cyclins D1, D2 and E1, not pRb, were further validated to be modulated post-transcriptionally by miR-16. In addition, the signal transducer and activator of transcription-3 and c-Myc were activated during myocardial hypertrophy, and inhibitions of them prevented miR-16 attenuation. Therefore, attenuation of miR-16 provoke cardiomyocyte hypertrophy via derepressing the cyclins D1, D2 and E1, and activating cyclin/Rb pathway, revealing that miR-16 might be a target to manage cardiac hypertrophy. PMID:25583328

  16. 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.

  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. Hawthorn (Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay.

    Science.gov (United States)

    Salehi, Satin; Long, Shannon R; Proteau, Philip J; Filtz, Theresa M

    2009-01-01

    Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation.

  19. 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.

  20. Transmural Differences in Mechanical Properties of Isolated Subendocardial and Subepicardial Cardiomyocytes.

    Science.gov (United States)

    Khokhlova, A D; Iribe, G

    2016-11-01

    We studied the differences in twitch force of subendocardial and subepicardial cardiomyocytes isolated from mouse left ventricular wall at different preloads using an original single cell stretch method recently developed by us. Then, we used our mathematical models of subendocardial and subepicardial cells to predict underlying cellular mechanisms. Transmural differences in the amplitudes of active tension of subendocardial and subepicardial cardiomyocytes were revealed that could be related to the differences in cooperative end-to-end interaction between the neighboring regulatory units of the thin filament.

  1. Evidence for Cardiomyocyte Renewal in Humans

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. 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

  3. High glucose suppresses embryonic stem cell differentiation into cardiomyocytes : High glucose inhibits ES cell cardiogenesis.

    Science.gov (United States)

    Yang, Penghua; Chen, Xi; Kaushal, Sunjay; Reece, E Albert; Yang, Peixin

    2016-12-09

    Babies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. ESC maintenance and differentiation are currently performed under high glucose conditions, whose adverse effects have never been clarified. We investigated the effect of high glucose on cardiomyocyte differentiation from a well-characterized ESC line, E14, derived from mouse blastocysts. E14 cells maintained under high glucose (25 mM) failed to generate any beating cardiomyocytes using the hanging-drop embryonic body method. We created a glucose-responsive E14 cell line (GR-E14) through a graduated low glucose adaptation. The expression of stem cell markers was similar in the parent E14 cells and the GR-E14 cells. Glucose transporter 2 gene was increased in GR-E14 cells. When GR-E14 cells were differentiated into cardiomyocytes under low (5 mM) or high (25 mM) glucose conditions, high glucose significantly delayed the appearance and reduced the number of TNNT2 (Troponin T Type 2)-positive contracting cardiomyocytes. High glucose suppressed the expression of precardiac mesoderm markers, cardiac transcription factors, mature cardiomyocyte markers, and potassium channel proteins. High glucose impaired the functionality of ESC-derived cardiomyocytes by suppressing the frequencies of Ca 2+ wave and contraction. Our findings suggest that high glucose inhibits ESC cardiogenesis by suppressing key developmental genes essential for the cardiac program.

  4. Telmisartan suppresses cardiac hypertrophy by inhibiting cardiomyocyte apoptosis via the NFAT/ANP/BNP signaling pathway.

    Science.gov (United States)

    Li, Xiurong; Lan, Yuhuai; Wang, Yan; Nie, Minghao; Lu, Yanhong; Zhao, Eryang

    2017-05-01

    Telmisartan, a type of angiotensin II (Ang II) receptor inhibitor, is a common agent used to treat hypertension in the clinic. Hypertension increases cardiac afterload and promotes cardiac hypertrophy. However, the ventricular Ang II receptor may be activated in the absence of hypertension. Therefore, telmisartan may reduce cardiac hypertrophy by indirectly ameliorating hypertensive symptoms and directly inhibiting the cardiac Ang II receptor. Nuclear factor of activated T‑cells (NFAT) contributes to cardiac hypertrophy via nuclear translocation, which induces a cascade of atrial natriuretic peptide (ANP) and brain/B‑type natriuretic peptide (BNP) expression and cardiomyocyte apoptosis. However, NFAT-mediated inhibition of cardiac hypertrophy by telmisartan remains poorly understood. The present study demonstrated that telmisartan suppressed cardiomyocyte hypertrophy in a mouse model of cardiac afterload and in cultured cardiomyocytes by inhibiting NFAT nuclear translocation, as well as by inhibiting ANP and BNP expression and cardiomyocyte apoptosis, in a dose‑dependent manner. The present study provides a novel insight into the potential underlying mechanisms of telmisartan-induced inhibition of cardiomyocyte hypertrophy, which involves inhibition of NFAT activation, nuclear translocation and the ANP/BNP cascade.

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

    Science.gov (United States)

    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

  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. 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.

  8. Testosterone Antagonizes Doxorubicin-Induced Senescence of Cardiomyocytes.

    Science.gov (United States)

    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

  9. Physiological testosterone retards cardiomyocyte aging in Tfm mice via androgen receptor-independent pathway.

    Science.gov (United States)

    Zhang, Li; Lei, Da; Zhu, Gui-Ping; Hong, Lei; Wu, Sai-Zhu

    2013-06-01

    To determine whether testosterone modulates markers of cardiomyocytes aging via its classic androgen receptor (AR)-dependent pathway or conversion to estradiol. Male littermates and testicular feminized (Tfm) mice were randomly separated into 4 experimental groups littermate controls (n=8), Tfm mice (n=7), testosterone-treated Tfm mice (n=8), and Tfm mice treated with testosterone in combination with the aromatase inhibitor anastrazole (n=7). Cardiomyocytes were isolated from mouse left ventricles, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the amount of malondialdehyde (MDA) were measured using colorimetry method, and expression of p16(INK4α) and retinoblastoma (Rb) proteins were detected by Western blotting. The SOD and GSH-Px enzyme activities of cardiomyocytes were decreased, and the MDA levels and the expression of p16(INK4α) and Rb proteins were increased in Tfm mice compared with control mice. An increase was observed in the activities of SOD and GSH-Px enzyme as well as a decrease in MDA levels and the expression of p16(INK4α) and Rb proteins in the testosterone-treated Tfm mice. After co-treatment with anastrazole in Tfm mice, these improvement were partly inhibited. Physiological testosterone replacement can delay cardiomyocyte aging in Tfm mice, an effect that is independent of the AR pathway and in part conversion to estradiol.

  10. Testosterone therapy delays cardiomyocyte aging via an androgen receptor-independent pathway.

    Science.gov (United States)

    Zhang, L; Wu, S Z; Ruan, Y J; Hong, L; Xing, X W; Lai, W Y

    2011-11-01

    The testicular feminized (Tfm) mouse carries a nonfunctional androgen receptor (AR) and reduced circulating testosterone levels. We used Tfm and castrated mice to determine whether testosterone modulates markers of aging in cardiomyocytes via its classic AR-dependent pathway or conversion to estradiol. Male littermates and Tfm mice were divided into 6 experimental groups. Castrated littermates (group 1) and sham-operated Tfm mice (group 2, N = 8 each) received testosterone. Sham-operated Tfm mice received testosterone in combination with the aromatase inhibitor anastrazole (group 3, N = 7). Castrated littermates (group 4) and sham-operated untreated Tfm mice (group 5) were used as controls (N = 8 and 7, respectively). An additional control group (group 6) consisted of age-matched non-castrated littermates (N = 8). Cardiomyocytes were isolated from the left ventricle, telomere length was measured by quantitative PCR and expression of p16INK4α, retinoblastoma (Rb) and p53 proteins was detected by Western blot 3 months after treatment. Compared with group 6, telomere length was short (P testosterone deficiency contributes to cardiomyocyte aging. Physiological testosterone can delay cardiomyocyte aging via an AR-independent pathway and in part by conversion to estradiol.

  11. Role of tumour necrosis factor-a in the regulation of T-type calcium channel current in HL-1 cells.

    Science.gov (United States)

    Rao, Fang; Xue, Yu-Mei; Wei, Wei; Yang, Hui; Liu, Fang-Zhou; Chen, Shao-Xian; Kuang, Su-Juan; Zhu, Jie-Ning; Wu, Shu-Lin; Deng, Chun-Yu

    2016-07-01

    Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation (AF). Although tumour necrosis factor (TNF)-α levels are increased in patients with AF, the role of TNF-α in the pathogenesis of AF remains unclear. Besides L-type Ca(2+) currents (IC a,L ), T-type Ca(2+) currents (IC a,T ) also plays an important role in the pathogenesis of AF. This study was designed to use the whole-cell voltage-clamp technique and biochemical assays to explore if TNF-α is involved in the pathogenesis of AF through regulating IC a,T in atrial myocytes. It was found that compared with sinus rhythm (SR) controls, T-type calcium channel (TCC) subunit mRNA levels were decreased, while TNF-α expression levels were increased, in human atrial tissue from patients with AF. In murine atrial myocyte HL-1 cells, after culturing for 24 h, 12.5, 25 and 50 ng/mL TNF-α significantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner. The peak current was reduced by the application of 12.5 or 25 ng/mL TNF-α in a concentration-dependent manner (from -15.08 ± 1.11 pA/pF in controls to -11.89 ± 0.83 pA/pF and -8.54 ± 1.55 pA/pF in 12.5 or 25 ng/mL TNF-α group respectively). TNF-α application also inhibited voltage-dependent inactivation of IC a,T, shifted the inactivation curve to the left. These results suggest that TNF-α is involved in the pathogenesis of AF, probably via decreasing IC a,T current density in atrium-derived myocytes through impaired channel function and down-regulation of channel protein expression. This pathway thus represents a potential pathogenic mechanism in AF. © 2016 John Wiley & Sons Australia, Ltd.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. 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

  14. The primary cilium coordinates early cardiogenesis and hedgehog signaling in cardiomyocyte differentiation

    DEFF Research Database (Denmark)

    Clement, Christian A; Kristensen, Stine G; Møllgård, Kjeld

    2009-01-01

    Defects in the assembly or function of primary cilia, which are sensory organelles, are tightly coupled to developmental defects and diseases in mammals. Here, we investigated the function of the primary cilium in regulating hedgehog signaling and early cardiogenesis. We report that the pluripotent...... P19.CL6 mouse stem cell line, which can differentiate into beating cardiomyocytes, forms primary cilia that contain essential components of the hedgehog pathway, including Smoothened, Patched-1 and Gli2. Knockdown of the primary cilium by Ift88 and Ift20 siRNA or treatment with cyclopamine......, an inhibitor of Smoothened, blocks hedgehog signaling in P19.CL6 cells, as well as differentiation of the cells into beating cardiomyocytes. E11.5 embryos of the Ift88(tm1Rpw) (Ift88-null) mice, which form no cilia, have ventricular dilation, decreased myocardial trabeculation and abnormal outflow tract...

  15. 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.

  16. 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.

  17. Microcurrent stimulation promotes reverse remodelling in cardiomyocytes.

    Science.gov (United States)

    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.

  18. Analysis of cardiomyocyte movement in the developing murine heart

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. 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...

  20. 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.

  1. Natural Antioxidant-Isoliquiritigenin Ameliorates Contractile Dysfunction of Hypoxic Cardiomyocytes via AMPK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xiaoyu Zhang

    2013-01-01

    Full Text Available Isoliquiritigenin (ISL, a simple chalcone-type flavonoid, is derived from licorice compounds and is mainly present in foods, beverages, and tobacco. Reactive oxygen species (ROS is a critical factor involved in modulating cardiac stress response signaling during ischemia and reperfusion. We hypothesize that ISL as a natural antioxidant may protect heart against ischemic injury via modulating cellular redox status and regulating cardioprotective signaling pathways. The fluorescent probe H2DCFDA was used to measure the level of intracellular ROS. The glucose uptake was determined by 2-deoxy-D-glucose-3H accumulation. The IonOptix System measured the contractile function of isolated cardiomyocytes. The results demonstrated that ISL treatment markedly ameliorated cardiomyocytes contractile dysfunction caused by hypoxia. ISL significantly stimulated cardioprotective signaling, AMP-activated protein kinase (AMPK, and extracellular signal-regulated kinase (ERK signaling pathways. The ROS fluorescent probe H2DCFDA determination indicated that ISL significantly reduced cardiac ROS level during hypoxia/reoxygenation. Moreover, ISL reduced the mitochondrial potential (Δψ of isolated mouse cardiomyocytes. Taken together, ISL as a natural antioxidant demonstrated the cardioprotection against ischemic injury that may attribute to the activation of AMPK and ERK signaling pathways and balance of cellular redox status.

  2. 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.

  3. Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Steve O Winitsky

    2005-04-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  4. Adult Murine Skeletal Muscle Contains Cells That Can Differentiate into Beating Cardiomyocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Winitsky Steve O

    2005-01-01

    Full Text Available It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

  5. Targeting Dynamin 2 as a Novel Pathway to Inhibit Cardiomyocyte Apoptosis Following Oxidative Stress.

    Science.gov (United States)

    Gao, Danchen; Yang, Jian; Wu, Yutao; Wang, Qiwen; Wang, Qiaoling; Lai, En Yin; Zhu, Jianhua

    2016-01-01

    Inhibition of Drp-1-mediated mitochondrial fission limits reactive oxygen species (ROS) production and apoptosis in cardiomyocytes subjected to ischemia/reperfusion injury. It remains unknown if Dynamin 2 inhibition results in similar protective effects. Here we studied the role of Dynamin 2 in cardiomyocyte oxidative stress-induced apoptosis and ROS production. The effect of lentiviral shRNA (lv5-shRNA) mediated Dynamin 2 knockdown on apopotosis, mitochondria, and ROS production were studied in neonatal mouse cardiomycytes, which were further treated with either selective Drp1 inhibitor mdivi-1 or the Dynamin 2/Drp1 inhibitor Dynasore. Apoptosis was evaluated by flow cytometry. Mitochondrial morphology and transmembrane potential (ΔΨm) were studied by confocal microscopy, and ROS production was detected by dichlorofluorescein diacetate. Inhibition of Drp1 and Dynamin 2 protected against mitochondrial fragmentation, maintained ΔΨm, attenuated cellular ROS production and limited apoptosis. Moreover, Lv5-shRNA mediated knockdown of Dynamin 2 alleviated mitochondrial fragmentation, and reduced both ROS production and oxidative stress-induced apoptosis. The protective effects of Dynamin 2 knockdown were enhanced by Dynasore, indicating an added benefit. Oxidative stress-induced apoptosis and ROS production are attenuated by not only Drp1 inhibition but also Dynamin 2 inhibition, implicating Dynamin 2 as a mediator of oxidative stress in cardiomyocytes. © 2016 The Author(s) Published by S. Karger AG, Basel.

  6. Histone hyperacetylation and exon skipping: a calcium-mediated dynamic regulation in cardiomyocytes

    Science.gov (United States)

    Sharma, Alok; Nguyen, Hieu; Cai, Lu; Lou, Hua

    2015-01-01

    In contrast to cell type-specific pre-mRNA alternative splicing, mechanisms controlling activity-dependent alternative splicing is under-studied and not well understood. In a recent study, we conducted a comprehensive analysis of calcium-mediated mechanism that regulates alternative exon skipping in mouse cardiomyocytes. Our results reveal a strong link between histone hyperacetylation and skipping of cassette exons, and provide support to the kinetic coupling model of the epigenetic regulation of alternative splicing at the chromatin level. PMID:26325491

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  1. File list: NoD.CDV.50.AllAg.Cardiomyocytes [Chip-atlas[Archive

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  2. Assessment of the cellular and electrophysiological response of cardiomyocytes to radiation

    Science.gov (United States)

    Helm, Alexander; Ritter, Sylvia; Durante, Marco; Friess, Johannes; Thielemann, Christiane; Mr; Frank, Simon

    Cardiac disease is considered as a late effect resulting from an exposure during long-term space missions. Yet, the underlying mechanisms and the impact of radiation quality and dose are not well understood. To address this topic, we used cardiomyocytes derived from mouse embryonic stem cells (mESC) as a model system. This model has already been successfully used for cardiotoxicity screening of new drugs. Both, the cellular and electrophysiological response to X-ray irradiation were examined. Cellular endpoints such as the induction of micronuclei, apoptosis, number of binucleated cells and expression of connexin43 (Cx 43) were analyzed by standard techniques. For electrophysiological studies a microelectrode array (MEA) was used allowing non-invasive recordings of electrical signals such as signal amplitude and shape, beat rate and conduction velocity. Data analysis was performed using the MATLAB based software DrCell. As a first approach, cardiomyocytes were generated by differentiation of mESC via the formation of embryoid bodies. However, the system proved to be unsuitable due to large intra- and inter-sample variations. In consecutive experiments we used commercially available Cor.At cells, i.e. a pure culture of mESC derived cardiomyocytes. For the analysis of cellular and electrophysiological endpoints Cor.At cells were seeded onto chamber slides or MEA chips, respectively. Irradiation with 0.5 and 2 Gy X-rays (250 kV, 16 mA) was performed two days after seeding. At that time cardiomyocytes are electrically coupled through gap junctions and form a spontaneously beating network. Samples were examined up to four days after exposure. Analysis of the electrophysiological data revealed only minor differences between controls and X-irradiated samples indicating the functionality of cardiomyocytes is not within the dose range examined. Currently, further experiments are performed to statistically verify this finding. Additionally, the expression of Cx 43, a major

  3. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.

    Directory of Open Access Journals (Sweden)

    Anders Waldenström

    Full Text Available BACKGROUND: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. METHODOLOGY/PRINCIPAL FINDINGS: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. CONCLUSIONS/SIGNIFICANCE: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted "cardiosomes", can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Acoustical sensing of cardiomyocyte cluster beating

    Energy Technology Data Exchange (ETDEWEB)

    Tymchenko, Nina; Kunze, Angelika [Dept. of Applied Physics, Chalmers University of Technology, 412 96 Göteborg (Sweden); Dahlenborg, Kerstin [Cellectis, 413 46 Göteborg (Sweden); Svedhem, Sofia, E-mail: sofia.svedhem@chalmers.se [Dept. of Applied Physics, Chalmers University of Technology, 412 96 Göteborg (Sweden); Steel, Daniella [Cellectis, 413 46 Göteborg (Sweden)

    2013-06-14

    Highlights: •An example of the application of QCM-D to live cell studies. •Detection of human pluripotent stem cell-derived cardiomyocyte cluster beating. •Clusters were studied in a thin liquid film and in a large liquid volume. •The QCM-D beating profile provides an individual fingerprint of the hPS-CMCs. -- Abstract: Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cell quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to μm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66–168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.

  7. 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

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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. Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

    Directory of Open Access Journals (Sweden)

    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

  11. BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes.

    Science.gov (United States)

    Hong, Ting-Ting; Smyth, James W; Chu, Kevin Y; Vogan, Jacob M; Fong, Tina S; Jensen, Brian C; Fang, Kun; Halushka, Marc K; Russell, Stuart D; Colecraft, Henry; Hoopes, Charles W; Ocorr, Karen; Chi, Neil C; Shaw, Robin M

    2012-05-01

    Heart failure is a growing epidemic, and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T tubules. Bridging integrator 1 (BIN1) is a membrane scaffolding protein that causes Cav1.2 to traffic to T tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. Intact myocardium and freshly isolated cardiomyocytes from nonfailing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch-clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking-competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after small hairpin RNA-mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino-mediated knockdown of BIN1. BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced to 42% by imaging, and a biochemical T-tubule fraction of Cav1.2 is reduced to 68%. The total calcium current is reduced to 41% in a cell line expressing a nontrafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility. Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  12. 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...

  13. Regulation of the sodium pump during cardiomyocyte adaptation to pregnancy.

    Science.gov (United States)

    Elzwiei, F; Bassien-Capsa, V; St-Louis, J; Chorvatova, A

    2013-01-01

    Regulation of the sodium pump during normal pregnancy and its effect on the function of cardiomyocytes is poorly understood. Our objective was to evaluate the possible implication of the Na(+)-K(+)-ATPase, the sodium pump which controls cellular ionic and metabolic homeostasis, in the adaptations of cardiomyocytes to normal pregnancy. We have used Western blots and patch-clamp measurements to identify changes in the sodium pump proteins. Confocal microscopy was applied to estimate intracellular sodium concentration. Time-resolved spectroscopy was employed to measure mitochondrial NAD(P)H fluorescence and estimate oxidative metabolic state. Optical microscopy was adopted to study the contractility responses of cardiomyocytes. Cells from non-pregnant and pregnant rats (1 day prior parturition) were studied. Our results showed lower protein expression of the α1 Na(+)-K(+)-ATPase isoform in cardiomyocytes in pregnant rats, decreased sodium pump membrane current and elevated steady-state sodium concentration. In addition, ouabain, the inhibitor of the sodium pump capable of increasing cardiomyocyte contractility in non-pregnant rats in a concentration-dependent manner, failed to affect cell contractions in pregnant rats. We also noted modified responsiveness of the mitochondrial metabolic state to ouabain in cardiac cells. The gathered data confirmed that in pregnant rats, the sodium pump protein content and transmembrane flux are decreased, while the sensitivity of cardiomyocyte contractility and the sensitivity of mitochondrial metabolic redox state to ouabain are modified, pointing to regulation of the Na(+)-K(+)-ATPase during cardiac cell adaptations to normal pregnancy.

  14. A non-cardiomyocyte autonomous mechanism of cardioprotection involving the SLO1 BK channel

    Directory of Open Access Journals (Sweden)

    Andrew P. Wojtovich

    2013-03-01

    Full Text Available Opening of BK-type Ca2+ activated K+ channels protects the heart against ischemia-reperfusion (IR injury. However, the location of BK channels responsible for cardioprotection is debated. Herein we confirmed that openers of the SLO1 BK channel, NS1619 and NS11021, were protective in a mouse perfused heart model of IR injury. As anticipated, deletion of the Slo1 gene blocked this protection. However, in an isolated cardiomyocyte model of IR injury, protection by NS1619 and NS11021 was insensitive to Slo1 deletion. These data suggest that protection in intact hearts occurs by a non-cardiomyocyte autonomous, SLO1-dependent, mechanism. In this regard, an in-situ assay of intrinsic cardiac neuronal function (tachycardic response to nicotine revealed that NS1619 preserved cardiac neurons following IR injury. Furthermore, blockade of synaptic transmission by hexamethonium suppressed cardioprotection by NS1619 in intact hearts. These results suggest that opening SLO1 protects the heart during IR injury, via a mechanism that involves intrinsic cardiac neurons. Cardiac neuronal ion channels may be useful therapeutic targets for eliciting cardioprotection.

  15. Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Feng Cao

    Full Text Available Human embryonic stem cells (hESCs can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies.

  16. Loss of epidermal growth factor receptor in vascular smooth muscle cells and cardiomyocytes causes arterial hypotension and cardiac hypertrophy.

    Science.gov (United States)

    Schreier, Barbara; Rabe, Sindy; Schneider, Bettina; Bretschneider, Maria; Rupp, Sebastian; Ruhs, Stefanie; Neumann, Joachim; Rueckschloss, Uwe; Sibilia, Maria; Gotthardt, Michael; Grossmann, Claudia; Gekle, Michael

    2013-02-01

    The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, contributes to parainflammatory dysregulation, possibly causing cardiovascular dysfunction and remodeling. The physiological role of cardiovascular EGFR is not completely understood. To investigate the physiological importance of EGFR in vascular smooth muscle cells and cardiomyocytes, we generated a mouse model with targeted deletion of the EGFR using the SM22 (smooth muscle-specific protein 22) promoter. While the reproduction of knockout animals was not impaired, life span was significantly reduced. Systolic blood pressure was not different between the 2 genotypes-neither in tail cuff nor in intravascular measurements-whereas total peripheral vascular resistance, diastolic blood pressure, and mean blood pressure were reduced. Loss of vascular smooth muscle cell-EGFR results in a dilated vascular phenotype with minor signs of fibrosis and inflammation. Echocardiography, necropsy, and histology revealed a dramatic eccentric cardiac hypertrophy in knockout mice (2.5-fold increase in heart weight), with increased stroke volume and cardiac output as well as left ventricular wall thickness and lumen. Cardiac hypertrophy is accompanied by an increase in cardiomyocyte volume, a strong tendency to cardiac fibrosis and inflammation, as well as enhanced NADPH-oxidase 4 and hypertrophy marker expression. Thus, in cardiomyocytes, EGFR prevents excessive hypertrophic growth through its impact on reactive oxygen species balance, whereas in vascular smooth muscle cells EGFR contributes to the appropriate vascular wall architecture and vessel reactivity, thereby supporting a physiological vascular tone.

  17. Testosterone therapy delays cardiomyocyte aging via an androgen receptor-independent pathway

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    L. Zhang

    2011-11-01

    Full Text Available The testicular feminized (Tfm mouse carries a nonfunctional androgen receptor (AR and reduced circulating testosterone levels. We used Tfm and castrated mice to determine whether testosterone modulates markers of aging in cardiomyocytes via its classic AR-dependent pathway or conversion to estradiol. Male littermates and Tfm mice were divided into 6 experimental groups. Castrated littermates (group 1 and sham-operated Tfm mice (group 2, N = 8 each received testosterone. Sham-operated Tfm mice received testosterone in combination with the aromatase inhibitor anastrazole (group 3, N = 7. Castrated littermates (group 4 and sham-operated untreated Tfm mice (group 5 were used as controls (N = 8 and 7, respectively. An additional control group (group 6 consisted of age-matched non-castrated littermates (N = 8. Cardiomyocytes were isolated from the left ventricle, telomere length was measured by quantitative PCR and expression of p16INK4α, retinoblastoma (Rb and p53 proteins was detected by Western blot 3 months after treatment. Compared with group 6, telomere length was short (P < 0.01 and expression of p16INK4α, Rb and p53 proteins was significantly (P < 0.05 up-regulated in groups 4 and 5. These changes were improved to nearly normal levels in groups 1 and 2 (telomere length = 0.78 ± 0.05 and 0.80 ± 0.08; p16INK4α = 0.13 ± 0.03 and 0.15 ± 0.04; Rb = 0.45 ± 0.05 and 0.39 ± 0.06; p53 = 0.16 ± 0.04 and 0.13 ± 0.03, but did not differ between these two groups. These improvements were partly inhibited in group 3 compared with group 2 (telomere length = 0.65 ± 0.08 vs 0.80 ± 0.08, P = 0.021; p16INK4α = 0.28 ± 0.05 vs 0.15 ± 0.04, P = 0.047; Rb = 0.60 ± 0.06 vs 0.39 ± 0.06, P < 0.01; p53 = 0.34 ± 0.06 vs 0.13 ± 0.03, P = 0.004. In conclusion, testosterone deficiency contributes to cardiomyocyte aging. Physiological testosterone can delay cardiomyocyte aging via an AR-independent pathway and in part by conversion to estradiol.

  18. Collagen scaffolds with or without the addition of RGD peptides support cardiomyogenesis after aggregation of mouse embryonic stem cells.

    Science.gov (United States)

    Dawson, Jennifer; Schussler, Olivier; Al-Madhoun, Ashraf; Menard, Claudine; Ruel, Marc; Skerjanc, Ilona S

    2011-10-01

    Embryonic stem (ES) cell-based cardiac muscle repair using tissue-engineered scaffolds is an attractive prospective treatment option for patients suffering from heart disease. In this study, our aim was to characterize mouse ES cell-derived cardiomyocytes growing on collagen I/III scaffolds, modified with the adhesion peptides arginine-glycine-aspartic acid (RGD). Mouse ES-derived embryoid bodies (EBs) differentiated efficiently into beating cardiomyocytes on the collagen scaffolds. QPCR analysis and immunofluorescent staining showed that cardiomyocytes expressed cardiac muscle-related transcripts and proteins. Analysis of cardiomyocytes by electron microscopy identified muscle fiber bundles and Z bands, typical of ES-derived cardiomyocytes. No differences were detected between the collagen + RGD and collagen control scaffolds. ES cells that were not differentiated as EBs prior to seeding on the scaffold, did not differentiate into cardiomyocytes. These results indicate that a collagen I/III scaffold supports cardiac muscle development and function after EB formation, and that this scaffold appears suitable for future in vivo testing. The addition of the RGD domain to the collagen scaffold did not improve cardiomyocyte development or viability, indicating that RGD signaling to integrins was not a rate-limiting event for cardiomyogenesis from EBs seeded on a collagen scaffold.

  19. GFRA2 Identifies Cardiac Progenitors and Mediates Cardiomyocyte Differentiation in a RET-Independent Signaling Pathway

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    Hidekazu Ishida

    2016-07-01

    Full Text Available A surface marker that distinctly identifies cardiac progenitors (CPs is essential for the robust isolation of these cells, circumventing the necessity of genetic modification. Here, we demonstrate that a Glycosylphosphatidylinositol-anchor containing neurotrophic factor receptor, Glial cell line-derived neurotrophic factor receptor alpha 2 (Gfra2, specifically marks CPs. GFRA2 expression facilitates the isolation of CPs by fluorescence activated cell sorting from differentiating mouse and human pluripotent stem cells. Gfra2 mutants reveal an important role for GFRA2 in cardiomyocyte differentiation and development both in vitro and in vivo. Mechanistically, the cardiac GFRA2 signaling pathway is distinct from the canonical pathway dependent on the RET tyrosine kinase and its established ligands. Collectively, our findings establish a platform for investigating the biology of CPs as a foundation for future development of CP transplantation for treating heart failure.

  20. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    Science.gov (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  1. Cardiomyocyte formation by skeletal muscle-derived multi-myogenic stem cells after transplantation into infarcted myocardium.

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    Tetsuro Tamaki

    Full Text Available BACKGROUND: Cellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported. METHODS AND RESULTS: Skeletal muscle interstitium-derived CD34(+/CD45(- (Sk-34 cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI model (left ventricle. Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed. CONCLUSIONS AND SIGNIFICANCE: Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a

  2. cardiomyocyte-like cells after being co-cultured with cardiomyocytes

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    Jie FAN

    2014-01-01

    Full Text Available Objective To explore the gap junction function and pacing function transformed myocardial cells, rat bone marrow mesenchymal stem cells (BMSCs transfected by hyperpolarization activated cyclic nucleotide-gated cation channel 4 (HCN4 gene were co-cultured with neonatal rat cardiomyocytes (CM. Methods After being labelled with DAPI, neonatal rat CM were co-cultured with BMSCs, HCN4+ BMSCs or HCN4– BMSCs for 4 days, respectively. The spontaneous beating frequency of cells in each group was then detected, and the anatomical location of two types of cells was observed by fluorescence microscopy. Western blotting was used to detect the expression of connexin 43 (Cx43, and the intercellular gap junction was observed by transmission electron microscopy. Results The spontaneous beating rate of ventricular myocardium was significantly higher after being cultured. The anatomical location of BMSCs was found to be adjacent to CM under fluorescence microscope. Western blotting showed that Cx43 expression was significantly higher in HCN4+ MSC co-cultured with CM group than in HCN4– BMSCs cultured with CM group. Transmission electron microscopy revealed that HCN4+ BMSCs formed to be interlaced with adjacent CM. Conclusion The rat BMSCs transfected by HCN4 gene may establish an effective electro-mechanical connection with cardiomyocytes, the spontaneous pacing function is close to that of CM, and the result provides an experimental basis for promoting biological pacemaker therapy. DOI: 10.11855/j.issn.0577-7402.2013.12.02

  3. 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.

  4. Epigenetic revival of a dead cardiomyocyte through mitochondrial interventions.

    Science.gov (United States)

    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.

  5. Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription

    Science.gov (United States)

    Murray, Thomas V.A.; Smyrnias, Ioannis; Schnelle, Moritz; Mistry, Rajesh K.; Zhang, Min; Beretta, Matteo; Martin, Daniel; Anilkumar, Narayana; de Silva, Shana M.; Shah, Ajay M.; Brewer, Alison C.

    2015-01-01

    Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell

  6. 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.

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  14. 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.

  15. 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.

  16. Techniques for the induction of human pluripotent stem cell differentiation towards cardiomyocytes.

    Science.gov (United States)

    Lewandowski, Jarosław; Kolanowski, Tomasz J; Kurpisz, Maciej

    2017-05-01

    The derivation of pluripotent stem cells from human embryos and the generation of induced pluripotent stem cells (iPSCs) from somatic cells opened a new chapter in studies on the regeneration of the post-infarction heart and regenerative medicine as a whole. Thus, protocols for obtaining iPSCs were enthusiastically adopted and widely used for further experiments on cardiac differentiation. iPSC-mediated cardiomyocytes (iPSC-CMs) under in vitro culture conditions are generated by simulating natural cardiomyogenesis and involve the wingless-type mouse mammary tumour virus integration site family (WNT), transforming growth factor beta (TGF-β) and fibroblast growth factor (FGF) signalling pathways. New strategies have been proposed to take advantage of small chemical molecules, organic compounds and even electric or mechanical stimulation. There are three main approaches to support cardiac commitment in vitro: embryoid bodis (EBs), monolayer in vitro cultures and inductive co-cultures with visceral endoderm-like (END2) cells. In EB technique initial uniform size of pluripotent stem cell (PSC) colonies has a pivotal significance. Hence, some methods were designed to support cells aggregation. Another well-suited procedure is based on culturing cells in monolayer conditions in order to improve accessibility of growth factors and nutrients. Other distinct tactics are using visceral endoderm-like cells to culture them with PSCs due to secretion of procardiac cytokines. Finally, the appropriate purification of the obtained cardiomyocytes is required prior to their administration to a patient under the prospective cellular therapy strategy. This goal can be achieved using non-genetic methods, such as the application of surface markers and fluorescent dyes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. 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

  18. Clonality of mouse and human cardiomyogenesis in vivo

    OpenAIRE

    Hosoda, Toru; D'Amario, Domenico; Cabral-Da-Silva, Mauricio Castro; Zheng, Hanqiao; Padin-Iruegas, M. Elena; Ogorek, Barbara; Ferreira-Martins, João; Yasuzawa-Amano, Saori; Amano, Katsuya; Ide-Iwata, Noriko; Cheng, Wei; Rota, Marcello; Urbanek, Konrad; Kajstura, Jan; Anversa, Piero

    2009-01-01

    An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1–5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cel...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. 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

  2. Robust Cardiomyocyte-Specific Gene Expression Following Systemic Injection of AAV: In Vivo Gene Delivery Follows a Poisson Distribution

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    Prasad, Konkal-Matt R.; Xu, Yaqin; Yang, Zequan; Acton, Scott T.; French, Brent A

    2010-01-01

    Newly-isolated serotypes of AAV readily cross the endothelial barrier to provide efficient transgene delivery throughout the body. However, tissue-specific expression is preferred in most experimental studies and gene therapy protocols. Previous efforts to restrict gene expression to the myocardium often relied on direct injection into heart muscle or intracoronary perfusion. Here, we report an AAV vector system employing the cardiac troponin T promoter (cTnT). Using luciferase and eGFP, the efficiency and specificity of cardiac reporter gene expression using AAV serotype capsids: AAV-1, 2, 6, 8 or 9 were tested after systemic administration to 1 week old mice. Luciferase assays showed that the cTnT promoter worked in combination with each of the AAV serotype capsids to provide cardiomyocyte-specific gene expression, but AAV-9 followed closely by AAV-8 was the most efficient. AAV9-mediated gene expression from the cTnT promoter was 640-fold greater in the heart compared to the next highest tissue (liver). eGFP fluorescence indicated a transduction efficiency of 96% using AAV-9 at a dose of only 3.15×1010 viral particles per mouse. Moreover, the intensity of cardiomyocyte eGFP fluorescence measured on a cell-by-cell basis revealed that AAV-mediated gene expression in the heart can be modeled as a Poisson distribution; requiring an average of nearly two vector genomes per cell to attain an 85% transduction efficiency. PMID:20703310

  3. Ultrastructural and functional alterations of EC coupling elements in mdx cardiomyocytes: an analysis from membrane surface to depth.

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    Lorin, Charlotte; Gueffier, Mélanie; Bois, Patrick; Faivre, Jean-François; Cognard, Christian; Sebille, Stéphane

    2013-07-01

    A dilated cardiomyopathy (DCM) is associated with Duchenne muscular dystrophy (DMD). The loss of dystrophin leads to membrane instability and calcium dysregulation in skeletal muscle but effects of such a loss are not elucidated at cardiomyocytes level. We sought to examine whether membrane and transverse tubules damages occur in ventricular myocytes from mdx mouse model of DMD and how they impact the function of single excitation-contraction coupling elements. Scanning ion conductance microscopy (SICM) was used to characterize the integrity loss of living mdx cardiomyocytes surface. 2D Fourier transform analysis of labeled internal networks (transverse tubules, alpha-actinin, dihydropyridine receptors, ryanodine receptors) was performed to evaluate internal alterations. During calcium measurements, "smart microperfusions" of depolarizing solutions were applied through SICM nanopipette, stimulating single tubules elements. These approaches revealed structural membrane surface (39% decrease for Z-groove ratio) and transverse tubules disorganization (21% transverse tubules ratio decrease) in mdx as compared to control. These disruptions were associated with functional alterations (sixfold increase of calcium signal duration and twofold increase of sparks frequency). In DCM associated with DMD, myocytes display evident membrane alterations at the surface level but also in the cell depth with a disruption of transverse tubules network as observed in other cases of heart failure. These ultrastructural changes are associated with changes in the function of some coupling elements. Thus, these profound disruptions may play a role in calcium dysregulation through excitation-contraction coupling elements perturbation and suggest a transverse tubules stabilizing role for dystrophin.

  4. Shock Wave Therapy Promotes Cardiomyocyte Autophagy and Survival during Hypoxia

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    Ling Du

    2017-06-01

    Full Text Available Background: Autophagy plays an important role in cardiovascular disease. Controversy still exists regarding the effect of autophagy on ischemic/hypoxic myocardium. Cardiac shock wave therapy (CSWT is an effective alternative treatment for refractory ischemic heart disease. Whether CSWT can regulate cardiomyocyte autophagy under hypoxic conditions is not clear. We established a myocardial hypoxia model using the H9c2 cell line and performed shock waves (SWs treatment to evaluate the effect of SW on autophagy. Methods: The H9c2 cells were incubated under hypoxic conditions, and SW treatment was then performed at energies of 0.02, 0.05, or 0.10 mJ/mm2. The cell viability and intracellular ATP level were examined. Western blot analysis was used to assess the expression of LC3B, AMPK, mTOR, Beclin-1, Sirt1, and HIF-1α. Autophagic vacuoles were visualized by monodansylcadaverine staining. Results: After the 24-hour hypoxic period, cardiomyocyte viability and ATP levels were decreased and autophagy was significantly increased in H9c2 cells. SW treatment with an energy of 0.05 mJ/mm2 significantly increased the cellular viability, ATP level, LC3B-II/I, and number of autophagic vacuoles. In addition, phosphorylated AMPK and Sirt1 were increased and phosphorylated mTOR and HIF-1α were decreased after SW treatment. Conclusion: SW treatment can potentially promote cardiomyocyte autophagy during hypoxia and protect cardiomyocyte function by regulating the AMPK/mTOR pathway.

  5. Modeling Fatty Acid Transfer from Artery to Cardiomyocyte.

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    Theo Arts

    2015-12-01

    Full Text Available Despite the importance of oxidation of blood-borne long-chain fatty acids (Fa in the cardiomyocytes for contractile energy of the heart, the mechanisms underlying the transfer of Fa from the coronary plasma to the cardiomyocyte is still incompletely understood. To obtain detailed insight into this transfer process, we designed a novel model of Fa transfer dynamics from coronary plasma through the endothelial cells and interstitium to the cardiomyocyte, applying standard physicochemical principles on diffusion and on the chemical equilibrium of Fa binding to carrier proteins Cp, like albumin in plasma and interstitium and Fatty Acid-Binding Proteins within endothelium and cardiomyocytes. Applying these principles, the present model strongly suggests that in the heart, binding and release of Fa to and from Cp in the aqueous border zones on both sides of the cell membranes form the major hindrance to Fa transfer. Although often considered, the membrane itself appears not to be a significant hindrance to diffusion of Fa. Proteins, residing in the cellular membrane, may facilitate transfer of Fa between Cp and membrane. The model is suited to simulate multiple tracer dilution experiments performed on isolated rabbit hearts administrating albumin and Fa as tracer substances into the coronary arterial perfusion line. Using parameter values on myocardial ultrastructure and physicochemical properties of Fa and Cp as reported in literature, simulated washout curves appear to be similar to the experimentally determined ones. We conclude therefore that the model is realistic and, hence, can be considered as a useful tool to better understand Fa transfer by evaluation of experimentally determined tracer washout curves.

  6. 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...

  7. Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

    Science.gov (United States)

    Chen, Zhiheng; Tang, Yamei; Yang, Zuocheng; Liu, Shaojun; Liu, Yong; Li, Yan; He, Wei

    2013-09-01

    Endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are involved in the pathogenesis of cardiac dysfunction. The Mas receptor is a functional binding site for angiotensin (Ang)‑(1-7), which is now considered a critical component of the RAS and exerts cardioprotective effects. To the best of our knowledge, the present study aimed to examine, for the first time, the effects of ET-1 on Mas expression in cultured human cardiomyocytes. Human cardiomyocytes were treated with ET-1 at different concentrations (1, 5, 10, 20 and 30 nM) for varied time periods (0.5, 1.5, 3, 4.5 or 6 h) with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123 and ETB receptor blocker BQ788, or different kinase inhibitors. ET-1 decreased the Mas mRNA level in a statistically significant dose- and time-dependent manner within 4.5 h, which was reflected in the dose-dependent downregulation of Mas promoter activity, Mas protein levels and Ang-(1-7) binding on the cell membrane. Actinomycin D (1 mg/ml), BQ123 (1 µM), p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 µM), completely eliminated the inhibitory effects of ET-1 on Mas expression in human cardiomyocytes. In conclusion, the present study demonstrated that ET-1 downregulates Mas expression at the transcription level in human cardiomyocytes via the ETA receptor by a p38 MAPK‑dependent mechanism. This study provides novel insights into the function of ET-1 and the Ang‑(1-7)/Mas axis in cardiac pathophysiology.

  8. Mechanosensitive Kinases Regulate Stiffness-Induced Cardiomyocyte Maturation

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    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

  9. Atrial Fibrillation and Fibrosis: Beyond the Cardiomyocyte Centric View

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    Miragoli, Michele; Glukhov, Alexey V.

    2015-01-01

    Atrial fibrillation (AF) associated with fibrosis is characterized by the appearance of interstitial myofibroblasts. These cells are responsible for the uncontrolled deposition of the extracellular matrix, which pathologically separate cardiomyocyte bundles. The enhanced fibrosis is thought to contribute to arrhythmias “indirectly” because a collagenous septum is a passive substrate for propagation, resulting in impulse conduction block and/or zigzag conduction. However, the emerging results demonstrate that myofibroblasts in vitro also promote arrhythmogenesis due to direct implications upon cardiomyocyte electrophysiology. This electrical interference may be considered beneficial as it resolves any conduction blocks; however, the passive properties of myofibroblasts might cause a delay in impulse propagation, thus promoting AF due to discontinuous slow conduction. Moreover, low-polarized myofibroblasts reduce, via cell-density dependence, the fast driving inward current for cardiac impulse conduction, therefore resulting in arrhythmogenic uniformly slow propagation. Critically, the subsequent reduction in cardiomyocytes resting membrane potential in vitro significantly increases the likelihood of ectopic activity. Myofibroblast densities and the degree of coupling at cellular border zones also impact upon this likelihood. By considering future in vivo studies, which identify myofibroblasts “per se” as a novel targets for cardiac arrhythmias, this review aims to describe the implications of noncardiomyocyte view in the context of AF. PMID:26229964

  10. 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

  11. Clinically relevant strategies for lowering cardiomyocyte glucose uptake for 18F-FDG imaging of myocardial inflammation in mice.

    Science.gov (United States)

    Thackeray, James T; Bankstahl, Jens P; Wang, Yong; Wollert, Kai C; Bengel, Frank M

    2015-04-01

    Myocardial inflammation is an emerging target for novel therapies and thus for molecular imaging. Positron emission tomography (PET) with (18)F-fluorodeoxyglucose (FDG) has been employed, but requires an approach for suppression of cardiomyocyte uptake. We tested clinically viable strategies for their suitability in mouse models in order to optimize preclinical imaging protocols. C57BL/6 mice (n = 56) underwent FDG PET under various conditions. In healthy animals, the effect of low-dose (5 units/kg) or high-dose (500 units/kg, 15 min prior) intravenous heparin, extended fasting (18 h) and the impact of conscious injection with limited, late application of isoflurane anaesthesia after 40 min of conscious uptake were examined in comparison to ketamine/xylazine anaesthesia. Conscious injection/uptake strategies were further evaluated at 3 days after permanent coronary artery occlusion. Under continuous isoflurane anaesthesia, neither heparin administration nor extended fasting significantly impacted myocardial (18)F-FDG accumulation. Injection with 40 min uptake in awake mice resulted in a marked reduction of global myocardial (18)F-FDG uptake compared to standard isoflurane anaesthesia (5.7 ± 1.1 %ID/g vs 30.2 ± 7.9 %ID/g, p < 0.01). Addition of heparin and fasting further reduced uptake compared to conscious injection alone (3.8 ± 1.5 %ID/g, p < 0.01) similar to ketamine/xylazine (2.4 ± 2.2 %ID/g, p < 0.001). In the inflammatory phase, 3 days after myocardial infarction, conscious injection/uptake with and without heparin/fasting identified a marked increase in myocardial (18)F-FDG accumulation that was similar to that observed under ketamine/xylazine. Continuous isoflurane anaesthesia obscures any suppressive effect of heparin or fasting on cardiomyocyte glucose utilization. Conscious injection of FDG in rodents significantly reduces cardiomyocyte uptake and enables further suppression by heparin and fasting, similar

  12. Clinically relevant strategies for lowering cardiomyocyte glucose uptake for {sup 18}F-FDG imaging of myocardial inflammation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Thackeray, James T.; Bankstahl, Jens P.; Bengel, Frank M. [Hanover Medical School, Department of Nuclear Medicine, Hanover (Germany); Wang, Yong; Wollert, Kai C. [Hanover Medical School, Department of Cardiology and Angiology, Hanover (Germany)

    2015-04-01

    Myocardial inflammation is an emerging target for novel therapies and thus for molecular imaging. Positron emission tomography (PET) with {sup 18}F-fluorodeoxyglucose (FDG) has been employed, but requires an approach for suppression of cardiomyocyte uptake. We tested clinically viable strategies for their suitability in mouse models in order to optimize preclinical imaging protocols. C57BL/6 mice (n = 56) underwent FDG PET under various conditions. In healthy animals, the effect of low-dose (5 units/kg) or high-dose (500 units/kg, 15 min prior) intravenous heparin, extended fasting (18 h) and the impact of conscious injection with limited, late application of isoflurane anaesthesia after 40 min of conscious uptake were examined in comparison to ketamine/xylazine anaesthesia. Conscious injection/uptake strategies were further evaluated at 3 days after permanent coronary artery occlusion. Under continuous isoflurane anaesthesia, neither heparin administration nor extended fasting significantly impacted myocardial {sup 18}F-FDG accumulation. Injection with 40 min uptake in awake mice resulted in a marked reduction of global myocardial {sup 18}F-FDG uptake compared to standard isoflurane anaesthesia (5.7 ± 1.1 %ID/g vs 30.2 ± 7.9 %ID/g, p < 0.01). Addition of heparin and fasting further reduced uptake compared to conscious injection alone (3.8 ± 1.5 %ID/g, p < 0.01) similar to ketamine/xylazine (2.4 ± 2.2 %ID/g, p < 0.001). In the inflammatory phase, 3 days after myocardial infarction, conscious injection/uptake with and without heparin/fasting identified a marked increase in myocardial {sup 18}F-FDG accumulation that was similar to that observed under ketamine/xylazine. Continuous isoflurane anaesthesia obscures any suppressive effect of heparin or fasting on cardiomyocyte glucose utilization. Conscious injection of FDG in rodents significantly reduces cardiomyocyte uptake and enables further suppression by heparin and fasting, similar to clinical observations. In

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

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    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

  14. DYRK2 negatively regulates cardiomyocyte growth by mediating repressor function of GSK-3β on eIF2Bε.

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    Celine S Weiss

    Full Text Available BACKGROUND: A prerequisite of hypertrophic response of the myocardium is an increase in protein synthesis. A central regulator of translation initiation is Eukaryotic initiation factor 2B (eIF2B. Here we assessed the hypothesis that regulation of protein synthesis via eIF2Bε is essential to cardiac hypertrophic response in vivo. METHODS: Two transgenic mouse lines were generated with cardiac restricted overexpression of eIF2Bε or its mutant eIF2Bε-eIFS(535A, which cannot be inactivated by phosphorylation through GSK-3β. RESULTS: (1 Under baseline conditions eIF2Bε transgenic mice showed no difference in cardiac phenotype compared to wild type, whereas in the mutant eIF2Bε-S(535A an increase in LV/tibia length (7.5 ± 0.4 mg/mm vs. 6.2 ± 0.2 mg/mm, p<0.001 and cardiomyocyte cross sectional area (13004 ± 570 vs. 10843 ± 347 RU, p<0.01 was observed. (2 Cardiac overexpression of eIF2Bε did not change the response of the heart to pathologic stress induced by chronic isoproterenol treatment. (3 Cardiac overexpression of the eIF2Bε transgene was followed by overexpression of DYRK2 which is known to prime the inhibitory action of GSK-3β on eIF2Bε, while DYRK1A and GSK-3β itself were not increased. (4 In C57BL/6 mice after 48 h of isoproterenol-stimulation or aortic banding, eIF2Bε was increased and DYRK2 was concomitantly decreased. (5 In line with these in vivo findings, siRNA knockdown of DYRK2 in cultured cardiomyocytes resulted in decreased levels of p(S535- eIF2Bε, (6 whereas adenoviral induced overexpression of DYRK2 was accompanied by clearly increased phosphorylation of eIF2Bε, indicating a coordinated response pattern (7 Adenoviral induced overexpression of DYRK2 leads to significantly reduced cardiomyocyte size and diminishes hypertrophic response to adrenergic stimulation. CONCLUSIONS: The interaction of GSK-3β and its priming kinase DYRK2 regulate the activity of eIF2Bε in cardiac myocytes. DYRK2 is a novel negative

  15. Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes.

    Science.gov (United States)

    Ruiz-Hurtado, Gema; Li, Linwei; Fernández-Velasco, María; Rueda, Angélica; Lefebvre, Florence; Wang, Yueyi; Mateo, Philippe; Cassan, Cécile; Gellen, Barnabas; Benitah, Jean Pierre; Gómez, Ana María

    2015-10-01

    Abnormalities in cardiomyocyte Ca2+ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca2+ sparks (brief, localized increased in [Ca2+]i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca2+]i transient amplitude and sarcoplasmic reticulum (SR) Ca2+ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca2+ sparks were less frequent, whereas the fractional release and the frequency of Ca2+ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca2+ sparks were more frequent in PMI cells (under conditions of similar SR Ca2+ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca2+ sparks frequency normalized by the SR Ca2+ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca2+ spark frequency. Indeed, using an internal "failing solution" with less ATP (as found in HF), we observed a dramatic decrease in Ca2+ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca2+ load may mask these effects at the Ca2+ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca2+ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients. © 2015 Ruiz

  16. Interaction of annexin A6 with alpha actinin in cardiomyocytes

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    Park Woo J

    2011-01-01

    Full Text Available Abstract Background Annexins are calcium dependent phospholipid binding proteins that are expressed in a wide variety of tissues and implicated in various extra- and intracellular processes. In myocardial tissue, annexins A2, A5 and A6 are particularly abundant, of which the expression levels of annexin A6 has been found to be maximal. Conflicting reports from transgenic mice overexpressing annexin A6 or null mice lacking annexin A6 showed imbalances in intracellular calcium turnover and disturbed cardiac contractility. However, few studies have focussed on the signalling module of annexin A6 in the heart either in normal or in pathological state. Results To identify the putative binding partners of annexin A6 in the heart, ventricular extracts were subjected to glutathione S-transferase (GST- annexin A6 pull down assay and the GST- annexin A6 bound proteins were identified by mass spectrometry. The pull down fractions of ventricular extracts with GST-full length annexin A6 as well as GST-C terminus deleted annexin A6 when immunoblotted with anti sarcomeric alpha (α-actinin antibody showed the presence of α-actinin in the immunoblot which was absent when GST-N terminus deleted annexin A6 was used for pull down. Overexpression of green fluorescent protein (GFP tagged full length annexin A6 showed z-line like appearance in cardiomyocytes whereas GFP-N termimus deleted annexin A6 was mostly localized to the nucleus. Overexpression of GFP-C terminus deleted annexin A6 in cardiomyocytes showed aggregate like appearance in the cytoplasm. Double immunofluorescent staining of cardiomyocytes with anti annexin A6 and anti sarcomeric α-actinin antibodies showed perfect co-localization of these two proteins with annexin A6 appearing like a component of sarcomere. Transient knockdown of annexin A6 in cardiomyocytes by shRNA significantly enhances the contractile functions but does not affect the z-band architecture, as revealed by

  17. Usefulness of field potential as a marker of embryonic stem cell-derived cardiomyocytes, and endpoint analysis of embryonic stem cell test.

    Science.gov (United States)

    Koseki, Naoteru; Deguchi, Jiro; Yamada, Toru; Funabashi, Hitoshi; Seki, Takaki

    2010-12-01

    The embryonic stem cell test (EST) is a validated method and a useful screening tool for drug discovery. EST requires microscopic observation of beating cells to be considered cardiomyocytes as an endpoint assay. However, this procedure is time-consuming and limits the throughput performance. Instead of microscopic observation, we previously established a novel assay method based on cardiac field potential as an endpoint. However, cardiac specificity of this field potential is not yet clarified, because beating cells have not been rigorously evaluated as skeletal or cardiomyocyte. Here, we investigated the relationships between field potential, beating, and cardiac troponin T (cTnT) expression, selected as a cardiomyocyte-specific marker, and evaluated suitability of the field potential as a marker for cardiomyocyte in vehicle or 5-fluorouracil treated embryo bodies. Embryoid bodies of mouse embryonic stem cells (D3) were differentiated in a chamber with multi-electrode array for 5 days, and field potential and beating were measured at the end of differentiation. In addition, these chambers were immunohistochemically stained with anti-cTnT antibody, and the correlation between field potential, beating, and cTnT expression was examined. These results indicated the area of field potential or beating mainly coincided with that of cTnT expression. 5-fluorouracil treatment decreased not only the number of field potential detecting electrodes and beating area, but also cTnT expression, and the area of these parameters was also nearly identical. These results indicate that field potential can be used as a suitable cardiac differentiation marker, and can be a promising parameter of EST.

  18. Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming.

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    Kouris, Nicholas A; Schaefer, Jeremy A; Hatta, Masato; Freeman, Brian T; Kamp, Timothy J; Kawaoka, Yoshihiro; Ogle, Brenda M

    2012-01-01

    Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies.

  19. Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

    Science.gov (United States)

    Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

    2013-01-01

    Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

  20. 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

  1. Transient activation of PKC results in long-lasting detrimental effects on systolic [Ca2+]i in cardiomyocytes by altering actin cytoskeletal dynamics and T-tubule integrity.

    Science.gov (United States)

    Guo, Ang; Chen, Rong; Wang, Yihui; Huang, Chun-Kai; Chen, Biyi; Kutschke, William; Hong, Jiang; Song, Long-Sheng

    2018-01-04

    Protein kinase C (PKC) isozymes contribute to the development of heart failure through dysregulation of Ca2+ handling properties and disruption of contractile function in cardiomyocytes. However, the mechanisms by which PKC activation leads to Ca2+ dysfunction are incompletely understood. Shortly upon ventricular pressure overload in mice, we detected transient PKC activation that was associated with pulsed actin cytoskeletal rearrangement. In cultured cardiomyocytes, transient activation of PKC promoted long-term deleterious effects on the integrity of the transverse (T)- tubule system, resulting in a significant decrease in the amplitude and increase in the rising kinetics of Ca2+ transients. Treatment with a PKCα/β inhibitor restored the synchronization of Ca2+ transients and maintained T-tubule integrity in cultured cardiomyocytes. Supporting these data, PKCα/β inhibition protected against T-tubule remodeling and cardiac dysfunction in a mouse model of pressure overload-induced heart failure. Mechanistically, transient activation of PKC resulted in biphasic actin cytoskeletal rearrangement, consistent with in vivo observations in the pressure overloaded mouse model. Transient inhibition of actin polymerization or depolymerization resulted in severe T-tubule damage, recapitulating the T-tubule damage induced by PKC activation. Moreover, inhibition of stretch activated channels (SAC) protected against T-tubule remodeling and E-C coupling dysfunction induced by transient PKC activation and actin cytoskeletal rearrangement. These data identify a key mechanistic link between transient PKC activation and long-term Ca2+ handling defects through PKC-induced actin cytoskeletal rearrangement and resultant T-tubule damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. 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.

  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. 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.

  5. 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.

  6. Cellular Interplay between Cardiomyocytes and Nonmyocytes in Cardiac Remodeling

    Directory of Open Access Journals (Sweden)

    Norifumi Takeda

    2011-01-01

    Full Text Available Cardiac hypertrophy entails complex structural remodeling involving rearrangement of muscle fibers, interstitial fibrosis, accumulation of extracellular matrix, and angiogenesis. Many of the processes underlying cardiac remodeling have features in common with chronic inflammatory processes. During these processes, nonmyocytes, such as endothelial cells, fibroblasts, and immune cells, residing in or infiltrating into the myocardial interstitium play active roles. This paper mainly addresses the functional roles of nonmyocytes during cardiac remodeling. In particular, we focus on the communication between cardiomyocytes and nonmyocytes through direct cell-cell interactions and autocrine/paracrine-mediated pathways.

  7. Imaging alterations of cardiomyocyte cAMP microdomains in disease

    Directory of Open Access Journals (Sweden)

    Alexander eFroese

    2015-08-01

    Full Text Available 3’,5’-cyclic adenosine monophosphate (cAMP is an important second messenger which regulates heart function by acting in distinct subcellular microdomains. Recent years have provided deeper mechanistic insights into compartmentalized cAMP signaling and its link to cardiac disease. In this mini review, we summarize newest developments in this field achieved by cutting-edge biochemical and biophysical techniques. We further compile the data from different studies into a bigger picture of so far uncovered alterations in cardiomyocyte cAMP microdomains which occur in compensated cardiac hypertrophy and chronic heart failure. Finally, future research directions and translational perspectives are briefly discussed.

  8. 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.

  9. Cardiomyocyte MEA data analysis (CardioMDA)--a novel field potential data analysis software for pluripotent stem cell derived cardiomyocytes.

    Science.gov (United States)

    Pradhapan, Paruthi; Kuusela, Jukka; Viik, Jari; Aalto-Setälä, Katriina; Hyttinen, Jari

    2013-01-01

    Cardiac safety pharmacology requires in-vitro testing of all drug candidates before clinical trials in order to ensure they are screened for cardio-toxic effects which may result in severe arrhythmias. Micro-electrode arrays (MEA) serve as a complement to current in-vitro methods for drug safety testing. However, MEA recordings produce huge volumes of data and manual analysis forms a bottleneck for high-throughput screening. To overcome this issue, we have developed an offline, semi-automatic data analysis software, 'Cardiomyocyte MEA Data Analysis (CardioMDA)', equipped with correlation analysis and ensemble averaging techniques to improve the accuracy, reliability and throughput rate of analysing human pluripotent stem cell derived cardiomyocyte (CM) field potentials. With the program, true field potential and arrhythmogenic complexes can be distinguished from one another. The averaged field potential complexes, analysed using our software to determine the field potential duration, were compared with the analogous values obtained from manual analysis. The reliability of the correlation analysis algorithm, evaluated using various arrhythmogenic and morphology changing signals, revealed a mean sensitivity and specificity of 99.27% and 94.49% respectively, in determining true field potential complexes. The field potential duration of the averaged waveforms corresponded well to the manually analysed data, thus demonstrating the reliability of the software. The software has also the capability to create overlay plots for signals recorded under different drug concentrations in order to visualize and compare the magnitude of response on different ion channels as a result of drug treatment. Our novel field potential analysis platform will facilitate the analysis of CM MEA signals in semi-automated way and provide a reliable means of efficient and swift analysis for cardiomyocyte drug or disease model studies.

  10. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Helen P McWilliams-Koeppen

    Full Text Available Light chain (AL amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(PH-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  11. Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover

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    Bergmann, Olaf; Zdunek, Sofia [Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Alkass, Kanar; Druid, Henrik [Department of Forensic Medicine, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Bernard, Samuel [CNRS UMR5208, Institut Camille Jordan, Universite Claude Bernard Lyon 1 (France); Frisen, Jonas, E-mail: jonas.frisen@ki.se [Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden)

    2011-01-15

    Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously {sup 14}C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that {sup 14}C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Novel insights into cardiomyocytes provided by atomic force microscopy.

    Science.gov (United States)

    Borin, Daniele; Pecorari, Ilaria; Pena, Brisa; Sbaizero, Orfeo

    2017-07-04

    Cardiovascular diseases (CVDs) are the number one cause of death globally, therefore interest in studying aetiology, hallmarks, progress and therapies for these disorders is constantly growing. Over the last decades, the introduction and development of atomic force microscopy (AFM) technique allowed the study of biological samples at the micro- and nanoscopic level, hence revealing noteworthy details and paving the way for investigations on physiological and pathological conditions at cellular scale. The present work is aimed to collect and review the literature on cardiomyocytes (CMs) studied by AFM, in order to emphasise the numerous potentialities of this approach and provide a platform for researchers in the field of cardiovascular diseases. Original data are also presented to highlight the application of AFM to characterise the viscoelastic properties of CMs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. A ketogenic diet increases succinic dehydrogenase activity in aging cardiomyocytes.

    Science.gov (United States)

    Balietti, Marta; Fattoretti, Patrizia; Giorgetti, Belinda; Casoli, Tiziana; Di Stefano, Giuseppina; Solazzi, Moreno; Platano, Daniela; Aicardi, Giorgio; Bertoni-Freddari, Carlo

    2009-08-01

    Impairment of energy metabolism and an increase of reactive oxygen species (ROS) production seem to play a major role in age-related apoptotic loss of cardiomyocytes. Succinic dehydrogenase (SDH) is an important marker of the mitochondrial capability to provide an adequate amount of ATP. Moreover, because of its unique redox properties, SDH activity contributes to maintain the reduced state of the ubiquinone pool. Recent reports have shown that ketone body intake improves cardiac metabolic efficiency and exerts a cardioprotective antioxidant action, we therefore performed a cytochemical investigation of SDH activity in cardiomyocytes of late-adult (19-month-old) rats fed for 8 weeks with a medium-chain triglycerides ketogenic diet (MCT-KD). Young, age-matched and old animals fed with a standard chow were used as controls. The overall area of the precipitates (PA) from SDH activity and the area of the SDH-positive mitochondria (MA) were measured. The percent ratios PA/MA and MA/total myocardial tissue area (MA/TA) were the parameters taken into account. We found that PA/MA was significantly higher in young control rats and in MCT-KD-fed rats versus late-adult and old control rats and in young control versus MCT-KD-fed rats. MA/TA of MCT-KD-fed rats was significantly higher versus age-matched and old control rats and tended to be higher versus young control rats; this parameter was significantly higher in young versus old control rats. Thus, MCT-KD intake partially recovers age-related decrease of SDH activity and increases the myocardial area occupied by metabolically active mitochondria. These effects might counteract metabolic alterations leading to apoptosis-induced myocardial atrophy and failure during aging.

  18. The location of energetic compartments affects energetic communication in cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Rikke eBirkedal

    2014-09-01

    Full Text Available The heart relies on accurate regulation of mitochondrial energy supply to match energy demand. The main regulators are Ca2+ and feedback of ADP and Pi. Regulation via feedback has intrigued for decades. First, the heart exhibits a remarkable metabolic stability. Second, diffusion of ADP and other molecules is restricted specifically in heart and red muscle, where a fast feedback is needed the most. To explain the regulation by feedback, compartmentalization must be taken into account. Experiments and theoretical approaches suggest that cardiomyocyte energetic compartmentalization is elaborate with barriers obstructing diffusion in the cytosol and at the level of the mitochondrial outer membrane (MOM. A recent study suggests the barriers are organized in a lattice with dimensions in agreement with those of intracellular structures. Here, we discuss the possible location of these barriers. The more plausible scenario includes a barrier at the level of MOM. Much research has focused on how the permeability of MOM itself is regulated, and the importance of the creatine kinase system to facilitate energetic communication. We hypothesize that at least part of the diffusion restriction at the MOM level is not by MOM itself, but due to the close physical association between the sarcoplasmic reticulum (SR and mitochondria. This will explain why animals with a disabled creatine kinase system exhibit rather mild phenotype modifications. Mitochondria are hubs of energetics, but also ROS production and signaling. The close association between SR and mitochondria may form a diffusion barrier to ADP added outside a permeabilised cardiomyocyte. But in vivo, it is the structural basis for the mitochondrial-SR coupling that is crucial for the regulation of mitochondrial Ca2+-transients to regulate energetics, and for avoiding Ca2+-overload and irreversible opening of the mitochondrial permeability transition pore.

  19. 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.

  20. 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

  1. 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.

  2. Protective Effects of the Mechanistic Target of Rapamycin against Excess Iron and Ferroptosis in Cardiomyocytes.

    Science.gov (United States)

    Baba, Yuichi; Higa, Jason K; Shimada, Briana K; Horiuchi, Kate M; Suhara, Tomohiro; Kobayashi, Motoi; Woo, Jonathan D; Aoyagi, Hiroko; Marh, Karra S; Kitaoka, Hiroaki; Matsui, Takashi

    2017-11-10

    Clinical studies suggest that myocardial iron is a risk factor for left ventricular remodeling in patients after myocardial infarction (MI). Ferroptosis was recently reported as a mechanism of iron-dependent non-apoptotic cell death. However, ferroptosis in the heart is not well understood. The mechanistic target of rapamycin (mTOR) protects the heart against pathological stimuli such as ischemia. To define the role of cardiac mTOR on cell survival in iron-mediated cell death, we examined cardiomyocyte (CM) cell viability under excess iron and ferroptosis conditions. Adult mouse CMs were isolated from cardiac-specific mTOR transgenic (mTOR-Tg), cardiac-specific mTOR knockout (mTOR-KO), or control mice. CMs were treated with ferric iron [Fe (III)]-citrate, erastin, a class 1 ferroptosis inducer, or Ras Selective Lethal 3 (RSL3), a class 2 ferroptosis inducer. Live/Dead Cell Viability Assays revealed that Fe (III)-citrate, erastin, and RSL3 induced cell death. Co-treatment with ferrostatin-1, a ferroptosis inhibitor, inhibited cell death in all conditions. mTOR overexpression suppressed Fe (III)-citrate, erastin, and RSL3-induced cell death, while mTOR deletion exaggerated cell death in these conditions. H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) measurement of reactive oxygen species (ROS) production showed that erastin-induced ROS production was significantly lower in mTOR-Tg versus control CMs. These findings suggest that ferroptosis is a significant type of cell death in CMs, and that mTOR plays an important role in protecting CMs against excess iron and ferroptosis, at least in part by regulating ROS production. Understanding the effects of mTOR in preventing iron-mediated cell death will provide a new therapy for patients with MI. Copyright © 2017, American Journal of Physiology-Heart and Circulatory Physiology.

  3. Mouse phenotyping.

    Science.gov (United States)

    Fuchs, Helmut; Gailus-Durner, Valérie; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Calzada-Wack, Julia; Da Silva-Buttkus, Patricia; Neff, Frauke; Götz, Alexander; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Kastenmüller, Gabi; Kemter, Elisabeth; Lengger, Christoph; Maier, Holger; Matloka, Mikolaj; Möller, Gabriele; Naton, Beatrix; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Römisch-Margl, Werner; Rozman, Jan; Wang-Sattler, Rui; Schrewe, Anja; Stöger, Claudia; Tost, Monica; Adamski, Jerzy; Aigner, Bernhard; Beckers, Johannes; Behrendt, Heidrun; Busch, Dirk H; Esposito, Irene; Graw, Jochen; Illig, Thomas; Ivandic, Boris; Klingenspor, Martin; Klopstock, Thomas; Kremmer, Elisabeth; Mempel, Martin; Neschen, Susanne; Ollert, Markus; Schulz, Holger; Suhre, Karsten; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Hrabě de Angelis, Martin

    2011-02-01

    Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]). Copyright © 2010 Elsevier Inc. All rights reserved.

  4. 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...

  5. 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...

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

    National Research Council Canada - National Science Library

    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

    ...; 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...

  7. Automated multi-parameter measurement of cardiomyocytes dynamics with digital holographic microscopy.

    Science.gov (United States)

    Rappaz, Benjamin; Moon, Inkyu; Yi, Faliu; Javidi, Bahram; Marquet, Pierre; Turcatti, Gerardo

    2015-05-18

    Compounds tested during drug development may have adverse effects on the heart; therefore all new chemical entities have to undergo extensive preclinical assessment for cardiac liability. Conventional intensity-based imaging techniques are not robust enough to provide detailed information for cell structure and the captured images result in low-contrast, especially to cell with semi-transparent or transparent feature, which would affect the cell analysis. In this paper we show, for the first time, that digital holographic microscopy (DHM) integrated with information processing algorithms automatically provide dynamic quantitative phase profiles of beating cardiomyocytes. We experimentally demonstrate that relevant parameters of cardiomyocytes can be obtained by our automated algorithm based on DHM phase signal analysis and used to characterize the physiological state of resting cardiomyocytes. Our study opens the possibility of automated quantitative analysis of cardiomyocyte dynamics suitable for further drug safety testing and compounds selection as a new paradigm in drug toxicity screens.

  8. Electrophysiological properties and calcium handling of embryonic stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jae Boum Youm

    2016-03-01

    Full Text Available Embryonic stem cell-derived cardiomyocytes (ESC-CMs hold great interest in many fields of research including clinical applications such as stem cell and gene therapy for cardiac repair or regeneration. ESC-CMs are also used as a platform tool for pharmacological tests or for investigations of cardiac remodeling. ESC-CMs have many different aspects of morphology, electrophysiology, calcium handling, and bioenergetics compared with adult cardiomyocytes. They are immature in morphology, similar to sinus nodal-like in the electrophysiology, higher contribution of trans-sarcolemmal Ca2+ influx to Ca2+ handling, and higher dependence on anaerobic glycolysis. Here, I review a detailed electrophysiology and Ca2+ handling features of ESC-CMs during differentiation into adult cardiomyocytes to gain insights into how all the developmental changes are related to each other to display cardinal features of developing cardiomyocytes.

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

    Science.gov (United States)

    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.

  10. Apoptosis of cardiomyocytes in children with right ventricular pressure overload with and without hypoxemia.

    Science.gov (United States)

    Tsang, Flora Hau Fung; Chow, Pak-Cheong; Ma, Yuen-Yuen; Man, Kwan; Cheng, Lik-Cheung; Cheung, Yiu-Fai

    2014-07-01

    Cardiomyocyte apoptosis has been implicated in ventricular remodeling and initiation of cardiac failure. We sought to determine the severity of right ventricular (RV) cardiomyocyte apoptosis in cyanotic and acyanotic children with RV pressure overload. Fourteen patients, seven with tetralogy of Fallot (group I) and seven with pulmonary stenosis and ventricular septal defect (group II), undergoing open-heart surgery were studied. Right ventricular biopsies were examined for cardiomyocyte apoptosis by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling. The magnitude of cardiomyocyte apoptosis was related to preoperative oxygen saturation and postoperative inotrope use and hospital stay. Compared with group I patients, group II patients were significantly older at operation (p = 0.002) and had a larger body size (p right ventricle is related to the severity of hypoxia and may have an impact on postoperative course in terms of early postoperative use of inotropes and duration of hospital stay. © 2014 Wiley Periodicals, Inc.

  11. 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 generation shrunk those cells after peeling off the substrate. The results indicate the importance of fabrication technology of thickness control of cellular networks for effective extracellular stimulation and the potential concerning thick cardiomyocyte networks for long-term cultivation.

  12. Smads, Tak1, and Their Common Target Atf-2 Play a Critical Role in Cardiomyocyte Differentiation

    Science.gov (United States)

    Monzen, Koshiro; Hiroi, Yukio; Kudoh, Sumiyo; Akazawa, Hiroshi; Oka, Toru; Takimoto, Eiki; Hayashi, Doubun; Hosoda, Toru; Kawabata, Masahiro; Miyazono, Kohei; Ishii, Shunsuke; Yazaki, Yoshio; Nagai, Ryozo; Komuro, Issei

    2001-01-01

    We previously demonstrated that bone morphogenetic proteins (BMPs) induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1. Transcription factors Smads mediate transforming growth factor-β signaling and the ATF/CREB family transcription factor ATF-2 has recently been shown to act as a common target of the Smad and the TAK1 pathways. We here examined the role of Smads and ATF-2 in cardiomyocyte differentiation of P19CL6, a clonal derivative of murine P19 cells. Although P19CL6 efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide, P19CL6noggin, a P19CL6 cell line constitutively overexpressing the BMP antagonist noggin, did not differentiate into cardiomyocytes. Cooverexpression of Smad1, a ligand-specific Smad, and Smad4, a common Smad, restored the ability of P19CL6noggin to differentiate into cardiomyocytes, whereas stable overexpression of Smad6, an inhibitory Smad, completely blocked differentiation of P19CL6, suggesting that the Smad pathway is necessary for cardiomyocyte differentiation. ATF-2 stimulated the βMHC promoter activity by the synergistic manner with Smad1/4 and TAK1 and promoted terminal cardiomyocyte differentiation of P19CL6noggin, whereas overexpression of the dominant negative form of ATF-2 reduced the promoter activities of several cardiac-specific genes and inhibited differentiation of P19CL6. These results suggest that Smads, TAK1, and their common target ATF-2 cooperatively play a critical role in cardiomyocyte differentiation. PMID:11352931

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

    Science.gov (United States)

    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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. 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.

  2. 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.

  3. New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique.

    Science.gov (United States)

    Takahashi, Yuto; Tomotsune, Daihachiro; Takizawa, Sakiko; Yue, Fengming; Nagai, Mika; Yokoyama, Tadayuki; Hirashima, Kanji; Sasaki, Katsunori

    2015-06-26

    To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. Cardiomyocytes (1.5 × 10(6)) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm(2) including 2.1 × 10(6) cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or NaOH maceration: G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+), NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. The present results show that close contacts were acquired and facilitated the beating function

  4. Mechanical properties of non-sarcomeric cytoskeleton of mice cardiomyocytes and skeletal muscle fibers after 30-day spaceflight biosatellite BION-M1

    Science.gov (United States)

    Ogneva, Irina; Maximova, Maria; Larina, Irina

    The aim of this study was to determine transversal stiffness of the cortical cytoskeleton and cytoskeletal protein desmin content of the left ventricle cardiomyocytes, soleus and tibialis anterior muscle fibers of the mice after 30-days space flight biosatellite «BION-M1» (Russia, 2013). The dissection was made after 13-16.5 hours after landing. Transversal stiffness was measured in relaxed and calcium activated state by atomic force microscope. Desmin content was estimated by using western-blot, expression level of the gene, coding desmin, - by real time PCR. The transversal stiffness of the cortical cytoskeleton of the cardiomyocytes and fibers of the skeletal muscles (as measured using the atomic force microscopy) did not differ significantly within the study groups in comparison to the vivarium control group, except for its slight increase in tibialis anterior fibers muscle in the post-flight group of animals. The content of beta- and gamma-actin in the membranous fraction of proteins in the left ventricular cardiomyocytes (as detected using the western blot technique) did not differ significantly within all study groups and correlated with the transversal stiffness. Similar situation was revealed in fibers of the soleus muscle and tibialis anterior muscle, as well as correlation with the transversal stiffness of their cortical cytoskeleton was noted. At the same time, the content of beta-actin in the cytoplasmic fraction of proteins was found to be decreased in all types of studied tissues in comparison to the control levels in the post-flight group, as well as lowered beta-actin gene expression rates in the post-flight group of animals (as detected using the RT-PCR technique). After completion of the space flight, content of alpha-actinin-4 was found to be reduced in the membranous fraction of proteins of mouse cardiomyocytes, while its content in the cytoplasmic fraction of proteins did not change significantly. Furthermore, gene expression rates of this

  5. Echinochrome A Protects Mitochondrial Function in Cardiomyocytes against Cardiotoxic Drugs

    Directory of Open Access Journals (Sweden)

    Seung Hun Jeong

    2014-05-01

    Full Text Available Echinochrome A (Ech A is a naphthoquinoid pigment from sea urchins that possesses antioxidant, antimicrobial, anti-inflammatory and chelating abilities. Although Ech A is the active substance in the ophthalmic and cardiac drug Histochrome®, its underlying cardioprotective mechanisms are not well understood. In this study, we investigated the protective role of Ech A against toxic agents that induce death of rat cardiac myoblast H9c2 cells and isolated rat cardiomyocytes. We found that the cardiotoxic agents tert-Butyl hydroperoxide (tBHP, organic reactive oxygen species (ROS inducer, sodium nitroprusside (SNP; anti-hypertension drug, and doxorubicin (anti-cancer drug caused mitochondrial dysfunction such as increased ROS level and decreased mitochondrial membrane potential. Co-treatment with Ech A, however, prevented this decrease in membrane potential and increase in ROS level. Co-treatment of Ech A also reduced the effects of these cardiotoxic agents on mitochondrial oxidative phosphorylation and adenosine triphosphate level. These findings indicate the therapeutic potential of Ech A for reducing cardiotoxic agent-induced damage.

  6. 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.

  7. Altered Cardiomyocyte Function and Trypanosoma cruzi Persistence in Chagas Disease.

    Science.gov (United States)

    Cruz, Jader Santos; Santos-Miranda, Artur; Sales-Junior, Policarpo Ademar; Monti-Rocha, Renata; Campos, Paula Peixoto; Machado, Fabiana Simão; Roman-Campos, Danilo

    2016-05-04

    Chagas disease, caused by the triatominae Trypanosoma cruzi, is one of the leading causes of heart malfunctioning in Latin America. The cardiac phenotype is observed in 20-30% of infected people 10-40 years after their primary infection. The cardiac complications during Chagas disease range from cardiac arrhythmias to heart failure, with important involvement of the right ventricle. Interestingly, no studies have evaluated the electrical properties of right ventricle myocytes during Chagas disease and correlated them to parasite persistence. Taking advantage of a murine model of Chagas disease, we studied the histological and electrical properties of right ventricle in acute (30 days postinfection [dpi]) and chronic phases (90 dpi) of infected mice with the Colombian strain of T. cruzi and their correlation to parasite persistence. We observed an increase in collagen deposition and inflammatory infiltrate at both 30 and 90 dpi. Furthermore, using reverse transcriptase polymerase chain reaction, we detected parasites at 90 dpi in right and left ventricles. In addition, we observed action potential prolongation and reduced transient outward K(+) current and L-type Ca(2+) current at 30 and 90 dpi. Taking together, our results demonstrate that T. cruzi infection leads to important modifications in electrical properties associated with inflammatory infiltrate and parasite persistence in mice right ventricle, suggesting a causal role between inflammation, parasite persistence, and altered cardiomyocyte function in Chagas disease. Thus, arrhythmias observed in Chagas disease may be partially related to altered electrical function in right ventricle. © The American Society of Tropical Medicine and Hygiene.

  8. The spatial pattern of atrial cardiomyocyte calcium signalling modulates contraction.

    Science.gov (United States)

    Mackenzie, Lauren; Roderick, H Llewelyn; Berridge, Michael J; Conway, Stuart J; Bootman, Martin D

    2004-12-15

    We examined the regulation of calcium signalling in atrial cardiomyocytes during excitation-contraction coupling, and how changes in the distribution of calcium impacts on contractility. Under control conditions, calcium transients originated in subsarcolemmal locations and showed local regeneration through activation of calcium-induced calcium release from ryanodine receptors. Despite functional ryanodine receptors being expressed at regular (approximately 2 microm) intervals throughout atrial myocytes, the subsarcolemmal calcium signal did not spread in a fully regenerative manner through the interior of a cell. Rather, there was a diminishing centripetal propagation of calcium. The lack of regeneration was due to mitochondria and SERCA pumps preventing the inward movement of calcium. Inhibiting these calcium buffering mechanisms allowed the globalisation of action potential-evoked responses. In addition, physiological positive inotropic agents, such as endothelin-1 and beta-adrenergic agonists, as well as enhanced calcium current, calcium store loading and inositol 1,4,5-trisphosphate infusion also led to regenerative global responses. The consequence of globalising calcium signals was a significant increase in cellular contraction. These data indicate how calcium signals and their consequences are determined by the interplay of multiple subcellular calcium management systems.

  9. Human cardiomyocyte progenitor cells: a short history of nearly everything.

    Science.gov (United States)

    van Vliet, Patrick; Goumans, Marie-José; Doevendans, Pieter A; Sluijter, Joost P G

    2012-08-01

    The high occurrence of cardiac disease in the Western world has driven clinicians and cardiovascular biologists to look for alternative strategies to treat patients. A challenging approach is the use of stem cells to repair the heart, in itself an inspiring thought. In the past 10 years, stem cells from different sources have been under intense investigation and, as a result, a multitude of studies have been published on the identification, isolation, and characterization, of cardiovascular progenitor cells and repair in different animal models. However, relatively few cardiovascular progenitor populations have been identified in human hearts, including, but not limited to, cardiosphere-derived cells, cKit+ human cardiac stem cells , Isl1+ cardiovascular progenitors, and, in our lab, cardiomyocyte progenitor cells (CMPCs). Here, we aim to provide a comprehensive summary of the past findings and present challenges for future therapeutic potential of CMPCs. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  10. 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

  11. Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes.

    Science.gov (United States)

    Shao, Zuo-Hui; Becker, Lance B; Vanden Hoek, Terry L; Schumacker, Paul T; Li, Chang-Qing; Zhao, Danhong; Wojcik, Kim; Anderson, Travis; Qin, Yimin; Dey, Lucy; Yuan, Chun-Su

    2003-06-01

    This study sought to test whether grape seed proanthocyanidin extract (GSPE) attenuates exogenous and endogenous oxidant stress induced in chick cardiomyocytes and whether this cytoprotection is mediated by PKC activation, mito K(ATP) channel opening, NO production, oxidant scavenging, or iron chelating effects. Cells were exposed to hydrogen peroxide (H(2)O(2)) (exogenous oxidant stress, 0.5mM) or antimycin A (endogenous oxidant stress, 100 micro M) for 2h following pretreatment with GSPE at various concentrations for 2h. Cells were also pretreated with GSPE or with inhibitors of PKC (chelerytherine), mito K(ATP) channel (5-hydroxydecanoate), nitric oxide synthase (nitro-L-arginine methyl ester) for 2h. Oxidant stress was measured by 2',7'-dichlorofluorescin diacetate and cell viability was assessed using propidium iodide. Free radical scavenging and iron chelating ability was tested in vitro. GSPE dose-dependently attenuated oxidant formation and significantly improved cell survival and contractile function. However, inhibitors of PKC, mito K(ATP) channel or NO synthase failed to abolish the protective action of GSPE during H(2)O(2) or antimycin A exposure. In vitro studies suggested that GSPE scavenges H(2)O(2), hydroxyl radical and superoxide, and may chelate iron. These results indicate that GSPE confers cardioprotection against exogenous H(2)O(2)- or antimycin A-induced oxidant injury. Its effect does not require PKC, mito K(ATP) channel, or NO synthase, presumably because it acts by reactive oxygen species scavenging and iron chelating directly.

  12. α-B Crystallin Reverses High Diastolic Stiffness of Failing Human Cardiomyocytes.

    Science.gov (United States)

    Franssen, Constantijn; Kole, Jeroen; Musters, René; Hamdani, Nazha; Paulus, Walter J

    2017-03-01

    Cardiomyocytes with a less distensible titin and interstitial collagen contribute to the high diastolic stiffness of failing myocardium. Their relative contributions and mechanisms underlying loss of titin distensibility were assessed in failing human hearts. Left ventricular tissue was procured in patients with aortic stenosis (AS, n=9) and dilated cardiomyopathy (DCM, n=6). Explanted donor hearts (n=8) served as controls. Stretches were performed in myocardial strips, and an extraction protocol differentiated between passive tension (Fpassive) attributable to cardiomyocytes or to collagen. Fpassive-cardiomyocytes was higher in AS and DCM at shorter muscle lengths, whereas Fpassive-collagen was higher in AS at longer muscle lengths and in DCM at shorter and longer muscle lengths. Cardiomyocytes were stretched to investigate titin distensibility. Cardiomyocytes were incubated with alkaline phosphatase, subsequently reassessed after a period of prestretch and finally treated with the heat shock protein α-B crystallin. Alkaline phosphatase shifted the Fpassive-sarcomere length relation upward only in donor. Prestretch shifted the Fpassive-sarcomere length relation further upward in donor and upward in AS and DCM. α-B crystallin shifted the Fpassive-sarcomere length relation downward to baseline in donor and to lower than baseline in AS and DCM. In failing myocardium, confocal laser microscopy revealed α-B crystallin in subsarcolemmal aggresomes. High cardiomyocyte stiffness contributed to stiffness of failing human myocardium because of reduced titin distensibility. The latter resulted from an absent stiffness-lowering effect of baseline phosphorylation and from titin aggregation. High cardiomyocyte stiffness was corrected by α-B crystallin probably through relief of titin aggregation. © 2017 American Heart Association, Inc.

  13. Resveratrol attenuates ischemia/reperfusion injury in neonatal cardiomyocytes and its underlying mechanism.

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    Min Shen

    Full Text Available This study was designed to investigate whether Resveratrol (Res could be a prophylactic factor in the prevention of I/R injury and to shed light on its underlying mechanism. Primary culture of neonatal rat cardiomyocytes were randomly distributed into three groups: the normal group (cultured cardiomyocytes were in normal conditions, the I/R group (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion, and the Res+I/R group (100 µmol/L Res was administered before cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion. To test the extent of cardiomyocyte injury, several indices were detected including cell viability, LDH activity, Na(+-K(+-ATPase and Ca(2+-ATPase activity. To test apoptotic cell death, caspase-3 activity and the expression of Bcl-2/Bax were detected. To explore the underlying mechanism, several inhibitors, intracellular calcium, SOD activity and MDA content were used to identify some key molecules involved. Res increased cell viability, Na(+-K(+-ATPase and Ca(2+-ATPase activity, Bcl-2 expression, and SOD level. While LDH activity, capase-3 activity, Bax expression, intracellular calcium and MDA content were decreased by Res. And the effect of Res was blocked completely by either L-NAME (an eNOS inhibitor or MB (a cGMP inhibitor, and partly by either DS (a PKC inhibitor or Glybenclamide (a K(ATP inhibitor. Our results suggest that Res attenuates I/R injury in cardiomyocytes by preventing cell apoptosis, decreasing LDH release and increasing ATPase activity. NO, cGMP, PKC and K(ATP may play an important role in the protective role of Res. Moreover, Res enhances the capacity of anti-oxygen free radical and alleviates intracellular calcium overload in cardiomyocytes.

  14. Diabetes increases stiffness of live cardiomyocytes measured by atomic force microscopy nanoindentation.

    Science.gov (United States)

    Benech, Juan C; Benech, Nicolás; Zambrana, Ana I; Rauschert, Inés; Bervejillo, Verónica; Oddone, Natalia; Damián, Juan P

    2014-11-15

    Stiffness of live cardiomyocytes isolated from control and diabetic mice was measured using the atomic force microscopy nanoindentation method. Type 1 diabetes was induced in mice by streptozotocin administration. Histological images of myocardium from mice that were diabetic for 3 mo showed disorderly lineup of myocardial cells, irregularly sized cell nuclei, and fragmented and disordered myocardial fibers with interstitial collagen accumulation. Phalloidin-stained cardiomyocytes isolated from diabetic mice showed altered (i.e., more irregular and diffuse) actin filament organization compared with cardiomyocytes from control mice. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) pump expression was reduced in homogenates obtained from the left ventricle of diabetic animals compared with age-matched controls. The apparent elastic modulus (AEM) for live control or diabetic isolated cardiomyocytes was measured using the atomic force microscopy nanoindentation method in Tyrode buffer solution containing 1.8 mM Ca(2+) and 5.4 mM KCl (physiological condition), 100 nM Ca(2+) and 5.4 mM KCl (low extracellular Ca(2+) condition), or 1.8 mM Ca(2+) and 140 mM KCl (contraction condition). In the physiological condition, the mean AEM was 112% higher for live diabetic than control isolated cardiomyocytes (91 ± 14 vs. 43 ± 7 kPa). The AEM was also significantly higher in diabetic than control cardiomyocytes in the low extracellular Ca(2+) and contraction conditions. These findings suggest that the material properties of live cardiomyocytes were affected by diabetes, resulting in stiffer cells, which very likely contribute to high diastolic LV stiffness, which has been observed in vivo in some diabetes mellitus patients. Copyright © 2014 the American Physiological Society.

  15. Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Zhao, Shaomin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Song, Langying [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Manyuan [School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Jiao, Kai, E-mail: kjiao@uab.edu [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2013-11-29

    Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, the activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.

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

    Directory of Open Access Journals (Sweden)

    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

    Science.gov (United States)

    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. 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. 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.

  20. 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

  1. 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

  2. Early Administration of Glutamine Protects Cardiomyocytes from Post-Cardiac Arrest Acidosis

    Directory of Open Access Journals (Sweden)

    Yan-Ren Lin

    2016-01-01

    Full Text Available Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH < 7.2 were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine, n=20 and control (normal saline, n=20 groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition, cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5 or to culture medium (control. Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group (p<0.05. In addition, in pH 6.5 or pH 7.3 HBSS buffer, the expression levels of cell stress (p53 and apoptosis (caspase-3, Bcl-xL markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR. L-glutamine also increased the beating function of cardiomyocytes, especially at the lower pH level (6.5. More importantly, glutamine decreased cardiomyocyte apoptosis and increased these cells’ beating function at a low pH level.

  3. 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.

  4. Etanercept protects rat cardiomyocytes against hypertrophy by regulating inflammatory cytokines secretion and cell apoptosis.

    Science.gov (United States)

    Li, Q; Yu, Q; Na, R; Liu, B

    2017-05-15

    We aimed to investigate the effect of etanercept, a tumor necrosis factor-α (TNF-α) inhibitor, on rat cardiomyocyte hypertrophy and its underlying mechanism. Primary neonatal rat cardiomyocytes were isolated from Sprague-Dawley rats. The model of rat cardiomyocyte hypertrophy was induced by endothelin, and then treated with different concentrations of etanercept (1, 10, and 50 μM). After treatment, cell counts, viability and cell apoptosis were evaluated. The mRNA levels of myocardial hypertrophy marker genes, including atrial natriuretic factor (ANF), matrix metalloproteinase (MMP)-9 and MMP-13, were detected by qRT-PCR, and the expressions of apoptosis-related proteins (Bcl-2 and Bax) were measured by western blotting. The protein levels of transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β, IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) were determined using enzyme linked immunosorbent assay (ELISA) kits. In the present study, TNF-α level in cardiomyocytes with hypertrophy was significantly enhanced (Petanercept (Petanercept remarkably reduced the mRNA levels of ANF, MMP-9 and MMP-13, inhibited the expression of Bax, and increased the expression of Bcl-2 compared to the model group (Petanercept lowered the levels of IL-1β, IL-6, LIF and CT-1 but not TGF-β1 compared to the model group (PEtanercept may protect rat cardiomyocytes from hypertrophy by inhibiting inflammatory cytokines secretion and cell apoptosis.

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

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    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.

  6. Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes.

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    Mao, Kai; Kobayashi, Satoru; Jaffer, Zahara M; Huang, Yuan; Volden, Paul; Chernoff, Jonathan; Liang, Qiangrong

    2008-02-01

    Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes.

  7. Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells.

    Science.gov (United States)

    Burridge, Paul W; Holmström, Alexandra; Wu, Joseph C

    2015-10-06

    Since the first discovery that human pluripotent stem cells (hPS cells) can differentiate to cardiomyocytes, efforts have been made to optimize the conditions under which this process occurs. One of the most effective methodologies to optimize this process is reductionist simplification of the medium formula, which eliminates complex animal-derived components to help reveal the precise underlying mechanisms. Here we describe our latest, cost-effective and efficient methodology for the culture of hPS cells in the pluripotent state using a modified variant of chemically defined E8 medium. We provide exact guidelines for cell handling under these conditions, including non-enzymatic EDTA passaging, which have been optimized for subsequent cardiomyocyte differentiation. We describe in depth the latest version of our monolayer chemically defined small molecule differentiation protocol, including metabolic selection-based cardiomyocyte purification and the addition of triiodothyronine to enhance cardiomyocyte maturation. Finally, we describe a method for the dissociation of hPS cell-derived cardiomyocytes, cryopreservation, and thawing. Copyright © 2015 John Wiley & Sons, Inc.

  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. 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.

  10. Synthesis and characterization of some bi, tri and tetravalent transition metal complexes of N'-(furan-2-yl-methylene)-2-(p-tolylamino)acetohydrazide HL1 and N'-(thiophen-2-yl-methylene)-2-(p-tolylamino)acetohydrazide HL2

    Science.gov (United States)

    Emam, S. M.; AbouEl-Enein, S. A.; El-Saied, F. A.; Alshater, S. Y.

    2012-06-01

    The tetradentate Schiff bases hydrazone ligands HL1, HL2 and their metal complexes have been prepared and characterized by analytical, spectral (IR, UV-vis, 1H NMR and ESR), molar conductivity, magnetic and TGA measurements. The results show that all the metal complexes are non-electrolytes, except (2, 10 and 20) which have ionic nature. The ligands coordinate in keto-neutral form and act as bidentate or tridentate for all metal complexes, except complexes (4 and 12). The ligands react as monobasic tetradentate and tridentate for complexes (4 and 12), respectively. Octahedral/tetrahedral Co(II) and Ni(II), octahedral/square planar Cu(II), and octahedral Mn(II), Fe(III), Cr(III), Ru(III), Hf(IV) and Zr(IV)O geometries were proposed. The ESR spectra of copper complexes (12 and 14) indicate d ground state with covalent bond character. The thermal decomposition and the types of crystallized water for some metal complexes were studied. The studied metal complexes are very weakly active against the tested microorganisms.

  11. Herpesvirus-Mediated Delivery of a Genetically Encoded Fluorescent Ca2+ Sensor to Canine Cardiomyocytes

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    János Prorok

    2009-01-01

    Full Text Available We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca2+ sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (Ito, kinetics of the intracellular Ca2+ transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the Ito current was significantly changed and no major shifts occurred in parameters of [Ca2+]i transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

  12. Fatty acids composition of inner mitochondrial membrane of rat cardiomyocytes and hepatocytes during hypoxia-hypercapnia

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    S. V. Khyzhnyak

    2016-06-01

    Full Text Available We studied the influence of hypoxic-hypercapnic environment under the effect of hypothermia (artificial hibernation on fatty acids spectrum of inner mitochondrial membrane (IMM lipids of rat cardiomyocytes and hepatocytes. Specific for cellular organelles redistribution of IMM fatty acids was determined. It led to the reduction of total amount of saturated fatty acids (SFAs and increase of unsaturated fatty acids (UFAs in cardiomyocytes and to the increase of SFAs and decrease of UFAs in hepatocytes. The decrease in the content of oleic acid and increased content of arachidonic and docosahexaenoic acids in IMM were shown. This may be due to their role in the regulatory systems during hibernation, as well as following exit therefrom. It is assumed that artificial hibernation state is characterized by the stress reaction leading to optimal readjustment of fatty acids composition of membrane lipids, which supports functional activity of mitochondria in hepatocytes and cardiomyocytes.

  13. Essential role of STIM1 in the development of cardiomyocyte hypertrophy

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    Ohba, Takayoshi [Department of Physiology, Akita University Graduate School of Medicine, Akita (Japan); Watanabe, Hiroyuki [Department of Internal Medicine Division of Cardiovascular and Respiratory Medicine, Akita University Graduate School of Medicine, Akita (Japan); Murakami, Manabu [Department of Physiology, Akita University Graduate School of Medicine, Akita (Japan); Sato, Takako [Department of Internal Medicine Division of Cardiovascular and Respiratory Medicine, Akita University Graduate School of Medicine, Akita (Japan); Ono, Kyoichi [Department of Physiology, Akita University Graduate School of Medicine, Akita (Japan); Ito, Hiroshi, E-mail: hitomed2@gipc.akita-u.ac.jp [Department of Internal Medicine Division of Cardiovascular and Respiratory Medicine, Akita University Graduate School of Medicine, Akita (Japan)

    2009-11-06

    Store-operated Ca{sup 2+} entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48 h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.

  14. 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

  15. Fructose modulates cardiomyocyte excitation-contraction coupling and Ca²⁺ handling in vitro.

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    Kimberley M Mellor

    Full Text Available BACKGROUND: High dietary fructose has structural and metabolic cardiac impact, but the potential for fructose to exert direct myocardial action is uncertain. Cardiomyocyte functional responsiveness to fructose, and capacity to transport fructose has not been previously demonstrated. OBJECTIVE: The aim of the present study was to seek evidence of fructose-induced modulation of cardiomyocyte excitation-contraction coupling in an acute, in vitro setting. METHODS AND RESULTS: The functional effects of fructose on isolated adult rat cardiomyocyte contractility and Ca²⁺ handling were evaluated under physiological conditions (37°C, 2 mM Ca²⁺, HEPES buffer, 4 Hz stimulation using video edge detection and microfluorimetry (Fura2 methods. Compared with control glucose (11 mM superfusate, 2-deoxyglucose (2 DG, 11 mM substitution prolonged both the contraction and relaxation phases of the twitch (by 16 and 36% respectively, p<0.05 and this effect was completely abrogated with fructose supplementation (11 mM. Similarly, fructose prevented the Ca²⁺ transient delay induced by exposure to 2 DG (time to peak Ca²⁺ transient: 2 DG: 29.0±2.1 ms vs. glucose: 23.6±1.1 ms vs. fructose +2 DG: 23.7±1.0 ms; p<0.05. The presence of the fructose transporter, GLUT5 (Slc2a5 was demonstrated in ventricular cardiomyocytes using real time RT-PCR and this was confirmed by conventional RT-PCR. CONCLUSION: This is the first demonstration of an acute influence of fructose on cardiomyocyte excitation-contraction coupling. The findings indicate cardiomyocyte capacity to transport and functionally utilize exogenously supplied fructose. This study provides the impetus for future research directed towards characterizing myocardial fructose metabolism and understanding how long term high fructose intake may contribute to modulating cardiac function.

  16. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Science.gov (United States)

    2011-01-01

    Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within

  17. 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.

  18. 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.

  19. Virtual population generator for human cardiomyocytes parameters: in silico drug cardiotoxicity assessment.

    Science.gov (United States)

    Polak, Sebastian; Fijorek, Kamil; Glinka, Anna; Wisniowska, Barbara; Mendyk, Aleksander

    2012-01-01

    The anatomical and histological parameters of the human ventricle depend on many factors including age and sex. Myocyte volume and electric capacitance are significant physiological parameters of left ventricle cardiomyocyte mathematical models. They allow the assessment of inter-individual variability during in vitro-in vivo extrapolation of the drug cardiotoxic effect. The current research was carried out to analyze the relationship between age, human left ventricle cardiomyocyte volume, and electric capacitance in a healthy population. In order to collect data describing cardiomyocyte volume and membrane area, literature searches were performed. It was assumed that the cardiomyocyte volume (VOL) and area (AREA) distribution have non-negative support and are skewed to the right. A log-linear model with constant variance was used. A simulation study was run to assess the influence of physiological parameters on action potential duration. The coefficient of determination for the proposed model R(2) = 0.95, that is, 95% of the variability observed in log cardiomyocyte volume can be explained by the estimated regression equation. To allow simple calculation and model performance validation, a simple Excel file was developed (Supplementary material). To the best of our knowledge, there is no other model available, combining age, cardiomyocyte volume, and area. The main limitations of the proposed models result from the assumptions made at the data analysis stage. The limited amount of information available in the literature and the lack of differentiation between sexes results in one common equation. The developed model is a part of the computational system for drug cardiotoxicity assessment.

  20. 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

    Full Text Available Advanced glycation end-products (AGEs have been implicated in diverse pathological settings including diabetes, inflammation and acute ischemia/reperfusion injury in the heart. AGEs interact with the receptor for AGEs (RAGE and transduce signals through activation of MAPKs and proapoptotic pathways. In the current study, adult cardiomyocytes were studied in an in vitro ischemia/reperfusion (I/R injury model to delineate the molecular mechanisms underlying RAGE-mediated injury due to hypoxia/reoxygenation (H/R.Cardiomyocytes isolated from adult wild-type (WT, homozygous RAGE-null (RKO, and WT mice treated with soluble RAGE (sRAGE were subjected to hypoxia for 30 minutes alone or followed by reoxygenation for 1 hour. In specific experiments, RAGE ligand carboxymethyllysine (CML-AGE (termed "CML" in this manuscript was evaluated in vitro. LDH, a marker of cellular injury, was assayed in the supernatant in the presence or absence of signaling inhibitor-treated cardiomyocytes. Cardiomyocyte levels of heterogeneous AGEs were measured using ELISA. A pronounced increase in RAGE expression along with AGEs was observed in H/R vs. normoxia in WT cardiomyocytes. WT cardiomyocytes after H/R displayed increased LDH release compared to RKO or sRAGE-treated cardiomyocytes. Our results revealed significant increases in phospho-JNK in WT cardiomyocytes after H/R. In contrast, neither RKO nor sRAGE-treated cardiomyocytes exhibited increased phosphorylation of JNK after H/R stress. The impact of RAGE deletion on GSK-3beta phosphorylation in the cardiomyocytes subjected to H/R revealed significantly higher levels of phospho-GSK-3beta/total GSK-3beta in RKO, as well as in sRAGE-treated cardiomyocytes versus WT cardiomyocytes after H/R. Further investigation established a key role for Akt, which functions upstream of GSK-3beta, in modulating H/R injury in adult cardiomyocytes.These data illustrate key roles for RAGE-ligand interaction in the pathogenesis of

  1. Global protein quantification of mouse heart tissue based on the SILAC mouse.

    Science.gov (United States)

    Konzer, Anne; Ruhs, Aaron; Braun, Thomas; Krüger, Marcus

    2013-01-01

    Metabolic labeling of living organisms with stable isotopes has become a powerful tool for global protein quantitation. The SILAC (stable isotope labeling with amino acids in cell culture) approach is based on the incorporation of nonradioactive-labeled isotopic forms of amino acids into cellular proteins. The effective SILAC labeling of immortalized cells and single-cell organisms (e.g., yeast and bacteria) was recently extended to more complex organisms, including worms, flies, and even rodents. The administration of a (13)C6-lysine (heavy) containing diet for one mouse generation leads to a complete exchange of the natural (light) isotope (12)C6-lysine. SILAC-labeled organisms are mainly used as a heavy "spike-in" standard into nonlabeled counterparts, and the combination with high-performance mass spectrometers allows for global proteomic screening. Here we used the fully labeled SILAC mice to identify proteins based on SILAC pairs from isolated cardiomyocytes, and we analyzed β-parvin-deficient hearts. Our approach confirmed the absence β-parvin and revealed simultaneously a clear up regulation of α-parvin in heart tissue. In this protocol, we describe the generation of a SILAC mouse colony and show two approaches to perform a proteome-wide analysis of heart tissue. Thus, the SILAC mouse spike-in approach is a readily available procedure and allows for a straightforward systematic analysis of disease models and knockout mice.

  2. 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.

  3. MEMS piezoresistive cantilever for the direct measurement of cardiomyocyte contractile force

    Science.gov (United States)

    Matsudaira, Kenei; Nguyen, Thanh-Vinh; Hirayama Shoji, Kayoko; Tsukagoshi, Takuya; Takahata, Tomoyuki; Shimoyama, Isao

    2017-10-01

    This paper reports on a method to directly measure the contractile forces of cardiomyocytes using MEMS (micro electro mechanical systems)-based force sensors. The fabricated sensor chip consists of piezoresistive cantilevers that can measure contractile forces with high frequency (several tens of kHz) and high sensing resolution (less than 0.1 nN). Moreover, the proposed method does not require a complex observation system or image processing, which are necessary in conventional optical-based methods. This paper describes the design, fabrication, and evaluation of the proposed device and demonstrates the direct measurements of contractile forces of cardiomyocytes using the fabricated device.

  4. Mouse adhalin

    DEFF Research Database (Denmark)

    Liu, L; Vachon, P H; Kuang, W

    1997-01-01

    Adhalin, or alpha-sarcoglycan, is a 50-kDa glycoprotein that was originally characterized as a muscle membrane protein. The importance of adhalin is suggested by the diseases associated with its absence, notably the limb-girdle muscular dystrophies. However, the function of adhalin is unknown...... was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation...... in vitro. The proper localization of adhalin to the muscle cell membrane was observed only in late stages of myotube maturation, coincident with the re-distribution of caveolin-3 and dystrophin. These data suggest that adhalin is highly specific for striated muscle and that it is linked with the formation...

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

    Science.gov (United States)

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

    2011-01-01

    Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell-based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue-derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant DiI and then treated with fusion-inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte-like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole-cell voltage-clamp analysis demonstrated action potentials in beating fused cells. RT-PCR analysis using rat- or human-specific myosin heavy chain primers revealed that the myosin heavy-chain expression in fused cells was derived from rat cardiomyocytes. Real-time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory

  6. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

    Science.gov (United States)

    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  7. Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development

    Directory of Open Access Journals (Sweden)

    Silvia Mazzotta

    2016-10-01

    Full Text Available Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.

  8. Inflammatory and mitochondrial gene expression data in GPER-deficient cardiomyocytes from male and female mice

    Directory of Open Access Journals (Sweden)

    Hao Wang

    2017-02-01

    Full Text Available We previously showed that cardiomyocyte-specific G protein-coupled estrogen receptor (GPER gene deletion leads to sex-specific adverse effects on cardiac structure and function; alterations which may be due to distinct differences in mitochondrial and inflammatory processes between sexes. Here, we provide the results of Gene Set Enrichment Analysis (GSEA based on the DNA microarray data from GPER-knockout versus GPER-intact (intact cardiomyocytes. This article contains complete data on the mitochondrial and inflammatory response-related gene expression changes that were significant in GPER knockout versus intact cardiomyocytes from adult male and female mice. The data are supplemental to our original research article “Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER leads to left ventricular dysfunction and adverse remodeling: a sex-specific gene profiling” (Wang et al., 2016 [1]. Data have been deposited to the Gene Expression Omnibus (GEO database repository with the dataset identifier GSE86843.

  9. Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Xueming; Chen, Aihua, E-mail: aihuachen2012@sina.com; Yang, Pingzhen; Song, Xudong; Liu, Yingfeng; Li, Zhiliang; Wang, Xianbao; Wang, Lizi; Li, Yunpeng

    2013-11-29

    Highlights: •We observed the cell viability and death subjected to H/R in H9c2 cardiomyocytes. •We observed the degree of autophagy subjected to H/R in H9c2 cardiomyocytes. •LA inhibited the degree of autophagy in parallel to the enhanced cell survival. •LA inhibited the autophagy in parallel to the decreased total cell death. •We concluded that LA protected cardiomyocytes against H/R by inhibiting autophagy. -- Abstract: Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.

  10. Fluorescence assay for mitochondrial permeability transition in cardiomyocytes cultured in a microtiter plate

    DEFF Research Database (Denmark)

    Christensen, Marie Louise Muff; Braunstein, Thomas Hartig; Treiman, Marek

    2008-01-01

    neonatal cardiomyocytes in a 96-well microtiter plate format. In the presence of mitochondrial membrane potential Delta Psi m, accumulation of rhodamine-123 in mitochondria (40,000 cells/well, 2.6 microM rhodamine-123) caused fluorescence signal quenching. Following substitution of dye-free buffer...

  11. Evaluation of electrical propagation delay with cardiomyocytes by photosensitization reaction in vitro

    Science.gov (United States)

    Doi, Marika; Ogawa, Emiyu; Arai, Tsunenori

    2017-02-01

    In order to study cardiomyocyte electrical conduction damage by a photosensitization reaction (PR) mostly comes from outside of the cells in a few minutes after the PR, we studied propagation delay of contact action potential with cardiomyocyte by the PR. To determine appropriate PR condition for tachyarrhythmia ablation, a precise electrophysiological experiment in vitro has been preferable. We measured the contact action potential using a microelectrode array system of which information may be correct than conventional Ca2+ measurement. We investigated the propagation delays of an evoked potential to evaluate the electrical conduction damage by the PR. Rat cardiomyocytes were cultivated for 5-7 days on a dish with which 64 electrodes were patterned, in an incubator controlled to 37°C, 5% CO2. The following conditions were used for the PR: 40 μg/ml talapordfin sodium and 290 mW/cm2, 40-78 J/cm2 for an irradiation. A 2D map was obtained to visualize the propagation delays of the evoked potential. The propagation speed, which was calculated based on the measured propagation delays, was decreased by about 30-50% on average of all electrodes after the PR. Therefore, we think 2D propagation delays measurement of the evoked potential with contact action potential measuring system might be available to evaluate the acute electrical conduction damage of cardiomyocyte by the PR.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

    Science.gov (United States)

    Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

    2016-02-01

    Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes Under Defined Conditions.

    Science.gov (United States)

    van den Berg, Cathelijne W; Elliott, David A; Braam, Stefan R; Mummery, Christine L; Davis, Richard P

    2016-01-01

    Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate to cardiomyocytes in vitro, offering unique opportunities to investigate cardiac development and disease as well as providing a platform to perform drug and toxicity tests. Initial cardiac differentiation methods were based on either inductive co-culture or aggregation as embryoid bodies, often in the presence of fetal calf serum. More recently, monolayer differentiation protocols have evolved as feasible alternatives and are often performed in completely defined culture medium and substrates. Thus, our ability to efficiently and reproducibly generate cardiomyocytes from multiple different hESC and hiPSC lines has improved significantly.We have developed a directed differentiation monolayer protocol that can be used to generate cultures comprising ~50% cardiomyocytes, in which both the culture of the undifferentiated human pluripotent stem cells (hPSCs) and the differentiation procedure itself are defined and serum-free. The differentiation method is also effective for hPSCs maintained in other culture systems. In this chapter, we outline the differentiation protocol and describe methods to assess cardiac differentiation efficiency as well as to identify and quantify the yield of cardiomyocytes.

  15. KCNQ channels are involved in the regulatory volume decrease response in primary neonatal rat cardiomyocytes

    DEFF Research Database (Denmark)

    Calloe, Kirstine; Nielsen, Morten Schak; Grunnet, Morten

    2007-01-01

    inhibited in the presence of the KCNQ channel blocker XE-991 (10 and 100 microM). Electrophysiological experiments confirmed the presence of an XE-991 sensitive current and Western blotting analysis revealed that KCNQ1 channel protein was present in the neonatal rat cardiomyocytes. Hypoosmotic cell swelling...

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

    Science.gov (United States)

    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.

  17. Subcellular trafficking of the substrate transporters GLUT4 and CD36 in cardiomyocytes

    NARCIS (Netherlands)

    Steinbusch, L.K.M.; Schwenk, R.W.; Ouwens, D.M.; Diamant, M.; Glatz, J.F.C.; Luiken, J.J.F.P.

    2011-01-01

    Cardiomyocytes use glucose as well as fatty acids for ATP production. These substrates are transported into the cell by glucose transporter 4 (GLUT4) and the fatty acid transporter CD36. Besides being located at the sarcolemma, GLUT4 and CD36 are stored in intracellular compartments. Raised plasma

  18. Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Susi Zatti

    2014-01-01

    Full Text Available Duchenne muscular dystrophy (DMD–associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients' somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient–derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.

  19. Cardiomyocyte Hypocontractility and Reduced Myofibril Density in End-Stage Pediatric Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Ilse A. E. Bollen

    2017-12-01

    Full Text Available Dilated cardiomyopathy amongst children (pediatric cardiomyopathy, pediatric CM is associated with a high morbidity and mortality. Because little is known about the pathophysiology of pediatric CM, treatment is largely based on adult heart failure therapy. The reason for high morbidity and mortality is largely unknown as well as data on cellular pathomechanisms is limited. Here, we assessed cardiomyocyte contractility and protein expression to define cellular pathomechanisms in pediatric CM. Explanted heart tissue of 11 pediatric CM patients and 18 controls was studied. Contractility was measured in single membrane-permeabilized cardiomyocytes and protein expression was assessed with gel electrophoresis and western blot analysis. We observed increased Ca2+-sensitivity of myofilaments which was due to hypophosphorylation of cardiac troponin I, a feature commonly observed in adult DCM. We also found a significantly reduced maximal force generating capacity of pediatric CM cardiomyocytes, as well as a reduced passive force development over a range of sarcomere lengths. Myofibril density was reduced in pediatric CM compared to controls. Correction of maximal force and passive force for myofibril density normalized forces in pediatric CM cardiomyocytes to control values. This implies that the hypocontractility was caused by the reduction in myofibril density. Unlike in adult DCM we did not find an increase in compliant titin isoform expression in end-stage pediatric CM. The limited ability of pediatric CM patients to maintain myofibril density might have contributed to their early disease onset and severity.

  20. Cardiomyocyte behavior on biodegradable polyurethane/gold nanocomposite scaffolds under electrical stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Ganji, Yasaman [Faculty of Biomedical Engineering, Amirkabir University of Technology, 424 Hafez Ave, Tehran (Iran, Islamic Republic of); Institute for Materials Science, Dept. Biocompatible Nanomaterials, University of Kiel, Kaiserstr. 2, D-24143 Kiel (Germany); Li, Qian [Institute for Materials Science, Dept. Biocompatible Nanomaterials, University of Kiel, Kaiserstr. 2, D-24143 Kiel (Germany); Quabius, Elgar Susanne [Dept. of Otorhinolaryngology, Head and Neck Surgery, University of Kiel, Arnold-Heller-Str. 3, Building 27, D-24105 Kiel (Germany); Institute of Immunology, University of Kiel, Arnold-Heller-Str. 3, Building 17, D-24105 Kiel (Germany); Böttner, Martina [Department of Anatomy, University of Kiel, Otto-Hahn-Platz 8, 24118 Kiel (Germany); Selhuber-Unkel, Christine, E-mail: cse@tf.uni-kiel.de [Institute for Materials Science, Dept. Biocompatible Nanomaterials, University of Kiel, Kaiserstr. 2, D-24143 Kiel (Germany); Kasra, Mehran [Faculty of Biomedical Engineering, Amirkabir University of Technology, 424 Hafez Ave, Tehran (Iran, Islamic Republic of)

    2016-02-01

    Following a myocardial infarction (MI), cardiomyocytes are replaced by scar tissue, which decreases ventricular contractile function. Tissue engineering is a promising approach to regenerate such damaged cardiomyocyte tissue. Engineered cardiac patches can be fabricated by seeding a high density of cardiac cells onto a synthetic or natural porous polymer. In this study, nanocomposite scaffolds made of gold nanotubes/nanowires incorporated into biodegradable castor oil-based polyurethane were employed to make micro-porous scaffolds. H9C2 cardiomyocyte cells were cultured on the scaffolds for one day, and electrical stimulation was applied to improve cell communication and interaction in neighboring pores. Cells on scaffolds were examined by fluorescence microscopy and scanning electron microscopy, revealing that the combination of scaffold design and electrical stimulation significantly increased cell confluency of H9C2 cells on the scaffolds. Furthermore, we showed that the gene expression levels of Nkx2.5, atrial natriuretic peptide (ANF) and natriuretic peptide precursor B (NPPB), which are functional genes of the myocardium, were up-regulated by the incorporation of gold nanotubes/nanowires into the polyurethane scaffolds, in particular after electrical stimulation. - Highlights: • Biodegradable polyurethane/gold nanocomposites for cardiomyocyte adhesion are proposed. • The nanocomposite scaffolds are porous and electrical stimulation enhances cell adhesion. • Expression levels of functional myocardium genes were upregulated after electrical stimulation.

  1. Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Susann Björk

    2017-11-01

    Full Text Available Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs, cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and

  2. Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a

    Science.gov (United States)

    Kamkin, Andre; Kiseleva, Irina; Theres, Heinz; Eulert-Grehn, Jaime-Jürgen; Wagner, Kay-Dietrich; Scholz, Holger; Vetter, Roland

    2010-01-01

    BACKGROUND: Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca2+-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca2+ uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart. OBJECTIVE: To explore whether enhanced Ca2+ cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca2+ (ICa-L) currents. METHODS: The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals. RESULTS: The amplitudes of ICa-L currents at depolarizing pulses ranging from −45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of ICa-L showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced ICa-L currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca2+ with BAPTA. CONCLUSIONS: These findings indicate that enhanced Ca2+ cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in ICa-L currents. Modified ICa-L kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats. PMID:21264068

  3. On-chip constructive cell-network study (I): contribution of cardiac fibroblasts to cardiomyocyte beating synchronization and community effect.

    Science.gov (United States)

    Kaneko, Tomoyuki; Nomura, Fumimasa; Yasuda, Kenji

    2011-05-23

    To clarify the role of cardiac fibroblasts in beating synchronization, we have made simple lined-up cardiomyocyte-fibroblast network model in an on-chip single-cell-based cultivation system. The synchronization phenomenon of two cardiomyocyte networks connected by fibroblasts showed (1) propagation velocity of electrophysiological signals decreased a magnitude depending on the increasing number of fibroblasts, not the lengths of fibroblasts; (2) fluctuation of interbeat intervals of the synchronized two cardiomyocyte network connected by fibroblasts did not always decreased, and was opposite from homogeneous cardiomyocyte networks; and (3) the synchronized cardiomyocytes connected by fibroblasts sometimes loses their synchronized condition and recovered to synchronized condition, in which the length of asynchronized period was shorter less than 30 beats and was independent to their cultivation time, whereas the length of synchronized period increased according to cultivation time. The results indicated that fibroblasts can connect cardiomyocytes electrically but do not significantly enhance and contribute to beating interval stability and synchronization. This might also mean that an increase in the number of fibroblasts in heart tissue reduces the cardiomyocyte 'community effect', which enhances synchronization and stability of their beating rhythms.

  4. Pro-survival effect of Dock180 overexpression on rat-derived H9C2 cardiomyocytes.

    Science.gov (United States)

    Yan, An; Li, Gang; Zhang, Xu; Zhu, Bingbao; Linghu, Hua

    2013-01-14

    Integrin â1 subunit and its downstream molecule, focal adhesion kinase (FAK), have been demonstrated to be indispensible to the promotion of cell proliferation and survival and anti-apoptosis in cardiomyocytes via activation of their downstream pro-survival signaling molecule, AKT. As a component of the integrin pathway, Dock180 (dedicator of cytokinesis 1) protein is also thought to be involved in the promotion of cell proliferation and survival and anti-apoptosis in the H9C2 cardiomyocytes. Rat-derived H9C2 cardiomyocytes were transfected with pCXN2-flag-hDock180, a human Dock180 overexpression eukaryotic recombinant plasmid. The rat and human Dock180 mRNA and protein expression, apoptosis and cell proliferation and survival were analyzed in the H9C2 cardiomyocytes treated with either hypoxia/reoxygenation (H/R) or not, respectively. Human Dock180 mRNA overexpression could significantly increase the Dock180 protein expression in the H9C2 cardiomyocytes, no matter whether treated with H/R or not. Dock180 overexpression could promote the cell proliferation and survival and anti-apoptosis, and relieve the cell proliferative and survival inhibition and apoptosis induced by H/R in the H9C2 cardiomyocytes via activation of its downstream pro-survival signaling molecule AKT. Dock180 could act as a pro-survival molecule in H9C2 cardiomyocytes via activation of its downstream pro-survival signaling molecule, AKT.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Determination of the exact molecular requirements for type 1 angiotensin receptor epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy.

    Science.gov (United States)

    Smith, Nicola J; Chan, Hsiu-Wen; Qian, Hongwei; Bourne, Allison M; Hannan, Katherine M; Warner, Fiona J; Ritchie, Rebecca H; Pearson, Richard B; Hannan, Ross D; Thomas, Walter G

    2011-05-01

    Major interest surrounds how angiotensin II triggers cardiac hypertrophy via epidermal growth factor receptor transactivation. G protein-mediated transduction, angiotensin type 1 receptor phosphorylation at tyrosine 319, and β-arrestin-dependent scaffolding have been suggested, yet the mechanism remains controversial. We examined these pathways in the most reductionist model of cardiomyocyte growth, neonatal ventricular cardiomyocytes. Analysis with [(32)P]-labeled cardiomyocytes, wild-type and [Y319A] angiotensin type 1 receptor immunoprecipitation and phosphorimaging, phosphopeptide analysis, and antiphosphotyrosine blotting provided no evidence for tyrosine phosphorylation at Y319 or indeed of the receptor, and mutation of Y319 (to A/F) did not prevent either epidermal growth factor receptor transactivation in COS-7 cells or cardiomyocyte hypertrophy. Instead, we demonstrate that transactivation and cardiomyocyte hypertrophy are completely abrogated by loss of G-protein coupling, whereas a constitutively active angiotensin type 1 receptor mutant was sufficient to trigger transactivation and growth in the absence of ligand. These results were supported by the failure of the β-arrestin-biased ligand SII angiotensin II to transactivate epidermal growth factor receptor or promote hypertrophy, whereas a β-arrestin-uncoupled receptor retained these properties. We also found angiotensin II-mediated cardiomyocyte hypertrophy to be attenuated by a disintegrin and metalloprotease inhibition. Thus, G-protein coupling, and not Y319 phosphorylation or β-arrestin scaffolding, is required for epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy via the angiotensin type 1 receptor.

  7. Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

    Science.gov (United States)

    Wang, Xiaohong; Huang, Wei; Liu, Guansheng; Cai, Wenfeng; Millard, Ronald W.; Wang, Yigang; Chang, Jiang; Peng, Tianqing; Fan, Guo-Chang

    2014-01-01

    Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells

  8. Sphingosine-1-phosphate promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes under the designated culturing conditions

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    Zhang Henggui

    2011-06-01

    Full Text Available Abstract Background It is of growing interest to develop novel approaches to initiate differentiation of mesenchymal stem cells (MSCs into cardiomyocytes. The purpose of this investigation was to determine if Sphingosine-1-phosphate (S1P, a native circulating bioactive lipid metabolite, plays a role in differentiation of human umbilical cord mesenchymal stem cells (HUMSCs into cardiomyocytes. We also developed an engineered cell sheet from these HUMSCs derived cardiomyocytes by using a temperature-responsive polymer, poly(N-isopropylacrylamide (PIPAAm cell sheet technology. Methods Cardiomyogenic differentiation of HUMSCs was performed by culturing these cells with either designated cardiomyocytes conditioned medium (CMCM alone, or with 1 μM S1P; or DMEM with 10% FBS + 1 μM S1P. Cardiomyogenic differentiation was determined by immunocytochemical analysis of expression of cardiomyocyte markers and patch clamping recording of the action potential. Results A cardiomyocyte-like morphology and the expression of α-actinin and myosin heavy chain (MHC proteins can be observed in both CMCM culturing or CMCM+S1P culturing groups after 5 days' culturing, however, only the cells in CMCM+S1P culture condition present cardiomyocyte-like action potential and voltage gated currents. A new approach was used to form PIPAAm based temperature-responsive culture surfaces and this successfully produced cell sheets from HUMSCs derived cardiomyocytes. Conclusions This study for the first time demonstrates that S1P potentiates differentiation of HUMSCs towards functional cardiomyocytes under the designated culture conditions. Our engineered cell sheets may provide a potential for clinically applicable myocardial tissues should promote cardiac tissue engineering research.

  9. Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

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    Billy Chen

    Full Text Available Doxorubicin (Adriamycin is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1 is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4 in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent

  10. Exogenous Nitric Oxide Protects Human Embryonic Stem Cell-Derived Cardiomyocytes against Ischemia/Reperfusion Injury

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    János Pálóczi

    2016-01-01

    Full Text Available Background and Aims. Human embryonic stem cell- (hESC- derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days and then on more differentiated cardiomyocytes (6 + 24 days, both derived from hESCs. Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP (10−7, 10−6, and 10−5 M, BNP (10−9, 10−8, and 10−7 M, and the nonspecific NO synthase inhibitor Nω-nitro-L-arginine (L-NNA, 10−5 M. Results. SNAP (10−6, 10−5 M significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10−6 M also protected hESC-derived cardiomyocytes. Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms.

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

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    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.

  12. The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system.

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    Ahir, Bhavesh K; Pratten, Margaret K

    2016-07-01

    Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 μm (P cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 μm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 μm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  13. The protective effect of picroside II against hypoxia/reoxygenation injury in neonatal rat cardiomyocytes.

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    Meng, Fan-Ji; Hou, Zhi-Wen; Li, Yang; Yang, Ying; Yu, Bo

    2012-10-01

    Picroside II, an iridoid glucoside found in the root of Picrorhiza scrophulariiflora Pennell (Scrophulariaceae), has been demonstrated to possess potent antioxidant activity. However, whether picroside II has a protective effect against hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury is poorly understood. To explore the cardioprotective role of picroside II against oxidative stress induced by H/R injury in neonatal rat cardiacmyocytes. The viability and cellular damage of cardiomyocytes were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide (MTT) and lactate dehydrogenase (LDH) assays, respectively. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), the levels of reduced (GSH) and oxidized glutathione (GSSG), and the contents of malondialdehyde (MDA) were determined by a colorimetric method. The levels of intracellular reactive oxygen species (ROS) and calcium were evaluated by flow cytometric analysis. We analyzed the effective half-maximal concentration for protection from the dose-response curves and obtained the concentration of 50 µg/mL as EC(50). Pretreated cardiomyocytes with picroside II (50-200 µg/mL), prior to H/R exposure, inhibited LDH activity in culture media and increased cell viability in a dose-dependent manner. This protective effect was accompanied by significantly increasing reduced GSH contents and the activities of SOD and GSH-Px and attenuating MDA and GSSG contents in response to H/R injury. Furthermore, treatment with picroside II also inhibited ROS production and calcium accumulation in cardiomyocytes. The present study demonstrates that picroside II protects cardiomyocytes against oxidative-stress injury induced by H/R through reduction of ROS production and calcium accumulation and enhancement of the activity of antioxidant defense.

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

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    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.

  15. 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.

  16. Beat Rate Variability in Murine Embryonic Stem Cell-Derived Cardiomyocytes: Effect of Antiarrhythmic Drugs

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    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.

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

    Science.gov (United States)

    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. Reciprocal regulation of transcription factors and PLC isozyme gene expression in adult cardiomyocytes.

    Science.gov (United States)

    Singal, Tushi; Dhalla, Naranjan S; Tappia, Paramjit S

    2010-06-01

    By employing a pharmacological approach, we have shown that phospholipase C (PLC) activity is involved in the regulation of gene expression of transcription factors such as c-Fos and c-Jun in cardiomyocytes in response to norepinephrine (NE). However, there is no information available regarding the identity of specific PLC isozymes involved in the regulation of c-Fos and c-Jun or on the involvement of these transcription factors in PLC isozyme gene expression in adult cardiomyocytes. In this study, transfection of cardiomyocytes with PLC isozyme specific siRNA was found to prevent the NE-mediated increases in the corresponding PLC isozyme gene expression, protein content and activity. Unlike PLC gamma(1) gene, silencing of PLC beta(1), beta(3) and delta(1) genes with si RNA prevented the increases in c-Fos and c-Jun gene expression in response to NE. On the other hand, transfection with c-Jun si RNA suppressed the NE-induced increase in c-Jun as well as PLC beta(1), beta(3) and delta(1) gene expression, but had no effect on PLC gamma(1) gene expression. Although transfection of cardiomyocytes with c-Fos si RNA prevented NE-induced expression of c-Fos, PLC beta(1) and PLC beta(3) genes, it did not affect the increases in PLC delta(1) and PLC gamma(1) gene expression. Silencing of either c-Fos or c-Jun also depressed the NE-mediated increases in PLC beta(1), beta(3) and gamma(1) protein content and activity in an isozyme specific manner. Furthermore, silencing of all PLC isozymes as well as of c-Fos and c-Jun resulted in prevention of the NE-mediated increase in atrial natriuretic factor gene expression. These findings, by employing gene silencing techniques, demonstrate that there occurs a reciprocal regulation of transcription factors and specific PLC isozyme gene expression in cardiomyocytes.

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

    Science.gov (United States)

    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.

  20. 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.

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

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    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.

  2. [Electron microscope analysis of cardiomyocytes in the rat left ventricle under simulation of weightlessness effects and artificial gravitation].

    Science.gov (United States)

    Varenik, E N; Lipina, T V; Shornikova, M V; Krasnov, I B; Chentsov, Iu S

    2012-01-01

    Electron microscopic study of left ventricle cardiomyocytes and quantitative analysis of their mitochondriom was performed in rats exposed to tail-suspension, as a model of weightlessness effects, to artificial gravity produced by intermittent 2G centrifugation and a combination of these effects. It was found that the cardiomyocytes ultrastructure changed slightly after tail-suspension and after intermittent 2G influence, as well as under a combination of these effects. However, the number of intermitochondrial junctions increased significantly in the interfibrillar zone of cardiomyocytes under a combination of tail-suspension and intermittent 2G influence, which agrees with the cell hypertrophy described earlier.

  3. Localization of the transmembrane proteoglycan syndecan-4 and its regulatory kinases in costameres of rat cardiomyocytes: a deconvolution microscopic study

    DEFF Research Database (Denmark)

    VanWinkle, W Barry; Snuggs, Mark B; De Hostos, Eugenio L

    2002-01-01

    redifferentiation of myofibrils in cultured neonatal cardiomyocytes, syn-4 is present only in costameres, not in focal adhesions. In cultured adult cardiomyocytes, it is present in both costameres and focal adhesions-the latter in two distinct regions of the spread cardiomyocytes, reflecting localization with two...... types of actin-containing filaments. The fact that syn-4 is observed early in the costameric regions, as opposed to later in the focal adhesions, suggests that it may play an initial role in early adhesion/signal transduction mechanisms in close proximity to the contractile apparatus, as well...

  4. 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

  5. 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.

  6. Evaluation of MYBPC3 trans-Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes

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    Maksymilian Prondzynski

    2017-06-01

    Full Text Available Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C. Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5′ or 3′ pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1 the feasibility of trans-splicing, although with low efficiency, and (2 efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

  7. Stimulus-dependent regulation of nuclear Ca2+ signaling in cardiomyocytes: a role of neuronal calcium sensor-1.

    Science.gov (United States)

    Nakao, Shu; Wakabayashi, Shigeo; Nakamura, Tomoe Y

    2015-01-01

    In cardiomyocytes, intracellular calcium (Ca2+) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca2+levels ([Ca2+]) also occur in the nucleus, the precise mechanism of nuclear [Ca2+] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca2+] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca2+ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca2+ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca2+ transients are slower than cytoplasmic Ca2+ transients, and chelating cytoplasmic Ca2+ abolished nuclear Ca2+ transients, suggesting that nuclear Ca2+ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca2+] compared to cytoplasmic [Ca2+]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca2+ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca2+ transient amplitude was significantly lower in myocytes lacking neuronal Ca2+ sensor-1 (NCS-1), a Ca2+ binding protein implicated in IP3R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP3R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca2+] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca2+ signal regulation.

  8. Cardiomyopathy in Offspring of Pregestational Diabetic Mouse Pregnancy

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    Daniel Dowling

    2014-01-01

    Full Text Available Purpose. To investigate cardiomyopathy in offspring in a mouse model of pregestational type 1 diabetic pregnancy. Methods. Pregestational diabetes was induced with STZ administration in female C57BL6/J mice that were subsequently mated with healthy C57BL6/J males. Offspring were sacrificed at embryonic day 18.5 and 6-week adolescent and 12-week adult stages. The size and number of cardiomyocyte nuclei and also the extent of collagen deposition within the hearts of diabetic and control offspring were assessed following cardiac tissue staining with either haematoxylin and eosin or Picrosirius red and subsequently quantified using automated digital image analysis. Results. Offspring from diabetic mice at embryonic day 18.5 had a significantly higher number of cardiomyocyte nuclei present compared to controls. These nuclei were also significantly smaller than controls. Collagen deposition was shown to be significantly increased in the hearts of diabetic offspring at the same age. No significant differences were found between the groups at 6 and 12 weeks. Conclusions. Our results from offspring of type 1 diabetic mice show increased myocardial collagen deposition in late gestation and have increased myocardial nuclear counts (hyperplasia as opposed to increased myocardial nuclear size (hypertrophy in late gestation. These changes normalize postpartum after removal from the maternal intrauterine environment.

  9. TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

    Science.gov (United States)

    Kirschmer, Nadine; Bandleon, Sandra; von Ehrlich-Treuenstätt, Viktor; Hartmann, Sonja; Schaaf, Alice; Lamprecht, Anna-Karina; Miranda-Laferte, Erick; Langsenlehner, Tanja; Ritter, Oliver; Eder, Petra

    2016-01-01

    The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an

  10. TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

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    Nadine Kirschmer

    Full Text Available The Transient Receptor Potential Channel Subunit 4 (TRPC4 has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II stimulation. NRCs infected with TRPC4β (Ad-C4β also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not

  11. 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

  12. 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.

  13. In EXOG-depleted cardiomyocytes cell death is marked by a decreased mitochondrial reserve capacity of the electron transport chain

    NARCIS (Netherlands)

    Tigchelaar, Wardit; De Jong, Anne Margreet; van Gilst, Wiek H.; De Boer, Rudolf A.; Sillje, Herman H. W.

    Depletion ofmitochondrial endo/exonuclease G-like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease

  14. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

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    Jordan S. Leyton-Mange

    2014-02-01

    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  15. Centralized mouse repositories.

    Science.gov (United States)

    Donahue, Leah Rae; Hrabe de Angelis, Martin; Hagn, Michael; Franklin, Craig; Lloyd, K C Kent; Magnuson, Terry; McKerlie, Colin; Nakagata, Naomi; Obata, Yuichi; Read, Stuart; Wurst, Wolfgang; Hörlein, Andreas; Davisson, Muriel T

    2012-10-01

    Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world.

  16. 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.

  17. 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.

  18. SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

    OpenAIRE

    Dubois, Nicole C.; Craft, April M; Sharma, Parveen; Elliott, David A.; Stanley, Edouard G.; Elefanty, Andrew G.; Gramolini, Anthony; Keller, Gordon

    2011-01-01

    To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody...

  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. Urotensin II induction of neonatal cardiomyocyte hypertrophy involves the CaMKII/PLN/SERCA 2a signaling pathway.

    Science.gov (United States)

    Shi, Hongtao; Han, Qinghua; Xu, Jianrong; Liu, Wenyuan; Chu, Tingting; Zhao, Li

    2016-05-25

    Although studies have shown that Urotensin II (UII) can induce cardiomyocyte hypertrophy and UII-induced cardiomyocyte hypertrophy model has been widely used for hypertrophy research, but its precise mechanism remains unknown. Recent researches have demonstrated that UII-induced cardiomyocyte hypertrophy has a relationship with the changes of intracellular Ca(2+) concentration. Therefore, the aim of this study was to investigate the mechanisms of cardiomyocyte hypertrophy induced by UII and to explore whether the calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulating of phospholamban (PLN) Thr17-phosphorylation signaling pathway contributed to UII-induced cardiomyocyte hypertrophy. Primary cultures of neonatal rat cardiomyocytes were stimulated for 48h with UII. Cell size, protein/DNA contents and intracellular Ca(2+) were determined. Phosphorylated and total forms of CaMKII, PLN and the total amount of serco/endo-plasmic reticulum ATPases (SERCA 2a) were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the CaMKII inhibitor, KN-93. These results showed that UII increased cell size, protein/DNA ratio and intracellular Ca(2+), consistent with a hypertrophic response. Furthermore, the phosphorylation of CaMKII and its downstream target PLN (Thr17), SERCA 2a levels were up-regulated by UII treatment. Conversely, treatment with KN-93 reversed all those effects of UII. Taken together, the results suggest that UII can induce cardiomyocyte hypertrophy through CaMKII-mediated up-regulating of PLN Thr17-phosphorylation signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

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    Chuanyu Wei

    Full Text Available 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. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage.Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated.Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4.This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the myocardium under oxidative stress.

  2. Cardiac peroxisome proliferator-activated receptor-γ expression is modulated by oxidative stress in acutely infrasound-exposed cardiomyocytes.

    Science.gov (United States)

    Pei, Zhaohui; Meng, Rongsen; Zhuang, Zhiqiang; Zhao, Yiqiao; Liu, Fangpeng; Zhu, Miao-Zhang; Li, Ruiman

    2013-12-01

    The aim of the present study was to examine the effects of acute infrasound exposure on oxidative damage and investigate the underlying mechanisms in rat cardiomyocytes. Neonatal rat cardiomyocytes were cultured and exposed to infrasound for several days. In the study, the expression of CAT, GPx, SOD1, and SOD2 and their activities in rat cardiomyocytes in infrasound exposure groups were significantly decreased compared to those in the various time controls, along with significantly higher levels of O2 (-) and H2O2. Decreased cardiac cell viability was not observed in various time controls. A significant reduction in cardiac cell viability was observed in the infrasound group compared to the control, while significantly increased cardiac cell viability was observed in the infrasound exposure and rosiglitazone pretreatment group. Compared to the control, rosiglitazone significantly upregulated CAT, GPx, SOD1, and SOD2 expression and their activities in rat cardiomyocytes exposed to infrasound, while the levels of O2 (-) or H2O2 were significantly decreased. A potential link between a significant downregulation of PPAR-γ expression in rat cardiomyocytes in the infrasound group was compared to the control and infrasound-induced oxidative stress. These findings indicate that infrasound can induce oxidative damage in rat cardiomyocytes by inactivating PPAR-γ.

  3. Insulin protects apoptotic cardiomyocytes from hypoxia/reoxygenation injury through the sphingosine kinase/sphingosine 1-phosphate axis.

    Directory of Open Access Journals (Sweden)

    Huan Yu

    Full Text Available OBJECTIVE: Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the mechanisms underlying this effect are still unknown. In this study, the ability of insulin to protect apoptotic cardiomyocytes from hypoxia/reoxygenation injury using the sphingosine kinase/sphingosine 1-phosphate axis was investigated. METHODS AND RESULTS: Rat cardiomyocytes were isolated and subjected to hypoxia and reoxygenation. [γ-32P] ATP was used to assess sphingosine kinase activity. Insulin was found to increase sphingosine kinase activity. Immunocytochemistry and Western blot analysis showed changes in the subcellular location of sphingosine kinase 1 from cytosol to the membrane in cardiomyocytes. Insulin caused cardiomyocytes to accumulate of S1P in a dose-dependent manner. FRET efficiency showed that insulin also transactivates the S1P1 receptor. TUNEL staining showed that administration of insulin during reoxygenation could to reduce the rate of reoxygenation-induced apoptosis, which is a requirement for SphK 1 activity. It also reduced the rate of activation of the S1P receptor and inhibited hypoxia/reoxygenation-induced cell death in cardiomyocytes. CONCLUSION: The sphingosine kinase 1/sphingosine 1-phosphate/S1P receptor axis is one pathway through which insulin protects rat cardiomyocytes from apoptosis induced by hypoxia/reoxygenation injury.

  4. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing.

    Science.gov (United States)

    Nicolas, Jonathan; Hendriksen, Peter J M; de Haan, Laura H J; Koning, Rosella; Rietjens, Ivonne M C M; Bovee, Toine F H

    2015-03-01

    The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na(+), K(+), or Ca(2+) channels or the Na(+)/K(+) ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K(+) channel blockers, amiodarone, clofilium and sematilide, and the Na(+)/K(+) ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na(+) and Ca(2+) channels, but less sensitive for the compounds affecting K(+) channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Yusuke Kuroda

    2017-03-01

    Full Text Available Andersen-Tawil syndrome (ATS is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs. Multi-electrode arrays (MEAs were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca2+ imaging of cells loaded with the Ca2+ indicator Fluo-4 enabled us to examine intracellular Ca2+ handling properties, and we found a significantly higher incidence of irregular Ca2+ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca2+ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na+/Ca2+exchanger (NCX inhibitor, KB-R7943, was also found to suppress the irregular Ca2+ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (INCX. ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of INCX.

  6. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

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    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

  7. Enhanced Nanomagnetic Gene Transfection of Human Prenatal Cardiac Progenitor Cells and Adult Cardiomyocytes

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    Subramanian, Mahendran; Lim, Jenson; Dobson, Jon

    2013-01-01

    Magnetic nanoparticle-based gene transfection has been shown to be an effective, non-viral technique for delivery of both plasmid DNA and siRNA into cells in culture. It has several advantages over other non-viral delivery techniques, such as short transfection times and high cell viability. These advantages have been demonstrated in a number of primary cells and cell lines. Here we report that oscillating magnet array-based nanomagnetic transfection significantly improves transfection efficiency in both human prenatal cardiac progenitor cells and adult cardiomyocytes when compared to static magnetofection, cationic lipid reagents and electroporation, while maintaining high cell viability. In addition, transfection of adult cardiomyocytes was improved further by seeding the cells onto Collagen I-coated plates, with transfection efficiencies of up to 49% compared to 24% with lipid reagents and 19% with electroporation. These results demonstrate that oscillating nanomagnetic transfection far outperforms other non-viral transfection techniques in these important cells. PMID:23936108

  8. 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

  9. Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis.

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    Angeloni, Cristina; Turroni, Silvia; Bianchi, Laura; Fabbri, Daniele; Motori, Elisa; Malaguti, Marco; Leoncini, Emanuela; Maraldi, Tullia; Bini, Luca; Brigidi, Patrizia; Hrelia, Silvana

    2013-01-01

    Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.

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

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    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.

  11. 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.

  12. Sumo E2 enzyme UBC9 is required for efficient protein quality control in cardiomyocytes.

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    Gupta, Manish K; Gulick, James; Liu, Ruijie; Wang, Xuejun; Molkentin, Jeffery D; Robbins, Jeffrey

    2014-09-26

    Impairment of proteasomal function is pathogenic in several cardiac proteinopathies and can eventually lead to heart failure. Loss of proteasomal activity often results in the accumulation of large protein aggregates. The ubiquitin proteasome system (UPS) is primarily responsible for cellular protein degradation, and although the role of ubiquitination in this process is well studied, the function of an ancillary post-translational modification, SUMOylation, in protein quality control is not fully understood. To determine the role of ubiquitin-conjugating enzyme 9 (UBC9), a small ubiquitin-like modifier-conjugating enzyme, in cardiomyocyte protein quality control. Gain- and loss-of-function approaches were used to determine the importance of UBC9. Overexpression of UBC9 enhanced UPS function in cardiomyocytes, whereas knockdown of UBC9 by small interfering RNA caused significant accumulations of aggregated protein. UPS function and relative activity was analyzed using a UPS reporter protein consisting of a short degron, CL1, fused to the COOH-terminus of green fluorescent protein (GFPu). Subsequently, the effects of UBC9 on UPS function were tested in a proteotoxic model of desmin-related cardiomyopathy, caused by cardiomyocyte-specific expression of a mutated αB crystallin, CryAB(R120G). CryAB(R120G) expression leads to aggregate formation and decreased proteasomal function. Coinfection of UBC9-adenovirus with CryAB(R120G) virus reduced the proteotoxic sequelae, decreasing overall aggregate concentrations. Conversely, knockdown of UBC9 significantly decreased UPS function in the model and resulted in increased aggregate levels. UBC9 plays a significant role in cardiomyocyte protein quality control, and its activity can be exploited to reduce toxic levels of misfolded or aggregated proteins in cardiomyopathy. © 2014 American Heart Association, Inc.

  13. Sumo E2 Ligase UBC9 is Required for Efficient Protein Quality Control in Cardiomyocytes

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    Gupta, Manish K.; Gulick, James; Liu, Ruijie; Wang, Xuejun; Molkentin, Jeffery D.; Robbins, Jeffrey

    2014-01-01

    Rationale Impairment of proteasomal function is pathogenic in a number of cardiac proteinopathies and can eventually lead to heart failure. Loss of proteasomal activity often results in the accumulation of large protein aggregates. The ubiquitin proteasome system (UPS) is primarily responsible for cellular protein degradation and, while the role of ubiquitination in this process is well studied, the function of an ancillary post-translational modification, SUMOylation, in protein quality control (PQC) is not fully understood. Objective To determine the role of UBC9, a SUMO-conjugating enzyme, in cardiomyocyte PQC. Methods and Results Gain- and loss-of-function approaches were used to determine the importance of UBC9. Overexpression of UBC9 enhanced UPS function in cardiomyocytes while knockdown of UBC9 by siRNA caused significant accumulations of aggregated protein. UPS function and relative activity was analyzed using a UPS reporter protein, GFPu. Subsequently, UBC9's effects on UPS function were tested in a proteotoxic model of desmin-related cardiomyopathy, caused by cardiomyocyte specific expression of a mutated alpha B crystallin, CryABR120G. CryABR120G expression leads to aggregate formation and decreased proteasomal function. Co-infection of UBC9-adenovirus with CryABR120G virus reduced the proteotoxic sequelae, decreasing overall aggregate concentrations. Conversely, knockdown of UBC9 significantly decreased UPS function in the model and resulted in increased aggregate levels. Conclusions UBC9 plays a significant role in cardiomyocyte PQC and its activity can be exploited to reduce toxic levels of misfolded or aggregated proteins in cardiomyopathy. PMID:25097219

  14. Familial Hypertrophic Cardiomyopathy: Functional variance among individual cardiomyocytes as a trigger of FHC-phenotype development

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    Bernhard eBrenner

    2014-10-01

    Full Text Available Familial hypertrophic cardiomyopathy (FHC is the most frequent inherited cardiac disease. It has been related to numerous mutations in many sarcomeric and even some non-sarcomeric proteins. So far, however, no common mechanism has been identified by which the many different mutations in different sarcomeric and non-sarcomeric proteins trigger development of the FHC phenotype. Here we show for different MYH7 mutations variance in force pCa-relations from normal to highly abnormal as a feature common to all mutations we studied, while direct functional effects of the different FHC-mutations, e.g., on force generation, ATPase or calcium sensitivity of the contractile system, can be quite different. The functional variation among individual M. soleus fibers of FHC-patients is accompanied by large variation in mutant vs. wildtype β-MyHC-mRNA. Preliminary results show a similar variation in mutant vs. wildtype β-MyHC-mRNA among individual cardiomyocytes. We discuss our previously proposed concept as to how different mutations in the β-MyHC and possibly other sarcomeric and non-sarcomeric proteins may initiate an FHC-phenotype by functional variation among individual cardiomyocytes that results in structural distortions within the myocardium, leading to cellular and myofibrillar disarray. In addition, distortions can activate stretch-sensitive signalling in cardiomyocytes and non-myocyte cells which is known to induce cardiac remodelling with interstitial fibrosis and hypertrophy. Such a mechanism will have major implications for therapeutic strategies to prevent FHC-development, e.g., by reducing functional imbalances among individual cardiomyocytes or by inhibition of their triggering of signalling paths initiating remodelling. Targeting increased or decreased contractile function would require selective targeting of mutant or wildtype protein to reduce functional imbalances.

  15. Electrospun Gelatin–Chondroitin Sulfate Scaffolds Loaded with Platelet Lysate Promote Immature Cardiomyocyte Proliferation

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    Francesca Saporito

    2018-02-01

    Full Text Available The aim of the present work was the development of heart patches based on gelatin (G and chondroitin sulfate (CS to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD. Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system’s general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

  16. ISL1 protein transduction promotes cardiomyocyte differentiation from human embryonic stem cells.

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    Hananeh Fonoudi

    Full Text Available BACKGROUND: Human embryonic stem cells (hESCs have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes. METHODS: We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1 recombinant protein into the cells. RESULTS: We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1-8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4 doubled. This protocol was also reproducible for another hESC line. CONCLUSIONS: This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.

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

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    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.

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

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    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.

  19. In Situ Real-Time Chemiluminescence Imaging of Reactive Oxygen Species Formation from Cardiomyocytes

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    Yunbo Li

    2008-01-01

    Full Text Available We have applied the highly sensitive chemiluminescence (CL imaging technique to investigate the in situ ROS formation in cultured monolayers of rat H9c2 cardiomyocytes. Photon emission was detected via an innovative imaging system after incubation of H9c2 cells in culture with luminol and horseradish peroxidase (HRP, suggesting constitutive formation of ROS by the cardiomyocytes. Addition of benzo(apyrene-1,6-quinone (BPQ to cultured H9c2 cells resulted in a 4-5-fold increase in the formation of ROS, as detected by the CL imaging. Both constitutive and BPQ-stimulated CL responses in cultured H9c2 cells were sustained for up to 1 hour. The CL responses were completely abolished in the presence of superoxide dismutase and catalase, suggesting the primary involvement of superoxide and hydrogen peroxide (H2O2. In contrast to BPQ-mediated redox cycling, blockage of mitochondrial electron transport chain by either antimycin A or rotenone exerted marginal effects on the ROS formation by cultured H9c2 cells. Upregulation of cellular antioxidants for detoxifying both superoxide and H2O2 by 3H-1,2-dithiole-3-thione resulted in marked inhibition of both constitutive and BPQ-augmented ROS formation in cultured H9c2 cells. Taken together, we demonstrate the sensitive detection of ROS by CL imaging in cultured cardiomyocytes.

  20. 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.

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

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    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.

  2. 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.

  3. Ibutilide protects against cardiomyocytes injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways.

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    Wang, Yu; Wang, Yi-Li; Huang, Xia; Yang, Yang; Zhao, Ya-Jun; Wei, Cheng-Xi; Zhao, Ming

    2017-02-01

    Atrial fibrillation (AF) is a complex disease with multiple inter-relating causes culminating in rapid atrial activation and atrial structural remodeling. The contribution of endoplasmic reticulum and mitochondria stress to AF has been highlighted. As the class III antiarrhythmic agent, ibutilide are widely used to AF. This study was designed to explore whether ibutilide could treat AF by inhibiting endoplasmic reticulum stress pathways and mitochondria stress. The neonatal rat cardiomyocytes were isolated and exposed to H2O2, ibutilide was add to the culture medium 12 h. Then the cell viability, oxidative stress levels and apoptotic rate were analyzed. In addition, endoplasmic reticulum stress related protein (GRP78, GRP94, CHOP), mitochondria-dependent protein (Bax, Bcl-2) and caspase-3/9/12 were identified by real-time PCR and western blot analysis. In our results, remarkable decreased cell viability and oxidative stress levels were detected in cardiomyocytes after treating with H2O2. The apoptotic rate and the expression of proteins involved in mitochondrial stress and endoplasmic reticulum stress pathways increased. While ibutilide significantly inhibited these changes. These data suggested that ibutilide serves a protective role against H2O2-induced apoptosis of neonatal rat cardiomyocytes, and the mechanism is related to suppression of mitochondrial stress and endoplasmic reticulum stress.

  4. Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes.

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    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.

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

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    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.

  6. Comparison of nonviral transfection and adeno-associated viral transduction on cardiomyocytes.

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    Djurovic, Srdjan; Iversen, Nina; Jeansson, Stig; Hoover, Frank; Christensen, Geir

    2004-09-01

    Cardiomyocytes are terminally differentiated cells that to date have been characterized as poor targets for nonviral gene transfer. This study was therefore designed to determine the optimal nonviral gene transfer parameters in cell cultures of neonatal rat cardiomyocytes and to compare them with the efficiency of gene transfer using adeno-associated viral vectors (AAV). Transfection efficiency was measured by quantitative chloramphenicol acetyltransferase type I (CAT)-enzyme-linked immunosorbent assay and beta-galactosidase staining, based on overexpression of reporter genes (CAT and LacZ). The efficiency of CAT/LacZ overexpression was assessed using the following techniques: (1) liposomal reagents, such as: FuGENE 6, LipofectAMINE 2000, LipofectAMINE PLUS, GenePORTER, Metafectene, and LipoGen; (2) electroporation and nucleofector techniques; and (3) an AAV2 vector harboring a lacZ reporter gene. Toxicity was monitored by total protein measurement and by analyzing cell metabolism. On average, Lipofectamine 2000 was the most effective nonviral method examined yielding consistently high transfection rates (8.1% beta-galactosidase-positive cells) combined with low toxicity. Electroporation also resulted in high transfection values (7.5%); however, cellular toxicity was higher than that of Lipofectamine 2000. Finally, transduction with AAV2 vectors provided the highest levels of transduction (88.1%) with no cellular toxicity. We conclude that although transduction with AAV is more efficient (88.1%), transfections with nonviral techniques, when optimized, may provide a useful alternative for overexpression of therapeutic genes in neonatal cardiomyocytes.

  7. Recent Insights in the Paracrine Modulation of Cardiomyocyte Contractility by Cardiac Endothelial Cells

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    Jacques Noireaud

    2014-01-01

    Full Text Available The cardiac endothelium is formed by a continuous monolayer of cells that line the cavity of the heart (endocardial endothelial cells (EECs and the luminal surface of the myocardial blood vessels (intramyocardial capillary endothelial cells (IMCEs. EECs and IMCEs can exercise substantial control over the contractility of cardiomyocytes by releasing various factors such as nitric oxide (NO via a constitutive endothelial NO-synthase (eNOS, endothelin-1, prostaglandins, angiotensin II, peptide growth factors, and neuregulin-1. The purpose of the present paper is actually to shortly review recent new information concerning cardiomyocytes as effectors of endothelium paracrine signaling, focusing particularly on contractile function. The modes of action and the regulatory paracrine role of the main mediators delivered by cardiac endothelial cells upon cardiac contractility identified in cardiomyocytes are complex and not fully described. Thus, careful evaluation of new therapeutic approaches is required targeting important physiological signaling pathways, some of which have been until recently considered as deleterious, like reactive oxygen species. Future works in the field of cardiac endothelial cells and cardiac function will help to better understand the implication of these mediators in cardiac physiopathology.

  8. EFFECTS OF AEROBIC TRAINING ON THE CARDIOMYOCYTES OF THE RIGHT ATRIUM OF MICE

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    Vanessa Gonçalves Coutinho de Oliveira

    Full Text Available ABSTRACT Introduction: Polypeptide hormones (natriuretic peptides, NPs are secreted by the cardiac atria and play an important role in the regulation of blood pressure. Objective: To evaluate the effects of aerobic training on the secretory apparatus of NPs in cardiomyocytes of the right atrium. Methods: Nine-month-old mice were divided in two groups (n=10: control group (CG and trained group (TG. The training protocol was performed on a motor treadmill for 8 weeks. Systolic blood pressure was measured at the beginning of the experiment (9 months of age and at moment of the sacrifice (11 months of age. Electron micrographs were used to quantify the following variables: the quantitative density and area of NP granules, the relative volumes of the mitochondria, endoplasmic reticulum, and Golgi complex and the relative volume of euchromatin in the nucleus and the number of pores per 10 µm of the nuclear membrane. The results were compared by Student's t test (p< 0.05. Results: The cardiomyocytes obtained from TG mice showed increased density and sectional area of secretory granules of NP, higher relative volume of endoplasmic reticulum, mitochondria, and Golgi complex compared with the CG mice. Furthermore, the quantitative density of nuclear pores and the relative volume of euchromatin in the nucleus were significantly higher compared with the CG mice. Conclusion: Aerobic training caused hypertrophy of the secretory apparatus in the cardiomyocytes of right atrium, which could explain the intense synthesis of natriuretic peptides in trained mice with respect to the untrained mice.

  9. 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.

  10. 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

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

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    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

  12. 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

  13. Garlic extracts prevent oxidative stress, hypertrophy and apoptosis in cardiomyocytes: a role for nitric oxide and hydrogen sulfide

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    Louis Xavier

    2012-08-01

    Full Text Available Abstract Background In ancient times, plants were recognized for their medicinal properties. Later, the arrival of synthetic drugs pushed it to the backstage. However, from being merely used for food, plants are now been widely explored for their therapeutic value. The current study explores the potential of skin and flesh extracts from a hard-necked Rocambole variety of purple garlic in preventing cardiomyocyte hypertrophy and cell death. Methods Norepinephrine (NE was used to induce hypertrophy in adult rat cardiomyocytes pretreated with garlic skin and flesh extracts. Cell death was measured as ratio of rod to round shaped cardiomyocytes. Fluorescent probes were used to measure apoptosis and oxidative stress in cardiomyocytes treated with and without extracts and NE. Pharmacological blockade of nitric oxide (NO and hydrogen sulfide (H2S were used to elucidate the mechanism of action of garlic extracts. Garlic extract samples were also tested for alliin and allicin concentrations. Results Exposure of cardiomyocytes to NE induced an increase in cell size and cell death; this increase was significantly prevented upon treatment with garlic skin and flesh extracts. Norepinephrine increased apoptosis and oxidative stress in cardiomyocytes which was prevented upon pretreatment with skin and flesh extracts; NO, and H2S blockers significantly inhibited this beneficial effect. Allicin and alliin concentration were significantly higher in garlic flesh extract when compared to the skin extract. Conclusion These results suggest that both skin and flesh garlic extracts are effective in preventing NE induced cardiomyocyte hypertrophy and cell death. Reduction in oxidative stress may also play an important role in the anti-hypertrophic and anti-apoptotic properties of garlic extracts. These beneficial effects may in part be mediated by NO and H2S.

  14. Garlic extracts prevent oxidative stress, hypertrophy and apoptosis in cardiomyocytes: a role for nitric oxide and hydrogen sulfide

    Science.gov (United States)

    2012-01-01

    Background In ancient times, plants were recognized for their medicinal properties. Later, the arrival of synthetic drugs pushed it to the backstage. However, from being merely used for food, plants are now been widely explored for their therapeutic value. The current study explores the potential of skin and flesh extracts from a hard-necked Rocambole variety of purple garlic in preventing cardiomyocyte hypertrophy and cell death. Methods Norepinephrine (NE) was used to induce hypertrophy in adult rat cardiomyocytes pretreated with garlic skin and flesh extracts. Cell death was measured as ratio of rod to round shaped cardiomyocytes. Fluorescent probes were used to measure apoptosis and oxidative stress in cardiomyocytes treated with and without extracts and NE. Pharmacological blockade of nitric oxide (NO) and hydrogen sulfide (H2S) were used to elucidate the mechanism of action of garlic extracts. Garlic extract samples were also tested for alliin and allicin concentrations. Results Exposure of cardiomyocytes to NE induced an increase in cell size and cell death; this increase was significantly prevented upon treatment with garlic skin and flesh extracts. Norepinephrine increased apoptosis and oxidative stress in cardiomyocytes which was prevented upon pretreatment with skin and flesh extracts; NO, and H2S blockers significantly inhibited this beneficial effect. Allicin and alliin concentration were significantly higher in garlic flesh extract when compared to the skin extract. Conclusion These results suggest that both skin and flesh garlic extracts are effective in preventing NE induced cardiomyocyte hypertrophy and cell death. Reduction in oxidative stress may also play an important role in the anti-hypertrophic and anti-apoptotic properties of garlic extracts. These beneficial effects may in part be mediated by NO and H2S. PMID:22931510

  15. Garlic extracts prevent oxidative stress, hypertrophy and apoptosis in cardiomyocytes: a role for nitric oxide and hydrogen sulfide.

    Science.gov (United States)

    Louis, Xavier Lieben; Murphy, Ryan; Thandapilly, Sijo Joseph; Yu, Liping; Netticadan, Thomas

    2012-08-29

    In ancient times, plants were recognized for their medicinal properties. Later, the arrival of synthetic drugs pushed it to the backstage. However, from being merely used for food, plants are now been widely explored for their therapeutic value. The current study explores the potential of skin and flesh extracts from a hard-necked Rocambole variety of purple garlic in preventing cardiomyocyte hypertrophy and cell death. Norepinephrine (NE) was used to induce hypertrophy in adult rat cardiomyocytes pretreated with garlic skin and flesh extracts. Cell death was measured as ratio of rod to round shaped cardiomyocytes. Fluorescent probes were used to measure apoptosis and oxidative stress in cardiomyocytes treated with and without extracts and NE. Pharmacological blockade of nitric oxide (NO) and hydrogen sulfide (H₂S) were used to elucidate the mechanism of action of garlic extracts. Garlic extract samples were also tested for alliin and allicin concentrations. Exposure of cardiomyocytes to NE induced an increase in cell size and cell death; this increase was significantly prevented upon treatment with garlic skin and flesh extracts. Norepinephrine increased apoptosis and oxidative stress in cardiomyocytes which was prevented upon pretreatment with skin and flesh extracts; NO, and H₂S blockers significantly inhibited this beneficial effect. Allicin and alliin concentration were significantly higher in garlic flesh extract when compared to the skin extract. These results suggest that both skin and flesh garlic extracts are effective in preventing NE induced cardiomyocyte hypertrophy and cell death. Reduction in oxidative stress may also play an important role in the anti-hypertrophic and anti-apoptotic properties of garlic extracts. These beneficial effects may in part be mediated by NO and H₂S.

  16. 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.

  17. 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

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

    Science.gov (United States)

    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

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

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    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.

  20. 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.

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

    Science.gov (United States)

    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.

  2. 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

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

    Science.gov (United States)

    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.

  4. Late Sodium Current in Human Atrial Cardiomyocytes from Patients in Sinus Rhythm and Atrial Fibrillation.

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    Claire Poulet

    Full Text Available Slowly inactivating Na+ channels conducting "late" Na+ current (INa,late contribute to ventricular arrhythmogenesis under pathological conditions. INa,late was also reported to play a role in chronic atrial fibrillation (AF. The objective of this study was to investigate INa,late in human right atrial cardiomyocytes as a putative drug target for treatment of AF. To activate Na+ channels, cardiomyocytes from transgenic mice which exhibit INa,late (ΔKPQ, and right atrial cardiomyocytes from patients in sinus rhythm (SR and AF were voltage clamped at room temperature by 250-ms long test pulses to -30 mV from a holding potential of -80 mV with a 100-ms pre-pulse to -110 mV (protocol I. INa,late at -30 mV was not discernible as deviation from the extrapolated straight line IV-curve between -110 mV and -80 mV in human atrial cells. Therefore, tetrodotoxin (TTX, 10 μM was used to define persistent inward current after 250 ms at -30 mV as INa,late. TTX-sensitive current was 0.27±0.06 pA/pF in ventricular cardiomyocytes from ΔKPQ mice, and amounted to 0.04±0.01 pA/pF and 0.09±0.02 pA/pF in SR and AF human atrial cardiomyocytes, respectively. With protocol II (holding potential -120 mV, pre-pulse to -80 mV TTX-sensitive INa,late was always larger than with protocol I. Ranolazine (30 μM reduced INa,late by 0.02±0.02 pA/pF in SR and 0.09±0.02 pA/pF in AF cells. At physiological temperature (37°C, however, INa,late became insignificant. Plateau phase and upstroke velocity of action potentials (APs recorded with sharp microelectrodes in intact human trabeculae were more sensitive to ranolazine in AF than in SR preparations. Sodium channel subunits expression measured with qPCR was high for SCN5A with no difference between SR and AF. Expression of SCN8A and SCN10A was low in general, and lower in AF than in SR. In conclusion, We confirm for the first time a TTX-sensitive current (INa,late in right atrial cardiomyocytes from SR and AF patients at room

  5. pH recovery from a proton load in rat cardiomyocytes: effects of chronic exercise.

    Science.gov (United States)

    Danes, Vennetia R; Anthony, Josephine; Rayani, Kaveh; Spitzer, Kenneth W; Tibbits, Glen F

    2018-02-01

    The ability of cardiomyocytes to recover from a proton load was examined in the hearts of exercise-trained and sedentary control rats in CO 2 /[Formula: see text]-free media. Acidosis was created by the NH 4 Cl prepulse technique, and intracellular pH (pH i ) was determined using fluorescence microscopy on carboxy-SNARF-1 AM-loaded isolated cardiomyocytes. CO 2 -independent pH i buffering capacity (β i ) was measured by incrementally reducing the extracellular NH 4 Cl concentration in steps of 50% from 20 to 1.25 mM. β i increased as pH i decreased in both exercise-trained and sedentary control groups. However, the magnitude of increase in β i as a function of pH i was found to be significantly ( P trained group compared with the sedentary control group. The rate of pH i recovery from an imposed proton load was found to not be different between the exercise-trained and control groups. The Na + /H + exchanger-dependent H + extrusion rate during the recovery from an imposed proton load, however, was found to be significantly greater in the exercise-trained group compared with the control group. By increasing β i and subsequently the Na + /H + exchanger-dependent H + extrusion rate, exercise training may provide cardiomyocytes with the ability to better handle an intracellular excess of H + generated during hypoxia/ischemic insults and may serve in a cardioprotective role. These data may be predictive of two positive outcomes: 1) increased exercise tolerance by the heart and 2) a protective mechanism that limits the degree of myocardial acidosis and subsequent damage that accompanies ischemia-reperfusion stress. NEW & NOTEWORTHY The enhanced ability to deal with acidosis conferred by exercise training is likely to improve exercise tolerance and outcomes in response to myocardial ischemia-reperfusion injury.

  6. Scaffold protein enigma homolog activates CREB whereas a short splice variant prevents CREB activation in cardiomyocytes.

    Science.gov (United States)

    Ito, Jumpei; Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Kuroda, Shun'ichi; Maturana, Andrés D

    2015-12-01

    Enigma Homolog (ENH1 or Pdlim5) is a scaffold protein composed of an N-terminal PDZ domain and three LIM domains at the C-terminal end. The enh gene encodes for several splice variants with opposing functions. ENH1 promotes cardiomyocytes hypertrophy whereas ENH splice variants lacking LIM domains prevent it. ENH1 interacts with various Protein Kinase C (PKC) isozymes and Protein Kinase D1 (PKD1). In addition, the binding of ENH1's LIM domains to PKC is sufficient to activate the kinase without stimulation. The downstream events of the ENH1-PKC/PKD1 complex remain unknown. PKC and PKD1 are known to phosphorylate the transcription factor cAMP-response element binding protein (CREB). We tested whether ENH1 could play a role in the activation of CREB. We found that, in neonatal rat ventricular cardiomyocytes, ENH1 interacts with CREB, is necessary for the phosphorylation of CREB at ser133, and the activation of CREB-dependent transcription. On the contrary, the overexpression of ENH3, a LIM-less splice variant, inhibited the phosphorylation of CREB. ENH3 overexpression or shRNA knockdown of ENH1 prevented the CREB-dependent transcription. Our results thus suggest that ENH1 plays an essential role in CREB's activation and dependent transcription in cardiomyocytes. At the opposite, ENH3 prevents the CREB transcriptional activity. In conclusion, these results provide a first molecular explanation to the opposing functions of ENH splice variants. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Science.gov (United States)

    Kucera, Jan P.; Prudat, Yann; Marcu, Irene C.; Azzarito, Michela; Ullrich, Nina D.

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias. PMID:26442264

  8. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jan Pavel Kucera

    2015-09-01

    Full Text Available Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs. However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands.CV was significantly lower in strands composed purely of SCMs (5.5±1.5 cm/s, n=11 as compared to PCMs (34.9±2.9 cm/s, n=21 at similar refractoriness (100% SCMs: 122±25 ms, n=9; 100% PCMs: 139±67 ms, n=14. In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV.These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

  9. Moderate vs. high exercise intensity: differential effects on aerobic fitness, cardiomyocyte contractility, and endothelial function.

    Science.gov (United States)

    Kemi, Ole J; Haram, Per M; Loennechen, Jan P; Osnes, Jan-Bjørn; Skomedal, Tor; Wisløff, Ulrik; Ellingsen, Øyvind

    2005-07-01

    Current guidelines are controversial regarding exercise intensity in cardiovascular prevention and rehabilitation. Although high-intensity training induces larger increases in fitness and maximal oxygen uptake (VO(2max)), moderate intensity is often recommended as equally effective. Controlled preclinical studies and randomized clinical trials are required to determine whether regular exercise at moderate versus high intensity is more beneficial. We therefore assessed relative effectiveness of 10-week HIGH versus moderate (MOD) exercise intensity on integrative and cellular functions. Sprague-Dawley rats performed treadmill running intervals at either 85%-90% (HIGH) or 65%-70% (MOD) of VO2max 1 h per day, 5 days per week. Weekly VO2max-testing adjusted exercise intensity. HIGH and MOD increased VO2max by 71% and 28%, respectively. This was paralleled by intensity-dependent cardiomyocyte hypertrophy, 14% and 5% in HIGH and MOD, respectively. Cardiomyocyte function (fractional shortening) increased by 45% and 23%, contraction rate decreased by 43% and 39%, and relaxation rate decreased by 20% and 10%, in HIGH and MOD, respectively. Ca2+ transient time-courses paralleled contraction/relaxation, whereas Ca2+ sensitivity increased 40% and 30% in HIGH and MOD, respectively. Carotid artery endothelial function improved similarly with both intensities. EC50 for acetylcholine-induced relaxation decreased 4.3-fold in HIGH (p hypertrophy, contractility and vasorelaxation also correlated significantly with VO2max. The present study demonstrates that cardiovascular adaptations to training are intensity-dependent. A close correlation between VO2max, cardiomyocyte dimensions and contractile capacity suggests significantly higher benefit with high intensity, whereas endothelial function appears equivalent at moderate levels. Thus, exercise intensity emerges as an important variable in future preclinical and clinical investigations.

  10. 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.

  11. Alterations in cardiomyocyte function after pulmonary treatment with stainless steel welding fume in rats.

    Science.gov (United States)

    Popstojanov, Risto; Antonini, James M; Salmen, Rebecca; Ye, Morgan; Zheng, Wen; Castranova, Vincent; Fekedulegn, Desta B; Kan, Hong

    2014-01-01

    Welding fume is composed of a complex of different metal particulates. Pulmonary exposure to different welding fumes may exert a negative impact on cardiac function, although the underlying mechanisms remain unclear. To explore the effect of welding fumes on cardiac function, Sprague-Dawley rats were exposed by intratracheal instillation to 2 mg/rat of manual metal arc hard surfacing welding fume (MMA-HS) once per week for 7 wk. Control rats received saline. Cardiomyocytes were isolated enzymatically at d 1 and 7 postexposure. Intracellular calcium ([Ca(2+)]i) transients (fluorescence ratio) were measured on the stage of an inverted phase-contrast microscope using a myocyte calcium imaging/cell length system. Phosphorylation levels of cardiac troponin I (cTnI) were determined by Western blot. The levels of nonspecific inflammatory marker C-reactive protein (CRP) and proinflammatory cytokine interleukin-6 (IL-6) in serum were measured by enzyme-linked immunosorbent assay (ELISA). Contraction of isolated cardiomyocytes was significantly reduced at d 1 and d 7 postexposure. Intracellular calcium levels were decreased in response to extracellular calcium stimulation at d 7 postexposure. Changes of intracellular calcium levels after isoprenaline hydrochloride (ISO) stimulation were not markedly different between groups at either time point. Phosphorylation levels of cTnI in the left ventricle were significantly lower at d 1 postexposure. The serum levels of CRP were not markedly different between groups at either time point. Serum levels of IL-6 were not detectable in both groups. Cardiomyocyte alterations observed after welding fume treatment were mainly due to alterations in intracellular calcium handling and phosphorylation levels of cTnI.

  12. 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

  13. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Kucera, Jan P; Prudat, Yann; Marcu, Irene C; Azzarito, Michela; Ullrich, Nina D

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

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

    Directory of Open Access Journals (Sweden)

    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

  15. 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.

  16. 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

  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. Cardiomyocyte Differentiation Promotes Cell Survival During Nicotinamide Phosphoribosyltransferase Inhibition Through Increased Maintenance of Cellular Energy Stores.

    Science.gov (United States)

    Kropp, Erin M; Broniowska, Katarzyna A; Waas, Matthew; Nycz, Alyssa; Corbett, John A; Gundry, Rebekah L

    2017-04-01

    To address concerns regarding the tumorigenic potential of undifferentiated human pluripotent stem cells (hPSC) that may remain after in vitro differentiation and ultimately limit the broad use of hPSC-derivatives for therapeutics, we recently described a method to selectively eliminate tumorigenic hPSC from their progeny by inhibiting nicotinamide phosphoribosyltransferase (NAMPT). Limited exposure to NAMPT inhibitors selectively removes hPSC from hPSC-derived cardiomyocytes (hPSC-CM) and spares a wide range of differentiated cell types; yet, it remains unclear when and how cells acquire resistance to NAMPT inhibition during differentiation. In this study, we examined the effects of NAMPT inhibition among multiple time points of cardiomyocyte differentiation. Overall, these studies show that in vitro cardiomyogenic commitment and continued culturing provides resistance to NAMPT inhibition and cell survival is associated with the ability to maintain cellular ATP pools despite depletion of NAD levels. Unlike cells at earlier stages of differentiation, day 28 hPSC-CM can survive longer periods of NAMPT inhibition and maintain ATP generation by glycolysis and/or mitochondrial respiration. This is distinct from terminally differentiated fibroblasts, which maintain mitochondrial respiration during NAMPT inhibition. Overall, these results provide new mechanistic insight into how regulation of cellular NAD and energy pools change with hPSC-CM differentiation and further inform how NAMPT inhibition strategies could be implemented within the context of cardiomyocyte differentiation. Stem Cells Translational Medicine 2017;6:1191-1201. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  19. α1A-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

    Science.gov (United States)

    Sato, Masaaki; Evans, Bronwyn A; Sandström, Anna L; Chia, Ling Yeong; Mukaida, Saori; Thai, Bui San; Nguyen, Anh; Lim, Linzi; Tan, Christina Y R; Baltos, Jo-Anne; White, Paul J; May, Lauren T; Hutchinson, Dana S; Summers, Roger J; Bengtsson, Tore

    2018-02-01

    The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α 1A -adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α 1A -AR signalling in CHO-K1 cells co-expressing the human α 1A -AR and GLUT4 (CHOα 1A GLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca 2+ mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α 1A -AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α 1 -AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOα 1A GLUT4myc cells, siRNA directed against rictor but not raptor suppressed α 1A -AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α 1A -AR agonists. Our findings identify a novel link between the α 1A -AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α 1A -AR selective agonists as tools in the treatment of cardiac dysfunction. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. 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.

  1. 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.

  2. A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer.

    Directory of Open Access Journals (Sweden)

    Hideyuki Terazono

    Full Text Available A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

  3. A distribution analysis of action potential parameters obtained from patch-clamped human stem cell-derived cardiomyocytes

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    Fernando López-Redondo

    2016-06-01

    Full Text Available We investigated electrophysiological properties of human induced-pluripotent-stem-cell-derived and embryonic-stem-cell-derived cardiomyocytes, and analyzed action potential parameters by plotting their frequency distributions. In the both cell lines, the distribution analysis revealed that histograms of maximum upstroke velocity showed two subpopulations with similar intersection values. Sub-populations with faster maximum upstroke velocity showed significant prolongation of action potential durations by application of E-4031, whereas others did not, which may be partly due to shallower maximum diastolic potentials. We described electrophysiological and pharmacological properties of stem-cell-derived cardiomyocytes in the respective sub-populations, which provides a way to characterize diverse electrical properties of stem-cell-derived cardiomyocytes systematically.

  4. 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...

  5. Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes

    Directory of Open Access Journals (Sweden)

    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.

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

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    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

  7. 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

  8. 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.

  9. In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

    Science.gov (United States)

    Shijun, Xu; Junsheng, Mu; Jianqun, Zhang; Ping, Bo

    2016-03-01

    Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture. © The Author(s) 2016.

  10. 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.

  11. Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP-2/MLCK-mediated cardiomyocyte protection from hypoxic injury.

    Science.gov (United States)

    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.

  12. Analysis of mitochondrial 3D-deformation in cardiomyocytes during active contraction reveals passive structural anisotropy of orthogonal short axes.

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    Yael Yaniv

    Full Text Available The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the isolated cell in an elongated cylindrical shape with an elliptical cross-section, even during contraction-relaxation cycles. Cardiomyocyte mitochondria are micron-sized, fluid-filled passive spheres distributed throughout the cell in a crystal-like lattice, arranged in pairs sandwiched between the sarcomere contractile machinery, both longitudinally and radially. Their shape represents the extant 3-dimensional (3D force-balance. We developed a novel method to examine mitochondrial 3D-deformation in response to contraction and relaxation to understand how dynamic forces are balanced inside cardiomyocytes. The variation in transmitted light intensity induced by the periodic lattice of myofilaments alternating with mitochondrial rows can be analyzed by Fourier transformation along a given cardiomyocyte axis to measure mitochondrial deformation along that axis. This technique enables precise detection of changes in dimension of ∼1% in ∼1 µm (long-axis structures with 8 ms time-resolution. During active contraction (1 Hz stimulation, mitochondria deform along the length- and width-axes of the cell with similar deformation kinetics in both sarcomere and mitochondrial structures. However, significant deformation anisotropy (without hysteresis was observed between the orthogonal short-axes (i.e., width and depth of mitochondria during electrical stimulation. The same degree of deformation anisotropy was also found between the myocyte orthogonal short-axes during electrical stimulation. Therefore, the deformation of the mitochondria reflects the overall deformation of the cell, and the apparent stiffness and stress/strain characteristics of the cytoskeleton differ appreciably between the two cardiomyocyte orthogonal short-axes. This method may be applied to obtaining a better understanding of the dynamic force-balance inside cardiomyocytes and of changes in the

  13. A Non-invasive Platform for Functional Characterization of Stem-Cell-Derived Cardiomyocytes with Applications in Cardiotoxicity Testing

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    Mahnaz Maddah

    2015-04-01

    Full Text Available We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes.

  14. 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

  15. 5'-Hydroxymethylcytosine Precedes Loss of CpG Methylation in Enhancers and Genes Undergoing Activation in Cardiomyocyte Maturation.

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    David K Kranzhöfer

    Full Text Available Cardiomyocytes undergo major changes in DNA methylation during maturation and transition to a non-proliferative state after birth. 5'-hydroxylation of methylated cytosines (5hmC is not only involved in DNA loss of CpG methylation but is also thought to be an epigenetic mark with unique distribution and functions. Here, we sought to get insight into the dynamics of 5'-hydroxymethylcytosine in newborn and adult cardiomyocytes.Cardiomyocyte nuclei from newborn and adult C57BL/6 mice were purified by flow cytometric sorting. 5hmC-containing DNA was captured by selective chemical labeling, followed by deep sequencing. Sequencing reads of library replicates were mapped independently (n = 3 for newborn, n = 2 for adult mice and merged for further analysis steps. 5hmC coverage was normalized to read length and the total number of mapped reads (RPKM. MethylC-Seq, ChIP-Seq and RNA-Seq data sets of newborn and adult cardiomyocytes served to elucidate specific features of 5hmC at gene bodies and around low methylated regions (LMRs representing regulatory genomic regions with enhancer function.163,544 and 315,220 5hmC peaks were identified in P1 and adult cardiomyocytes, respectively. Of these peaks, 66,641 were common between P1 and adult cardiomyocytes with more than 50% reciprocal overlap. P1 and adult 5hmC peaks were overrepresented in genic features such as exons, introns, 3'- and 5'-untranslated regions (UTRs, promotors and transcription end sites (TES. During cardiomyocyte maturation, 5hmC was found to be enriched at sites of subsequent DNA loss of CpG methylation such as gene bodies of upregulated genes (i.e. Atp2a2, Tnni3, Mb, Pdk4. Additionally, centers of postnatally established enhancers were premarked by 5hmC before DNA loss of CpG methylation.Simultaneous analysis of 5hmC-Seq, MethylC-Seq, RNA-Seq and ChIP-Seq data at two defined time points of cardiomyocyte maturation demonstrates that 5hmC is positively associated with gene expression and

  16. Loss of Mouse P2Y6 Nucleotide Receptor Is Associated with Physiological Macrocardia and Amplified Pathological Cardiac Hypertrophy.

    Science.gov (United States)

    Clouet, Sophie; Di Pietrantonio, Larissa; Daskalopoulos, Evangelos-Panagiotis; Esfahani, Hrag; Horckmans, Michael; Vanorlé, Marion; Lemaire, Anne; Balligand, Jean-Luc; Beauloye, Christophe; Boeynaems, Jean-Marie; Communi, Didier

    2016-07-22

    The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced postnatal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Mouse models of cataract

    Indian Academy of Sciences (India)

    2009-12-31

    Dec 31, 2009 ... Much of our knowledge about the function of genes in cataracts has been derived from the molecular analysis of spontaneous or induced mutations in the mouse. Mutations affecting the mouse lens can be identified easily by visual inspection, and a remarkable number of mutant lines have been ...

  18. Gaze beats mouse

    DEFF Research Database (Denmark)

    Mateo, Julio C.; San Agustin, Javier; Hansen, John Paulin

    2008-01-01

    Facial EMG for selection is fast, easy and, combined with gaze pointing, it can provide completely hands-free interaction. In this pilot study, 5 participants performed a simple point-and-select task using mouse or gaze for pointing and a mouse button or a facial-EMG switch for selection. Gaze po...

  19. Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions.

    Science.gov (United States)

    Mollerup, Sarah; Hofgaard, Johannes P; Braunstein, Thomas H; Kjenseth, Ane; Leithe, Edward; Rivedal, Edgar; Holstein-Rathlou, Niels-Henrik; Nielsen, Morten Schak

    2011-08-01

    Gαq-stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α-adrenergic Gαq-coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se.

  20. 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

  1. RNA expression profiling of human iPSC-derived cardiomyocytes in a cardiac hypertrophy model.

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    Praful Aggarwal

    Full Text Available Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs play an important role in the regulation of messenger RNA (mRNA and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1 stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.

  2. 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.

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

    Science.gov (United States)

    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.

  4. Corticosteroids and redox potential modulate spontaneous contractions in isolated rat ventricular cardiomyocytes.

    Science.gov (United States)

    Rossier, Michel F; Lenglet, Sébastien; Vetterli, Laurène; Python, Magaly; Maturana, Andrés

    2008-10-01

    The mineralocorticoid receptor has been implicated in the development of several cardiac pathologies and could participate in the high incidence of lethal ventricular arrhythmias associated with hyperaldosteronism. We have observed previously that aldosterone markedly increases in vitro the rate of spontaneous contractions of isolated neonate rat ventricular myocytes, a putative proarrhythmogenic condition if occurring in vivo. In the present study, we investigated the effect of glucocorticoids, the involvement of the glucocorticoid receptor, and the modulation of their action by redox agents. Aldosterone and glucocorticoids exerted in vitro a similar, concentration-dependent chronotropic action on cardiomyocytes, which was mediated by both the mineralocorticoid and glucocorticoid receptors. However, the relative contribution of each receptor was different for each agonist, at each concentration. Angiotensin II induced a similar response that was entirely dependent on the activity of the glucocorticoid receptor. Corticosteroid action was modulated by the redox state of the cells, with oxidation increasing the response while reducing conditions partially preventing it. When only the mineralocorticoid receptor was functionally present in the cells, oxidation was necessary to reveal glucocorticoid action, but no obvious competition with mineralocorticoids was observed when both agonists where simultaneously present. In conclusion, corticosteroids exert a strong chronotropic action in ventricular cardiomyocytes, mediated by both the mineralocorticoid and glucocorticoid receptors and modulated by the redox state of the cell. This phenomenon is believed to be because of cell electric remodeling and could contribute in vivo to the deleterious consequence of inappropriate receptor activation, leading to increased susceptibility of patients to arrhythmias.

  5. 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.

  6. 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

    Science.gov (United States)

    Correia, Cláudia; Koshkin, Alexey; Duarte, Patrícia; Hu, Dongjian; Carido, Madalena; Sebastião, Maria J; Gomes-Alves, Patrícia; Elliott, David A; Domian, Ibrahim J; Teixeira, Ana P; Alves, Paula M; Serra, Margarida

    2017-11-27

    Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs. © 2017 Wiley Periodicals, Inc.

  7. Spatiotemporal stability of neonatal rat cardiomyocyte monolayers spontaneous activity is dependent on the culture substrate.

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    Jonathan Boudreau-Béland

    Full Text Available In native conditions, cardiac cells must continuously comply with diverse stimuli necessitating a perpetual adaptation. Polydimethylsiloxane (PDMS is commonly used in cell culture to study cellular response to changes in the mechanical environment. The aim of this study was to evaluate the impact of using PDMS substrates on the properties of spontaneous activity of cardiomyocyte monolayer cultures. We compared PDMS to the gold standard normally used in culture: a glass substrate. Although mean frequency of spontaneous activity remained unaltered, incidence of reentrant activity was significantly higher in samples cultured on glass compared to PDMS substrates. Higher spatial and temporal instability of the spontaneous rate activation was found when cardiomyocytes were cultured on PDMS, and correlated with decreased connexin-43 and increased CaV3.1 and HCN2 mRNA levels. Compared to cultures on glass, cultures on PDMS were associated with the strongest response to isoproterenol and acetylcholine. These results reveal the importance of carefully selecting the culture substrate for studies involving mechanical stimulation, especially for tissue engineering or pharmacological high-throughput screening of cardiac tissue analog.

  8. Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes.

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    Sheyda R Frolova

    Full Text Available The ability of azobenzene trimethylammonium bromide (azoTAB to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav, calcium (ICav, and potassium (IKv currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+ and calcium (Ca2+ currents and potentiation of net potassium (K+ currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.

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

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    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.

  10. Semantics-Based Composition of Integrated Cardiomyocyte Models Motivated by Real-World Use Cases.

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    Neal, Maxwell L; Carlson, Brian E; Thompson, Christopher T; James, Ryan C; Kim, Karam G; Tran, Kenneth; Crampin, Edmund J; Cook, Daniel L; Gennari, John H

    2015-01-01

    Semantics-based model composition is an approach for generating complex biosimulation models from existing components that relies on capturing the biological meaning of model elements in a machine-readable fashion. This approach allows the user to work at the biological rather than computational level of abstraction and helps minimize the amount of manual effort required for model composition. To support this compositional approach, we have developed the SemGen software, and here report on SemGen's semantics-based merging capabilities using real-world modeling use cases. We successfully reproduced a large, manually-encoded, multi-model merge: the "Pandit-Hinch-Niederer" (PHN) cardiomyocyte excitation-contraction model, previously developed using CellML. We describe our approach for annotating the three component models used in the PHN composition and for merging them at the biological level of abstraction within SemGen. We demonstrate that we were able to reproduce the original PHN model results in a semi-automated, semantics-based fashion and also rapidly generate a second, novel cardiomyocyte model composed using an alternative, independently-developed tension generation component. We discuss the time-saving features of our compositional approach in the context of these merging exercises, the limitations we encountered, and potential solutions for enhancing the approach.

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

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    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.

  12. Generation of Cardiomyocytes in Pipe-Based Microbioreactor Under Segmented Flow

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    Dimitry Spitkovsky

    2016-05-01

    Full Text Available Background/Aims: Embryonic stem (ES cells have got a broad range differentiation potential. The differentiation is initiated via aggregation of non-differentiated ES cells into embryoid body (EB capable of multi-lineage development. However experimental variables present in standard differentiation techniques lead to high EB heterogeneity, affecting development into the cells of desired lineage, and do not support the process automatization and scalability. Methods: Here we present a novel pipe based microbioreactor (PBM setup based on segmented flow, designed for spatial maintenance of temperature, nutrition supply, gas supply and sterility. Results: We verified PBM feasibility for continuous process generating cardiac cells starting from single ES cell suspension followed by EB formation for up to 10 days. The ES cells used in the study were genetically modified for cardiac-specific EGFP expression allowing optical monitoring of cardiomyocytes while EBs remained within PBM for up to 10 days. Efficiency of cardiac cells formation within PBM was similar compared to a standard hanging drop based protocol. Conclusion: Our findings ensure further development of microfluidic bioreactor technology to enable robust cardiomyocytes production for needs of drug screening, tissue engineering and other applications.

  13. Exercise training prior to myocardial infarction attenuates cardiac deterioration and cardiomyocyte dysfunction in rats.

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    Bozi, Luiz Henrique Marchesi; Maldonado, Izabel Regina dos Santos Costa; Baldo, Marcelo Perim; Silva, Márcia Ferreira da; Moreira, José Bianco Nascimento; Novaes, Rômulo Dias; Ramos, Regiane Maria Soares; Mill, José Geraldo; Brum, Patricia Chakur; Felix, Leonardo Bonato; Gomes, Thales Nicolau Prímola; Natali, Antônio José

    2013-04-01

    The present study was performed to investigate 1) whether aerobic exercise training prior to myocardial infarction would prevent cardiac dysfunction and structural deterioration and 2) whether the potential cardiac benefits of aerobic exercise training would be associated with preserved morphological and contractile properties of cardiomyocytes in post-infarct remodeled myocardium. Male Wistar rats underwent an aerobic exercise training protocol for eight weeks. The rats were then assigned to sham surgery (SHAM), sedentary lifestyle and myocardial infarction or exercise training and myocardial infarction groups and were evaluated 15 days after the surgery. Left ventricular tissue was analyzed histologically, and the contractile function of isolated myocytes was measured. Student's t-test was used to analyze infarct size and ventricular wall thickness, and the other parameters were analyzed by the Kruskal-Wallis test followed by Dunn's test or a one-way analysis of variance followed by Tukey's test (pmyocardial infarction extension, a thicker infarcted wall and less collagen accumulation as compared to myocardial infarctions in sedentary animals. Myocardial infarction-induced left ventricular dilation and cardiac dysfunction, as evaluated by +dP/dt and -dP/dt, were both prevented by previous aerobic exercise training. Moreover, aerobic exercise training preserved cardiac myocyte shortening, improved the maximum shortening and relengthening velocities in infarcted hearts and enhanced responsiveness to calcium. Previous aerobic exercise training attenuated the cardiac dysfunction and structural deterioration promoted by myocardial infarction, and such benefits were associated with preserved cardiomyocyte morphological and contractile properties.

  14. Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness.

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    Ribeiro, Alexandre J S; Ang, Yen-Sin; Fu, Ji-Dong; Rivas, Renee N; Mohamed, Tamer M A; Higgs, Gadryn C; Srivastava, Deepak; Pruitt, Beth L

    2015-10-13

    Single cardiomyocytes contain myofibrils that harbor the sarcomere-based contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetal-like misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-µm(2) rectangles with length:width aspect ratios of 5:1-7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.

  15. 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.

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

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    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.

  17. Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury

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    Yi-Wen Chen

    2013-01-01

    Full Text Available Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3, caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP, Ena/VASP-like protein (Evl, and isopentenyl-diphosphate delta-isomerase 1 (Idi-1 were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.

  18. Mitochondrial function in permeabilized cardiomyocytes is largely preserved in the senescent rat myocardium.

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    Martin Picard

    Full Text Available The aging heart is characterized by a progressive decline in contractile function and diastolic relaxation. Amongst the factors implicated in these changes is a progressive replacement fibrosis secondary to cardiomyocyte death, oxidative damage, and energetic deficit, each of which may be secondary to impaired mitochondrial function. Here, we performed an in-depth examination of mitochondrial function in saponin-permeabilized cardiomyocyte bundles, a preparation where all mitochondria are represented and their structure intact, from young adult (YA and senescent (SEN rats (n = 8 per group. When accounting for increased fibrosis (+19%, P<0.01 and proportional decrease in citrate synthase activity in the SEN myocardium (-23%, P<0.05, mitochondrial respiration and reactive oxygen species (H(2O(2 emission across a range of energized states was similar between age groups. Accordingly, the abundance of electron transport chain proteins was also unchanged. Likewise, except for CuZnSOD (-37%, P<0.05, the activity of antioxidant enzymes was unaltered with aging. Although time to mitochondrial permeability transition pore (mPTP opening was decreased (-25%, P<0.05 in the SEN heart, suggesting sensitization to apoptotic stimuli, this was not associated with a difference in apoptotic index measured by ELISA. Collectively, our results suggest that the function of existing cardiac ventricular mitochondria is relatively preserved in SEN rat heart when measured in permeabilized cells.

  19. Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis.

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    Xiaohu Fan

    Full Text Available Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk disassembly precedes that of titin (M-line, suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1 induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.

  20. Changes in the mitochondrial function and in the efficiency of energy transfer pathways during cardiomyocyte aging.

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    Tepp, Kersti; Puurand, Marju; Timohhina, Natalja; Adamson, Jasper; Klepinin, Aleksandr; Truu, Laura; Shevchuk, Igor; Chekulayev, Vladimir; Kaambre, Tuuli

    2017-08-01

    The role of mitochondria in alterations that take place in the muscle cell during healthy aging is a matter of debate during recent years. Most of the studies in bioenergetics have a focus on the model of isolated mitochondria, while changes in the crosstalk between working myofibrils and mitochondria in senescent cardiomyocytes have been less studied. The aim of our research was to investigate the modifications in the highly regulated ATP production and energy transfer systems in heart cells in old rat cardiomyocytes. The results of our work demonstrated alterations in the diffusion restrictions of energy metabolites, manifested by changes in the apparent Michaelis-Menten constant of mitochondria to exogenous ADP. The creatine kinase (CK) phosphotransfer pathway efficiency declines significantly in senescence. The ability of creatine to stimulate OXPHOS as well as to increase the affinity of mitochondria for ADP is falling and the most critical decline is already in the 1-year group (middle-age model in rats). Also, a moderate decrease in the adenylate kinase phosphotransfer system was detected. The importance of glycolysis increases in senescence, while the hexokinase activity does not change during healthy aging. The main result of our study is that the decline in the heart muscle performance is not caused by the changes in the respiratory chain complexes activity but mainly by the decrease in the energy transfer efficiency, especially by the CK pathway.

  1. Mitochondrial dynamics in the adult cardiomyocytes: which roles for a highly specialized cell?

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    Jerome ePiquereau

    2013-05-01

    Full Text Available Mitochondrial dynamics is a recent topic of research in the field of cardiac physiology. The study of mechanisms involved in the morphological changes and in the motility of mitochondria is legitimate since the adult cardiomyocytes possess numerous mitochondria which occupy at least 30% of cell volume. However, architectural constraints exist in the cardiomyocyte that limit mitochondrial movements and communication between adjacent mitochondria. Still, the proteins involved in mitochondrial fusion and fission are highly expressed in these cells and could be involved in different processes important for the cardiac function. For example, they are required for mitochondrial biogenesis to synthesize new mitochondria and for the quality-control of the organelles. They are also involved in inner membrane organization and may play a role in apoptosis. More generally, change in mitochondrial morphology can have consequences in the functioning of the respiratory chain, in the regulation of the mitochondrial permeability transition pore (MPTP, and in the interactions with other organelles. Furthermore, the proteins involved in fusion and fission of mitochondria are altered in cardiac pathologies such as ischemia/reperfusion or heart failure, and appear to be valuable targets for pharmacological therapies. Thus, mitochondrial dynamics deserves particular attention in cardiac research. The present review draws up a report of our knowledge on these phenomena.

  2. 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.

  3. Human Cardiomyocyte Progenitor Cells in Co-culture with Rat Cardiomyocytes Form a Pro-arrhythmic Substrate: Evidence for Two Different Arrhythmogenic Mechanisms

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    Nicoline W. Smit

    2017-10-01

    Full Text Available Background: Cardiomyocyte progenitor cells (CMPCs are a promising cell source for regenerative cell therapy to improve cardiac function after myocardial infarction. However, it is unknown whether undifferentiated CMPCs have arrhythmogenic risks. We investigate whether undifferentiated, regionally applied, human fetal CMPCs form a pro-arrhythmic substrate in co-culture with neonatal rat ventricular myocytes (NRVMs.Method: Unipolar extracellular electrograms, derived from micro-electrode arrays (8 × 8 electrodes containing monolayers of NRVMs (control, or co-cultures of NRVMs and locally seeded CMPCs were used to determine conduction velocity and the incidence of tachy-arrhythmias. Micro-electrodes were used to record action potentials. Conditioned medium (Cme of CMPCs was used to distinguish between coupling or paracrine effects.Results: Co-cultures demonstrated conduction slowing (5.6 ± 0.3 cm/s, n = 50 compared to control monolayers (13.4 ± 0.4 cm/s, n = 26 and monolayers subjected to Cme (13.7 ± 0.6 cm/s, n = 11, all p < 0.001. Furthermore, co-cultures had a more depolarized resting membrane than control monolayers (−47.3 ± 17.4 vs. −64.8 ± 7.7 mV, p < 0.001 and monolayers subjected to Cme (−64.4 ± 8.1 mV, p < 0.001. Upstroke velocity was significantly decreased in co-cultures and action potential duration was prolonged. The CMPC region was characterized by local ST-elevation in the recorded electrograms. The spontaneous rhythm was faster and tachy-arrhythmias occurred more often in co-cultured monolayers than in control monolayers (42.0 vs. 5.4%, p < 0.001.Conclusion: CMPCs form a pro-arrhythmic substrate when co-cultured with neonatal cardiomyocytes. Electrical coupling between both cell types leads to current flow between a, slowly conducting, depolarized and the normal region leading to local ST-elevations and the occurrence of tachy-arrhythmias originating from the non-depolarized zone.

  4. Contribution of bone marrow-derived hematopoietic stem/progenitor cells to the generation of donor-marker⁺ cardiomyocytes in vivo.

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    Mitsuhiro Fukata

    Full Text Available BACKGROUND: Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker(+ cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: Purified BM stem/progenitor cells from green fluorescence protein (GFP mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP-transgenic mice. Purified human hematopoietic stem cells (HSCs from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγ(null mice. GFP(+ cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP(+ donor-derived cells, GFP(+CFP(+ fused cells, and CFP(+ recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin(-/lowCD45(+ hematopoietic cells generated greater number of GFP(+ cardiomyocytes than Lin(-/lowCD45(- mesenchymal cells (37.0+/-23.9 vs 0.00+/-0.00 GFP(+ cardiomyocytes per a recipient, P = 0.0095. The number of transplanted purified HSCs (Lin(-/lowSca-1(+ or Lin(-Sca-1(+c-Kit(+ or CD34(-Lin(-Sca-1(+c-Kit(+ showed correlation to the number of GFP(+ cardiomyocytes (P<0.05 in each cell fraction, and the incidence of GFP(+ cardiomyocytes per injected cell dose was greatest in CD34(-Lin(-Sca-1(+c-Kit(+ recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP(+ cardiomyocytes than common lymphoid progenitors (12.8+/-10.7 vs 0.67+/-1.00 GFP(+ cardiomyocytes per a recipient, P = 0.0021. In CFP recipients, all GFP(+ cardiomyocytes examined coexpressed CFP

  5. Transcriptional effects of E3 ligase atrogin-1/MAFbx on apoptosis, hypertrophy and inflammation in neonatal rat cardiomyocytes.

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    Yong Zeng

    Full Text Available Atrogin-1/MAFbx is an ubiquitin E3 ligase that regulates myocardial structure and function through the ubiquitin-dependent protein modification. However, little is known about the effect of atrogin-1 activation on the gene expression changes in cardiomyocytes. Neonatal rat cardiomyocytes were infected with adenovirus atrogin-1 (Ad-atrogin-1 or GFP control (Ad-GFP for 24 hours. The gene expression profiles were compared with microarray analysis. 314 genes were identified as differentially expressed by overexpression of atrogin-1, of which 222 were up-regulated and 92 were down-regulated. Atrogin-1 overexpression significantly modulated the expression of genes in 30 main functional categories, most genes clustered around the regulation of cell death, proliferation, inflammation, metabolism and cardiomyoctye structure and function. Moreover, overexpression of atrogin-1 significantly inhibited cardiomyocyte survival, hypertrophy and inflammation under basal condition or in response to lipopolysaccharide (LPS. In contrast, knockdown of atrogin-1 by siRNA had opposite effects. The mechanisms underlying these effects were associated with inhibition of MAPK (ERK1/2, JNK1/2 and p38 and NF-κB signaling pathways. In conclusion, the present microarray analysis reveals previously unappreciated atrogin-1 regulation of genes that could contribute to the effects of atrogin-1 on cardiomyocyte survival, hypertrophy and inflammation in response to endotoxin, and may provide novel insight into how atrogin-1 modulates the programming of cardiac muscle gene expression.

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

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    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.

  7. Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

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    Kiso, Hironori [Department of Internal Medicine, Division of Cardiovascular and Respiratory Medicine, Akita University Graduate School of Medicine (Japan); Ohba, Takayoshi [Department of Cell Physiology, Akita University Graduate School of Medicine (Japan); Iino, Kenji; Sato, Kazuhiro; Terata, Yutaka [Department of Internal Medicine, Division of Cardiovascular and Respiratory Medicine, Akita University Graduate School of Medicine (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University Graduate School of Medicine (Japan); Ono, Kyoichi [Department of Cell Physiology, Akita University Graduate School of Medicine (Japan); Watanabe, Hiroyuki, E-mail: hirow@doc.med.akita-u.ac.jp [Department of Internal Medicine, Division of Cardiovascular and Respiratory Medicine, Akita University Graduate School of Medicine (Japan); Ito, Hiroshi [Department of Internal Medicine, Division of Cardiovascular and Respiratory Medicine, Akita University Graduate School of Medicine (Japan)

    2013-07-05

    Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.

  8. 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

  9. 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

  10. Engineered Biomaterials Control Differentiation and Proliferation of Human-Embryonic-Stem-Cell-Derived Cardiomyocytes via Timed Notch Activation

    Directory of Open Access Journals (Sweden)

    Jason C. Tung

    2014-03-01

    Full Text Available For cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes.

  11. In vitro analysis of SERCA2 gene regulation in hypertrophic cardiomyocytes and increasing transfection efficiency by gene-gun biolistics

    NARCIS (Netherlands)

    K. Eizema (Karin); H.A. van Heugten (Han); K. Bezstarosti (Karel); M.C. van Setten (Marga); J.M.J. Lamers (Jos)

    1999-01-01

    textabstractThe transcriptional downregulation of the SERCA2 gene is studied using neonatal rat cardiomyocytes stimulated with endothelin-1 to induce hypertrophy. Liposome-based transfection of cells with a 1.9 kb SERCA2 promoter fragment directed expression of a reporter gene identical to the

  12. Stroma cell-derived factor-1α signaling enhances calcium transients and beating frequency in rat neonatal cardiomyocytes.

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    Ielham Hadad

    Full Text Available Stroma cell-derived factor-1α (SDF-1α is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP3R blocker, but not with a ryanodine receptor (RyR antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP3R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP3-gated fashion leading to a positive chronotropic and inotropic effect.

  13. MUSCLEMOTION : A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo

    NARCIS (Netherlands)

    Sala, Luca; van Meer, Berend J; Tertoolen, Leon T; Bakkers, Jeroen; Bellin, Milena; Davis, Richard P; Denning, Chris N; Dieben, Michel A; Eschenhagen, Thomas; Giacomelli, Elisa; Grandela, Catarina; Hansen, Arne; Holman, Eduard; Jongbloed, Monique R; Kamel, Sarah M; Koopman, Charlotte D; Lachaud, Quentin; Mannhardt, Ingra; Mol, Mervyn P; Mosqueira, Diogo; Orlova, Valeria V; Passier, Robert; Ribeiro, Marcelo C; Saleem, Umber; Smith, Godfrey; Burton, Francis L L; Mummery, Christine L

    2017-01-01

    Rationale: There are several methods to measure cardiomyocyte (CM) and muscle contraction but these require customized hardware, expensive apparatus and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across

  14. 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.

  15. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing

    NARCIS (Netherlands)

    Nicolas, J.A.Y.; Hendriksen, P.J.M.; Haan, de L.H.J.; Koning, R.; Rietjens, I.M.C.M.; Bovee, T.F.H.

    2015-01-01

    The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on

  16. 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

  17. 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

  18. Mechanical stress triggers cardiomyocyte autophagy through angiotensin II type 1 receptor-mediated p38MAP kinase independently of angiotensin II.

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    Li Lin

    Full Text Available Angiotensin II (Ang II type 1 (AT1 receptor is known to mediate a variety of physiological actions of Ang II including autophagy. However, the role of AT1 receptor in cardiomyocyte autophagy triggered by mechanical stress still remains elusive. The aim of this study was therefore to examine whether and how AT1 receptor participates in cardiomyocyte autophagy induced by mechanical stresses. A 48-hour mechanical stretch and a 4-week transverse aorta constriction (TAC were imposed to cultured cardiomyocytes of neonatal rats and adult male C57B/L6 mice, respectively, to induce cardiomyocyte hypertrophy prior to the assessment of cardiomyocyte autophagy using LC3b-II. Losartan, an AT1 receptor blocker, but not PD123319, the AT2 inhibitor, was found to significantly reduce mechanical stretch-induced LC3b-II upregulation. Moreover, inhibition of p38MAP kinase attenuated not only mechanical stretch-induced cardiomyocyte hypertrophy but also autophagy. To the contrary, inhibition of ERK and JNK suppressed cardiac hypertrophy but not autophagy. Intriguingly, mechanical stretch-induced autophagy was significantly inhibited by Losartan in the absence of Ang II. Taken together, our results indicate that mechanical stress triggers cardiomyocyte autophagy through AT1 receptor-mediated activation of p38MAP kinase independently of Ang II.

  19. Multi-parameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Doherty, Kimberly R., E-mail: kimberly.doherty@quintiles.com [Quintiles, 777 Oakmont Lane Suite 100, Westmont, IL 60559 (United States); Wappel, Robert L.; Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M. [Quintiles, 777 Oakmont Lane Suite 100, Westmont, IL 60559 (United States); Kramer, James W.; Brown, Arthur M. [ChanTest Corporation, 14656 Neo Parkway, Cleveland, OH 44128 (United States); Shell, Scott A.; Bacus, Sarah [Quintiles, 777 Oakmont Lane Suite 100, Westmont, IL 60559 (United States)

    2013-10-01

    Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development. - Highlights: • TKi with known adverse effects show unique cardiotoxicity profiles in this panel. • Crizotinib increases ROS, apoptosis, and

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  1. 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.

  2. 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

  3. 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.

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. Engineered myocardial tissues constructed in vivo using cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells in rats

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    Xing Yujie

    2012-01-01

    Full Text Available Abstract Background To explore the feasibility of constructing engineered myocardial tissues (EMTs in vivo, using polylactic acid -co-glycolic acid (PLGA for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs for seeded cells. Methods BMMSCs were isolated from femur and tibia of Sprague-Dawley (SD rats by density-gradient centrifugation. The third passage cells were treated with 10 μmol/L 5-azacytidine (5-aza and 0.1 μmol/L angiotensin II (Ang II for 24 h, followed by culturing in complete medium for 3 weeks to differentiated into cardiomyocyte-like cells. The cardiomyocyte-like cells were seeded into PLGA scaffolds to form the grafts. The grafts were cultured in the incubator for three days and then implanted into the peritoneal cavity of SD rats. Four weeks later, routine hematoxylin-eosin (HE staining, immunohistochemical staining for myocardium-specific cardiac troponin I (cTnI, scanning electron microscopy and transmission electron microscopy were used to analyze the morphology and microconstruction of the EMTs in host rats. Results HE staining showed that the cardiomyocyte-like cells distributed equally in the PLGA scaffold, and the nuclei arranged in the spindle shape. Immunohistochemical staining revealed that majority of engrafted cells in the PLGA -Cardiomyocyte-like cells group were positive for cTnI. Scanning electron microscopy showed that the inoculated cells well attached to PLGA and grew in 3 dimensions in construct. Transmission electron microscopy showed that the EMTs contained well arranged myofilaments paralleled to the longitudinal cell axis, the cells were rich in endoplasmic reticulum and mitochondria, while desmosomes, gap junction and Z line-like substances were also can be observed as well within the engrafted cells. Conclusion We have developed an in vivo method to construct engineered myocardial tissue. The in vivo microenvironment helped engrafted cells/tissue survive and

  6. 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.

  7. 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.

  8. Mouse Genome Informatics (MGI)

    Data.gov (United States)

    U.S. Department of Health & Human Services — MGI is the international database resource for the laboratory mouse, providing integrated genetic, genomic, and biological data to facilitate the study of human...

  9. Mouse Phenome Database (MPD)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Mouse Phenome Database (MPD) has characterizations of hundreds of strains of laboratory mice to facilitate translational discoveries and to assist in selection...

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

    Science.gov (United States)

    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

  11. Gαi2- and Gαi3-specific regulation of voltage-dependent L-type calcium channels in cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Sara Dizayee

    Full Text Available BACKGROUND: Two pertussis toxin sensitive G(i proteins, G(i2 and G(i3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G(i isoforms are functionally distinct. To test for isoform-specific functions of G(i proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC. METHODS: Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα(i2 (Gα(i2 (-/- or Gα(i3 (Gα(i3 (-/-. mRNA levels of Gα(i/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gα(i and Ca(vα(1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2 phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. RESULTS: In cardiac tissue from Gα(i2 (-/- mice, Gα(i3 mRNA and protein expression was upregulated to 187 ± 21% and 567 ± 59%, respectively. In Gα(i3 (-/- mouse hearts, Gα(i2 mRNA (127 ± 5% and protein (131 ± 10% levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα(i2 (-/- mice was lowered (-7.9 ± 0.6 pA/pF, n = 11, p<0.05 compared to wild-type cells (-10.7 ± 0.5 pA/pF, n = 22, whereas it was increased in myocytes from Gα(i3 (-/- mice (-14.3 ± 0.8 pA/pF, n = 14, p<0.05. Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα(i2 (but not of Gα(i3 and following treatment with pertussis toxin in Gα(i3 (-/-. The pore forming Ca(vα(1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca(vα(1 and Ca(vβ(2 subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα(i2. CONCLUSION: Our data provide novel evidence for an isoform

  12. Substrate stiffness-modulated registry phase correlations in cardiomyocytes map structural order to coherent beating

    Science.gov (United States)

    Dasbiswas, K.; Majkut, S.; Discher, D. E.; Safran, Samuel A.

    2015-01-01

    Recent experiments show that both striation, an indication of the structural registry in muscle fibres, as well as the contractile strains produced by beating cardiac muscle cells can be optimized by substrate stiffness. Here we show theoretically how the substrate rigidity dependence of the registry data can be mapped onto that of the strain measurements. We express the elasticity-mediated structural registry as a phase-order parameter using a statistical physics approach that takes the noise and disorder inherent in biological systems into account. By assuming that structurally registered myofibrils also tend to beat in phase, we explain the observed dependence of both striation and strain measurements of cardiomyocytes on substrate stiffness in a unified manner. The agreement of our ideas with experiment suggests that the correlated beating of heart cells may be limited by the structural order of the myofibrils, which in turn is regulated by their elastic environment.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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

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

    Science.gov (United States)

    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. Reverse engineering life: physical and chemical mimetics for controlled stem cell differentiation into cardiomyocytes.

    Science.gov (United States)

    Skuse, Gary R; Lamkin-Kennard, Kathleen A

    2013-01-01

    Our ability to manipulate stem cells in order to induce differentiation along a desired developmental pathway has improved immeasurably in recent years. That is in part because we have a better understanding of the intracellular and extracellular signals that regulate differentiation. However, there has also been a realization that stem cell differentiation is not regulated only by chemical signals but also by the physical milieu in which a particular stem cell exists. In this regard we are challenged to mimic both chemical and physical environments. Herein we describe a method to induce stem cell differentiation into cardiomyocytes using a combination of chemical and physical cues. This method can be applied to produce differentiated cells for research and potentially for cell-based therapy of cardiomyopathies.

  17. Cardiomyocyte lipotoxicity is mediated by Il-6 and causes down-regulation of PPARs.

    Science.gov (United States)

    Haffar, Taha; Bérubé-Simard, Félix-Antoine; Bousette, Nicolas

    2015-03-27

    Here we sought to evaluate the effect of palmitate on cytokine and PPAR activity/expression. We investigated the effect of BSA conjugated palmitate and oleate on PPAR activity, PPAR-α and δ expression, as well as the expression of cytokines and key factors responsible for β-oxidation by qRT-PCR and western blotting in primary rat neonatal cardiomyocytes (NCMs). Furthermore we evaluated the effect of anti-inflammatory actions of AICAR and PPAR agonists on cytokine expression and cell death in palmitate treated NCMs. We found that palmitate caused down regulation of PPARs and increased cytokine expression and cell death, all of which was significantly attenuated by the co-administration of either AICAR or PPAR agonists. This work supports the pro-inflammatory actions of intracellular lipid and provides further insight into the pathological mechanism of cardiac lipotoxicity as occurs in diabetic hearts. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. 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

  19. The influence of proteasome inhibitor on the expression of cardiomyocytes damage markers after incubation with doxorubicin

    Directory of Open Access Journals (Sweden)

    Tereszkiewicz Sylwia

    2014-06-01

    Full Text Available The aim of the study was to verify the thesis that the cardiotoxic effects of doxorubicin are connected with activation of the ubiquitin - proteasome pathway followed by protein degradation. The expression of myocardial damage markers - fatty acid binding protein (H-FABP and brain natriuretic peptide (BNP was evaluated in rat fetal cardiomyocytes simultaneously treated with doxorubicin and the proteasome inhibitor - bortezomib. The level of H-FABP and BNP protein under the influence of doxorubicin was decreased below the detection threshold with unchanged (H-FABP or elevated (BNP mRNA expression level. Against the expectations, the inhibitor of proteasome did not abolish this effect. The observed abnormal expression of BNP and H-FABP protein after doxorubicin treatment makes their diagnostic significance in anthracycline cardiotoxicity questionable.

  20. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

    Directory of Open Access Journals (Sweden)

    Joshua D. Tompkins

    2016-02-01

    Full Text Available The directed differentiation of human cardiomyocytes (CMs from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.

  1. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

    Science.gov (United States)

    Tompkins, Joshua D.; Jung, Marc; Chen, Chang-yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

    2016-01-01

    The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors. PMID:26981572

  2. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation "Memories".

    Science.gov (United States)

    Tompkins, Joshua D; Jung, Marc; Chen, Chang-Yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A; Riggs, Arthur D

    2016-02-01

    The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation "memories" persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors.

  3. L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes.

    Science.gov (United States)

    Himes, Ryan D; Smolin, Nikolai; Kukol, Andreas; Bossuyt, Julie; Bers, Donald M; Robia, Seth L

    2016-11-08

    To determine if mutations introduced into phospholemman (PLM) co