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Background: Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear diploid cardiomyocytes. On maturation, mononuclear diploid cardiomyocytes become binucleated or polyploid and exit the cell cycle. Cardiomyocyte metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative mononuclear diploid cardiomyocytes enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism in cardiac repair after injury. Methods: LIN28a overexpression was tested using mouse transgenesis on postnatal cardiomyocyte numbers, cell cycle, and response to apical resection injury. With the use of neonatal and adult cell culture systems and adult and Mosaic Analysis with Double Markers myocardial injury models in mice, the effect of LIN28a overexpression on cardiomyocyte cell cycle and metabolism was tested. Last, isolated adult cardiomyocytes from LIN28a and wild-type mice 4 days after myocardial injury were used for RNA-immunoprecipitation sequencing. Results: LIN28a was found to be active primarily during cardiac development and rapidly decreases after birth. LIN28a reintroduction at postnatal day (P) 1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing cardiomyocyte cell cycle activity in LIN28a transgenic pups compared with wild-type littermates. Moreover, LIN28a overexpression extended cardiomyocyte cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated cardiomyocyte apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared with wild-type littermate controls. Instead, LIN28a small molecule inhibitor attenuated the proreparative effects of LIN28a on the heart. Neonatal rat ventricular myocytes overexpressing LIN28a mechanistically showed increased glycolysis, ATP production, and levels of metabolic enzymes compared with control. LIN28a immunoprecipitation followed by RNA-immunoprecipitation sequencing in cardiomyocytes isolated from LIN28a-overexpressing hearts after injury identified long noncoding RNA-H19 as its most significantly altered target. Ablation of long noncoding RNA-H19 blunted LIN28a-induced enhancement on cardiomyocyte metabolism and cell cycle activity. Conclusions: Collectively, LIN28a reprograms cardiomyocyte metabolism and promotes persistence of mononuclear diploid cardiomyocytes in the injured heart, enhancing proreparative processes, thereby linking cardiomyocyte metabolism to regulation of ploidy/nucleation and repair in the heart.

Developmental cardiac tissue holds a remarkable capacity to regenerate after injury and consists of regenerative mononuclear and diploid cardiomyocytes (MNDCMs). Upon maturation, MNDCMs become binucleated or polyploid and exit the cell cycle. Interestingly, CM metabolism undergoes a profound shift that coincides with the cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative MNDCMs enhancing cardiac function and regeneration after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism, in cardiac regeneration following injury. LIN28a was found as primarily active during cardiac developmental stages and rapidly decreases after birth. LIN28a reintroduction at P0, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size enhancing CM cell cycle activity (Ki67+, pHH3+, and AurB+ cells) in LIN28a transgenic pups compared to WT littermates. Moreover, LIN28a overexpression extended CM cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated CM apoptosis, enhanced CM cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared to WT littermate controls. Alternatively, LIN28a small molecule inhibitor attenuated the pro-regenerative effects of LIN28a on the heart. Mechanistically, Neonatal rat ventricular myocytes (NRVMs) overexpressing LIN28a showed increased glycolysis, ATP production, and activity of metabolic enzymes compared to control. Collectively, LIN28a reprograms CM metabolism and promotes persistence of regenerative MNDCMs in the injured heart enhancing pro-regenerative processes thereby linking CM metabolism to the regulation of ploidy/nucleation and regeneration in the heart.