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

Sen Li, Heping Cheng, Gordon F Tomaselli, Ronald A Li
Heart Rhythm: the Official Journal of the Heart Rhythm Society 2014, 11 (1): 133-40

BACKGROUND: 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.

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

METHODS: 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.

RESULTS: 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.

CONCLUSION: 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.

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