Abstract 1427: Pathogenesis of Hypertrophic Cardiomyopathy Caused by the Myozenin 2 Mutations Involves Calcineurin-Dependent and -Independent Mechanisms
Identification of myozenin-2 mutations (MYOZ2) has expanded the spectrum of causal mutations for hypertrophic cardiomyopathy (HCM) to include the Z disk proteins. The molecular mechanism(s) by which MYOZ2 mutations cause HCM are unknown. MYOZ2 is known to inhibit calcineurin phosphatase (PP2B). We posit mutant MYOZ2 lose their inhibitory effects on PP2B and hence, the ensuing cardiac phenotype results from enhanced activity of PP2B. To test this hypothesis, we generated lines of transgenic mice expressing either N-terminally flag-tagged wild type (MYOZ2-WT) or mutant MYOZ2-P48 or MYOZ2-M246 regulated by the α-myosin heavy chain promoter. Expression levels of the transgenic and endogenous MYOZ2 proteins were detected by immunoblotting using pan-specific anti-MYOZ2 and anti-Flag antibodies. The transgene proteins constituted 15 to 35% of the total MYOZ2 protein. The total MYOZ2 levels remained unchanged. Immunofluorescence staining showed both WT and mutant MYOZ2 proteins were incorporated into the Z disks. To determine the effects of the WT and mutant MYOZ2 proteins on PP2B activity, we measured PP2B phosphatase and Nuclear Factor of Activated T Cells (NFATc), a downstream target of PP2B, activities in myocardial protein extracts by ELISA-based assays. Expression of MYOZ2-WT reduced PP2B phosphatase and NFAT activities by ~90% and 20%, respectively while equal levels of expression of the mutant MYOZ2 reduced these activities by ~ 50% and 10%, respectively, i.e., a 2-fold reduction in inhibitory effect of the mutant as compared to WT. The mutant MYOZ2-P48 and MYOZ2-M246 showed increased mortality, cardiac hypertrophy, preserved cardiac systolic function, increased myocytes size and interstitial fibrosis. The MYOZ2-WT mice also exhibited cardiac hypertrophy and interstitial fibrosis. Thus, the mutation impart a loss of function effect on MYOZ2, suggesting a possible gain of function of PP2B in humans with with autosomal dominant HCM. However, excess inhibition of PP2B in the heart, as observed in the MYOZ2-WT mice, also led to cardiac hypertrophy and fibrosis. Thus both excess inhibition as well as relative enhancement of PP2B activity in the heart could cause cardiac hypertrophy and fibrosis.