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J Biol Chem. 2013 May 24;288(21):14780-7. doi: 10.1074/jbc.M113.450668. Epub 2013 Apr 11.

Transgenic mouse α- and β-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic differences.

Author information

  • 1Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA. susan.lowey@uvm.edu

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.

KEYWORDS:

Cardiac Myosin Isoforms; Cardiomyopathy; Cardiovascular Disease; Familial Hypertrophic Cardiomyopathy; Kinetics; Myosin; Myosin Heavy Chain Mutations; Transgenic Mice; Transient Kinetics

PMID:
23580644
[PubMed - indexed for MEDLINE]
PMCID:
PMC3663502
Free PMC Article
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