Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

The R403Q mutation in the β-myosin heavy chain (MHC) was the first mutation to be linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle. The initial studies with R403Q myosin, isolated from biopsies of patients, showed a large decrease in myosin motor function, leading to the hypothesis that hypertrophy was a compensatory response. The introduction of the mouse model for FHC (the mouse expresses predominantly α-MHC as opposed to the β-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q α-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous α-MHC was largely replaced with transgenically encoded β-MHC. A His6 tag was cloned at the N terminus of the α-and β-MHC to facilitate protein isolation by Ni2+-chelating chromatography. Characterization of the R403Q α-MHC by the in vitro motility assay showed a 30-40% increase in actin filament velocity compared with wild type, consistent with published studies. In contrast, the R403Q mutation in a β-MHC backbone showed no enhancement in velocity. Cleavage of the His-tagged myosin by chymotrypsin made it possible to isolate homogeneous myosin subfragment 1 (S1), uncontaminated by endogenous myosin. We find that the actin-activated MgATPase activity for R403Q α-S1 is ∼30% higher than for wild type, whereas the enzymatic activity for R403Q β-S1 is reduced by ∼10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.