The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production

Reactive oxygen species are important signaling molecules crucial for muscle differentiation and adaptation to exercise. However, their uncontrolled generation is associated with an array of pathological conditions. To identify and quantify the sources of superoxide and hydrogen peroxide in skeletal muscle we used site-specific suppressors (S1QELs, S3QELs and NADPH oxidase inhibitors). We measured the rates of hydrogen peroxide release from isolated rat muscle mitochondria incubated in media mimicking the cytosol of intact muscle. By measuring the extent of inhibition caused by the addition of different site-specific suppressors of mitochondrial superoxide/hydrogen peroxide production (S1QELs for site I_Q and S3QELs for site III_Qo), we determined the contributions of these sites to the total signal. In media mimicking resting muscle, their contributions were each 12-18%, consistent with a previous method. In C2C12 myoblasts, site I_Q contributed 12% of cellular hydrogen peroxide production and site III_Qo contributed about 30%. When C2C12 myoblasts were differentiated to myotubes, hydrogen peroxide release increased five-fold, and the proportional contribution of site I_Q doubled. The use of S1QELs and S3QELs is a powerful new way to measure the relative contributions of different mitochondrial sites to muscle hydrogen peroxide production under different conditions. Our results show that mitochondrial sites I_Q and III_Qo make a substantial contribution to superoxide/hydrogen peroxide production in muscle mitochondria and C2C12 myoblasts. The total hydrogen peroxide release rate and the relative contribution of site I_Q both increase substantially upon differentiation to myotubes.