title: Calcium handling in human embryonic stem cell-derived cardiomyocytes.
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 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.
authors.affiliation: The Sohnis Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Satin
authors.forename: Ilanit
authors.initials: I
authors.lastname: Itzhaki
authors.forename: Sophia
authors.initials: S
authors.lastname: Rapoport
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Schroder
authors.forename: Leighton
authors.initials: L
authors.lastname: Izu
authors.forename: Gil
authors.initials: G
authors.lastname: Arbel
authors.forename: Rafael
authors.initials: R
authors.lastname: Beyar
authors.forename: C William
authors.initials: CW
authors.lastname: Balke
authors.forename: Jackie
authors.initials: J
authors.lastname: Schiller
authors.forename: Lior
authors.initials: L
authors.lastname: Gepstein
mesh_terms: 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
chemical_list: D053496:Inositol 1,4,5-Trisphosphate Receptors; D010726:Phosphodiesterase Inhibitors; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium