Abstract 18363: Activation of the Hippo Pathway, Associated With Suppression the Canonical Wnt Signaling, Contributes to the Pathogenesis of Arrhythmogenic Right Ventricular Cardiomyopathy

Abstract

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an enigmatic disease characterized by excess fibro-adipocytes in the heart, cardiac arrhythmias and heart failure. Molecular genetic studies have identified mutations in several desmosome proteins as causes of ARVC. Our objective is to delineate the molecular links between the desmosomes and fibro-adiposis in ARVC. Expression levels and localization of plakophilin 2 (PKP2), plakoglobin (JUP), desmocollin 2 (DSC2), desmosome proteins and connexin 43 (GJA1), a member of Gap junction, in the human hearts with ARVC, were reduced. To test whether molecular remodeling of the junctions affected signaling pathways, we analyzed the Hippo kinase pathway, which is regulated by cell-cell contact and known to suppress the canonical Wnt signaling, the latter implicated in the pathogenesis of ARVC. Accordingly, Merlin, an upstream regulator of the Hippo kinase cascade, was activated and YAP, the downstream effector was inactivated by phosphorylation. To gain mechanistic insights, expression of Pkp2, the most common causal gene for ARVC, was stably knocked down using shRNA in the HL-1 cells. Transcriptome analysis showed reduced mRNA levels of the canonical Wnt and Hippo targets including Ctgf, an inhibitor of adipogenesis. KD of PKP2 was associated with a dramatic reduction in pPKC-α level, in accord with the known function of PKP2 as a platform for phosphorylation of PKC-α. Reduced pPKCα, an inhibitor of Merlin through phosphorylation, was associated with activation of Merlin and cascade phosphorylation of MST1, LATS1/2 and YAP, constituents of the Hippo pathway. Consequently, signaling through TEAD, the transcription factor of the Hippo pathway, was reduced and sequestration of pβ-catenin in the cytoplasm was increased, leading to suppression of the canonical Wnt signaling and enhanced adipogenesis. KD of pLAT1/2 using shRNAs partially rescued the molecular phenotype. Thus, human ARVC is characterized by molecular remodeling of the intercalated discs, which results in inactivation of PKCα, phosphorylation of the Hippo kinases, suppression of the canonical Wnt signaling and enhanced adipogenesis. The findings implicate the Hippo pathway in the pathogenesis of ARVC.