Mutations in MyHC genes have been associated with a number of skeletal myopathies. However, the mechanisms by which these mutations cause disease are not understood.
Aims I and II of this renewal will test the hypotheses that disease-causing mutations in MyHC genes lead to: i) catalytic, mechanical and structural changes and/or ii) changes in protein turnover or sarcomere dynamics. In the previous grant period, we achieved the first production of enzymatically active recombinant human striated muscle myosin motor domains. Using this system, we have determined the detailed kinetic properties of 8 of them and have begun studies on the functional changes caused by 2 disease-causing mutations. These resources will be used to assess which steps of the ATPase cycle and mechanical properties are affected by mutation. Collaboratively, we have also obtained the first crystals of the human Type I/?-MyHC motor domain and propose to determine the impact of mutations on the structure of the myosin motor by X-ray crystallographic studies. We will measure whether myosin mutations affect half-life and sarcomere dynamics using a novel approach based on the controlled expression of MyHC linked to fluorescent timer (FT) proteins. Using live cell imaging, we have demonstrated as proof of principle that we can detect altered turnover of MyHCs in sarcomeres. The myosin composition of muscle is dynamic and responsive to a wide variety of stimuli, including disease, contributing to resulting functional diversity. The discovery of 2 phylogenetically distant MyHC genes that likely have novel cellular roles leads logically to Aims III and IV.
These Aims will test the hypotheses that the catalytic an mechanical properties of MYH7b and MYH15 are unique and that MYH7b has specialized functions in muscle and brain. We will use the kinetic and biochemical assays employed in Aim I to characterize MyH7b and MyH15 which have unique alternate N-terminal amino acid extensions. The in vitro motility and single molecule mechanics of the MyH7b and MyH15 motor domains will also be measured. Comparison with the other characterized myosins will help elucidate why expression of these two phylogenetically distant myosins is limited to specialized muscles (extraocular muscles and the sensory muscle spindles) and brain. Since the cerebellum controls motor skills and coordination and muscle spindles are important for posture and gait, we hypothesize that MYH7B plays important biological roles in these functions. To test this hypothesis, we have made mice null for the expression of MYH7b. At the same time, we are generating transgenic mice with forced expression of MYH7b in fast skeletal muscle to test the hypothesis that mammals evolved mechanisms to protect fast skeletal muscles from MYH7b protein expression because MYH7b expression in those tissues would be harmful. These studies will allow us to integrate the in vitro biochemical/biophysical data into the context of functioning muscle.
Numerous molecular motors drive muscle contraction. Mutations in these motors are common causes of muscle diseases. However, how these mutations cause disease has yet to be determined. We are also studying 2 new myosins which will complete the functional analysis of all such genes. This project uses novel approaches to pursue the systematic studies of the muscle motor, myosin and its function and dysfunction. New insights into the function of the human myosins and deficits in disease will be invaluable in developing new molecular targets for therapeutic interventions.
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