Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression

Am J Physiol Cell Physiol. 2009 Jul;297(1):C152-9. doi: 10.1152/ajpcell.00060.2009. Epub 2009 Apr 8.

Abstract

Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca(2+) handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic Ca(2+) transient amplitudes, slower kinetics, and reduced Ca(2+) content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca(2+) handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, Ca(2+)-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQDelta-IRES-GFP (Ad-CSQDelta, which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca(2+) content but did not alter L-type Ca(2+) current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQDelta-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the Ca(2+) transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca(2+) handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics
  • Calcium Channels, L-Type / metabolism
  • Calcium Signaling* / drug effects
  • Calsequestrin / genetics
  • Calsequestrin / metabolism*
  • Cell Differentiation*
  • Cell Line
  • Embryonic Stem Cells / drug effects
  • Embryonic Stem Cells / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Genetic Vectors
  • Humans
  • Kinetics
  • Membrane Potentials
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Ryanodine / pharmacology
  • Ryanodine Receptor Calcium Release Channel / metabolism
  • Sarcoplasmic Reticulum / metabolism
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism
  • Thapsigargin / pharmacology
  • Transduction, Genetic

Substances

  • CASQ2 protein, human
  • Calcium Channels, L-Type
  • Calsequestrin
  • Enzyme Inhibitors
  • Recombinant Fusion Proteins
  • Ryanodine Receptor Calcium Release Channel
  • Ryanodine
  • Thapsigargin
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases