Poloxamer 188 reduces the contraction-induced force decline in lumbrical muscles from mdx mice

Duchenne Muscular Dystrophy is a genetic disease caused by the lack of the protein dystrophin. Dystrophic muscles are highly susceptible to contraction-induced injury, and following contractile activity, have disrupted plasma membranes that allow leakage of calcium ions into muscle fibers. Because of the direct relationship between increased intracellular calcium concentration and muscle dysfunction, therapeutic outcomes may be achieved through the identification and restriction of calcium influx pathways. Our purpose was to determine the contribution of sarcolemmal lesions to the force deficits caused by contraction-induced injury in dystrophic skeletal muscles. Using isolated lumbrical muscles from dystrophic (mdx) mice, we demonstrate for the first time that poloxamer 188 (P188), a membrane-sealing poloxamer, is effective in reducing the force deficit in a whole mdx skeletal muscle. A reduction in force deficit was also observed in mdx muscles that were exposed to a calcium-free environment. These results, coupled with previous observations of calcium entry into mdx muscle fibers during a similar contraction protocol, support the interpretation that extracellular calcium enters through sarcolemmal lesions and contributes to the force deficit observed in mdx muscles. The results provide a basis for potential therapeutic strategies directed at membrane stabilization of dystrophin-deficient skeletal muscle fibers.     

Increased calcium influx in dystrophic muscle

We examined pathways which might result in the elevated resting free calcium [( Ca2+]i) levels observed in dystrophic mouse (mdx) skeletal muscle fibers and myotubes and human Duchenne muscular dystrophy myotubes. We found that mdx fibers, loaded with the calcium indicator fura-2, were less able to regulate [Ca2+]i levels in the region near the sarcolemma. Increased calcium influx or decreased efflux could lead to elevated [Ca2+]i levels. Calcium transient decay times were identical in normal and mdx fibers if resting [Ca2+]i levels were similar, suggesting that calcium-sequestering mechanisms are not altered in dystrophic muscle, but are slowed by the higher resting [Ca2+]i. The defect appears to be specific for calcium since resting free sodium levels and sodium influx rates in the absence of Na+/K(+)-ATPase activity were identical in normal and dystrophic cells when measured with sodium-binding benzofuran isophthalate. Calcium leak channels, whose opening probabilities (Po) were voltage independent, could be the major calcium influx pathway at rest. We have shown previously that calcium leak channel Po is significantly higher in dystrophic myotubes. These leak channels were selective for calcium over sodium under physiological conditions. Agents that increased leak channel activity also increased [Ca2+]i in fibers and myotubes. These results suggest that increased calcium influx, as a result of increased leak channel activity, could result in the elevated [Ca2+]i in dystrophic muscle.     

Regulation of TRPC1 and TRPC4 Cation Channels Requires an ��1-Syntrophin-dependent Complex in Skeletal Mouse Myotubes

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and α1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of α1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of α1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by α1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by α1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the α1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and α1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.     

Stretch-activated calcium channel protein TRPC1 is correlated with the different degrees of the dystrophic phenotype in mdx mice

In Duchenne muscular dystrophy (DMD) and in the mdx mouse model of DMD, the lack of dystrophin is related to enhanced calcium influx and muscle degeneration. Stretch-activated channels (SACs) might be directly involved in the pathology of DMD, and transient receptor potential cation channels have been proposed as likely candidates of SACs. We investigated the levels of transient receptor potential canonical channel 1 (TRPC1) and the effects of streptomycin, a SAC blocker, in muscles showing different degrees of the dystrophic phenotype. Mdx mice (18 days old, n = 16) received daily intraperitoneal injections of streptomycin (182 mg/kg body wt) for 18 days, followed by removal of the diaphragm, sternomastoid (STN), biceps brachii, and tibialis anterior muscles. Control mdx mice (n = 37) were injected with saline. Western blot analysis showed higher levels of TRPC1 in diaphragm muscle compared with STN and limb muscles. Streptomycin reduced creatine kinase and prevented exercise-induced increases of total calcium and Evans blue dye uptake in diaphragm and in STN muscles. It is suggested that different levels of the stretch-activated calcium channel protein TRPC1 may contribute to the different degrees of the dystrophic phenotype seen in mdx mice. Early treatment designed to regulate the activity of these channels may ameliorate the progression of dystrophy in the most affected muscle, the diaphragm.     

Increased expression of deltaCaMKII isoforms in skeletal muscle regeneration: Implications in dystrophic muscle disease

The expression of delta isoforms of calcium-calmodulin/dependent protein kinase II (CaMKII) has been reported in mammalian skeletal muscle; however, their functions in this tissue are largely unknown. This study was conducted to determine if deltaCaMKII expression was altered during regeneration of skeletal muscle fibers in two distinct models. In the first model, necrosis and regeneration were induced in quadriceps of normal mice by intramuscular administration of 50% glycerol. Immunostaining and confocal microscopy revealed that deltaCaMKII expression was clearly enhanced in fibers showing centralized nuclei. The second model was the mdx mouse, which undergoes enhanced muscle necrosis and regeneration due to a mutation in the dystrophin gene. sern blot analysis of hind leg extracts from 4 to 6 week old mdx mice revealed that deltaCaMKII content was decreased when compared to age-matched control mice. This loss in delta kinase content was seen in myofibrillar and membrane fractions and was in contrast to unchanged deltaCaMKII levels in cardiac and brain extracts from dystrophic mice. Confocal microscopy of mdx quadriceps and tibialis muscle showed that deltaCaMKII expression was uniformly decreased in most fibers from dystrophic mice; however, enhanced kinase expression was observed in regenerating muscle fibers. These data support a fundamental role for deltaCaMKII in the regeneration process of muscle fibers in normal and mdx skeletal muscle and may have important implications in the reparative process following muscle death.     

IGF-II ameliorates the dystrophic phenotype and coordinately down-regulates programmed cell death

Duchenne muscular dystrophy (DMD) is a fatal and crippling disease of skeletal muscle which displays increased fibre turnover and elevated levels of programmed cell death (PCD) in muscle stem cells. Previously we showed that this cell death is inhibited by the growth factor IGF-II. To determine the functional significance of PCD to the dystrophic phenotype, we used a transgene to over-express IGF-II in the mdx mouse. We found that ectopic expression of IGF-II inhibited the elevated PCD observed in skeletal muscles in the absence of functional dystrophin and significantly ameliorates the early gross histopathological changes in skeletal muscles characteristic of the dystrophic phenotype. Replacement of the dystrophin gene abolished abnormal skeletal muscle cell PCD levels in vivo in a dose-dependent manner and in dystrophic SMS cell lines cultured in vitro. Thus elevation of stem cell PCD in dystrophic skeletal muscle is a direct consequence of the loss of functional dystrophin. Together these data demonstrate that elevated skeletal muscle cell PCD is a critical component of dystrophic pathology and is inversely correlated with both dystrophin gene dosage and with muscle fibre pathology. Targeting PCD in dystrophic muscles reduces both PCD and the classical features of dystrophic pathology in the mdx mouse suggesting that IGF-II is a strong candidate for therapeutic intervention in the dystrophinopathies.     

Alterations in the permeability of dystrophic fibers during neuromuscular junction development

In the mdx mice, lack of dystrophin leads to increases in calcium influx and myonecrosis, followed by muscle regeneration. Synapse elimination is faster in mdx than in controls, suggesting that increases in calcium influx during development could be involved. In the present study, we evaluated whether dystrophic fibers display changes in permeability to Evans Blue Dye (EBD) during development of the neuromuscular junction. EBD is a sensitive label for the early detection of increased myofiber permeability and sarcolemmal damage. After intraperitoneal injection of EBD, sternomastoid (STN) and tibialis anterior (T. anterior) muscles were analyzed with fluorescence microscopy. At 01, 07 and 14 days of age, STN and TA mdx myofibers were not stained with EBD. At 21 days of age, positive labeling of TA and STN mdx myofibers was seen, suggesting permeability modification and myonecrosis. Adult muscles showed a decrease (T. anterior) or no changes (STN) in the amount of EBD-positive fibers. These results suggest that there is no sarcolemmal damage detected by EBD during development of dystrophic neuromuscular junctions and other factors may contribute to the earlier synapse elimination seen in dystrophic muscle.     

Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle.

Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.     

The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin

The absence of dystrophin complex leads to disorganization of the force-transmitting costameric cytoskeleton and disruption of sarcolemmal membrane integrity in skeletal muscle. However, it has not been determined whether the dystrophin complex can form a mechanically strong bond with any costameric protein. We performed confocal immunofluorescence analysis of isolated sarcolemma that were mechanically peeled from skeletal fibers of mouse hindlimb muscle. A population of gamma-actin filaments was stably associated with sarcolemma isolated from normal muscle and displayed a costameric pattern that precisely overlapped with dystrophin. However, costameric actin was absent from all sarcolemma isolated from dystrophin-deficient mdx mouse muscle even though it was localized to costameres in situ. Vinculin, alpha-actinin, beta-dystroglycan and utrophin were all retained on mdx sarcolemma, indicating that the loss of costameric actin was not due to generalized membrane instability. Our data demonstrate that the dystrophin complex forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton through interaction with gamma-actin filaments. Destabilization of costameric actin filaments may also be an important precursor to the costamere disarray observed in dystrophin-deficient muscle. Finally, these methods will be broadly useful in assessing the mechanical integrity of the membrane cytoskeleton in dystrophic animal models lacking other costameric proteins.     

Involvement of inositol 1,4,5-trisphosphate in nicotinic calcium responses in dystrophic myotubes assessed by near-plasma membrane calcium measurement

In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.     

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle.

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.     

Fiber regeneration is not persistent in dystrophic (MDX) mouse skeletal muscle

Fiber replacement has been measured in adult mdx mouse limb skeletal muscles. During the first 10 days after birth all fibers appear normal; between Week 3 and 4 there is massive fiber degeneration followed by regeneration in which close to 100% of the fibers are repaired or replaced. New fibers arising in adult mice are characterized by expression of fetal myosin mRNAs in whole muscle extracts, and by staining of individual fibers with an embryonic myosin heavy chain-specific antibody. By 10 weeks of age new fiber replacement rate, indicated by frequency of fibers reacting with antibody, is reduced to about 10%, and by 1 year of age less than 1% of the fibers are being replaced at rates above control. Total fiber number also remains fairly constant. We conclude that the fibers regenerating up to 10 weeks of age become stabilized and do not undergo further rounds of degeneration and regeneration. This is consistent with the observed benign phenotype of adult mdx animals and with the idea that once-regenerated fibers escape the catastrophic dystrophic phenotype by acquiring a function that compensates for their mdx mutation. The mechanism by which regenerated mdx fibers restore adequate function in the absence of dystrophin may, when understood, provide clues to effective nongenetic interventions for muscular dystrophy in humans where regenerated fibers continue to degenerate and where the disease is often fatal.     

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

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

Alterations in dihydropyridine receptors in dystrophin-deficient cardiac muscle

The deficiency of dystrophin, a critical membrane stabilizing protein, in the mdx mouse causes an elevation in intracellular calcium in myocytes. One mechanism that could elicit increases in intracellular calcium is enhanced influx via the L-type calcium channels. This study investigated the effects of the dihydropyridines BAY K 8644 and nifedipine and alterations in dihydropyridine receptors in dystrophin-deficient mdx hearts. A lower force of contraction and a reduced potency of extracellular calcium (P < 0.05) were evident in mdx left atria. The dihydropyridine agonist BAY K 8644 and antagonist nifedipine had 2.7- and 1.9-fold lower potencies in contracting left atria (P < 0.05). This corresponded with a 2.0-fold reduction in dihydropyridine receptor affinity evident from radioligand binding studies of mdx ventricular homogenates (P < 0.05). Increased ventricular dihydropyridine receptor protein was evident from both radioligand binding studies and Western blot analysis and was accompanied by increased mRNA levels (P < 0.05). Patch-clamp studies in isolated ventricular myocytes showed no change in L-type calcium current density but revealed delayed channel inactivation (P < 0.05). This study indicates that a deficiency of dystrophin leads to changes in dihydropyridine receptors and L-type calcium channel properties that may contribute to enhanced calcium influx. Increased influx is a potential mechanism for the calcium overload observed in dystrophin-deficient cardiac muscle.     

Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice

Dystrophin, the protein product of the human Duchenne muscular dystrophy gene, exists in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kD and a protein of 59 kD. Here, we investigated the relative abundance of each of the components of the dystrophin-glycoprotein complex in skeletal muscle from normal and mdx mice, which are missing dystrophin. Immunoblot analysis using total muscle membranes from control and mdx mice of ages 1 d to 30 wk found that all of the dystrophin-associated proteins were greatly reduced (80-90%) in mdx mouse skeletal muscle. The specificity of the loss of the dystrophin-associated glycoproteins was demonstrated by the finding that the major glycoprotein composition of skeletal muscle membranes from normal and mdx mice was identical. Furthermore, skeletal muscle membranes from the dystrophic dy/dy mouse exhibited a normal density of dystrophin and dystrophin-associated proteins. Immunofluorescence microscopy confirmed the results from the immunoblot analysis and showed a drastically reduced density of dystrophin-associated proteins in mdx muscle cryosections compared with normal and dy/dy mouse muscle. Therefore, our results demonstrate that all of the dystrophin-associated proteins are significantly reduced in mdx skeletal muscle and suggest that the loss of dystrophin-associated proteins is due to the absence of dystrophin and not due to secondary effects of muscle fiber degradation.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

The NO way to increase muscular utrophin expression?

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder that results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to the search for a treatment is to compensate for dystrophin loss by utrophin, another cytoskeletal protein. During development, in normal as in dystrophic embryos, utrophin is found at the membrane surface of immature skeletal fibres and is progressively replaced by dystrophin. Thus, it is possible to consider utrophin as a 'foetal homologue' of dystrophin. In a previous work, we studied the effect of L-arginine, the substrate of nitric oxide synthetase (NOS), on utrophin expression at the muscle membrane. Using a novel antibody, we confirm here that the immunocytochemical staining was indeed due to an increase in utrophin at the sarcolemma. The result is observed not only on mdx (an animal model of DMD) myotubes in culture but also in mdx mice treated with L-arginine. In addition, we show here the utrophin increase in muscle extracts of mdx mice treated with L-arginine, after electrophoretic separation and western-blotting using this novel antibody, and thus extending the electrophoretic results previously obtained on myotube cultures to muscles of treated mice.     

Calcium homeostasis and cell death in Sol8 dystrophin-deficient cell line in culture

Abnormalities of calcium homeostasis are involved in the process of cell injuries such as Duchenne muscular dystrophy characterized by the absence of the protein dystrophin. But how the absence of dystrophin leads to cytosolic calcium overload is as yet poorly understood. This question has been addressed with skeletal muscle cells from human DMD muscles or mdx mice. Although easier to obtain than human muscles, mdx muscle cells have provided controversial data concerning the resting intracellular calcium level ([Ca2+](i)). This work describes the culture of Sol8 cell line that expresses neither dystrophin nor adhalin, a dystrophin-associated protein. The [Ca2+](i)and intracellular calcium transients induced by different stimuli (acetylcholine, caffeine and high potassium) are normal during the first days of culture. At later stages, calcium homeostasis exhibits drastic alterations with a breaking down of the calcium responses and a large [Ca2+](i)elevation. Concomitantly, Sol8 cells exhibit morphological signs of cell death like cytoplasmic shrinkage and incorporation of propidium iodide. Cell death could be significantly reduced by blocking the activity of calpains, a type of calcium-regulated proteases. These results suggest that Sol8 cell line provides an alternative model of dystrophin-deficient skeletal muscle cells for which a clear disturbance of the calcium homeostasis is observed in culture in association with calpain-dependent cell death. It is shown that transfection with a plasmid cDNA permits the forced expression of dystrophin in Sol8 myotubes as well as a correct sorting of the protein. This approach could be used to explore possible interactions between dystrophin deficiency, calcium homeostasis alteration, and dystrophic cell death.     

A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy.