Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the beta-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express alpha-MHC, whereas the ventricles of larger mammals express predominantly beta-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous alpha-MHC was partially replaced with transgenically encoded beta-MHC or alpha-MHC. A His(6) tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for alpha-S1 (100 mu M) is similar to 4-fold weaker than for beta-S1 (25 mu M). Correspondingly, the MgADP release rate for alpha-S1 (350 s(-1)) is similar to 3-fold greater than for beta-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for beta-S1, but R403Q in alpha-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.