Key result
The R403Q mutation enhanced the ADP release rate by 20% (to 430 s−1) in mouse α-cardiac myosin but caused only a minor reduction in kinetics for β-cardiac myosin.
The functional impact of the R403Q mutation on cardiac myosin kinetics depends on the isoform backbone, indicating that mouse models expressing α-MHC may not fully recapitulate the human hypertrophic cardiomyopathy phenotype driven by β-MHC.
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Isoform-dependent R403Q effects caution against α-MHC mouse models for human β-MHC HCM; leaves open need for matched isoform studies.
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 His 6 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. Background: The mouse model for cardiac disease has been the focus for mutational studies on the α-cardiac myosin heavy chain (MHC). Results: Transient kinetics showed that R403Q enhanced ADP release in mouse α-MHC but not in mouse β-MHC. Conclusion: The functional impact of a mutation depends on the myosin isoform. Significance: The R403Q mutation in human β-cardiac myosin likely compromises its function leading to hypertrophic cardiomyopathy.
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Lowey et al. (2013) studied Familial hypertrophic cardiomyopathy. R403Q mutation in α- and β-cardiac myosin heavy chain vs. Wild-type isoforms was evaluated on Equilibrium constants and rates of nucleotide binding and release. The R403Q mutation enhanced the ADP release rate by 20% (to 430 s−1) in mouse α-cardiac myosin but caused only a minor reduction in kinetics for β-cardiac myosin.
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