Project Summary/Abstract Current available clinical treatments greatly reduced the acute mortality of myocardial infarction. However, lost cardiomyocytes during myocardial injury still cannot be replenished, leading to a steady increase of heart failure patients. In contrast, adult zebrafish and newborn mammals are capable of robust cardiac regeneration. This process has been shown to rely on proliferation of preexisting cardiomyocytes after injury. In rodents, such an ability is lost within the first week after birth when the majority of cardiomyocytes undergo permanent cell-cycle arrest. However, the physiological triggers of this transition remain largely unknown. Our recently published work suggests that activation of thermogenic pathways during the acquisition of endothermy in ontogeny and phylogeny may cause a loss of cardiomyocyte proliferative and regenerative capacity (Hirose et al., 2019 Science). Following this direction, we discover that increases of neurohormonal activities associated with postnatal thermogenesis drive cardiomyocyte cell-cycle exit, at least in part by turning on B cell lymphoma 6 (Bcl6), a transcription factor previously unappreciated in the heart field. Moreover, Bcl6 is also identified in our RNA-seq analysis of all 1179 annotated mouse transcription factors as one of the top candidates that may induce postnatal loss of cardiac regenerative potential. Intriguingly, cardiac expression of Bcl6 increases 19 folds after birth in mice but not in naked mole-rats (Heterocephalus glaber), a poikilothermic rodent. The function of Bcl6 in cardiomyocytes have never been reported. Our preliminary data show that cardiomyocyte- specific deletion of Bcl6 in mice leads to enhanced cardiomyogenesis both in heart growth and after ischemic injury. We further identify a putative direct target gene of Bcl6, and demonstrate its major contribution to the phenotypes through genetic rescue experiments in mice. Based on these results, this grant proposal will apply a novel method that integrates whole-heart clearing, immunostaining and volume imaging by light-sheet microscopy to accurately assess the total cardiomyocyte number, and exploit mouse genetic models to elucidate the functions of Bcl6 and its downstream target in cardiomyocyte cell-cycle control during postnatal heart growth and adult myocardial injury repair. The regulation of Bcl6 in ontogeny and its expression in phylogeny will be further investigated to understand whether and how its expression increases in parallel with the development and evolution of endothermy. Altogether, this work will yield significant knowledge about the physiological brake of cardiac regeneration, and may offer novel treatment strategies to enhance heart regenerative repair in adult mammals.