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Heart Rhythm. 2014 Jan;11(1):133-40. doi: 10.1016/j.hrthm.2013.10.006. Epub 2013 Oct 3.

Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: direct evidence of functional Ca2+-induced Ca2+ release.

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Stem Cell and Regenerative Medicine Consortium; Department of Physiology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong.
Institute of Molecular Medicine, Peking University, Beijing, People's Republic of China.
Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, Maryland.
Stem Cell and Regenerative Medicine Consortium; Department of Physiology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong; Center of Cardiovascular Research, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address:



Human embryonic stem cells (hESCs) serve as a potential unlimited ex vivo source of cardiomyocytes for disease modeling, cardiotoxicity screening, drug discovery, and cell-based therapies. Despite the fundamental importance of Ca(2+)-induced Ca(2+) release in excitation-contraction coupling, the mechanistic basis of Ca(2+) handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive.


To study Ca(2+) sparks as unitary events of Ca(2+) handling for mechanistic insights.


To avoid ambiguities owing to the heterogeneous nature, we experimented with hESC-VCMs, purified on the basis of zeocin resistance and signature ventricular action potential after LV-MLC2v-tdTomato-T2A-Zeo transduction.


Ca(2+) sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca(2+)-ATPase (thapsigargin and cyclopiazonic acid) and ryanodine receptor (RyR; ryanodine, tetracaine) but not inositol trisphosphate receptors (xestospongin C and 2-aminoethyl diphenylborinate) could be recorded. In a permeabilization model, we further showed that RyRs could be sensitized by Ca(2+). Increasing external Ca(2+) dramatically escalated the basal Ca(2+) and spark frequency. Furthermore, RyR-mediated Ca(2+) release sensitized nearby RyRs, leading to compound Ca(2+) sparks. Depolarization or L-type Ca(2+) channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca(2+)-dependent cytosolic Ca(2+) increase and reduced the sarcoplasmic reticulum content. By contrast, removal of external Na(+) or the addition of the Na(+)-Ca(2+) exchanger inhibitor (KB-R7943 and SN-6) had no effect, suggesting that the Na(+)-Ca(2+) exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca(2+) waves.


Taken collectively, our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca(2+)-induced Ca(2+) release. However, the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development.


AP; CCCP; CICR; CM; Ca(2+) sparks; Ca(2+) waves; Ca(2+)-induced Ca(2+) release; EC; FDHM; FWHM; I(Ca,L); IP3R; L-type Ca(2+) current; MLC2v; NCX; Na(+)-Ca(2+) exchanger; RyR; SERCA; SR; Stem cell; VCM; Ventricular cardiomyocytes; action potential; carbonyl cyanide m-chlorophenyl hydrazone; cardiomyocyte; excitation-contraction; full duration at half maximum; full width at half maximum; hESC; human embryonic stem cell; iPSC; induced pluripotent stem cell; inositol trisphosphate receptor; myosin light chain 2v; ryanodine receptor; sarco/endoplasmic reticulum Ca(2+)-ATPase; sarcoplasmic reticulum; ventricular cardiomyocyte

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