Expression of recombinant dystrophin and its localization to the cell membrane.

Duchenne's muscular dystrophy (DMD) is an X-linked progressive myopathy caused by a defect in the DMD gene locus. The gene corresponding to the DMD locus produces a 14-kilobase (kb) messenger RNA that codes for a large cytoskeletal membrane protein, dystrophin. DMD and Becker's muscular dystrophy are the consequences of dystrophin mutations. The exact biological function of dystrophin remains unknown but it has been demonstrated that it is localized to the cytoplasmic face of the cell membrane and has direct interaction with several other membrane proteins. We report here the synthesis of a 14-kb full-length complementary DNA for the mouse muscle dystrophin mRNA and the expression of this cDNA in COS cells. The recombinant dystrophin is indistinguishable from mouse muscle dystrophin by western blot analysis with anti-dystrophin antibodies and was shown by an immunofluorescent technique to be localized in the cell membrane. Our successful construction of a functional full-length cDNA opens opportunities for the study of structure and function of dystrophin and provides an opportunity to initiate gene therapy studies.     

Deficiency of the 50K dystrophin-associated glycoprotein in severe childhood autosomal recessive muscular dystrophy.

X-linked recessive Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoprotein. The dystrophin-glycoprotein complex has been proposed to span the sarcolemma to provide a link between the subsarcolemmal cytoskeleton and the extracellular matrix component, laminin. In DMD, the absence of dystrophin leads to a large reduction in all of the dystrophin-associated protein. We have investigated the possibility that a deficiency of a dystrophin-associated protein could be the cause of severe childhood autosomal recessive muscular dystrophy (SCARMD) with a DMD-like phenotype. Here we report the specific deficiency of the 50K dystrophin-associated glycoprotein (M(r) 50,000) in sarcolemma of SCARMD patients. Therefore, the loss of this glycoprotein is a common denominator of the pathological process leading to muscle cell necrosis in two forms of muscular dystrophy, DMD and SCARMD.     

The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle

Duchenne muscular dystrophy (DMD) and its milder form, Becker muscular dystrophy (BMD), are allelic X-linked muscle disorders in man. The gene responsible for the disease has been cloned from knowledge of its map location at band Xp21 on the short arm of the X chromosome. The product of the DMD gene, a protein of relative molecular mass 400,000 (Mr 400K) recently named dystrophin, has been reported to co-purify with triads of mouse and rabbit skeletal muscle when assayed using polyclonal antibodies raised against fusion proteins encoded by regions of mouse DMD complementary DNA. Here we show that antibodies directed against synthetic peptides and fusion proteins derived from the N-terminal region of human DMD cDNA strongly react with an antigen present in skeletal muscle sarcolemma on cryostat sections of normal human muscle biopsies. This immunoreactivity is reduced or absent in muscle fibres from DMD patients but appears normal in muscle fibres from patients with other myopathic diseases. The same antibodies specifically react with a 400K protein in sodium dodecyl sulphate (SDS) extracts of normal human muscle subjected to Western blot analysis. We conclude that the product of the DMD gene is associated with the sarcolemma rather than with the triads and speculate that it strengthens the sarcolemma by anchoring elements of the internal cytoskeleton to the surface membrane.     

Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs

Duchenne's muscular dystrophy (DMD), which affects one in 3,500 males, causes progressive myopathy of skeletal and cardiac muscles and premature death. One approach to treatment would be to introduce the normal dystrophin gene into diseased muscle cells. When pure plasmid DNA is injected into rodent skeletal or cardiac muscle, the cells express reporter genes. We now show that a 12-kilobase full-length human dystrophin complementary DNA gene and a 6.3-kilobase Becker-like gene can be expressed in cultured cells and in vivo. When the human dystrophin expression plasmids are injected intramuscularly into dystrophin-deficient mdx mice, the human dystrophin proteins are present in the cytoplasm and sarcolemma of approximately 1% of the myofibres. Myofibres expressing human dystrophin contain an increased proportion of peripheral nuclei. The results indicate that transfer of the dystrophin gene into the myofibres of DMD patients could be beneficial, but a larger number of genetically modified myofibres will be necessary for clinical efficacy.     

Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle.

Dystrophin is associated with a complex of muscle membrane (sarcolemmal) glycoproteins that provide a linkage to the extracellular matrix protein, laminin. The absence of dystrophin leads to a dramatic reduction of the dystrophin-associated proteins (156DAG, 59DAP, 50DAG, 43DAG and 35DAG) in the sarcolemma of patients with Duchenne muscular dystrophy and mdx mice. Here we demonstrate that dystrophin-related protein (DRP, utrophin), an autosomal homologue of dystrophin, is associated with an identical or antigenically similar complex of sarcolemmal proteins and that DRP and the dystrophin/DRP-associated proteins colocalize to the neuromuscular junction in Duchenne muscular dystrophy and mdx muscle. The DRP and dystrophin/DRP-associated proteins are found throughout the sarcolemma in small-calibre skeletal muscles and cardiac muscle of adult mdx mice. Because these muscles show minimal pathological changes, our results could provide a basis for the upregulation of DRP as a potential therapeutic approach.     

Localization of dystrophin to postsynaptic regions of central nervous system cortical neurons

Moderate non-progressive cognitive impairment is a consistent feature of Duchenne muscular dystrophy (DMD), although no central nervous system (CNS) abnormality has been identified. Recent studies have elucidated the molecular defect in DMD, including the absence of the protein dystrophin in affected individuals. Normal brain tissue contains dystrophin messenger RNA and dystrophin is present in low abundance in the brain and seems to be regulated in this tissue, at least in part, by a promoter that differs from that in muscle. Until now, antibodies and immunocytochemical methods used to demonstrate dystrophin at the plasma membrane of mouse and human muscle have proven inadequate to localize precisely dystrophin in the mammalian CNS. We have now made an antibody (anti 6-10) which is much more sensitive than those previously available to immunolabel dystrophin in the CNS. Using this antibody, we found that in the mouse, dystrophin is particularly abundant in the neurons of the cerebral and cerebellar cortices, and that it is localized at postsynaptic membrane specializations. Dystrophin may have a different role in neurons than in muscle, and an alteration at the synaptic level may be the basis of the cognitive impairment in DMD.     

Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle

Dystrophin, the protein encoded by the Duchenne muscular dystrophy (DMD) gene, exists in a large oligomeric complex. We show here that four glycoproteins are integral components of the dystrophin complex and that the concentration of one of these is greatly reduced in DMD patients. Thus, the absence of dystrophin may lead to the loss of a dystrophin-associated glycoprotein, and the reduction in this glycoprotein may be one of the first stages of the molecular pathogenesis of muscular dystrophy.     

Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide

Duchenne muscular dystrophy (DMD) is a debilitating X-linked muscle disease. We have used sequence information from complementary DNA clones, derived from the gene that is deleted in DMD patients, to generate an antiserum that stains the surface membrane of intact human and mouse skeletal muscle, but not that of DMD patients and mdx mice. Here we identify the protein reacting with this antiserum as a single component of relative molecular mass 210,000 (Mr = 210K) that fractionates with a low-ionic strength extract of intact human and mouse skeletal muscle. It is therefore distinct from the 400 K protein found in the heavy microsomal fraction of normal muscle and identified as a putative product of the DMD gene. We also analyse further the disease specificity of the antiserum. Positive staining is seen in normal controls, and in samples from patients with a wide range of muscular dystrophies other than DMD. Becker muscular dystrophy, which is allelically related to DMD, was the only other exception, and gave a sporadic staining pattern. The demonstration of a specific defect in the surface membrane of DMD muscle fibres substantiates the hypothesis that membrane lesions may initiate muscle degradation in DMD.     

Decreased osmotic stability of dystrophin-less muscle cells from the mdx mouse

Human X-linked Duchenne and Becker muscular dystrophies are due to defects in dystrophin, the product of an exceptionally large gene. Although dystrophin has been characterized as a spectrin-like submembranous cytoskeletal protein, there is no experimental evidence for its function in the structural maintenance of muscle. Current hypotheses attribute necrosis of dystrophin-less fibres in situ to mechanical weakening of the outer membrane, to an excessive influx of Ca2+ ions, or to a combination of these two mechanism, possibly mediated by stretch-sensitive ion channels. Using hypo-osmotic shock to determine stress resistance and a mouse model (mdx) for the human disease, we show that functional dystrophin contributes to the stability of both cultured myotubes and isolated mature muscle fibres.     

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 dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. 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 death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) 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.     

Calcium entry through stretch-inactivated ion channels in mdx myotubes.

Recent advances in understanding the molecular basis of human X-linked muscular dystrophies have come from the identification of dystrophin, a cytoskeletal protein associated with the surface membrane. Although there is little or virtually no dystrophin in affected individuals, it is not known how this causes muscle degeneration. One possibility is that the membrane of dystrophic muscle is weakened and becomes leaky to Ca2+. In muscle from mdx mice, an animal model of the human disease, intracellular Ca2+ is elevated and associated with a high rate of protein degradation. The possibility that a lack of dystrophin alters the resting permeability of skeletal muscle to Ca2+ prompted us to compare Ca2(+)-permeable ionic channels in muscle cells from normal and mdx mice. We now show that recordings of single-channel activity from mdx myotubes are dominated by the presence of Ca2(+)-permeable mechano-transducing ion channels. Like similar channels in normal skeletal muscle, they are rarely open at rest, but open when the membrane is stretched by applying suction to the electrode. Other channels in mdx myotubes, however, are often open for extended periods of time at rest and close when suction is applied to the electrode. The results show a novel type of mechano-transducing ion channel in mdx myotubes that could provide a pathway for Ca2+ to leak into the cell.     

Immunoelectron microscopic localization of dystrophin in myofibres

Duchenne muscular dystrophy, a common X-linked recessive human disease, has recently been shown to be caused by the deficiency of a large, low abundance protein called 'dystrophin'. Biochemical techniques have shown dystrophin to be membrane-associated in skeletal muscle, with enrichment of dystrophin in the t-tubules of 'triads'. Other studies using immunohistochemistry on thick (10 micron) sections have shown dystrophin to be located at the periphery of muscle fibres, possibly at the plasma membrane. These results have been interpreted as being either consistent and complementary, or contradictory. To localize dystrophin more precisely relative to these membrane systems we have employed highly sensitive and spatially accurate immuno-gold electron microscopy of ultra-thin (70-100 nm) cryosections. The major distribution of dystrophin was on the cytoplasmic face of the plasma membrane of muscle fibres, and possibly on the contiguous t-tubule membranes. The presented data, taken together with recently accumulated information regarding the primary structure of dystrophin, suggests that dystrophin is a component of the membrane cytoskeleton in myogenic cells. Thus, myofibre necrosis in patients affected with Duchenne muscular dystrophy is likely the result of plasma membrane instability.     

Dystrophic heart failure blocked by membrane sealant poloxamer

Dystrophin deficiency causes Duchenne muscular dystrophy (DMD) in humans, an inherited and progressive disease of striated muscle deterioration that frequently involves pronounced cardiomyopathy. Heart failure is the second leading cause of fatalities in DMD. Progress towards defining the molecular basis of disease in DMD has mostly come from studies on skeletal muscle, with comparatively little attention directed to cardiac muscle. The pathophysiological mechanisms involved in cardiac myocytes may differ significantly from skeletal myofibres; this is underscored by the presence of significant cardiac disease in patients with truncated or reduced levels of dystrophin but without skeletal muscle disease. Here we show that intact, isolated dystrophin-deficient cardiac myocytes have reduced compliance and increased susceptibility to stretch-mediated calcium overload, leading to cell contracture and death, and that application of the membrane sealant poloxamer 188 corrects these defects in vitro. In vivo administration of poloxamer 188 to dystrophic mice instantly improved ventricular geometry and blocked the development of acute cardiac failure during a dobutamine-mediated stress protocol. Once issues relating to optimal dosing and long-term effects of poloxamer 188 in humans have been resolved, chemical-based membrane sealants could represent a new therapeutic approach for preventing or reversing the progression of cardiomyopathy and heart failure in muscular dystrophy.