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Oxidative stress plays a critical role in cellular dysfunction associated with cigarette smoke exposure and aging. Some chemicals from tobacco smoke have the potential to amplify mitochondrial ROS (mROS) production, which, in turn, may impair mitochondrial respiratory function. Accordingly, the present study tested the hypothesis that a mitochondria-targeted antioxidant (MitoTEMPO, MT) would attenuate the inhibitory effects of cigarette smoke on skeletal muscle respiratory capacity of middle-aged mice. Specifically, mitochondrial oxidative phosphorylation was assessed using high-resolution respirometry in permeabilized fibers from the fast-twitch gastrocnemius muscle of middle-aged C57Bl/6J mice. Before the assessment of respiration, tissues were incubated for 1hr with a control buffer (CON), cigarette smoke condensate (2 % dilution, SMOKE), or MitoTEMPO (10 μM) combined with cigarette smoke condensate (MT + SMOKE). Cigarette smoke condensate (CSC) decreased maximal-ADP stimulated respiration (CON: 60 ± 15 pmolO 2.s−1.mg−1 and SMOKE: 33 ± 8 pmolO 2.s−1.mg−1; p = 0.0001), and this effect was attenuated by MT (MT + SMOKE: 41 ± 7 pmolO 2.s−1.mg−1; p = 0.02 with SMOKE). Complex-I specific respiration was inhibited by CSC, with no significant effect of MT (p = 0.35). Unlike CON, the addition of glutamate (ΔGlutamate) had an additive effect on respiration in fibers exposed to CSC (CON: 0.9 ± 1.1 pmolO 2.s−1.mg−1 and SMOKE: 5.4 ± 3.7 pmolO 2.s−1.mg−1; p = 0.008) and MT (MT + SMOKE: 8.2 ± 3.8 pmolO 2.s−1.mg−1; p ≤ 0.01). Complex-II specific respiration was inhibited by CSC but was partially restored by MT (p = 0.04 with SMOKE). Maximal uncoupled respiration induced by FCCP was inhibited by CSC, with no significant effect of MT. These findings underscore that mROS contributes to cigarette smoke condensate-induced inhibition of mitochondrial respiration in fast-twitch gastrocnemius muscle fibers of middle-aged mice thus providing a potential target for therapeutic treatment of smoke-related diseases. In addition, this study revealed that CSC largely impaired muscle respiratory capacity by decreasing metabolic flux through mitochondrial pyruvate transporter (MPC) and/or the enzymes upstream of α -ketoglutarate in the Krebs cycle. [Display omitted] • MitoTEMPO attenuated cigarette smoke condensate-induced inhibition of mitochondrial respiration in the skeletal muscle of middle-aged mice. • MitoTEMPO treatment only partially restored, maximal muscle respiratory capacity. • A rate-limiting step in the TCA cycle upstream of α-ketoglutarate was identified. • Mitochondria plays a role as both a source of oxidative stress, and a target of cigarette smoke toxicity, contributing to organ dysfunction. [ABSTRACT FROM AUTHOR]