Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for ϳ50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining ϳ50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result.

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Oxidative stress has been implicated in the etiology of age-related muscle loss (sarcopenia). However, the underlying mechanisms by which oxidative stress contributes to sarcopenia have not been thoroughly investigated. To directly examine the role of chronic oxidative stress in vivo, we used a mouse model that lacks the antioxidant enzyme CuZnSOD (Sod1). Sod1(-/-) mice are characterized by high levels of oxidative damage and an acceleration of sarcopenia. In the present study, we demonstrate that muscle atrophy in Sod1(-/-) mice is accompanied by a progressive decline in mitochondrial bioenergetic function and an elevation of mitochondrial generation of reactive oxygen species. In addition, Sod1(-/-) muscle exhibits a more rapid induction of mitochondrial-mediated apoptosis and loss of myonuclei. Furthermore, aged Sod1(-/-) mice show a striking increase in muscle mitochondrial content near the neuromuscular junctions (NMJs). Despite the increase in content, the function of mitochondria is significantly impaired, with increased denervated NMJs and fragmentation of acetylcholine receptors. As a consequence, contractile force in aged Sod1(-/-) muscles is greatly diminished. Collectively, we show that Sod1(-/-) mice display characteristics of normal aging muscle in an accelerated manner and propose that the superoxide-induced NMJ degeneration and mitochondrial dysfunction are potential mechanisms of sarcopenia.

Van Remmen H. Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production. Am J Physiol Regul Integr Comp Physiol 293: R1159-R1168, 2007. First published June 20, 2007; doi:10.1152/ajpregu.00767.2006.-Reactive oxygen species (ROS), especially mitochondrial ROS, are postulated to play a significant role in muscle atrophy. We report a dramatic increase in mitochondrial ROS generation in three conditions associated with muscle atrophy: in aging, in mice lacking CuZn-SOD (Sod1 Ϫ/Ϫ ), and in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). ROS generation in muscle mitochondria is nearly threefold higher in 28-to 32-mo-old than in 10-mo-old mice and is associated with a 30% loss in gastrocnemius mass. In Sod1 Ϫ/Ϫ mice, muscle mitochondrial ROS production is increased Ͼ100% in 20-mo compared with 5-mo-old mice along with a Ͼ50% loss in muscle mass. ALS G93A mutant mice show a 75% loss of muscle mass during disease progression and up to 12-fold higher muscle mitochondrial ROS generation. In a second ALS mutant model, H46RH48Q mice, ROS production is approximately fourfold higher than in control mice and is associated with a less dramatic loss (30%) in muscle mass. Thus ROS production is strongly correlated with the extent of muscle atrophy in these models. Because each of the models of muscle atrophy studied are associated to some degree with a loss of innervation, we were interested in determining whether denervation plays a role in ROS generation in muscle mitochondria isolated from hindlimb muscle following surgical sciatic nerve transection. Seven days postdenervation, muscle mitochondrial ROS production increased nearly 30-fold. We conclude that enhanced generation of mitochondrial ROS may be a common factor in the mechanism underlying denervation-induced atrophy. mitochondria; reactive oxygen species; amyotrophic lateral sclerosis; copper, zinc superoxide dismutase SKELETAL MUSCLE ATROPHY is a debilitating phenotype that is associated with a variety of conditions, including neurodegenerative diseases, cancer cachexia, and immobilization or disuse (49,56,76,77). Muscle atrophy is also an unavoidable consequence of normal human aging (43, 68). Despite the importance and impact of losing muscle mass, the biochemical and molecular mechanisms leading to muscle atrophy are still poorly understood. Several potential contributing factors in loss of muscle mass have been identified, including neuromuscular alterations, changes in protein synthesis and degradation, and loss of fibers due to apoptosis (15,52,58). Oxidative stress and mitochondrial dysfunction have also been implicated in sarcopenia (27, 62), hindlimb unloading (3, 46, 65), and in atrophic mouse muscle from amyotrophic lateral sclerosis (ALS) transgenic mice (54). Because mitochondria are an important source of reactive oxygen species (ROS) in cells, we were interested in delineating the role of muscle mitochondrial ROS generation in muscle atrophy. In this study, we measured mitochondrial ROS prod...

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Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpressing MnSOD in young (4-6 months) and old (26-28 months) mice on mitochondrial function, levels of oxidative damage or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression did not alter either life span or age-related pathology.

Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme important in reducing hydroperoxides in membrane lipids and lipoproteins. Gpx4 is essential for survival of embryos and neonatal mice; however, whether Gpx4 is required for adult animals remains unclear. In this study, we generated a floxed Gpx4 mouse (Gpx4(f/f)), in which exons 2–4 of Gpx4 gene are flanked by loxP sites. We then cross-bred the Gpx4(f/f) mice with a tamoxifen (tam)-inducible Cre transgenic mouse (R26CreER mice) to obtain mice in which the Gpx4 gene could be ablated by tam administration (Gpx4(f/f)/Cre mice). After treatment with tam, adult Gpx4(f/f)/Cre mice (6–9 months of age) showed a significant reduction of Gpx4 levels (a 75–85 % decrease) in tissues such as brain, liver, lung and kidney. Tam-treated Gpx4(f/f)/Cre mice lost body weight and died within 2 weeks, indicating that Gpx4 is essential for survival of adult animals. Tam-treated Gpx4(f/f)/Cre mice exhibited increased mitochondrial damage, as evidenced by the elevated 4-hydroxylnonenal (4-HNE) level, decreased activities of electron transport chain complex I and IV, and reduced ATP production in liver. Tam treatment also significantly elevated apoptosis in Gpx4(f/f)/Cre mice. Moreover, tam-treated Gpx4(f/f)/Cre mice showed neuronal loss in hippocampus region and had increased astrogliosis. These data indicate that Gpx4 is essential for mitochondria integrity and survival of neurons in adult animals.

Previously, we demonstrated that mitochondria from denervated muscle exhibited dramatically higher Amplex Red dependent fluorescence (thought to be highly specific for hydrogen peroxide) compared with control muscle mitochondria. We now demonstrate that catalase only partially inhibits the Amplex Red signal in mitochondria from denervated muscle. In contrast, ebselen (a glutathione peroxidase mimetic and inhibitor of fatty acid hydroperoxides) significantly inhibits the Amplex Red signal. This suggests that the majority of the Amplex Red signal in mitochondria from denervated muscle is not derived from hydrogen peroxide. Because Amplex Red cannot react with substrates in the lipid environment, we hypothesize that lipid hydroperoxides formed within the mitochondrial lipid bilayer are released as fatty acid hydroperoxides and react with the Amplex Red probe. We also suggest that the release of fatty acid hydroperoxides from denervated muscle mitochondria may be an important determinant of muscle atrophy. In support of this, muscle atrophy and the Amplex Red signal are inhibited in caloric restricted mice and in transgenic mice that overexpress the lipid hydroperoxide-detoxifying enzyme glutathione peroxidase 4. Finally, we propose that cytosolic phospholipase A 2 may be a potential source of these hydroperoxides.A progressive loss of muscle mass leading to a decline in both strength and function is a normal consequence of biological aging (1, 2). Although several mechanisms have been implicated in age-related muscle atrophy (2-5), the loss of motor neurons or innervation may be one of the most important factors responsible for muscle atrophy observed during aging and in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) 3 (6 -8). The sciatic nerve transection model of skeletal muscle denervation leads to rapid decline in muscle mass and has been extensively used to investigate the mechanisms of muscle atrophy following the loss of innervation (9 -11). Recent studies using this denervation model in rodents point to a role of mitochondrial oxidative stress in the mechanism of muscle atrophy (11,12). Studies from our laboratory and others point to oxidative stress and mitochondrial dysfunction as key players in the mechanisms underlying loss of muscle mass during aging and in neurodegenerative diseases, which are characterized by the loss of muscle mass (12-17). We recently reported a significant elevation in mitochondrial production of reactive oxygen species (ROS) using the Amplex Red probe in various mouse models that exhibit muscle atrophy associated with loss of innervation aging, copper-zinc superoxide dismutase knockout (Sod1 Ϫ/Ϫ ) mice, and the G93A Sod1 mutant mouse model of ALS (13). In addition, we demonstrated that ROS were significantly elevated in muscle mitochondria isolated from mice 7 days after surgical sciatic nerve transection (13). ROS production was positively correlated with the extent of muscle atrophy, indicating that mitochondrial oxidative stress may have a major role in mu...