Calcium Handling in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes

Abstract

BackgroundThe ability to establish human induced pluripotent stem cells (hiPSCs) by reprogramming of adult fibroblasts and to coax their differentiation into cardiomyocytes opens unique opportunities for cardiovascular regenerative and personalized medicine. In the current study, we investigated the Ca2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).Methodology/Principal FindingsRT-PCR and immunocytochemistry experiments identified the expression of key Ca2+-handling proteins. Detailed laser confocal Ca2+ imaging demonstrated spontaneous whole-cell [Ca2+]i transients. These transients required Ca2+ influx via L-type Ca2+ channels, as demonstrated by their elimination in the absence of extracellular Ca2+ or by administration of the L-type Ca2+ channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca2+ store, contributing to [Ca2+]i transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca2+) and ryanodine (decreasing [Ca2+]i). Similarly, the importance of Ca2+ reuptake into the SR via the SR Ca2+ ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca2+]i transients elimination. Finally, the presence of an IP3-releasable Ca2+ pool in hiPSC-CMs and its contribution to whole-cell [Ca2+]i transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phosopholipase C inhibitor U73122.Conclusions/SignificanceOur study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca2+ store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca2+]i transients in hiPSC-CMs on both sarcolemmal Ca2+ entry via L-type Ca2+ channels and intracellular store Ca2+ release.

Determination of Appropriate Stage of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes for Drug Screening and Pharmacological Evaluation In Vitro

“…In the previous report, nifedipine at 1000 nM stopped contraction of hiPS-CMs at day 60. 27 Several reports have demonstrated that hiPS-CMs at day 20 to 40 of differentiation have electrophysiological properties and respond to ion channel blockers. [12][13][14] In the present study, electrophysiological responses to ion channel blockers in d30CMs were similar to those in d60CMs and d90CMs, although the sensitivity to the calcium channel blocker was weaker.…”

Modeling of Arrhythmogenic Right Ventricular Cardiomyopathy With Human Induced Pluripotent Stem Cells

“…The hiPSCs were established by retroviral delivery of Oct4, Sox-2, and Klf-4 followed by valproic acid treatment as described. 11,16 Several ARVC-hiPSC clones, which were positively stained with vital Tra-I-60 staining, were selected and expanded. Undifferentiated hiPSC colonies were cultured on mouse embryonic fibroblasts as previously described.…”

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

“…Therefore, interpretation of data regarding cardiomyogenesis efficiency and subtype identity must not only consider the presence and quantity of reference marker levels, but must consider the developmental stage(s) to which the timepoints of differentiation that are analyzed correspond. This is especially important considering that the maturation stage of cardiomyogenic cells generated by in vitro differentiation of hPSCs resembles most closely those of embryonic/fetal development [21][22][23][24][25] . Thus, relying on a marker's spatial expression in the postnatal heart may not be appropriate for the assessment of hPSCderived cells, at least in some cases.…”