Abstract
Duchenne Muscular Dystrophy (DMD) is a lethal X-linked disease, leading to progressive weakness and mortality in the third decade of life. Reactive oxygen species (ROS) and Ca2+ levels are elevated prior to phases of muscle degradation and regeneration, suggesting these two signalling pathways play an important role in dystrophic pathophysiology. NADPH oxidases (NOX) are a major source of ROS generation within skeletal muscle, with NOX2 and NOX4 being the predominant isoforms. Notably, NOX4-ROS have been shown to mediate changes to cellular structure and metabolism within muscle, influencing tissue function through modification of subcellular components including Ca2+ handling proteins and signalling pathways. NOX4-ROS mediated changes to Ca2+ homeostasis may impede the functional capacity of skeletal muscle, and contribute to DMD pathophysiology.
This project aims to determine the impact of systemic NOX4 inhibition on whole muscle function, and on ROS and Ca2+ levels within isolated DMD myocytes. mdx and C57BL/6 (CTRL) mice were allocated to one of 3 experimental groups; mdx treated, mdx vehicle and WT CTRL Treated mdx mice received dual NOX1/4 inhibitor GKT137831 via oral gavage for 28 days. Functional assessment was undertaken ex-vivo using the soleus muscle, with individual myocytes isolated and cultured from flexor digitorum brevis to assess resting cytoplasmic ROS, mitochondrial ROS and Ca2+ levels with DCF, MitoSox Green and FURA2 dyes respectively. Isolated myocytes were imaged live using fluorescence microscopy. Western blotting was performed to probe expression levels of both NOX4 and COX4.
Selective inhibition of NOX4 may prove beneficial in attenuating ROS-derived damage within DMD muscle. Repurposing of the drug GKT137831, which has previously been used in clinical trials for the treatment of hepatic, renal and pulmonary fibrosis, may provide a new therapeutic avenue for patients with DMD. Findings from this project will help to establish the functional and molecular effects of systemic NOX4 inhibition in DMD muscle and inform future strategies in the treatment of DMD.
Results showed significant reductions in both cytoplasmic and mitochondrial ROS within treated mdx myocytes when compared to untreated vehicle counterparts. Further, NOX4 expression was shown to decrease in GKT137831-treated muscle, and COX IV expression remained unchanged. No functional differences were observed between WT CTRL, mdx vehicle or mdx treated muscle. Pathological ROS production is a crucial pathophysiological feature of DMD, findings from this project demonstrate the efficacy of specific NOX4 inhibition using GKT137831 in ameliorating elevated cytoplasmic and mitochondrial ROS and suggest that GKT137831 proves an exciting therapeutic avenue for the treatment of DMD.