Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy.

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1-/-) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1-/-) or -2 (CAPN2-/-) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.

Dusp6 is a genetic modifier of growth through enhanced ERK activity.

Like other single-gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole-genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the dual-specificity phosphatase 6 (Dusp6) gene from the DBA/2 J (D2) mouse strain. DUSP6 is a broadly expressed dual-specificity phosphatase protein, which binds and dephosphorylates extracellular-signal-regulated kinase (ERK), leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DUSP6 and ERK resulting in increased ERK phosphorylation and ERK activity. In dystrophic muscle, DUSP6 Met62Ile is strongly upregulated to counteract its reduced activity. We found that myoblasts from the D2 background were insensitive to a specific small molecule inhibitor of DUSP6, while myoblasts expressing the canonical DUSP6 displayed enhanced proliferation after exposure to DUSP6 inhibition. These data identify DUSP6 as an important regulator of ERK activity in the setting of muscle growth and muscular dystrophy.

Xanthine oxidase is hyper-active in Duchenne muscular dystrophy.

Generation of superoxide by xanthine oxidase can be stimulated under ischemic and aberrant calcium homeostasis. Because patients and mice with Duchenne muscular dystrophy (DMD) suffer from ischemia and excessive calcium influx, we tested the hypothesis that xanthine oxidase activity is elevated and contributes to disease pathology. Xanthine oxidase activity was measured by urinary isoxanthopterin in DMD patients at rest and in response to exercise. Urinary isoxanthopterin/creatinine was elevated compared to age-matched controls and Becker muscular dystrophy (BMD) patients. Concentrations were also increased after a six minute walk test in ambulatory patients. We also measured urinary isoxanthopterin in wildtype mice and a number of dystrophic mouse models; the DMD mouse model (mdx), mdx mice overexpressing a variety of transgenic miniaturized and chimeric skeletal muscle-specific dystrophins and utrophin and the ß-sarcoglycan deficient (Scgb-/-) mouse which represents type 2E human limb-girdle muscular dystrophy. Mdx and Scgb-/-mice had greater urinary isoxanthopterin/creatinine than wildtype mice while mdx mice expressing dystrophin or utrophin linking the extracellular matrix to the actin cytoskeleton were not different than wildtype. We also measured higher levels of urinary ortho-tyrosine in humans and mice deficient for dystrophin to confirm elevated oxidative stress. Surprisingly, mdx had lower xanthine oxidase protein levels and higher mRNA in gastrocnemius muscle compared to wildtype mice, however, the enzymatic activity of skeletal muscle xanthine oxidase was elevated above wildtype and a transgenic rescued mdx mouse (DysΔMTB-mdx). Downhill treadmill running also caused significant increases in mdx urinary isoxanthopterin that was prevented with the xanthine oxidase inhibitor allopurinol. Similarly, in vitro eccentric contraction-induced force drop of mdx muscle was attenuated by the allopurinol metabolite, oxypurinol. Together, our data suggests hyper-activity of xanthine oxidase in DMD, identifies xanthine oxidase activity as a contributing factor in eccentric contraction-induced force drop of dystrophin-deficient skeletal muscle and highlights the potential of isoxanthopterin as a noninvasive biomarker in DMD.

Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy.

Chronic muscle inflammation is a critical feature of Duchenne muscular dystrophy and contributes to muscle fibre injury and disease progression. Although previous studies have implicated T cells in the development of muscle fibrosis, little is known about their role during the early stages of muscular dystrophy. Here, we show that T cells are among the first cells to infiltrate mdx mouse dystrophic muscle, prior to the onset of necrosis, suggesting an important role in early disease pathogenesis. Based on our comprehensive analysis of the kinetics of the immune response, we further identify the early pre-necrotic stage of muscular dystrophy as the relevant time frame for T-cell-based interventions. We focused on protein kinase C θ (PKCθ, encoded by Prkcq), a critical regulator of effector T-cell activation, as a potential target to inhibit T-cell activity in dystrophic muscle. Lack of PKCθ not only reduced the frequency and number of infiltrating T cells but also led to quantitative and qualitative changes in the innate immune cell infiltrate in mdx/Prkcq-/- muscle. These changes were due to the inhibition of T cells, since PKCθ was necessary for T-cell but not for myeloid cell infiltration of acutely injured muscle. Targeting T cells with a PKCθ inhibitor early in the disease process markedly diminished the size of the inflammatory cell infiltrate and resulted in reduced muscle damage. Moreover, diaphragm necrosis and fibrosis were also reduced following treatment. Overall, our findings identify the early T-cell infiltrate as a therapeutic target and highlight the potential of PKCθ inhibition as a therapeutic approach to muscular dystrophy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

Pharmacological Inhibition of PKCθ Counteracts Muscle Disease in a Mouse Model of Duchenne Muscular Dystrophy.

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease. RESEARCH IN CONTEXT: Duchenne muscular dystrophy (DMD) is a severe muscle disease affecting 1:3500 male births. DMD is caused by a mutation in dystrophin gene, coding for a protein required for skeletal and cardiac muscle integrity. Lack of a functional dystrophin is primarily responsible for the muscle eccentric contraction-induced muscle damage, observed in dystrophic muscle. However, inflammation plays a considerable role in the progression of DMD. Glucocorticoids, which have anti-inflammatory properties, are being used to treat DMD with some success; however, long term treatment with these drugs induces muscle atrophy and wasting, outweighing their benefit. The identification of specific targets for anti-inflammatory therapies is one of the ongoing therapeutic options. Although blunting inflammation would not be a "cure" for the disease, the emerging clue is that multiple strategies, addressing different aspects of the pathology, which may eventually converge, may be successful. In this context, we previously showed that genetic ablation of Protein Kinase C θ (PKCθ), an enzyme known to be involved in immune response, in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease.

Use of the six-minute walk test to characterize golden retriever muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder in which loss of the dystrophin protein causes progressive skeletal/cardiac muscle degeneration and death within the third decade. For clinical trials and supportive animal studies, DMD disease progression and response to treatment must be established using outcome parameters (biomarkers). The 6-minute walk test (6MWT), defined as the distance an individual can walk in 6 minutes, is commonly used in DMD clinical trials and has been employed in dogs to characterize cardiac and respiratory disease severity. Building on methods established in DMD and canine clinical studies, we assessed the 6MWT in dogs with the DMD genetic homolog, golden retriever muscular dystrophy (GRMD). Twenty-one cross-bred golden retrievers were categorized as affected (DMD mutation and GRMD phenotype), carrier (female heterozygous for DMD mutation and no phenotype), and normal (wild type DMD gene and normal phenotype). When compared to grouped normal/carrier dogs, GRMD dogs walked shorter height-adjusted distances at 6 and 12 months of age and their distances walked declined with age. Percent change in creatine kinase after 6MWT was greater in GRMD versus normal/carrier dogs at 6 months, providing another potential biomarker. While these data generally support use of the 6MWT as a biomarker for preclinical GRMD treatment trials, there were certain limitations. Results of the 6MWT did not correlate with other outcome parameters for GRMD dogs when considered alone and an 80% increase in mean distance walked would be necessary to achieve satisfactory power.

Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and ß-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD.

Muscular dystrophy due to a sarcoglycan deficiency in a female Dobermann dog.

A four-month-old female Dobermann presented with myalgia, dysphagia, progressive weakness and loss of body condition. Diagnostic evaluation at nine months of age revealed markedly elevated serum creatine kinase activity, electromyographic abnormalities and histological evidence of chronic-active muscle necrosis. Imaging confirmed dysphagia and aspiration pneumonia. Muscular dystrophy was suspected and immunohistochemical staining of muscle cryosections demonstrated reduced sarcoglycans. Treatment consisted of gastrostomy, and over the next 5 months the dog gained weight, despite continued loss of muscle mass. The dog died at 14 months of age after developing clinical signs of aspiration pneumonia. To the authors' knowledge, this is the first report of muscular dystrophy in a Dobermann and only the second detailed report of a canine sarcoglycanopathy. Supportive care resulted in an acceptable quality of life for 10 months after clinical signs were first observed.

Immunoproteasome in animal models of Duchenne muscular dystrophy.

Increased proteasome activity has been implicated in the atrophy and deterioration associated with dystrophic muscles of Duchenne muscular dystrophy (DMD). While proteasome inhibitors show promise in the attenuation of muscle degeneration, proteasome inhibition-induced toxicity was a major drawback of this therapeutic strategy. Inhibitors that selectively target the proteasome subtype that is responsible for the loss in muscle mass and quality would reduce side effects and be less toxic. This study examined proteasome activity and subtype populations, along with muscle function, morphology and damage in wild-type (WT) mice and two murine models of DMD, dystrophin-deficient (MDX) and dystrophin- and utrophin-double-knockout (DKO) mice. We found that immunoproteasome content was increased in dystrophic muscles while the total proteasome content was unchanged among the three genotypes of mice. Proteasome proteolytic activity was elevated in dystrophic muscles, especially in DKO mice. These mice also exhibited more severe muscle atrophy than either WT or MDX mice. Muscle damage and regeneration, characterized by the activity of muscle creatine kinase in the blood and the percentage of central nuclei were equally increased in dystrophic mice. Accordingly, the overall muscle function was similarly reduced in both dystrophic mice compared with WT. These data demonstrated that there was transformation of standard proteasomes to immunoproteasomes in dystrophic muscles. In addition, DKO that showed greatest increase in proteasome activities also demonstrated more severe atrophy compared with MDX and WT. These results suggest a putative role for the immunoproteasome in muscle deterioration associated with DMD and provide a potential target for therapeutic intervention.

Conditional knockout of pik3c3 causes a murine muscular dystrophy.

Abnormalities in phosphoinositide metabolism are an emerging theme in human neurodegenerative disease. Myotubular myopathy is a prototypical disorder of phosphoinositide dysregulation that is characterized by profound muscle pathology and weakness and that is caused by mutations in MTM1, which encodes a phosphatase that targets 3-position phosphoinositides, including phosphatidylinositol 3-phosphate. Although the association between MTM1 and muscle disease has become increasingly clarified, the normal role(s) of phosphatidylinositol 3-phosphate metabolism in muscle development and homeostasis remain poorly understood. To begin to address the function of phosphatidylinositol 3-phosphate in skeletal muscle, we focused on the primary kinase responsible for its production, and created a muscle-specific conditional knockout of the class III phosphatidylinositol 3-kinase, Pik3c3. Muscle-specific deletion of Pik3c3 did not disturb embryogenesis or early postnatal development, but resulted in progressive disease characterized by reduced activity and death by 2 months of age. Histopathological analysis demonstrated changes consistent with a murine muscular dystrophy. Examination for cellular mechanism(s) responsible for the dystrophic phenotype revealed significant alterations in the autophagolysosomal pathway with mislocation of known dystrophy proteins to the lysosomal compartment. In all, we present the first analysis of Pik3c3 in skeletal muscle, and report a novel association between deletion of Pik3c3 and muscular dystrophy.

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-ß-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.

Muscle carbonic anhydrase III levels in normal and muscular dystrophia afflicted chickens.

BACKGROUND: The levels and immunohistochemical localization of muscle carbonic anhydrase III (CA-III) in healthy chickens and in muscular dystrophia affected (DA) chickens show that the muscles of diseased animal undergo a progressive increase of enzyme activity. METHODS: An enzyme-linked immunoassay was used to assess the CA-III levels in the muscles and other tissues from eight normal White Leghorn chickens and in two chickens with muscular dystrophy. Immunohistochemical localization of the enzyme in the muscles of these animals was also determined. RESULTS: The levels of CA-III in the tensor fasciae latae and the superficial pectoral muscles of the DA chickens were higher than the level in normal chickens. The concentrations of CA-III in erythrocytes and plasma from diseased chickens were approximately 15-fold and 1.4-fold higher than in the normal chickens, respectively. In the superficial pectoral and the tensor fasciae latae muscles of diseased chickens, the numbers of strongly stained and weakly stained fibers were greater than that in the normal chickens. CONCLUSION: The levels of CA-III in the superficial pectoral muscle, the tensor fasciae latae muscle, plasma and erythrocytes from the chickens with muscular dystrophy were higher than found in normal chickens.

Novel mini-dystrophin gene dual adeno-associated virus vectors restore neuronal nitric oxide synthase expression at the sarcolemma.

Six- to 8-kb mini-dystrophin genes are promising candidates for Duchenne muscular dystrophy (DMD) gene therapy. Several dual adeno-associated virus (AAV) mini-dystrophin vectors have been tested in dystrophin-deficient mice. Despite the encouraging preclinical results, none of the existing dual AAV vectors can restore sarcolemmal neuronal nitric oxide synthase (nNOS) expression. Localization of nNOS to the sarcolemma may greatly improve the therapeutic outcome in DMD (Lai, Y., Thomas, G.D., Yue, Y., et al. [2009]. J. Clin. Invest. 119, 624-635). In this study, we developed a series of dual AAV expression vectors to express a synthetic minigene that carries the nNOS localization domain. To help validate dual vector reconstitution, we also included a FLAG tag and a GFP reporter at different ends of the minigene. These dual AAV vectors were packaged in Y445F tyrosine mutant AAV-6 and tested in dystrophin-null mdx4cv mice by direct muscle injection. All dual vectors expressed GFP/FLAG-tagged mini-dystrophin and restored sarcolemmal nNOS. However, the reconstitution efficiency was significantly different among different sets. The dual vector set YZ27/YZ22 yielded the highest transduction efficiency (∼90%). Further development of this set dual vector may lead to more effective DMD gene therapy.

Reduced resting potentials in dystrophic (mdx) muscle fibers are secondary to NF-κB-dependent negative modulation of ouabain sensitive Na+-K+ pump activity.

To examine potential mechanisms for the reduced resting membrane potentials (RPs) of mature dystrophic (mdx) muscle fibers, the Na(+)-K(+) pump inhibitor ouabain was added to freshly isolated nondystrophic and mdx fibers. Ouabain produced a 71% smaller depolarization in mdx fibers than in nondystrophic fibers, increased the [Na(+)](i) in nondystrophic fibers by 40%, but had no significant effect on the [Na(+)](i) of mdx fibers, which was approximately double that observed in untreated nondystrophic fibers. Western blots indicated no difference in total and phosphorylated Na(+)-K(+) ATPase catalytic α1 subunit between nondystrophic and mdx muscle. Examination of the effects of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) indicated that direct application of the drug slowly hyperpolarized mdx fibers (7 mV in 90 min) but had no effect on nondystrophic fibers. Pretreatment with ouabain abolished this hyperpolarization, and pretreatment with PDTC restored ouabain-induced depolarization and reduced [Na(+)](i). Administration of an NF-κB inhibitor that utilizes a different mechanism for reducing nuclear NF-κB activation, ursodeoxycholic acid (UDCA), also hyperpolarized mdx fibers. These results suggest that in situ Na(+)-K(+) pump activity is depressed in mature dystrophic fibers by NF-κB dependent modulators, and that this reduced pump activity contributes to the weakness characteristic of dystrophic muscle.

MicroRNAs involved in molecular circuitries relevant for the Duchenne muscular dystrophy pathogenesis are controlled by the dystrophin/nNOS pathway.

In Duchenne muscular dystrophy (DMD) the absence of dystrophin at the sarcolemma delocalizes and downregulates nitric oxide synthase (nNOS); this alters S-nitrosylation of HDAC2 and its chromatin association. We show that the differential HDAC2 nitrosylation state in Duchenne versus wild-type conditions deregulates the expression of a specific subset of microRNA genes. Several circuitries controlled by the identified microRNAs, such as the one linking miR-1 to the G6PD enzyme and the redox state of cell, or miR-29 to extracellular proteins and the fibrotic process, explain some of the DMD pathogenetic traits. We also show that, at variance with other myomiRs, miR-206 escapes from the dystrophin-nNOS control being produced in activated satellite cells before dystrophin expression; in these cells, it contributes to muscle regeneration through repression of the satellite specific factor, Pax7. We conclude that the pathway activated by dystrophin/nNOS controls several important circuitries increasing the robustness of the muscle differentiation program.

Arginine metabolism by macrophages promotes cardiac and muscle fibrosis in mdx muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD. METHODOLOGY: We use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial effects of short-term treatments. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that arginine metabolism by arginase promotes fibrosis of muscle in muscular dystrophy and contributes to kyphosis. Our findings also show that long-term, dietary supplementation with arginine exacerbates fibrosis of dystrophic heart and muscles. Thus, commonly-practiced dietary supplementation with arginine by DMD patients has potential risk for increasing pathology when performed for long periods, despite reports of benefits acquired with short-term supplementation.

A growth stimulus is needed for IGF-1 to induce skeletal muscle hypertrophy in vivo.

Here, we characterise new strains of normal and dystrophic (mdx) mice that overexpress Class 2 IGF-1 Ea in skeletal myofibres. We show that transgenic mice have increased muscle levels of IGF-1 (approximately 13-26 fold) and show striking muscle hypertrophy (approximately 24-56% increase in mass). Adult normal muscles were resistant to elevated IGF-1; they reached adult steady state and maintained the same mass from 3 to 12 months. By contrast, dystrophic muscles from mdx/IGF-1(C2:Ea) mice continued to increase in mass during adulthood. IGF-1 signalling was evident only in muscles that were growing as a result of normal postnatal development (23-day-old mice) or regenerating in response to endogenous necrosis (adult mdx mice). Increased phosphorylation of Akt at Ser473 was not evident in fasted normal adult transgenic muscles, but was 1.9-fold higher in fasted normal young transgenic muscles compared with age-matched wild-type controls and fourfold higher in fasted adult mdx/IGF-1(C2:Ea) compared with mdx muscles. Muscles of adult mdx/IGF-1(C2:Ea) mice showed higher p70(S6K)(Thr421/Ser424) phosphorylation and both young transgenic and adult mdx/IGF-1(C2:Ea) mice had higher phosphorylation of rpS6(Ser235/236). The level of mRNA encoding myogenin was increased in normal young (but not adult) transgenic muscles, indicating enhanced myogenic differentiation. These data demonstrate that elevated IGF-1 has a hypertrophic effect on skeletal muscle only in growth situations.

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse.

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase beta is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase alpha is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb(-/-) mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A(2) in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb(-/-) mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb(-/-) mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb(-/-) mice is due to abundant choline kinase alpha and the stable homeostasis of phosphatidylcholine.

Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy.

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.