Transgenic expression of {alpha}7{beta}1 integrin maintains muscle integrity, increases regenerative capacity, promotes hypertrophy, and reduces cardiomyopathy in dystrophic mice

We previously reported that enhanced expression of the alpha7beta1 integrin ameliorates the development of muscular dystrophy and extends longevity in alpha7BX2-mdx/utr(-/-) transgenic mice (Burkin DJ, Wallace GQ, Nicol KJ, Kaufman DJ, Kaufman SJ: Enhanced expression of the alpha7beta1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. We now report on the mechanism by which these mice were rescued by the integrin. As a result of increased integrin in alpha7BX2-mdx/utr(-/-) mice the structural integrity of the myotendinous and neuromuscular junctions are maintained. A twofold increase in satellite cells in alpha7BX2-mdx/utr(-/-) skeletal muscle was detected by immunofluorescence using the satellite cell marker c-met. These cells enhanced the regenerative capacity of muscle in the transgenic animals as determined by fusion of BrdUrd-labeled cells into muscle fibers. Increased integrin also leads to hypertrophy. Finally, transgenic expression of alpha7BX2 integrin chain in skeletal muscle secondarily reduces the development of cardiomyopathy, the ultimate cause of death in these animals. We believe this multiplicity of responses to increased alpha7beta1 integrin collectively inhibits the development of muscle disease and increases longevity in these mice.     

Organization of the myotendinous junction is dependent on the presence of alpha7beta1 integrin

The laminin receptor alpha7beta1 is enriched at the myotendinous junctions, and mice with a targeted inactivation of the alpha7 gene develop a form of muscular dystrophy that primarily affects this structure. By ultrastructural analysis of alpha7-deficient mice, in comparison with wild-type and mdx mice, we attempted to elucidate the role of alpha7 integrin for the integrity and function of the myotendinous junctions. Ultrastructurally, myotendinous junctions of alpha7-deficient myofibers lose their interdigitations and the myofilaments retract from the sarcolemmal membrane, whereas the lateral side of the myofibers remains morphologically normal. The basement membrane at the myotendinous junctions in alpha7 -/- mice is significantly broadened, and immunogold-histochemistry has demonstrated that the laminin alpha2 chain is not localized here but, instead, in the matrix of the neighboring tendon. In contrast, mdx mice have normal myotendinous junctions, with a matrix protein pattern also found in wild-type mice, however the lateral sides of the myofibers are severely damaged. These results suggest that the alpha7beta1 integrin is a major receptor connecting the muscle cell to the tendon and helps to organize the myotendinous junction, whereas the dystrophin-glycoprotein complex is necessary for the lateral integrity of the muscle cell.     

Absence of alpha 7 integrin in dystrophin-deficient mice causes a myopathy similar to Duchenne muscular dystrophy

Both the dystrophin-glycoprotein complex and alpha7beta1 integrin have critical roles in the maintenance of muscle integrity via the provision of mechanical links between muscle fibres and the basement membrane. Absence of either dystrophin or alpha7 integrin results in a muscular dystrophy. To clarify the role of alpha7 integrin and dystrophin in muscle development and function, we generated integrin alpha7/dystrophin double-mutant knockout (DKO) mice. Surprisingly, DKO mice survived post-natally and were indistinguishable from wild-type, integrin alpha7-deficient and mdx mice at birth, but died within 24-28 days. Histological analysis revealed a severe muscular dystrophy in DKO mice with endomysial fibrosis and ectopic calcification. Weight loss was correlated with the loss of muscle fibres, indicating that progressive muscle wasting in the double mutant was most likely due to inadequate muscle regeneration. The data further support that premature death of DKO mice is due to cardiac and/or respiratory failure. The integrin alpha7/dystrophin-deficient mouse model, therefore, resembles the pathological changes seen in Duchenne muscular dystrophy and suggests that the different clinical severity of dystrophin deficiency in human and mouse may be due to a fine-tuned difference in expression of dystrophin and integrin alpha7 in both species. Together, these findings indicate an essential role for integrin alpha7 in the maintenance of dystrophin-deficient muscles.     

Defective integrin switch and matrix composition at alpha 7-deficient myotendinous junctions precede the onset of muscular dystrophy in mice

Force transmission at the myotendinous junction requires a strong link between the muscle cytoskeleton and the extracellular matrix. At the adult junction, two splice variants of the laminin-binding integrins, alpha7Abeta1D and alpha7Bbeta1D, are highly enriched. The alpha7 subunits are critical for the integrity of the junctional sarcolemma because integrin alpha7-deficient mice develop muscular dystrophy, primarily affecting this site of the muscle. Here, we report that beta1D integrin coimmunoprecipitates and colocalizes with the alpha5 subunit at alpha7-deficient junctions, but does not associate with alpha3, alpha6 or alphav integrins. By immunogold labelling we show that the basement membranes of integrin alpha7-deficient muscles recruit abnormally high levels of fibronectin, the ligand of alpha5beta1D. Finally, we demonstrate that alpha5beta1D is down-regulated at the normal postnatal junction and is displaced by alpha7beta1D. These results suggest that the alpha7 subunit is implicated in the down-regulation of alpha5beta1D and in the removal of fibronectin from the maturing myotendinous junction, thus providing an alpha7beta1D-based link to laminin. We propose that the persistence of alpha5beta1D in alpha7-deficient mice is not compatible with normal muscle function and leads to muscle wasting.     

Transgenic expression of Laminin α1 chain does not prevent muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder, and one of the most frequently encountered, but one for which there is as yet no treatment. Laminin-111 protein therapy was recently shown to be a promising approach to prevent muscle disease in the mdx mouse model of DMD. The present study demonstrated that transgenic expression of laminin α1 chain in mdx animals, resulting in laminin-111 heterotrimer formation in mdx muscle, does not improve the dystrophic phenotype. The mdx mice overexpressing laminin-111 (mdxLMα1) display features of mdx littermates: dystrophic pattern of muscle biopsy, elevated creatine kinase levels, reduced muscle strength, and decreased sarcolemmal integrity. Increased expression of integrin α7 is not beneficial for mdxLMα1 muscle, and components of the dystrophin-glycoprotein complex are not restored at the sarcolemma on laminin-111 overexpression. In summary, further studies are needed to verify the functionality of laminin-111 protein therapy in DMD and to describe the molecular events resulting from this approach.     

Transgenic overexpression of dystroglycan does not inhibit muscular dystrophy in mdx mice

Recently, there have been a number of studies demonstrating that overexpression of molecules in skeletal muscle can inhibit or ameliorate aspects of muscular dystrophy in the mdx mouse, a model for Duchenne muscular dystrophy. Several such studies involve molecules that increase the expression of dystroglycan, an important component of the dystrophin-glycoprotein complex. To test whether dystroglycan itself inhibits muscular dystrophy in mdx mice, we created dystroglycan transgenic mdx mice (DG/mdx). The alpha and beta chains of dystroglycan were highly overexpressed along the sarcolemmal membrane in most DG/mdx muscles. Increased dystroglycan expression, however, did not correlate with increased expression of utrophin or sarcoglycans, but rather caused their decreased expression. In addition, the percentage of centrally located myofiber nuclei and the level of serum creatine kinase activity were not decreased in DG/mdx mice relative to mdx animals. Therefore, dystroglycan overexpression does not cause the concomitant overexpression of a utrophin-glycoprotein complex in mdx muscles and has no effect on the development of muscle pathology associated with muscular dystrophy.     

Transgenic overexpression of ADAM12 suppresses muscle regeneration and aggravates dystrophy in aged mdx mice

Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy.     

Expression of extracellular matrix ligands and receptors in the muscular tissue and draining lymph nodes of mdx dystrophic mice.

The mdx mouse, an animal model of Duchenne muscular dystrophy, develops an X-linked recessive inflammatory myopathy. During onset of disease and height of myonecrosis, mdx mice also display important changes in the microenvironment of lymphoid tissues. Draining lymph nodes showed reduced cellularity and atrophy accompanied by intense immunolabeling for fibronectin, laminin, and type-IV collagen. Following clinical amelioration of dystrophy, mdx mice showed enhanced cellularity and a consistent increase in the absolute numbers of CD4(+) and CD8(+) cells expressing alpha4(high) and alpha5(high) extracellular matrix receptors. Furthermore, infiltrating cells in the proximity of myonecrosis expressed alpha4, alpha5, and alpha6 integrin chains during both height of myonecrosis and muscular tissue regeneration. Such results indicate that during distinct phases of muscular dystrophy, altered expression of extracellular matrix ligands and receptors may be influencing myonecrosis by promoting adhesion and migration of mononuclear cells into the altered skeletal muscle and toward local draining lymphoid tissue.     

Characterization of ARC, apoptosis repressor interacting with CARD, in normal and dystrophin-deficient skeletal muscle

Duchenne muscular dystrophy is an X-linked recessive disorder, primarily characterized by progressive muscle weakness and wasting. The disease results from the absence of dystrophin, however the precise molecular mechanisms leading to muscle pathology are poorly understood. Dystrophic muscles undergo increased oxidative stress and altered calcium homeostasis, which may contribute to myofiber loss by triggering both necrosis and apoptosis. Recent studies have identified ARC (apoptosis repressor with caspase recruitment domain) as an abundant protein in human muscle that can inhibit both hypoxia and caspase-8-induced apoptosis as well as protect cells from oxidative stress. To explore a potential role for ARC in protecting muscle fibers from dystrophic breakdown, we have cloned and characterized murine ARC and studied its expression in normal and dystrophic mouse muscle. ARC is expressed at high levels in striated muscle and displays fiber-type restricted expression patterns. ARC expression levels are normal in dystrophic mdx mice, although the intracellular localization pattern of ARC is slightly altered compared with normal muscles. Overexpression of ARC in transgenic mdx mice failed to alleviate the dystrophic pathology in skeletal muscles, suggesting that misregulation of the molecular pathways regulated by ARC does not significantly contribute to myofiber death.     

Platelet-derived growth factor and its receptors are related to the progression of human muscular dystrophy: an immunohistochemical study

This study has examined the immunological localization of platelet-derived growth factor (PDGF)-A, PDGF-B, and PDGF receptor (PDGFR) alpha and beta to clarify their role in the progression of muscular dystrophy. Biopsied frozen muscles from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD) were analysed immunohistochemically using antibodies raised against PDGF-A, PDGF-B, and PDGFR alpha and beta. Muscles from two dystrophic mouse models (dy and mdx mice) were also immunostained with antibodies raised against PDGFR alpha and beta. In normal human control muscle, neuromuscular junctions and vessels were positively stained with antibodies against PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta. In human dystrophic muscles, PDGF-A, PDGF-B, PDGFR alpha and PDGFR beta were strongly immunolocalized in regenerating muscle fibres and infiltrating macrophages. PDGFR alpha was also immunolocalized to the muscle fibre sarcolemma and necrotic fibres. The most significant finding in this study was a remarkable overexpression of PDGFR beta and, to a lesser extent, PDGFR alpha in the endomysium of DMD and CMD muscles. PDGFR was also overexpressed in the interstitium of muscles from dystrophic mice, particularly dy mice. Double immunolabelling revealed that activated interstitial fibroblasts were clearly positive for PDGFR alpha and beta. However, DMD and CMD muscles with advanced fibrosis showed very poor reactivity against PDGF and PDGFR. Those findings were confirmed by immunoblotting with PDGFR beta. These findings indicate that PDGF and its receptors are significantly involved in the active stage of tissue destruction and are associated with the initiation or promotion of muscle fibrosis. They also have roles in muscle fibre regeneration and signalling at neuromuscular junctions in both normal and diseased muscle.     

Suppression of revertant fibers in mdx mice by expression of a functional dystrophin.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration that results from the absence of dystrophin. Despite null mutations in the dystrophin gene, many DMD patients display a low percentage of dystrophin-positive fibers. These "revertant fibers" are also present in the dystrophin-deficient mdx mouse and are believed to result from alternative splicing or second mutation events that bypass the mutation and restore an open reading frame. However, it is unclear what role dystrophin and the dystrophic pathology might play in revertant fiber formation and accumulation. We have analyzed the role of dystrophin expression and the dystrophic pathology in this process by monitoring revertant fibers in transgenic mdx mice that express truncated dystrophins. We found that newborn transgenic mice displayed approximately the same number of revertant fibers as newborn mdx mice, indicating that expression of a functional dystrophin does not suppress the initiation of revertant fiber formation. Surprisingly, when the transgene encoded a functional dystrophin, revertant fibers were not detected in adult or old mdx mice. In contrast, adult transgenic mice expressing a non-functional dystrophin accumulated increasing numbers of revertant fibers, similar to mdx mice, suggesting that positive selection is required for the persistence of revertant fibers. Finally, we provide evidence that the loss of revertant dystrophin in transgenic mdx muscle fibers overexpressing a functional dystrophin results from displacement of the revertant protein by the transgene-encoded dystrophin.     

Preservation of muscle force in Mdx3cv mice correlates with low-level expression of a near full-length dystrophin protein

The complete absence of dystrophin causes Duchenne muscular dystrophy. Its restoration by greater than 20% is needed to reduce muscle pathology and improve muscle force. Dystrophin levels lower than 20% are considered therapeutically irrelevant but are associated with a less severe phenotype in certain Becker muscular dystrophy patients. To understand the role of low-level dystrophin expression, we compared muscle force and pathology in mdx3cv and mdx4cv mice. Dystrophin was eliminated in mdx4cv mouse muscle but was expressed in mdx3cv mice as a near full-length protein at approximately 5% of normal levels. Consistent with previous reports, we found dystrophic muscle pathology in both mouse strains. Surprisingly, mdx3cv extensor digitorium longus muscle showed significantly higher tetanic force and was also more resistant to eccentric contraction-induced injury than mdx4cv extensor digitorium longus muscle. Furthermore, mdx3cv mice had stronger forelimb grip strength than mdx4cv mice. Immunostaining revealed utrophin up-regulation in both mouse strains. The dystrophin-associated glycoprotein complex was also restored in the sarcolemma in both strains although at levels lower than those in normal mice. Our results suggest that subtherapeutic expression levels of near full-length, membrane-bound dystrophin, possibly in conjunction with up-regulated utrophin levels, may help maintain minimal muscle force but not arrest muscle degeneration or necrosis. Our findings provide valuable insight toward understanding delayed clinical onset and/or slow disease progression in certain Becker muscular dystrophy patients.     

两种不同Duchenne型肌营养不良症模型鼠神经肌肉接头结构的比较

BACKGROUND: Neuromuscular junction structure has defects in patients with Duchenne muscular dystrophy, mainly presenting acetylcholine receptor structure fragmentation and the reduction of spine-like processes on the  postsynaptic membrane. It is generally recognized that the structural defects are induced by structural damage of muscle cells and muscle fiber necrosis. OBJECTIVE: To explore the reasons of damage on neuromuscular junction in mouse models of Duchenne muscular dystrophy.  METHODS: We introduced Duchenne muscular dystrophy models of male mdx mice and male Dko mice. After gene identification, they were used for tests. Male C57BL/6 mice were selected as normla controls. Hematoxylin-eosin staining was utilized to detect pathological changes in muscles. Neuromuscular junction structure was revealed using immunofluorescence staining. The differences in dystrophin expression, pathological features and neuromuscular junction structure were compared in mouse models of two kinds of Duchenne muscular dystrophy.  RESULTS AND CONCLUSION: The introduced mouse models were accorded with the requirement of our experiment in aspects of genotype and protein expression levels. The number of acetylcholine receptor was apparently reduced in the neuromuscular junction of two kinds of mouse models. Although dko mouse muscles presented more obvious inflammatory infiltration and muscle fiber damage compared with mdx mice, but there was no significant difference in the damage to neuromuscular junction between them, and acetylcholine receptor fragmentation was identical. The evidence suggested that structural damage of neuromuscular junction and inflammatory pathological response are independent events. There is no direct relationship between them. Dystrophin gene deficiency is the main cause of the fragmentation of the acetylcholine receptor.        

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated β-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.     

Role for the alpha7beta1 integrin in vascular development and integrity.

The alpha7beta1 integrin is a laminin receptor that has been implicated in muscle disease and the development of neuromuscular and myotendinous junctions. Studies have shown the alpha7beta1 integrin is also expressed in nonskeletal muscle tissues. To identify the expression pattern of the alpha7 integrin in these tissues during embryonic development, alpha7 integrin chain knockout mice were generated by a LacZ knockin strategy. In these mice, expression from the alpha7 promoter is reported by beta-galactosidase. From embryonic day (ED) 11.5 to ED14.5, beta-galactosidase was detected in the developing central and peripheral nervous systems and vasculature. The loss of the alpha7 integrin gene resulted in partial embryonic lethality. Several alpha7 null embryos were identified with cerebrovascular hemorrhages and showed reduced vascular smooth muscle cells and cerebral vascularization. The alpha7 null mice that survived to birth exhibited vascular smooth muscle defects, including hyperplasia and hypertrophy. In addition, altered expression of alpha5 and alpha6B integrin chains was detected in the cerebral arteries of alpha7 null mice, which may contribute to the vascular phenotype. Our results demonstrate for the first time that the alpha7beta1 integrin is important for the recruitment or survival of cerebral vascular smooth muscle cells and that this integrin plays an important role in vascular development and integrity.     

Age-dependent effect of myostatin blockade on disease severity in a murine model of limb-girdle muscular dystrophy

Myostatin (MSTN) is a muscle-specific secreted peptide that functions to limit muscle growth through an autocrine regulatory feedback loop. Loss of MSTN activity in cattle, mice, and humans leads to a profound phenotype of muscle overgrowth, associated with more and larger fibers and enhanced regenerative capacity. Deletion of MSTN in the mdx mouse model of Duchenne muscular dystrophy enhances muscle mass and reduces disease severity. In contrast, loss of MSTN activity in the dyW/dyW mouse model of laminin-deficient congenital muscular dystrophy, a much more severe and lethal disease model, does not improve all aspects of muscle pathology. Here we examined disease severity associated with myostatin (mstn-/-) deletion in mice nullizygous for delta-sarcoglycan (scgd-/-), a model of limb-girdle muscular dystrophy. Early loss of MSTN activity achieved either by monoclonal antibody administration or by gene deletion each improved muscle mass, regeneration, and reduced fibrosis in scgd-/- mice. However, antibody-mediated inhibition of MSTN in late-stage dystrophic scgd-/- mice did not improve disease. These findings suggest that MSTN inhibition may benefit muscular dystrophy when instituted early or if disease is relatively mild but that MSTN inhibition in severely affected or late-stage disease may be ineffective.     

Expression of a NOS transgene in dystrophin-deficient muscle reduces muscle membrane damage without increasing the expression of membrane-associated cytoskeletal proteins

Muscular dystrophy that is caused by mutation of the membrane-associated, cytoskeletal protein called dystrophin, is accompanied by loss of a dystrophin-associated protein complex (DPC) that includes neuronal nitric oxide synthase (nNOS). Previous work showed that expression of a nNOS transgene in the dystrophin-deficient, mdx mouse greatly reduces muscle membrane damage. In this investigation, we test whether expression of a nNOS transgene in wild-type or mdx muscle increases expression of DPC proteins, or functionally related proteins in the integrin complex that are upregulated in dystrophin-deficiency, or affects expression of the dystrophin homolog, utrophin. Many members of the DPC are enriched in Western blots of cell membranes isolated from NOS transgenic muscle, compared to wild-type. Similarly, alpha7-integrin and the associated cytoskeletal proteins talin and vinculin are increased in NOS transgenic, non-dystrophic muscle. However, utrophin expression is unaffected by elevated NOS expression in healthy muscle. A similar trend in mRNA levels for these proteins was observed by expression profiling. Analysis of membrane preparations from mdx mice and NOS transgenic mdx mice shows that expression of the NOS transgene causes significant reductions in utrophin, talin, and vinculin. Expression profiling of mRNA from mdx and NOS transgenic mdx muscles also shows reduced expression of talin. Immunohistochemistry of mdx and NOS transgenic mdx muscle indicates that reduction in utrophin in NOS transgenic mdx muscle results from a decrease in regenerative fibers that express high levels of utrophin. Together, these findings indicate that the NOS transgene does not reduce dystrophinopathy by increasing the expression of compensatory, structural proteins.