Human Embryonic Cell Consortium, Stem Cell Program, University of California, Davis, Sacramento, CA 95817, USA.
Stem Cells Dev. 2009 Dec;18(10):1493-500. doi: 10.1089/scd.2009.0052.
Mouse (m) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are known to exhibit immature Ca(2+) dynamics such as small whole-cell peak amplitude and slower kinetics relative to those of adult. In this study, we examined the maturity and efficiency of Ca(2+)-induced Ca(2+) release in m and hESC-CMs, the presence of transverse (t) tubules and its effects on the regional Ca(2+) dynamics. In m and hESC-CMs, fluorescent staining and atomic force microscopy (AFM) were used to detect the presence of t-tubules, caveolin-3, amphiphysin-2 and colocalization of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs). To avoid ambiguities, regional electrically-stimulated Ca(2+) dynamics of single ESC-CMs, rather than spontaneously beating clusters, were measured using confocal microscopy. m and hESC-CMs showed absence of dyads, with neither t-tubules nor colocalization of DHPRs and RyRs. Caveolin-3 and amphiphysin-2, crucial for the biogenesis of t-tubules with robust expression in adult CMs, were also absent. Single m and hESC-CMs displayed non-uniform Ca(2+) dynamics across the cell that is typical of CMs deficient of t-tubules. Local Ca(2+) transients exhibited greater peak amplitude at the peripheral than at the central region for m (3.50 +/- 0.42 vs. 3.05 +/- 0.38) and hESC-CMs (2.96 +/- 0.25 vs. 2.72 +/- 0.25). Kinetically, both the rates of rise to peak amplitude and transient decay were faster for the peripheral relative to the central region. Immature m and hESC-CMs display unsynchronized Ca(2+) transients due to the absence of t-tubules and gene products crucial for their biogenesis. Our results provide insights for driving the maturation of ESC-CMs.
鼠（m）和人胚胎干细胞衍生的心肌细胞（hESC-CMs）已知表现出不成熟的 Ca（2+）动力学，例如全细胞峰值幅度较小和动力学较慢，与成人相比。在这项研究中，我们检查了 m 和 hESC-CMs 中 Ca（2+）诱导的 Ca（2+）释放的成熟度和效率、横管（t）管的存在及其对区域 Ca（2+）动力学的影响。在 m 和 hESC-CMs 中，荧光染色和原子力显微镜（AFM）用于检测 t 管、窖蛋白-3、 amphiphysin-2 和二氢吡啶受体（DHPRs）和兰尼碱受体（RyRs）的共定位。为了避免混淆，使用共聚焦显微镜测量单个 ESC-CMs 的区域电刺激 Ca（2+）动力学，而不是自发跳动的簇。m 和 hESC-CMs 显示不存在二联体，既没有 t 管，也没有 DHPRs 和 RyRs 的共定位。窖蛋白-3 和 amphiphysin-2，对于具有强大成年 CM 表达的 t 管生物发生至关重要，也不存在。单个 m 和 hESC-CMs 显示细胞内 Ca（2+）动力学不均匀，这是 t 管缺乏的 CM 的典型特征。局部 Ca（2+）瞬变在细胞的外周区域比中央区域具有更大的峰值幅度，对于 m（3.50 +/- 0.42 对 3.05 +/- 0.38）和 hESC-CMs（2.96 +/- 0.25 对 2.72 +/- 0.25）。动力学上，外周相对于中央区域的峰值幅度上升速度和瞬态衰减速度都更快。不成熟的 m 和 hESC-CMs 由于缺乏 t 管和对其生物发生至关重要的基因产物，显示出不同步的 Ca（2+）瞬变。我们的结果为推动 ESC-CMs 的成熟提供了见解。
