Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency-force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency-force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program

Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.

HSP72 preserves muscle function and slows progression of severe muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin¹, ², ³. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca²⁺, which activates inflammatory and muscle degenerative pathways⁴, ⁵, ⁶. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death⁷, ⁸, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca²⁺) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

Stimulation of calcineurin Aα activity attenuates muscle pathophysiology in mdx dystrophic mice

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin A transgene (CnA) was overexpressed in skeletal muscles of mdx (mdx CnA*) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnA* and mdx mice was examined in vitro. Hindlimb muscles from mdx CnA* mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnA* than mdx mice. In the diaphragm, despite a slower phenotype and a 35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnA* compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity.

The calcineurin signal transduction pathway is essential for successful muscle regeneration in mdx dystrophic mice

Although mdx mice share the same genetic defect and lack dystrophin expression as in Duchenne muscular dystrophy (DMD), their limb muscles have a high regenerative capacity that ensures a more benign phenotype and essentially normal function. The cellular pathways responsible for this enhanced regenerative capacity are unknown. We tested the hypothesis that the calcineurin signal transduction pathway is essential for the successful regeneration following severe degeneration observed in the limb muscles of young mdx mice (2–4 weeks old) and that inhibition of this pathway using cyclosporine A (CsA) would exacerbate the dystrophic pathology. Eighteen-day-old mdx and C57BL/10 mice were treated with CsA for 16 days. CsA administration severely disrupted muscle regeneration in mdx mice, but had minimal effect in C57BL/10 mice. Muscles from CsA-treated mdx mice had fewer centrally nucleated fibers and extensive collagen, connective tissue, and mononuclear cell infiltration than muscles from vehicle-treated littermates. The deleterious effects of CsA on muscle morphology were accompanied by a 30–35% decrease in maximal force producing capacity. Taken together, these observations indicate that the calcineurin signal transduction pathway is a significant determinant of successful skeletal muscle regeneration in young mdx mice. Up-regulating this pathway may have clinical significance for DMD.

G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle

Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In mdx mice, a model of human Duchenne muscular dystrophy (DMD), G-CSF mRNA and protein was down-regulated in limb and diaphragm muscle, but circulating G-CSF ligand levels were elevated. G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy.

MicroRNAs in skeletal muscle and their regulation with exercise, ageing, and disease

Skeletal muscle makes up approximately 40% of the total body mass, providing structural support and enabling the body to maintain posture, to control motor movements and to store energy. It therefore plays a vital role in whole body metabolism. Skeletal muscle displays remarkable plasticity and is able to alter its size, structure and function in response to various stimuli; an essential quality for healthy living across the lifespan. Exercise is an important stimulator of extracellular and intracellular stress signals that promote positive adaptations in skeletal muscle. These adaptations are controlled by changes in gene transcription and protein translation, with many of these molecules identified as potential therapeutic targets to pharmacologically improve muscle quality in patient groups too ill to exercise. MicroRNAs (miRNAs) are recently identified regulators of numerous gene networks and pathways and mainly exert their effect by binding to their target messenger RNAs (mRNAs), resulting in mRNA degradation or preventing protein translation. The role of exercise as a regulatory stimulus of skeletal muscle miRNAs is now starting to be investigated. This review highlights our current understanding of the regulation of skeletal muscle miRNAs with exercise and disease as well as how they may control skeletal muscle health.

Differential calcineurin signalling activity and regeneration efficacy in diaphragm and limb muscles of dystrophic mdx mice

Calcineurin activity is essential for successful skeletal muscle regeneration in young mdx mice and in wild type mice following myotoxic injury and cryodamage. In mature myofibres of adult mdx mice, calcineurin stimulation can ameliorate the dystrophic pathology. The aim of this study was to test the hypothesis that the more severe dystrophic pathology of the diaphragm compared with hindlimb muscles of mdx mice could be attributed to aberrant calcineurin signalling and that due to ongoing regeneration calcineurin activity would be greater in muscles of adult mdx than wild type mice. Differences in markers of regeneration between tibialis anterior and diaphragm muscles were also characterised, to determine whether there was an association between regeneration efficacy and calcineurin activity in dystrophic muscles. In diaphragm muscles of adult mdx mice, the proportion of centrally nucleated fibres and developmental myosin heavy chain protein expression was lower and myogenin protein expression was higher than in tibialis anterior muscles. Calcineurin and activated NFATc1 protein content and calcineurin phosphatase activity were higher in muscles from mdx than wild type mice and calcineurin activation was greater in diaphragm than tibialis anterior muscles of mdx mice. Thus, despite greater calcineurin activity in diaphragm compared to hindlimb muscles, regeneration events downstream of myoblast differentiation and mediated by the injured myofibre were severely compromised.

Versican processing by a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats proteinases-5 and -15 facilitates myoblast fusion

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.

Pursuit of the muscular ideal: Physical and psychological consequences and putative risk factors

Developing a lean muscular figure for the purposes of sports and/or appearance has become a central issue for males. Concern has been raised because the desire to develop such a body build may lead to the adoption of numerous health-threatening behaviors. Consequently, this review presents a comprehensive analysis of the physical and psychological consequences that result from the use of steroids (legal and illegal), ephedrine, and deleterious dieting strategies specific to males. Putative risk factors for these behaviors will be identified, and the clinical disorder associated with the extreme abuse of these behaviors, muscle dysmorphia, will be examined.

Joint range of motion limitations in children and young adults with spinal muscular atrophy

Objectives
To elicit descriptive data about limited joint range of motion (ROM) in subjects with type II or III spinal muscular atrophy (SMA) and to examine the relation between the number of motions with limited range and both age and functional ability.
Design
Descriptive cross-sectional study.
Setting
Neurologic pediatric outpatient clinic at a hospital in Taiwan.
Participants
Twenty-seven subjects with SMA type II (mean age, 9.8±6.5y) and 17 with SMA type III (mean age, 12.2±8.7y).
Intervention
Measurement with transparent goniometers of joint ROM bilaterally of the shoulder, elbow, wrist, hip, knee, and ankle.
Main outcome measures
The proportion of participants with each ROM limitation compared with all participants with the same SMA type, age distribution of the participants with each ROM limitation, mean range loss of each motion limitation, and the contracture index (risk index of joint contracture).
Results
Eighty-nine percent of the participants with SMA type II experienced knee extension limitation. Approximately 50% of the participants with both types of SMA had ankle dorsiflexion limitation. The motions of knee and hip extension and ankle dorsiflexion also had a relatively high contracture index. The number of motions with limited range positively correlated (P<.001) with age and upper-extremity functional grade (the higher the functional grade, the poorer the functional ability) for SMA type II.
Conclusions
We found varying degrees of joint ROM limitation. Certain motions were noted to be high risks for the development of contractures. This risk was higher mostly in younger children.

Effects of systemic hypoxia on human muscular adaptations to resistance exercise training

Hypoxia is an important modulator of endurance exercise-induced oxidative adaptations in skeletal muscle. However, whether hypoxia affects resistance exercise-induced muscle adaptations remains unknown. Here, we determined the effect of resistance exercise training under systemic hypoxia on muscular adaptations known to occur following both resistance and endurance exercise training, including muscle cross-sectional area (CSA), one-repetition maximum (1RM), muscular endurance, and makers of mitochondrial biogenesis and angiogenesis, such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), citrate synthase (CS) activity, nitric oxide synthase (NOS), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF-1), and capillary-to-fiber ratio. Sixteen healthy male subjects were randomly assigned to either a normoxic resistance training group (NRT, n = 7) or a hypoxic (14.4% oxygen) resistance training group (HRT, n = 9) and performed 8 weeks of resistance training. Blood and muscle biopsy samples were obtained before and after training. After training muscle CSA of the femoral region, 1RM for bench-press and leg-press, muscular endurance, and skeletal muscle VEGF protein levels significantly increased in both groups. The increase in muscular endurance was significantly higher in the HRT group. Plasma VEGF concentration and skeletal muscle capillary-to-fiber ratio were significantly higher in the HRT group than the NRT group following training. Our results suggest that, in addition to increases in muscle size and strength, HRT may also lead to increased muscular endurance and the promotion of angiogenesis in skeletal muscle.