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

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

Abstract

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.

Keywords: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arrhythmias, Cardiac / metabolism*
  • Arrhythmias, Cardiac / pathology
  • Calcium / metabolism*
  • Calcium Channels, L-Type / metabolism*
  • Cells, Cultured
  • Excitation Contraction Coupling
  • Heart Ventricles / cytology*
  • Heart Ventricles / metabolism
  • Humans
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism*
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism*
  • Sarcoplasmic Reticulum / metabolism*

Substances

  • Calcium Channels, L-Type
  • Calcium