Chamber oxygen concentration impacts mitochondrial function and hydrogen peroxide appearance in permeabilized human...

Skeletal muscle mitochondrial respiration is commonly assessed ex vivo using permeabilized fibers in media with high oxygen (O₂) concentrations to ensure that O₂ availability does not limit respiration. However, high O₂ concentrations also increase the production of reactive O₂ species that can negatively affect respiration. In this study, we tested the hypotheses that permeabilized fiber mitochondria in a high, compared to low, O₂ concentration would (i) not be different at maximal state 3 respiration rate (V_max), (ii) have lower submaximal respiration rates at submaximal O₂ concentrations, and (iii) have greater total cumulative hydrogen peroxide (H₂O₂) appearance. We continuously monitored mitochondrial state 3 respiration and H₂O₂ appearance rates using high-resolution respirometry in permeabilized skeletal muscle fibers (12 untrained participants; 22 ± 4 yrs) with either control (~127 mmHg; CON) or high (~327 mmHg; HIGH) partial pressures of O₂ (PO₂). V_max was not different between conditions (HIGH: 80.7 ± 16.7 vs. CON: 82.3 ± 18.7 pmol/s/mg, p = 0.695). The PO₂ at 80 % V_max (P₈₀) was greater in HIGH (73.9 ± 25.5 vs. 28.0 ± 7.1 mmHg, p < 0.001) and respiration rates at 5-60 mmHg PO₂ were lower for HIGH than CON (all p < 0.001). Additionally, the total cumulative H₂O₂ appearance was greater in HIGH than CON (n = 11; 51.5 ± 23.2 vs. 18.3 ± 10.3 pmol/mg, p < 0.001), and this difference was directly correlated with the difference in P₈₀ (r = 0.655, p = 0.029). The current findings support that a high O₂ concentration, by itself, does not appear to affect V_max in the permeabilized skeletal muscle fiber preparation, but the corollary increase in H₂O₂ exposure may diminish mitochondrial state 3 respiratory function.