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.     

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.     

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.     

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.     

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.     

Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface

Dystrophin is the altered gene product in Duchenne muscular dystrophy (DMD). We used polyclonal antibodies against dystrophin to immunohistochemically localize the protein in human muscle. In normal individuals and in patients with myopathies other than DMD, dystrophin was localized to the sarcolemma of the fibers. The protein was absent or markedly deficient in DMD. The sarcolemmal localization of dystrophin is consistent with other evidence that there are structural and functional abnormalities of muscle surface membranes in DMD.     

Acute pathophysiological effects of muscle-expressed Dp71 transgene on normal and dystrophic mouse muscle.

products of the dystrophin gene range from the 427-kDa full-length dystrophin to the 70.8-kDa Dp71. Dp427 is expressed in skeletal muscle, where it links the actin cytoskeleton with the extracellular matrix via a complex of dystrophin-associated proteins (DAPs). Dystrophin deficiency disrupts the DAP complex and causes muscular dystrophy in humans and the mdx mouse. Dp71, the major nonmuscle product, consists of the COOH-terminal part of dystrophin, including the binding site for the DAP complex but lacks binding sites for microfilaments. Dp71 transgene (Dp71tg) expressed in mdx muscle restores the DAP complex but does not prevent muscle degeneration. In wild-type (WT) mouse muscle, Dp71tg causes a mild muscular dystrophy. In this study, we tested, using isolated extensor digitorum longus muscles, whether Dp71tg exerts acute influences on force generation and sarcolemmal stress resistance. In WT muscles, there was no effect on isometric twitch and tetanic force generation, but with a cytomegalovirus promotor-driven transgene, contraction with stretch led to sarcolemmal ruptures and irreversible loss of tension. In MDX muscle, Dp71tg reduced twitch and tetanic tension but did not aggravate sarcolemmal fragility. The adverse effects of Dp71 in muscle are probably due to its competition with dystrophin and utrophin (in MDX muscle) for binding to the DAP complex.     

Animal Models for Muscular Dystrophy Show Different Patterns of Sarcolemmal Disruption

Genetic defects in a number of components of the dystrophin–glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy^2J/dy^2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin α2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.     

Regulation of TRPC1 and TRPC4 Cation Channels Requires an ��1-Syntrophin-dependent Complex in Skeletal Mouse Myotubes

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and α1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of α1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of α1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by α1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by α1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the α1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and α1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.     

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.     

Fukutin-related protein associates with the sarcolemmal dystrophin-glycoprotein complex

Mutations in fukutin-related protein (FKRP) give rise to mild and more severe forms of muscular dystrophy. FKRP patients have reduced glycosylation of the extracellular protein dystroglycan, and FKRP itself shows sequence similarity to glycosyltransferases, implicating FKRP in the processing of dystroglycan. However, FKRP localization is controversial, and no FKRP complexes are known, so any FKRP-dystroglycan link remains elusive. Here, we demonstrate a novel FKRP localization in vivo; in mouse, both endogenous and recombinant FKRP are present at the sarcolemma. Biochemical analyses revealed that mouse muscle FKRP and dystroglycan co-enrich and co-fractionate, indicating that FKRP coexists with dystroglycan in the native dystrophin-glycoprotein complex. Furthermore, FKRP sedimentation shifts with dystroglycan in disease models involving the dystrophin-glycoprotein complex, and sarcolemmal FKRP immunofluorescence mirrors that of dystroglycan in muscular dystrophy mice, suggesting that FKRP localization may be mediated by dystroglycan. These data offer the first evidence of an FKRP complex in muscle and suggest that FKRP may influence the glycosylation status of dystroglycan from within the sarcolemmal dystrophin-glycoprotein complex.     

A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel

Disruption of the dystrophin-glycoprotein complex caused by genetic defects of dystrophin or sarcoglycans results in muscular dystrophy and/or cardiomyopathy in humans and animal models. However, the key early molecular events leading to myocyte degeneration remain elusive. Here, we observed that the growth factor-regulated channel (GRC), which belongs to the transient receptor potential channel family, is elevated in the sarcolemma of skeletal and/or cardiac muscle in dystrophic human patients and animal models deficient in dystrophin or delta-sarcoglycan. However, total cell GRC does not differ markedly between normal and dystrophic muscles. Analysis of the properties of myotubes prepared from delta-sarcoglycan-deficient BIO14.6 hamsters revealed that GRC is activated in response to myocyte stretch and is responsible for enhanced Ca2+ influx and resultant cell damage as measured by creatine phosphokinase efflux. We found that cell stretch increases GRC translocation to the sarcolemma, which requires entry of external Ca2+. Consistent with these findings, cardiac-specific expression of GRC in a transgenic mouse model produced cardiomyopathy due to Ca2+ overloading, with disease expression roughly parallel to sarcolemmal GRC levels. The results suggest that GRC is a key player in the pathogenesis of myocyte degeneration caused by dystrophin-glycoprotein complex disruption.     

Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from <Emphasis Type="Italic">mdx</Emphasis> mouse muscle

The dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.     

Ankyrin-B interactions with spectrin and dynactin-4 are required for dystrophin-based protection of skeletal muscle from exercise injury

Costameres are cellular sites of mechanotransduction in heart and skeletal muscle where dystrophin and its membrane-spanning partner dystroglycan distribute intracellular contractile forces into the surrounding extracellular matrix. Resolution of a functional costamere interactome is still limited but likely to be critical for understanding forms of muscular dystrophy and cardiomyopathy. Dystrophin binds a set of membrane-associated proteins (the dystrophin-glycoprotein complex) as well as γ-actin and microtubules and also is required to align sarcolemmal microtubules with costameres. Ankyrin-B binds to dystrophin, dynactin-4, and microtubules and is required for sarcolemmal association of these proteins as well as dystroglycan. We report here that ankyrin-B interactions with β2 spectrin and dynactin-4 are required for localization of dystrophin, dystroglycan, and microtubules at costameres as well as protection of muscle from exercise-induced injury. Knockdown of dynactin-4 in adult mouse skeletal muscle phenocopied depletion of ankyrin-B and resulted in loss of sarcolemmal dystrophin, dystroglycan, and microtubules. Moreover, mutations of ankyrin-B and of dynactin-4 that selectively impaired binary interactions between these proteins resulted in loss of their costamere-localizing activity and increased muscle fiber fragility as a result of loss of costamere-associated dystrophin and dystroglycan. In addition, costamere-association of dynactin-4 did not require dystrophin but did depend on β2 spectrin and ankyrin-B, whereas costamere association of ankyrin-B required β2 spectrin. Together, these results are consistent with a functional hierarchy beginning with β2 spectrin recruitment of ankyrin-B to costameres. Ankyrin-B then interacts with dynactin-4 and dystrophin, whereas dynactin-4 collaborates with dystrophin in coordinating costamere-aligned microtubules.     

Creatine kinase, cell membrane and Duchenne muscular dystrophy

In 1958 Professor Setsuro Ebashi found that serum creatine kinase activity is increased in patients suffering from various muscular dystrophies, especially Duchenne muscular dystrophy (DMD). He and others proposed that creatine kinase passes through the cell membrane as it is released from DMD muscle fibers.Since then, it has been found that dystrophin and dystrophin-associated proteins are connected to several other components, including the basal lamina and subsarcolemmal cytoskeletal networks on the cell membrane, while dystrophin anchors these dystrophin-associated proteins to the actin filaments inside the muscle cell. In DMD muscle, dystrophin has been found to be absent and dystroglycans and sarcoglycans decreased. However, how creatine kinase molecules can pass through the DMD muscle cell membrane still remains unanswered.On the basis of recent findings on the structure of the protein layers which sandwich the lipid bilayer of muscle cell membranes, this essay stresses the importance of these lipid bilayers in protecting creatine kinase release from protoplasma in normal muscle. It further indicates the possibility that the absence of dystrophin in DMD muscle during muscle contraction may result in temporal damage to the lipid bilayer.     

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.     

Amelioration of muscular dystrophy by transgenic expression of Niemann-Pick C1

Duchenne muscular dystrophy (DMD) and other types of muscular dystrophies are caused by the loss or alteration of different members of the dystrophin protein complex. Understanding the molecular mechanisms by which dystrophin-associated protein abnormalities contribute to the onset of muscular dystrophy may identify new therapeutic approaches to these human disorders. By examining gene expression alterations in mouse skeletal muscle lacking α-dystrobrevin (Dtna^−/−), we identified a highly significant reduction of the cholesterol trafficking protein, Niemann-Pick C1 (NPC1). Mutations in NPC1 cause a progressive neurodegenerative, lysosomal storage disorder. Transgenic expression of NPC1 in skeletal muscle ameliorates muscular dystrophy in the Dtna^−/− mouse (which has a relatively mild dystrophic phenotype) and in the mdx mouse, a model for DMD. These results identify a new compensatory gene for muscular dystrophy and reveal a potential new therapeutic target for DMD.     

Caveolin-3 directly interacts with the C-terminal tail of beta -dystroglycan. Identification of a central WW-like domain within caveolin family members

Caveolin-3, the most recently recognized member of the caveolin gene family, is muscle-specific and is found in both cardiac and skeletal muscle, as well as smooth muscle cells. Several independent lines of evidence indicate that caveolin-3 is localized to the sarcolemma, where it associates with the dystrophin-glycoprotein complex. However, it remains unknown which component of the dystrophin complex interacts with caveolin-3. Here, we demonstrate that caveolin-3 directly interacts with beta-dystroglycan, an integral membrane component of the dystrophin complex. Our results indicate that caveolin-3 co-localizes, co-fractionates, and co-immunoprecipitates with a fusion protein containing the cytoplasmic tail of beta-dystroglycan. In addition, we show that a novel WW-like domain within caveolin-3 directly recognizes the extreme C terminus of beta-dystroglycan that contains a PPXY motif. As the WW domain of dystrophin recognizes the same site within beta-dystroglycan, we also demonstrate that caveolin-3 can effectively block the interaction of dystrophin with beta-dystroglycan. In this regard, interaction of caveolin-3 with beta-dystroglycan may competitively regulate the recruitment of dystrophin to the sarcolemma. We discuss the possible implications of our findings in the context of Duchenne muscular dystrophy.     

Dystrophin is tightly associated with the sarcolemma of mammalian skeletal muscle fibers

Sarcolemmal vesicles with right-side-out configuration were prepared from normal fresh human and rabbit skeletal muscle bundles by incubation in 140 mM KCl solution containing collagenase. The vesicles were used to examine the association of dystrophin, the protein product of the Duchenne muscular dystrophy gene, with the sarcolemma. Western blot analysis, indirect immunofluorescence, and immunoperoxidase staining using specific antibodies raised against the N-terminal and the C-terminal domains show that dystrophin remains associated with the membrane of sarcolemmal vesicles. Indirect immunofluorescence microscopy using permeabilized and unpermeabilized vesicles indicated that both the N-terminus and the C-terminus of dystrophin are localized to the cytoplasmic surface of the sarcolemma. These results suggest that dystrophin has much stronger attachment to the surface membrane than it has to the internal domain of skeletal muscle fibers. Sarcolemmal vesicles thus represent a new system for studying the function of dystrophin and the molecular basis of its association with the sarcolemma.