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 â-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 á-MHC, whereas the ventricles of larger mammals express predominantly â-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous á-MHC was partially replaced with transgenically encoded â-MHC or á-MHC. A His6 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 á-S1 (100 ìM) is ~ 4-fold weaker than for â-S1 (25 ìM). Correspondingly, the MgADP release rate for á-S1 (350 s(-1)) is ~3-fold greater than for â-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for â-S1, but R403Q in á-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.
Adenosine Diphosphate Amino Acid Substitution