Calcium Handling in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
Calcium Handling in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
复制标题
DOI：
10.1371/journal.pone.0018037
复制
发表时间：
2011-04-01
期刊：
影响因子：
3.7
通讯作者：
Gepstein, Lior
中科院分区：
文献类型：
--
作者：
Itzhaki, Ilanit;Rapoport, Sophia;Gepstein, Lior
背景：通过对成人成纤维细胞重新编程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提供了独特的机会。在目前的研究中，我们研究了HiPSC来源的心肌细胞(HiPSC-CMS)的钙处理特性。方法/主要发现：RT-PCR和免疫细胞化学实验证实了关键的钙处理蛋白的表达。详细的激光共聚焦钙成像显示自发的全细胞[Ca~(2+)](I)瞬间。这些瞬变需要钙离子通过L型钙通道内流，在无细胞外钙离子的情况下消除它们或给予L型钙通道阻滞剂硝苯地平证明了这一点。通过应用咖啡因(触发胞浆内钙的快速升高)和兰诺定(降低[钙](I))，建立了功能兰诺定受体(RyR)介导的肌浆网(SR)钙存储，从而促进了[Ca~(2+)](I)瞬变。同样，通过肌质网钙三磷酸腺苷酶(SERCA)泵进入SR的钙再摄取的重要性也被其阻滞剂(Thapsigargin)抑制，从而导致[Ca+](I)瞬时消除。最后，IP3受体阻断剂2-氨基乙氧基二苯硼酸酯(2-APB)和磷脂酶C抑制剂U73122的抑制作用证实了HiPSC-CMS中存在IP3可释放的钙库及其对全细胞[Ca~(2+)](I)瞬变的贡献。结论/意义：我们的研究证实了HiPSC-CMS中存在一个功能性的、SERCA-隔离的、RyR介导的SR钙库。此外，它还证实了HIPSC-CMS中全细胞[Ca~(2+)](I)的瞬变依赖于肌膜上L钙通道的钙进入和细胞内储存钙的释放。
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 Ca2+-handling properties of hiPSCs derived-cardiomyocytes (hiPSC-CMs).Methodology/Principal Findings: RT-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/Significance: Our 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.