Rationale: Embryonic heart is characterized of rapidly dividing cardiomyocytes required to build a working myocardium. Cardiomyocytes retain some proliferative capacity in the neonates but lose it in adulthood. Consequently, a number of signaling hubs including microRNAs are altered during cardiac development that adversely impacts regenerative potential of cardiac tissue. Embryonic stem cell cycle miRs are a class of microRNAs exclusively expressed during developmental stages; however, their effect on cardiomyocyte proliferation and heart function in adult myocardium has not been studied previously. Objective: To determine whether transient reintroduction of embryonic stem cell cycle miR-294 promotes cardiomyocyte cell cycle reentry enhancing cardiac repair after myocardial injury. Methods and Results: miR-294 is expressed in the heart during development, prenatal stages, lost in the neonate, and adult heart confirmed by qRT-PCR and in situ hybridization. Neonatal ventricular myocytes treated with miR-294 showed elevated expression of Ki67, p-histone H3, and Aurora B confirmed by immunocytochemistry compared with control cells. miR-294 enhanced oxidative phosphorylation and glycolysis in Neonatal ventricular myocytes measured by seahorse assay. Mechanistically, miR-294 represses Wee1 leading to increased activity of the cyclin B1/CDK1 complex confirmed by qRT-PCR and immunoblot analysis. Next, a doxycycline-inducible AAV9-miR-294 vector was delivered to mice for activating miR-294 in myocytes for 14 days continuously after myocardial infarction. miR-294–treated mice significantly improved left ventricular functions together with decreased infarct size and apoptosis 8 weeks after MI. Myocyte cell cycle reentry increased in miR-294 hearts analyzed by Ki67, pH3, and AurB (Aurora B kinase) expression parallel to increased small myocyte number in the heart. Isolated adult myocytes from miR-294 hearts showed increased 5-ethynyl-2′-deoxyuridine+ cells and upregulation of cell cycle markers and miR-294 targets 8 weeks after MI. Conclusions: Ectopic transient expression of miR-294 recapitulates developmental signaling and phenotype in cardiomyocytes promoting cell cycle reentry that leads to augmented cardiac function in mice after myocardial infarction.

Rationale: Embryonic heart is characteristic of rapidly dividing cardiomyocytes required to build a working myocardium. Cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Consequently, a number of signaling hubs including microRNAs are altered during cardiac development. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs exclusively expressed during developmental stages but their effect on cardiomyocyte proliferation and heart function has not been previously studied. Objective: To determine whether miR-294 promotes cardiomyocyte cell cycle reentry enhancing cardiac repair after myocardial infarction. Methods and Results: miR expression analysis in the heart during development revealed elevated levels of miR-294 in the prenatal stages but was lost in the neonate and adult heart as confirmed by qRT-PCR and in situ hybridization. Neonatal ventricular cardiomyocytes (NRVMs) treated with miR-294 mimic showed elevated expression of Ki67 (7-fold), p-histone 3 (7.5-fold) and Aurora B kinase (11.8 fold) as confirmed by immunocytochemistry compared to control cells. Similar results were confirmed in adult cardiomyocytes with p-histone staining and mRNA analysis. Moreover, miR-294 enhanced oxidative phosphorylation and glycolysis in NRVMs as measured by seahorse assay. Mechanistically, array analysis revealed upregulation of cell cycle markers including blunting of negative regulators as confirmed by qRT-pCR and immunoblot analysis. Next, an inducible miR-294 AAV-9-αMHC vector was delivered to mice fed dox chow for 2 wks following myocardial infarction. Increased EF, FS and hemodynamic parameters together with decreased infarct size and apoptosis was observed 8 wks after MI. Myocyte proliferation increased in miR-294 hearts analyzed by Ki67, p-H3 and AurB expression parallel to increase in small myocyte number in the heart. Isolated adult myocytes from miR-294 hearts showed increased EdU+ cells and upregulation of cell cycle markers/miR targets 8 wks after MI. Conclusion: Ectopic expression of miR-294 recapitulates developmental signaling and phenotype in cardiomyocyte promoting cell cycle reentry that leads to augmented cardiac function in mice after myocardial infarction.