title: Transgenic mouse α- and β-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic differences.
abstract: 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 β-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 β-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 β-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 β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-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.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA. susan.lowey@uvm.edu
authors.forename: Susan
authors.initials: S
authors.lastname: Lowey
authors.forename: Vera
authors.initials: V
authors.lastname: Bretton
authors.forename: James
authors.initials: J
authors.lastname: Gulick
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Robbins
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Trybus
mesh_terms: D000244:Adenosine Diphosphate / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D007700:Kinetics; D051379:Mice; D008822:Mice, Transgenic; D020125:Mutation, Missense; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D024723:Ventricular Myosins / Q000235:genetics / Q000378:metabolism*
chemical_list: C551613:Myh7 protein, mouse; D000244:Adenosine Diphosphate; D024723:Ventricular Myosins; D018995:Myosin Heavy Chains
keywords: Cardiac Myosin Isoforms; Cardiomyopathy; Cardiovascular Disease; Familial Hypertrophic Cardiomyopathy; Kinetics; Myosin; Myosin Heavy Chain Mutations; Transgenic Mice; Transient Kinetics