58 - LED Therapy Plus Idebenone Treatment Targeting Signaling Pathways in Dystrophic Muscle Cells

Poster Board Number: 58
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Abstract Body : Introduction and Objective: Duchenne muscular dystrophy (DMD) is a lethal genetic disorder characterized by mutations in the gene that encodes the 427-kDa cytoskeletal protein dystrophin. Intracellular calcium dysregulation; oxidative stress; and mitochondrial dysfunction are some of the main mechanism contributors to disease progression in DMD. There is no effective treatment for DMD, therefore there is the need to search for new therapies that can improve muscle function; quality of life; and survival of dystrophic patients. This study is aimed at investigating the effects of light emitting diode therapy (LEDT) applied together with Idebenone antioxidant treatment on the signaling pathways in the dystrophic primary muscle cells of mdx mice, the experimental model of DMD.
Materials and Methods: Dystrophic primary muscle cells were submitted to LEDT (850nm wavelengths; 0.5J dose) and Idebenone treatment (0.06µM), and evaluated after 48h for cytotoxic effects; intracellular calcium content; hydrogen peroxide (H2O2) production; and mitochondrial superoxide levels. In addition, 4-HNE (a lipid peroxidation marker); PGC-1α (a master regulator of mitochondrial biogenesis); and PPARδ (main regulator of PGC-1α); calpain (ubiquitous calcium-dependent cysteine protease); and calsequestrin (calcium regulation) levels were evaluated in dystrophic primary muscle cells by western blotting. The untreated dystrophic muscle cells were used as controls. All animal protocols followed the Ethics Commission on Animal Use (CEUA-UNICAMP; 5603-1/2020)
Results: The MTT assays showed that LEDT plus Idebenone treatment presented no cytotoxic effect on dystrophic muscle cells. After 48h of LEDT plus Idebenone treatment, a significant reduction on oxidative stress levels markers, such as H2O2; mitochondrial superoxide; and 4-HNE levels, was observed. In addition, a significant decrease in calpain and calsequestrin levels was also verified in dystrophic muscle cells after treatment with LEDT plus Idebenone. On the other hand, the PGC-1α and PPARδ levels were significantly higher in the LEDT plus Idebenone treated-dystrophic muscle cells.
Conclusion: Our findings demonstrated that LEDT plus Idebenone treatment decreased intracellular calcium and oxidative stress in primary dystrophic muscle cells, targeting signaling pathways, such as calpain; calsequestrin; PGC-1α; and PPARδ.
Significance/Implication: The development of research in experimental mdx muscle cell culture is very important, as this could provide useful platforms for future therapeutic strategies for muscular dystrophies and pave the way towards potential drug screening and the testing of efficacy and toxicity. Thus, the current study provides muscle cell-specific insights into the alternative therapy effect in signaling pathways in the dystrophic primary muscle cell culture.