Dystrophin-deficient Mdx Mice Research Articles

Chronic exercise accelerates the degeneration–regeneration cycle and downregulates insulin‐like growth factor‐1 in muscle of mdx mice

The aim of this study was to examine the effects of chronic running exercise on degenerative-regenerative processes in the hindlimb muscles of dystrophin-deficient mdx mice. The number of large-sized degenerative-regenerative groups (DRGs) was markedly decreased, whereas that of small-sized DRGs was unchanged by exercise. Expression of insulin-like growth factor-1 (Igf1), as well as a myogenic factor MyoD (Myod1), was downregulated in mdx muscles by exercise. The downregulation of Igf1 may well correlate with the decrease in the population of early regenerating fibers, which existed predominantly in DRGs, because IGF-1 was mainly localized in these fibers. Our data indicate that chronic exercise may accelerate the active cycle of degeneration-regeneration in mdx skeletal muscles. This means that mdx skeletal muscles can temporarily cope with work-induced injury by enhancing muscle regeneration and repair, but we speculate that an early decline of IGF-1 will accelerate age-dependent muscle wasting and weakness in the later stage of life in mdx mice.

In Vivo Fusion of Circulating Fluorescent Cells with Dystrophin-Deficient Myofibers Results in Extensive Sarcoplasmic Fluorescence Expression but Limited Dystrophin Sarcolemmal Expression

To investigate the therapeutic potential of bone marrow transplantation in Duchenne muscular dystrophy, green fluorescent protein-positive (GFP+) bone marrow cells were transplanted into irradiated wild-type and dystrophin-deficient mdx mice. Tibialis anterior muscles showed fivefold to sixfold more GFP+ mononucleated cells and threefold to fourfold more GFP+ myofibers in mdx than in wild-type mice. In contrast, dystrophin expression in mdx mice remained within the level of nontransplanted mdx mice, and co-expression with GFP was rare. Longitudinal sections of 5000 myofibers showed 160 GFP+ fibers, including 9 that co-expressed dystrophin. GFP was always visualized as full-length sarcoplasmic fluorescence that exceeded the span of sample length (up to 1500 microm), whereas dystrophin expression was restricted to 11 to 28% of this length. Dystrophin expression span was much shorter in GFP+ fibers (116 +/- 46 microm) than in revertant fibers (654 +/- 409 microm). These data suggest that soluble GFP diffuses far from the fusion site with a pre-existing dystrophin(-) myofiber whereas dystrophin remains mainly expressed close to the site of fusion. Because restoration of dystrophin in whole muscle fiber length is required to expect functional improvement and clinical benefits for Duchenne muscular dystrophy, future applications of cell therapies to neuromuscular disorders could be more appropriately envisaged for replacement of defective soluble sarcoplasmic proteins.

Cardiomyopathy in dystrophin-deficient hearts is prevented by expression of a neuronal nitric oxide synthase transgene in the myocardium

Null mutation of dystrophin causes the lethal pathology of Duchenne muscular dystrophy (DMD) in which there is progressive pathology of skeletal and cardiac muscles. A large proportion of DMD patient deaths are attributable to cardiac dysfunction associated with ventricular fibrosis, arrhythmias and conduction abnormalities, although the relationships between the dystrophin mutation and the cardiac defects are unknown. Here, we tested whether cardiac pathology in dystrophin-deficient mdx mice can be corrected by the elevated production of nitric oxide (NO) by the myocardium. Dystrophin-deficient mdx mice were produced in which there was myocardial expression of a neuronal nitric oxide synthase (nNOS) transgene. Expression of the transgene prevented the progressive ventricular fibrosis of mdx mice and greatly reduced myocarditis. Electrocardiographs (ECG) attained by radiotelemetry of freely ambulatory mice showed that mdx mice displayed cardiac abnormalities that are characteristic of DMD patients, including deep Q-waves, diminished S:R ratios, polyphasic R-waves and frequent premature ventricular contractions. All of these ECG abnormalities in mdx mice were improved or corrected by nNOS transgene expression. In addition, defects in mdx cardiac autonomic function, which were reflected by decreased heart rate variability, were significantly reduced by nNOS transgene expression. These findings indicate that increasing NO production by dystrophic hearts may have therapeutic value.

253. An AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb. We recently demonstrated that the AAV vector-mediated micro-dystrophin cDNA transfer could ameliorate dystrophic phenotypes in skeletal muscles of dystrophin-deficient mdx mice (Mol Ther 10: 821-828, 2004). For clinical application, it is important to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp Anim. 52: 93-97, 2003). To investigate transduction efficiency in canine skeletal muscle using an AAV vector, we injected the AAV vector encoding the LacZ gene driven by a CMV promoter (1.0-2.0 |[times]| 1013 vg/ml, 100-500 |[mu]|l/muscle) into skeletal muscles of normal dogs. |[beta]|-galactosidase (|[beta]|-gal) was expressed only in few fibers at 2 weeks after the injection, and not detected at 4 or 8 weeks after the injection. Instead, large numbers of mononuclear cells appeared around |[beta]|-gal-expressing fibers. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of the AAV vector, we examined viral infectivity in vitro, cytotoxicity and immune responses of AAV vector transduction in vivo. First, we infected the AAV vector into canine primary myotubes. This in vitro study showed that the AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particles elicit cytotoxicity or not. When a promoter-less AAV vector expressing no transgene (5 |[times]| 1012 vg/muscle) was injected into canine muscle, almost no infiltrating cells was observed in injected muscle. Third, we investigated immune responses. A lot of CD4- or CD8-positive cells were detected in clusters of infiltrating cells, together with elevated serum level of anti-|[beta]|-gal IgG. To confirm low transduction depending on immune response, dogs received daily oral administration of cyclosporine (20 mg/kg/day) from |[ndash]|5 day of the introduction of the AAV vector. Immunosuppression largely but not completely improves transduction efficiency of the AAV vector. These results suggest that AAV vector-mediated gene transfer elicits stronger immune responses in canine muscle, but immune responses against transgene products can not thoroughly explain the phenomenon. Cellular toxicity of transgene products might also participate in these infiltrations, while cytotoxicity and immunity of the AAV particles themselves can be negligible based on the result of a promoter-less AAV vectors. It is indispensable to know the molecular background of excess immune responses and cellular toxicity in canine models to establish AAV vector-mediated gene transfer in dystrophic patients.

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative non-myocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft-host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.

Regenerating More Than Muscle in Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder of skeletal and cardiac muscle. Mutations in the dystrophin gene produce DMD, but much is still unknown about the pathophysiology of DMD. Dystrophic muscle is defined by replacement of normal myofibers by connective tissue and adipocytes. Normal skeletal muscle is highly regenerative and, in the face of continued degeneration, muscle regeneration in DMD is robust, although insufficient to match the pace of degeneration. The dystrophin protein is a subsarcolemmal, spectrin repeat–containing protein of skeletal muscle myofibers and cardiomyocytes. Dystrophin associates tightly with a constellation of transmembrane proteins forming the dystrophin-glycoprotein complex (DGC). Extracellularly, the DGC binds to laminin 2, and intracellularly, the DGC binds to cytoplasmic γ-actin, a myocyte-specific form of filamin and neuronal nitric oxide synthase (nNOS). Positioned at the plasma membrane, the DGC is a mechanosignaling complex that connects the extracellular matrix with the cytoskeleton, and dystrophin is central to this role.1 When dystrophin is absent, as is the case in DMD, the entire DGC is destabilized. The dissolution of the DGC produces plasma membrane instability associated with increased intracellular calcium and myofiber degeneration. See p 3341 The ongoing and widespread muscle degeneration in DMD produces a number of systemic responses. In the present issue of Circulation , Straino and colleagues2 now show that arteriogenesis is enhanced in mdx mice, a mouse model of DMD. Under ischemic conditions, an increase in hindlimb perfusion was seen in mdx as compared with control mice. As an explanation of increased perfusion, muscle from mdx mice was found to have longer arterioles compared with control muscle. Interestingly, capillary density was not increased, and the observed difference in blood flow was attributed solely to arteriole length differences consistent with enhanced arteriogenesis. Given the prominent regeneration that is a feature of mdx muscle, …

Evolving Therapeutic Strategies for Duchenne Muscular Dystrophy: Targeting Downstream Events

Duchenne muscular dystrophy (DMD) is a progressive, lethal, muscle wasting disease that affects 1 of 3500 boys born worldwide. The disease results from mutation of the dystrophin gene that encodes a cytoskeletal protein associated with the muscle cell membrane. Although gene therapy will likely provide the cure for DMD, it remains on the distant horizon, emphasizing the need for more rapid development of palliative treatments that build on improved understanding of the complex pathology of dystrophin deficiency. In this review, we have focused on therapeutic strategies that target downstream events in the pathologic progression of DMD. Much of this work has been developed initially using the dystrophin-deficient mdx mouse to explore basic features of the pathophysiology of dystrophin deficiency and to test potential therapeutic interventions to slow, reverse, or compensate for functional losses that occur in muscular dystrophy. In some cases, the initial findings in the mdx model have led to clinical treatments for DMD boys that have produced improvements in muscle function and quality of life. Many of these investigations have concerned interventions that can affect protein balance in muscle, by inhibiting specific proteases implicated in the DMD pathology, or by providing anabolic factors or depleting catabolic factors that can contribute to muscle wasting. Other investigations have exploited the use of anti-inflammatory agents that can reduce the contribution of leukocytes to promoting secondary damage to dystrophic muscle. A third general strategy is designed to increase the regenerative capacity of dystrophic muscle and thereby help retain functional muscle mass. Each of these general approaches to slowing the pathology of dystrophin deficiency has yielded encouragement and suggests that targeting downstream events in dystrophinopathy can yield worthwhile, functional improvements in DMD.

Albumin targeting of damaged muscle fibres in the mdx mouse can be monitored by MRI

Increased sarcolemmal permeability has been implicated as a major pathological event in muscular dystrophies. In our study, we evaluated whether damaged muscle fibres can be specifically targeted using albumin as a carrier. We tagged human serum albumin (HSA) with Gadolinium (Gd) and systemically applied this compound (Gd-DTPA-HSA) to wildtype and dystrophin-deficient mdx mice. We performed magnetic resonance imaging before and after intravenous administration of Gd-DTPA-HSA and found localised signal enhancement only in mdx skeletal muscle. We also examined skeletal muscle after contrast enhanced magnetic resonance imaging using anti-human albumin antibodies and demonstrated intracellular accumulation of Gd-DTPA-HSA in clusters of damaged mdx muscle fibres. Comparison of magnetic resonance imaging and histological data emphasised the value of contrast agent enhanced magnetic resonance imaging for the in vivo assessment of fibre damage in muscular dystrophies. Furthermore, our data provide evidence that albumin can be used as a carrier to target covalently bound molecules to degenerating muscle fibres.

Identification of a zebrafish model of muscular dystrophy.

1. Large-scale mutagenic screens of the zebrafish genome have identified a number of different classes of mutations that disrupt skeletal muscle formation. Of particular interest and relevance to human health is a class of recessive lethal mutations in which muscle differentiation occurs normally, but is followed by tissue-specific degeneration reminiscent of human muscular dystrophies. 2. We have shown that one member of this class of mutations, sapje (sap), results from mutations within the zebrafish orthologue of the 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 by disrupting the link between the actin cytoskeleton and the extracellular matrix in skeletal muscle cells. 3. We have found that the progressive muscle degeneration phenotype of sapje-mutant zebrafish embryos is caused by the failure of somitic muscle attachments at the embryonic myotendinous junction (MTJ). 4. Although a role for dystrophin at the MTJ 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 is thus far lacking. Therefore, the sapjre mutation may provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases. In the present review, we discuss this finding in light of previously postulated models of dystrophin function.

Long-term depression is reduced in cerebellar Purkinje cells of dystrophin-deficient mdx mice

The mdx (muscular dystrophy X-linked) mouse is a model for human Duchenne muscular dystrophy (DMD) and is characterized by the absence of the cytoskeletal protein dystrophin. Using a cerebellar slice preparation, we show that postsynaptically mediated long-term depression (LTD) is significantly reduced in mdx Purkinje cells, while presynaptically mediated paired-pulse facilitation (PPF) is normal. This disruption of LTD could contribute to the cognitive deficit in boys with Duchenne muscular dystrophy.

Impaired long-term spatial and recognition memory and enhanced CA1 hippocampal LTP in the dystrophin-deficient Dmdmdx mouse

Duchenne muscular dystrophy (DMD) is associated with cognitive deficits that may result from dystrophin deficiency in neurons. However, in the dystrophin-deficient Dmd mdx mouse model of DMD, the nature of the memory impairment is not well characterised and its biological substrate is uncertain. Here, we demonstrate that dystrophin deficiency in Dmd mdx mice impairs long-term, but not short-term, object recognition memory and impairs long-term spatial memory, but not acquisition, following massed training in the water maze. Furthermore, we show that the abnormal enhancement of CA1 hippocampal LTP in Dmd mdx mice is not restricted to short-lasting mechanisms, but also affects the maintenance phase of LTP of both synaptic efficacy and neuronal excitability. We conclude that dystrophin loss alters memory consolidation in both spatial and nonspatial learning tasks, at least in part due to altered synaptic plasticity mechanisms, and suggest that the severity of the deficits may depend on the nature of the training procedure.

244. Successful AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle Required Suppression of Excess Immune Responses

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by a mutation in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb. To find a short but functional dystrophin cDNA, we have previously constructed three micro-dystrophin cDNAs, and generated transgenic (Tg) dystrophin-deficient mdx mice expressing micro-dystrophin. Among them, CS1-Tg mdx mice showed lowest levels of serum creatine kinase, complete amelioration of muscle pathology, and nearly full restoration of contractile force (BBRC. 293:1265, 2002). We also showed that muscle-specific MCK promoter in AAV vector could drive longer expression of the LacZ gene than the CMV promoter in skeletal muscle (Gene Ther. 23:1576, 2002). Furthermore, we constructed the AAV2 vector expressing ΔCS1 micro-dystrophin driven by MCK promoter, and demonstrated that AAV vector-mediated ΔCS1 transfer could ameliorate dystrophic phenotypes in mdx muscles (7th ASGT Annual Meeting 2004, in submission). For the application of this strategy to DMD patients, however, it is necessary to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We recently established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp. Anim. 52: 93, 2003). When the AAV vector encoding the LacZ gene driven by a CMV promoter was introduced into skeletal muscles of dogs, β-galactosidase (β-gal) was expressed only in few fibers of injected muscle after 2 weeks of injection. No β-gal-positive fiver was detected in canine muscle at 4 and 8 weeks post-injection. Instead, large numbers of mononuclear cells appeared around β-gal-expressing fibers in injected muscle. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of AAV vector, we examined viral infectivity, cytotoxicity and immune responses. First, we infected AAV vector into canine primary myotubes. This in vitro study showed that AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particle elicit cytotoxicity or not. When the AAV vector expressing no transgene was injected into canine muscle, almost no infiltrating cells was observed in injected muscle. Third, we investigated immune responses. A lot of CD4- or CD8-positive cells were detected in clusters of infiltrating cells, together with elevated serum level of anti-β-gal IgG. To confirm low transduction depending on immune response, dogs received daily oral administration of cyclosporine (20 mg/kg/day) from -5 day of the introduction of the AAV vector. Immunosuppression considerably improved transduction efficiency by an AAV vector introduction in canine muscle. These results suggested that AAV vector-mediated gene transfer elicited stronger immune responses in canine muscle, and it was necessary to know the molecular background of excess immune responses and to find the way to minimize and suppress immune responses.

Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector.

Dystrophin gene transfer using helper-dependent adenoviral vectors (HDAd) deleted of all viral genes is a promising option to treat muscles in Duchenne muscular dystrophy (DMD). Previously, we reported high-level dystrophin expression and functional correction of dystrophin-deficient (mdx) mouse muscle 60 days after gene transfer with an HDAd encoding two full-length murine dystrophin cDNAs (referred to as HDCBDysM). In the present study, we tested the long-term efficacy of HDCBDysM by examining muscle contractility parameters and the stability of dystrophin expression 1 year after injection into neonatal mdx muscles. At this point, HDCBDysM-treated muscles averaged 52% dystrophin-positive fibers. Treated muscles also displayed significantly greater isometric force production as well as greater resistance to the force deficits and damage caused by eccentric contractions. The level of protection against eccentric contraction-induced force deficits correlated with the percentage of dystrophin-positive fibers. Furthermore, HDCBDysM treatment restored the dystrophin-glycoprotein complex (DGC) to the sarcolemma and improved other aspects of mdx muscle histopathology examined (central nucleation, muscle hypertrophy, and mononuclear [phagocytic] cell infiltration). These improvements occurred despite the induction of a humoral response against murine dystrophin. Our results indicate that major therapeutic benefits of HDCBDysM are maintained for a long period of the animals' lifespan and suggest that HDCBDys holds promise for treating DMD by gene therapy.

Loss of dystrophin causes aberrant mechanotransduction in skeletal muscle fibers.

Dystrophin is a cytoskeletal protein found at the inner surface of skeletal and cardiac muscle fibers. We hypothesize that deficiency of dystrophin increases muscle compliance and causes an aberrant mechanotransduction in muscle fibers. To test this hypothesis, we measured the length-tension relationships and determined intracellular signaling leading to the activation of mitogen-activated protein (MAP) kinases in diaphragm muscle fibers from dystrophin-deficient mdx mice. Compared with controls, length-tension curves of the mdx mice were shifted to the right. A higher level of activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun N-terminal kinase-1 or p38 MAP kinase was observed in the mdx muscle compared with the normal muscle in response to mechanical stretch. Removal of Ca2+ from the medium inhibited stretch-induced ERK1/2 activation only in mdx muscle fibers but not in the normal fibers. Conversely, pretreatment with TMB-8 (an antagonist of intracellular Ca2+ blocked the mechanical stretch-induced ERK1/2 activation in normal but not in mdx muscle fibers. Pretreatment of muscle with nifedipine (L-type calcium channel antagonist) marginally decreased the activation of ERK1/2 in normal or mdx muscle whereas pretreatment with gadolinium (III) chloride (an inhibitor of stretch-activated channels) only blocked the activation of ERK1/2 in mdx muscle, with no significant effect on normal muscle. A higher basal level of activation of activator protein-1 (AP-1) transcription factor was observed in dystrophin-deficient diaphragm, which was further augmented by mechanical stretch. Mechanical stretch-induced activation of AP-1 was decreased by pretreatment of muscle fibers with PD98059 (ERK1/2 inhibitor) and removal of Ca2+ ions from incubation medium. Our results show that dystrophin is a load-bearing element and its deficiency leads to loss of muscle stiffness and aberrant mechanotransduction in skeletal muscle fibers.

Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells.

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca2+ and inhibited by blocking electrogenic Ca2+ uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient (mdx) mice, elevated intracellular Ca2+, increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting. mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial "umbrella" cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO-) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental.

Temporal gene expression profiling of dystrophin-deficient (mdx) mouse diaphragm identifies conserved and muscle group-specific mechanisms in the pathogenesis of muscular dystrophy

Mutations in dystrophin are the proximate cause of Duchenne muscular dystrophy (DMD), but pathogenic mechanisms linking the absence of dystrophin from the sarcolemma to myofiber necrosis are not fully known. The muscular dystrophies also have properties not accounted for by current disease models, including the temporal delay to disease onset, broad species differences in severity, and diversity of skeletal muscle responses. To address the mechanisms underlying the differential targeting of muscular dystrophy, we characterized temporal expression profiles of the diaphragm in dystrophin-deficient (mdx) mice between postnatal days 7 and 112 using oligonucleotide microarrays and contrasted these data with published hindlimb muscle data. Although the diaphragm and hindlimb muscle groups differ in severity of response to dystrophin deficiency, and exhibited substantial divergence in some transcript categories including inflammation and muscle-specific genes, our data show that the general mechanisms operative in muscular dystrophy are highly conserved. The two muscle groups principally differed in expression levels of differentially regulated genes, as opposed to the non-conserved induced/repressed transcripts defining fundamentally distinct mechanisms. We also identified a postnatal divergence of the two wild-type muscle group expression profiles that temporally correlated with the onset and progression of the dystrophic process. These findings support the hypothesis that conserved disease mechanisms interacting with baseline differences in muscle group-specific transcriptomes underlie their differential responses to DMD. We further suggest that muscle group-specific transcriptional profiles contribute toward the muscle targeting and sparing patterns observed for a variety of metabolic and neuromuscular diseases.

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.

Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis.

Altered inhibitory input to Purkinje cells of dystrophin-deficient mice

We investigated evoked EPSPs and spontaneous IPSPs in cerebellar slices from dystrophin-deficient mdx mice. In the presence of the GABA A antagonist bicuculline the increase in EPSP amplitude was less in mdx Purkinje cells compared to control, and the amplitude of miniature IPSCs in mdx cells was also significantly less than in controls. This reduced inhibitory input is most likely due to the reported reduction in the size of GABA A channel clusters.

Prolonged dystrophin expression and functional correction of mdx mouse muscle following gene transfer with a helper-dependent (gutted) adenovirus-encoding murine dystrophin.

Dystrophin gene transfer using helper-dependent adenoviruses (HDAd), which are deleted of all viral genes, is a promising option to treat muscles in Duchenne muscular dystrophy. We investigated the benefits of this approach by injecting the tibialis anterior (TA) muscle of neonatal and juvenile (4-6-week-old) dystrophin-deficient (mdx) mice with a fully deleted HDAd (HDCBDysM). This vector encoded two full-length murine dystrophin cDNAs regulated by the powerful cytomegalovirus enhancer/beta-actin promoter. At 10 days post-injection of neonatal muscles, 712 fibers (42% of the total number of TA fibers) were dystrophin-positive (dys+), a value that did not decrease for 6 months (the study duration). In treated juveniles, maximal transduction occurred at 30 days post-injection (414 dys+ fibers, 24% of the total number of TA fibers), but decreased by 51% after 6 months. All studied aspects of the pathology were improved in neonatally treated muscles: the percentage of dys+ fibers with centrally localized myonuclei remained low, localization of the dystrophin associated protein complex was restored at the plasma membrane, muscle hypertrophy was reduced, and maximal force-generating capacity and resistance to contraction-induced injuries were increased. The same pathological aspects were improved in the treated juveniles, except for reduction of muscle hypertrophy and maximal force-generating capacity. We demonstrated a strong humoral response against murine dystrophin in both animal groups, but mild inflammatory response occurred only in the treated juveniles. HDCBDysM is thus one of the most promising and efficient vectors for treating DMD by gene therapy.