Effect of Catalase/Superoxide Dismutase Mimetic EUK-134 on Damage, Inflammation, and Force Generation of the Diaphragm Muscle in mdx Mice

Abstract

Duchenne muscular dystrophy (DMD) is the most devastating form of muscular dystrophy caused by a mutation in the dystrophin gene. Defects in the dystrophin gene in DMD, are homologous to that found in mdx mice, and result in profound muscle damage, inflammation and weakness in diaphragm and limb muscles. Dystrophin, a scaffolding protein located in the sarcolemmal cytoskeleton, helps cells to maintain their structural integrity and associates with critical cell signaling molecules that regulate cell growth and repair (e.g., nNOS). While the contributing mechanisms leading to DMD-induced degenerative muscle function and damage are multi-factorial, elevated oxidative stress has been proposed as a central mechanism. In contrast, antioxidants can attenuate muscle damage as well as improve contractile function in dystrophin-deficient muscles. However, it is unknown if oxidative stress is a causal factor in dystrophin-deficient diaphragm muscle pathology and specifically targeted antioxidant (e.g., EUK-134) treated early in the course of the disease (3-4 weeks) can modulate oxidative stress, functional damage and weakness in mdx diaphragm. Therefore, the purpose of this study was to determine the effects of catalase/superoxide dismutase mimetic EUK-134 on damage, inflammation, and contractile function of the diaphragm muscle in mdx mice. We hypothesized that (a) EUK-134 would attenuate muscle damage and oxidative stress in mdx diaphragm, (b) EUK-134 would reduce inflammatory cells and an important transcription factor including nuclear factor-kappaB (NF-kB) in mdx diaphragm and (c) EUK-134 would restore proteins that attach to dystrophin such as nNOS and cytoskeletal proteins back to sarcolemmal region and improve muscle contractility in mdx diaphragm. C57BL/10ScSn wild type and mdx mice were given EUK-134 (30mg/kg, i.p., injection) beginning at 20 days of age for 8 days. The mice were euthanized and the diaphragm muscle was harvested at 4 weeks of age, the time of peak inflammation, and analyzed to measure myofiber inflammation, NF-kB activation, cytoskeletal proteins and oxidative stress markers using Western immunoblotting, ELISA, immunofluoresence, and immunohistochemistry. We found that EUK-134 ameliorated muscle damage and oxidative stress in mdx diaphragm. EUK-134 protected against inflammation by decreasing NF-kB activation in the nucleosome fraction of mdx diaphragm. Further, EUK-134 partially rescued nNOS and k-1 syntrophin back to sarcolemmal membranes and recovered force generation even in acute application in vitro in mdx diaphragm. These results are the first to demonstrate a causal relationship between oxidative stress and pathology caused by dystrophin-deficient diaphragm muscle. Moreover, the data indicate that EUK-134 has a protective effect against muscle damage, inflammation, and contractility in mdx diaphragm. We believe that the results from our investigation will provide clinical significance, as we expect to elucidate mechanisms by which oxidative stress contribute to tissue damage and weakness in dystrophic diaphragm.