Calcium handling in human embryonic stem cell-derived cardiomyocytes

Jonathan Satin, Ilanit Itzhaki, Sophia Rapoport, Elizabeth A. Schroder, Leighton Izu, Gil Arbel, Rafael Beyar, C. William Balke, Jackie Schiller, Lior Gepstein

Research output: Contribution to journalArticle

138 Scopus citations

Abstract

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events ("sparks") that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30-40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 μM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies.

Original languageEnglish
Pages (from-to)1961-1972
Number of pages12
JournalSTEM CELLS
Volume26
Issue number8
DOIs
StatePublished - Aug 1 2008
Externally publishedYes

Keywords

  • Calcium transients
  • Embryoid body
  • Fluorescence microscope
  • Human embryonic stem cells
  • Myogenesis

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    Satin, J., Itzhaki, I., Rapoport, S., Schroder, E. A., Izu, L., Arbel, G., Beyar, R., Balke, C. W., Schiller, J., & Gepstein, L. (2008). Calcium handling in human embryonic stem cell-derived cardiomyocytes. STEM CELLS, 26(8), 1961-1972. https://doi.org/10.1634/stemcells.2007-0591