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

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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 Max Guillot, MD, Anne-Laure Charles, PhD, Thien Nga Chamaraux-Tran, MD, Jamal Bouitbir, PhD, Alain Meyer, MD, Joffrey Zoll, PhD, Francis Schneider, MD, PhD, Bernard Geny, MD, PhD Journal of Vascular Surgery Volume 60, Issue 4, Pages 1043-1051.e5 (October 2014) DOI: 10.1016/j.jvs.2013.07.100 Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 1 Ischemia alone and ischemia followed by 2 hours of reperfusion enhance reactive oxygen species (ROS) production in skeletal muscles. a, Dihydroethidium (DHE) fluorescence intensity (expressed as percentage of sham group). The error bars indicate the standard error of the mean. *P < .05. b, Examples of DHE staining. I3, 3 hours of ischemia; I3R10′, 3 hours of ischemia, followed by 10 minutes of reperfusion; I3R2, 3 hours of ischemia, followed 2 hours of reperfusion. Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 2 Impaired mitochondrial maximal oxidative capacity and mitochondrial coupling after 3 hours of ischemia and 2 hours of reperfusion. a, Maximal fiber mitochondrial respiration rates (Vmax) in complexes I, III, and IV. b, Combined activity of mitochondrial complexes II, III, and IV after stimulation with succinate (Vsucc). c, Complex IV activity after stimulation with N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (Vtmpd). d, Acceptor control ratio (ACR). The error bars indicate the standard error of the mean. I3, 3 hours of ischemia; I3R10′, 3 hours of ischemia, followed by 10 minutes of reperfusion; I3R2, 3 hours of ischemia, followed 2 hours of reperfusion.*P < .05; **P < .01. Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 3 Lack of deleterious effects of lower limb ischemia-reperfusion (IR) on lung, heart, brain, liver, and kidney mitochondrial respiratory chain complexes I, II, III, and IV activities. Volume of mitochondrial oxygen consumption (Vo2) in (a) lung, (b) heart, (c) brain, (d) liver, and (e) kidney. I3, 3 hours of ischemia; I3R10′, 3 hours of ischemia, followed by 10 minutes of reperfusion; I3R2, 3 hours of ischemia, followed 2 hours of reperfusion; Vmax, maximal fiber mitochondrial respiration; Vsucc, activity after stimulation with succinate; Vtmpd, activity after stimulation with N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride. The error bars indicate the standard error of the mean. Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 1 (online only) Top, Schematic representation shows the mitochondrial respiratory chain with specific substrates and inhibitors. ADP, adenosine diphosphate; I, complex I (nicotinamide adenine dinucleotide reductase [NADH]-coenzyme Q [CoQ] reductase); II, complex II (succinate-CoQ reductase); III, complex III (CoQH2-c reductase); IV, complex IV (cytochrome-c oxidase, COX); V, adenosine triphosphate (ATP) synthase; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride. Bottom, Schematic oxygraph trace shows oxygen consumption by the permeabilized skeletal myofibers, using indicated substrates and inhibitors: Vo, before ADP; Vmax, complexes I, III, IV activities, using glutamate and malate; Vsucc, complexes II, III, IV activities, using succinate; VTMPD, complex IV activity using TMPD/ascorbate. Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 2 (online only) Lactate production after an ischemia of 3 hours. The error bars indicate the standard error of the mean. I3, 3 hours of ischemia; I3R10′, 3 hours of ischemia, followed by 10 minutes of reperfusion; I3R2, 3 hours of ischemia, followed 2 hours of reperfusion.****P < .0001. Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 3 (online only) Dihydroethidium (DHE) fluorescence intensity (expressed as percentage of sham). Ischemia for 3 hours, followed by 2 hours of reperfusion (I32H) does not enhance reactive oxygen species (ROS) production in (A) heart, (B) kidney and (C) liver. Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 4 (online only) Compared with sham, 3 hours of ischemia and 2 hours of reperfusion (I3R2) of skeletal muscle activates inflammation in (C) liver but not (A) in heart or (B) kidney (original magnification ×200). Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 5 (online only) Three hours of ischemia, followed by 2 hours of reperfusion (I3R2), increases circulating (a) aspartate aminotransferase (ASAT), (b) alanine aminotransferase (ALAT), and (c) serum creatinine. The error bars indicate the standard error of the mean. **P < .01; ***P < .001; ****P < .0001. Journal of Vascular Surgery 2014 60, 1043-1051.e5DOI: (10.1016/j.jvs.2013.07.100) Copyright © 2014 Society for Vascular Surgery Terms and Conditions