Desmoplakin's Regulation of Gap Junction Dynamics in Cardiocutaneous Model Systems

Desmoplakin (DP) is an essential component of desmosomal junctions, tethering intermediate filaments to sites of strong cell-cell adhesion. Reflecting its importance in tissues that experience mechanical stress, DP is a frequent target for mutation in cardiocutaneous disorders. In particular, DP mutations cause arrhythmogenic cardiomyopathy (AC), an inherited disorder characterized by replacement of healthy myocardium by fibro-fatty deposits. During AC pathogenesis, a loss of electrical conduction between cardiomyocytes can occur prior to fibro-fatty infiltration, an event that has been dubbed the “concealed phase”. Providing a possible explanation for the conduction defect, DP deficiency has been reported to be associated with decreased levels of the gap junction protein Connexin-43 (Cx43). However, the mechanism by which loss of DP decreases Cx43 expression is unknown. Here, we utilized in vitro and in vivo models of DP deficiency in heart and skin to elucidate the mechanism by which DP regulates Cx43 protein levels. Depletion of DP in neonatal rat ventricular cardiomyocytes (NRVCMs) resulted in decreased Cx43 protein levels, consistent with previous in vivo observations. Cx43-based gap junction dynamics are regulated by a number of phosphorylation sites on the Cx43 intracellular C-terminal tail. Systematic analysis of known Cx43 phosphorylation sites in DP-deficient NRVCMs revealed a specific increase in the MAPK sites S279/282, previously shown to signal internalization and degradation of Cx43. Accordingly, elevated Cx43 p-S279/282 in DP-deficient cardiac myocytes was accompanied by a significant increase in p-ERK1/2. Similar changes in Cx43 and ERK were observed in cardiac and epidermal tissues obtained from mice with conditional ablation of DP. Cx43 levels were restored in NRVCMs using pharmacologic MAPK inhibitors, supporting a mechanistic connection between Erk activation and Cx43 expression. To address whether p-S279/282 Cx43 regulates Cx43 degradation, we treated cells with cycloheximide to block protein synthesis and measured Cx43 expression over time. DP depletion accelerated loss of Cx43, which was prevented by inhibitors of lysosomal, but not proteosomal, degradation. DP loss was also associated with increased Cx43 ubiquitination. Ubiquitination mediated internalization of Cx43 was previously shown to involve Myosin VI, an unconventional myosin known for its role in vesicular trafficking. Indeed, we observed increased co-immunoprecipitation of Myosin VI and Cx43 upon DP knockdown in NRVCMs, implicating Myosin VI in internalization of Cx43 that occurs in DP-deficient cells. Collectively, our results expose a novel mechanism for the regulation of Cx43 levels in cardiocutaneous disease caused by loss of DP.