Mitophagy In Skeletal Muscle Research Articles

Bone metastases induce metabolic changes and mitophagy in mice.

What is the central question of this study? Cachexia causes severe changes in skeletal muscle metabolism and function and is a key predictor of negative outcomes in cancer patients: what are the changes in whole animal energy metabolism and mitochondria in skeletal muscle? What is the main finding and its importance? There is decreased whole animal energy expenditure in mice with cachexia. They displayed highly dysmorphic mitochondria and mitophagy in skeletal muscle. Cachexia causes changes in skeletal muscle metabolism. Mice with MDA-MB-231 breast cancer bone metastases and cachexia have decreased whole animal energy metabolism and increased skeletal muscle mitophagy. We examined whole animal energy metabolism by indirect calorimetry in mice with MDA-MB-231 breast cancer bone metastases, and showed decreased energy expenditure. We also examined skeletal muscle mitochondria and found that mitochondria in mice with MDA-MB-231 bone metastases are highly dysmorphic and have altered protein markers of mitochondrial biogenesis and dynamics. In addition, LC3B protein was increased in mitochondria of skeletal muscle from cachectic mice, and colocalized with the mitochondrial protein Tom20. Our data demonstrate the importance of mitophagy in cachexia. Understanding these changes will help contribute to defining treatments for cancer cachexia.

Bufei Jianpi Formula Improves Mitochondrial Function and Suppresses Mitophagy in Skeletal Muscle via the Adenosine Monophosphate-Activated Protein Kinase Pathway in Chronic Obstructive Pulmonary Disease.

Skeletal muscle dysfunction, a striking systemic comorbidity of chronic obstructive pulmonary disease (COPD), is associated with declines in activities of daily living, reductions in health status and prognosis, and increases in mortality. Bufei Jianpi formula (BJF), a traditional Chinese herbal formulation, has been shown to improve skeletal muscle tension and tolerance via inhibition of cellular apoptosis in COPD rat models. This study aimed to investigate the mechanisms by which BJF regulates the adenosine monophosphate-activated protein kinase (AMPK) pathway to improve mitochondrial function and to suppress mitophagy in skeletal muscle cells. Our study showed that BJF repaired lung function and ameliorated pathological impairment in rat lung and skeletal muscle tissues. BJF also improved mitochondrial function and reduced mitophagy via the AMPK signaling pathway in rat skeletal muscle tissue. In vitro, BJF significantly improved cigarette smoke extract-induced mitochondrial functional impairment in L6 skeletal muscle cells through effects on mitochondrial membrane potential, mitochondrial permeability transition pores, adenosine triphosphate production, and mitochondrial respiration. In addition, BJF led to upregulated expression of mitochondrial biogenesis markers, including AMPK-α, PGC-1α, and TFAM and downregulation of mitophagy markers, including LC3B, ULK1, PINK1, and Parkin, with increased expression of downstream markers of the AMPK pathway, including mTOR, PPARγ, and SIRT1. In conclusion, BJF significantly improved skeletal muscle and mitochondrial function in COPD rats and L6 cells by promoting mitochondrial biogenesis and suppressing mitophagy via the AMPK pathway. This study suggests that BJF may have therapeutic potential for prophylaxis and treatment of skeletal muscle dysfunction in patients with COPD.

Rhodiola Crenulata ameliorates exhaustive exercise‑induced fatigue in mice by suppressing mitophagy in skeletal muscle

The aim of present study was to evaluate the potential effects of Rhodiola crenulata oral liquid (RCOL) on exhaustive exercise (EE)-induced fatigue in mice. Male Institute of Cancer Research mice from five treatment groups (n=10 per group) were orally administered with sterilized water for the Control and EE groups and/or RCOL at doses of 1.02, 3.03 and 6.06 ml/kg/day, once daily for 2 weeks. Anti-fatigue activity was subsequently evaluated by measuring the levels of creatine kinase (CK), lactic acid (LA), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and total anti-oxidative capability (T-AOC). Histopathology was assessed using hematoxylin and eosin staining. Ultrastructures of mitochondria were observed by transmission electron microscopy. Energy supply capacity was assessed using citrate synthase (CS), succinate dehydrogenase (SDH), Na+-K+-ATPase, and liver and quadriceps glycogen content assays. Expression levels of mRNA and protein associated with mitophagy in the skeletal muscle were measured by reverse transcription-quantitative PCR and western blotting, respectively. RCOL was observed to markedly inhibit fatigue-induced oxidative stress by increasing the activities of SOD, CAT and T-AOC, whilst reducing the accumulation of LA, CK, LDH and MDA. Histological analysis of the quadriceps femoris tissue suggested increased numbers of muscle fibers in the RCOL groups compared with those in the EE group. RCOL administration was found to reverse EE-induced mitochondrial structural damage and alleviated defects inflicted onto the energy supply mechanism by increasing CS, SDH, Na+-K+-ATPase and glycogen levels. Additionally, RCOL reduced the protein expression of PTEN-induced kinase 1 (PINK1), Parkin, microtubule-associated proteins 1A/1B light chain 3, sequestosome 1 and ubiquitin, whilst lowering the gene expression of PINK1 and Parkin. Taken together, results from the present study clarified the anti-fatigue effect of RCOL, where the underlying mechanism may be associated with increased antioxidant activity, enhanced energy production and the inhibition of mitophagy by suppressing the PINK1/Parkin signaling pathway.

Skeletal Muscle Atrophy Was Alleviated by Salidroside Through Suppressing Oxidative Stress and Inflammation During Denervation

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. Oxidative stress and inflammation are two main molecular mechanisms involved in muscle atrophy. In the current study, we want to explore whether and how salidroside, with antioxidant and anti-inflammatory properties, protects against skeletal muscle atrophy induced by denervation. First, oxidative stress and inflammatory response were examined during myotube atrophy induced by nutrition deprivation. The results demonstrated that oxidative stress and inflammatory response were induced in cultured myotubes suffered from nutrition deprivation, and salidroside not only inhibited oxidative stress and inflammatory response but also attenuated nutrition deprivation-induced myotube atrophy, as evidenced by an increased myotube diameter. The antioxidant, anti-inflammatory, and antiatrophic properties of salidroside in cultured myotubes were confirmed in denervated mouse models. The mice treated with salidroside showed less oxidative stress and less inflammatory cytokines, as well as higher skeletal muscle wet weight ratio and larger average cross sectional areas of myofibers compared with those treated with saline only during denervation-induced skeletal muscle atrophy. Moreover, salidroside treatment of denervated mice resulted in an inhibition of the activation of mitophagy in skeletal muscle. Furthermore, salidroside reduced the expression of atrophic genes, including MuRF1 and MAFbx, autophagy genes, including PINK1, BNIP3, LC3B, ATG7, and Beclin1, and transcription factor forkhead box O3 A (Foxo3A), and improved the expression of myosin heavy chain and transcriptional factor phosphorylated Foxo3A. Taken together, these results suggested that salidroside alleviated denervation-induced muscle atrophy by suppressing oxidative stress and inflammation.

Exercise-Induced Mitophagy in Skeletal Muscle and Heart.

Regular exercise enhances mitochondrial function by promoting healthy mitochondrial remodeling, but the underlying mechanisms are not thoroughly understood. An emerging hypothesis suggests that, in addition to anabolic events such as mitochondria biogenesis, the selective degradation of dysfunctional mitochondria (i.e., mitophagy) also is a key component of exercise-mediated adaptations in striated muscle, which eventually leads to better mitochondrial functions.

Parkin the progression of sarcopenia.

Parkin the progression of sarcopenia.

Intensified mitophagy in skeletal muscle with aging is downregulated by PGC-1alpha overexpression in vivo

Mitochondrial dysfunction plays an important role in the etiology of age-related muscle atrophy known as sarcopenia. PGC-1α is positioned at the center of crosstalk in regulating mitochondrial quality control, but its role in mitophagy in aged skeletal muscle is currently unclear. The present study investigated the effects of aging and PGC-1α overexpression via in vivo DNA transfection on key mitophagy protein markers, as well as mitochondrial dynamics related proteins, metabolic function and antioxidant capacity in mouse muscle. C57BL/6J mice at the age of 2 mo (young, Y; N = 14) and 24 mo (old, O; N = 14) were transfected in vivo with either PGC-1α DNA (OE, N = 7) or GFP (N = 7) into the tibialis anterior (TA) muscle followed by electroporation. PINK1 and Parkin protein contents were 3.6 and 1.4-fold higher (P < 0.01), whereas mitochondrial ubiquitination (Ub) increased 1.5-fold (P < 0.05), in O vs. Y mice. PGC-1 OE suppressed PINK and Parkin protein levels by 50–60% (P < 0.01), and decreased Ub by 20% (P < 0.05) in old mice. Aging significantly increased the protein content of LC3II (30%, P < 0.05), p62 (42%, P < 0.05), RheB (5.5-fold, P < 0.01), Beclin-1 (3-fold, P < 0.01) and Mfn2 (~4-fold, P < 0.01) in the TA muscle. However, these age-related increases in mitophagy markers were attenuated by PGC-1α OE. Furthermore, aging dramatically increased Fis-1 protein content by 14-fold (P < 0.01), along with a severe reduction of citrate synthase activity (64%, P < 0.01) and cytochrome c oxidase subunit IV (COXIV) protein content (85%, P < 0.01). PGC-1α OE mitigated the age effects on Fis-1 and Drp-1 (P < 0.05). Moreover, PGC-1α OE enhanced mitochondrial oxidative function and antioxidant enzyme activities, and decreased lipid peroxidation and inner membrane damage found in old mice (P < 0.01). In summary, our data demonstrate that mitophagy protein expression in skeletal muscle was enhanced at old age driven possibly by increased mitochondrial dysfunction, damage, and fission. PGC-1α OE was effective in ameliorating mitochondrial deficits but did not restore muscle fiber atrophy.

Contractile activity attenuates autophagy suppression and reverses mitochondrial defects in skeletal muscle cells

ABSTRACT Macroautophagy/autophagy is a survival mechanism that facilitates protein turnover in post-mitotic cells in a lysosomal-dependent process. Mitophagy is a selective form of autophagy, which arbitrates the selective recognition and targeting of aberrant mitochondria for degradation. Mitochondrial content in cells is the net balance of mitochondrial catabolism via mitophagy, and organelle biogenesis. Although the latter process has been well described, mitophagy in skeletal muscle is less understood, and it is currently unknown how these two opposing mechanisms converge during contractile activity. Here we show that chronic contractile activity (CCA) in muscle cells induced mitochondrial biogenesis and coordinately enhanced the expression of TFEB (transcription factor EB) and PPARGC1A/PGC-1α, master regulators of lysosome and mitochondrial biogenesis, respectively. CCA also enhanced the expression of PINK1 and the lysosomal protease CTSD (cathepsin D). Autophagy blockade with bafilomycin A1 (BafA) reduced mitochondrial state 3 and 4 respiration, increased ROS production and enhanced the accumulation of MAP1LC3B-II/LC3-II and SQSTM1/p62. CCA ameliorated this mitochondrial dysfunction during defective autophagy, increased PPARGC1A, normalized LC3-II levels and reversed mitochondrially-localized SQSTM1 toward control levels. NAC emulated the LC3-II reductions induced by contractile activity, signifying that a decrease in oxidative stress could represent a mechanism of autophagy normalization brought about by CCA. CCA enhances mitochondrial biogenesis and lysosomal activity, and normalizes autophagy flux during autophagy suppression, partly via ROS-dependent mechanisms. Thus, contractile activity represents a potential therapeutic intervention for diseases in which autophagy is inhibited, such as vacuolar myopathies in skeletal muscle, by establishing a healthy equilibrium of anabolic and catabolic pathways. Abbreviations: AMPK: AMP-activated protein kinase; BafA: bafilomycin A1; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CCA: chronic contractile activity; COX4I1: cytochrome c oxidase subunit 4I1; DMEM: Dulbecco’s modified Eagle’s medium; GFP: green fluorescent protein; LSD: lysosomal storage diseases; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB

Resveratrol Improves Muscle Atrophy by Modulating Mitochondrial Quality Control in STZ-Induced Diabetic Mice.

ScopeIn this study, we aim to determine the effects of resveratrol (RSV) on muscle atrophy in streptozocin‐induced diabetic mice and to explore mitochondrial quality control (MQC) as a possible mechanism.Methods and resultsThe experimental mice were fed either a control diet or an identical diet containing 0.04% RSV for 8 weeks. Examinations were subsequently carried out, including the effects of RSV on muscle atrophy and muscle function, as well as on the signaling pathways related to protein degradation and MQC processes. The results show that RSV supplementation improves muscle atrophy and muscle function, attenuates the increase in ubiquitin and muscle RING‐finger protein‐1 (MuRF‐1), and simultaneously attenuates LC3‐II and cleaved caspase‐3 in the skeletal muscle of diabetic mice. Moreover, RSV treatment of diabetic mice results in an increase in mitochondrial biogenesis and inhibition of the activation of mitophagy in skeletal muscle. RSV also protects skeletal muscle against excess mitochondrial fusion and fission in the diabetic mice.ConclusionThe results suggest that RSV ameliorates diabetes‐induced skeletal muscle atrophy by modulating MQC.

Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle.

Apobec2 is a member of the activation-induced deaminase/apolipoprotein B mRNA editing enzyme catalytic polypeptide cytidine deaminase family expressed in differentiated skeletal and cardiac muscle. We previously reported that Apobec2 deficiency in mice leads to a shift in muscle fiber type, myopathy, and diminished muscle mass. However, the mechanisms of myopathy caused by Apobec2 deficiency and its physiologic functions are unclear. Here we show that, although Apobec2 localizes to the sarcomeric Z-lines in mouse tissue and cultured myotubes, the sarcomeric structure is not affected in Apobec2-deficient muscle. In contrast, electron microscopy reveals enlarged mitochondria and mitochondria engulfed by autophagic vacuoles, suggesting that Apobec2 deficiency causes mitochondrial defects leading to increased mitophagy in skeletal muscle. Indeed, Apobec2 deficiency results in increased reactive oxygen species generation and depolarized mitochondria, leading to mitophagy as a defensive response. Furthermore, the exercise capacity of Apobec2−/− mice is impaired, implying Apobec2 deficiency results in ongoing muscle dysfunction. The presence of rimmed vacuoles in myofibers from 10-mo-old mice suggests that the chronic muscle damage impairs normal autophagy. We conclude that Apobec2 deficiency causes mitochondrial defects that increase muscle mitophagy, leading to myopathy and atrophy. Our findings demonstrate that Apobec2 is required for mitochondrial homeostasis to maintain normal skeletal muscle function.—Sato, Y., Ohtsubo, H., Nihei, N., Kaneko, T., Sato, Y., Adachi, S.-I., Kondo, S., Nakamura, M., Mizunoya, W., Iida, H., Tatsumi, R., Rada, C., Yoshizawa, F. Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle.

Influence of endurance training on skeletal muscle mitophagy regulatory proteins in type 2 diabetic men

ABSTRACTBackground: Mitophagy is a form of autophagy for the elimination of mitochondria. Mitochondrial content and function are reduced in the skeletal muscle of patients with type 2 diabetes mellitus (T2DM). Physical training has been shown to restore mitochondrial capacity in T2DM patients, but the role of mitophagy has not been examined in this context. This study analyzes the impact of a 3-month endurance training on important skeletal muscle mitophagy regulatory proteins and oxidative phosphorylation (OXPHOS) complexes in T2DM patients. Methods: Muscle biopsies were obtained from eight overweight/obese T2DM men (61±10 years) at T1 (6 weeks pre-training), T2 (1 week pre-training), and T3 (3 to 4 days post-training). Protein contents were determined by Western blotting. Results: The training increased mitochondrial complex II significantly (T2–T3: +29%, p = 0.037). The protein contents of mitophagy regulatory proteins (phosphorylated form of forkhead box O3A (pFOXO3A), mitochondrial E3 ubiquitin protein ligase-1 (MUL1), Bcl-2/adenovirus E1B 19-kD interacting protein-3 (BNIP3), microtubule-associated protein 1 light chain-3B (the ratio LC3B-II/LC3B-I was determined)) did not differ significantly between T1, T2, and T3. Conclusions: The results imply that training-induced changes in OXPHOS subunits (significant increase in complex II) are not accompanied by changes in mitophagy regulatory proteins in T2DM men. Future studies should elucidate whether acute exercise might affect mitophagic processes in T2DM patients (and whether a transient regulation of mitophagy regulatory proteins is evident) to fully clarify the role of physical activity and mitophagy for mitochondrial health in this particular patient group.

Regulation of Exercise-Induced Autophagy in Skeletal Muscle.

Physical exercise is a highly effective method to prevent several pathogenic conditions, such as obesity, type 2 diabetes and cardiovascular diseases, largely due to metabolic adaptations induced by exercise in skeletal muscle. Yet how exercise induces the beneficial effects in muscle remains to be fully elucidated. Autophagy is a lysosomal degradation pathway that regulates nutrient recycling, energy production and organelle quality control. The autophagy pathway is upregulated in response to stress during exercise and muscle contraction, and may be an important mechanism mediating exercise-induced health benefits. A number of studies have indicated that physical exercise induces non-selective autophagy and selective mitophagy in skeletal muscle in animal models and humans. The AMPK-ULK1 and the FoxO3 signaling pathways play an essential role in the activation of the upstream autophagy machinery in skeletal muscle during exercise. The autophagy activity is required for health benefits of exercise, as in different autophagy-deficient mouse lines exercise-induced effects are abolished. This review aims to summarize and highlight the most recent findings on the role of autophagy in muscle maintenance, the molecular pathways that upregulate autophagy during exercise, and the potential functions of exercise-induced autophagy and mitophagy in skeletal muscle.

Androgens alter mitophagy in the refed metabolic state

We previously showed that depletion of androgens via castration increased markers of autophagy activation (i.e. increased LC3 II/I ratio and reduced p62 protein content) in the tibialis anterior (TA) following nutrient consumption. Importantly, this androgen‐mediated effect occurred despite no differences in measures of known putative autophagy regulatory factors. Because of this, it is likely that the sustained elevation in autophagy‐activation was specific to a cellular organelle(s) rather than global autophagy. As others have shown that castration reduced skeletal muscle oxidative capacity, we sought to determine whether autophagic clearance of mitochondria, termed mitophagy, could be contributing to the sustained increase in autophagic markers. To answer this, mice were randomized to sham or castration surgery. After 8 weeks of recovery, all mice were fasted overnight. The next morning, mice remained fasted or were refed for 30 min, and sacrificed 4.5 hr following completion of the refeeding. In the TA muscle, castration reduced mitochondrial protein content as the mitochondria specific proteins cytochrome c oxidase subunit IV (COX IV) and voltage‐dependent anion channel (VDAC) were decreased. An inverse relationship was observed between the protein content of either COX IV or VDAC with p62, suggesting enhanced mitophagy. Castration also reduced the protein expression of the mitophagy regulatory protein, BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3), despite no change in BNIP3 mRNA suggesting enhanced turnover of the protein during mitophagy. Indeed, direct relationships were observed between the protein content of BNIP3 and either p62, COX IV, or VDAC supporting a role of BNIP3 in castration‐induced mitophagy. Additional analysis also supports a role of Pink1 and Parkin as castration enhanced the mRNA levels of each without a corresponding change in the protein expression, consistent with enhanced turnover of these proteins during mitophagy. Lastly, fasting conditions that enhance global autophagy prevented the castration‐induced reduction in mitochondrial proteins and enhancement of mitophagy markers. In all, these data support a model in which androgens prevent BNIP3/Pink1/Parkin‐mediated mitophagy in skeletal muscle during the refed, but not the fasted, metabolic state.Support or Funding InformationNIH Grant F32 AA‐023422 to JLS

Parkin‐Mediated Mitophagy in Skeletal Muscle with Endurance Training

Skeletal muscle is a highly adaptive tissue that is responsive to environmental cues. With exercise training, intracellular signaling pathways are activated that promote the synthesis of mitochondria, a process termed biogenesis. The increased number of mitochondria allows for efficient substrate utilization and greater muscle endurance. However, little is known regarding mitochondrial turnover with training. Mitophagy is a process involved in the specific elimination of dysfunctional mitochondria. It is unclear if mitophagy undergoes adaptive responses in muscle with training. Parkin is a ubiquitin ligase involved in neuronal mitophagy but its role in muscle remains inconclusive. To investigate Parkin's role in muscle with exercise training, we subjected 3‐month‐old wild‐type (WT) and Parkin knock out (KO) mice to a 6‐week voluntary wheel running training paradigm. Overall, WT mice ran 28% longer than KO mice in average total running distance. Endurance training elicited 1.5‐ and 1.4‐fold increases in whole muscle mitochondrial content in WT and KO mice, respectively. Furthermore, subunits I and IV of cytochrome c oxidase and mitochondrial transcription factor A were augmented on intermyofibrillar mitochondria (IMF) isolated from hindlimb muscles of trained WT animals. This was supported by a 1.7‐fold increase in State 3 (active) IMF respiration in trained WT mice. Following 6 weeks of training, both Parkin KO and WT mice exhibited 2‐fold increases in running performance during an exhaustive bout of exercise, compared to their untrained counterparts. Interestingly, basal mitophagy flux and mitochondrial localization of LC3II were augmented by 1.5‐fold in muscle of trained WT mice. However, this increase was abolished in KO animals. Acute exercise‐induced a 2‐fold increase in LC3‐II flux in untrained WT mice, which was attenuated to a 1.6‐fold increase with training. In contrast, acute exercise decreased mitophagy flux in untrained and trained KO muscle by 10–20%. Our findings indicate that the localization of autophagy proteins to mitochondria is increased in trained muscle, but is reduced when Parkin is absent. However, during exercise‐induced mitophagy, an attenuation of autophagy proteins localized on mitochondria occurs, which is partly Parkin‐mediated. With training, exercise‐induced mitophagic signaling is reduced, likely due to enhanced mitochondrial biogenesis, leading to improved endurance.Support or Funding InformationSupported by NSERC.

Skeletal muscle mitophagy in chronic disease: implications for muscle oxidative capacity?

Loss of skeletal muscle oxidative capacity is a common feature of chronic diseases such as chronic obstructive pulmonary disease, type 2 diabetes, and congestive heart failure. It may lead to physical impairments and has been suggested to contribute to metabolic inflexibility-induced cardiometabolic risk. The mechanism underlying loss of muscle oxidative capacity is incompletely understood. This review discusses the role of mitophagy as a driving force behind the loss of skeletal muscle oxidative capacity in these patients. Mitophagy has been studied to a very limited extent in human skeletal muscle. There are, however, clear indications that disease-related factors, including hypoxia, systemic inflammation, muscle inactivity, and iron deficiency are able to induce mitophagy, and that these factors trigger mitophagy via different regulatory mechanisms. Although mitophagy may lead to mitochondrial loss, it is also required to maintain homeostasis through clearance of damaged mitochondria. Based on available evidence, we propose that enhanced mitophagy is involved in chronic disease-induced loss of muscle oxidative capacity. Clearly more research is required to confirm this role and to establish to what extent mitophagy is pathological or a part of physiological adaptation to maintain muscle health.

Parkin‐Mediated Mitophagy in Skeletal Muscle with Aging and Exercise

In skeletal muscle, the progressive age‐related loss of muscle mass and strength is known as sarcopenia. Alterations in skeletal muscle mitochondria are considered to be a contributing factor in this process. When mitochondria are no longer able to sustain the energetic requirements of the cell, selective autophagy (mitophagy) pathways are induced to promote the clearance of damaged mitochondria. In aged muscle, dysfunctional mitochondria generate increased amounts of reactive oxygen species (ROS) and can impair quality control mechanisms that facilitate their removal. It remains unclear if mitophagy mediates this reduced response in aged muscle. Parkin is an ubiquitin ligase involved in mitophagy, but little is known about its function with aging. To evaluate mitophagic signaling in aged muscle, young (3 months) and aged (18 months) Parkin deficient (KO) mice and wild‐type (WT) animals were assigned to three groups: control, acute exercise, or acute exercise plus 2 hours of recovery. Muscle mass was reduced by 46% and 65% respectively in aged Parkin KO and WT mice when compared to their young counterparts. Furthermore, aged WT animals displayed a 25% decrease in whole muscle cytochrome c oxidase activity. This was accompanied by a 40% reduction in State 3 (active) intermyofibrillar mitochondrial respiration in aged WT animals. In response to exercise, both aged Parkin KO and WT mice exhibited ~30% declines in running performance, and increased lactic acidosis by 2.1‐ and 1.4‐fold, respectively. Interestingly, basal mitophagy flux and subsarcolemmal mitochondrial localization of LC3II, p62 and ubiquitin were augmented in both young and aged Parkin KO muscle. Acute exercise increased mitochondrial flux in young animals, but reduced flux in older muscle. Our findings indicate that the expression of mitophagy proteins and their localization to mitochondria are not decreased in aged muscle, even in the absence of Parkin. However, attenuation of exercise‐induced mitophagy in response to aging may contribute to mitochondrial accumulation and dysfunction in aged muscle.Support or Funding InformationSupported by NSERC.

The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase) and Other Eukaryotic BAM/GIDE Domain Proteins.

MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1) is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane) domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings. Our work paves the way for future studies into MAPL function in alternative model organisms like Capsaspora owczarzaki and Chlamydomonas reinhardtii that will help to answer the question of MAPL’s ancestral function in ways that cannot be answered by studying animal cells alone.

Ulk1 is Required for Lysosome Targeting to Damaged Mitochondria Following Acute Exercise

Mitochondrial health is critical for skeletal muscle function and is improved by exercise training. Using our newly developed mitochondrial reporter gene, pMitoTimer, with somatic gene transfer in mouse flexor digitorum brevis (FDB) muscle, we observed a transient increase of mitochondrial stress following an acute bout of treadmill running (peak at 6 hrs post exercise). This was accompanied by increased mitophagy as shown by co-localization of lysosome associated membrane protein 1 (Lamp1)-YFP with MitoTimer. We also observed increased phosphorylation of unc-51 like autophagy activating kinase 1 (Ulk1) at serine 555 (S555) immediately after exercise suggesting its activation. To determine the role of Ulk1 in exercise-induced mitophagy, we co-transfected the FDB muscle with pMitoTimer and pLamp1-YFP in muscle-specific Ulk1 knockout mice (MKO). Compared with WT mice, MKO mice had similar levels of Lamp1-positive puncta, but they did not co-localize with MitoTimer. WT and MKO mice had increased Lamp1 puncta following exercise, but in MKO mice the signals did not co-localize with MitoTimer either. These findings suggest that an acute bout of exercise induces a transient increase in mitophagy in skeletal muscle. In addition, our observations in MKO mice suggest that targeting of lysosome to the stressed/damaged mitochondria is dependent on Ulk1. This signaling pathway may play an important role in exercise training-induced mitochondrial quality control in skeletal muscle. Supported by NIH grant R01AR050429

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.

The effects of doxorubicin on the mitochondrial dynamics and mitophagy machinery in varying types of skeletal muscles (1164.1)

Doxorubicin (DOX) is an effective chemotherapeutic agent, known to cause cardiotoxicity via induction of oxidative stress and apoptosis. Despite toxic effects of DOX, few studies have investigated its effects in skeletal muscles. Multiple studies have examined DOX induced oxidative stress and mitochondrial dysfunction. However, no studies have investigated the effects of Dox on mitochondrial morphology and mitophagy. Thus, the first aim of the study was to determine the effects of DOX on the protein expression of primary regulators of mitochondrial morphology and mitophagy in skeletal muscle. The second aim was to investigate expression differences between three types of skeletal muscles varying in fiber type composition: diaphragm (DIA), extensor digitorum longus (EDL), and soleus (SOL). Male F344 rats were injected IP with 20 mg/kg of DOX or saline. Animals were fasted with no food or water until sacrifice 24 hours later. DOX treatment increased the protein content of DRP1, MFN2, and LC3‐I with no effect on FIS1, OPA1, MFN1, Parkin, PINK1, or p62. These effects were only observed in the DIA. Differences in expression levels between muscle types were present in FIS1, Parkin, LC3‐I, LC3‐II, and P62. In conclusion, DIA is more sensitive to the effects of DOX when compared to the EDL and SOL. Furthermore, the expression of regulators of mitochondria morphology and mitophagy varies among skeletal muscle types.