Exhaustive exercise alters native and site-specific H₂O₂ emission in red and white skeletal muscle mitochondria

Mitochondrial reactive oxygen species (ROS) homeostasis is intricately linked to energy conversion reactions and entails regulation of the mechanisms of ROS production and removal. However, there is limited understanding of how energy demand modulates ROS balance. Skeletal muscle experiences a wide range of energy requirements depending on the intensity and duration of exercise and therefore is an excellent model to probe the effect of altered energy demand on mitochondrial ROS production. Because in most fish skeletal muscle exists essentially as pure spatially distinct slow-twitch red oxidative and fast-twitch white glycolytic fibers, it provides a natural system for investigating how functional specialization affects ROS homeostasis. We tested the hypothesis that acute increase in energy demand imposed by exhaustive exercise will increase mitochondrial H₂O₂ emission to a greater extent in red muscle mitochondria (RMM) compared with white muscle mitochondria (WMM). We found that native H₂O₂ emission rates varied by up to 6-fold depending on the substrate being oxidized and muscle fiber type, with RMM emitting at higher rates with glutamate-malate and palmitoylcarnitine while WMM emitted at higher rates with succinate and glyceral-3-phosphate. Exhaustive exercise increased the native and site-specific H₂O₂ emission rates; however, the maximal emission rates depended on the substrate, fiber type and redox site. The H₂O₂ consumption capacity and activities of individual antioxidant enzymes including the glutathione- and thioredoxin-dependent peroxidases as well as catalase were higher in RMM compared with WMM indicating that the activity of antioxidant defense system does not explain the differences in H₂O₂ emission rates in RMM and WMM. Overall, our study suggests that substrate selection and oxidation may be the key factors determining the rates of ROS production in RMM and WMM following exhaustive exercise.