Identification and Characterization of Calcium Sparks in Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells

IntroductionCa2+ spark constitutes the elementary units of cardiac excitation-contraction (E-C) coupling in mature cardiomyocytes. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are known to have electrophysiological properties similar to mature adult cardiomyocytes. However, it is unclear if they share similar calcium handling property. We hypothesized that Ca2+ sparks in human induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs) may display unique structural and functional properties than mature adult cardiomyocytes.
Methods and resultsCa2+ sparks in hiPSC-CMs were recorded with Ca2+ imaging assay with confocal laser scanning microscopy. Those sparks were stochastic with a tendency of repetitive occurrence at the same site. Nevertheless, the spatial-temporal properties of Ca2+ spark were analogous to that of adult CMs. Inhibition of L-type Ca2+ channels by nifedipine caused a 61% reduction in calcium spark frequency without affecting amplitude of those sparks and magnitude of caffeine releasable sarcoplasmic reticulum (SR) Ca2+ content. In contrast, high extracellular Ca2+ and ryanodine increased the frequency, full width at half maximum (FWHM) and full duration at half maximum (FDHM) of spontaneous Ca2+ sparks.
ConclusionsFor the first time, spontaneous Ca2+ sparks were detected in hiPSC-CMs. The Ca2+ sparks are predominately triggered by L-type Ca2+ channels mediated Ca2+ influx, which is comparable to sparks detected in adult ventricular myocytes in which cardiac E-C coupling was governed by a Ca2+-induced Ca2+ release (CICR) mechanism. However, focal repetitive sparks originated from the same intracellular organelle could reflect an immature status of the hiPSC-CMs.

引言：钙火花（Ca²⁺ spark）是成熟心肌细胞心脏兴奋-收缩耦联（excitation-contraction coupling, E-C coupling）的基本功能单位。已知人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）诱导分化的心肌细胞（hiPSC-derived cardiomyocytes, hiPSC-CMs）具备与成熟成年心肌细胞相似的电生理特性，但二者的钙处理特性是否一致仍未明确。本研究提出假说：hiPSC-CMs中的钙火花可能表现出与成熟成年心肌细胞存在差异的独特结构与功能特性。

材料与方法及结果：采用共聚焦激光扫描显微镜（confocal laser scanning microscopy）开展钙成像实验，记录hiPSC-CMs中的钙火花。该类火花呈随机分布特征，且倾向于在同一部位重复触发。尽管如此，钙火花的时空特性与成年心肌细胞的钙火花并无显著差异。使用硝苯地平（nifedipine）抑制L型钙通道（L-type Ca²⁺ channels）可使钙火花频率降低61%，但不会改变火花振幅及咖啡因可释放的肌浆网（sarcoplasmic reticulum, SR）钙负荷。与之相反，升高细胞外钙浓度及加入芮丹碱（ryanodine）可提升自发性钙火花的频率、半高宽（full width at half maximum, FWHM）及半高时程（full duration at half maximum, FDHM）。

结论：本研究首次在hiPSC-CMs中检测到自发性钙火花。这类钙火花主要由L型钙通道介导的钙内流触发，其触发机制与成年心室肌细胞中以钙诱导钙释放（Ca²⁺-induced Ca²⁺ release, CICR）为核心的心脏E-C耦联机制下检测到的钙火花相当。然而，源自同一细胞内细胞器的局灶性重复火花，可反映hiPSC-CMs仍处于未成熟状态。