Familial Hypertrophic Cardiomyopathy-related myosin mutations. Functional effects studied by single molecule approaches

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Point mutations in the beta-cardiac myosin heavy chain (beta-MHC) are the most frequent cause of Familial Hypertrophic Cardiomyopathy (FHC) in humans. While a large number of different mutations were identified in the myosin head domain and other sarcomeric proteins, the pathogenic path to a common FHC-phenotype is not understood. For FHC mutations in the myosin head domain we hypothesize that they induce changes in motor activity that act as the trigger for the development of the FHC-phenotype. Thus, it is conceivable that selective rectification of the primary change in the functional behaviour of mutant myosin heads may prevent the development of a noticeable FHC-phenotype. The main goal of our proposed research is to gain comprehensive understanding of functional domains and altered communication pathways in the myosin head, as a consequence of point mutations. The project aims to characterize functional changes in the beta-MHC head domain, induced by FHC-mutations, by investigating specific effects of FHC mutations on motor properties, such as ATPase kinetics, stroke size, force production, and movement of actin filaments. Previous work from our group reported e.g., reduced calcium sensitivity of affected cells for some mutations while other FHC-mutations in the converter domain increased contractile forces due to increased head stiffness. This illustrates the need to distinguish the widely varying effects of FHC mutations on myosin function. Most FHC-patients produce both mutant and wild-type beta-MHC. Using single molecule techniques, we can probe samples, molecule by molecule, and thus study wildtype and mutant molecules separately even in mixed samples. We investigate ATPase kinetics via Cy3-EDA-ATP dwell- and waiting-times by total internal reflection fluorescence microscopy (TIRFM) and zero mode waveguide (ZMWG) based assay. We use optical trapping of beta-MHC individual head domains to directly measure parameters that are difficult to derive from fibres or cells, such as force generation, stroke size, and resistance to elastic deformation (stiffness). To overcome restrictions due to the limited availability of native protein from patient/donor biopsies, we produce the beta-MHC head domain with FHC-mutations in human cardiomyocytes. The major advantage of establishing this new expression system is the correct composition of myosin heavy chain with its endogenous accessory proteins (essential and regulatory light chains), which is vital for precise comparison of the native beta-MHC function with the recombinant counterpart. Additionally, rationally designed mutant proteins will be produced in cardiomyocytes to further elucidate communication pathways and their importance for chemo-mechanical coupling in the myosin motor.