Calcium handling in human embryonic stem cell-derived cardiomyocytes.
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 microM 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.
Satin
Jonathan
J
The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Itzhaki
Ilanit
I
Rapoport
Sophia
S
Schroder
Elizabeth A
EA
Izu
Leighton
L
Arbel
Gil
G
Beyar
Rafael
R
Balke
C William
CW
Schiller
Jackie
J
Gepstein
Lior
L
D000200:Action Potentials; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D053496:Inositol 1,4,5-Trisphosphate Receptors; D010726:Phosphodiesterase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium