Mechanical Regulation of Striated Muscle Nitric Oxide Signaling in Muscular Dystrophy

Abstract

The dystrophin-glycoprotein complex (DGC) is a transmembrane structure that links the cytoskeleton of muscle cells to the extracellular matrix. Genetic disruption of this complex in muscular dystrophies causes sarcolemmal instability that results in injury and death of the muscle cells and causes altered activation of mechanosensitive signaling pathways. These features suggest dual structural and signaling roles for the dystrophin-glycoprotein complex. While much research has focused on protein-protein interactions that enable the DGC’s structural function, less is known about how the complex regulates signaling within muscle cells. Therefore, the goal of this thesis was to investigate the mechanisms whereby the dystrophin-glycoprotein complex regulates muscle nitric oxide (NO) production, a phenomenon that is crucial to normal muscle function and is disrupted in several forms of muscular dystrophy. A novel live cell imaging assay was developed to measure the mechanical activation of NO production in isolated muscle cells and investigate the biochemical signaling pathways involved in this process. This investigation identified dystrophin-dependent mechanoregulation of AMP-activated protein kinase (AMPK) as a key component of mechanosensitive NO production in striated muscle. Since defective muscle NO production contributes to diminished exercise tolerance in muscular dystrophy, subsequent studies investigated the therapeutic potential for acute pharmacologic AMPK activation to restore striated muscle NO production and improve exercise tolerance in a mouse model of dystrophin-deficient muscular dystrophy. Acute AMPK activation stimulated NO production in isolated dystrophin-deficient striated muscle cells in vitro, and increased the exercise capacity of dystrophin-deficient mice in vivo. These results suggest that acute AMPK activation may be a viable therapeutic strategy to improve exercise tolerance in muscular dystrophy patients. Finally, a novel transgenic mouse model was generated in order to test the contribution of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, to poor exercise tolerance in muscular dystrophy. These experiments suggested that ADMA contributes to exercise-induced fatigue in female dystrophin-heterozygous mice, a model for female carriers of Duchenne muscular dystrophy mutations. They also indicated that ADMA may affect exercise tolerance via effects to promote hypertrophy and impair the contractile function of the dystrophin-heterozygous heart. Considered together, the findings of this thesis support the idea that nitric oxide production is impaired in dystrophin-deficient muscle due to combined effects of disrupted intracellular signaling cascades and increased release of endogenous nitric oxide synthase inhibitors from damaged cells. Thus, this research provides evidence for the hypothesis that both the structural and signaling functions of the dystrophin-glycoprotein complex are critical for the appropriate regulation of striated muscle nitric oxide production.