Costamere Defects in Muscular Dystrophies

PROJECT SUMMARY/ABSTRACT While abundant skeletal (ask-actin) and cardiac (aca-actin) actin isoforms are famous for their essential role in striated muscle contraction, low abundance non-muscle “cytoplasmic” actin isoforms (bcyto- and gcyto-actin) are also emerging as important in the maintenance of specialized structures (and functions) in normal and diseased skeletal muscle. During this project, we generated and characterized muscle-specific mouse lines either lacking or overexpressing bcyto-actin or gcyto-actin to understand their endogenous functions and role(s) in dystrophin-deficient muscular dystrophy. Interestingly, each bcyto-actin or gcyto-actin single knockout develops a qualitatively similar phenotype characterized by a progressive myopathy with significant myofiber degeneration/regeneration and muscle weakness. We have shown that skeletal muscle-specific overexpression of bcyto-actin or gcyto-actin in dystrophin-deficient mdx mice affords significant protection from eccentric contraction-induced force drop while overexpression of a C272A mutant of gcyto-actin affords no protection. These and other data suggest that eccentric contraction drives a rapidly-reversible, reactive oxygen species (ROS)-mediated inhibition of sarcomeric contractility that may function to protect dystrophic muscles from damage caused by repeated, high force contractions. Our new preliminary data show that muscle-specific ablation of bcyto-actin or gcyto-actin from wildtype muscle results in eccentric contraction-induced force drop that is reversed by the nonspecific antioxidant N-acetylcysteine. Finally, we have obtained new data suggesting that gcyto-actin is important for repair of membrane damage. Going forward, we will make use of our unique animal models, isoform-specific reagents, and biochemical and physiological methodologies to address new fundamental questions about cytoplasmic actins in normal skeletal muscle function and in dystrophin-deficient muscular dystrophy. In aim 1, we will identify the sources of ROS contributing to eccentric contraction-induced force drop in dystrophic mdx skeletal muscle as well as the downstream targets of ROS that ultimately inhibit force production. In aim 2, we will investigate the role of oxidative stress in driving the myopathy and eccentric contraction-induced force drop associated with genetic ablation of bcyto- or gcyto-actin in skeletal muscle. In aim 3, the interplay between cytoplasmic actin isoforms and ROS in membrane repair will be investigated using state-of-the-art imaging approaches to analyze muscles from the same mouse lines used in aims 1 and 2. The results of the proposed studies will further delineate the unique and important contributions of cytoplasmic actin isoforms to the function of normal and dystrophic skeletal muscle.