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.     

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.     

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.     

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.     

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.     

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.     

Congenital muscular dystrophies involving the O-mannose pathway

A number of forms of congenital muscular dystrophy (CMD) have been identified that involve defects in the glycosylation of dystroglycan with O-mannosyl-linked glycans. There are at least six genes that can affect this type of glycosylation, and defects in these genes give rise to disorders that have many aspects of muscle and brain pathology in common. Overexpression of one gene implicated in CMD, LARGE, was recently shown to increase dystroglycan glycosylation and restore its function in cells taken from CMD patients. Overexpression of Galgt2, a glycosyltransferase not implicated in CMD, also alters dystroglycan glycosylation and inhibits muscular dystrophy in a mouse model of Duchenne muscular dystrophy. These findings suggest that a common approach to therapy in muscular dystrophies may be to increase the glycosylation of dystroglycan with particular glycan structures.     

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.     

A rostrocaudal muscular dystrophy caused by a defect in choline kinase beta, the first enzyme in phosphatidylcholine biosynthesis

Muscular dystrophies include a diverse group of genetically heterogeneous disorders that together affect 1 in 2000 births worldwide. The diseases are characterized by progressive muscle weakness and wasting that lead to severe disability and often premature death. Rostrocaudal muscular dystrophy (rmd) is a new recessive mouse mutation that causes a rapidly progressive muscular dystrophy and a neonatal forelimb bone deformity. The rmd mutation is a 1.6-kb intragenic deletion within the choline kinase beta (Chkb) gene, resulting in a complete loss of CHKB protein and enzymatic activity. CHKB is one of two mammalian choline kinase (CHK) enzymes (alpha and beta) that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid phosphatidylcholine. While mutant rmd mice show a dramatic decrease of CHK activity in all tissues, the dystrophy is only evident in skeletal muscle tissues in an unusual rostral-to-caudal gradient. Minor membrane disruption similar to dysferlinopathies suggest that membrane fusion defects may underlie this dystrophy, because severe membrane disruptions are not evident as determined by creatine kinase levels, Evans Blue infiltration, and unaltered levels of proteins in the dystrophin-glycoprotein complex. The rmd mutant mouse offers the first demonstration of a defect in a phospholipid biosynthetic enzyme causing muscular dystrophy, representing a unique model for understanding mechanisms of muscle degeneration.     

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.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells.     

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     

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy.

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-delta, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-delta (sgcd-/-) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd-/-, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd-/- arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd-/- arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd-/- arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.     

Viral-mediated gene therapy for the muscular dystrophies: successes, limitations and recent advances

Much progress has been made over the past decade elucidating the molecular basis for a variety of muscular dystrophies (MDs). Accordingly, there are examples of mouse models of MD whose disease progression has been halted in large part with the use of viral vector technology. Even so, we must acknowledge significant limitations of present vector systems that must be overcome prior to successful treatment of humans with such approaches. This review will present a variety of viral-mediated therapeutic strategies aimed at counteracting the muscle-wasting symptoms associated with muscular dystrophy. We include viral vector systems used for muscle gene transfer, with a particular emphasis on adeno-associated virus. Findings of several encouraging studies focusing on repair of the mutant dystrophin gene are also included. Lastly, we present a discussion of muscle compensatory therapeutics being considered that include pathways involved in the up-regulation of utrophin, promotion of cellular adhesion, enhancement of muscle mass, and antagonism of the inflammatory response. Considering the complexity of the muscular dystrophies, it appears likely that a multilayered approach tailored to a patient sub-group may be warranted in order to effectively contest the progression of this devastating disease.