Age-related declines in α-Klotho drive progenitor cell mitochondrial dysfunction and impaired muscle regeneration.

While young muscle is capable of restoring the original architecture of damaged myofibers, aged muscle displays a markedly reduced regeneration. We show that expression of the "anti-aging" protein, α-Klotho, is up-regulated within young injured muscle as a result of transient Klotho promoter demethylation. However, epigenetic control of the Klotho promoter is lost with aging. Genetic inhibition of α-Klotho in vivo disrupted muscle progenitor cell (MPC) lineage progression and impaired myofiber regeneration, revealing a critical role for α-Klotho in the regenerative cascade. Genetic silencing of Klotho in young MPCs drove mitochondrial DNA (mtDNA) damage and decreased cellular bioenergetics. Conversely, supplementation with α-Klotho restored mtDNA integrity and bioenergetics of aged MPCs to youthful levels in vitro and enhanced functional regeneration of aged muscle in vivo in a temporally-dependent manner. These studies identify a role for α-Klotho in the regulation of MPC mitochondrial function and implicate α-Klotho declines as a driver of impaired muscle regeneration with age.

Preventing oxidative stress: a new role for XBP1.

Antioxidant molecules reduce oxidative stress and protect cells from reactive oxygen species (ROS)-mediated cellular damage and probably the development of cancer. We have investigated the contribution of X-box-binding protein (XBP1), a major endoplasmic reticulum stress-linked transcriptional factor, to cellular resistance to oxidative stress. After exposure to hydrogen peroxide (H(2)O(2)) or a strong ROS inducer parthenolide, loss of mitochondrial membrane potential (MMP) and subsequent cell death occurred more extensively in XBP1-deficient cells than wild-type mouse embryonic fibroblast cells, whereas two other anticancer agents induced death similarly in both cells. In XBP1-deficient cells, H(2)O(2) exposure induced more extensive ROS generation and prolonged p38 phosphorylation, and expression of several antioxidant molecules including catalase was lower. Knockdown of XBP1 decreased catalase expression, enhanced ROS generation and MMP loss after H(2)O(2) exposure, but extrinsic catalase supply rescued them. Overexpression of XBP1 recovered catalase expression in XBP1-deficient cells and diminished ROS generation after H(2)O(2) exposure. Mutation analysis of the catalase promoter region suggests a pivotal role of CCAAT boxes, NF-Y-binding sites, for the XBP1-mediated enhancing effect. Taken together, these results indicate a protective role of XBP1 against oxidative stress, and its positive regulation of catalase expression may at least in part account for this function.

Inhibition of dystroglycan binding to laminin disrupts the PI3K/AKT pathway and survival signaling in muscle cells.

Dystroglycan is a component of the dystrophin-glycoprotein complex (DGC) in muscle and a cell surface receptor for laminin. Numerous muscular dystrophies are the result of disruption of proteins comprising the DGC, but the underlying pathogenetic mechanisms are unknown. Because apoptosis is an early feature of muscular dystrophy in vivo, and perturbation of cell-extracellular matrix associations is known to induce apoptosis, we investigated the role of dystroglycan-laminin interactions in the propagation and maintenance of cell survival signals in muscle cells. We found that disrupting the interaction between alpha-dystroglycan and the extracellular matrix protein laminin induces apoptosis in muscle cells. This increase in apoptosis is mediated in part by caspase activation and can be blocked by a caspase-3 inhibitor. We demonstrate a role for the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in muscle cell-survival signaling using a pharmacological inhibitor of PI3K. Treatment with this inhibitor resulted in decreased phosphorylation of AKT and its downstream effector glycogen synthase kinase (GSK)-3beta and induced apoptosis in muscle cell cultures. Disruption of dystroglycan-laminin interactions resulted in decreased phosphorylation of AKT and GSK-3beta. Furthermore, activation of AKT prior to the disruption of dystroglycan-laminin protected the muscle cells from the induction of apoptosis. These results support a role for the PI3K/AKT pathway in the propagation of cell-survival signals mediated by the DGC and provide new insight into the molecular pathogenesis associated with the development of muscular dystrophies.

The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies.

Mutations of different components of the dystrophin-glycoprotein complex (DGC) cause muscular dystrophies that vary in terms of severity, age of onset, and selective involvement of muscle groups. Although the primary pathogenetic processes in the muscular dystrophies have clearly been identified as apoptotic and necrotic muscle cell death, the pathogenetic mechanisms that lead to cell death remain to be determined. Studies of components of the DGC in muscle and in nonmuscle tissues have revealed that the DGC is undoubtedly a multifunctional complex and a highly dynamic structure, in contrast to the unidimensional concept of the DGC as a mechanical component in the cell. Analysis of the DGC reveals compelling analogies to two other membrane-associated protein complexes, namely integrins and caveolins. Each of these complexes mediates signal transduction cascades in the cell, and disruption of each complex causes muscular dystrophies. The signal transduction cascades associated with the DGC, like those associated with integrins and caveolins, play important roles in cell survival signaling, cellular defense mechanisms, and regulation of the balance between cell survival and cell death. This review focuses on the functional components of the DGC, highlighting the evidence of their participation in cellular signaling processes important for cell survival. Elucidating the link between these functional components and the pathogenetic processes leading to cell death is the foremost challenge to understanding the mechanisms of disease expression in the muscular dystrophies due to defects in the DGC.

Role of nitric oxide in the pathogenesis of muscular dystrophies: a "two hit" hypothesis of the cause of muscle necrosis.

Although the genetic and biochemical bases of many of the muscular dystrophies have been elucidated, the pathophysiological mechanisms leading to muscle cell death and degeneration remain elusive. Among the most well studied of the dystrophies are those due to defects in proteins that make up the dystrophin-glycoprotein complex (DGC). There has been much interest in the role of nitric oxide (NO(*)) in the pathogenesis of these diseases because the enzyme that synthesizes NO(*), nitric oxide synthase (NOS), is associated with the DGC. Recent studies of dystrophies related to DGC defects suggest that one mechanism of cellular injury is functional ischemia related to alterations in cellular NOS and disruption of a normal protective action of NO(*). This protective action is the prevention of local ischemia during contraction-induced increases in sympathetic vasoconstriction. However, the loss of this protection, alone, does not explain the subsequent muscle cell death and degeneration since mice lacking neuronal NOS (the predominant isoform expressed in muscle) do not develop a muscular dystrophy. Thus, there must be additional biochemical changes conferred upon the cells by these DGC defects, and these changes are discussed in terms of a proposed "two hit" hypothesis of the pathogenetic mechanisms that underlie the muscular dystrophies. According to this hypothesis, pathogenic defects in the DGC have at least two biochemical consequences: a reduction in NO(*)-mediated protection against ischemia, and an increase in cellular susceptibility to metabolic stress. Either one alone may be insufficient to lead to muscle cell death. However, in combination, the biochemical consequences are sufficient to cause muscle degeneration. The role of oxidative stress as a final common pathophysiologic pathway is discussed in terms of data showing that oxidative injury precedes pathologic changes and that muscle cells with defects in the DGC have an increased susceptibility to oxidant challenges. Accordingly, this "two hit" hypothesis may explain many of the complex spatial and temporal variations in disease expression that characterize the muscular dystrophies, such as grouped necrosis, a pre-necrotic phase of the disease, and selective muscle involvement.

NF kappa B and AP-1 mediate transcriptional responses to oxidative stress in skeletal muscle cells.

The ability to induce cellular defense mechanisms in response to environmental challenges is a fundamental property of eukaryotic and prokaryotic cells. We have previously shown that oxidative challenges lead to an increase in antioxidant enzymes, particularly glutathione peroxidase (GPx) and catalase (CAT), in mouse skeletal muscle. The focus of the current studies is the transcriptional regulatory mechanisms responsible for these increases. Sequence analysis of the mouse GPx and CAT genes revealed putative binding motifs for NF kappa B and AP-1, transcriptional regulators that are activated in response to oxidative stress in various tissues. To test whether NF kappa B or AP-1 might be mediating the induction of GPx and CAT in muscle cells subjected to oxidative stress, we first characterized their activation by pro-oxidants. Electrophoretic mobility shift assays showed that oxidative stress led to increases in the DNA binding of NF kappa B in differentiated muscle cells. The NF kappa B complexes included a p50/p65 heterodimer, a p50 homodimer, and a p50/RelB heterodimer. AP-1 was also activated, but with slower kinetics than that of NF kappa B. The major component of the AP-1 complexes was a heterodimer composed of c-jun/fos. To test for redox regulation of NF kappa B- or AP-1-dependent transcriptional activation, muscle cells expressing either kappa B/luciferase or TRE/luciferase reporter constructs were subjected to oxidative stress. Pro-oxidant treatment resulted in increased luciferase activity in cells expressing either construct. To test whether NF kappa B mediates oxidant-induced increases of GPx and CAT expression, we transfected cells with either a transdominant inhibitor (I kappa B alpha) or a dominant-negative inhibitor (Delta SP) of NF kappa B. Both inhibitors blocked the induction of antioxidant gene expression by more than 50%. In summary, our results suggest that NF kappa B and AP-1 are important mediators of redox-responsive gene expression in skeletal muscle, and that at least NF kappa B is actively involved in the upregulation of the GPx and CAT in response to oxidative stress.

The susceptibility of muscle cells to oxidative stress is independent of nitric oxide synthase expression.

The free radical, nitric oxide (NO.), has been implicated in the pathogenesis of muscular dystrophies because the enzyme, nitric oxide synthase (NOS), which produces NO., binds to the dystrophin-glycoprotein complex (DGC). In various studies of tissue samples from human and animal muscular dystrophies due to DGC defects, correlations between reductions of NOS activity and disease severity have been reported. To test for any direct effect of NOS expression on muscle cell susceptibility, we examined muscle cells in vitro under conditions of experimentally altered NOS activity. There were no differences in susceptibility to oxidative stress between differentiated myotube cultures from wild-type and from neuronal NOS (nNOS)-deficient mice. Likewise, pharmacological inhibition of NOS did not alter cellular susceptibility to oxidative challenges. Overexpression of NOS neither enhanced nor diminished cellular susceptibility to oxidative stress. Finally, we assessed the effect of NOS overexpression on myotube cultures from dystrophin-deficient (mdx) mice. NOS protein was localized to both membrane and cytosolic compartments in the transduced cells. Still, no difference in susceptibility to oxidative stress was found between the NOS-overexpressing cells and control cells. These data suggest that muscle cell susceptibility to oxidative challenges is independent of the level of NOS expression. Therefore, any role NO. may play in the pathogenesis of muscular dystrophies is likely to be independent of its effect on the redox state of the cell.

Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides.

Chimeric RNA/DNA oligonucleotides ("chimeraplasts") have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated "MDX1") designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1-2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were "revertant fibers." Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription-PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.

Dystrophin mutations predict cellular susceptibility to oxidative stress.

Mutations in the dystrophin gene that lead to the expression of truncated forms of the dystrophin protein cause muscular dystrophies of varying severities both in humans and in mice. We have shown previously that dystrophin-deficient muscle is more susceptible to oxidative injury than is normal muscle. In this report, we have used muscle cells derived from mdx mice, which express no dystrophin, and mdx-transgenic strains that express full-length dystrophin or truncated forms of dystrophin to explore further the relationship between dystrophin expression and susceptibility of muscle to oxidative injury. We show that, when differentiated into myotubes, the relative susceptibility of the cell populations to oxidative stress correlates with the severity of the dystrophy in the strain from which the cells were isolated. The most susceptible populations exhibited the greatest oxidative damage as assessed by protein oxidation. Thus, the relative efficacy of truncated dystrophin proteins to protect muscle from necrotic degeneration in vivo is predicted by their ability to protect muscle cells from free radical mediated injury. These findings support the hypothesis that the dystrophin protein complex may have important regulatory or signaling properties in terms of cell survival and antioxidant defense mechanisms.

Neuromuscular disorders of childhood.

Neuromuscular disorders are common causes of weakness and hypotonia in the infantile period and in childhood. Accurate diagnosis of specific neuromuscular disorders depends first on identification of which aspect of the peripheral neuromuscular system is affected--the motor neuron in the spinal cord, the nerve root or peripheral nerve, the neuromuscular junction, or the muscle--and then on the determination of the etiology and specific clinical entity. This review provides an overview of the major neuromuscular disorders of childhood with attention to recent advances and emerging areas of research.

Integrin-mediated muscle cell spreading. The role of protein kinase c in outside-in and inside-out signaling and evidence of integrin cross-talk.

Muscle cell survival depends upon the presence of various integrins with affinities for different extracellular matrix proteins. The absence of either alpha(5) or alpha(7) integrins leads to degenerative disorders of skeletal muscle, muscular dystrophies. To understand the cell survival signals that are mediated by integrin engagement with matrix proteins, we studied the early signaling events initiated by the attachment of muscle cells to fibronectin, an interaction that is mediated primarily by alpha(5) integrins. Cells that express alpha(5) integrin rapidly spread on fibronectin, and this process is associated with the phosphorylation of focal adhesion kinase (FAK). Cells deficient in alpha(5) integrin failed to spread or promote FAK phosphorylation when plated on fibronectin. For alpha(5)-expressing cells, both spreading and FAK phosphorylation could be blocked by inhibitors of protein kinase C (PKC), indicating that PKC is necessary for this "outside-in signaling" mediated by alpha(5) integrin. Surprisingly, activators of PKC could promote spreading and FAK phosphorylation in alpha(5)-deficient muscle cells plated on fibronectin. This PKC-induced cell spreading appeared to be due to activation of alpha(4) integrins ("inside-out signaling") since it could be blocked by peptides that specifically inhibit alpha(4) integrin binding to fibronectin. A model of integrin signaling in muscle cells is presented in which there is a positive feedback loop involving PKC in both outside-in and inside-out signaling, and the activation of this cycle is essential for cell spreading and downstream signaling to promote cell survival. In addition, the data indicate a cross-talk that occurs between integrins in which the outside-in signaling via one integrin can promote the activation of another integrin via inside-out signaling.

Regulation of antioxidant enzyme gene expression in response to oxidative stress and during differentiation of mouse skeletal muscle.

Various properties of skeletal muscle, including high metabolic activity and high levels of heme-containing proteins, render it particularly susceptible to free radical injury. Indeed, cellular injury from reactive oxygen species (ROS) has been implicated in many muscle disorders. Thus muscle cell survival is critically dependent on the ability of the cell to respond to periods of oxidative stress. To investigate this important homeostatic response, we studied the effect of oxidative challenges on the expression of genes encoding the antioxidant enzymes Cu,Zn-superoxide dismutase (CuZnSOD), Mn-superoxide dismutase (MnSOD), glutathione peroxidase (GPx), and catalase (CAT) in myotube cultures. Using Northern blot analysis, we found that treatment with the pro-oxidant paraquat resulted in time- and dose-dependent increases of transcript levels that were greatest for GPx and CAT (approximately 4-5 fold). CuZnSOD and MnSOD transcripts were also increased, albeit more modestly (approximately 2-3 fold). Transcript levels were also induced by treatment of the cells with two other pro-oxidants, menadione and H2O2, and correlated with the level of oxidative injury to the cells, measured as protein carbonyl group formation. Activities of all of the enzymes increased in response to the oxidative challenges, although the magnitudes of the increases were less robust than the increases of the respective transcript levels. In studying the effect of cellular differentiation on antioxidant gene expression and susceptibility to oxidative stress, we found that pro-oxidant treatment resulted in greater oxidative injury to differentiated myotubes than to undifferentiated myoblasts. Furthermore, the increased susceptibility of myotubes correlated with decreased antioxidant defenses-as muscle cells differentiated, both transcript and activity levels of antioxidant enzymes decreased. These data suggest that muscle cells regulate antioxidant defenses in response to oxidative stress and cellular differentiation.

Dystrophic muscle in mice chimeric for expression of alpha5 integrin.

alpha5-deficient mice die early in embryogenesis (). To study the functions of alpha5 integrin later in mouse embryogenesis and during adult life we generated alpha5 -/-;+/+ chimeric mice. These animals contain alpha5-negative and positive cells randomly distributed. Analysis of the chimerism by glucose- 6-phosphate isomerase (GPI) assay revealed that alpha5 -/- cells contributed to all the tissues analyzed. High contributions were observed in the skeletal muscle. The perinatal survival of the mutant chimeras was lower than for the controls, however the subsequent life span of the survivors was only slightly reduced compared with controls (). Histological analysis of alpha5 -/-;+/+ mice from late embryogenesis to adult life revealed an alteration in the skeletal muscle structure resembling a typical muscle dystrophy. Giant fibers, increased numbers of nuclei per fiber with altered position and size, vacuoli and signs of muscle degeneration-regeneration were observed in head, thorax and limb muscles. Electron microscopy showed an increase in the number of mitochondria in some muscle fibers of the mutant mice. Increased apoptosis and immunoreactivity for tenascin-C were observed in mutant muscle fibers. All the alterations were already visible at late stages of embryogenesis. The number of altered muscle fibers varied in different animals and muscles and was often increased in high percentage chimeric animals. Differentiation of alpha5 -/- ES cells or myoblasts showed that in vitro differentiation into myotubes was achieved normally. However proper adhesion and survival of myoblasts on fibronectin was impaired. Our data suggest that a novel form of muscle dystrophy in mice is alpha5-integrin-dependent.

Overexpression of copper/zinc superoxide dismutase: a novel cause of murine muscular dystrophy.

Oxidative injury underlies the cellular injury and cell death in a variety of disease states. In muscular dystrophies, evidence from in vivo and in vitro studies suggests that muscle degeneration may be secondary to an increased susceptibility to oxidative stress. To address the role of free radical metabolism in the pathogenetic process of muscular dystrophies, we examined the muscle of transgenic mice that overexpress copper/zinc (Cu/Zn) superoxide dismutase. Overexpression of this enzyme can sensitize cells to oxidative injury, and Cu/Zn superoxide dismutase activity was elevated approximately fourfold above control levels in skeletal muscle of the transgenic strain. Examination of serum creatine phosphokinase levels in these mice revealed significant elevations after 2 months of age, indicative of active muscle breakdown. By 8 months of age, there was gross atrophy of the quadriceps muscle, and other hindlimb muscles were variably affected. Histologically, there was evidence of widespread muscle necrosis and regeneration, fiber splitting, and replacement of muscle with adipose and fibrous connective tissue, typical of a muscular dystrophy. Associated with the development of this degeneration was an increase in the levels of lipid peroxidation in the muscle of Cu/Zn superoxide dismutase transgenic mice, highlighting the central role of oxidative injury in this pathogenetic process. These results demonstrate that oxidative damage can be the primary pathogenetic process underlying a muscular dystrophy.

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.

Two regions of the ryanodine receptor involved in coupling with L-type Ca2+ channels.

Ryanodine receptors (RyRs) are present in the endoplasmic reticulum of virtually every cell type and serve critical roles, including excitation-contraction (EC) coupling in muscle cells. In skeletal muscle the primary control of RyR-1 (the predominant skeletal RyR isoform) occurs via an interaction with plasmalemmal dihydropyridine receptors (DHPRs), which function as both voltage sensors for EC coupling and as L-type Ca2+ channels (Rios, E., and Brum, G. (1987) Nature 325, 717-720). In addition to "receiving" the EC coupling signal from the DHPR, RyR-1 also "transmits" a retrograde signal that enhances the Ca2+ channel activity of the DHPR (Nakai, J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam, K. G., and Allen, P. D. (1996) Nature 380, 72-76). A similar kind of retrograde signaling (from RyRs to L-type Ca2+ channels) has also been reported in neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature 382, 719-722). To investigate the molecular mechanism of reciprocal signaling, we constructed cDNAs encoding chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We found that a chimera that contained residues 1,635-2,636 of RyR-1 both mediated skeletal-type EC coupling and enhanced Ca2+ channel function, whereas a chimera containing adjacent RyR-1 residues (2, 659-3,720) was only able to enhance Ca2+ channel function. These results demonstrate that two distinct regions are involved in the reciprocal interactions of RyR-1 with the skeletal DHPR.

Muscle cells from mdx mice have an increased susceptibility to oxidative stress.

Several lines of evidence suggest that free radical mediated injury and oxidative stress may lead to muscle necrosis in the muscular dystrophies, including those related to defects in the dystrophin gene. We have examined muscle cell death using an in vitro assay in which the processes that lead to myofiber necrosis in vivo may be amenable to investigation in a simplified cell culture system. Using myotube cultures from normal and dystrophin-deficient (mdx) mice, we have examined the susceptibilities of the cells to different metabolic stresses. Dystrophin-deficient cells were more susceptible to free radical induced injury when compared to normal cells, but the two populations were equally susceptible to other forms of metabolic stress. The differential response appeared to be specifically related to dystrophin expression since undifferentiated myoblasts (which do not express dystrophin) from normal and mdx mice were equally sensitive to oxidative stress. Thus, the absence of dystrophin appears to render muscle specifically more susceptible to free radical induced injury. These results support the hypothesis that oxidative stress may lead to myofiber necrosis in these disorders. Elucidating the mechanisms leading to cell death may help to explain the variabilities in disease expression that are seen as a function of age, among different muscles, and across species in animals with muscular dystrophy due to dystrophin deficiency.

Evidence of oxidative stress in mdx mouse muscle: studies of the pre-necrotic state.

Considerable evidence indicates that free radical injury may underlie the pathologic changes in muscular dystrophies from mammalian and avian species. We have investigated the role of oxidative injury in muscle necrosis in mice with a muscular dystrophy due to a defect in the dystrophin gene (the mdx strain). In order to avoid secondary consequences of muscle necrosis, all experiments were done on muscle prior to the onset of the degenerative process (i.e. during the 'pre-necrotic' phase) which lasted up to 20 days of age in the muscles examined. In pre-necrotic mdx muscle, there was an induction of expression of genes encoding antioxidant enzymes, indicative of a cellular response to oxidative stress. In addition, the levels of lipid peroxidation were greater in mdx muscle than in the control. Since the free radical nitric oxide (NO*) has been shown to mediate oxidative injury in various disease states, and because dystrophin has been shown to form a complex with the enzyme nitric oxide synthase, we examined pre-necrotic mdx muscle for evidence of NO*-mediated injury by measuring cellular nitrotyrosine formation. By both immunohistochemical and electrochemical analyses, no evidence of increased nitrotyrosine levels in mdx muscle was detected. Therefore, although no relationship with NO*-mediated toxicity was found, we found evidence of increased oxidative stress preceding the onset of muscle cell death in dystrophin-deficient mice. These results lend support to the hypothesis that free radical-mediated injury may contribute to the pathogenesis of muscular dystrophies.