Mutations in the ?-myosin heavy chain (MHC) are among the major causes of familial hypertrophic cardiomyopathy (FHC), a relatively widespread heart disease in humans. Despite many decades of study, the effect of a single point mutation in the ? -MHC on the functional and structural properties of the myosin molecule, the filament and the complex muscle cell are not well understood. The mouse has been the most popular animal model for FHC because of its advanced genetics and the relative ease in generating mutant strains. Approaches ranging from single molecule mechanics to cardiac muscle physiology have led to the hypothesis that a """"""""gain of function"""""""" arises from FH mutations in mice, in contrast to earlier studies on human tissue suggesting a """"""""loss of function"""""""". However, the transgenic mouse model differs significantly in protein composition from rabbits and humans, insofar as ? -MHC is the major myosin isoform in the mouse heart, whereas ? -MHC predominates in the ventricles of all larger mammals. We have therefore generated a transgenic rabbit model in which a severe FHC mutation, R403Q in the ? -MHC, was over-expressed in the ventricles. To facilitate isolation and quantification, a His-tag was cloned at the N-terminus of the rabbit transgene along with the R403Q mutation. We hypothesize that the functional effects of a mutation are dependent on the isoform backbone, and R403Q will lead to a """"""""loss of function"""""""" in ? - MHC.
Aim 1 will determine how an FHC mutation in rabbit cardiac ? -MHC affects the stopped-flow kinetics and in vitro motility properties of the cross-bridge cycle.
Aim 2 will determine the effect of load on actin filament velocity and force generation by R403Q myosin using a force clamp/laser trap assay.
Aim 3 will determine the structural consequences of an FHC mutation on the actomyosin interaction and the relaxed state of the myosin filament by advanced high resolution cryo-electron microscopy and single particle imaging techniques. These studies should serve to increase our knowledge of the basic molecular mechanism of this disease by examining the effects of an FHC mutation at increasing levels of complexity from the molecule to the filament.
Familial hypertrophic cardiomyopathy (FHC) is an inherited disease of heart muscle that can cause sudden death in young adults. Mutations in the heavy chain of the muscle protein, myosin, account for almost half of the several hundred mutations linked to FHC. It is hoped that molecular insights gained from a genetically engineered rabbit model will lead to a better understanding of the disease with the ultimate goal of developing new targets for therapeutic intervention.
|Lowey, Susan; Bretton, Vera; Gulick, James et al. (2013) Transgenic mouse Î±- and Î²-cardiac myosins containing the R403Q mutation show isoform-dependent transient kinetic differences. J Biol Chem 288:14780-7|