Mdx Muscle Research Articles

M.I.4 Progress in gene therapy for muscular dystrophy using vascular delivery of GALGT2

The myomatrix present at the neuromuscular junction, as well as the transmembrane receptor complexes linking it to the F-actin cytoskeleton, are distinct from those present along the rest of the skeletal myofiber sarcolemmal membrane. Covalent protein modifications, including glycosylation, also differ between these two muscle regions. We have shown that overexpression of GALGT2, a glycosyltransferase normally confined to the neuromuscular synapse, in mouse or macaque skeletal muscle leads to ectopic glycosylation of alpha dystroglycan and the overexpression of normally synaptic dystroglycan-binding proteins, including utrophin, laminin alpha5 and agrin. As a likely result of these and related changes, GALGT2 overexpression can inhibit the development of muscle disease in the mdx model of Duchenne muscular dystrophy, the dyW model of Congenital Muscular Dystrophy 1A, and the Sgca−/− model of Limb Girdle Muscular Dystrophy 2D. In addition, GALGT2 overexpression can increase binding of myomatrix proteins to alpha dystroglycan and prevent muscle damage induced by eccentric contractions. Moreover, elimination of endogenous GALGT2 upregulation in mdx muscles leads to more severe disease. These proof of concept studies suggest that GALGT2 is a surrogate gene therapy with potentially broad applications in muscle disease. With help from a team of investigators in the Center for Gene Therapy at Nationwide Children's Hospital, we are developing methods for vascular delivery of gene therapies, using both gene replacement and surrogate gene strategies, with the goal of performing clinical trials in multiple forms of muscular dystrophy. This work was supported by grants from the NIH U54 NS055958 and R01 AR060949.

Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

BackgroundPresently, there is no effective treatment for the lethal muscle wasting disease Duchenne muscular dystrophy (DMD). Here we show that increased sphingosine-1-phoshate (S1P) through direct injection or via the administration of the small molecule 2-acetyl-4(5)-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, has beneficial effects in acutely injured dystrophic muscles of mdx mice.MethodsWe treated mdx mice with and without acute injury and characterized the histopathological and functional effects of increasing S1P levels. We also tested exogenous and direct administration of S1P on mdx muscles to examine the molecular pathways under which S1P promotes regeneration in dystrophic muscles.ResultsShort-term treatment with THI significantly increased muscle fiber size and extensor digitorum longus (EDL) muscle specific force in acutely injured mdx limb muscles. In addition, the accumulation of fibrosis and fat deposition, hallmarks of DMD pathology and impaired muscle regeneration, were lower in the injured muscles of THI-treated mdx mice. Furthermore, increased muscle force was observed in uninjured EDL muscles with a longer-term treatment of THI. Such regenerative effects were linked to the response of myogenic cells, since intramuscular injection of S1P increased the number of Myf5nlacz/+ positive myogenic cells and newly regenerated myofibers in injured mdx muscles. Intramuscular injection of biotinylated-S1P localized to muscle fibers, including newly regenerated fibers, which also stained positive for S1P receptor 1 (S1PR1). Importantly, plasma membrane and perinuclear localization of phosphorylated S1PR1 was observed in regenerating muscle fibers of mdx muscles. Intramuscular increases of S1P levels, S1PR1 and phosphorylated ribosomal protein S6 (P-rpS6), and elevated EDL muscle specific force, suggest S1P promoted the upregulation of anabolic pathways that mediate skeletal muscle mass and function.ConclusionsThese data show that S1P is beneficial for muscle regeneration and functional gain in dystrophic mice, and that THI, or other pharmacological agents that raise S1P levels systemically, may be developed into an effective treatment for improving muscle function and reducing the pathology of DMD.

Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves cell therapy

In Duchenne muscular dystrophy (DMD) and the mdx mouse model, the absence of the cytoskeletal protein dystrophin causes defective anchoring of myofibres to the basal lamina. The resultant myofibre degeneration and necrosis lead to a progressive loss of muscle mass, increased fibrosis and ultimately fatal weakness. Connective tissue growth factor (CTGF/CCN-2) is critically involved in several chronic fibro-degenerative diseases. In DMD, the role of CTGF might extend well beyond replacement fibrosis secondary to loss of muscle fibres, since its overexpression in skeletal muscle could by itself induce a dystrophic phenotype. Using two independent approaches, we here show that mdx mice with reduced CTGF availability do indeed have less severe muscular dystrophy. Mdx mice with hemizygous CTGF deletion (mdx-Ctgf+/-), and mdx mice treated with a neutralizing anti-CTGF monoclonal antibody (FG-3019), performed better in an exercise endurance test, had better muscle strength in isolated muscles and reduced skeletal muscle impairment, apoptotic damage and fibrosis. Transforming growth factor type-β (TGF-β), pERK1/2 and p38 signalling remained unaffected during CTGF suppression. Moreover, both mdx-Ctgf+/- and FG-3019 treated mdx mice had improved grafting upon intramuscular injection of dystrophin-positive satellite cells. These findings reveal the potential of targeting CTGF to reduce disease progression and to improve cell therapy in DMD.

Treatment with the cysteine precursor l-2-oxothiazolidine-4-carboxylate (OTC) implicates taurine deficiency in severity of dystropathology in mdx mice

Oxidative stress has been implicated in the pathology of the lethal skeletal muscle disease Duchenne muscular dystrophy (DMD), and various antioxidants have been investigated as a potential therapy. Recently, treatment of the mdx mouse model for DMD with the antioxidant and cysteine and glutathione (GSH) precursor n-acetylcysteine (NAC) was shown to decrease protein thiol oxidation and improve muscle pathology and ex vivo muscle strength. This study further investigates the mechanism for the benefits of NAC on dystrophic muscle by administering l-2-oxothiazolidine-4-carboxylate (OTC) which also upregulates intracellular cysteine and GSH, but does not directly function as an antioxidant. We observed that OTC, like NAC, decreases protein thiol oxidation, decreases pathology and increases strength, suggesting that the both NAC and OTC function via increasing cysteine and GSH content of dystrophic muscle. We demonstrate that mdx muscle is not deficient in either cysteine or GSH and that these are not increased by OTC treatment. However, we show that dystrophic muscle of 12 week old mdx mice is deficient in taurine, a by-product of disposal of excess cysteine, a deficiency that is ameliorated by OTC treatment. These data suggest that in dystrophic muscles, apart from the strong association of increased oxidative stress and protein thiol oxidation with dystropathology, another major issue is an insufficiency in taurine that can be corrected by increasing the availability of cysteine. This study provides new insight into the molecular mechanism underlying the benefits of NAC in muscular dystrophy and supports the use of OTC as an alternative drug for potential clinical applications to DMD.

Delayed Bone Regeneration Is Linked to Chronic Inflammation in Murine Muscular Dystrophy

Duchenne muscular dystrophy (DMD) patients exhibit skeletal muscle weakness with continuous cycles of muscle fiber degeneration/regeneration, chronic inflammation, low bone mineral density, and increased risks of fracture. Fragility fractures and associated complications are considered as a consequence of the osteoporotic condition in these patients. Here, we aimed to establish the relationship between muscular dystrophy and fracture healing by assessing bone regeneration in mdx mice, a model of DMD with absence of osteoporosis. Our results illustrate that muscle defects in mdx mice impact the process of bone regeneration at various levels. In mdx fracture calluses, both cartilage and bone deposition were delayed followed by a delay in cartilage and bone remodeling. Vascularization of mdx fracture calluses was also decreased during the early stages of repair. Dystrophic muscles are known to contain elevated numbers of macrophages contributing to muscle degeneration. Accordingly, we observed increased macrophage recruitment in the mdx fracture calluses and abnormal macrophage accumulation throughout the process of bone regeneration. These changes in the inflammatory environment subsequently had an impact on the recruitment of osteoclasts and the remodeling phase of repair. Further damage to the mdx muscles, using a novel model of muscle trauma, amplified both the chronic inflammatory response and the delay in bone regeneration. In addition, PLX3397 treatment of mdx mice, a cFMS (colony stimulating factor receptor 1) inhibitor in monocytes, partially rescued the bone repair defect through increasing cartilage deposition and decreasing the number of macrophages. In conclusion, chronic inflammation in mdx mice contributes to the fracture healing delay and is associated with a decrease in angiogenesis and a transient delay in osteoclast recruitment. By revealing the role of dystrophic muscle in regulating the inflammatory response during bone repair, our results emphasize the implication of muscle in the normal bone repair process and may lead to improved treatment of fragility fractures in DMD patients.

Acute failure of action potential conduction in mdx muscle reveals new mechanism of contraction‐induced force loss

A primary feature of skeletal muscle lacking the protein dystrophin, as occurring in Duchenne muscular dystrophy, is a hypersensitivity to contraction-induced strength loss. We tested the hypothesis that the extensive strength loss results from an impairment in the electrophysiological function of the plasmalemma specifically impaired action potential development. Anterior crural muscles from mdx and wildtype mice performed a single bout of 100 electrically stimulated eccentric contractions in vivo. Electromyography, specifically the M-wave, was analysed during muscle contraction to assess the ability of the tibialis anterior muscle plasmalemma to generate and conduct action potentials. During eccentric contractions, wildtype mice exhibited a 36% loss in torque about the ankle but mdx mice exhibited a greater torque loss of 73% (P < 0.001). Despite the loss of torque, there was no reduction in M-wave root mean square (RMS) for wildtype mice, which was in stark contrast to mdx mice that had a 55% reduction in M-wave RMS (P < 0.001). This impairment resolved within 24 h and coincided with a significant improvement in strength and membrane integrity. Intracellular measurements of resting membrane potential (RMP) in uninjured and injured extensor digitorum longus muscles were made to determine if a chronic depolarization had occurred, which could lead to impaired fibre excitability and/or altered action potential conduction properties. The distributions of RMP were not different between wildtype uninjured and injured muscle cells (median: -73.2 mV vs. -72.7 mV, P = 0.46) whereas there was a significant difference between mdx uninjured and injured cells (median: -71.5 mV vs. -56.6 mV, P < 0.001). These data show that mdx muscle fibres are depolarized after an injurious bout of eccentric contractions. These findings (i) suggest a major plasmalemma-based mechanism of strength loss underlying contraction-induced injury in Duchenne muscular dystrophy distinctly different from that for healthy muscle, and (ii) demonstrate dystrophin is critical for maintaining action potential generation and conduction after eccentric contractions.

Comparative proteomic profiling of soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscles from the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy is due to genetic abnormalities in the dystrophin gene and represents one of the most frequent genetic childhood diseases. In the X-linked muscular dystrophy (mdx) mouse model of dystrophinopathy, different subtypes of skeletal muscles are affected to a varying degree albeit the same single base substitution within exon 23 of the dystrophin gene. Thus, to determine potential muscle subtype-specific differences in secondary alterations due to a deficiency in dystrophin, in this study, we carried out a comparative histological and proteomic survey of mdx muscles. We intentionally included the skeletal muscles that are often used for studying the pathomechanism of muscular dystrophy. Histological examinations revealed a significantly higher degree of central nucleation in the soleus and extensor digitorum longus muscles compared with the flexor digitorum brevis and interosseus muscles. Muscular hypertrophy of 20–25% was likewise only observed in the soleus and extensor digitorum longus muscles from mdx mice, but not in the flexor digitorum brevis and interosseus muscles. For proteomic analysis, muscle protein extracts were separated by fluorescence two-dimensional (2D) gel electrophoresis. Proteins with a significant change in their expression were identified by mass spectrometry. Proteomic profiling established an altered abundance of 24, 17, 19 and 5 protein species in the dystrophin-deficient soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscle, respectively. The key proteomic findings were verified by immunoblot analysis. The identified proteins are involved in the contraction-relaxation cycle, metabolite transport, muscle metabolism and the cellular stress response. Thus, histological and proteomic profiling of muscle subtypes from mdx mice indicated that distinct skeletal muscles are differentially affected by the loss of the membrane cytoskeletal protein, dystrophin. Varying degrees of perturbed protein expression patterns in the muscle subtypes from mdx mice may be due to dissimilar downstream events, including differences in muscle structure or compensatory mechanisms that counteract pathophysiological processes. The interosseus muscle from mdx mice possibly represents a naturally protected phenotype.

Fatty Acid Profile of Cardiac Muscle Phospholipid and Triacylglycerol in MDX Mice and C57BL/10ScSnJ Controls

The mdx mouse is a model for Duchenne muscular dystrophy (DMD), a debilitating disease affecting striated muscle. It is established that the fatty acid (FA) composition of skeletal muscle phospholipid (PL) is altered in mdx mice, but it is not known if cardiac muscle is similarly affected by dystrophin-deficiency. We tested FA profiles in PL and triacylglycerol (TAG) in cardiac muscle of 12-week old mdx and control (con) mice. Of 22 different FA, similar to our previous finding for skeletal muscle, the most abundant FA in heart PL were palmitic, stearic, cis-vaccenic, linoleic, and docosahexaenoic acid, while for TAG the most abundant FA were palmitic, oleic, cis-vaccenic, and linoleic acid. In comparing mdx and con, no significant group differences were detected for any FA in PL or TAG. Thus, unlike skeletal muscle, FA composition in cardiac muscle PL is not different between mdx and con at the age studied. The results can be understood in the context of tissue-specific disease severity in mdx mice, as pathology is quite modest in cardiac compared with skeletal muscle.

Linking cytoarchitecture to metabolism: sarcolemma-associated plectin affects glucose uptake by destabilizing microtubule networks in mdx myofibers.

BackgroundDuchenne muscular dystrophy (DMD) is one of the most frequent forms of muscular disorders. It is caused by the absence of dystrophin, a core component of the sarcolemma-associated junctional complex that links the cytoskeleton to the extracellular matrix. We showed previously that plectin 1f (P1f), one of the major muscle-expressed isoforms of the cytoskeletal linker protein plectin, accumulates at the sarcolemma of DMD patients as well as of mdx mice, a widely studied animal model for DMD.Based on plectin’s dual role as structural protein and scaffolding platform for signaling molecules, we speculated that the dystrophic phenotype observed after loss of dystrophin was caused, at least to some extent, by excess plectin. Thus, we hypothesized that elimination of plectin expression in mdx skeletal muscle, while probably resulting in an overall more severe phenotype, may lead to a partial phenotype rescue. In particular, we wanted to assess whether excess sarcolemmal plectin contributes to the dysregulation of sugar metabolism in mdx myofibers.MethodsWe generated plectin/dystrophin double deficient (dKO) mice by breeding mdx with conditional striated muscle-restricted plectin knockout (cKO) mice. The phenotype of these mice was comparatively analyzed with that of mdx, cKO, and wild-type mice, focusing on structural integrity and dysregulation of glucose metabolism.ResultsWe show that the accumulation of plectin at the sarcolemma of mdx muscle fibers hardly compensated for their loss of structural integrity. Instead, it led to an additional metabolic deficit by impairing glucose uptake. While dKO mice suffered from an overall more severe form of muscular dystrophy compared to mdx or plectin-deficient mice, sarcolemmal integrity as well as glucose uptake of their myofibers were restored to normal levels upon ablation of plectin. Furthermore, microtubule (MT) networks in intact dKO myofibers, including subsarcolemmal areas, were found to be more robust than those in mdx mice. Finally, myotubes differentiated from P1f-overexpressing myoblasts showed an impairment of glucose transporter 4 translocation and a destabilization of MT networks.ConclusionsBased on these results we propose that sarcolemma-associated plectin acts as an antagonist of MT network formation in myofibers, thereby hindering vesicle-mediated (MT-dependent) transport of glucose transporter 4. This novel role of plectin throws a bridge between extra-sarcomeric cytoarchitecture and metabolism of muscle fibers. Our study thus provides new insights into pathomechanisms of plectinopathies and muscular dystrophies in general.

Increased sarcolipin expression and decreased sarco(endo)plasmic reticulum Ca2+ uptake in skeletal muscles of mouse models of Duchenne muscular dystrophy

Abnormal intracellular Ca(2+) handling is an important factor in the progressive functional decline of dystrophic muscle. In the present study, we investigated the function of sarco(endo)plasmic reticulum (SR) Ca(2+) ATPase (SERCA) in various dystrophic muscles of mouse models of Duchenne muscular dystrophy. Our studies show that the protein expression of sarcolipin, a key regulator of the SERCA pump is abnormally high and correlates with decreased maximum velocity of SR Ca(2+) uptake in the soleus, diaphragm and quadriceps of mild (mdx) and severe (mdx:utr-/-) dystrophic mice. These changes are more pronounced in the muscles of mdx:utr-/- mice. We also found increased expression of SERCA2a and calsequestrin specifically in the dystrophic quadriceps. Immunostaining analysis further showed that SERCA2a expression is associated both with fibers expressing slow-type myosin and regenerating fibers expressing embryonic myosin. Together, our data suggest that sarcolipin upregulation is a common secondary alteration in all dystrophic muscles and contributes to the abnormal elevation of intracellular Ca(2+) concentration via SERCA inhibition.

Theoretical and Experimental SHG Angular Intensity Patterns from Healthy and Proteolysed Muscles

SHG angular intensity pattern (SHG-AIP) of healthy and proteolysed muscle tissues are simulated and imaged here for the first time to our knowledge. The role of the spatial distribution of second-order nonlinear emitters on SHG-AIP is highlighted. SHG-AIP with two symmetrical spots is found to be a signature of healthy muscle whereas SHG-AIP with one centered spot in pathological mdx muscle is found to be a signature of myofibrillar disorder. We also show that SHG-AIP provides information on the three-dimensional structural organization of myofibrils in physiological and proteolysed muscle. Our results open an avenue for future studies aimed at unraveling more complex physiological and pathological fibrillar tissues organization.

NF‐kB–dependent IL‐6 expression, induced by electrical stimulation, is reduced in dystrophic mdx skeletal muscle cells.

In skeletal muscle cells, membrane depolarization increases reactive oxygen species (ROS) due to NOX2 activation; this is related with changes in gene expression. Our aim was to study IL‐6 gene in wt and mdx muscle cells, and its relationship with NOX2 and NF‐kB activity. Differentiated wt and mdx myotubes were stimulated with 250p, 0.5 ms at 20 Hz. ROS production was assessed with CM‐DCFA. NOX2 was assessed using apocynin and DPI. NF‐kB transcriptional activity was studied with a luciferase reporter, p65 phosphorylation by western blot and gene expression by real time PCR.NOX2 subunits (gp91 and p47) were upregulated in mdx myotubes. After stimulation, ROS production was higher in mdx compared to wt. NF‐kB activity after both electrical and ROS stimulation increased in wt myotubes, but not in mdx. NOX2 inhibition reduced NF‐kB activation. IL‐6 gene expression induced by both electrical and ROS stimulation was higher in wt compared to mdx muscle cells.ROS production and NF‐kB activity are linked in normal cells. Altered NF‐kB, excess ROS production and differential gene expression could explain oxidative stress and progressive damage reported in DMD.Fondecyt 1110467 (EJ), AT‐24100066 (FA).

Protective effects of Coenzyme Q10 on dystrophic muscle cells

Duchenne muscular dystrophy (DMD) is a progressive and lethal X‐linked myopathy characterized by a deficiency of the dystrophin protein. Oxidative stress and abnormal production of reactive oxygen species (ROS) plays a key role in the pathophysiology of DMD. We investigated the effects of the antioxidant Coenzyme Q10 (CoQ10) on the primary cultures of dystrophic muscle cells from mdx mice, the experimental model of DMD. Mdx muscle cells were treated with CoQ10 (5 μM) for 24 h. The muscle cells of C57BL/10 mice were used as controls. Drug toxicity was assessed for using an MTT cell viability assay. H2O2 (a marker of cellular ROS production) was measured by using the Amplex red assay kit. The levels of 4‐HNE (a lipid peroxidation marker) were analyzed by western blotting. The MTT results showed that CoQ10 not had cytotoxic effect on mdx and C57BL/10 muscle cells. H2O2 production and 4‐HNE levels were significantly higher in mdx muscle cells compared to C57BL/10 muscle cells (P≤0.05, Student's t‐test). CoQ10 treatment decreased H2O2 production by 51% and 4‐HNE levels by 18% in the dystrophic muscle cells. Based on the results, it can be seen that CoQ10 has protective effects on mdx muscle cells and this in turn would support further investigations for its use as a potential therapy for dystrophinopathies.

Architecture of healthy and dystrophic muscles detected by optical coherence tomography

The ability to view individual myofibers is possible with many histological techniques, but not yet with standard in vivo imaging. Optical coherence tomography (OCT) is an emerging technology that can generate high resolution 1-10 μm cross-sectional imaging of tissue in vivo and in real time. We used OCT to determine architectural differences of tibialis anterior muscles in situ from healthy mice (wild-type [WT], n = 4) and dystrophic mice (mdx, n = 4). After diffusion tensor imaging (DTI) and OCT, muscles were harvested, snap-frozen, and sectioned for staining with wheat germ agglutinin. DTI suggested differences in pennation and OCT was used to confirm this supposition. OCT indicated a shorter intramuscular tendon (WT/mdx ratio of 1.2) and an 18% higher degree of pennation in mdx. Staining confirmed these architectural changes. Architectural changes in mdx muscles, which could contribute to reduction of force, are detectable with OCT.

From chaos to split‐ups – SHG microscopy reveals a specific remodelling mechanism in ageing dystrophic muscle

Duchenne muscular dystrophy (DMD) is a common inherited muscle disease showing chronic inflammation and progressive muscle weakness. Absent dystrophin renders sarcolemma more Ca(2+) -permeable, disturbs signalling and triggers inflammation. Sustained degeneration/regeneration cycles render muscle cytoarchitecture susceptible to remodelling. Quantitative morphometry was introduced in living cells using second-harmonic generation (SHG) microscopy of myosin. As the time course of cellular remodelling is not known, we used SHG microscopy in mdx muscle fibres over a wide age range for three-dimensional (3D) rendering and detection of verniers and cosine angle sums (CASs). Wild-type (wt) and transgenic mini-dystrophin mice (MinD) were also studied. Vernier densities (VDs) declined in wt and MinD fibres until adulthood, while in mdx fibres, VDs remained significantly elevated during the life span. CAS values were close to unity in adult wt and MinD fibres, in agreement with tight regular myofibril orientation, while always smaller in mdx fibres. Using SHG 3D morphometry, we identified two types of altered ultrastructure: branched fibres and a novel, previously undetected 'chaotic' fibre type, both of which can be classified by distinct CAS and VD combinations. We present a novel model of tissue remodelling in dystrophic progression with age that involves the transition from normal to chaotic to branched fibres. Our model predicts a ~50% contribution of altered cytoarchitecture to progressive force loss with age. We also provide an improved automated image algorithm that is suitable for future ageing studies in human myopathies.

Prosaposin expression in the regenerated muscles of mdx and cardiotoxin-treated mice.

The trophic factor prosaposin (PS) is strongly expressed in skeletal muscle, and reportedly, a PS-derived peptide attenuates loss of muscle mass after nerve injury in vivo and increases myoblast fusion into myotubes in vitro. However, few studies have focused on the role of PS during muscle regeneration. We examined the expression of PS in the skeletal muscles in normal, mdx, and cardiotoxin (CTX)-treated mice using immunofluorescence staining, Western blotting, and in situ hybridisation. Immunofluorescence showed intense PS immunoreactivity in the peripheral cytoplasm of uninjured myofibres of normal mice and regenerated myofibres of 8 weeks post-CTX-injection mice. In early stage CTX-treated mice (14 days and earlier), intense PS immunoreactivity was also detected in the immune cells that infiltrated damaged muscle, but it was weak for regenerating myofibres. Western blot confirmed these findings. In contrast, PS was continuously low in mdx mice in both immunofluorescence and Western blotting. In situ hybridisation confirmed the decrease of PS mRNA in regenerated myofibres and revealed the main form of PS mRNA as Pro+0 without a 9-base insertion both in normal and mdx mice. The embryonic myosin (MYH3) was clearly localized in the newly regenerated myofibres at 3, 7, and 14 days of post-CTX-injection and mdx mice, but was lower in the late stage of regenerated myofibres (28 and 56 days post-CTX injection). The inverse distribution of MYH3 and PS indicates that the PS expression is closely related to the differentiation of regenerated myofibres. Investigation of the mitogen-activated protein (MAP) kinase signal pathway showed the inversely synchronous correlation of phosphorylated ERK1/2 with myofibre PS and the synchronous correlation of phosphorylated p-38 with myofibre PS. These data suggest that PS is involved in the regulation of muscle differentiation of regenerated fibres.

AAV-mediated Overexpression of Human α7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the DMD gene, with loss of its gene product, dystrophin. Dystrophin helps link integral membrane proteins to the actin cytoskeleton and stabilizes the sarcolemma during muscle activity. We investigated an alternative therapeutic approach to dystrophin replacement by overexpressing human α7 integrin (ITGA7) using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal and cardiac muscle that links the extracellular matrix (ECM) to the actin skeleton. It is modestly upregulated in DMD muscle and has been proposed to be an important modifier of dystrophic symptoms. We delivered rAAV8.MCK.ITGA7 to the lower limb of mdx mice through isolated limb perfusion (ILP) of the femoral artery. We demonstrated ~50% of fibers in the tibialis anterior (TA) and extensor digitorum longus (EDL) overexpressing α7 integrin at the sarcolemma following AAV gene transfer. The increase in ITGA7 in skeletal muscle significantly protected against loss of force following eccentric contraction-induced injury compared with untreated (contralateral) muscles while specific force following tetanic contraction was unchanged. Reversal of additional dystrophic features included reduced Evans blue dye (EBD) uptake and increased muscle fiber diameter. Taken together, this data shows that rAAV8.MCK.ITGA7 gene transfer stabilizes the sarcolemma potentially preserving mdx muscle from further damage. This therapeutic approach demonstrates promise as a viable treatment for DMD with further implications for other forms of muscular dystrophy.

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca2+ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca2+ deregulation and Ca2+ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca2+ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca2+ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca2+ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca2+ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca2+ store contributes to the disrupted Ca2+ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.

Levels of α7 integrin and laminin-α2 are increased following prednisone treatment in themdxmouse and GRMD dog models of Duchenne muscular dystrophy

SUMMARYDuchenne muscular dystrophy (DMD) is a fatal neuromuscular disease for which there is no cure and limited treatment options. Prednisone is currently the first line treatment option for DMD and studies have demonstrated that it improves muscle strength. Although prednisone has been used for the treatment of DMD for decades, the mechanism of action of this drug remains unclear. Recent studies have shown that the α7β1 integrin is a major modifier of disease progression in mouse models of DMD and is therefore a target for drug-based therapies. In this study we examined whether prednisone increased α7β1 integrin levels in mdx mouse and GRMD dog models and myogenic cells from humans with DMD. Our results show that prednisone promotes an increase in α7 integrin protein in cultured myogenic cells and in the muscle of mdx and GRMD animal models of DMD. The prednisone-mediated increase in α7 integrin was associated with increased laminin-α2 in prednisone-treated dystrophin-deficient muscle. Together, our results suggest that prednisone acts in part through increased merosin in the muscle basal lamina and through sarcolemmal stabilization of α7β1 integrin in dystrophin-deficient muscle. These results indicate that therapies that target an increase in muscle α7β1 integrin, its signaling pathways and/or laminin could be therapeutic in DMD.

Nox2-Dependent Redox Regulation of Calcium Influx in Dystrophic Skeletal Muscle

Duchenne Muscular Dystrophy (DMD) is an X-linked, muscle-wasting disease caused by deletions in the gene that encodes dystrophin, an integral muscle membrane protein that links the contractile proteins to the muscle membrane. Recent studies have suggested that increased Ca2+ influx into the muscle and increased production of free radicals (or reactive oxygen species, ROS) are essential for increased susceptibility of mdx muscle to damage. However, the source of the ROS, the Ca2+ channels affected, and the mechanism(s) of how mechanical stress results in altered regulation of these signaling pathways have yet to be determined. We hypothesis that NADPH oxidase (Nox2) drives excessive ROS production, increased Ca2+ influx, and muscle damage. Manganes (Mn2+) quench was used to assess the role of Nox2 dependent ROS on sarcolemmal Ca2+ influx in response to a physiological stretch and depolarization. Our results show that stretch-activated Ca2+ entry in mdx is significantly increased 4.2-fold (p<0.001) compared to WT, while K+-induced depolarization results in 20-fold (p<0.001) increase in Mn2+ quench in mdx skeletal muscle. Administration of either gp91ds-TAT, an inhibitor of Nox2, or reduced glutathione ethyl ester, a glutathione analogue, in mdx muscle reduces the Mn2+ quench rates back to the WT levels for both passive stretch and K+depolarization. Addition of BTP2, a calcium release activated calcium channel inhibitor, also significantly attenuates Mn2+ quench rates in mdx compared to WT. Our data supports a model in which Nox2 dependent redox modifications of stretch and depolarization activated Ca2+ channels leads to exuberant Ca2+ influx. Our results identify Nox2 as a potential therapeutic approach to minimize or delay the progressive DMD disease.