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AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

      Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.
      Duchenne muscular dystrophy (DMD), the most common X-linked lethal disorder in humans, is caused by defects in the dystrophin gene.
      • Koenig M.
      • Hoffman E.P.
      • Bertelson C.J.
      • Monaco A.P.
      • Feener C.
      • Kunkel L.M.
      Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals.
      Absence of dystrophin is associated with muscle fiber death involving both apoptosis and necrosis.
      • Tidball J.G.
      • Albrecht D.E.
      • Lokensgard B.E.
      • Spencer M.J.
      Apoptosis precedes necrosis of dystrophin-deficient muscle.
      Because DMD affects the diaphragm and other respiratory muscles, many patients die of respiratory failure. Although the ideal treatment for DMD would be restoration of dystrophin expression to all muscles of the body, this is not currently feasible, and there is an urgent need for new therapies. The mdx mouse also lacks dystrophin, and is a commonly used animal model for studying the disease and its potential responsiveness to new treatments.
      • Stedman H.H.
      • Sweeney H.L.
      • Shrager J.B.
      • Maguire H.C.
      • Panettieri R.A.
      • Petrof B.
      • Narusawa M.
      • Leferovich J.M.
      • Sladky J.T.
      • Kelly A.M.
      The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.
      Muscles lacking dystrophin exhibit multiple cellular defects, including abnormal fragility of the sarcolemma, increased oxidative stress, and elevated cytosolic calcium levels.
      • Petrof B.J.
      Molecular pathophysiology of myofiber injury in deficiencies of the dystrophin-glycoprotein complex.
      DMD muscles also show mitochondrial dysfunction and diminished expression of energy-producing metabolic genes.
      • Kuznetsov A.V.
      • Winkler K.
      • Wiedemann F.R.
      • von B.P.
      • Dietzmann K.
      • Kunz W.S.
      Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse.
      • Chen Y.W.
      • Zhao P.
      • Borup R.
      • Hoffman E.P.
      Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology.
      Conversely, forced expression of the mitochondrial biogenesis factor peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α; official symbol, PPARGC-1-α) ameliorates dystrophic pathology in mdx mice.
      • Handschin C.
      • Kobayashi Y.M.
      • Chin S.
      • Seale P.
      • Campbell K.P.
      • Spiegelman B.M.
      PGC-1alpha regulates the neuromuscular junction program and ameliorates Duchenne muscular dystrophy.
      AMP-activated protein kinase (AMPK), a major sensor of cellular energy status, switches on mechanisms favoring ATP generation under conditions of energetic deficiency.
      • Hardie D.G.
      AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy.
      Thus, AMPK can stimulate mitochondrial biogenesis through the PGC-1-α pathway, as well as through catabolic pathways that fuel energy production. Chief among these catabolic mechanisms is the lysosomally mediated process of macroautophagy (henceforth referred to simply as autophagy), which targets cellular constituents that are too large to be removed by other degradative pathways.
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      • Mizushima N.
      • Levine B.
      • Cuervo A.M.
      • Klionsky D.J.
      Autophagy fights disease through cellular self-digestion.
      In addition to its energy-producing function, autophagy plays a critical role in cellular quality control by preferentially eliminating proteins and organelles that are nonessential or dysfunctional, including defective mitochondria.
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      • Mizushima N.
      • Levine B.
      • Cuervo A.M.
      • Klionsky D.J.
      Autophagy fights disease through cellular self-digestion.
      Recent work has linked defects in autophagic removal of mitochondria (mitophagy) to Parkinson's disease and other neurodegenerative diseases, as well as to muscular dystrophy associated with collagen VI deficiency.
      • Youle R.J.
      • Narendra D.P.
      Mechanisms of mitophagy.
      • Grumati P.
      • Coletto L.
      • Sabatelli P.
      • Cescon M.
      • Angelin A.
      • Bertaggia E.
      • Blaauw B.
      • Urciuolo A.
      • Tiepolo T.
      • Merlini L.
      • Maraldi N.M.
      • Bernardi P.
      • Sandri M.
      • Bonaldo P.
      Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration.
      Impaired mitophagy permits the accumulation of damaged mitochondria, which not only perform poorly in their energy-generating role but also have a greater propensity to undergo opening of the mitochondrial membrane permeability transition pore (PTP) complex.
      • Grumati P.
      • Coletto L.
      • Sabatelli P.
      • Cescon M.
      • Angelin A.
      • Bertaggia E.
      • Blaauw B.
      • Urciuolo A.
      • Tiepolo T.
      • Merlini L.
      • Maraldi N.M.
      • Bernardi P.
      • Sandri M.
      • Bonaldo P.
      Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration.
      • Palma E.
      • Tiepolo T.
      • Angelin A.
      • Sabatelli P.
      • Maraldi N.M.
      • Basso E.
      • Forte M.A.
      • Bernardi P.
      • Bonaldo P.
      Genetic ablation of cyclophilin D rescues mitochondrial defects and prevents muscle apoptosis in collagen VI myopathic mice.
      This leads to mitochondrial swelling, collapse of the mitochondrial membrane potential, and release of proapoptotic factors, which have been implicated not only in cell death but also in muscle fiber atrophy and injury.
      • Marzetti E.
      • Hwang J.C.
      • Lees H.A.
      • Wohlgemuth S.E.
      • Dupont-Versteegden E.E.
      • Carter C.S.
      • Bernabei R.
      • Leeuwenburgh C.
      Mitochondrial death effectors: relevance to sarcopenia and disuse muscle atrophy.
      • Powers S.K.
      • Kavazis A.N.
      • McClung J.M.
      Oxidative stress and disuse muscle atrophy.
      In the present study, we postulated that therapeutic activation of AMPK might stimulate autophagic removal of defective mitochondria in mdx mice, thereby leading to beneficial effects on mitochondrial PTP opening, as well as on the overall muscular dystrophy phenotype. To test this hypothesis, we treated mdx mice with AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), an established pharmacological activator of AMPK
      • Narkar V.A.
      • Downes M.
      • Yu R.T.
      • Embler E.
      • Wang Y.X.
      • Banayo E.
      • Mihaylova M.M.
      • Nelson M.C.
      • Zou Y.
      • Juguilon H.
      • Kang H.
      • Shaw R.J.
      • Evans R.M.
      AMPK and PPARdelta agonists are exercise mimetics.
      that has been shown to promote mitophagy.
      • Egan D.F.
      • Shackelford D.B.
      • Mihaylova M.M.
      • Gelino S.
      • Kohnz R.A.
      • Mair W.
      • Vasquez D.S.
      • Joshi A.
      • Gwinn D.M.
      • Taylor R.
      • Asara J.M.
      • Fitzpatrick J.
      • Dillin A.
      • Viollet B.
      • Kundu M.
      • Hansen M.
      • Shaw R.J.
      Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.
      We focused the present study on the mdx mouse diaphragm, because this model closely mimics human DMD with respect to both fiber loss and weakness.
      • Stedman H.H.
      • Sweeney H.L.
      • Shrager J.B.
      • Maguire H.C.
      • Panettieri R.A.
      • Petrof B.
      • Narusawa M.
      • Leferovich J.M.
      • Sladky J.T.
      • Kelly A.M.
      The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.
      Here, we show that chronic AICAR treatment effectively stimulates autophagy, increases the ability of muscle fiber mitochondria to resist PTP opening, and ameliorates histological features of muscular dystrophy, as well as muscle strength in the mdx diaphragm. These results support the concept that promoting the autophagic removal of defective mitochondria via AMPK stimulation could be a useful therapeutic strategy in DMD patients.

      Materials and Methods

      Animals and Cell Culture

      Six-week-old male mdx mice (Jackson Laboratories, Bar Harbour, ME) received intraperitoneal injections of AICAR (Toronto Research Chemicals, Toronto, ON, Canada) at a daily dose of 500 mg/kg body weight (5 consecutive days per week) for 4 weeks.
      • Narkar V.A.
      • Downes M.
      • Yu R.T.
      • Embler E.
      • Wang Y.X.
      • Banayo E.
      • Mihaylova M.M.
      • Nelson M.C.
      • Zou Y.
      • Juguilon H.
      • Kang H.
      • Shaw R.J.
      • Evans R.M.
      AMPK and PPARdelta agonists are exercise mimetics.
      Untreated littermate mdx mice were injected in the same manner with vehicle (0.9% NaCl); wild-type (WT) (C57BL6) mice (Janvier SAS, Le Genest Saint Isle, France) did not receive any treatment. Primary myotube cultures from mdx mice were derived from single living myofibers, as described previously.
      • Demoule A.
      • Divangahi M.
      • Danialou G.
      • Gvozdic D.
      • Larkin G.
      • Bao W.
      • Petrof B.J.
      Expression and regulation of CC class chemokines in the dystrophic (mdx) diaphragm.
      The investigation complied with the Guide for the Care and Use of Laboratory Animals (2011 edition).

      Immunoblotting and Gene Expression Analysis

      Immunoblotting was performed by standard methods using antibodies against phospho-AMPKα (Thr172), total AMPKα, phospho-ACC (Ser79), total ACC, LC3, Beclin-1, phospho-mTOR (Ser2448), total mTOR, phospho-raptor (Ser792), total raptor, phospho-p70S6K (Thr389), and total p70S6K from Cell Signaling Technology (Danvers, MA); Ulk1, Bnip3, β-actin, and tubulin from Sigma-Aldrich (St. Louis, MO); mitochondrial respiratory complex subunits (I-20 kDa; II-30 kDa; III-47 kDa; V-53 kDa) from MitoSciences (Eugene, OR); GAPDH from Abcam (Cambridge, UK); and utrophin, with a polyclonal antibody previously characterized by our laboratory.
      • Chazalette D.
      • Hnia K.
      • Rivier F.
      • Hugon G.
      • Mornet D.
      alpha7B integrin changes in mdx mouse muscles after L-arginine administration.
      Analysis and quantification (normalized in all cases to indicated loading control proteins) were performed with ImageJ software version 1.43u (NIH, Bethesda, MD). Real-time PCR to quantify mRNA levels was performed using Fast SYBR Green (Applied Biosystems) and the cycle threshold method.

      Immunohistochemistry and Morphometry

      Transverse cryosections stained by H&E were used for determination of fiber size and the percentage of centrally nucleated fibers, with a minimum of five cross sections per muscle, each containing an average of 100 fibers.
      • Jaber S.
      • Petrof B.J.
      • Jung B.
      • Chanques G.
      • Berthet J.P.
      • Rabuel C.
      • Bouyabrine H.
      • Courouble P.
      • Koechlin-Ramonatxo C.
      • Sebbane M.
      • Similowski T.
      • Scheuermann V.
      • Mebazaa A.
      • Capdevila X.
      • Mornet D.
      • Mercier J.
      • Lacampagne A.
      • Philips A.
      • Matecki S.
      Rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans.
      For determination of fiber types, adjacent sections were immunostained with anti-slow type 1 (M-8421; Sigma-Aldrich) and anti-fast type 2a (mAb SC-71; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. Electron microscopy was performed on muscle tissues as described previously.
      • Jaber S.
      • Petrof B.J.
      • Jung B.
      • Chanques G.
      • Berthet J.P.
      • Rabuel C.
      • Bouyabrine H.
      • Courouble P.
      • Koechlin-Ramonatxo C.
      • Sebbane M.
      • Similowski T.
      • Scheuermann V.
      • Mebazaa A.
      • Capdevila X.
      • Mornet D.
      • Mercier J.
      • Lacampagne A.
      • Philips A.
      • Matecki S.
      Rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans.

      Mitochondrial Enzyme and Respiration Assays

      Mitochondrial enzyme and respiration assays were performed as described previously.
      • Daussin F.N.
      • Godin R.
      • Ascah A.
      • Deschênes S.
      • Burelle Y.
      Cyclophilin-D is dispensable for atrophy and mitochondrial apoptotic signalling in denervated muscle.
      Briefly, activities of citrate synthase and cytochrome c oxidase (COX) were determined spectrophotometrically using standard coupled enzyme assays. Mitochondrial respiration was analyzed in an oximeter equipped with a Clarke type of electrode. The chamber was filled with the following solution: 2.77 mmol/L CaK2-EGTA, 7.23 mmol/L K2-EGTA (100 nmol/L free Ca2+), 6.56 mmol/L MgCl2 (1 mmol/L free Mg2+), 20 mmol/L taurine, 0.5 mmol/L dithiothreitol, 50 mmol/L potassium-methane sulfonate (160 mmol/L ionic strength), and 20 mmol/L imidazole (pH 7.1). After the baseline oxygen content in the chamber had been recorded, one bundle of 1 to 2 mg dry weight of saponin-permeabilized myofibers was placed into the chamber. Readings were taken of the rate of O2 consumption per unit time, first basally in the presence of glutamate plus malate (10:5, mmol/L), and then after the addition of ADP (2 mmol/L). Respiration rates were measured at 23°C under continuous stirring. At the end of each test, fibers were carefully removed from the oxygraphic cell, blotted, and dried for determination of fiber weight. Rates of O2 consumption were expressed in nmol O2/minute per per milligram dry weight.

      Mitochondrial H2O2 Release and Scavenging

      Mitochondrial H2O2 dynamics were measured in dissected fiber bundles with the fluorescent probe Amplex Red (20 μmol/L; Invitrogen), as described previously.
      • Daussin F.N.
      • Godin R.
      • Ascah A.
      • Deschênes S.
      • Burelle Y.
      Cyclophilin-D is dispensable for atrophy and mitochondrial apoptotic signalling in denervated muscle.
      After saponin permeabilization, the fibers were rinsed three times in the following buffer maintained at 4°C and at pH 7.3: 110 mmol/L K-2-(N-morpholino)ethanesulfonic acid, 35 mmol/L KCl, 1 mmol/L EGTA, 5 mmol/L K2HPO4, 3 mmol/L MgCl2 · 6H2O, and 0.5 mg/mL BSA. Fiber bundles (0.3 to 1.0 mg dry weight) were then incubated in a quartz microcuvette with continuous magnetic stirring in the same buffer supplemented with 1.2 U/mL horseradish peroxidase at 37°C. Baseline fluorescence readings were taken in the absence of any exogenous respiratory substrates. The following additions were then made sequentially: 5 mmol/L succinate, 10 mmol/L ADP, and 8 μmol/L antimycin A. To determine H2O2 scavenging capacity, permeabilized fiber bundles were placed in the above buffer containing additionally 50 μmol/L pyruvate and 20 μmol/L malate in a thermally controlled chamber set at 37°C with continuous stirring. An aliquot of the buffer was removed immediately after adding 40 μmol/L of H2O2 and subsequently at 20, 40, and 60 seconds. H2O2 content in aliquots was determined immediately on a fluorescence plate reader in a buffer containing 10 μmol/L of Amplex Red and 0.5 U/mL horseradish peroxidase. The rate of H2O2 scavenging by mitochondria was determined as the difference between fluorescence levels obtained at time t = 0. Rates of H2O2 production and scavenging were calculated from standard curves established under corresponding experimental conditions. All measurements were performed at least in duplicate, and results were expressed in nmol H2O2 scavenged per minute per milligram dry weight.

      Mitochondrial PTP Function

      The PTP opening time and calcium retention capacity were determined by fluorimetrically monitoring changes in extramitochondrial calcium concentration, using the probe Calcium Green-5N (Invitrogen-Life Technologies, Carlsbad, CA), after exposing fibers to a single pulse of external calcium.
      • Daussin F.N.
      • Godin R.
      • Ascah A.
      • Deschênes S.
      • Burelle Y.
      Cyclophilin-D is dispensable for atrophy and mitochondrial apoptotic signalling in denervated muscle.
      • Ascah A.
      • Khairallah M.
      • Daussin F.
      • Bourcier-Lucas C.
      • Godin R.
      • Allen B.G.
      • Petrof B.J.
      • Des Rosiers C.
      • Burelle Y.
      Stress-induced opening of the permeability transition pore in the dystrophin-deficient heart is attenuated by acute treatment with sildenafil.
      Ghost fibers were first prepared by incubating saponin-permeabilized bundles in a high-KCl medium, to extract myosin. The ghost fibers were then incubated at 23°C in a quartz microcuvette under continuous stirring in the following buffer: 250 mmol/L sucrose, 10 mmol/L 3-(N-morpholino)propanesulfonic acid, 0.005 mmol/L EGTA, and 10 mmol/L propidium iodide-Tris (pH 7.3), supplemented with glutamate plus malate (5:2.5, mmol/L) and 0.5 nmol/L oligomycin. After adding 20 nmol Ca2+ to the buffer, PTP opening time was taken as the time lapse between addition of the Ca2+ pulse and the time at which Ca2+ release was first noted to occur. Calcium retention capacity was defined as the total amount of Ca2+ accumulated by mitochondria before Ca2+ release caused by PTP opening, expressed per milligram wet fiber weight. Ca2+ concentration in the cuvette was calculated from a standard curve relating [Ca2+] to the fluorescence of Calcium Green-5N.

      Measurement of Skeletal Muscle Contractile Properties

      Diaphragm strips were electrically stimulated to determine intrinsic contractile properties, as described previously.
      • Bellinger A.M.
      • Reiken S.
      • Carlson C.
      • Mongillo M.
      • Liu X.
      • Rothman L.
      • Matecki S.
      • Lacampagne A.
      • Marks A.R.
      Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle.
      After euthanasia, the diaphragm was surgically excised and immediately transferred to chilled Krebs solution perfused with 95% O2:5% CO2 (pH 7.4). From the central tendon to the rib, a 2-mm-wide muscle strip was dissected free and mounted between two electrodes within a jacketed tissue bath chamber filled with continuously perfused Krebs solution warmed to 25°C. A 4-0 silk thread was used to secure the central tendon to an isometric force transducer. After a 15-minute thermoequilibration period, muscle length was gradually adjusted to optimal length (Lo, the length at which maximal twitch force is obtained). The force-frequency relationship was determined by sequential supramaximal stimulation for 1 second over a range of stimulation frequencies (from 10 to 120 Hz), with 2 minutes between each stimulation train. Fatigue resistance was assessed by measuring the rate of loss of muscle force during repetitive stimulation at 30 Hz over a 10-minute period. At the end of the experiment, Lo was directly measured with a microcaliper and the muscle was blotted dry and weighed. Specific force (force/cross-sectional area) was calculated and expressed in newtons per square centimeter, assuming a muscle density of 1.056 g/cm3.

      Statistical Analysis

      Data are expressed as means ± SEM. Statistical significance was defined as P < 0.05, using Student's unpaired t-test or analysis of variance (one- or two-way), followed by a Bonferroni selected-comparison test.

      Results

      Premature Mitochondrial PTP Opening and Energetic Deficiency in Dystrophic Muscle

      Dystrophic diaphragms contained numerous fibers with morphologically abnormal mitochondria, characterized by a rounded swollen appearance and grossly disrupted internal structure (Figure 1A). To examine the ability to resist PTP opening, which is a critically important aspect of mitochondrial function, permeabilized fibers were exposed to an external calcium load. The mitochondria of mdx diaphragms underwent significantly earlier PTP opening than observed in WT controls (Figure 1B). In keeping with these morphological and functional signs of defective mitochondria, baseline levels of AMPK activation (phosphorylation) were also higher in mdx than in WT diaphragms (Figure 1, C and D), which is consistent with cellular sensing of relative energetic deficiency within dystrophic myofibers.
      Figure thumbnail gr1
      Figure 1Defective mitochondria and premature PTP opening in mdx diaphragm fibers. A: Electron micrographs demonstrating differences in mitochondrial morphology between normal WT (CTL) and mdx diaphragms. Note numerous swollen mitochondria with abnormal cristae (arrow) in the mdx group. Scale bar = 1 µ. B: Time to mitochondrial permeability transition pore (PTP) opening after calcium challenge in mdx versus WT diaphragm fibers. C: Representative immunoblots showing baseline levels of phosphorylated and total forms of AMPK in WT and mdx diaphragms. D: Optical density quantification of p-AMPKα/total AMPKα protein levels (arbitrary units normalized to actin). Data are expressed as means ± SEM. *P < 0.05. n = 7 to 9 per group (B and D).

      AICAR Treatment Activates AMPK and ACC in Dystrophic Muscle

      To determine whether short-term AICAR treatment could further boost AMPK activation in dystrophic muscle, we first examined AICAR effects on phosphorylation of AMPK and its downstream target, ACC (acetyl CoA carboxylase) in primary mdx myotubes. AICAR induced phosphorylation of the α-subunit of AMPK and to an even greater extent ACC in vitro (Figure 2A). As expected, AICAR also induced increased activation of AMPK in vivo, such that increased phosphorylation levels of both AMPK and ACC were observed in the diaphragms of mdx mice within 48 hours after AICAR treatment was initiated (Figure 2B). Food intake did not differ significantly between the untreated and AICAR-treated mdx groups for the study period as a whole, from 6 to 10 weeks of age, although it was briefly increased in the AICAR-treated group from days 10 to 12. Similarly, there were no significant differences in body weight between the untreated and AICAR-treated mdx mice over the total treatment period (Figure 2C).
      Figure thumbnail gr2
      Figure 2AICAR promotes AMPK activation in dystrophic muscles in vitro and in vivo. A and B: Phosphorylated and total forms of AMPK and acetyl CoA carboxylase (ACC) in primary myotube cultures derived from mdx mice, either without or with AICAR treatment (1 mmol/L for 24 hours) (A) and in dystrophic mdx diaphragms in vivo, either without or with AICAR treatment (500 mg/kg/day intraperitoneally for 48 hours) (B). Optical density quantification is in arbitrary units, normalized to actin or tubulin. C: Food intake and body weight of mdx mice during 4 weeks of receiving AICAR treatment or vehicle alone. Data are expressed as means ± SEM. n = 8 per group. *P < 0.05.

      AICAR Treatment Induces Autophagy and Improves Mitochondrial PTP Function

      After 4 weeks of AICAR treatment in vivo, there was a major up-regulation of multiple components of the autophagy program in mdx diaphragms. This was reflected by increases in the mammalian Atg1 homolog, Ulk1 (an autophagy-initiating kinase that is directly activated by AMPK), as well as in Beclin-1, a component of the class III phosphoinositide 3-kinase complex involved in the initiation of autophagosomes (Figure 3A). In addition, the AICAR-treated group demonstrated higher levels of LC3-II, the lipidated form of LC3 that is generated during the process of autophagosome formation, and in the ratio of LC3-II to LC3-I (Figure 3B). Bnip3, a mitochondria-associated protein that plays a crucial role in the autophagic removal mitochondria in skeletal muscle was also found to be significantly up-regulated in the diaphragms of AICAR-treated mdx mice (Figure 3C). The levels of Ulk1, LC3, and Bnip3 were all mildly increased above WT levels in untreated mdx mice, suggesting that a degree of autophagic induction occurs basally in mdx diaphragms, most likely as an adaptive response to the dystrophic disease process itself.
      Figure thumbnail gr3
      Figure 3AICAR activates the autophagy pathway in mdx diaphragms. Representative immunoblots showing protein expression levels of Ulk1 and Beclin-1 (A), LC3-I and LC3-II (B), and Bnip3 (C) within the diaphragms of WT mice (CTL), untreated mdx mice, and AICAR-treated mdx mice. All quantifications of autophagy pathway proteins are normalized to GAPDH in terms of fold-change relative to WT mice. Data are expressed as means ± SEM. n = 4 to 6. *P < 0.05 versus wild type; P < 0.05 versus mdx.
      The mammalian target of rapamycin (mTOR) exists within two functional complexes, mTORC1 and mTORC2, with the former being directly regulated by cellular energy status.
      • Inoki K.
      • Kim J.
      • Guan K.L.
      AMPK and mTOR in cellular energy homeostasis and drug targets.
      To determine whether the stimulation of autophagy by AICAR was associated with mTORC1 modulation in mdx diaphragms, we measured total and phosphorylated protein levels of key mTORC1 members, mTOR and raptor, as well as p70S6K, the downstream target of activated mTOR (Figure 4A). Levels of total mTOR, raptor, and p70S6K were all increased above WT levels in the two groups of mdx mice (Figure 4, B–D). However, the phosphorylated forms of these proteins increased in a proportionate fashion, and the ratios of phosphorylated to total forms of these proteins did not differ significantly between the untreated and AICAR-treated mdx mice (Figure 4, E–G).
      Figure thumbnail gr4
      Figure 4AICAR treatment does not alter mTORC1 activation in mdx diaphragms. Representative immunoblots (A) and quantification of protein levels for the total forms of mTOR (B), raptor (C), and p70S6K (D), as well as their relative phosphorylation levels (E–G), within the diaphragms of WT mice (CTL), untreated mdx mice, and AICAR-treated mdx mice. Optical density quantification is in arbitrary units, normalized to GAPDH in all cases. Data are expressed as means ± SEM. n = 4 or 5. *P < 0.05 versus wild type.
      Given that autophagy is an important mechanism for eliminating damaged mitochondria, we next sought to determine whether AICAR treatment led to improved mitochondrial PTP function. In AICAR-treated mdx mice, electron microscopy suggested qualitative improvements in mitochondrial morphology (Figure 5A). Importantly, the abnormal susceptibility to PTP opening observed in mdx diaphragm fibers was reversed in AICAR-treated mdx mice (Figure 5B). There were no statistically significant differences in global calcium retention capacity of mitochondria between WT, mdx, and mdx+AICAR groups (1.17 ± 0.18, 1.37 ± 0.27, and 1.69 ± 0.28 nmol Ca2+ per milligram, respectively). Thus, the major findings are that the autophagic process in dystrophic muscles is greatly boosted by chronic administration of AICAR, and that this is associated with a significantly greater ability of mitochondria to resist calcium-induced membrane permeabilization.
      Figure thumbnail gr5
      Figure 5AICAR improves mitochondrial PTP function in mdx diaphragm fibers. A: Electron microscopic images of mitochondria in mdx and mdx+AICAR diaphragms. B: Effects of AICAR treatment on PTP opening time in mdx diaphragms (left) in WT mice (CTL), untreated mdx mice, and AICAR-treated mdx mice, as well as a representative tracing of mitochondrial calcium uptake and release within permeabilized fibers after calcium challenge (right). Data are expressed as means ± SEM. n = 7 to 10 per group. *P < 0.05 versus WT; **P < 0.05 versus mdx. Scale bar = 500 nm.

      Lack of AICAR Effects on Mitochondrial Biogenesis and Oxidative Functions

      In addition to autophagy, improved mitochondrial PTP function after AICAR treatment could be the result of enhanced mitochondrial biogenesis and its accompanying antioxidant effects. Therefore, we evaluated whether AICAR reprogrammed the mdx diaphragm to a more oxidative phenotype. At 4 weeks after initiation of AICAR treatment, the mRNA levels for PGC-1-α and PGC-1-β, two transcription factors typically associated with activation of the mitochondrial biogenesis program, were unchanged compared with untreated mice (Figure 6A). In AICAR-treated mdx mice, there was a small increase in type 1 (slow oxidative) fibers, but without any alteration in type 2a (fast oxidative) fibers in the diaphragm (Figure 6B). Immunoblotting revealed increased mitochondrial complex I protein expression without changes in complexes II, III, or V (Figure 6C). Levels of the dystrophin homolog protein utrophin, which is up-regulated in mdx muscles and typically is expressed at higher levels in oxidative fibers,
      • Ljubicic V.
      • Miura P.
      • Burt M.
      • Boudreault L.
      • Khogali S.
      • Lunde J.A.
      • Renaud J.M.
      • Jasmin B.J.
      Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle.
      were similarly unaltered by AICAR treatment in mdx diaphragms (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr6
      Figure 6AICAR effects on oxidative fiber types and mitochondrial complexes. A: mRNA levels of transcription factor genes associated with mitochondrial biogenesis, reported in terms of fold change relative to untreated mdx diaphragms. Scale bars: 20 µ. B: Comparison of relative proportion of fiber types in the diaphragms of untreated mdx and AICAR-treated mdx mice; micrographs show serial transverse muscle sections (with same fibers indicated by small circle and square) immunostained for type 1 or type 2a myosin heavy chain (MHC) expression. C: Quantification of respiratory chain complexes in untreated mdx and AICAR-treated mdx mice, with immunoblotting for the same complexes (CI, CII, CIII, and CV). Protein and mRNA levels are normalized to housekeeping GAPDH. Data are expressed as means ± SEM. n = 6 to 10 per group. *P < 0.05.
      The above findings suggesting minimal effects of chronic AICAR administration on the overall oxidative capacity of mdx diaphragms were further examined at a functional level. Enzymatic activity levels of both mitochondrial citrate synthase and cytochrome c oxidase (COX) in the diaphragm were unaffected by AICAR administration (Figure 7A). In addition, basal (V0) and maximal (Vmax) rates of oxygen consumption by mdx diaphragm fibers were unchanged (Figure 7B). Antioxidant function was determined by measuring H2O2 fluxes from permeabilized fibers using Amplex Red. Neither the H2O2 scavenging capacity (Figure 7C) nor H2O2 release under conditions of altered substrate supply or of inhibition of the mitochondrial electron transport chain (Figure 7D) was affected by AICAR treatment. Overall, these findings indicate that improvements in mitochondrial PTP function in the mdx diaphragm after AICAR treatment were not linked to the development of a substantially more oxidative fiber type.
      Figure thumbnail gr7
      Figure 7Lack of AICAR treatment effects on oxidative function. A: Citrate synthase (CS) and cytochrome c oxidase (COX) activity levels. B: Rate of O2 consumption per unit of time, basally in the presence of glutamate plus malate (V0) and after the addition of ADP (Vmax). C: H2O2 scavenging as indicated by levels at 60 seconds after addition of H2O2 (40 μmol/L) to permeabilized fibers. D: Net rate of mitochondrial H2O2 release from permeabilized diaphragm fibers under basal conditions (Fiber) and after the addition of succinate, ADP, or antimycin A. Data are expressed as means ± SEM. n = 8 to 10 per group.

      AICAR Treatment Improves Dystrophic Muscle Structure and Contractile Function

      To establish whether autophagic induction and improved mitochondrial PTP function were associated with any amelioration of the muscular dystrophy phenotype in the diaphragm, we first performed histological analysis of untreated versus AICAR-treated mdx mice. Muscle fibers with centrally located nuclei are a hallmark of previously regenerated muscle, and their relative proportion is therefore considered reflective of prior episodes of muscle fiber necrosis in mdx mice.
      • Karpati G.
      • Carpenter S.
      • Prescott S.
      Small-caliber skeletal muscle fibers do not suffer necrosis in mdx mouse dystrophy.
      • Yang L.
      • Lochmüller H.
      • Luo J.
      • Massie B.
      • Nalbantoglu J.
      • Karpati G.
      • Petrof B.J.
      Adenovirus-mediated dystrophin minigene transfer improves muscle strength in adult dystrophic (mdx) mice.
      In AICAR-treated mdx mice, the diaphragm showed a reduction in the percentage of centrally nucleated fibers (Figure 8A), consistent with a mitigation of prior necrosis. Moreover, despite the increased level of autophagy in the AICAR group, the mean cross-sectional area of individual diaphragmatic muscle fibers was unaffected (Figure 8B), indicating that chronic AICAR administration did not induce fiber atrophy. In keeping with the lack of atrophy, AICAR treatment also partially eliminated the pathological presence in mdx diaphragms of a 14-kDa actin cleavage product that has previously been shown to be a reliable biomarker of skeletal muscle wasting (Figure 8C).
      • Du J.
      • Wang X.
      • Miereles C.
      • Bailey J.L.
      • Debigare R.
      • Zheng B.
      • Price S.R.
      • Mitch W.E.
      Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions.
      • Workeneh B.T.
      • Rondon-Berrios H.
      • Zhang L.
      • Hu Z.
      • Ayehu G.
      • Ferrando A.
      • Kopple J.D.
      • Wang H.
      • Storer T.
      • Fournier M.
      • Lee S.W.
      • Du J.
      • Mitch W.E.
      Development of a diagnostic method for detecting increased muscle protein degradation in patients with catabolic conditions.
      Figure thumbnail gr8
      Figure 8Beneficial effects of AICAR-treated relative to untreated mdx diaphragms. A: Representative H&E staining and percentages of diaphragm myofibers with centrally located nuclei. B: Mean cross-sectional area of diaphragm myofibers. C: Immunoblotting for actin, as well as the ratio between cleaved (14 kDa) and full-length forms of the protein, relative to untreated mdx diaphragms. D: Maximal force-generating capacity of the diaphragm ex vivo at different frequencies of electrical stimulation. E: Fatigue resistance of the diaphragm during repetitive electrical stimulation. Data are expressed as means ± SEM. n = 8 per group (B, D, and E); n = 5 per group (C). *P < 0.05. CTL, WT control. Scale bar = 50 μm.
      Finally, to determine effects on contractile function, we compared the force-generating capacities of untreated and AICAR-treated mdx diaphragm muscle strips electrically stimulated ex vivo. AICAR-treated mdx mice demonstrated significantly greater diaphragmatic force production over a broad range of stimulation frequencies (30 to 120 Hz) (Figure 8D). In this regard, the maximal tetanic force production by the diaphragm increased by a mean of 21% (P < 0.005) in AICAR-treated mdx mice. On the other hand, endurance properties (fatigue resistance) of the diaphragm were unaffected by AICAR treatment (Figure 8E), which is consistent with its lack of physiologically important effects on oxidative capacity.

      Discussion

      Skeletal muscles lacking dystrophin have a reduced capacity for oxidative phosphorylation, and the diminished energy-producing potential of dystrophic muscle has been characterized as a metabolic crisis.
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      • Winkler K.
      • Wiedemann F.R.
      • von B.P.
      • Dietzmann K.
      • Kunz W.S.
      Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse.
      • Chen Y.W.
      • Zhao P.
      • Borup R.
      • Hoffman E.P.
      Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology.
      The metabolic sensor AMPK plays a key role in orchestrating the adaptive changes, including autophagy, that permit the survival of cells faced with energetic stress.
      • Hardie D.G.
      AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy.
      Autophagy not only supplies substrates for cellular energy production, but also provides a mechanism for more efficient use of these substrates via the removal of dysfunctional mitochondria.
      • Youle R.J.
      • Narendra D.P.
      Mechanisms of mitophagy.
      • Egan D.F.
      • Shackelford D.B.
      • Mihaylova M.M.
      • Gelino S.
      • Kohnz R.A.
      • Mair W.
      • Vasquez D.S.
      • Joshi A.
      • Gwinn D.M.
      • Taylor R.
      • Asara J.M.
      • Fitzpatrick J.
      • Dillin A.
      • Viollet B.
      • Kundu M.
      • Hansen M.
      • Shaw R.J.
      Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.
      In the present study, we postulated that pharmacological activation of AMPK would promote this normal adaptive response and thus have beneficial effects on the physiological function of the mdx diaphragm. The diaphragm is the primary muscle of respiration, and its involvement by the disease in DMD is responsible for the majority of patient deaths. In addition, in the mdx mouse model the diaphragm is the most severely affected muscle and bears the greatest resemblance to the human DMD phenotype.
      • Stedman H.H.
      • Sweeney H.L.
      • Shrager J.B.
      • Maguire H.C.
      • Panettieri R.A.
      • Petrof B.
      • Narusawa M.
      • Leferovich J.M.
      • Sladky J.T.
      • Kelly A.M.
      The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.
      Our investigation revealed several new findings. First, mdx mice show evidence for increased autophagy and augmented AMPK activation at baseline, which suggests an adaptive response to the presence of mitochondrial damage and energetic stress. Second, AICAR treatment led to a further major increase in activation of the autophagy pathway, as indicated by the characteristic biochemical changes of increased LC3-II content and up-regulation of other prototypical autophagy-associated proteins.
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      Third, in comparison with untreated mdx mice, the AICAR-treated group showed an improved ability to maintain mitochondrial integrity, as indicated by a greater resistance to PTP opening in the face of calcium overload. Fourth, and most importantly, AICAR treatment of mdx mice for 4 weeks led to significant improvements in both muscle structure and maximum force-generating capacity of the mdx diaphragm.
      Autophagy has been linked to situations associated with an inhibition of mTORC1 activation, such as nutrient deprivation, hypoxia, endoplasmic reticulum stress, and infections.
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      • Mizushima N.
      • Levine B.
      • Cuervo A.M.
      • Klionsky D.J.
      Autophagy fights disease through cellular self-digestion.
      However, autophagy can also be triggered without the direct participation of mTORC1. For example, Beclin-1 can be activated by the stress-responsive c-Jun amino-terminal kinase 1 (JNK1) and death-associated protein kinase (DAPK) in an mTOR-independent fashion.
      • Wei Y.
      • Pattingre S.
      • Sinha S.
      • Bassik M.
      • Levine B.
      JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy.
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      • Mizrachy L.
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      • Koren I.
      • Eisenstein M.
      • Sabanay H.
      • Pinkas-Kramarski R.
      • Kimchi A.
      DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy.
      Acute AMPK stimulation by AICAR has been shown to initiate autophagy through complex interrelated mechanisms involving activation of the tuberous sclerosis complex (TSC), inhibition of mTORC1, phosphorylation of raptor, and activation of Ulk1.
      • Egan D.F.
      • Shackelford D.B.
      • Mihaylova M.M.
      • Gelino S.
      • Kohnz R.A.
      • Mair W.
      • Vasquez D.S.
      • Joshi A.
      • Gwinn D.M.
      • Taylor R.
      • Asara J.M.
      • Fitzpatrick J.
      • Dillin A.
      • Viollet B.
      • Kundu M.
      • Hansen M.
      • Shaw R.J.
      Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.
      • Lee J.W.
      • Park S.
      • Takahashi Y.
      • Wang H.G.
      The association of AMPK with ULK1 regulates autophagy.
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.L.
      AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
      In the present study, chronic AICAR administration induced autophagy in mdx diaphragms, but this was not associated with evidence of mTORC1 inhibition. Thus, the phosphorylation status (ie, the phosphorylated fraction) was unaltered not only for mTOR, but also for its partner raptor and the downstream target p70S6K. We therefore speculate that AICAR-induced autophagy in our model may have occurred, at least in part, through an mTOR-independent mechanism. This could potentially involve the direct phosphorylation of Ulk1 by AMPK, as recently described by different groups of investigators.
      • Egan D.F.
      • Shackelford D.B.
      • Mihaylova M.M.
      • Gelino S.
      • Kohnz R.A.
      • Mair W.
      • Vasquez D.S.
      • Joshi A.
      • Gwinn D.M.
      • Taylor R.
      • Asara J.M.
      • Fitzpatrick J.
      • Dillin A.
      • Viollet B.
      • Kundu M.
      • Hansen M.
      • Shaw R.J.
      Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.
      • Lee J.W.
      • Park S.
      • Takahashi Y.
      • Wang H.G.
      The association of AMPK with ULK1 regulates autophagy.
      • Kim J.
      • Kundu M.
      • Viollet B.
      • Guan K.L.
      AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
      Rapamycin therapy from 6 to 12 weeks of age in mdx mice was recently reported to improve dystrophic histopathology in the diaphragm and tibialis anterior muscles, but once again with no consistent relationship to mTOR phosphorylation status.
      • Eghtesad S.
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      • Little S.R.
      • Clemens P.R.
      Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle.
      Whether autophagy is beneficial or harmful for skeletal muscle is dependent on its magnitude and on the specific context in which it occurs. Both excessive and inadequate autophagy can lead to muscle fiber atrophy in various disease states.
      • Sandri M.
      Autophagy in skeletal muscle.
      AICAR administration did not induce muscle fiber atrophy in the present study, suggesting that the autophagic process induced by AICAR was tightly regulated in its magnitude and/or was selective for dysfunctional cellular components. Although it may seem somewhat counterintuitive, previous work has shown that autophagy is actually required to maintain normal muscle mass.
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      Autophagy is required to maintain muscle mass.
      Thus, autophagy-deficient knockout mice exhibit fiber atrophy, as well as increases in abnormal mitochondria and apoptosis.
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      Autophagy is required to maintain muscle mass.
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      Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease.
      Along these same lines, muscular dystrophies linked to collagen VI deficiency have an autophagy defect leading to the accumulation of dysfunctional mitochondria and exaggerated apoptosis, which can be significantly ameliorated by the forced activation of autophagy.
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      • Bernardi P.
      • Sandri M.
      • Bonaldo P.
      Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration.
      At an appropriate level, therefore, autophagy and, more particularly, mitophagy appear to be important homeostatic mechanisms in skeletal muscle that are necessary for ensuring mitochondrial quality control through the removal of damaged or dysfunctional mitochondria, as well as for the maintenance of normal muscle mass.
      In addition to their primordial role in cellular energy production, mitochondria act as a calcium sink that can buffer and locally modulate cytosolic calcium levels.
      • Gunter T.E.
      • Sheu S.S.
      Characteristics and possible functions of mitochondrial Ca(2+) transport mechanisms.
      When this mechanism is overwhelmed, however, mitochondrial calcium overload induces opening of the PTP.
      • Gunter T.E.
      • Sheu S.S.
      Characteristics and possible functions of mitochondrial Ca(2+) transport mechanisms.
      • Rasola A.
      • Bernardi P.
      Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis.
      In the present study, we evaluated sensitivity to calcium-induced PTP opening in a skinned myofiber preparation, thereby eliminating the potential for selection bias or experimentally induced damage associated with mitochondrial isolation procedures.
      • Picard M.
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      • Hepple R.T.
      Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers.
      We found that mitochondria from mdx diaphragms exhibit premature PTP opening, compared with WT mice, when challenged with a calcium load. This is in keeping with the fact that damaged mitochondria, such as observed in mdx fibers, have a lower threshold for PTP opening. In addition, the pathological elevations of intracellular calcium and increased oxidative stress found in dystrophin-deficient muscles
      • Petrof B.J.
      Molecular pathophysiology of myofiber injury in deficiencies of the dystrophin-glycoprotein complex.
      are potent sensitizers of the PTP.
      • Rasola A.
      • Bernardi P.
      Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis.
      AICAR therapy improved the ability of mdx mitochondria to withstand an increased calcium load, as indicated by a normalization of the calcium exposure time needed to induce PTP opening under these conditions.
      In AICAR-treated mdx mice, there was significant up-regulation of Ulk1, which, as noted above, has recently been identified as a direct target of AMPK that links cellular energy sensing to the process of mitophagy.
      • Egan D.F.
      • Shackelford D.B.
      • Mihaylova M.M.
      • Gelino S.
      • Kohnz R.A.
      • Mair W.
      • Vasquez D.S.
      • Joshi A.
      • Gwinn D.M.
      • Taylor R.
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      • Fitzpatrick J.
      • Dillin A.
      • Viollet B.
      • Kundu M.
      • Hansen M.
      • Shaw R.J.
      Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.
      In addition, we observed increased expression of Bnip3, a mitochondrial BH3-only protein of the Bcl-2 family, which recruits the autophagy proteins LC3-II and Gabarap to mitochondria.
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      Nix is a selective autophagy receptor for mitochondrial clearance.
      Bnip3 has also been strongly implicated in the process of mitophagy, and inhibition of Bnip3 impedes autophagosome formation in skeletal muscles.
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      • Kirshenbaum L.A.
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      Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy.
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      • Zhao J.
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      • Sandri M.
      FoxO3 controls autophagy in skeletal muscle in vivo.
      Furthermore, Bnip3 is capable of stimulating mitophagy even without the requirement for mitochondrial membrane permeabilization.
      • Quinsay M.N.
      • Thomas R.L.
      • Lee Y.
      • Gustafsson A.B.
      Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore.
      Thus, the combined up-regulation of the mitophagy-associated proteins Ulk1 and Bnip3 in the AICAR-treated group, together with an improvement in mitochondrial PTP function, suggests that increased autophagy in treated mdx mice may have preferentially eliminated the mitochondrial population with a lower threshold for PTP opening.
      In addition to stimulating mitophagy, AMPK activation by AICAR has the potential to induce mitochondrial biogenesis, which could also have beneficial effects on muscle function.
      • Narkar V.A.
      • Downes M.
      • Yu R.T.
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      • Wang Y.X.
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      • Mihaylova M.M.
      • Nelson M.C.
      • Zou Y.
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      • Kang H.
      • Shaw R.J.
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      AMPK and PPARdelta agonists are exercise mimetics.
      In this regard, forced expression of PGC-1-α has been shown to mitigate aging-associated muscle atrophy,
      • Wenz T.
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      • Rotundo R.L.
      • Spiegelman B.M.
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      Increased muscle PGC-1alpha expression protects from sarcopenia and metabolic disease during aging.
      as well as muscle pathology in mdx mice.
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      PGC-1alpha regulates the neuromuscular junction program and ameliorates Duchenne muscular dystrophy.
      The latter effect could be, at least in part, related to the fact that utrophin, a protein capable of functionally compensating for the absence of dystrophin, is expressed at higher levels in fibers with a greater oxidative capacity.
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      PGC-1alpha regulates the neuromuscular junction program and ameliorates Duchenne muscular dystrophy.
      In the present study, however, there was no increase in utrophin protein within the diaphragms of AICAR-treated mdx mice. This is consistent with the absence of any detectable change in mitochondrial content (as reflected by citrate synthase) or functional indices of oxidative capacity (as determined by several complementary molecular and physiological assays, including direct measurements of fatigue resistance during repetitive muscle contractions induced by electrical stimulation). Our data are thus in keeping with prior observations that AICAR effects on mitochondrial biogenesis are minimal in muscles that are already highly oxidative,
      • Narkar V.A.
      • Downes M.
      • Yu R.T.
      • Embler E.
      • Wang Y.X.
      • Banayo E.
      • Mihaylova M.M.
      • Nelson M.C.
      • Zou Y.
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      • Kang H.
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      AMPK and PPARdelta agonists are exercise mimetics.
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      PGC-1{alpha} is required for AICAR-induced expression of GLUT4 and mitochondrial proteins in mouse skeletal muscle.
      such as the diaphragm. Accordingly, we conclude that the beneficial effects of AICAR treatment on the mdx diaphragm were not the result of increased mitochondrial biogenesis or other factors specifically linked to oxidative metabolism (although it has recently been reported that this could be an additional advantage of activating AMPK in more glycolytic mdx muscles
      • Ljubicic V.
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      • Burt M.
      • Boudreault L.
      • Khogali S.
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      • Renaud J.M.
      • Jasmin B.J.
      Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle.
      ). Furthermore, we do not exclude the possibility that AICAR exerts other biological effects beyond autophagy that could be beneficial for dystrophic muscles (eg, altered glucose uptake), nor that the relative importance of these effects may also vary among different muscles.
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      • Jasmin B.J.
      Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle.
      Given that many DMD patients ultimately die of respiratory muscle failure, the most clinically relevant finding of the present study is that AICAR administration improved mdx diaphragm force-generating capacity. There are several ways in which the autophagic removal of damaged mitochondria could account for this finding. Although we initially postulated that eliminating damaged mitochondria would reduce oxidative stress arising from dystrophic fibers, diminished reactive oxygen species production from mitochondria (as determined by direct H2O2 release measurements) could not be demonstrated in the AICAR-treated group. However, damaged mitochondria are also impaired in their ability to effectively buffer elevated cytosolic calcium levels, which has been implicated in numerous aspects of dystrophic pathophysiology.
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      Molecular pathophysiology of myofiber injury in deficiencies of the dystrophin-glycoprotein complex.
      This can include activation of calpains and other proteolytic enzymes,
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      Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle.
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      In addition, mitochondrial membrane permeabilization leads to the release of several apoptosis and muscle injury-promoting factors.
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      By initiating disassembly of the sarcomeric apparatus, these factors play an important role in the early phases of muscle atrophy and in depressing specific force production even when atrophy is not yet present.
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      • Callahan L.A.
      Caspase activation contributes to endotoxin-induced diaphragm weakness.
      In this regard, we observed that levels of a 14-kDa actin cleavage product previously associated with caspase-3 activation in muscle-wasting conditions
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      • Zheng B.
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      Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions.
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      • Ferrando A.
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      • Fournier M.
      • Lee S.W.
      • Du J.
      • Mitch W.E.
      Development of a diagnostic method for detecting increased muscle protein degradation in patients with catabolic conditions.
      was elevated in mdx but not in WT mice in vivo, and that the presence of this cleavage product was also significantly attenuated by AICAR treatment. Our results are thus compatible with previous studies showing muscle fiber necrosis and wasting to be attenuated by interventions that inhibit or desensitize the PTP in dystrophic mice.
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      • Dorchies O.M.
      • Patthey-Vuadens O.
      • Vuagniaux G.
      • Ruegg U.T.
      Investigation of Debio 025, a cyclophilin inhibitor, in the dystrophic mdx mouse, a model for Duchenne muscular dystrophy.
      In summary, treatment of mdx mice with the AMPK agonist AICAR significantly mitigated histological signs of pathology and improved contractile function of the diaphragm in the mdx mouse model of DMD. These beneficial effects were associated with induction of the autophagy program and evidence for improved mitochondrial integrity in dystrophic muscle fibers. Given the current availability of commonly used drugs (eg, metformin) that stimulate AMPK, our findings suggest that AMPK could represent a useful therapeutic target in DMD. Indeed, at a dose of metformin often prescribed for diabetes therapy (2 g/day), provided over 10 weeks, both AMPK activity and phospho-AMPK levels increased by approximately 80% over baseline levels in skeletal muscle of human subjects.
      • Musi N.
      • Hirshman M.F.
      • Nygren J.
      • Svanfeldt M.
      • Bavenholm P.
      • Rooyackers O.
      • Zhou G.
      • Williamson J.M.
      • Ljunqvist O.
      • Efendic S.
      • Moller D.E.
      • Thorell A.
      • Goodyear L.J.
      Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes.
      This is similar to the magnitude of increase in phospho-AMPK levels observed with AICAR treatment in the present study. We therefore propose that pharmacological stimulation of AMPK to enhance the autophagic removal of damaged cellular constituents, including mitochondria, is worthy of further clinical exploration as a therapy in DMD, and may also have broader applicability to other forms of skeletal muscle pathology.

      Acknowledgments

      We thank Johanne Bourdon and Christian Lemaire for expert technical assistance.

      Supplementary data

      • Supplemental Figure S1

        Lack of AICAR treatment effect on utrophin expression. Representative immunoblots show utrophin protein levels within diaphragms of WT control (CTL), untreated mdx, and AICAR-treated mdx mice. All quantifications are normalized to housekeeping GAPDH. Data are expressed as means ± SEM. n = 6 per group. *P < 0.05 wild type.

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