Abstract 20311: MYBPC3 Mutations Causative for Hypertrophic Cardiomyopathy Result in Locus-Dependent Alterations in Cellular Localization and Contractility

Abstract

Introduction: Mutations in MYBPC3 resulting in truncated proteins are the most common genetic cause of hypertrophic cardiomyopathy (HCM). Whether lack of sufficient MYBPC3 protein is the primary disease mechanism is controversial. We hypothesize instead that truncated MYBPC3 proteins exert dominant-negative effects on sarcomere structure and function. Methods and Results: MYBPC3 mutations resulting in short (Ile154LeuFs*5, 17 kD) or long (Asp1076Valfs*6 and Trp1098*, each ~120 kD) truncated proteins were studied. For each truncated protein, in silico analysis (I-TASSER) predicted marked deviation from the predicted wild-type (WT) structure. Immunofluorescence of adenoviral-expressed proteins in neonatal rat ventricular cardiomyocytes revealed that WT MYBPC3 was correctly localized at sarcomere A-bands. Near-complete mislocalization (predominantly nuclear) was observed for Ile154LeuFs*5 MYBPC3. Both Asp1076Valfs*6 and Trp1098*MYBPC3 mutant proteins localized imprecisely within the sarcomere, but also were diffusely present in the cytosol. Western blot of myofilament preparations confirmed no sarcomere incorporation of Ile154LeuFs*5, and reduced incorporation of Asp1076Valfs*6 and Trp1098* truncated MYBPC3 (Asp1076Valfs*6 28.1+0.4% and Trp1098* 20.3+0.2% vs. WT 72.4+0.2%, p<0.001). Ile154LeuFs*5 MYBPC3 was exclusively present in the non-myofilament fraction, while Asp1076Valfs*6 and Trp1098* MYBPC3 were 3-fold enriched in this fraction (p=0.01). Analysis of sarcomere shortening (Ionoptix) in adenoviral-infected adult rat ventricular cardiomyocytes indicated reduced peak shortening for Trp1098* (5.30+0.92% vs. WT 8.35+0.49%, p<0.05) and time to peak contraction (43+2 ms vs. WT 67+3 ms, p<0.05). In contrast, no significant effect of Ile154LeuFs*5 MYBPC3 on cellular contractility was observed. Conclusions: Truncating MYBPC3 mutations are predicted to markedly alter protein structure. Longer truncated proteins partially incorporate into the sarcomere lattice and alter contractile function while shorter truncated proteins accumulate in other cellular compartments. These results suggest distinct mutation-specific negative effects of truncating MYBPC3 mutations on myocyte function.