Abstract 2724: Disparate Biomechanical Effects Of Two Arrhythmogenic Right Ventricular Cardiomyopathy-causing Mutations In Plakoglobin
Mutations in desmosomal proteins cause arrhythmogenic right ventricular cardiomyopathy (ARVC), implicating a defect in cell-cell adhesion. To gain insights into disease mechanisms, we characterized the cell biological and biomechanical effects of 2 distinct ARVC-causing mutations in plakoglobin, an autosomal dominant mutation (S39 K40insS) and a recessive deletion (2057del2) known to underlie the cardiocutaneous syndrome Naxos disease. HEK293 cells, stably transfected to express wildtype or mutant plakoglobin, were subjected to magnetic micromanipulation to measure cell stiffness, drag deformation to assess cell-cell adhesion strength, linear scraping to measure wound healing rate, and uniaxial pulsatile stretch to characterize changes in the expression of proteins at cell junctions. The effects on apoptosis were also investigated by measuring caspase-3 activity. In some assays, both mutant forms caused similar phenotypes. Wound healing was greatly accelerated in cells expressing either mutant plakoglobin, but without an increase in cell division rates. Cells expressing either mutant failed to normally up-regulate expression of mechanical and electrical junction proteins in response to stretch. In other assays, however, the mutants caused disparate cellular phenotypes. Magnetic micromanipulation showed that S39 K40insS but not 2057del2 significantly decreased cell stiffness compared with cells expressing wildtype (wt) plakoglobin (bead displacement: 2.16 ± 1.53 vs. 2.26 ± 1.24μm in wt and 2057del2, n = 6, p = NS; 1.88 ± 1.31 vs. 2.34 ± 1.23μm in wt and S39 K40insS, n = 6, p = 0.05). In contrast, drag deformation revealed a marked reduction in the strength of cell-cell adhesion in cells expressing 2057del2, but no change with S39 K40insS (smearing distance: 452 ± 124 vs. 503 ± 146μm in wt and S39 K40insS; n = 6, p = NS; vs. 224 ± 36 μm in 2057del2, n = 6, p = 0.05). Caspase activity was decreased in cells expressing S39 K40insS, but increased in cells expressing 2057del2 (1261 ± 87 vs. 961 ± 95 units in wt and S39 K40insS, n = 8, p = 0.05; vs. 1903 ± 64 in 2057del2, n = 8, p = 0.05). Thus, different ARVC-causing mutations in the same gene have disparate effects on cellular biomechanics. These results suggest multiple, complex mechanisms in ARVC pathogenesis.