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.     

Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.     

Dystroglycan is not required for localization of dystrophin, syntrophin, and neuronal nitric-oxide synthase at the sarcolemma but regulates integrin alpha 7B expression and caveolin-3 distribution.

Dystroglycan is part of the dystrophin-associated protein complex, which joins laminin in the extracellular matrix to dystrophin within the subsarcolemmal cytoskeleton. We have investigated how mutations in the components of the laminin-dystroglycan-dystrophin axis affect the organization and expression of dystrophin-associated proteins by comparing mice mutant for merosin (alpha(2)-laminin, dy), dystrophin (mdx), and dystroglycan (Dag1) using immunohistochemistry and immunoblots. We report that syntrophin and neuronal nitric-oxide synthase are depleted in muscle fibers lacking both dystrophin and dystroglycan. Some fibers deficient in dystroglycan, however, localize dystrophin at the cell surface at levels similar to that in wild-type muscle. Nevertheless, these fibers have signs of degeneration/regeneration including increased cell surface permeability and central nuclei. In these fibers, syntrophin and nitric-oxide synthase are also localized to the plasma membrane, whereas the sarcoglycan complex is disrupted. These results suggest a mechanism of membrane attachment for dystrophin independent of dystroglycan and that the interaction of sarcoglycans with dystrophin requires dystroglycan. The distribution of caveolin-3, a muscle-specific component of caveolae recently found to bind dystroglycan, was affected in dystroglycan- and dystrophin-deficient mice. We also examined alternative mechanisms of cell-extracellular matrix attachment to elucidate how the muscle basement membrane may subsist in the absence of dystroglycan, and we found the alpha(7B) splice variant of the alpha(7) integrin receptor subunit to be up-regulated. These results support the possibility that alpha(7B) integrin compensates in mediating cell-extracellular matrix attachment but cannot rescue the dystrophic phenotype.     

Laminin polymerization induces a receptor-cytoskeleton network

The transition of laminin from a monomeric to a polymerized state is thought to be a crucial step in the development of basement membranes and in the case of skeletal muscle, mutations in laminin can result in severe muscular dystrophies with basement membrane defects. We have evaluated laminin polymer and receptor interactions to determine the requirements for laminin assembly on a cell surface and investigated what cellular responses might be mediated by this transition. We found that on muscle cell surfaces, laminins preferentially polymerize while bound to receptors that included dystroglycan and alpha7beta1 integrin. These receptor interactions are mediated through laminin COOH-terminal domains that are spatially and functionally distinct from NH2-terminal polymer binding sites. This receptor-facilitated self-assembly drives rearrangement of laminin into a cell-associated polygonal network, a process that also requires actin reorganization and tyrosine phosphorylation. As a result, dystroglycan and integrin redistribute into a reciprocal network as do cortical cytoskeleton components vinculin and dystrophin. Cytoskeletal and receptor reorganization is dependent on laminin polymerization and fails in response to receptor occupancy alone (nonpolymerizing laminin). Preferential polymerization of laminin on cell surfaces, and the resulting induction of cortical architecture, is a cooperative process requiring laminin- receptor ligation, receptor-facilitated self-assembly, actin reorganization, and signaling events.     

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.     

Forced expression of dystrophin deletion constructs reveals structure- function correlations

Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH- terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.     

Dystrophin constitutes 5% of membrane cytoskeleton in skeletal muscle

Dystrophin, which is absent in skeletal muscle of Duchenne muscular dystrophy patients, has not been considered to play a major structural role in the cell membrane of skeletal muscle because of its low abundance (approximately 0.002% of total muscle protein). Here, we have determined the relative abundance of dystrophin in a membrane cytoskeleton preparation and found that dystrophin constitutes approximately 5% of the total membrane cytoskeleton fraction of skeletal muscle sarcolemma. In addition, dystrophin can be removed from sarcolemma by alkaline treatment. Thus, our results have demonstrated that dystrophin is a major component of the subsarcolemmal cytoskeleton in skeletal muscle and suggest that dystrophin could play a major structural role in the cell membrane of skeletal muscle.     

S-myotrophin promotes the hypertrophy of skeletal muscle of mice in vivo

S-myotrophin is a newly discovered muscle growth factor. Effects of crude S-myotrophin injection on the growth and morphology of skeletal muscle of normal, ScN and mdx mice were investigated in the present study. Total dose of crude S-myotrophin was 100 microg (100 microg protein/ml x 50 microl x 20 times). In the case of normal mice (Sea:ddY), body weight and the weight of M. gluteus major of crude S-myotrophin injected mice was significantly heavier than that of control (PBS-injected) mice after 5 weeks' feeding. Antibody staining of laminin and dystrophin showed clear sarcolemmal and basement membrane structure surrounding each muscle fibre. The numbers of muscle fibres per 100 microm(2) was less in crude S-myotrophin-injected normal mice than in PBS-injected mice. Quite similar observations as in the case of normal mice were obtained in the case of ScN mice having heterogeneous gene of dystrophin. In the case of mdx mice, body weight and the weight of M. gluteus major of crude S-myotrophin injected mdx mice was significantly heavier than that of PBS-injected mdx mice. Antibody staining of laminin showed almost intact structure of the basement membrane containing laminin even in skeletal muscle of mdx mice subjected to crude S-myotrophin injection, while irregular and incompletely developed structure of muscle fibres or necrosis were observed in muscle fibres of PBS-injected mdx mice. In spite of crudeness of the preparation, the present animal experiments indicate that S-myotrophin has a strong growth promoting activity of muscle cells of normal and dystrophic mice.     

Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma

mAbs specific for protein components of the surface membrane of rabbit skeletal muscle have been used as markers in the isolation and characterization of skeletal muscle sarcolemma membranes. Highly purified sarcolemma membranes from rabbit skeletal muscle were isolated from a crude surface membrane preparation by wheat germ agglutination. Immunoblot analysis of subcellular fractions from skeletal muscle revealed that dystrophin and its associated glycoproteins of 156 and 50 kD are greatly enriched in purified sarcolemma vesicles. The purified sarcolemma was also enriched in novel sarcolemma markers (SL45, SL/TS230) and Na+/K(+)-ATPase, whereas t-tubule markers (alpha 1 and alpha 2 subunits of dihydropyridine receptor, TS28) and sarcoplasmic reticulum markers (Ca2(+)-ATPase, ryanodine receptor) were greatly diminished in this preparation. Analysis of isolated sarcolemma by SDS-PAGE and densitometric scanning demonstrated that dystrophin made up 2% of the total protein in the rabbit sarcolemma preparation. Therefore, our results demonstrate that although dystrophin is a minor muscle protein it is a major constituent of the sarcolemma membrane in skeletal muscle. Thus the absence of dystrophin in Duchenne muscular dystrophy may result in a major disruption of the cytoskeletal network underlying the sarcolemma in dystrophic muscle.     

The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.     

The role of defective glycosylation in congenital muscular dystrophy

The dystrophin glycoprotein complex (DGC) is an assembly of proteins spanning the sarcolemma of skeletal muscle cells. Defects in the DGC appear to play critical roles in several muscular dystrophies due to disruption of basement membrane organization. O -mannosyl oligosaccharides on alpha-dystroglycan, a major extracellular component of the DGC, are essential for normal binding of alpha-dystroglycan to ligands (such as laminin) in the extracellular matrix and subsequent signal transmission to actin in the cytoskeleton of the muscle cell. Muscle-Eye-Brain disease (MEB) and Walker-Warburg Syndrome (WWS) have mutations in genes encoding glycosyltransferases needed for O -mannosyl oligosaccharide synthesis. Myodystrophic myd mice and humans with Fukuyama Congenital Muscular Dystrophy (FCMD), congenital muscular dystrophy due to defective fukutin-related protein (FKRP) and MDC1D have mutations in putative glycosyltransferases. These human congenital muscular dystrophies and the myd mouse are associated with defective glycosylation of alpha-dystroglycan. It is expected other congenital muscular dystrophies will prove to have mutations in genes involved in glycosylation.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.     

Immunohistochemical distribution of laminin-5 gamma2 chain and its developmental change in human embryonic and foetal tissues

Immunohistochemical distribution of laminin gamma2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin gamma2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5-6 weeks of gestation. Between 6-7 and 12-13 weeks, laminin gamma2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin gamma2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin gamma2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin gamma2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its gamma2 chain in the morphogenesis of human tissues.     

Supramolecular organization of the subsarcolemmal cytoskeleton of adult skeletal muscle fibers. A review

This review is focused on the composition and organization of the junctional subsarcolemmal cytoskeleton of adult muscle fibers. The cytoskeleton of muscle fibers is organized in functionally distinct compartments and the subsarcolemmal cytoskeleton itself can be broadly divided into junctional (myotendinous junction, neuromuscular junction and costameres) and non-junctional domains. In junctional zones three different multimolecular cytoskeletal complexes coexist: the focal adhesion-type, the spectrin-based and the dystrophin vs utrophin-based membrane skeleton systems. These complexes extend over several levels, from intracytoplasmic to subsarcolemmal and transmembranous; their common feature is the anchorage of actin filaments emanating from the intracytoplasmic level. The different cytoskeletal proteins, their putative roles and their interactions with various signaling pathways are presented here in detail. The subsarcolemmal cytoskeleton complexes are thought to play distinct physiological roles (membrane stabilization, force transmission to extracellular matrix, ionic channel anchorage, etc) but their colocalization on the three sarcolemmal junctional domains strongly suggests interrelated or common functions.     

Phagocytosis of cytoplasmic blebs derived from smooth muscle cells by fibroblast-like cells and macrophages in the post-partum rat uterus

Numerous blebs were observed in contact with smooth muscle cells (SMC) by light microscopy in the myometrium of the rat uterus after parturition. Electron-microscopically the cell surface of SMC showed bulbous protrusions, which often lacked a basement membrane and were less electron-dense than the surrounding cytoplasm or sometimes nearly electron-lucent. Many bulbous protrusions were separated from SMC and became the isolated structures which we called cytoplasmic blebs. These bulbous protrusions and cytoplasmic blebs were often found to be phagocytosed by fibroblast-like cells and macrophages. A series of these tissue changes in the uterine myometrium after delivery, possibly due to hypoxic conditions, contribute to a rapid involution of SMC which have enlarged during pregnancy.     

Expression of the skeletal muscle dystrophin-dystroglycan complex and syntrophin-nitric oxide synthase complex is severely affected in the type 2 diabetic Goto-Kakizaki rat

The inability of insulin to stimulate glucose metabolism in skeletal muscle fibres is a classic characteristic of type 2 diabetes. Using the non-obese Goto-Kakizaki rat as an established animal model of this type of diabetes, sucrose gradient centrifugation studies were performed and confirmed the abnormal subcellular location of the glucose transporter GLUT4. In addition, this analysis revealed an unexpected drastic reduction in the surface membrane marker beta-dystroglycan, a dystrophin-associated glycoprotein. Based on this finding, a comprehensive immunoblotting survey was conducted which showed a dramatic decrease in the Dp427 isoform of dystrophin and the alpha/beta-dystroglycan subcomplex, but not in laminin, sarcoglycans, dystrobrevin, and excitation-contraction-relaxation cycle elements. Thus, the backbone of the trans-sarcolemmal linkage between the extracellular matrix and the actin membrane cytoskeleton might be structurally impaired in diabetic fibres. Immunohistochemical studies revealed that the reduction in the dystrophin-dystroglycan complex does not induce obvious signs of muscle pathology, and is neither universal in all fibres, nor fibre-type specific. Most importantly, the expression of alpha-syntrophin and the syntrophin-associated neuronal isoform of nitric oxide synthase, nNOS, was demonstrated to be severely reduced in diabetic fibres. The loss of the dystrophin-dystroglycan complex and the syntrophin-nNOS complex in selected fibres suggests a weakening of the sarcolemma, abnormal signalling and probably a decreased cytoprotective mechanism in diabetes. Impaired anchoring of the cortical actin cytoskeleton via dystrophin might interfere with the proper recruitment of the glucose transporter to the surface membrane, following stimulation by insulin or muscle contraction. This may, at least partially, be responsible for the insulin resistance in diabetic skeletal muscles.     

Organization of intestinal epithelial cells into multicellular structures requires laminin and functional actin microfilaments

Epithelial cell organization into multicellular structures is a critical biological process required for both organogenesis and repair following injury. The basement membrane and the cytoskeleton have important roles in this process; however, the functions of individual components of basement membrane and cytoskeleton are poorly understood. We used IEC-6 cells, a rat intestinal crypt cell line, grown on a three-dimensional gel of reconstituted basement membrane as a model system to determine which extracellular matrix and cytoskeletal components mediate intestinal epithelial cell organization. The cells entered the gel and formed hollow, tubular structures that resembled intestinal crypts. These structures were characterized by a single layer of polarized cells with apical tight junctions and microvilli on the luminal surface. Antiserum to laminin and the pentapeptide Tyr-Ile-Gly-Ser-Arg (which prevents cell attachment to laminin) inhibited this organization, but a control pentapeptide (Tyr-Tyr-Gly-Asp-Ala) and antiserum to collagen IV did not. Cytochalasin B, which interferes with actin microfilament polymerization, also inhibited organization of cells into multicellular structures, but vinblastine and Colcemid, which disrupt microtubules, and cycloheximide, which inhibits protein synthesis, did not. We conclude that organization of intestinal epithelial cells on a basement membrane into multicellular structures results from specific interactions between cells and laminin and requires intact actin microfilaments.     

Basement membrane component changes in skeletal muscle transplants undergoing regeneration or rejection

The basement membrane of myofibers plays an important role during orderly regeneration of skeletal muscle after injury. In this report, changes in various basement membrane components were analyzed in skeletal muscle grafts undergoing regeneration (autografts) or immune rejection (allografts). The immunofluorescence technique using specific antibodies against laminin, types IV and V collagen, heparan sulfate proteoglycan, fibronectin, in combination with binding of concanavalin A (ConA) was used to monitor basement membranes. In normal muscle, these components were localized in the pericellular region of myofiber corresponding to its basement membrane. After transplantation, the majority of myofibers underwent degeneration as a result of ischemic injury, followed by regeneration from precursor myosatellite cells. Various components of basement membrane zone disappeared from the degenerating myofibers, leaving behind some unidentifiable component that still bound ConA. A new basement membrane appeared around the regenerated myotubes which persisted during maturation of the regenerating muscle. In rejected skeletal muscles, the immunoreactivity of various components persisted even after the disappearance of myotubes and myofiber cytoplasm. In addition, an accumulation of fibronectin was seen throughout the rejected muscle with the onset of immune rejection. These results demonstrate that the major basement membrane components disappear and reappear sequentially during myofiber degeneration and regeneration. Such a turnover is not seen in rejected skeletal muscles. Thus, the myofiber basement membrane is not a static structure as previously thought but one which changes chemically during degeneration and regeneration. This feature of basement membrane may be important in the orderly regeneration of skeletal muscle after injury.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.