Abstract 370: Hedgehog Signaling Regulates Cardiac Regeneration in vivo

Abstract

The adult mammalian heart has a limited regenerative capacity due primarily to reduced cardiomyocyte (CM) proliferation. Here, we demonstrated hedgehog (HH) signaling pathway as an essential regulator of heart regeneration and CM proliferation. We undertook genome-wide screening using a novel algorithm, bootstrap, which showed an induction of HH signals in the regenerating newt heart. Blockade of HH signaling in the resected newt heart resulted in complete ablation of cardiac regeneration and scar formation. EdU-labeling revealed that inhibition of the HH pathway significantly reduced CM proliferation by 3-fold (n=4 at each time period post-injury). In mammals, cardiac specific loss- and gain-of-function of HH signals demonstrated its role in CM proliferation and regeneration in the postnatal heart. Genetic deletion of floxed-Smoothened ( Smo L/L ) allele at postnatal day 2 (P2) inhibited neonatal heart regeneration with impaired cardiac function and scarring following injury. Conversely, induction of constitutively active Smoothened (SmoM2) at P7 stimulated CM proliferation by 2.5-fold (n=3) and regeneration after myocardial infarction during the non-regenerative window. Lineage-tracing experiments showed that activation of Smo contributed to heart regeneration by promoting proliferation of the pre-existing cardiomyocytes. Activation of HH signals in the cultured CM at P1 and P7 showed an increased proliferative response by 2- and 3-fold (n=4; 1900 cells evaluated for each condition), respectively. Mechanistically, ChIP-seq analysis revealed that HH signals promoted the proliferative program by directly regulating the expression of cyclin-dependent kinases including cyclinD2, cyclinE1 and Cdc7. Finally, activation of HH signaling in the terminally differentiated hiPSC-derived CM resulted in an increase in the number of α-Actinin + /EdU + and α-Actinin + /Ki67 + cells by 2.5-fold (n=3; 645 cells assessed for each condition) and 3-fold (n=3; 685 cells assessed for each condition), respectively. These studies defined an evolutionarily conserved function of HH signaling from newt to mouse to human, as a key regulator of cardiomyocyte proliferation and regeneration that may serve as a platform for regenerative therapies.