Abstract Or201: MYCN Regulates Neonatal Cardiomyocyte Metabolism

Deciphering molecular regulators of cardiomyocyte (CM) proliferation is necessary because CMs have a limited ability to regenerate injury in the adult heart. Of the molecular mechanisms known to promote CM proliferation, we have identified the Sonic Hedgehog (Shh) pathway and MYCN as a regulator of perinatal CM proliferation. In previous studies, we conditionally deleted MYCN (cKO) in perinatal CMs and observed a reduction in cell cycle activity. Additionally, left anterior descending (LAD) ligation on cKO hearts at P2 resulted in an absence of regeneration compared to the control. Therefore, our data provides strong evidence for MYCN as an essential regulator of CM proliferation. To define the underlying cellular and molecular mechanisms of MYCN, we performed single-nucleus-RNA and -ATAC sequencing (MultiOmics), on P4 uninjured and ischemic injured hearts from control and cKO animals. Bioinformatic clustering analysis revealed 6 cell states of CMs, one of which was highly enriched in cKO cells. As expected, the cKO hearts had a significant reduction in the cluster of proliferating CMs, while broadly cell-cycle regulators were significantly downregulated across all CM clusters. Unexpectedly, the high-throughput data revealed vast changes in glycolysis and fatty acid oxidation regulators, as well as electron transport chain genes. In CMs, cell cycle and metabolism are linked, therefore we postulated that MYCN had a direct role in CM metabolism. In a mitochondria stress assay, primary CMs isolated from the cKO heart had significantly reduced mitochondrial activity compared to control CMs. On a molecular level, the transcription factor Foxo3, and members of the PI3K-Akt pathway, had significantly increased expression in cKO CMs, which is a negative regulator of the cell cycle and metabolism in CMs. Further bioinformatics and molecular analyses revealed a MYCN binding site near the promoter of Foxo3, along with regulators of glycolysis and fatty-acid oxidation. Additionally, we observed binding sites near genes that promote cell cycle progression. These results solidify an essential role for MYCN in regulating perinatal CM proliferation and a new function in CM metabolism.