Abstract 545: A Novel Mutation in Plakoglobin Blocks Up-Regulation of Intercellular Junction Proteins in Response to Mechanical Load
Mutations in the desmosomal protein plakoglobin cause arrhythmogenic right ventricular cardiomyopathy (ARVC). These mutations likely alter intercellular adhesion and impair cellular responses to mechanical stress, but disease mechanisms in ARVC remain poorly understood. Here, we analyzed the effects of a novel mutation in plakoglobin (S39 K40insS), which is known to cause autosomal dominant ARVC, on cellular responses to defined mechanical load. HEK293 cells, which stably expressed either wild type or mutant plakoglobin, were seeded on silicone membranes and, after growing to confluence, were subjected to uniaxial, pulsatile stretch (110% of resting length; 3 Hz) for 4 hr. Changes in the distribution of the major cardiac gap junction protein, Cx43, and the mechanical junction proteins N-cadherin and plakoglobin were measured by immunohistochemistry and quantitative confocal microscopy. The amount of Cx43, N-cadherin and plakoglobin located at cell-cell junctions was increased by ~3-fold in wild type cells (p<0.01) after 4 hr of stretch. In contrast, stretch-induced up-regulation of both electrical and mechanical junction proteins was significantly blunted in cells expressing mutant plakoglobin. Electron microscopy revealed fewer and smaller intercellular junctions in mutant cells. Conditioned medium removed from wild type cells that had been stretched for 4 hr increased expression of Cx43, but not mechanical junction proteins, when added to unstretched wild type or mutant cells. Stretch-induced up-regulation of Cx43 in wild type cells was blocked by anti-VEGF antibodies. Thus, the ARVC-causing S39 K40insS mutation in plakoglobin inhibits up-regulation of both electrical and mechanical junction proteins in cells subjected to mechanical load. Enhanced expression of Cx43, but not mechanical junction proteins, depends on secretion of growth factors in response to stretch. Expression of mutant plakoglobin leads to changes in the number and structure of intercellular adhesion junctions. These results support the hypothesis that defects in intercellular adhesion and abnormal responses to mechanical stress contribute to the pathogenesis of ARVC caused by mutations in plakoglobin.