Mouse Embryonic Stem Cell-derived Cardiomyocytes Research Articles

In vitro effect of hydroxychloroquine on pluripotent stem cells and their cardiomyocytes derivatives.

Introduction: Hydroxychloroquine (HDQ) is an antimalarial drug that has also shown its effectiveness in autoimmune diseases. Despite having side effects such as retinopathy, neuromyopathy and controversial cardiac toxicity, HDQ has been presented and now intensively studied for the treatment and prevention of coronavirus disease 2019 (COVID-19). Recent works revealed both beneficial and toxic effects during HDQ treatment. The cardiotoxic profile of HDQ remains unclear and identifying risk factors is challenging. Methods: Here, we used well-established cell-cultured to study the cytotoxic effect of HDQ, mouse induced pluripotent stem cells (miPSC) and their cardiomyocytes (CMs) derivatives were exposed to different concentrations of HDQ. Cell colony morphology was assessed by microscopy whereas cell viability was measured by flow cytometry and impedance-based methods. The effect of HDQ on beating activity of mouse and human induced pluripotent stem cell-derived CMs (miPSC-CMs and hiPSC-CMs, respectively) and mouse embryonic stem cell-derived CMs (mESC-CMs) were captured by the xCELLigence RTCA and microelectrode array (MEA) systems. Results and discussion: Our results revealed that 20µM of HDQ promotes proliferation of stem cells used suggesting that if appropriately monitored, HDQ may have a cardioprotective effect and may also represent a possible candidate for tissue repair. In addition, the field potential signals revealed that higher doses of this medication caused bradycardia that could be reversed with a higher concentration of ß-adrenergic agonist, Isoproterenol (Iso). On the contrary, HDQ caused an increase in the beating rate of hiPSC-CMs, which was further helped upon application of Isoproterenol (Iso) suggesting that HDQ and Iso may also work synergistically. These results indicate that HDQ is potentially toxic at high concentrations and can modulate the beating activity of cardiomyocytes. Moreover, HDQ could have a synergistic inotropic effect with isoproterenol on cardiac cells.

Live cell screening identifies glycosides as enhancers of cardiomyocyte cell cycle activity.

Promoting cardiomyocyte proliferation is a promising strategy to regenerate the heart. Yet, so far, it is poorly understood how cardiomyocyte proliferation is regulated, and no factor identified to promote mammalian cardiomyocyte proliferation has been translated into medical practice. Therefore, finding a novel factor will be vital. Here, we established a live cell screening based on mouse embryonic stem cell-derived cardiomyocytes expressing a non-functional human geminin deletion mutant fused to Azami Green (CM7/1-hgem-derived cardiomyocytes). We screened for a subset of compounds of the small molecule library Spectrum Collection and identified 19 potential inducers of stem cell-derived cardiomyocyte proliferation. Furthermore, the pro-proliferative potential of identified candidate compounds was validated in neonatal and adult rat cardiomyocytes as well as human induced pluripotent stem cell-derived cardiomyocytes. 18 of these compounds promoted mitosis and cytokinesis in neonatal rat cardiomyocytes. Among the top four candidates were two cardiac glycosides, peruvoside and convallatoxin, the flavonoid osajin, and the selective α-adrenoceptor antagonist and imidazoline I1 receptor ligand efaroxan hydrochloride. Inhibition of PTEN and GSK-3β enhanced cell cycle re-entry and progression upon stimulation with cardiac glycosides and osajin, while inhibition of IP3 receptors inhibited the cell cycle-promoting effect of cardiac glycosides. Collectively, we established a screening system and identified potential compounds to promote cardiomyocyte proliferation. Our data suggest that modulation of calcium handling and metabolism promotes cardiomyocyte proliferation, and cardiac glycosides might, besides increasing myocardial contraction force, contribute to cardiac repair by inducing cardiomyocyte proliferation.

Cpmer: A new conserved eEF1A2-binding partner that regulates Eomes translation and cardiomyocyte differentiation.

SummaryPrevious studies have shown that eukaryotic elongation factor 1A2 (eEF1A2) serves as an essential heart-specific translation elongation element and that its mutation or knockout delays heart development and causes congenital heart disease and death among species. However, the function and regulatory mechanisms of eEF1A2 in mammalian heart development remain largely unknown. Here we identified the long noncoding RNA (lncRNA) Cpmer (cytoplasmic mesoderm regulator), which interacted with eEF1A2 to co-regulate differentiation of mouse and human embryonic stem cell-derived cardiomyocytes. Mechanistically, Cpmer specifically recognized Eomes mRNA by RNA-RNA pairing and facilitated binding of eEF1A2 with Eomes mRNA, guaranteeing Eomes mRNA translation and cardiomyocyte differentiation. Our data reveal a novel functionally conserved lncRNA that can specifically regulate Eomes translation and cardiomyocyte differentiation, which broadens our understanding of the mechanism of lncRNA involvement in the subtle translational regulation of eEF1A2 during mammalian heart development.

Enhancement of pacing function by HCN4 overexpression in human pluripotent stem cell-derived cardiomyocytes

BackgroundThe number of patients with bradyarrhythmia and the number of patients with cardiac pacemakers are increasing with the aging population and the increase in the number of patients with heart diseases. Some patients in whom a cardiac pacemaker has been implanted experience problems such as pacemaker infection and inconvenience due to electromagnetic interference. We have reported that overexpression of HCN channels producing a pacemaker current in mouse embryonic stem cell-derived cardiomyocytes showed enhanced pacing function in vitro and in vivo. The aim of this study was to determine whether HCN4 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can strengthen the pacing function of the cells.MethodsHuman HCN4 was transduced in the AAVS1 locus of human induced pluripotent stem cells by nucleofection and HCN4-overexpressing iPSC-CMs were generated. Gene expression profiles, frequencies of spontaneous contraction and pacing abilities of HCN4-overexpressing and non-overexpressing iPSC-CMs in vitro were compared.ResultsHCN4-overexpressing iPSC-CMs showed higher spontaneous contraction rates than those of non-overexpressing iPSC-CMs. They responded to an HCN channel blocker and β adrenergic stimulation. The pacing rates against parent iPSC line-derived cardiomyocytes were also higher in HCN4-overexpressing iPSC-CMs than in non-overexpressing iPSC-CMs.ConclusionsOverexpression of HCN4 showed enhancement of If current, spontaneous firing and pacing function in iPSC-CMs. These data suggest this transgenic cell line may be useful as a cardiac pacemaker.

Recombinant Activated Protein C (rhAPC) Affects Lipopolysaccharide-Induced Mechanical Compliance Changes and Beat Frequency of mESC-Derived Cardiomyocyte Monolayers.

ABSTRACTBackground:Septic cardiomyopathy increases mortality by 70% to 90% and results in mechanical dysfunction of cells.Methods:Here, we created a LPS-induced in-vitro sepsis model with mouse embryonic stem cell-derived cardiomyocytes (mESC-CM) using the CellDrum technology which simultaneously measures mechanical compliance and beat frequency of mESCs. Visualization of reactive oxygen species (ROS), actin stress fibers, and mRNA quantification of endothelial protein C receptor (EPCR) and protease-activated receptor 1 (PAR1) before/after LPS incubation were used for method validation. Since activated protein C (APC) has cardioprotective effects, samples were treated with human recombinant APC (rhAPC) with/-out LPS predamage to demonstrate the application in therapeutic studies.Results:Twelve hours LPS treatment (5 μg/mL) increased ROS and decreased actin stress fiber density and significantly downregulated EPCR and PAR1 compared to control samples (0.26, 0.39-fold respectively). rhAPC application (5 μg/mL, 12 h) decreased ROS and recovered actin density, EPCR, and PAR1 levels were significantly upregulated compared to LPS predamaged samples (4.79, 3.49-fold respectively). The beat frequencies were significantly decreased after 6- (86%) and 12 h (73%) of LPS application. Mechanical compliance of monolayers significantly increased in a time-dependent manner, up to eight times upon 12-h LPS incubation compared to controls. rhAPC incubation increased the beat frequency by 127% (6h-LPS) and 123% (12h-LPS) and decreased mechanical compliance by 68% (12h-LPS) compared to LPS predamaged samples.Conclusion:LPS-induced contraction dysfunction and the reversal effects of rhAPC were successfully assessed by the mechanical properties of mESC-CMs. The CellDrum technology proved a decent tool to simulate sepsis in-vitro.

Organisational alteration of cardiac myofilament proteins by hyperglycaemia in mouse embryonic stem cell-derived cardiomyocytes.

The exposure of the developing foetal heart to hyperglycaemia in mothers with diabetes mellitus is a major risk factor for foetal cardiac complications that lead to heart failure. We studied the effects of hyperglycaemia on the layout of cardiac myofilament proteins in stem cell-derived cardiomyocytes and their possible underlying mechanisms. Mouse embryonic stem cells (mESCs) were differentiated into cardiac-like cells and cultured in media containing baseline- or high glucose concentrations. Cellular biomarkers were detected using Western blot analysis, immunocytochemistry, 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay, and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. High glucose decreased the proportion of cardiac troponin T and α-actinin 2 positive mESCs as well as disrupted the α-actinin 2 striated pattern and the distribution of the cardiac myosin heavy chain α- and β isoforms. However, there was no alteration of the cellular EdU uptake nor the expression of the receptor of advanced glycation end-product (RAGE). High glucose also increased the presence of the oxidative stress marker nitrotyrosine as well as the number of TUNEL-stained nuclei in cardiac-like cells. Treatment with the antioxidant N-acetyl cysteine decreased the number of TUNEL-stained cells in high glucose and improved the α-actinin 2 striated pattern. Hyperglycaemia negatively impacted the expression and cellular organisation of cardiac myofilament proteins in mESC-derived cardiomyocytes through oxidative stress. The results add further insights into the pathophysiological mechanisms of cardiac contractile dysfunction in diabetic cardiac developmental disease.

Evaluation of in vitro models of stem cell-derived cardiomyocytes to screen for potential cardiotoxicity of chemicals

Cardiotoxicity is an important toxicological endpoint for chemical and drug safety assessment. The present study aims to evaluate two stemcell-based in vitro models for cardiotoxicity screening of chemicals. Eleven model compounds were used to evaluate responses of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) using beating arrest as a readout and the analysis of electrophysiological parameters measured with a multi-electrode array (MEA) platform of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Results revealed that the hiPSC-CM MEA assay responded to all compounds. The mESC-CM beating arrest assay was not responsive to potassium channel blockers and showed a lower sensitivity to sodium channel blockers and Na+/K+ ATPase inhibitors compared to the hiPSC-CM MEA assay. Calcium channel blockers and a β-adrenergic receptor agonist showed comparable potencies in both models. The in vitro response concentrations from hiPSC-CMs were highly concordant with human effective serum concentrations of potassium and sodium channel blockers. It is concluded that both in vitro models enable the cardiotoxicity screening with different applicability domains. The mESC-CM beating arrest assay may be used as a first step in a tiered approach while the hiPSC-CM MEA assay may be the best starting point for quantitative in vitro to in vivo extrapolations.

Role of Parkin-mediated mitophagy in glucocorticoid-induced cardiomyocyte maturation

Glucocorticoids can promote cardiomyocyte maturation. However, the mechanism underlying glucocorticoid-mediated cardiomyocyte maturation is still unclear. Mitophagy plays a key role in cardiomyocyte maturation. Based on current knowledge, our study evaluated the effects of the glucocorticoid dexamethasone (100 nM) on the maturation of mouse embryonic stem cell-derived cardiomyocytes and the role of mitophagy in this maturation. The results showed that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation, inhibit cardiomyocyte proliferation, and promote myocardial fiber arrangement. However, dexamethasone did not affect mitochondrial morphology in cardiomyocytes. Glucocorticoid receptor inhibitors (RU486, 1 nM) can inhibit dexamethasone-mediated cardiomyocyte maturation. Additionally, dexamethasone can promote mitophagy in embryonic stem cell-derived cardiomyocytes and induce LC3 and lysosomal aggregation in mitochondria. The inhibition of mitophagy can inhibit the cardiomyocyte maturation effect of dexamethasone. Furthermore, our research found that dexamethasone may mediate the occurrence of mitophagy in cardiomyocytes through Parkin. The siRNA-mediated inhibition of Parkin expression can inhibit mitochondrial autophagy caused by dexamethasone, thus inhibiting cardiomyocyte maturation. Overall, our study found that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation through Parkin-mediated mitophagy.

Study of time-dependent pharmacokinetics of mouse embryonic stem cell-derived cardiomyocytes for drug screening

Study of time-dependent pharmacokinetics of mouse embryonic stem cell-derived cardiomyocytes for drug screening

Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients.

We earlier established the mouse embryonic stem (ES) cell "GS-2" line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420-495), green (~510-575), and red (~600-700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

HCN4-Overexpressing Mouse Embryonic Stem Cell-Derived Cardiomyocytes Generate a New Rapid Rhythm in Rats with Bradycardia.

A biological pacemaker is expected to solve the persisting problems of an artificial cardiac pacemaker including short battery life, lead breaks, infection, and electromagnetic interference. We previously reported HCN4 overexpression enhances pacemaking ability of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) in vitro. However, the effect of these cells on bradycardia in vivo has remained unclear. Therefore, we transplanted HCN4-overexpressing mESC-CMs into bradycardia model animals and investigated whether they could function as a biological pacemaker. The rabbit Hcn4 gene was transfected into mouse embryonic stem cells and induced HCN4-overexpressing mESC-CMs. Non-cardiomyocytes were removed under serum/glucose-free and lactate-supplemented conditions. Cardiac balls containing 5 × 103 mESC-CMs were made by using the hanging drop method. One hundred cardiac balls were injected into the left ventricular free wall of complete atrioventricular block (CAVB) model rats. Heart beats were evaluated using an implantable telemetry system 7 to 30 days after cell transplantation. The result showed that ectopic ventricular beats that were faster than the intrinsic escape rhythm were often observed in CAVB model rats transplanted with HCN4-overexpressing mESC-CMs. On the other hand, the rats transplanted with non-overexpressing mESC-CMs showed sporadic single premature ventricular contraction but not sustained ectopic ventricular rhythms. These results indicated that HCN4-overexpressing mESC-CMs produce rapid ectopic ventricular rhythms as a biological pacemaker.

Hypoxia favors myosin heavy chain beta gene expression in an Hif-1alpha-dependent manner

The potentiation of the naturally limited regenerative capacity of the heart is dependent on an understanding of the mechanisms that are activated in response to pathological conditions such as hypoxia. Under these conditions, the expression of genes suggested to support cardiomyocyte survival and heart adaptation is triggered. Particularly important are changes in the expression of myosin heavy chain (MHC) isoforms. We propose here that alterations in the expression profiles of MHC genes are induced in response to hypoxia and are primarily mediated by hypoxia inducible factor (HIF). In in vitro models of mouse embryonic stem cell-derived cardiomyocytes, we showed that hypoxia (1% O2) or the pharmacological stabilization of HIFs significantly increased MHCbeta (Myh7) gene expression. The key role of HIF-1alpha is supported by the absence of these effects in HIF-1alpha-deficient cells, even in the presence of HIF-2alpha. Interestingly, ChIP analysis did not confirm the direct interaction of HIF-1alpha with putative HIF response elements predicted in the MHCalpha and beta encoding DNA region. Further analyses showed the significant effect of the mTOR signaling inhibitor rapamycin in inducing Myh7 expression and a hypoxia-triggered reduction in the levels of antisense RNA transcripts associated with the Myh7 gene locus. Overall, the recognized and important role of HIF in the regulation of heart regenerative processes could be highly significant for the development of novel therapeutic interventions in heart failure.

Mitochondrial toxicity of perfluorooctane sulfonate in mouse embryonic stem cell-derived cardiomyocytes.

Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may cause cardiotoxicity in animals and humans. However, little is known about the underlying mechanism by which it affects the organelle toxicity in cardiomyocytes during the cardiogenesis. Our previous proteomic study showed that differences of protein expression mainly existed in mitochondria of cardiomyocytes differentiated from embryonic stem (ES) cells after exposure to PFOS. Here, we focused on mitochondrial toxicity of PFOS in ES cell-derived cardiomyocytes. The cardiomyogenesis from ES cells in vitro was inhibited, and the expression of L-type Ca2+ channel (LTCC) was decreased to interrupt [Ca2+]c transient amplitude in cardiomyocytes after PFOS treatment. Transmission electron microscope revealed that swollen mitochondrion with vacuole in PFOS-treated cells. Meanwhile, mitochondrial transmembrane potential (ΔΨm) was declined and ATP production was lowered. These changes were related to the increased EGFR phosphorylation, activated Rictor signaling, then mediated HK2 binding to mitochondrial membrane. Furthermore, PFOS reduced the interaction of IP3R-Grp75-VDAC and accumulated intracellular fatty acids by activating Rictor, thereby attenuating PGC-1α and Mfn2 expressions, then destroying mitochondria-associated endoplasmic reticulum membrane (MAM), which resulted in the decrease of [Ca2+]mito transient amplitude triggered by ATP. In conclusion, mitochondrial structure damages and abnormal Ca2+ shuttle were the important aspects in PFOS-induced cardiomyocytes toxicity from ES cells by activating Rictor signaling pathway.

Developmental stage-dependent effects of cardiac fibroblasts on function of stem cell-derived engineered cardiac tissues

We investigated whether the developmental stage of mouse cardiac fibroblasts (CFs) influences the formation and function of engineered cardiac tissues made of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs). Engineered cardiac tissue patches were fabricated by encapsulating pure mESC-CMs, mESC-CMs + adult CFs, or mESC-CMs + fetal CFs in fibrin-based hydrogel. Tissue patches containing fetal CFs exhibited higher velocity of action potential propagation and contractile force amplitude compared to patches containing adult CFs, while pure mESC-CM patches did not form functional syncytium. The functional improvements in mESC-CM + fetal CF patches were associated with differences in structural remodeling and increased expression of proteins involved in cardiac function. To determine role of paracrine signaling, we cultured pure mESC-CMs within miniature tissue “micro-patches” supplemented with media conditioned by adult or fetal CFs. Fetal CF-conditioned media distinctly enhanced CM spreading and contractile activity, which was shown by pathway inhibitor experiments and Western blot analysis to be mediated via MEK-ERK signaling. In mESC-CM monolayers, CF-conditioned media did not alter CM spreading or MEK-ERK activation. Collectively, our studies show that 3D co-culture of mESC-CMs with embryonic CFs is superior to co-culture with adult CFs for in vitro generation of functional myocardium. Ensuring consistent developmental stages of cardiomyocytes and supporting non-myocytes may be a critical factor for promoting functional maturation of engineered cardiac tissues.

Generation of Induced Cardiospheres via Reprogramming of Skin Fibroblasts for Myocardial Regeneration.

Recent pre-clinical and clinical studies have suggested that endogenous cardiospheres (eCS) are potentially safe and effective for cardiac regeneration following myocardial infarction (MI). Nevertheless the preparation of autologous eCS requires invasive myocardial biopsy with limited yield. We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from mouse adult skin fibroblasts treated with Gsk3β inhibitor-(2'Z,3'E)- 6-Bromoindirubin-3'-oxime and Oncostatin M. They resembled eCS, but contained a higher percentage of cells expressing Mesp1, Isl1, and Nkx2.5. They were differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to eCS and mouse embryonic stem cell-derived cardiomyocytes. Transplantation of iCS (1 × 106 cells) into mouse myocardium following MI had similar effects to transplantation of eCS but significantly better than saline or fibroblast in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness and capillary density in the infarcted region 4 weeks after transplantation. No tumor formation was observed. iCS generated from adult skin fibroblasts by somatic reprogramming and a cocktail of Gsk3β inhibitor-6-Bromoindirubin-3'-oxime and Oncostatin M may represent a novel source for cell therapy in MI. Stem Cells 2016;34:2693-2706.

The effect of PVDF-TrFE scaffolds on stem cell derived cardiovascular cells.

Recently, electrospun polyvinylidene fluoride (PVDF) and polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) scaffolds have been developed for tissue engineering applications. These materials have piezoelectric activity, wherein they can generate electric charge with minute mechanical deformations. Since the myocardium is an electroactive tissue, the unique feature of a piezoelectric scaffold is attractive for cardiovascular tissue engineering applications. In this study, we examined the cytocompatibility and function of pluripotent stem cell derived cardiovascular cells including mouse embryonic stem cell-derived cardiomyocytes (mES-CM) and endothelial cells (mES-EC) on PVDF-TrFE scaffolds. MES-CM and mES-EC adhered well to PVDF-TrFE and became highly aligned along the fibers. When cultured on scaffolds, mES-CM spontaneously contracted, exhibited well-registered sarcomeres and expressed classic cardiac specific markers such as myosin heavy chain, cardiac troponin T, and connexin43. Moreover, mES-CM cultured on PVDF-TrFE scaffolds responded to exogenous electrical pacing and exhibited intracellular calcium handling behavior similar to that of mES-CM cultured in 2D. Similar to cardiomyocytes, mES-EC also demonstrated high viability and maintained a mature phenotype through uptake of low-density lipoprotein and expression of classic endothelial cell markers including platelet endothelial cell adhesion molecule, endothelial nitric oxide synthase, and the arterial specific marker, Notch-1. This study demonstrates the feasibility of PVDF-TrFE scaffold as a candidate material for developing engineered cardiovascular tissues utilizing stem cell-derived cells. Biotechnol. Bioeng. 2016;113: 1577-1585. © 2015 Wiley Periodicals, Inc.

TRPC3 regulates the automaticity of embryonic stem cell-derived cardiomyocytes

BackgroundCardiac pacemaking is a complex phenomenon that is not completely understood. Canonical transient receptor potential isoform 3 (TRPC3) channel is a cation channel that permeates both Ca2+ and Na+. TRPC3 was previously found to express in adult cardiomyocytes. However, its role in cardiac pacemaking is unexplored. Here we used mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) to investigate whether TRPC3 regulates the spontaneous automaticity and the underlying mechanism involved. Methods and resultsImmunocytochemistry results showed that TRPC3 is expressed at the T-tubules of mESC-CMs. Whole-cell patch clamping showed that single mESC-CMs contain TRPC3 current. Confocal Ca2+ imaging showed that the TRPC3-specific blocker Pyr3 decreased Ca2+ transients and local Ca2+ release (LCR) of mESC-CMs. Combined current and voltage clamp recordings from the same cell showed that reducing the TRPC3 current, either by Pyr3 or a dominant negative (loss-of-function) construct of TRPC3, decreased the pacemaker activity of mESC-CMs as reflected by a decrease in action potential rate, a depolarized maximum diastolic potential and a decrease in slope of phase 4 diastolic depolarization. Furthermore, decreasing the TRPC3 current diminished, while increasing the TRPC3 current augmented the sodium–calcium exchanger (NCX) current in mESC-CMs. Lastly, decrease in TRPC3 current decreased the phosphorylation of ryanodine receptor isoform 2 at Ser2809 and phospholamban at Thr17. ConclusionsTRPC3 positively regulates diastolic depolarization of spontaneous action potential by increasing LCR and NCX current and therefore is an important determinant in pacemaking of mESC-CMs.

Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker.

BackgroundEstablishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (I f) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K+ current (I K1) flowing through inward rectifier potassium (Kir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor I K1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro.Methods and ResultsThe rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding Kir2.1, which is essential for generation of I K1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger I f currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an I f inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs.ConclusionsWe generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an I f inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.

Role of TRPV1 in the Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes.

Cytosolic Ca2+ ([Ca2+]i) is an important signal that regulates cardiomyocyte differentiation during cardiogenesis. TRPV1 is a Ca2+-permeable channel that is expressed in cardiomyocytes. In the present study, we utilized mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) as a model to investigate the functional role of TRPV1 in cardiomyocyte differentiation. Induction of embryonic stem cells into cardiomyocytes was achieved using embryoid body (EB)-based differentiation method. Quantitative PCRs showed an increased TRPV1 expression during the differentiation process. In [Ca2+]i measurement study, application of TRPV1 agonists, capsaicin and camphor, elicited a [Ca2+]i rise in mESC-CMs, the effect of which was abolished by TRPV1-shRNA. In functional study, treatment of EBs with TRPV1 antagonists (capsazepine and SB366791) and TRPV1-shRNA reduced the size of the EBs and decreased the percentage of spontaneously beating EBs. TRPV1 antagonists and TRPV1-shRNA also suppressed the expression of cardiomyocyte marker genes, including cardiac actin, c-TnT, c-TnI, and α-MHC. Taken together, this study demonstrated an important functional role of TRPV1 channels in the differentiation of mESCs into cardiomyocytes.

Optical microscopy imaging for the diagnosis of the pharmacological reaction of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs).

Quantitative diagnosis of pharmacological chronotropic reactions on mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) was successfully performed by utilizing derivative imaging analysis of videos recorded with a microscope camera at 30 Hz frame rate and 680 × 510 pixel resolution. The imaging analysis algorithm, developed in our lab, generated the contractile profile of the cells which was exploited for drug effect profiling. Six drugs such as isoproterenol (0.01-1 μM), quinidine (2-200 μM), propranolol (0.03-30 μM), verapamil (0.01-1 μM), sotalol (1-100 μM), and acetylsalicylic acid (0.1-10 μM) were administered and the quantitative medication effect was determined. Among the negative chronotropic agents administered, verapamil was found to be the most potent while sotalol was found to be the least potent at the micromolar level. Simultaneous measurement of the field potential and contractile motion in the verapamil effect test showed a coherent result. Moreover, this approach can provide insights into the contraction-relaxation conditions which are not available in the common electrophysiological approach. With these findings, it is expected that this study can aid in providing a simple and reliable in vitro mESC-CM-based screening platform for cardiovascular effect profiling of candidate drugs.