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First published online May 15, 2008
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Submitted on July 23, 2007
Accepted on May 5, 2008

EMBRYONIC STEM CELLS

Calcium Handling in Human Embryonic Stem Cell Derived Cardiomyocytes

Jonathan Satin 1, Ilanit Itzhaki 2, Sophia Rappaport 3, Elizabeth A. Schroder 1, Leighton Izu 3, Gil Arbel 2, Rafael Beyar 2, C. William Balke 4, Jackie Schiler 3, Lior Gepstein 2*

1 Department of Physiology and Institute of Molecular Medicine, Lexington, KY.
2 The Sohnis Research Laboratory of Cardiac Electrophysiology and Regenerative Medicine, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
3 the Department of Physiology, Rappaport Institute, Technion-Israel Institute of Technology, Haifa, Israel
4 University of Kentucky College of Medicine, Lexington, KY.

* To whom correspondence should be addressed. E-mail: Israel.mdlior{at}tx.technion.ac.il.

Correspondence may also be addressed to Jonathan Satin at jsatin1@uky.edu.


  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 PCR, immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal Ca-imaging and surface membrane labeling with Di-8-ANEPPS were employed. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) Ca release channels, RyR2, as well as Inositol-1,4,5-trisphosphate (IP3) receptors. Store Ca function was manifested as action-potential (AP)-induced Ca transients. Time-to-target plots showed that these AP-Ca transients traverse the width of the cell via a propagated wave of intracellular store Ca release. The hESC-CMs also exhibited local Ca events ("sparks") that were localized to the surface membrane. The presence of Caffeine-sensitive intracellular Ca stores was manifested following application of focal, temporally-limited, puffs of caffeine in three different age groups: early- (with the initiation of beating), intermediate- (10 days post initiation of beating; dpb) and late- (30-40 dpb) stage hESC-CMs. Ca store gradually increased during in-vitro maturation. Similarly, Ryanodine application decreased the amplitude of the spontaneous Ca transients. Interestingly, the expression and function of an IP3-releasable Ca-pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2-APB,2µM).

In summary, our study establishes the presence of a functional SR Ca-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 for both developmental studies as well as for the development of future myocardial cell replacement strategies.

______________________________________________________________________________

Author contributions: J.S.: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; I.I.: Conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; S.R.: Collection and/or assembly of data, data analysis and interpretation; E.S.: Collection and/or assembly of data, data analysis and interpretation; L.I.: Data analysis and interpretation; G.A.: Provision of study material, collection and/or assembly of data; R.B.: Conception and design, final approval of manuscript; W.B.: Conception and design, final approval of manuscript; J.S.: Conception and design, collection and/or assembly of data, data analysis and interpretation; L.G.: Conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.

 Key Words. Human embryonic stem cells, calcium transients, myogenesis, fluorescence microscope, embryoid body




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O. Sedan, K. Dolnikov, N. Zeevi-Levin, N. Leibovich, M. Amit, J. Itskovitz-Eldor, and O. Binah
1,4,5-Inositol Trisphosphate-Operated Intracellular Ca2+ Stores and Angiotensin-II/Endothelin-1 Signaling Pathway Are Functional in Human Embryonic Stem Cell-Derived Cardiomyocytes
Stem Cells, December 1, 2008; 26(12): 3130 - 3138.
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