Department of Genetics and Genome Sciences, UConn Health, Farmington, CT 06030, USA.
Department of Bioengineering, University of Washington, Seattle, WA 98109, USA.
Cell Rep. 2021 May 4;35(5):109088. doi: 10.1016/j.celrep.2021.109088.
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.
人类心脏的再生能力受到心肌细胞复制率低和多倍体化的限制，但其机制尚不清楚。为了研究这一过程，我们构建了一种人类心肌细胞模型，使用荧光标记的细胞周期蛋白 B1 和心肌肌钙蛋白 T 来追踪细胞的复制和多倍体化。通过延时成像，体外心肌细胞的复制模式重现了体内观察到的单核多倍体化和复制停滞的进展。单细胞转录组学和染色质状态分析表明，多倍体化发生在肌节组装、氧化代谢增强、DNA 损伤反应和 p53 激活之前。CRISPR 敲除筛选显示 p53 是细胞周期停滞和多倍体化的驱动因素。抑制肌节功能或清除 ROS 可抑制细胞周期停滞和多倍体化。最后，我们发现肌钙蛋白敲除型心肌细胞的增殖可使肌钙蛋白敲入型大鼠心肌梗死模型中的心肌细胞移植效率提高 4 倍。因此，肌节通过 DNA 损伤反应抑制细胞分裂，该反应可作为靶点以改善心肌细胞替代策略。
