Oxidative stress precedes skeletal muscle mitochondrial dysfunction during experimental aortic cross-clamping but is not associated with early lung, heart, brain, liver, or kidney mitochondrial impairment

ObjectiveLower limb ischemia-reperfusion results in skeletal muscle mitochondrial alterations, production of reactive oxygen species (ROS), and remote organ impairments that are largely involved in patient prognosis. However, whether ischemia without reperfusion increases ROS production and precedes mitochondrial alteration and whether mitochondrial dysfunction occurs early in remote organs is unknown. This study determined muscle mitochondrial function and ROS production after ischemia alone, or followed by two periods of reperfusion, and investigated heart, lung, liver, kidney, and brain mitochondrial functions after lower limb ischemia-reperfusion.MethodsWistar rats were randomized into four groups: sham (aortic exposure but no ischemia, n = 9), I3 (ischemia alone induced by aortic cross-clamping for 3 hours, n = 9), I3R10′ and I3R2 (aortic cross-clamping, followed by reperfusion for 10 minutes [n = 8] or 2 hours [n = 9]). Blood lactate, alanine aminotransferase, aspartate aminotransferase, and creatinine were measured. Mitochondrial respiratory chain complexes I, II, III, and IV activities and mitochondrial coupling (acceptor control ratio) were analyzed using a Clark oxygen electrode in skeletal muscle, lung, heart, brain, liver, and kidney. ROS production was determined using dihydroethidium staining in muscle, heart, liver, and kidney. Inflammation was also investigated in remote organs (heart, liver, and kidney) using monocyte-macrophage-2 antibody staining.ResultsLactate level increased after ischemia in all groups. In muscle, ROS increased significantly after ischemia alone (+324% ± 66%; P = .038), normalized after 10 minutes of reperfusion, and increased again at 2 hours of reperfusion (+349.2 ± 67%; P = .024). Interestingly, mitochondrial function was unaffected by ischemia alone or followed by 10 minutes of reperfusion, but maximal mitochondrial oxidative capacity (6.10 ± 0.51 vs 4.24 ± 0.36 μmol/min/g, −30%; P < .05) and mitochondrial coupling decreased after 2 hours of reperfusion (1.93 ± 0.17 vs 1.33 ± 0.07, −45%; P < .01), in sham and I3R2 rats, respectively. Despite increased serum aspartate aminotransferase (×13; P < .0001), alanine aminotransferase (×6; P = .0019), and creatinine (×3; P = .0004), remote organs did not show mitochondrial alteration, inflammation, or ROS production enhancement after 2 hours of reperfusion.ConclusionsOxidative stress precedes skeletal muscle mitochondrial dysfunction during lower limb ischemia. Such a kinetic explains the efficacy of ischemic preconditioning and supports that therapy should be conducted even during ongoing ischemia, suggesting that ischemic preconditioning might be a successful approach.

Clinical RelevanceAortic cross-clamping increases reactive oxygen species (ROS) and impairs skeletal muscle and remote organs, which is involved in patient prognosis. However, the temporal relationship between ROS production and mitochondrial dysfunction during lower limb ischemia reperfusion is unknown. This study demonstrates for the first time that ROS production occurs during ischemia alone, without reperfusion, and precedes skeletal muscle mitochondrial impairments. Although involved in multiorgan failure, lung, heart, brain, liver, and kidney mitochondria are not affected early. These results support a need for muscle protection even during lower limb ischemia and that ischemic preconditioning (conditioning performed during ongoing ischemia) might be a successful approach.