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 Ca 2+ release

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 Ca²⁺-induced Ca²⁺ release in excitation-contraction coupling, the mechanistic basis of Ca²⁺ handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive. Objectives To study Ca²⁺ sparks as unitary events of Ca²⁺ 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 Ca²⁺ sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca²⁺-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 Ca²⁺. Increasing external Ca²⁺ dramatically escalated the basal Ca²⁺ and spark frequency. Furthermore, RyR-mediated Ca²⁺ release sensitized nearby RyRs, leading to compound Ca²⁺ sparks. Depolarization or L-type Ca²⁺ channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca²⁺-dependent cytosolic Ca²⁺ increase and reduced the sarcoplasmic reticulum content. By contrast, removal of external Na⁺ or the addition of the Na⁺-Ca²⁺ exchanger inhibitor (KB-R7943 and SN-6) had no effect, suggesting that the Na⁺-Ca²⁺ exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca²⁺ uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca²⁺ waves. Conclusion Taken collectively, our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca²⁺-induced Ca²⁺ release. However, the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development.