The R403Q mutation in the ␤-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 ␣-MHC as opposed to the ␤-isoform in larger mammals) created a new paradigm for FHC based on finding enhanced motor function for R403Q ␣-MHC. To help resolve these conflicting mechanisms, we used a transgenic mouse model in which the endogenous ␣-MHC was largely replaced with transgenically encoded ␤-MHC. A His 6 tag was cloned at the N terminus of the ␣-and ␤-MHC to facilitate protein isolation by Ni 2؉ -chelating chromatography. Characterization of the R403Q ␣-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 ␤-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 ␣-S1 is ϳ30% higher than for wild type, whereas the enzymatic activity for R403Q ␤-S1 is reduced by ϳ10%. Thus, the functional consequences of the mutation are fundamentally changed depending upon the context of the cardiac MHC isoform.The arginine to glutamine mutation at amino acid 403 (R403Q) in the ␤-myosin heavy chain (MHC) 2 was the first MHC missense mutation linked to familial hypertrophic cardiomyopathy (FHC), a primary disease of heart muscle (1). Since that time several hundred mutations in genes encoding sarcomeric proteins have been identified, leading to the designation of FHC as primarily a "disease of the sarcomere" (2, 3). However, the pathways linking the various genetic defects to the characteristic human disease phenotype remain largely unknown. Even the fundamental question of whether these mutations cause a gain or a loss of function in myosin remains unresolved. We have confined our study to the R403Q mutation, because it is one of the most extensively studied FHC mutations and because it has a poor clinical prognosis (2, 4). The majority of the earlier biochemical/biophysical results, derived from expression systems and biopsies, supported the hypothesis that the R403Q mutation leads to a large decrease in motor function, which ultimately results in a compensatory hypertrophic response (for review, see Ref. 5).The first murine model for FHC replaced one allele of the endogenous cardiac mouse ␣-MHC with the mutant ␣-MHC (R403Q) gene by homologous recombination (6); this heterozygous mouse (R403Q/ϩ) resembled the human cardiac phenotype in many ways, except cardiac hypertrophy was notably milder. Subsequent studies on myosin extracted from a Ͻ1-week-o...