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This Article Full Text Full Text (PDF) All Versions of this Article: 2007-0591v1 2007-0591v2 26/8/1961    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Google Scholar Articles by Satin, J. Articles by Gepstein, L. PubMed PubMed Citation Articles by Satin, J. Articles by Gepstein, L.

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

^aDepartment of Physiology and
^dInstitute of Molecular Medicine, University of Kentucky College of Medicine, Lexington, Kentucky, USA;
^bThe Sohnis Research Laboratory of Cardiac Electrophysiology and Regenerative Medicine and
^cDepartment of Physiology, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel

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

Correspondence: Lior Gepstein, M.D., Ph.D., 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; e-mail: mdlior{at}tx.technion.ac.il; or Jonathan Satin, Ph.D., 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; e-mail: jsatin1{at}uky.edu

Received July 23, 2007; accepted for publication May 5, 2008.
First published online in STEM CELLS EXPRESS   June 5, 2008.

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.