Correlation of Utrophin Levels with the Dystrophin Protein Complex and Muscle Fibre Regeneration in Duchenne and Becker Muscular Dystrophy Muscle Biopsies

Duchenne muscular dystrophy is a severe and currently incurable progressive neuromuscular condition, caused by mutations in the DMD gene that result in the inability to produce dystrophin. Lack of dystrophin leads to loss of muscle fibres and a reduction in muscle mass and function. There is evidence from dystrophin-deficient mouse models that increasing levels of utrophin at the muscle fibre sarcolemma by genetic or pharmacological means significantly reduces the muscular dystrophy pathology. In order to determine the efficacy of utrophin modulators in clinical trials, it is necessary to accurately measure utrophin levels and other biomarkers on a fibre by fibre basis within a biopsy section. Our aim was to develop robust and reproducible staining and imaging protocols to quantify sarcolemmal utrophin levels, sarcolemmal dystrophin complex members and numbers of regenerating fibres within a biopsy section. We quantified sarcolemmal utrophin in mature and regenerating fibres and the percentage of regenerating muscle fibres, in muscle biopsies from Duchenne, the milder Becker muscular dystrophy and controls. Fluorescent immunostaining followed by image analysis was performed to quantify utrophin intensity and β-dystrogylcan and ɣ –sarcoglycan intensity at the sarcolemma. Antibodies to fetal and developmental myosins were used to identify regenerating muscle fibres allowing the accurate calculation of percentage regeneration fibres in the biopsy. Our results indicate that muscle biopsies from Becker muscular dystrophy patients have fewer numbers of regenerating fibres and reduced utrophin intensity compared to muscle biopsies from Duchenne muscular dystrophy patients. Of particular interest, we show for the first time that the percentage of regenerating muscle fibres within the muscle biopsy correlate with the clinical severity of Becker and Duchenne muscular dystrophy patients from whom the biopsy was taken. The ongoing development of these tools to quantify sarcolemmal utrophin and muscle regeneration in muscle biopsies will be invaluable for assessing utrophin modulator activity in future clinical trials.     

IRES-mediated translation of utrophin A is enhanced by glucocorticoid treatment in skeletal muscle cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6alpha-methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5'UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5'UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5'UTR. Additional experiments identified a distinct region within the utrophin A 5'UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.     

Gender dimorphism influences extracellular matrix expression and regeneration of muscular tissue in mdx dystrophic mice

Mdx mouse, the animal model of Duchenne muscular dystrophy, lacks dystrophin and develops an X-linked recessive inflammatory myopathy characterized by degeneration of skeletal muscle fibers and connective tissue replacement. The present work aimed to assess whether gender dimorphism in mdx mice would influence skeletal muscle pathology at ages corresponding to main histological changes in the microenvironment of muscular tissue: myonecrosis, regeneration, and fibrosis. At the height of myonecrosis (6 weeks postnatal), skeletal muscles of male mdx mice showed increased sarcolemmal permeability, numerous inflammatory foci, and marked deposition of the extracellular matrix components (ECM) type I collagen and laminin. In contrast, age-matched mdx females showed mild ECM deposition, discrete myonecrosis, but increased numbers of regenerating fibers expressing the satellite cell marker NCAM. In contrast ovariectomized mdx females showed decreased numbers of regenerating fibers. Older (24 and 48 weeks postnatal) mdx females showed extensive fibrosis with increased sarcolemmal permeability and marked deposition of ECM components than corresponding males. These results suggest a role for female hormones in the control of myonecrosis probably by promoting regeneration of muscular tissue and mitigating inflammation especially at ages under the critical influence of sex hormones.     

Muscular nitric oxide synthase (muNOS) and utrophin.

Duchenne muscular dystrophy (DMD), the severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. Three types of treatment are envisaged: pharmacological (glucocorticoid), myoblast transplantation, and gene therapy. An alternative to the pharmacological approach is to compensate for dystrophin loss by the upregulation of another cytoskeletal protein, utrophin. Utrophin and dystrophin are part of a complex of proteins and glycoproteins, which links the basal lamina to the cytoskeleton, thus ensuring the stability of the muscle membrane. One protein of the complex, syntrophin, is associated with a muscular isoform of the neuronal nitric oxide synthase (nNOS). We have demonstrated an overexpression of utrophin, visualised by immunofluorescence and quantified by Western blotting, in normal myotubes and in mdx (the animal model of DMD) myotubes, as in normal (C57) and mdx mice, both treated with nitric oxide (NO) donor or L-arginine, the NOS substrate. There is evidence that utrophin may be capable of performing the same cellular functions as dystrophin and may functionally compensate for its lack. Thus, we propose to use NO donors, as palliative treatment of Duchenne and Becker muscular dystrophies, pending, or in combination with, gene and/or cellular therapy. Discussion has focussed on the various isoforms of NOS that could be implicated in the regeneration process. Dystrophic and healthy muscles respond to treatment, suggesting that although NOS is delocalised in the cytoplasm in the case of DMD, it conserves substantial activity. eNOS present in mitochondria and iNOS present in cytoplasm and the neuromuscular junction could also be activated. Lastly, production of NO by endothelial NOS of the capillaries would also be beneficial through increased supply of metabolites and oxygen to the muscles.     

Utrophin upregulation for treating Duchenne or Becker muscular dystrophy: how close are we?

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder for which there is currently no effective treatment. This disorder is caused by mutations or deletions in the gene encoding dystrophin that prevent expression of dystrophin at the sarcolemma. A promising pharmacological treatment for DMD aims to increase levels of utrophin, a homolog of dystrophin, in muscle fibers of affected patients to compensate for the absence of dystrophin. Here, we review recent developments in our understanding of the regulatory pathways that govern utrophin expression, and highlight studies that have used activators of these pathways to alleviate the dystrophic symptoms in DMD animal models. The results of these preclinical studies are promising and bring us closer to implementing appropriate utrophin-based drug therapies for DMD patients.     

Chemokine receptor CCR7 is expressed in muscle fibers in juvenile dermatomyositis

To better elucidate the pathogenesis of lymphocyte recruitment of memory CD4(+) T cells in inflammatory myopathies, we studied the expression of CCR5 and CCR7 on CD4 memory T cells in muscle tissue from 11 patients with juvenile dermatomyositis, six adult patients with polymyositis, two patients with Duchenne muscular dystrophy, and two patients with spinal muscular atrophy. A prevalent infiltration of CCR5(+) effector CD4 T memory cells is observed in inflammatory myopathies. Moreover, we found a strong expression of CCR7 in perifascicular atrophic and in degenerating/regenerating muscle fibers in juvenile dermatomyositis (JDM) but not in fibers from adult polymyositis and Duchenne muscular dystrophy. The selective expression of CCR7 in JDM may open new perspectives in the understanding of the pathogenesis of inflammatory myopathies, offering a new tool for the differential diagnosis of these disorders.     

Pharmacological treatments for Duchenne and Becker dystrophies

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disease affecting 1 boy out of 3500. DMD is due to the lack of a submembranous cytoskeletal protein named dystrophin, leading to the progressive degeneration of skeletal, cardiac and smooth muscle tissue. A milder form of the disease, Becker muscular dystrophy (BMD), is characterised by the presence of a semi-functional truncated dystrophin, or the full-length dystrophin at reduced level. Three different therapeutic approaches are currently under study, gene therapy, cellular therapy and pharmacological therapy. One of the chosen strategies consists of the overexpression of utrophin, a protein 80% homologous with dystrophin, and able to perform similar functions. In this review, we shall consider studies of pharmacological therapy, the aims of which can be classified in three categories: reversal of dystrophic phenotype, dystrophin expression, utrophin overexpression.     

Protein Turnover and Cellular Stress in Mildly and Severely Affected Muscles from Patients with Limb Girdle Muscular Dystrophy Type 2I

Patients with Limb girdle muscular dystrophy type 2I (LGMD2I) are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. Our aim was to investigate if wasting could be caused by impaired regeneration in the proximal compared to distal muscles. Biopsies were simultaneously obtained from proximal and distal muscles of the same patients with LGMD2I (n = 4) and healthy subjects (n = 4). The level of past muscle regeneration was evaluated by counting internally nucleated fibers and determining actively regenerating fibers by using the developmental markers embryonic myosin heavy chain (eMHC) and neural cell adhesion molecule (NCAM) and also assessing satellite cell activation status by myogenin positivity. Severe muscle histopathology was occasionally observed in the proximal muscles of patients with LGMD2I whereas distal muscles were always relatively spared. No difference was found in the regeneration markers internally nucleated fibers, actively regenerating fibers or activation status of satellite cells between proximal and distal muscles. Protein turnover, both synthesis and breakdown, as well as cellular stress were highly increased in severely affected muscles compared to mildly affected muscles. Our results indicate that alterations in the protein turnover and myostatin levels could progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.     

Oscillating kissing stem-loop interactions mediate 5' scanning-dependent translation by a viral 3'-cap-independent translation element

The 3'-untranslated regions (UTRs) of a group of novel uncapped viral RNAs allow efficient translation initiation at the 5'-proximal AUG. A well-characterized model is the Barley yellow dwarf virus class of cap-independent translation elements (BTE). It facilitates translation by forming kissing stem-loops between the BTE in the 3'-UTR and a BTE-complementary loop in the 5'-UTR. Here we investigate the mechanisms of the long-distance interaction and ribosome entry on the RNA. Upstream AUGs or 5'-extensions of the 5'-UTR inhibit translation, indicating that, unlike internal ribosome entry sites in many viral RNAs, the BTE relies on 5'-end-dependent ribosome scanning. Cap-independent translation occurs when the kissing sites are moved to different regions in either UTR, including outside of the BTE. The BTE can even confer cap-independent translation when fused to the 3'-UTR of a reporter RNA harboring dengue virus sequences that cause base-pairing between the 3'- and 5'-ends. Thus, the BTE serves as a functional sensor to detect sequences capable of long-distance base-pairing. We propose that the kissing interaction is repeatedly disrupted by the scanning ribosome and re-formed in an oscillating process that regulates ribosome entry on the RNA.