Sohnis Family Research Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
PLoS One. 2011 Apr 1;6(4):e18037. doi: 10.1371/journal.pone.0018037.
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+) 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+) transients, was established by application of caffeine (triggering a rapid increase in cytosolic Ca(2+)) and ryanodine (decreasing Ca(2+)). 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+) transients elimination. Finally, the presence of an IP3-releasable Ca(2+) pool in hiPSC-CMs and its contribution to whole-cell Ca(2+) 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+) transients in hiPSC-CMs on both sarcolemmal Ca(2+) entry via L-type Ca(2+) channels and intracellular store Ca(2+) release.
通过重编程成纤维细胞来建立人诱导多能干细胞（hiPSCs）并诱导其分化为心肌细胞的能力为心血管再生和个性化医学开辟了独特的机会。在本研究中，我们研究了源自 hiPSCs 的心肌细胞（hiPSC-CMs）的 Ca（2+）处理特性。
方法/主要发现：RT-PCR 和免疫细胞化学实验鉴定了关键 Ca（2+）处理蛋白的表达。详细的激光共聚焦 Ca（2+）成像显示自发的全细胞 [Ca（2+）]（i）瞬变。这些瞬变需要通过 L 型 Ca（2+）通道进行 Ca（2+）内流，这可以通过在不存在细胞外 Ca（2+）或给予 L 型 Ca（2+）通道阻滞剂硝苯地平的情况下消除它们来证明。通过应用咖啡因（触发细胞浆 Ca（2+）快速增加）和ryanodine（降低 [Ca（2+）]（i））来建立功能性ryanodine 受体（RyR）介导的肌浆网（SR）Ca（2+）库的存在，该库有助于 [Ca（2+）]（i）瞬变。同样，通过其阻滞剂（thapsigargin）的抑制作用证明了通过 SR Ca（2+）ATP 酶（SERCA）泵将 Ca（2+）再摄取到 SR 中的重要性，这导致 [Ca（2+）]（i）瞬变的消除。最后，通过 IP3 受体阻滞剂 2-氨基乙氧基二苯硼酸盐（2-APB）和磷脂酶 C 抑制剂 U73122 诱导的抑制作用证明了 hiPSC-CMs 中存在 IP3 可释放的 Ca（2+）库及其对全细胞 [Ca（2+）]（i）瞬变的贡献。
结论/意义：我们的研究确立了 hiPSC-CMs 中存在功能性、SERCA 隔离的、由 RyR 介导的 SR Ca（2+）库。此外，它表明 hiPSC-CMs 中的全细胞 [Ca（2+）]（i）瞬变依赖于通过 L 型 Ca（2+）通道的肌膜 Ca（2+）内流和细胞内储存 Ca（2+）释放。
