Direct comparison of mononucleated and binucleated cardiomyocytes reveals molecular mechanisms underlying distinct proliferative competencies

The mammalian heart is incapable of regenerating a sufficient number of cardiomyocytes to ameliorate the loss of contractile muscle after acute myocardial injury, often resulting in heart failure. Several reports have demonstrated that mononucleated (MoNuc) cardiomyocytes are more responsive to pro-proliferative stimuli than are binucleated (BiNuc) cardiomyocytes. However, techniques to isolate and characterize these two different cardiomyocyte populations have been lacking. We have developed a novel fluorescence associated cell sorting method to isolate highly enriched populations of MoNuc and BiNuc cardiomyocytes at various times of development. Transcriptome analysis reveals an underlying biological signature that defines the differences between MoNucs and BiNucs, including a central role for the E2f/Rb transcriptional network in establishing the divergent ability of these two populations to re-enter and complete the cell cycle. Moreover, inducing binucleation by genetically blocking the ability of cardiomyocytes to complete cytokinesis leads to a reduction in E2f target gene expression, linking the E2f pathway with nucleation. These data identify key molecular differences between MoNuc and BiNuc mammalian cardiomyocytes that can be used to leverage cardiomyocyte proliferation for promoting injury repair in the heart. Overall design: Cardiomyocytes were isolated from Mlc2vcre:R26REYFP mice and sorted by FACS to separate mononucleated and binucleated cardiomyocytes for transcriptome analysis

哺乳动物心脏无法再生足够数量的心肌细胞以改善急性心肌损伤后收缩性心肌丢失所引发的功能障碍，常导致心力衰竭。已有多项研究证实，单核心肌细胞（mononucleated cardiomyocyte, MoNuc）相较于双核心肌细胞（binucleated cardiomyocyte, BiNuc），对促增殖刺激信号的响应更为敏感。然而，此前始终缺乏能够分离并鉴定这两类心肌细胞群的技术手段。我们开发了一种新型荧光激活细胞分选（fluorescence associated cell sorting, FACS）方法，可在发育的不同阶段分离得到高度富集的单核与双核心肌细胞群。转录组分析揭示了区分单核与双核心肌细胞的潜在生物学特征谱，其中E2f/Rb转录网络在调控这两类细胞重新进入并完成细胞周期的差异化能力中发挥核心作用。此外，通过基因手段阻断心肌细胞完成胞质分裂以诱导双核化，会使E2f靶基因的表达水平下调，从而将E2f通路与细胞核形成过程关联起来。本研究明确了哺乳动物单核与双核心肌细胞之间的关键分子差异，可用于调控心肌细胞增殖以促进心脏损伤修复。实验整体设计：从Mlc2vcre:R26REYFP小鼠体内分离心肌细胞，通过FACS分离单核与双核心肌细胞，用于转录组分析。