Point mutations in both the beta-cardiac myosin heavy chain and in alpha-cardiac actin lead to either familial hypertrophic cardiomyopathy (FHC) or dilated cardiomyopathy (DCM). To arrive at a definitive mechanism for the primary cause of these two diseases, we propose to undertake an extensive kinetic, mechanical, and structural analysis of mutated cardiac myosins and actins.
Aim 1 will examine the effect of isoform backbone on function by comparing the R403Q mutation in an alpha- or a beta-murine cardiac myosin heavy chain (MHC) isoform obtained by over-expression in transgenic mice. Mutated MHCs will be HIS-tagged at the N terminus to facilitate isolation by metal chelate affinity chromatography. The mutant myosins will be characterized enzymatically by steady-state and transient kinetics, and mechanically by measurements of velocity and average force. Structural differences between wildtype and mutant myosin isoforms will be investigated by computer-based fitting of crystal structures into 3D-reconstructions of actomyosin complexes obtained by electron cryomicroscopy. Similar analyses will be extended to point mutations leading to FHC (G741 R, R453C) and DCM (S532 and F764). For a better understanding of the functional consequences of a point mutation in human myosin, Aim 2 will analyze myosin isolated from transgenic rabbits that express a human beta-cardiac myosin (R403Q) gene. In parallel, several new strategies for expression of human beta-cardiac myosin in vitro will be explored, including use of the Drosophila S2 expression system, and the addition of chaperones to increase the yield of striated muscle myosin isoforms.
Aim 3 will seek to characterize the effect of point mutations in actin that lead to FHC or DCM. alpha-cardiac actin expressed in the baculovirus/insect cell system allows us to investigate the effect of mutations in the correct backbone, rather than in the currently used yeast actin system. Alterations to actin's intrinsic filamentous structure, and to its interactions with myosin, will be assessed by many of the same approaches as described for the myosin mutations.The overall goal of the proposal is to elucidate how mutations implicated in FHC and in DCM affect the mechanical performance of myosin and actin, and to determine if any correlation can be made between the effect of the primary mutation and the ultimate disease phenotype.
