Abstract Body: The identification of new factors and mechanisms that can promote the repair and regeneration of the adult injured mammalian heart are necessary as the adult heart has limited ability to repair itself. We identified MYCN as a downstream effector of the sonic hedgehog (SHH) signaling cascade during cardiomyocyte proliferation in the neonatal heart. Although the protooncogene, MYCN, has been shown to be an essential factor during embryonic heart development, its role in regenerating the adult heart has not been established. Here, we define MYCN as a critical regulator of cardiomyocyte (CM) proliferation and regeneration using genetic mouse models and in vivo modified RNA (modRNA) delivery. In these studies, we conditionally deleted Mycn in the neonatal heart (P0). Immunohistochemical (IHC) analysis of P3 neonatal hearts demonstrated significantly (p < 0.05) decreased expression of Ki67 and pH3 in the absence of Mycn compared to control (no 4-OHT) hearts. This observation was further validated in isolated CMs and neonatal P7 hearts pulsed with EdU which demonstrated a significant decrease in EdU+ CMs in the absence of Mycn compared to control. Single-nucleus RNA-sequencing results from control and Mycn CKO P4 ventricles revealed a severe depletion of the proliferative and regenerative cardiomyocyte population in the Mycn CKO hearts. We then ligated the left anterior descending coronary artery (LAD) to produce an ischemic cardiac injury in control and Mycn CKO P2 neonatal mice. Echocardiography one-month following the LAD surgeries demonstrated that the Mycn CKO failed to recover baseline cardiac function compared to their control counterparts who achieved complete recovery. RNAseq analysis of control and Mycn CKO hearts 1 month following LAD ligations demonstrated that muscle developmental pathways were significantly downregulated in the Mycn CKO group compared to the control. We then delivered either GFP (control) or Mycn modRNA intramuscularly into the border zone following LAD coronary artery ligation surgeries in adult (2-6 month old) mice. We observed that the Mycn modRNA treated mice had significantly improved cardiac function at one month and two months following injury/delivery compared to controls. These results identify MYCN as an important regulator of CM proliferation and heart regeneration.