Mild deficiency of dystrophin-associated proteins in Becker muscular dystrophy patients having in-frame deletions in the rod domain of dystrophin.

The dystrophin-glycoprotein complex spans the sarcolemma to provide a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in skeletal muscle. In Duchenne muscular dystrophy (DMD), the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins in the sarcolemma, thus causing the disruption of the dystrophin-glycoprotein complex and the loss of the linkage to the extracellular matrix. The resulting sarcolemmal instability is presumed to render muscle fibers susceptible to necrosis. In the present study, we investigated the status of the dystrophin-associated proteins in the skeletal muscle from patients with Becker muscular dystrophy (BMD), a milder allelic form of DMD. BMD patients having in-frame deletions in the rod domain of dystrophin showed a mild to moderate reduction in all of the dystrophin-associated proteins in the sarcolemma, but this reduction was not as severe as that in DMD patients. The reduction of the immunostaining for the dystrophin-associated proteins showed a good correlation with that for dystrophin in both intensity and distribution. Our results indicate that (1) the abnormality of the sarcolemmal glycoprotein complex, which is similar to but milder than that in DMD patients, also exists in these BMD patients and (2) the rod domain of dystrophin is not crucial for the interaction with the dystrophin-associated proteins.     

Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene

Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extarcellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex.     

Diagnose und Therapie der Muskeldystrophie Duchenne und Becker

Duchenne muscular dystrophy (DMD) is the most common neuromuscular disorder during childhood. It shows x-linked inheritance and is clinically characterized by progressive muscle weakness starting during infancy and leading to loss of ambulation during early adolescence. Serum creatine kinase levels are markedly elevated and the diagnosis is confirmed through genetic testing of the dystrophin gene or a muscle biopsy. Therapy of DMD necessitates a multidisciplinary approach. Use of medications, physiotherapy, orthopaedic care and non-invasive ventilation can markedly improve life expectancy and quality of life. Causative treatment strategies are in clinical development, but it is too early for a conclusion about their effectiveness. Becker muscular dystrophy is a much rarer allelic disorder with partial dystrophin deficiency and a milder clinical phenotype.     

Expression of dystrophin on the cell surface membrane of intrafusal fibers of human skeletal muscle

Summary We examined the morphological expression of dystrophin in the intrafusal muscle fibers in skeletal muscle from normal human and Duchenne muscular dystrophy (DMD) patients, using antisera against the N-terminal and C-terminal regions of dystrophin. The intrafusal fibers of normal muscle express dystrophin on their cell surface membrane, but those of DMD muscle do not.Abbreviation DMD Duchenne muscular dystrophy     

Exploration of Lipid Metabolism in Relation with Plasma Membrane Properties of Duchenne Muscular Dystrophy Cells: Influence of L-Carnitine

Duchenne muscular dystrophy (DMD) arises as a consequence of mutations in the dystrophin gene. Dystrophin is a membrane-spanning protein that connects the cytoskeleton and the basal lamina. The most distinctive features of DMD are a progressive muscular dystrophy, a myofiber degeneration with fibrosis and metabolic alterations such as fatty infiltration, however, little is known on lipid metabolism changes arising in Duchenne patient cells. Our goal was to identify metabolic changes occurring in Duchenne patient cells especially in terms of L-carnitine homeostasis, fatty acid metabolism both at the mitochondrial and peroxisomal level and the consequences on the membrane structure and function. In this paper, we compared the structural and functional characteristics of DMD patient and control cells. Using radiolabeled L-carnitine, we found, in patient muscle cells, a marked decrease in the uptake and the intracellular level of L-carnitine. Associated with this change, a decrease in the mitochondrial metabolism can be seen from the analysis of mRNA encoding for mitochondrial proteins. Probably, associated with these changes in fatty acid metabolism, alterations in the lipid composition of the cells were identified: with an increase in poly unsaturated fatty acids and a decrease in medium chain fatty acids, mono unsaturated fatty acids and in cholesterol contents. Functionally, the membrane of cells lacking dystrophin appeared to be less fluid, as determined at 37°C by fluorescence anisotropy. These changes may, at least in part, be responsible for changes in the phospholipids and cholesterol profile in cell membranes and ultimately may reduce the fluidity of the membrane. A supplementation with L-carnitine partly restored the fatty acid profile by increasing saturated fatty acid content and decreasing the amounts of MUFA, PUFA, VLCFA. L-carnitine supplementation also restored muscle membrane fluidity. This suggests that regulating lipid metabolism in DMD cells may improve the function of cells lacking dystrophin.     

The molecular basis of activity-induced muscle injury in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common of the human muscular dystrophies, affecting approximately 1 in 3500 boys. Most DMD patients die in their late teens or early twenties due to involvement of the diaphragm and other respiratory muscles by the disease. The primary abnormality in DMD is an absence of dystrophin, a 427 kd protein normally found at the cytoplasmic face of the muscle cell surface membrane. Based upon the predicted structure and location of the protein, it has been proposed that dystrophin plays an important role in providing mechanical reinforcement to the sarcolemmal membrane of muscle fibers. Therefore, dystrophin could help to protect muscle fibers from potentially damaging tissue stresses developed during muscle contraction. In the present paper, the nature of mechanical stresses placed upon myofibers during various forms of muscle contraction are reviewed, along with current lines of evidence supporting a critical role for dystrophin as a subsarcolemmal membrane-stabilizing protein in this setting. In addition, the implications of these findings for exercise programs and other potential forms of therapy in DMD are discussed.     

Current knowledge of dystrophin and dystrophin-associated proteins in the retina

Dramatical development of molecular genetics has been disclosing the molecular mechanism of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). DMD gene product, dystrophin, is a submembranous cytoskeletal protein and many dystrophin-associated proteins (DAPs) have been identified, such as utrophin, dystroglycans, sarcoglycans, syntrophins and dystrobrevins. Dystrophin and DAPs are very important proteins not only for skeletal, cardiac, or smooth muscles but also for peripheral and central nervous systems including the retina. The retina has been extensively examined to demonstrate that dystrophin and beta-dystroglycan localize at the photoreceptor terminal, and their deficiency produces the abnormal neurotransmission between photoreceptor cells and ON-bipolar cells. Dystrophin has seven isoforms in variable tissues, and the retina contains full-length dystrophin (Dp427), Dp260, and Dp71. Recent studies have demonstrated that Dp71 localizes in the inner limiting membrane (INL) and around the blood vessel, and Dp260 is expressed in the outer plexiform layer (OPL). beta-dystroglycan is also expressed in the same regions as well as dystrophin, but it remains unclear whether other DAPs are expressed in the retina or not. It is generally assumed that dystrophin functions to stabilize muscle fibers with DAPs by linking the sarcolemma to the basement membrane, but its function in the retina is totally unknown so far.     

Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice

Dystrophin, the protein product of the human Duchenne muscular dystrophy gene, exists in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kD and a protein of 59 kD. Here, we investigated the relative abundance of each of the components of the dystrophin-glycoprotein complex in skeletal muscle from normal and mdx mice, which are missing dystrophin. Immunoblot analysis using total muscle membranes from control and mdx mice of ages 1 d to 30 wk found that all of the dystrophin-associated proteins were greatly reduced (80-90%) in mdx mouse skeletal muscle. The specificity of the loss of the dystrophin-associated glycoproteins was demonstrated by the finding that the major glycoprotein composition of skeletal muscle membranes from normal and mdx mice was identical. Furthermore, skeletal muscle membranes from the dystrophic dy/dy mouse exhibited a normal density of dystrophin and dystrophin-associated proteins. Immunofluorescence microscopy confirmed the results from the immunoblot analysis and showed a drastically reduced density of dystrophin-associated proteins in mdx muscle cryosections compared with normal and dy/dy mouse muscle. Therefore, our results demonstrate that all of the dystrophin-associated proteins are significantly reduced in mdx skeletal muscle and suggest that the loss of dystrophin-associated proteins is due to the absence of dystrophin and not due to secondary effects of muscle fiber degradation.     

Bortezomib (PS-341) Treatment Decreases Inflammation and Partially Rescues the Expression of the Dystrophin-Glycoprotein Complex in GRMD Dogs

Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane.     

Dystrophin Dp71: The Smallest but Multifunctional Product of the Duchenne Muscular Dystrophy Gene

Dystrophin Dp71 is expressed in all tissues, with the exception of skeletal muscle, and is the main Duchenne muscular dystrophy (DMD) gene product in brain. As full-length dystrophin does in skeletal muscle, Dp71 associates with dystroglycans, sarcoglycans, dystrobrevins, syntrophins, and accessory proteins to form the dystrophin-associated protein complex (DAPC) in non-muscle tissues. Although it has been nearly 20 years since the discovery of Dp71, its study has become relevant only recently due to its direct involvement with the two main DMD non-muscular phenotypes: cognitive impairment and abnormal retinal physiology. In this review, we describe the historical background of Dp71 and the experimental models developed for its study. Additionally, we present and discuss the experimental evidence supporting the participation of Dp71 in different cellular processes, including cell adhesion, water homeostasis, cell division, and nuclear architecture. The functional diversity of Dp71 is attributed to the formation of Dp71-containing DAPC in numerous cell types and different subcellular compartments, including in plasma membrane and nucleus, as well as to the capability of Dp71-containing DAPC to work as the scaffold for proper clustering and anchoring of structural and signaling proteins to the plasma membrane and of nuclear envelope proteins to the inner nuclear membrane.     

Immunocytochemical study of dystrophin in cultured mouse muscle cells by the quick-freezing and deep-etching method

Summary Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is deficient in patients with DMD and in mdx mice. It is immunocytochemically localized in skeletal muscle sarcolemma. However, little is known about the three-dimensional ultrastructural localization of dystrophin and its relationship with other cytoskeletal proteins. We found that dystrophin is localized irregularly, just underneath the plasma membrane in normal cultured mouse myotubes, by using the quick-freezing and deep-etching (QF-DE) method; it was found to be closely linked to actin-like filaments (8–10 nm in diameter), most of which were decorated with myosin subfragment 1, and was attached to the cytoplasmic side of the plasma membrane. These results suggest that dystrophin might play an important role in the preservation of cell membrane stability by connecting actin cytoskeletons with the cytoplasmic side of the plasma membrane.     

Dystrophin analysis in duchenne muscular dystrophy: use in fetal diagnosis and in genetic counseling.

In this report we describe the use of dystrophin analysis both in the diagnosis of Duchenne muscular dystrophy (DMD) in an aborted fetus and in genetic counseling. Our consultand's initial carrier risk, as based on family history and creatine kinase determinations, was calculated as 0.6%. DNA analysis of her family (and fetus) modified this risk to 8.5%. Skeletal muscle of the 23-wk male abortus was found to be histologically indistinguishable from that of age-matched controls. However, immunoblot testing for dystrophin indicated that the fetus had indeed inherited dystrophin deficiency. The carrier risk of the consultand was thus elevated to 100%. Dystrophin assays should be employed whenever the diagnosis of fetal DMD is equivocal (e.g., cases in which a gene deletion cannot be identified). Assay results are crucial for genetic counseling for subsequent pregnancies and for studies of the early pathogenesis of muscular dystrophy.     

Human multipotent adipose-derived stem cells restore dystrophin expression of Duchenne skeletal-muscle cells in vitro.

BACKGROUND INFORMATION: DMD (Duchenne muscular dystrophy) is a devastating X-linked disorder characterized by progressive muscle degeneration and weakness. The use of cell therapy for the repair of defective muscle is being pursued as a possible treatment for DMD. Mesenchymal stem cells have the potential to differentiate and display a myogenic phenotype in vitro. Since liposuctioned human fat is available in large quantities, it may be an ideal source of stem cells for therapeutic applications. ASCs (adipose-derived stem cells) are able to restore dystrophin expression in the muscles of mdx (X-linked muscular dystrophy) mice. However, the outcome when these cells interact with human dystrophic muscle is still unknown. RESULTS: We show here that ASCs participate in myotube formation when cultured together with differentiating human DMD myoblasts, resulting in the restoration of dystrophin expression. Similarly, dystrophin was induced when ASCs were co-cultivated with DMD myotubes. Experiments with GFP (green fluorescent protein)-positive ASCs and DAPI (4',6-diamidino-2-phenylindole)-stained DMD myoblasts indicated that ASCs participate in human myogenesis through cellular fusion. CONCLUSIONS: These results show that ASCs have the potential to interact with dystrophic muscle cells, restoring dystrophin expression of DMD cells in vitro. The possibility of using adipose tissue as a source of stem cell therapies for muscular diseases is extremely exciting.     

Mechanical function of dystrophin in muscle cells

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.     

Dystrophin and Utrophin Complexed with Different Associated Proteins in Cardiac Purkinje Fibres

Abnormal dystrophin expression is directly responsible for Duchenne and Becker muscular dystrophies. In skeletal muscle, dystrophin provides a link between the actin network and the extracellular matrix via the dystrophin-associated protein complex. In mature skeletal muscle, utrophin is a dystrophin-related protein localized mainly at the neuromuscular junction, with the same properties as dystrophin in terms of linking the protein complex. Utrophin could potentially overcome the absence of dystrophin in dystrophic skeletal muscles. In cardiac muscle, dystrophin and utrophin were both found to be present with a distinct subcellular distribution in Purkinje fibres, i.e. utrophin was limited to the cytoplasm, while dystrophin was located in the cytoplasmic membrane.In this study, we used this particular characteristic of cardiac Purkinje fibres and demonstrated that associated proteins of dystrophin and utrophin are different in this structure. We conclude, contrary to skeletal muscle, dystrophin-associated proteins do not form a complex in Purkinje fibres. In addition, we have indirect evidence of the presence of two different 400kDa dystrophins in Purkinje fibres.     

Membrane organization of the dystrophin-glycoprotein complex

The stoichiometry, cellular location, glycosylation, and hydrophobic properties of the components in the dystrophin-glycoprotein complex were examined. The 156, 59, 50, 43, and 35 kd dystrophin-associated proteins each possess unique antigenic determinants, enrich quantitatively with dystrophin, and were localized to the skeletal muscle sarcolemma. The 156, 50, 43, and 35 kd dystrophin-associated proteins contained Asn-linked oligosaccharides. The 156 kd dystrophin-associated glycoprotein contained terminally sialylated Ser/Thr-linked oligosaccharides. Dystrophin, the 156 kd, and the 59 kd dystrophin-associated proteins were found to be peripheral membrane proteins, while the 50 kd, 43 kd, and 35 kd dystrophin-associated glycoproteins and the 25 kd dystrophin-associated protein were confirmed as integral membrane proteins. These results demonstrate that dystrophin and its 59 kd associated protein are cytoskeletal elements that are tightly linked to a 156 kd extracellular glycoprotein by way of a complex of transmembrane proteins.     

Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle

Duchenne's muscular dystrophy (DMD) is caused by the absence or drastic decrease of the structural protein, dystrophin, and is characterized by sarcolemmal lesions in skeletal muscle due to the stress of contraction. Dystrophin has been localized to the sarcolemma, but its organization there is not known. We report immunofluorescence studies which show that dystrophin is concentrated, along with the major muscle isoform of beta-spectrin, in three distinct domains at the sarcolemma: in elements overlying both I bands and M lines, and in occasional strands running along the longitudinal axis of the myofiber. Vinculin, which has previously been found at the sarcolemma overlying the I bands and in longitudinal strands, was present in the same three structures as spectrin and dystrophin. Controls demonstrated that the labeling was intracellular. Comparison to labeling of the lipid bilayer and of the extracellular matrix showed that the labeling for spectrin and dystrophin is associated with the intact sarcolemma and is not a result of processing artifacts. Dystrophin is not required for this lattice-like organization, as similar domains containing spectrin but not dystrophin are present in muscle from the mdx mouse and from humans with Duchenne's muscular dystrophy. We discuss the possibility that dystrophin and spectrin, along with vinculin, may function to link the contractile apparatus to the sarcolemma of normal skeletal muscle.     

Neural integrity is maintained by dystrophin in C. elegans

The dystrophin protein complex (DPC), composed of dystrophin and associated proteins, is essential for maintaining muscle membrane integrity. The link between mutations in dystrophin and the devastating muscle failure of Duchenne's muscular dystrophy (DMD) has been well established. Less well appreciated are the accompanying cognitive impairment and neuropsychiatric disorders also presented in many DMD patients, which suggest a wider role for dystrophin in membrane-cytoskeleton function. This study provides genetic evidence of a novel role for DYS-1/dystrophin in maintaining neural organization in Caenorhabditis elegans. This neuronal function is distinct from the established role of DYS-1/dystrophin in maintaining muscle integrity and regulating locomotion. SAX-7, an L1 cell adhesion molecule (CAM) homologue, and STN-2/γ-syntrophin also function to maintain neural integrity in C. elegans. This study provides biochemical data that show that SAX-7 associates with DYS-1 in an STN-2/γ-syntrophin-dependent manner. These results reveal a recruitment of L1CAMs to the DPC to ensure neural integrity is maintained.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.