Myofiber Death Research Articles

Lipin1 as a therapeutic target for respiratory insufficiency of duchenne muscular dystrophy

In Duchenne muscular dystrophy (DMD), diaphragm muscle dysfunction results in respiratory insufficiency which is a leading cause of death in patients. Mutations to the dystrophin gene result in myocyte membrane instability, contributing to the structural deterioration of the diaphragm muscle tissues. With previous works suggesting the importance of lipin1 for maintaining skeletal muscle membrane integrity, we explored the roles of lipin1 in the dystrophic diaphragm. We found that the protein expression levels of lipin1 were reduced by 60% in the dystrophic diaphragm. While further knockdown of lipin1 in the dystrophic diaphragm leads to increased necroptosis, restoration of lipin1 in the dystrophic diaphragm results in reduced inflammation and fibrosis, decreased myofiber death, and improved respiratory function. Our results demonstrated that lipin1 restoration improved respiratory function by enhancing membrane integrity and suggested that lipin1 could be a potential therapeutic target for preventing respiratory insufficiency and respiratory failure in DMD. Continued investigation is required to better understand the mechanisms behind these findings, and to determine the role of lipin1 in maintaining muscle membrane stability.

Ectopic expression of Myomaker and Myomixer in slow muscle cells induces slow muscle fusion and myofiber death

Zebrafish embryos possess two major types of myofibers, the slow and fast fibers, with distinct patterns of cell fusion. The fast muscle cells can fuse, while the slow muscle cells cannot. Here, we show that myomaker is expressed in both slow and fast muscle precursors, whereas myomixer is exclusive to fast muscle cells. The loss of Prdm1a, a regulator of slow muscle differentiation, results in strong myomaker and myomixer expression and slow muscle cell fusion. This abnormal fusion is further confirmed by the direct ectopic expression of myomaker or myomixer in slow muscle cells of transgenic models. Using the transgenic models, we show that the heterologous fusion between slow and fast muscle cells can alter slow muscle cell migration and gene expression. Furthermore, the overexpression of myomaker and myomixer also disrupts membrane integrity, resulting in muscle cell death. Collectively, this study identifies that the fiber-type-specific expression of fusogenic proteins is critical for preventing inappropriate fusion between slow and fast fibers in fish embryos, highlighting the need for precise regulation of fusogenic gene expression to maintain muscle fiber integrity and specificity.

Systemic γ-sarcoglycan AAV gene transfer results in dose-dependent correction of muscle deficits in the LGMD 2C/R5 mouse model

Limb-girdle muscular dystrophy (LGMD) type 2C/R5 results from mutations in the γ-sarcoglycan (SGCG) gene and is characterized by muscle weakness and progressive wasting. Loss of functional γ-sarcoglycan protein in the dystrophin-associated protein complex destabilizes the sarcolemma, leading to eventual myofiber death. The SGCG knockout mouse (SGCG -/-) has clinical-pathological features that replicate the human disease, making it an ideal model for translational studies. We designed a self-complementary rAAVrh74 vector containing a codon-optimized human SGCG transgene driven by the muscle-specific MHCK7 promoter (SRP-9005) to investigate adeno-associated virus (AAV)-mediated SGCG gene transfer in SGCG -/- mice as proof of principle for LGMD 2C/R5. Gene transfer therapy resulted in widespread transgene expression in skeletal muscle and heart, improvements in muscle histopathology characterized by decreased central nuclei and fibrosis, and normalized fiber size. Histopathologic improvements were accompanied by functional improvements, including increased ambulation and force production and resistance to injury of the tibialis anterior and diaphragm muscles. This study demonstrates successful systemic delivery of the hSGCG transgene in SGCG -/- mice, with functional protein expression, reconstitution of the sarcoglycan complex, and corresponding physiological and functional improvements, which will help establish a minimal effective dose for translation of SRP-9005 gene transfer therapy in patients with LGMD 2C/R5.

VP.61 An AAV-shRNA DUX4-based therapy to treat facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies in adult and so far there is no curative or preventive treatment. FSHD is characterized by the aberrant expression of the <i>DUX4</i> transcription factor in muscle. DUX4 is a toxic protein, which has been implicated in myofiber death, increased sensitivity in oxidative stress, defects in myogenesis, muscle atrophy etc, ultimately leading to myofiber death. Here, we designed an AAV vector carrying a ShRNA directed against <i>DUX4</i> (AAV-shDUX4) to knock down <i>DUX4</i> expression in FSHD muscle cells and in the cre-inducible <i>DUX4</i> bi-transgenic mouse model (ACTA1-MCM/FLExDUX4). In this model, DUX4 is expressed upon Cre-mediated translocation in the nucleus, which occurs after tamoxifen injection. ACTA1-MCM/FLExDUX4 mice were injected with either an AVV-shScrambled or an AAV-shDUX4. After 4 weeks, we observed that all pathological signs of the <i>DUX4</i> expression were reduced in the AAV-shDUX4 animals compared to the AAV-shScrambled animals including: a dramatic derease in the expression of the genes downstream of <i>DUX4</i>, a decrease of the number of fibers with centrally located nuclei, a reduced inflammation etc. Our data suggest that AAV-shDUX4 based therapy is promising therapeutic strategy for FSHD.

Contribution of Necroptosis to Myofiber Death in Idiopathic Inflammatory Myopathies.

Myofiber necrosis is a significant pathologic characteristic of idiopathic inflammatory myopathies (IIMs), and its molecular mechanism is largely unknown. Necroptosis is a recently identified form of regulated necrotic cell death, and its activation might have crucial biologic consequences. The aim of the present study was to investigate the role of necroptosis in IIM muscle damage. Western blot and immunohistochemistry analyses were performed to examine the expression of receptor-interacting protein 3 (RIP-3) and mixed-lineage kinase domain-like (MLKL) proteins in 26 IIM patients and 4 healthy controls, as well as necroptosis-related damage-associated molecular pattern molecules. Tumor necrosis factor (TNF) was used to stimulate cultured C2C12 myoblasts, and the involvement of necroptosis in cell death of C2C12 cells was studied in vitro. The expression of RIP-3 and MLKL proteins and their phosphorylated forms was significantly increased in the muscle tissue of IIM patients compared to that of healthy controls. The expression levels of RIP-3 and MLKL proteins were associated with the severity of muscle damage in patients with IIM. Significant colocalization of MLKL with high mobility group box chromosomal protein 1 in necrotizing myofibers was observed in muscle biopsy tissue from patients with IIM. Stimulation of C2C12 myoblasts with TNF and a pan-caspase inhibitor, Z-VAD, resulted in the overactivation of necroptosis and significantly increased necrotic cell death. Strategies involving either inhibition of necroptosis with necrostatin-1 or knockdown of MLKL expression successfully prevented necroptosis-induced cell death of C2C12 cells. These findings demonstrate that overactivated necroptosis contributes to muscle damage in IIMs and suggest that necroptosis inhibitors could represent a new therapeutic target in the treatment of IIMs.

CLINICAL RESEARCH: O.5 A phase 2, randomized, double-blind, placebo-controlled, 48-Week study of the efficacy and safety of losmapimod in subjects with FSHD: ReDUX4

The main objective is to evaluate the efficacy of losmapimod in inhibiting the aberrant expression of DUX4, the root cause of FSHD. Secondary objectives are to evaluate the safety, tolerability, PK, and TE in blood and muscle, and muscle health with MRI. FSHD is caused by aberrant expression of DUX4 due to loss of repression at the D4Z4 locus. DUX4 activates a downstream transcriptional program that causes myofiber death, maladaptive tissue remodeling characterized by replacement of muscle with fat ultimately resulting in progressive motor disability.

The Interplay of Mitophagy and Inflammation in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a pathogenic disruption of the DYSTROPHIN gene that results in non-functional dystrophin protein. DMD patients experience loss of ambulation, cardiac arrhythmia, metabolic syndrome, and respiratory failure. At the molecular level, the lack of dystrophin in the muscle results in myofiber death, fibrotic infiltration, and mitochondrial dysfunction. There is no cure for DMD, although dystrophin-replacement gene therapies and exon-skipping approaches are being pursued in clinical trials. Mitochondrial dysfunction is one of the first cellular changes seen in DMD myofibers, occurring prior to muscle disease onset and progresses with disease severity. This is seen by reduced mitochondrial function, abnormal mitochondrial morphology and impaired mitophagy (degradation of damaged mitochondria). Dysfunctional mitochondria release high levels of reactive oxygen species (ROS), which can activate pro-inflammatory pathways such as IL-1β and IL-6. Impaired mitophagy in DMD results in increased inflammation and further aggravates disease pathology, evidenced by increased muscle damage and increased fibrosis. This review will focus on the critical interplay between mitophagy and inflammation in Duchenne muscular dystrophy as a pathological mechanism, as well as describe both candidate and established therapeutic targets that regulate these pathways.

Foxm1 Modulates Cell Non-Autonomous Response in Zebrafish Skeletal Muscle Homeostasis.

foxm1 is a master regulator of the cell cycle, contributing to cell proliferation. Recent data have shown that this transcription factor also modulates gene networks associated with other cellular mechanisms, suggesting non-proliferative functions that remain largely unexplored. In this study, we used CRISPR/Cas9 to disrupt foxm1 in the zebrafish terminally differentiated fast-twitching muscle cells. foxm1 genomic disruption increased myofiber death and clearance. Interestingly, this contributed to non-autonomous satellite cell activation and proliferation. Moreover, we observed that Cas9 expression alone was strongly deleterious to muscle cells. Our report shows that foxm1 modulates a muscle non-autonomous response to myofiber death and highlights underreported toxicity to high expression of Cas9 in vivo.

Loss of membrane integrity drives myofiber death in lipin1-deficient skeletal muscle.

Mutations in lipin1 are suggested to be a common cause of massive rhabdomyolysis episodes in children; however, the molecular mechanisms involved in the regulation of myofiber death caused by the absence of lipin1 are not fully understood. Loss of membrane integrity is considered as an effective inducer of cell death in muscular dystrophy. In this study, we utilized a mouse line with selective homozygous lipin1 deficiency in the skeletal muscle (Lipin1Myf5cKO) to determine the role of compromised membrane integrity in the myofiber death in lipin1‐deficient muscles. We found that Lipin1Myf5cKO muscles had significantly elevated proapoptotic factors (Bax, Bak, and cleaved caspase‐9) and necroptotic proteins such as RIPK1, RIPK3, and MLKL compared with WT mice. Moreover, Lipin1Myf5cKO muscle had significantly higher membrane disruptions, as evidenced by increased IgG staining and elevated uptake of Evans Blue Dye (EBD) and increased serum creatine kinase activity in Lipin1Myf5cKO muscle fibers. EBD‐positive fibers were strongly colocalized with apoptotic or necroptotic myofibers, suggesting an association between compromised plasma membrane integrity and cell death pathways. We further show that the absence of lipin1 leads to a significant decrease in the absolute and specific muscle force (normalized to muscle mass). Our work indicates that apoptosis and necroptosis are associated with a loss of membrane integrity in Lipin1Myf5cKO muscle and that myofiber death and dysfunction may cause a decrease in contractile force.

FSHD / OPMD / MYOTONIC DYSTROPHY: P.231 A biomarker of DUX4 activity to evaluate losmapimod treatment effect in FSHD Phase 2 trials

To identify a panel of DUX4-regulated gene transcripts pathologically expressed in FSHD muscle biopsies to measure treatment effect of losmapimod on the root cause of FSHD. Both FSHD1 and 2 are caused by the pathogenic expression of the homeobox transcription factor DUX4. Aberrant DUX4 expression in skeletal muscle results in profound transcriptional dysregulation and a characteristic DUX4-regulated transcriptional signature leading to myofiber death and the replacement of muscle with fat. Clinical evaluation of the p38a/b inhibitor losmapimod is ongoing in FSHD including assessment of efficacy for inhibition of DUX4 expression in affected skeletal muscle.

LIMB GIRDLE MUSCULAR DYSTROPHIES: P.138 Systemic dose-finding study with AAV-Mediated gamma-sarcoglycan gene therapy for treatment of muscle deficits in LGMD2C Mice

Limb-girdle muscular dystrophy (LGMD) type 2C results from mutations in the γ-sarcoglycan gene, causing loss of functional protein. It is characterized by muscle weakness and progressive muscle wasting. The sarcoglycans (α, β, γ, and δ-SG) are structural proteins localized at the cell membrane of muscle fibers that together with dystrophin and other proteins make up the dystrophin-associated protein complex (DAPC). Loss of functional protein in this complex results in an unstable sarcolemma leading to eventual myofiber death.

"The Social Network" and Muscular Dystrophies: The Lesson Learnt about the Niche Environment as a Target for Therapeutic Strategies.

The muscle stem cells niche is essential in neuromuscular disorders. Muscle injury and myofiber death are the main triggers of muscle regeneration via satellite cell activation. However, in degenerative diseases such as muscular dystrophy, regeneration still keep elusive. In these pathologies, stem cell loss occurs over time, and missing signals limiting damaged tissue from activating the regenerative process can be envisaged. It is unclear what comes first: the lack of regeneration due to satellite cell defects, their pool exhaustion for degeneration/regeneration cycles, or the inhibitory mechanisms caused by muscle damage and fibrosis mediators. Herein, Duchenne muscular dystrophy has been taken as a paradigm, as several drugs have been tested at the preclinical and clinical levels, targeting secondary events in the complex pathogenesis derived from lack of dystrophin. We focused on the crucial roles that pro-inflammatory and pro-fibrotic cytokines play in triggering muscle necrosis after damage and stimulating satellite cell activation and self-renewal, along with growth and mechanical factors. These processes contribute to regeneration and niche maintenance. We review the main effects of drugs on regeneration biomarkers to assess whether targeting pathogenic events can help to protect niche homeostasis and enhance regeneration efficiency other than protecting newly formed fibers from further damage.

Lipid composition and organization helps plasma membrane repair and offers novel therapeutic target to treat degenerative muscle disease

The plasma membrane of all cells is routinely injured, with considerable investigative effort focused on understanding how proteins help repair this injury. Despite being the most abundant component of the plasma membrane, the role of lipids in repairing injured mammalian cells has remained unexplored. Through systematic live cell imaging we have examined the involvement of lipids in plasma membrane repair following injury. This shows that change in composition and localization of plasma membrane lipids is crucial for repair. Specifically, we find that cell injury results in a rapid change in local cholesterol and sphingomyelin content, and failure of which increases membrane fluidity and compromises membrane repair. Plasma membrane injury also alters the local composition of phospholipids including PIP2 and diacylglycerol (DAG), which is required for the repair of the injured plasma membrane. Failure of these lipid changes in muscle fibers injured by mechanical or other activity, results in myofiber death and Limb Girdle Muscular Dystrophy (LGMD2B). To target this deficit we developed a gene therapy approach to exogenously modify the plasma membrane lipid composition in a mouse model of LGMD2B. This approach improved the repair of the dystrophic muscle, attenuated myofiber death and improved muscle strength and histopathology. These findings provide direct evidence of the diverse roles of lipids in not only maintaining the integrity of the plasma membrane, but also in re‐establishing its integrity following injury. Here, we establish the clinical relevance of the structural and signaling roles of lipids in cell repair, and identify therapeutic approaches that address these deficits in muscle diseases.Support or Funding InformationThis work is supported by R01AR055686 from NIH/NIAMS.

Mitochondrial dysfunction and role of harakiri in the pathogenesis of myositis.

The etiology of myositis is unknown. Although attempts to identify viruses in myositis skeletal muscle have failed, several studies have identified the presence of a viral signature in myositis patients. Here we postulate that in individuals with susceptible genetic backgrounds, viral infection alters the epigenome to activate the pathological pathways leading to disease onset. To identify epigenetic changes, methylation profiling of Coxsackie B infected human myotubes and muscle biopsies from polymyositis (PM) and dermatomyositis (DM) patients were compared to changes in global transcript expression induced by in vitro Coxsackie B infection. Gene and protein expression analysis and live cell imaging were performed to examine the mechanisms. Analysis of methylation and gene expression changes identified that a mitochondria-localized activator of apoptosis-harakiri (HRK) - is upregulated in myositis skeletal muscle cells. Muscle cells with higher HRK expression have reduced mitochondrial potential and poor ability to repair from injury as compared to controls. In cells from myositis patient toll-like receptor 7 (TLR7) activates and sustains high HRK expression. Forced over expression of HRK in healthy muscle cells is sufficient to compromise their membrane repair ability. Endurance exercise that is associated with improved muscle and mitochondrial function in PM and DM patients decreased TLR7 and HRK expression identifying these as therapeutic targets. Increased HRK and TLR7 expression causes mitochondrial damage leading to poor myofiber repair, myofiber death and muscle weakness in myositis patients and exercise induced reduction of HRK and TLR7 expression in patients is associated with disease amelioration. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DRP1-mediated mitochondrial shape controls calcium homeostasis and muscle mass

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca2+ handling.

Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy

Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers.

Genetic evidence in the mouse solidifies the calcium hypothesis of myofiber death in muscular dystrophy.

Muscular dystrophy (MD) refers to a clinically and genetically heterogeneous group of degenerative muscle disorders characterized by progressive muscle wasting and often premature death. Although the primary defect underlying most forms of MD typically results from a loss of sarcolemmal integrity, the secondary molecular mechanisms leading to muscle degeneration and myofiber necrosis is debated. One hypothesis suggests that elevated or dysregulated cytosolic calcium is the common transducing event, resulting in myofiber necrosis in MD. Previous measurements of resting calcium levels in myofibers from dystrophic animal models or humans produced equivocal results. However, recent studies in genetically altered mouse models have largely solidified the calcium hypothesis of MD, such that models with artificially elevated calcium in skeletal muscle manifest fulminant dystrophic-like disease, whereas models with enhanced calcium clearance or inhibited calcium influx are resistant to myofiber death and MD. Here, we will review the field and the recent cadre of data from genetically altered mouse models, which we propose have collectively mostly proven the hypothesis that calcium is the primary effector of myofiber necrosis in MD. This new consensus on calcium should guide future selection of drugs to be evaluated in clinical trials as well as gene therapy-based approaches.

G.P.227: Beta-Sarcoglycan gene transfer leads to functional improvement in a model of LGMD2E

Limb-girdle muscular dystrophy (LGMD) type 2E results from mutations in the beta-sarcoglycan gene causing loss of functional protein. It is characterized by muscle weakness and progressive muscle wasting. The sarcoglycans (alpha, beta, gamma, and delta-SG) are structural proteins localized at the cell membrane of muscle fibers that together with dystrophin and other proteins make up the dystrophin-associated protein complex (DAPC). Loss of functional protein in this complex results in an unstable sarcolemma leading to eventual myofiber death. To date, no effective therapy exists to treat this debilitating disease. The goal of this study is to demonstrate efficacy of AAV-mediated beta-sarcoglycan gene transfer in beta-SG knock-out mice to provide proof-of-principle for gene replacement therapy for LGMD2E. A previous study by our group investigating AAV gene replacement therapy to treat LGMD2D led to a successful clinical trial in LGMD2D patients (alpha-sarcoglycan deficiency), which will provide a solid platform for this study. We generated a self-complementary AAVrh74 vector containing a codon optimized human beta-Sarcoglycan gene (hSGCB) driven by the muscle specific tMCK promoter. Following direct intramuscular injection to demonstrate potency of our vector, we treated beta-SG KO mice via a clinically relevant vascular delivery model to deliver AAV.tMCK.hSGCB in two doses (1x1011 and 5x1011 vg) to the lower limb muscles. We had clear demonstration of dose dependent beta-SG expression and improvement in dystrophic pathology with greater than 90% beta-SG expression in lower limb muscles following vascular delivery at high dose following 3months of treatment. Functional outcome measures performed on the extensor digitorum longus (EDL) muscle from these mice showed an improvement in specific force generation and protection from eccentric contraction induced injury. This pre-clinical study is pivotal for establishing proof-of-principle for translation of AAV.hSGCB gene transfer in LGMD2E patients.

P38α MAPK underlies muscular dystrophy and myofiber death through a Bax-dependent mechanism.

Muscular dystrophies are a group of genetic diseases that lead to muscle wasting and, in most cases, premature death. Cytokines and inflammatory factors are released during the disease process where they promote deleterious signaling events that directly participate in myofiber death. Here, we show that p38α, a kinase in the greater mitogen-activated protein kinase (MAPK)-signaling network, serves as a nodal regulator of disease signaling in dystrophic muscle. Deletion of Mapk14 (p38α-encoding gene) in the skeletal muscle of mdx- (lacking dystrophin) or sgcd- (δ-sarcoglycan-encoding gene) null mice resulted in a significant reduction in pathology up to 6 months of age. We also generated MAPK kinase 6 (MKK6) muscle-specific transgenic mice to model heightened p38α disease signaling that occurs in dystrophic muscle, which resulted in severe myofiber necrosis and many hallmarks of muscular dystrophy. Mechanistically, we show that p38α directly induces myofiber death through a mitochondrial-dependent pathway involving direct phosphorylation and activation of the pro-death Bcl-2 family member Bax. Indeed, muscle-specific deletion of Bax, but not the apoptosis regulatory gene Tp53 (encoding p53), significantly reduced dystrophic pathology in the muscles of MKK6 transgenic mice. Moreover, use of a p38 MAPK pharmacologic inhibitor reduced dystrophic disease in Sgcd(-/-) mice suggesting a future therapeutic approach to delay disease.

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.