Dataset Information

Calcium handling in human induced pluripotent stem cell derived cardiomyocytes.

ABSTRACT:

Background

The 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 Ca(2+)-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).

Methodology/principal findings

RT-PCR and immunocytochemistry experiments identified the expression of key Ca(2+)-handling proteins. Detailed laser confocal Ca(2+) imaging demonstrated spontaneous whole-cell [Ca(2+)](i) transients. These transients required Ca(2+) influx via L-type Ca(2+) channels, as demonstrated by their elimination in the absence of extracellular Ca(2+) or by administration of the L-type Ca(2+) channel blocker nifedipine. The presence of a functional ryanodine receptor (RyR)-mediated sarcoplasmic reticulum (SR) Ca(2+) store, contributing to [Ca(2+)](i) transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca(2+)) and ryanodine (decreasing [Ca(2+)](i)). Similarly, the importance of Ca(2+) reuptake into the SR via the SR Ca(2+) ATPase (SERCA) pump was demonstrated by the inhibiting effect of its blocker (thapsigargin), which led to [Ca(2+)](i) transients elimination. Finally, the presence of an IP3-releasable Ca(2+) pool in hiPSC-CMs and its contribution to whole-cell [Ca(2+)](i) transients was demonstrated by the inhibitory effects induced by the IP3-receptor blocker 2-Aminoethoxydiphenyl borate (2-APB) and the phospholipase C inhibitor U73122.

Conclusions/significance

Our study establishes the presence of a functional, SERCA-sequestering, RyR-mediated SR Ca(2+) store in hiPSC-CMs. Furthermore, it demonstrates the dependency of whole-cell [Ca(2+)](i) transients in hiPSC-CMs on both sarcolemmal Ca(2+) entry via L-type Ca(2+) channels and intracellular store Ca(2+) release.

SUBMITTER: Itzhaki I

PROVIDER: S-EPMC3069979 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RationaleCardiomyocytes generated from human induced pluripotent stem cells (hiPSCs) are suggested as the most promising candidate to replenish cardiomyocyte loss in regenerative medicine. Little is known about their calcium homeostasis, the key process underlying excitation-contraction coupling.ObjectiveWe investigated the calcium handling properties of hiPSC-derived cardiomyocytes and compared with those from human embryonic stem cells (hESCs).Methods and resultsWe differentiated cardiomyocytes from hiPSCs (IMR90 and KS1) and hESCs (H7 and HES3) with established protocols. Beating outgrowths from embryoid bodies were typically observed 2 weeks after induction. Cells in these outgrowths were stained positively for tropomyosin and sarcomeric alpha-actinin. Reverse-transcription polymerase chain reaction studies demonstrated the expressions of cardiac-specific markers in both hiPSC- and hESC-derived cardiomyocytes. Calcium handling properties of 20-day-old hiPSC- and hESC-derived cardiomyocytes were investigated using fluorescence confocal microscopy. Compared with hESC-derived cardiomyocytes, spontaneous calcium transients from both lines of hiPSC-derived cardiomyocytes were of significantly smaller amplitude and with slower maximal upstroke velocity. Better caffeine-induced calcium handling kinetics in hESC-CMs indicates a higher sacroplasmic recticulum calcium store. Furthermore, in contrast with hESC-derived cardiomyocytes, ryanodine did not reduce the amplitudes, maximal upstroke and decay velocity of calcium transients of hiPSC-derived cardiomyocytes. In addition, spatial inhomogeneity in temporal properties of calcium transients across the width of cardiomyocytes was more pronounced in hiPSC-derived cardiomyocytes than their hESC counterpart as revealed line-scan calcium imaging. Expressions of the key calcium-handling proteins including ryanodine recptor-2 (RyR2), sacroplasmic recticulum calcium-ATPase (SERCA), junction (Jun) and triadin (TRDN), were significantly lower in hiPSC than in hESCs.ConclusionsThe results indicate the calcium handling properties of hiPSC-derived cardiomyocytes are relatively immature to hESC counterparts.