Sunday, May 18, 2025
5/18/2025
Myosin Gene Diversity and Function - PROJECT SUMMARY Muscles are highly plastic tissues that respond rapidly to a wide variety of stimuli by altering their composition, contractility, and metabolism. For example, myosin is the most abundant protein in muscle, and during development, 50% of embryonic myosin is replaced in 3 hours by adult myosins. This is remarkable given that the average half-life of muscle myosin in vivo at baseline can be >1 month. Also, in terminally differentiated cardiac and skeletal muscle cells, protein turnover is essential to repair and replace damaged proteins. Little is known about the rates and mechanisms of protein exchange and turnover in different muscles. We propose to study these processes in cardiac, fast and slow skeletal muscles, and determine how they are influenced by biological sex, activity, and disease-causing sarcomeric mutations. We have developed two powerful methods to measure protein homeostasis in muscle cells. First, we will use multi-isotope imaging mass spectrometry electron microscopy (MIMS-EM) in mice to define the spatiotemporal distributions of old and new sarcomeres. MIMS-EM will be complemented by stable isotope labeling to determine turnover rates of individual proteins. Our data indicate muscle-specific differences for sarcomeric protein turnover. The fastest rates occur in cardiac muscle, followed by slow skeletal muscles and then fast skeletal muscles. In a second approach, we developed single myosin molecule live cell imaging in muscle cells to directly measure sarcomere dynamics. We will also measure the contributions of the ubiquitin (Ub) proteasome system and autophagy to exchange and turnover. We will investigate which factors (biological sex and muscle use) regulate turnover rates between different muscles. We will determine the impact of ubiquitination of myosin on sarcomere dynamics. We will focus on myosin since it is a major determinant of muscle physiology and function. Moreover, >500 mutations in myosin genes cause a variety of cardiomyopathies or skeletal myopathies. Our data show that myosin is heavily ubiquitinated in the human heart, with 68 unique sites. We will carry out Ub-proteomics on human hearts with and without myosin mutations to assess whether ubiquitinated myosin and sarcomeric proteins are altered in disease. We have mutagenized the two most heavily ubiquitinated lysines to arginines and have shown that these mutations delay turnover in cardiac myocytes. Finally, we will define the mechanisms whereby the R1500P MYH7 rod mutation causes skeletal myopathy while R1500W causes cardiomyopathy. We have shown that the R1500P mutation affects myosin conformation energetic states and used a small molecule myosin inhibitor in our R1500P mouse model that improves its phenotypes. We will determine the turnover rates of both mutant myosin proteins in cardiac myocytes and skeletal myotubes. Finally, we will quantify sarcomere dynamics in these two cell types using single myosin molecule imaging. The long-term goal of this work is to understand how muscles remodel their sarcomeres to meet the demands of changing environments.
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