Influence Of Ex Vivo Muscle Stimulation On Oxidative Biomarker Production

Muscle contraction induces oxidant production that matches the magnitude, duration, and intensity of the contractile stimulus, however the effects on redox sensitive proteins are still not well understood. In the present study, we aimed to measure redox induced oligomerization in response to skeletal muscle stimulation from the same animals in an ex vivo model. We hypothesized that muscle stimulation would induce redox-dependent dimerization in antioxidant proteins. Extensor digitorum longus (EDL) muscles were removed from mice and one muscle was stimulated ex vivo. The stimulated muscle received thirty isometric tetanic stimulations (500ms train, 0.8ms pulse, 140Hz) delivered with 500ms breaks between each tetanus. This stimulation loop was delivered ten times for a total of 300 tetanic stimulations. On average, the force decreased by 50% percent following the protocol. The control EDL muscle was placed in an oxygenated Krebs bath for an equal amount of time without any stimulation. The muscles were flash frozen in liquid nitrogen, stored at -80C and later homogenized in reducing and non-reducing buffer. Levels of glutathione reductase (GSR), glutathione peroxidase 1 (GPx1), and Kelch-like ECH-associated protein 1 (Keap1) were measured via western blotting and redox western blotting. There were no significant changes in protein content between stimulation, however in redox blotting samples, there were shifts in banding pattern of GSR in response to stimulation, suggesting GSR forms dimer and larger oligomeric structures in response to muscle stimulation. As far as we are aware these are the first data to describe this phenomenon. It is unclear how this affects enzyme function, turnover, or localization.