Mitochondrial-derived reactive oxygen species influence ADP sensitivity, but not CPT-I substrate sensitivity

Abstract

The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP (decreased ADP sensitivity) ∼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial HO emission rates, as exercise decreased the suppression of HO only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.

Blood flow restricted resistance exercise and reductions in oxygen tension attenuate mitochondrial H2O2 emission rates in human skeletal muscle

“…Mitochondrial ROS emission rates were determined in permeabilized muscle fibres by measuring the rate of H 2 O 2 release using Amplex Red fluorescence quantification (Invitrogen, Carlsbad, CA, USA) at 37°C, as described previously (Barbeau et al . ). 5 µ m blebbistatin was added to all experiments to inhibit myosin ATPase, which has previously been shown to be more indicative of the in vivo environment when modelling ADP kinetics (Perry et al .…”

“…Freeze‐dried muscle fibres were digested in a lysis buffer containing 10% glycerol, 5% β‐mercaptoethanol, 2.3% SDS in 62.5 m m Tris‐HCl, and 0.01% bromophenol blue, for 1 h at 65°C with gentle shaking (Barbeau et al . ). Samples were vortexed briefly every 15 min to improve digestion, and 5 µL of digested lysate was then loaded onto SDS‐polyacrylamide gels for western blotting protein quantification.…”

“…Vastus lateralis muscle samples in ice‐cold BIOPS were trimmed of non‐muscle tissue, separated with fine‐tipped forceps under a microscope and then incubated in saponin prior to performing mitochondrial respiration experiments using high‐resolution respirometry (Oroboros Oxygraph‐2K: Oroboros Instruments, Innsbruck, Austria), as described previously (Barbeau et al . ; Miotto & Holloway, ). Briefly, ADP was titrated (concentrations of 25, 100, 175, 250, 500, 1000, 2000, 4000, 6000, 8000, 10000 and 12000 µ m ADP) in the presence of 5 m m pyruvate and 1 m m malate, and then 10 m m glutamate and 10 m m succinate were sequentially added following ADP titrations to determine maximal complex I and complex I/II‐linked respiration.…”

“…As a closely linked control point (Holloway, ), evidence also suggests that an increase in mitochondrial reactive oxygen species (ROS) emission rates in the presence of physiological ADP concentrations occurs acutely in response to aerobic exercise (Barbeau et al . ; Miotto & Holloway, ). Although it has been speculated that ROS production is an important mechanism involved in the acute responses to BFR‐RE, direct evidence in skeletal muscle is lacking because previous studies have mainly examined plasma markers of redox balance (Takarada et al .…”

“…; Barbeau et al . ; Dohlmann et al . ; Petrick & Holloway, ), an effect that appears required for cellular adaptations to occur (Miotto & Holloway, ).…”