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

Sen Li, Heping Cheng, Gordon F. Tomaselli, Ronald A. Li

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)133-140
Number of pages8
JournalHeart Rhythm
Volume11
Issue number1
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

Fingerprint

Excitation Contraction Coupling
Pluripotent Stem Cells
Cardiac Myocytes
Sarcoplasmic Reticulum
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Sarcoplasmic Reticulum Calcium-Transporting ATPases
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester
Tetracaine
Ryanodine
Ryanodine Receptor Calcium Release Channel
Thapsigargin
Inositol
Drug Discovery
Cell- and Tissue-Based Therapy
Action Potentials
Human Embryonic Stem Cells

Keywords

  • Ca sparks
  • Ca waves
  • Ca-induced Ca release
  • Stem cell
  • Ventricular cardiomyocytes

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

@article{08c35bdecbeb49d8a935a7c5848a1814,
title = "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",
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 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.",
keywords = "Ca sparks, Ca waves, Ca-induced Ca release, Stem cell, Ventricular cardiomyocytes",
author = "Sen Li and Heping Cheng and Tomaselli, {Gordon F.} and Li, {Ronald A.}",
year = "2014",
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TY - JOUR

T1 - Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics

T2 - Direct evidence of functional Ca2+-induced Ca 2+ release

AU - Li, Sen

AU - Cheng, Heping

AU - Tomaselli, Gordon F.

AU - Li, Ronald A.

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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.

AB - 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.

KW - Ca sparks

KW - Ca waves

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KW - Ventricular cardiomyocytes

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