November 22, 2006; doi:10.1152/japplphysiol.00768.2006.-Chronic muscle disuse induced by denervation reduces mitochondrial content and produces muscle atrophy. To investigate the molecular mechanisms responsible for these adaptations, we assessed 1) mitochondrial biogenesis-and apoptosis-related proteins and 2) apoptotic susceptibility and cell death following denervation. Rats were subjected to 5, 7, 14, 21, or 42 days of unilateral denervation of the sciatic or peroneal nerve. Muscle mass and mitochondrial content were reduced by 40 -65% after 21 and 42 days of denervation. Denervation-induced decrements in mitochondrial content occurred along with 60% and 70% reductions in transcription factor A (Tfam) and peroxisome proliferator-activated receptor-␥ coactivator (PGC)-1␣, respectively. After 42 days of denervation, Bax was elevated by 115% and Bcl-2 was decreased by 89%, producing a 16-fold increase in the Bax-toBcl-2 ratio. Mitochondrial reactive oxygen species production was markedly elevated by 5-to 7.5-fold in subsarcolemmal mitochondria after 7, 14, and 21 days of denervation, whereas reactive oxygen species production in intermyofibrillar (IMF) mitochondria was reduced by 40 -50%. Subsarcolemmal and IMF mitochondrial levels of MnSOD were also reduced by 40 -50% after 14 -21 days of denervation. The maximal rate of IMF mitochondrial pore opening (Vmax) was elevated by 25-35%, and time to V max was reduced by 20 -25% after 14 and 21 days, indicating increased apoptotic susceptibility. Myonuclear decay, assessed by DNA fragmentation, was elevated at 7-21 days of denervation. Our data indicate that PGC-1␣ and Tfam are important factors that likely contribute to the reduced mitochondrial content after chronic disuse. In addition, our results illustrate that, despite the reduced mitochondrial content, denervated muscle has greater mitochondrial apoptotic susceptibility, which coincided with elevated apoptosis, and these processes may contribute to denervation-induced muscle atrophy. mitochondrial biogenesis; muscle disuse; reactive oxygen species; mitochondrial respiration; peroxisome proliferator-activated receptor-␥ coactivator-1␣

Aging muscle exhibits a progressive decline in mass and strength, known as sarcopenia, and a decrease in the adaptive response to contractile activity. The molecular mechanisms mediating this reduced plasticity have yet to be elucidated. The purposes of this study were 1) to determine whether denervation-induced muscle disuse would increase the expression of autophagy genes and 2) to examine whether selective autophagy pathways (mitophagy) are altered in aged animals. Denervation reduced muscle mass in young and aged animals by 24 and 16%, respectively. Moreover, young animals showed a 50% decrease in mitochondrial content following denervation, an adaptation that was not matched by aged animals. Basal autophagy protein expression was higher in aged animals, whereas young animals exhibited a greater induction of autophagy proteins following denervation. Localization of LC3II, Parkin, and p62 was significantly increased in the mitochondrial fraction of young and aged animals following denervation. Moreover, the unfolded protein response marker CHOP and the mitochondrial dynamics protein Fis1 were increased by 17-and 2.5-fold, respectively, in aged animals. Lipofuscin granules within lysosomes were evident with aging and denervation. Thus reductions in the adaptive plasticity of aged muscle are associated with decreases in disuse-induced autophagy. These data indicate that the expression of autophagy proteins and their localization to mitochondria are not decreased in aged muscle; however, the induction of autophagy in response to disuse, along with downstream events such as lysosome function, is impaired. This may contribute to an accumulation of dysfunctional mitochondria in aged muscle. reactive oxygen species; muscle atrophy; mitochondria; mitophagy; apoptosis SKELETAL MUSCLE IS A REMARKABLY plastic tissue that undergoes a striking transformation in response to decreases in contractile activity. This distinctive response is attributable to the multinucleated composition of muscle fibers and the coordinated activation of several catabolic signaling pathways. Although muscle retains its adaptability throughout the life of an organism, tissue malleability is reduced with advancing age (4, 22). Aged muscle is further affected by an age-associated loss of skeletal muscle mass and strength, a condition known as sarcopenia (8,22,42). Although the precise cellular mechanisms responsible for mediating sarcopenia have yet to be fully elucidated, several studies have implicated decreases in mitochondrial function and a corresponding increase in mitochondrially mediated cell death (apoptosis) as factors contributing to this age-induced decline (4, 8). Indeed, mitochondrially mediated apoptosis can be activated by increases in reactive oxygen species (ROS), which have also been associated with several other deleterious effects, including the oxidation of mitochondrial DNA, lipids, and proteins (10, 39). Our laboratory has shown that mitochondria from aged muscle generate more ROS, possess a lower mitochondrial membrane pot...

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-Skeletal muscle undergoes remarkable adaptations in response to chronic decreases in contractile activity, such as a loss of muscle mass, decreases in both mitochondrial content and function, as well as the activation of apoptosis. Although these adaptations are well known, questions remain regarding the signaling pathways that mediated these changes. Autophagy is an organelle turnover pathway that could contribute to these adaptations. The purpose of this study was to determine whether denervation-induced muscle disuse would result in the activation of autophagy gene expression in both wild-type (WT) and Bax/Bak double knockout (DKO) animals, which display an attenuated apoptotic response. Denervation caused a reduction in muscle mass for WT and DKO animals; however, there was a 40% attenuation in muscle atrophy in DKO animals. Mitochondrial state 3 respiration was significantly reduced, and reactive oxygen species production was increased by two-to threefold in both WT and DKO animals. Apoptotic markers, including cytosolic AIF and DNA fragmentation, were elevated in WT, but not in DKO animals following denervation. Autophagy proteins including LC3II, ULK1, ATG7, p62, and Beclin1 were increased similarly following denervation for both WT and DKO. Interestingly, denervation markedly increased the localization of LC3II to subsarcolemmal mitochondria, and this was more pronounced in the DKO animals. Thus denervation-induced muscle disuse activates both apoptotic and autophagic signaling pathways in muscle, and autophagic protein expression does not exhibit a compensatory increase in the presence of attenuated apoptosis. However, the absence of Bax and Bak may represent a potential signal to trigger mitophagy in muscle.reactive oxygen species; muscle atrophy; mitochondria; mitophagy MACROAUTOPHAGY (henceforth referred to as autophagy) is a highly conserved lysosomal-dependent degradation pathway that coordinates and oversees the digestion of organelles, proteins, and intracellular pathogens (4, 34, 42). More than 30 AuTophaGy-related (ATG) regulatory genes products have been identified that are known to facilitate the engulfment of cytoplasmic material into double-membraned vesicles called autophagosomes (4, 42), and to assist in their degradation through fusion with lysosomes (10, 21). As a consequence, autophagy plays a prominent housekeeping role, which maintains homeostasis by selectively eliminating cellular debris. Moreover, although the activity of the autophagic pathway can be increased in response to stress stimuli, the importance of maintaining adequate levels of autophagy for cellular health and function is best demonstrated in several disease conditions, such as Parkinson's and Pompe's disease (27, 41), which are attributable, at least in part, to mutations in either ATG proteins or other autophagy-related genes.Currently there is great interest in determining the meaningful purposes that autophagy has in regulating cellular health, and in more fully understanding the factors that direct selective for...

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