Anna Vainshtein

Role of PGC-1α during acute exercise-induced autophagy and mitophagy in skeletal muscle.

Regular exercise leads to systemic metabolic benefits, which require remodeling of energy resources in skeletal muscle. During acute exercise, the increase in energy demands initiate mitochondrial biogenesis, orchestrated by the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Much less is known about the degradation of mitochondria following exercise, although new evidence implicates a cellular recycling mechanism, autophagy/mitophagy, in exercise-induced adaptations. How mitophagy is activated and what role PGC-1α plays in this process during exercise have yet to be evaluated. Thus we investigated autophagy/mitophagy in muscle immediately following an acute bout of exercise or 90 min following exercise in wild-type (WT) and PGC-1α knockout (KO) animals. Deletion of PGC-1α resulted in a 40% decrease in mitochondrial content, as well as a 25% decline in running performance, which was accompanied by severe acidosis in KO animals, indicating metabolic distress. Exercise induced significant increases in gene transcripts of various mitochondrial (e.g., cytochrome oxidase subunit IV and mitochondrial transcription factor A) and autophagy-related (e.g., p62 and light chain 3) genes in WT, but not KO, animals. Exercise also resulted in enhanced targeting of mitochondria for mitophagy, as well as increased autophagy and mitophagy flux, in WT animals. This effect was attenuated in the absence of PGC-1α. We also identified Niemann-Pick C1, a transmembrane protein involved in lysosomal lipid trafficking, as a target of PGC-1α that is induced with exercise. These results suggest that mitochondrial turnover is increased following exercise and that this effect is at least in part coordinated by PGC-1α.

Adaptive plasticity of autophagic proteins to denervation in aging skeletal muscle

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.

Denervation-induced mitochondrial dysfunction and autophagy in skeletal muscle of apoptosis-deficient animals.

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.

PGC-1α modulates denervation-induced mitophagy in skeletal muscle.

BackgroundAlterations in skeletal muscle contractile activity necessitate an efficient remodeling mechanism. In particular, mitochondrial turnover is essential for tissue homeostasis during muscle adaptations to chronic use and disuse. While mitochondrial biogenesis appears to be largely governed by the transcriptional co-activator peroxisome proliferator co-activator 1 alpha (PGC-1α), selective mitochondrial autophagy (mitophagy) is thought to mediate organelle degradation. However, whether PGC-1α plays a direct role in autophagy is currently unclear.MethodsTo investigate the role of the co-activator in autophagy and mitophagy during skeletal muscle remodeling, PGC-1α knockout (KO) and overexpressing (Tg) animals were unilaterally denervated, a common model of chronic muscle disuse.ResultsAnimals lacking PGC-1α exhibited diminished mitochondrial density alongside myopathic characteristics reminiscent of autophagy-deficient muscle. Denervation promoted an induction in autophagy and lysosomal protein expression in wild-type (WT) animals, which was partially attenuated in KO animals, resulting in reduced autophagy and mitophagy flux. PGC-1α overexpression led to an increase in lysosomal capacity as well as indicators of autophagy flux but exhibited reduced localization of LC3II and p62 to mitochondria, compared to WT animals. A correlation was observed between the levels of the autophagy-lysosome master regulator transcription factor EB (TFEB) and PGC-1α in muscle, supporting their coordinated regulation.ConclusionsOur investigation has uncovered a regulatory role for PGC-1α in mitochondrial turnover, not only through biogenesis but also via degradation using the autophagy-lysosome machinery. This implies a PGC-1α-mediated cross-talk between these two opposing processes, working to ensure mitochondrial homeostasis during muscle adaptation to chronic disuse.

Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity

Physical activity has been recently documented to play a fundamental physiological role in the regulation of autophagy in several tissues. It has also been reported that autophagy is required for exercise itself and for training-induced adaptations in glucose homeostasis. These autophagy-mediated metabolic improvements are thought to be largely dependent on the activation of the metabolic sensor PRKAA1/AMPK. However, it is unknown whether these important benefits stem from systemic adaptations or are due solely to alterations in skeletal muscle metabolism. To address this we utilized inducible, muscle-specific, atg7 knockout mice that we have recently generated. Our findings indicate that acute inhibition of autophagy in skeletal muscle just prior to exercise does not have an impact on physical performance, PRKAA1 activation, or glucose homeostasis. However, we reveal that autophagy is critical for the preservation of mitochondrial function during damaging muscle contraction. This effect appears to be gender specific affecting primarily females. We also establish that basal oxidative stress plays a crucial role in mitochondrial maintenance during normal physical activity. Therefore, autophagy is an adaptive response to exercise that ensures effective mitochondrial quality control during damaging physical activity.

Skeletal muscle, autophagy, and physical activity: the ménage à trois of metabolic regulation in health and disease

Metabolic homeostasis is essential for cellular survival and proper tissue function. Multi-systemic metabolic regulation is therefore vital for good health. A number of tissues have the task of maintaining appropriate metabolism, and skeletal muscle is the most abundant of them. Muscle possesses a remarkable plasticity and is able to rapidly adapt to changes in energetic demands by fine-tuning the balance between catabolic and anabolic processes. Autophagy is a catabolic process responsible for the degradation of protein aggregates and damaged organelles, through the autophagosome–lysosome system. Proper regulation of autophagy flux is fundamental for organism homeostasis under physiological conditions and even more in response to metabolic stress, such as during physical activity and nutritional deficits. Both deficient and excessive autophagy are harmful for health and have devastating consequences in a myriad of pathologies. The regulation of autophagy flux in various tissues, and in particular in skeletal muscle, is of great importance for health and tissue homeostasis and represents a feasible mechanism by which physical exercise exerts its beneficial effects on muscle and whole body metabolism. This review is focused on the key molecular mechanisms regulating macromolecule and organelle turnover in muscle during alterations in nutrient availability and energetic demands, as well as their involvement in disease pathogenesis.

Mechanisms of Exercise-Induced Mitochondrial Biogenesis in Skeletal Muscle: Implications for Health and Disease

Mitochondria have paradoxical functions within cells. Essential providers of energy for cellular survival, they are also harbingers of cell death (apoptosis). Mitochondria exhibit remarkable dynamics, undergoing fission, fusion, and reticular expansion. Both nuclear and mitochondrial DNA (mtDNA) encode vital sets of proteins which, when incorporated into the inner mitochondrial membrane, provide electron transport capacity for ATP production, and when mutated lead to a broad spectrum of diseases. Acute exercise can activate a set of signaling cascades in skeletal muscle, leading to the activation of the gene expression pathway, from transcription, to post-translational modifications. Research has begun to unravel the important signals and their protein targets that trigger the onset of mitochondrial adaptations to exercise. Exercise training leads to an accumulation of nuclear- and mtDNA-encoded proteins that assemble into functional complexes devoted to mitochondrial respiration, reactive oxygen species (ROS) production, the import of proteins and metabolites, or apoptosis. This process of biogenesis has important consequences for metabolic health, the oxidative capacity of muscle, and whole body fitness. In contrast, the chronic muscle disuse that accompanies aging or muscle wasting diseases provokes a decline in mitochondrial content and function, which elicits excessive ROS formation and apoptotic signaling. Research continues to seek the molecular underpinnings of how regular exercise can be used to attenuate these decrements in organelle function, maintain skeletal muscle health, and improve quality of life.

Effects of endurance training on apoptotic susceptibility in striated muscle

An increase in the production of reactive oxygen species occurs with muscle disuse, ischemic cardiomyopathy, and conditions that arise with senescence. The resulting oxidative stress is associated with apoptosis-related myopathies. Recent research has suggested that chronic exercise is protective against mitochondrially mediated programmed cell death. To further investigate this, we compared soleus (Sol) and cardiac muscles of voluntary wheel-trained (T; 10 wk) and untrained (C) animals. Training produced a 52% increase in muscle cytochrome c oxidase (COX) activity. Sol and left ventricle (LV) strips were isolated and incubated in vitro with H2O2 for 4 h. Strips were then fractionated into cytosolic and mitochondrial fractions. Whole muscle apoptosis-inducing factor (AIF) and Bax/Bcl-2 levels were reduced in both the Sol and LV from T animals. H2O2 treatment induced increases in JNK phosphorylation, cofilin-2 localization to the mitochondria, as well as cytosolic AIF in both Sol and LV of T and C animals, respectively. Mitochondrial Bax and cytosolic cytochrome c were augmented under oxidative stress in the LV only. The H2O2-induced increases in P-JNK, mitochondrial Bax, and cytosolic AIF were ablated in the LV of T animals. These data suggest that short-term oxidative stress can induce apoptotic signaling in striated muscles in vitro. In addition, training can attenuate oxidative stress-induced apoptotic signaling in a tissue-specific manner, with an effect that is most prominent in cardiac muscle.

The regulation of autophagy during exercise in skeletal muscle

The merits of exercise on muscle health and well-being are numerous and well documented. However, the mechanisms underlying the robust adaptations induced by exercise, particularly on mitochondria, are less clear and much sought after. Recently, an evolutionary conserved cellular recycling mechanism known as autophagy has been implicated in the adaptations to acute and chronic exercise. A basal level of autophagy is constantly ongoing in cells and tissues, ensuring cellular clearance and energy homeostasis. This pathway can be further induced, as a survival mechanism, by cellular perturbations, such as energetic imbalance and oxidative stress. During exercise, a biphasic autophagy response is mobilized, leading to both an acute induction and a long-term potentiation of the process. Posttranslational modifications arising from upstream signaling cascades induce an acute autophagic response during a single bout of exercise by mobilizing core autophagy machinery. A transcriptional program involving the regulators Forkhead box O, transcription factor EB, p53, and peroxisome proliferator coactivator-1α is also induced to fuel sustained increases in autophagic capacity. Autophagy has also been documented to mediate chronic exercise-induced metabolic benefits, and animal models in which autophagy is perturbed do not adapt to exercise to the same extent. In this review, we discuss recent developments in the field of autophagy and exercise. We specifically highlight the molecular mechanisms activated during acute exercise that lead to a prolonged adaptive response.

The effects of chronic muscle use and disuse on cardiolipin metabolism.

Cardiolipin (CL) is a phospholipid that maintains the integrity of mitochondrial membranes. We previously demonstrated that CL content increases with chronic muscle use, and decreases with denervation-induced disuse. To investigate the underlying mechanisms, we measured the mRNA expression of 1) CL synthesis enzymes cardiolipin synthase (CLS) and CTP:PA-cytidylyltransferase-1 (CDS-1); 2) remodeling enzymes tafazzin and acyl-CoA:lysocardiolipin acyltransferase-1 (ALCAT1); and 3) outer membrane CL enzymes, mitochondrial phospholipase D and phospholipid scramblase 3 (Plscr3), during chronic contractile activity (CCA)-induced mitochondrial biogenesis and denervation. With CCA, CDS-1 expression increased by 128%, parelleling CL levels. Surprisingly, denervation also led to large increases in CDS-1 and CLS, despite a decrease in mitochondria, possibly due to a compensatory mechanism to restore lost CL. ALCAT1 decreased by 32% with CCA, but increased by 290% following denervation, indicating that both CCA and denervation alter CL remodeling. CCA and denervation also elicited 60-90% increases in Plscr3, likely to facilitate CL movement to the outer membrane. The expression of these genes was not affected by aging, but changes due to CCA and denervation were attenuated compared with young animals. The absence of PPARγ coactivator-1α in knockout animals led to a decrease in CDS-1 and an increase in ALCAT1 mRNA levels, implicating PGC-1α in regulating both CL synthesis and remodeling. These data suggest that chronic muscle use and disuse modify the expression of mRNAs encoding CL metabolism enzymes. Our data also illustrate, for the first time, that PPARγ coactivator-1α regulates the CL metabolism pathway in muscle.