Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies. [Display omitted] •Replication and polyploidization are tracked using engineered human cardiomyocytes•Polyploidization is associated with transcriptomic and epigenomic signatures•The sarcomere inhibits mitosis through a p53-dependent G2/M checkpoint•Sarcomere knockout cardiomyocytes improve in vivo engraftment and replication Pettinato et al. engineer human cardiomyocyte models to study replication and polyploidization using single-cell transcriptomics, chromatin-state analysis, and a CRISPR screen. This reveals how the sarcomere promotes polyploidization through enhanced oxidative metabolism, DNA damage, and p53. Exploiting this pathway improves in vivo cardiomyocyte replacement strategies.