MYCN-FOXK1 cascade regulates cardiomyocyte proliferation and heart regeneration
While the adult mammalian heart has limited regenerative potential, the neonatal mouse heart has a remarkable regenerative capacity. Therefore, we focused on defining new factors and mechanisms that could enhance repair and regeneration in the adult mouse heart. FOXK1 is a transcription factor that regulates cell cycle kinetics, myogenic stem cell proliferation and skeletal muscle regeneration. During development, its expression is restricted to progenitors, somites and the heart. However, its role in the neonatal heart and regeneration is unknown and warrants investigation. Here, we identify FOXK1 as a downstream target of the SHH-GLI1-MYCN regulatory cascade and demonstrate that FOXK1 is a regulator of cardiomyocyte (CM) proliferation and regeneration. Using computational analysis, we identified FOXK1 as a downstream target of MYCN and further determined that FOXK1 was expressed in neonatal CMs. qPCR analysis of P1 hearts from control or Foxk1 null mice demonstrated that the absence of FOXK1 led to the dysregulation of Ccne1 and p21 expression, supporting the hypothesis that FOXK1 has a role in the regulation of cell cycle kinetics in the neonatal heart. Control or Foxk1 null mice were pulsed with EdU which demonstrated that the absence of FOXK1 led to a decrease in CM proliferation in vivo. Utilizing a transgenic mouse model that overexpressed FOXK1 using the muscle creatine kinase (MCK-Foxk1) promoter, we isolated and cultured control or MCK-Foxk1 CMs (72 hrs) and determined that overexpression of FOXK1 led to a significant increase in CM proliferation. Furthermore, EdU pulsing studies in adult control or MCK-Foxk1 mice demonstrated a significant increase in EdU incorporation in adult CMs of MCK-Foxk1 unperturbed mice compared to control. We then performed LAD ligation surgeries in control and MCK-Foxk1 adult mice. Echocardiography 2 months following LAD ligations showed a significant increase in both ejection fraction and fractional shortening in MCK-Foxk1 mice compared to controls. Single nucleus RNAseq analysis of control and MCK-Foxk1 adult hearts highlighted two distinct CM populations by UMAP analysis, with the latter demonstrating a higher expression of myoblast proliferative pathways. These results identify FOXK1 as a regulator of CM proliferation and heart regeneration by employing both knockout and transgenic mouse models.