Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

Authors

  • Jonathan Satin Ph.D.,

    Corresponding author
    1. Department of Physiology, University of Kentucky College of Medicine, Lexington, Kentucky, USA
    • Department of Physiology, MS508/University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0298, USA. Telephone: 859-323-1146; Fax: 859-323-1070
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  • Ilanit Itzhaki,

    1. The Sohnis Research Laboratory of Cardiac Electrophysiology and Regenerative Medicine, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
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  • Sophia Rapoport,

    1. Department of Physiology, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
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  • Elizabeth A. Schroder,

    1. Department of Physiology, University of Kentucky College of Medicine, Lexington, Kentucky, USA
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  • Leighton Izu,

    1. Institute of Molecular Medicine, University of Kentucky College of Medicine, Lexington, Kentucky, USA
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  • Gil Arbel,

    1. The Sohnis Research Laboratory of Cardiac Electrophysiology and Regenerative Medicine, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
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  • Rafael Beyar,

    1. The Sohnis Research Laboratory of Cardiac Electrophysiology and Regenerative Medicine, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
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  • C. William Balke,

    1. Institute of Molecular Medicine, University of Kentucky College of Medicine, Lexington, Kentucky, USA
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  • Jackie Schiller,

    1. Department of Physiology, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
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  • Lior Gepstein M.D., Ph.D.

    Corresponding author
    1. The Sohnis Research Laboratory of Cardiac Electrophysiology and Regenerative Medicine, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
    • The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. Telephone: 972-4-8295303; Fax: 972-4-8524758;
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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.

Disclosure of potential conflicts of interest is found at the end of this article.

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