Identification of New Dystroglycan Complexes in Skeletal Muscle

The dystroglycan complex contains the transmembrane protein β-dystroglycan and its interacting extracellular mucin-like protein α-dystroglycan. In skeletal muscle fibers, the dystroglycan complex plays an important structural role by linking the cytoskeletal protein dystrophin to laminin in the extracellular matrix. Mutations that affect any of the proteins involved in this structural axis lead to myofiber degeneration and are associated with muscular dystrophies and congenital myopathies. Because loss of dystrophin in Duchenne muscular dystrophy (DMD) leads to an almost complete loss of dystroglycan complexes at the myofiber membrane, it is generally assumed that the vast majority of dystroglycan complexes within skeletal muscle fibers interact with dystrophin. The residual dystroglycan present in dystrophin-deficient muscle is thought to be preserved by utrophin, a structural homolog of dystrophin that is up-regulated in dystrophic muscles. However, we found that dystroglycan complexes are still present at the myofiber membrane in the absence of both dystrophin and utrophin. Our data show that only a minority of dystroglycan complexes associate with dystrophin in wild type muscle. Furthermore, we provide evidence for at least three separate pools of dystroglycan complexes within myofibers that differ in composition and are differentially affected by loss of dystrophin. Our findings indicate a more complex role of dystroglycan in muscle than currently recognized and may help explain differences in disease pathology and severity among myopathies linked to mutations in DAPC members.     

The emerging family of dystrophin-related proteins

Duchenne and Becker muscular dystrophies are caused by mutations in the gene encoding dystrophin, a component of the subsarcolemmal cytoskeleton. Dystrophin-related proteins are identical or homologous to the cysteine-rich and C-terminal domains of dystrophin. This part of dystrophin binds to a membrane-spanning glycoprotein complex in muscle. At least five dystrophin-related proteins are encoded by the Duchenne muscular dystrophy locus. These proteins are found in many non-muscle tissues where dystrophin is not expressed and they are thought to be membrane-associated. Two other dystrophin-related proteins--utrophin and an 87 kDa postsynaptic protein--are encoded by separate loci and, like dystrophin, they are components of the neuromuscular junction.     

Calmodulin-binding profiles for nebulin and dystrophin in human skeletal muscle

Nebulin and dystrophin are two high-molecular-mass skeletal muscle proteins that have both been associated with the defective gene in Duchenne muscular dystrophy, although the function of neither protein is known. Other high-molecular-mass, calmodulin-binding proteins have recently been implicated in regulating calcium release from skeletal muscle. Western blots of human skeletal muscle biopsy samples were probed with biotinylated calmodulin; nebulin was identified as a prominent high-molecular-mass calmodulin-binding protein but dystrophin did not bind detectable amounts of biotinylated calmodulin. Dystrophin was absent in a Duchenne muscle biopsy.     

Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice

In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of?MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.     

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     

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.     

Just say NO to muscle degeneration?

The devastating muscle degeneration characteristic of Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. The dystrophin complex has two functions: a structural role in maintaining sarcolemmal integrity during contraction and a scaffolding function that recruits signaling proteins such as neuronal nitric oxide synthase to the membrane. New studies indicate that transgenic restoration of nitric oxide (NO) production in the mdx dystrophic mouse improves muscle pathology. Although NO-mediated killing of inflammatory cells might be involved, other mechanisms are also possible. These results point to the therapeutic potential of manipulating the signaling activity of the dystophin complex as a way to ameliorate the progression of muscle degeneration.     

Muscular degeneration in the absence of dystrophin is a calcium-dependent process.

Duchenne muscular dystrophy (DMD) is a progressive degenerative muscular disease that is due to mutations in the dystrophin gene. Neither the function of dystrophin nor the physiopathology of the disease have been clearly established yet. Several groups have reported elevated calcium concentrations in the mdx mouse model of DMD, but the effect of calcium levels on the progression of the disease continues to be a matter of debate. Here, we show that, in Caenorhabditis elegans, a gain-of-function mutation in the egl-19 calcium channel gene dramatically increases muscle degeneration in dystrophin mutants. Conversely, RNAi-mediated inhibition of egl-19 function reduces muscle degeneration by half. Therefore, our results demonstrate that calcium channel activity is a critical factor in the progression of dystrophin-dependent muscle degeneration.     

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.     

Muscular Dystrophy in mdx Mice Despite Lack of Neuronal Nitric Oxide Synthase

Abstract: Neuronal nitric oxide synthase (nNOS) is a component of the dystrophin complex in skeletal muscle. The absence of dystrophin protein in Duchenne muscular dystrophy and in mdx mouse causes a redistribution of nNOS from the plasma membrane to the cytosol in muscle cells. Aberrant nNOS activity in the cytosol can induce free radical oxidation, which is toxic to myofibers. To test the hypothesis that derangements in nNOS disposition mediate muscle damage in Duchenne dystrophy, we bred dystrophin-deficient mdx male mice and female mdx heterozygote mice that lack nNOS. We found that genetic deletion of nNOS does not itself cause detectable pathology and that removal of nNOS does not influence the extent of increased sarcolemmal permeability in dystrophin-deficient mice. Thus, histological analyses of nNOS-dystrophin double mutants show pathological changes similar to the dystrophin mutation alone. Taken together, nNOS defects alone do not produce muscular dystrophy in the mdx model.     

DNA damage, somatic aneuploidy, and malignant sarcoma susceptibility in muscular dystrophies

Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD-pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD-gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases.     

Problems and solutions in myoblast transfer therapy

Duchenne muscular dystrophy is a severe X-linked neuromuscular disease that affects approximately 1/3500 live male births in every human population, and is caused by a mutation in the gene that encodes the muscle protein dystrophin. The characterization and cloning of the dystrophin gene in 1987 was a major breakthrough and it was considered that simple replacement of the dystrophin gene would ameliorate the severe and progressive skeletal muscle wasting characteristic of Duchenne muscular dystrophy. After 20 years, attempts at replacing the dystrophin gene either experimentally or clinically have met with little success, but there have been many significant advances in understanding the factors that limit the delivery of a normal dystrophin gene into dystrophic host muscle. This review addresses the host immune response and donor myoblast changes underlying some of the major problems associated with myoblast-mediated dystrophin replacement, presents potential solutions, and outlines other novel therapeutic approaches.     

Sarcoglycan complex: a muscular supporter of dystroglycan-dystrophin interplay?

In striated muscle, the cytoskeletal protein dystrophin, the protein product of the Duchenne muscular dystrophy gene, is associated with a number of sarcolemmal glycoproteins to form a large oligomeric complex, the dystrophin-glycoprotein complex (DGC). Over the last 10 years, four of these sarcolemmal glycoproteins, alpha-, beta-, gamma- and delta-sarcoglycans, have been shown to form a distinct subcomplex, the sarcoglycan complex, in the DGC. Furthermore, the genetic defects of alpha-, beta-, gamma- and delta-sarcoglycans have been identified as the causes of four distinct forms of muscular dystrophies, which are now collectively called sarcoglycanopathy. Current studies are beginning to focus on the biological functions of the sarcoglycan complex and the molecular mechanism by which its dysfunction leads to muscle cell degeneration.     

Immunological detection of the dystrophin molecule with antibody directed against the synthetic peptide.

We synthesized a peptide designated R8 (amino acid residues 1157-1201) based on the primary structure presumed from the nucleotide sequence of the cDNA clone from the gene for Duchenne muscular dystrophy. Antibody to the synthetic R8 generated by immunization of rabbits was tested on human and mouse skeletal muscle by Western blotting analysis. The antibody reacted with a component of the 400K dystrophin of normal human and mouse skeletal muscles, but not with components of the muscles of Duchenne muscular dystrophy patients and mdx mice. Thus we established that this peptide sequence is in fact missing in the protein product 'dystrophin' encoded by the DMD gene. The antibody may prove useful for the diagnosis of the Duchenne types of muscular dystrophy.     

A Splice Site Mutation in Laminin-α2 Results in a Severe Muscular Dystrophy and Growth Abnormalities in Zebrafish

Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501), exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.     

Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.