The properties of the actin-myosin interaction in the heart muscle depend on the isoforms of myosin but not of α-actin.

In myocardium of mammals there are two isoforms of myosin heavy chains, α and ß. In ventricle, together with ventricular isoforms of light chains they form two isomyosins: V1 and V3, homodimers of α- and ß-heavy chains, respectively. In atria, α- and ß-heavy chains together with atrial light chains form A1 (αα) and A2 (ßß) isomyosins. Besides in myocardium two isoforms of α-actin, skeletal and cardiac, are expressed. We assume that the differences in the amino acid sequence of cardiac and skeletal actin may affect its interaction with myosin. To test this hypothesis, we investigated characteristics of actin-myosin interactions of cardiac and skeletal isoforms of α-actin with the isoforms of cardiac myosin using an optical trap technique and an in vitro motility assay. It was found that the mechanical and kinetic characteristics of the interactions of the isoforms of cardiac myosin with actin depend on the isoforms of myosin not α-actin.

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