Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness

Significance Human cardiomyocytes differentiated from pluripotent stem cells (hPSC-CMs) have potential as in vitro models of cardiac health and disease but differ from mature cardiomyocytes. In single live engineered hPSC-CMs with physiological shapes, we assayed the mechanical output and activity of sarcomeres and myofibrils in a nondestructive, noninvasive manner. Substrates with physiological stiffness improved contractile activity of patterned hPSC-CMs, as well as calcium flow, mitochondrial organization, electrophysiology, and transverse-tubule formation. The mechanical output and activity of sarcomeres and myofibrils varied as a function of mechanical cues and disrupted cell tension. This study establishes a high-throughput platform for modeling single-cell cardiac contractile activity and yields insight into environmental factors that drive maturation and sarcomere function in hPSC-CMs. 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-µm2 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.