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

University of Vermont, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2013; 288(21). DOI: 10.1074/jbc.M113.450668
Source: PubMed


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.

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