Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness
Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness
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DOI：
10.1073/pnas.1508073112
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发表时间：
2015-10-13
影响因子：
11.1
通讯作者：
Pruitt, Beth L.
中科院分区：
文献类型：
--
作者：
Ribeiro, Alexandre J. S.;Ang, Yen-Sin;Pruitt, Beth L.
单个心肌细胞含有肌原纤维，其具有基于肌节的心肌收缩机制。从人多能干细胞（hPSC-CM）分化的心肌细胞具有作为心脏活动的体外模型的潜力。然而，它们胎儿样的肌原纤维错位限制了它们对收缩活动建模的有用性。我们分析了细胞形状和基底刚度对标记的肌节的缩短和移动以及活工程化hPSC-CM中肌节活性转化为机械输出（收缩性）的影响。将单个hPSC-CM在生理硬度（10 kPa）的聚丙烯酰胺基质上培养，并使用Matrigel微图案产生生理形状（2，000 μ m（2）矩形，长宽比为5：1-7：1）和成熟的肌原纤维排列。肌节缩短转化为机械输出在7：1 hPSC-CM中最高。增加的基底刚度和施加的过度拉伸诱导7：1 hPSC-CM中的肌原纤维缺陷和降低的机械输出。非肌肉肌球蛋白活性的抑制剂抑制了肌原纤维的组装，表明亚细胞张力驱动这些工程化hPSC-CM中改善的收缩活性。与收缩力改善相关的其他因素包括轴向钙流、系统性线粒体分布、更成熟的电生理学和横小管形成的证据。这些发现支持了这些工程化的hPSC-CM作为在细胞水平上研究心肌收缩性的强大模型的潜力。
Single cardiomyocytes contain myofibrils that harbor the sarcomere-based contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetal-like misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-mu m(2) rectangles with length:width aspect ratios of 5:1-7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.