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 phosopholipase 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.