Oxygen availability and mitochondrial respiration: Impact on permeabilized and isolated in vitro preparations

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Skeletal muscle mitochondrial respiration assessed in vitro is typically performed in hyperoxia utilizing either permeabilized fibers or isolated mitochondria preparations. However, although it remains to be comprehensively assessed, growing evidence supports that mitochondrial respiration is likely influenced by oxygen availability and this may have a different impact in these two preparations. Therefore, we tested the hypotheses that hyperoxia would 1) increase peak mitochondrial respiration (Vmax) in permeabilized fibers, but not in isolated mitochondria, without altering submaximal respiration in either preparation, and 2) not alter the mitochondrial critical partial pressure of O2 (PO2) in either preparation.MethodsFiber permeablization and mitochondrial isolation techniques were performed on muscle samples, and mitochondrial respiration was measured by high‐resolution respirometery (O2k) in both moderate (MOD, 100–125 mmHg) and high (HIGH, 250–300 mmHg) initial PO2 conditions, for both preparations. Respiration and chamber PO2 were continuously measured until the cessation of respiration, allowing for the assessment of Vmax, submaximal respiration, and critical PO2.ResultsVmax was significantly greater in HIGH than MOD with permeabilized fibers (~57%, p<0.05, effect size (d) =2.8), but was not significantly different between conditions with isolated mitochondria (~1%, p>0.05, d=0.07). Submaximal respiration was not significantly different between HIGH and MOD conditions with permeabilized fibers (~4%, p>0.05, d=0.1), but was significantly lower in HIGH with isolated mitochondria (~33%, p<0.05, d=2.4). The critical PO2 with isolated mitochondria was lower in HIGH (0.48±0.25 mmHg) compared to MOD (0.97±0.08 mmHg, p=0.1, d=1.5). In contrast, in the permeabilized fibers, in both MOD and HIGH, there was no precipitous fall in respiration with falling oxygen availability, and respiration ceased despite 3–10 mmHg PO2 remaining in the chamber.ConclusionThese findings reveal differing effects of oxygen availability on mitochondrial respiration, dependent upon the mitochondrial preparation, which need to be considered when making such measurements.Support or Funding InformationNational Heart, Lung, and Blood Institute at the National Institute of Health (PO1 HL1091830) and Veterans Administration Rehabilitation Research and Development Service (E6910‐R, E1697‐R, E1433‐P, E9275‐L and E1572‐P)