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A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

ABSTRACT: The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ? control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.

SUBMITTER: Lewis KJ

PROVIDER: S-EPMC4361473 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

Lewis Kimberley J KJ Silvester Nicole C NC Barberini-Jammaers Steven S Mason Sammy A SA Marsh Sarah A SA Lipka Magdalena M George Christopher H CH

Journal of biomolecular screening 20141103 3

The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantifi ...[more]

PMID: 25367900

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Project description:BACKGROUND: Characterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes. RESULTS: A transgenic alpha-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-MHC promoter. A puromycin-resistant, EGFP-positive, alpha-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of alpha-MHC-positive cardiomyocytes in comparison with undifferentiated alpha-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in alpha-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the alpha-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway. CONCLUSION: The specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.

Project description:RationaleCardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.ObjectiveWe investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).Methods and resultsWe differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.ConclusionsThe results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.

Project description:Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.

Project description:BackgroundBiological pacemakers consisting of pluripotent stem cell-derived cardiomyocytes are potentially useful for treating bradycardia. However, tachyarrhythmia caused by derived cardiomyocytes themselves is one of main barriers hampering their clinical translation. An in-depth understanding of the mechanisms underlying the spontaneous action potential (a.k.a. automaticity) might provide potential approaches to solve this problem. The aim of this project is to study the role of canonical transient receptor potential isoform 7 (TRPC7) channels in regulating the automaticity of embryonic stem cell-derived cardiomyocytes (ESC-CMs).Methods and resultsBy Western blotting, the expression of TRPC7 was found to be increased during the differentiation of mouse ESC-CMs (mESC-CMs). Adenovirus-mediated TRPC7 knockdown decreased while overexpression increased the frequency of Ca2+ transients (CaTs), local Ca2+ releases (LCRs), and action potentials (APs) as detected by confocal microscopy and whole-cell patch-clamping. TRPC7 was found to be positively associated with the activity of ryanodine receptor 2 (RyR2), sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and sodium-calcium exchanger (NCX) but not hyperpolarization-activated, cyclic nucleotide-gated channel (HCN), and inositol trisphosphate receptor (IP3R). Knockdown or overexpression of TRPC7 did not alter the expression of HCN4, Cav1.3, Cav3.1, Cav3.2, IP3R1, RyR2, and SERCA but positively regulated the phosphorylation of RyR2 at S2814 and phospholamban (PLN) at T17. Moreover, the positive regulation of APs by TRPC7 was Ca2+-dependent, as overexpression of N-terminus of TRPC7 (dominant negative of TRPC7) which diminished the Ca2+ permeability of TRPC7 decreased the AP frequency.ConclusionsTRPC7 regulates the automaticity of mESC-CMs through two mechanisms. On the one hand, TRPC7 positively regulates the intracellular Ca2+ clock through the regulation of activities of both RyR2 and SERCA; on the other hand, TRPC7 also positively regulates the membrane clock via its influence on NCX activity. Altogether, our study reveals that TRPC7 is a potential drug target to manipulate the action potential firing rate of pluripotent stem cell-derived cardiomyocyte-based biological pacemakers to prevent tachyarrhythmia, a condition that might be encountered after transplantation.

Dataset Information

A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

Publications

A new system for profiling drug-induced calcium signal perturbation in human embryonic stem cell-derived cardiomyocytes.

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