More than 500 human sarcomeric myosin heavy chain mutations in multiple members of the 10 gene family cause 11 distinct heart and skeletal muscle diseases. Myosin consists of a globular motor domain and an ?- helical rod domain, with disease-causing mutations throughout. Despite having studied the myosin gene family for many years, we have recently discovered unexpected biology such as a ?sarcomeric? myosin in mammalian brains and inner ears and the rapid movement of single myosin molecules in and out of sarcomeres. Our goal is to understand the diversification of the myosin gene family, how different myosins drive muscle function, and how mutations in the same residue of the same myosin gene give rise to skeletal or cardiac muscle disease. To accomplish these goals, we propose integrated structural, biophysical, cellular and in vivo approaches with WT and mutant myosins. The unorthodox MYH7b protein is found in striated muscles of pythons and birds, but only a small number of mammalian muscles expresses the protein. We will compare the functions of python and human MYH7b proteins, testing the hypothesis that human MYH7b protein has evolved from the typical sarcomeric motor seen in python to its use in specialized mammalian muscles, where it is sarcomeric, and in a subset of cells in the brain and the inner ear. Its role in the inner ear is intriguing since compound heterozygous mutations in MYH7b are linked to hereditary hearing loss and we will study these mutant myosin?s. Most mammalian muscles express abundant MYH7b RNA that cannot produce protein due to an alternative splicing mechanism. Our hypothesis, backed up by strong preliminary data, is that mammalian non-coding MYH7b RNA regulates expression of the highly expressed Type I slow skeletal/? cardiac myosin. The myosin composition of muscles is dynamic and known to drive physiology, but very little is known about how myosin moves into and out of sarcomeres in health and disease. The much longer half-life of myosin protein (~10 days) compared to its sarcomeric replacement rates (5-14 hours) suggests that each molecule undergoes multiple rounds of thick filament entry, egress and re-entry. Myosin mutations, particularly ones in the rod, could affect each step of myosin?s movement into and out of sarcomeres. We propose to use single myosin molecule imaging of WT and mutant myosins in genome edited hiPS muscle cells to measure these processes. Specifically, mutations in the same amino acid in the Type I/? cardiac myosin rod cause either a skeletal myopathy (R1500P) or a cardiomyopathy (R1500W). In mouse models of these mutations, we will use multi-isotope mass spectroscopy electron microscopy (MIMS-EM) technology to measure sarcomere homeostasis in WT mice as well as how it is affected by the R1500P and R1500W mutations.
Mutations in myosin molecular motors are common causes of muscle diseases; one in 500 people have such a mutation. However, how these mutations cause disease is still to be determined. This project uses many types of approaches to understand these diseases. A novel finding is that one of these motors is involved in hereditary hearing loss and we propose to understand how a muscle motor functions in the ear.
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