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Updated: May 12, 2026
Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes
Published on: May 25, 2022
Transgenic mouse α- and β-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic
Susan Lowey1, Vera Bretton, James Gulick
1Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA. susan.lowey@uvm.edu
Insights
Familial hypertrophic cardiomyopathy (FHC) is linked to myosin mutations. This study shows that the impact of an FHC mutation on myosin function depends on the specific myosin isoform backbone, impacting nucleotide binding and release.
Area of Science:
- Cardiovascular Research
- Molecular Biology
- Biophysics
Background:
- Familial hypertrophic cardiomyopathy (FHC) is a genetic heart condition often caused by mutations in muscle proteins.
- A known mutation (R403Q) in beta-myosin heavy chain (MHC) causes severe FHC.
- Existing mouse models use alpha-MHC, but human ventricles primarily express beta-MHC, necessitating isoform-specific studies.
Purpose of the Study:
- To investigate the role of the myosin heavy chain (MHC) isoform backbone in the functional consequences of FHC-associated mutations.
- To compare the mechanochemical properties of alpha-MHC and beta-MHC isoforms with and without the R403Q mutation.
Main Methods:
- Generated transgenic mice expressing human beta-MHC or alpha-MHC isoforms.
- Utilized a His6 tag for myosin subfragment 1 (S1) isolation.
- Employed stopped-flow kinetics to measure nucleotide binding and release rates for actin-bound S1 isoforms.
Main Results:
- Wild-type beta-S1 exhibited higher MgADP affinity and slower MgADP release compared to wild-type alpha-S1.
- The R403Q mutation had a minor effect on beta-S1 kinetics.
- R403Q significantly increased the ADP release rate of alpha-S1 by 20%.
Conclusions:
- The functional impact of the R403Q FHC mutation on cardiac myosin is dependent on the MHC isoform backbone.
- These findings highlight the importance of considering isoform-specific differences in studying FHC pathogenesis and developing therapeutic strategies.
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.
