PO-705-06 DESMOSOME AND PLAKOPHILIN-2 (PKP2) DEFICITS ACCELERATE PLAKOGLOBIN (JUP) DEGRADATION LEADING TO CARDIAC PATHOLOGIES IN ACM

Abstract

Defective desmosome junctions had been considered the main pathogenic mechanism for Arrhythmogenic Cardiomyopathy (ACM). However, recent studies suggest non-junctional roles of desmosome component proteins, especially PKP2, JUP and desmoplakin (DSP) that exist in many types of cells. Desmosomal proteins had been implicated in metabolic, calcium handling, structural, and gene regulations. We previously showed that reduced JUP levels mediate the insulin pathway deregulation, leading to ACM pathologies. Here, we study how existence of desmosomes and/or mutations in PKP2 or DSP regulate the levels of JUP in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with desmosomes and iPSC-derived mesenchymal stromal cells (iPSC-MSCs, fibroblast-like cells) that have no desmosome. Elucidating how JUP protein is regulated may enable developing novel therapies. We generated iPSC-CMs and iPSC-MSCs from normal iPSCs or ACM iPSCs with a PKP2 c.2013delC mutation or 2 different DSP mutations. We then studied JUP levels by Pulse-chase labeling of JUP with [35S]methionine, Western/Co-IP, immunostaining, and qPCR to elucidate how JUP protein levels are regulated. To determine how existence of desmosomes affects JUP levels, we compared JUP levels between CMs (with desmosomes) to MSCs (without desmosome) derived from the same normal iPSCs by Pulse-chase methods. We find that JUP levels degrade slower [Decay time constant (t) > 40 hours (hrs)] in CMs than in MSCs (t=10.4±1.1 hrs) but rates of JUP synthesis are similar. In MSCs with no desmosome, PKP2, but not DSP, mutations also accelerate JUP degradation by 1.56 folds (t fell from 10.6±1.1 to 6.8±0.6 hrs) without changing rates of JUP synthesis. The rates of JUP synthesis are not changed by pathogenic induction in CMs. The different degrees of decreases in JUP degradation rates may explain differential pathologies between CMs and MSCs. Desmosome-containing CMs have slower JUP turnover than MSCs that have no desmosome, supporting that desmosome is a source of stable JUP supply for its non-junctional roles in insulin pathway, especially for metabolically active CMs. Without desmosome, PKP2, but not DSP, also slows down JUP degradation likely due to PKP2 is a common binding partner of JUP in many cell types.