Ribeiro Alexandre J S, Ang Yen-Sin, Fu Ji-Dong, Rivas Renee N, Mohamed Tamer M A, Higgs Gadryn C, Srivastava Deepak, Pruitt Beth L
Department of Mechanical Engineering, Stanford University, Stanford, CA 94305; Stanford Cardiovascular Institute, Stanford University, Stanford, CA 94305;
Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158; Roddenberry Center for Stem Cell Biology and Medicine at Gladstone Institutes, San Francisco, CA 94158;
Proc Natl Acad Sci U S A. 2015 Oct 13;112(41):12705-10. doi: 10.1073/pnas.1508073112. Epub 2015 Sep 28.
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-µm(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.
单个心肌细胞含有肌原纤维，其中蕴藏着心肌基于肌节的收缩机制。从人多能干细胞分化而来的心肌细胞（hPSC-CMs）有潜力作为心脏活动的体外模型。然而，它们肌原纤维类似胎儿的排列不齐限制了其在模拟收缩活动方面的用途。我们分析了细胞形状和底物硬度对活的工程化hPSC-CMs中标记肌节的缩短和运动以及肌节活动向机械输出（收缩性）转化的影响。将单个hPSC-CMs培养在生理硬度（10 kPa）的聚丙烯酰胺底物上，并使用基质胶微图案来生成生理形状（长：宽纵横比为5:1至7:1的2000-µm²矩形）和肌原纤维的成熟排列。在长宽比为7:1的hPSC-CMs中，肌节缩短向机械输出的转化最高。底物硬度增加和施加过度拉伸会在长宽比为7:1的hPSC-CMs中诱导肌原纤维缺陷并降低机械输出。非肌肉肌球蛋白活性抑制剂会抑制肌原纤维的组装，表明亚细胞张力驱动了这些工程化hPSC-CMs中收缩活性的改善。与收缩性改善相关的其他因素包括轴向钙流、线粒体的系统分布、更成熟的电生理学以及横管形成的证据。这些发现支持了这些工程化hPSC-CMs作为在细胞水平研究心肌收缩性的强大模型的潜力。
