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J Appl Physiol 102: 1677-1686, 2007. First published November 9, 2006; doi:10.1152/japplphysiol.01145.2006
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INVITED REVIEW

HIGHLIGHTED TOPIC
Free Radical Biology in Skeletal Muscle

The role of free radicals in the pathophysiology of muscular dystrophy

James G. Tidball1,2 and Michelle Wehling-Henricks1

Departments of 1Physiological Science and 2Pathology and Laboratory Medicine, University of California, Los Angeles, California


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Null mutation of any one of several members of the dystrophin protein complex can cause progressive, and possibly fatal, muscle wasting. Although these muscular dystrophies arise from mutation of a single gene that is expressed primarily in muscle, the resulting pathology is complex and multisystemic, which shows a broader disruption of homeostasis than would be predicted by deletion of a single-gene product. Before the identification of the deficient proteins that underlie muscular dystrophies, such as Duchenne muscular dystrophy (DMD), oxidative stress was proposed as a major cause of the disease. Now, current knowledge supports the likelihood that interactions between the primary genetic defect and disruptions in the normal production of free radicals contribute to the pathophysiology of muscular dystrophies. In this review, we focus on the pathophysiology that results from dystrophin deficiency in humans with DMD and the mdx mouse model of DMD. Current evidence indicates three general routes through which free radical production can be disrupted in dystrophin deficiency to contribute to the ensuing pathology. First, constitutive differences in free radical production can disrupt signaling processes in muscle and other tissues and thereby exacerbate pathology. Second, tissue responses to the presence of pathology can cause a shift in free radical production that can promote cellular injury and dysfunction. Finally, behavioral differences in the affected individual can cause further changes in the production and stoichiometry of free radicals and thereby contribute to disease. Unfortunately, the complexity of the free radical-mediated processes that are perturbed in complex pathologies such as DMD will make it difficult to develop therapeutic approaches founded on systemic administration of antioxidants. More mechanistic knowledge of the specific disruptions of free radicals that underlie major features of muscular dystrophy is needed to develop more targeted and successful therapeutic approaches.

nitric oxide; muscle disease

THE COMPLEX PATHOPHYSIOLOGIES of the muscular dystrophies have largely defied explanation in the context of monogenic diseases. This complexity has been especially evident in Duchenne muscular dystrophy (DMD), the most common of all the muscular dystrophies. The pathology of DMD involves a broad and not easily related set of defects that includes skeletal muscle weakness, inflammation, wasting and fibrosis, increased fatiguability, vascular dysfunction, cardiomyopathy, lower IQ, muscle metabolic defects, and synaptic dysfunction (27, 29, 31, 69, 81). Despite the broad, systemic pathophysiology of DMD, the primary defect is mutation of the dystrophin gene, which encodes a membrane-associated protein that is found primarily in skeletal muscle, but even there it is present at very low concentrations (11, 95). Furthermore, the only clearly established function of dystrophin is to serve as a cytoskeletal element (59). How can defects in expression of a low-prevalence structural protein in muscle produce such a complex pathology?

Before the identification of dystrophin mutations as the cause of DMD, early investigators speculated that DMD could result, at least in part, from oxidative stress experienced by muscle. The first suspicions of free radical-mediated pathology in DMD were founded on the similarities to muscle pathology that occurred in vitamin E deficiency (8), which was directly attributed to an increase in free radicals and increased oxidative damage. Subsequently, parallels were drawn between DMD histopathology and muscle damage that occurred during muscle reperfusion that followed periods of ischemia, in which oxidative damage was also an established feature of the pathogenic process (53). Nevertheless, controversy still surrounds the question of whether oxidative damage is a significant, pathogenic event in muscular dystrophies, or whether it is a consequence of other pathology that is unimportant in promoting muscle death or dysfunction.

Although early hypotheses that concerned the role of free radicals in muscular dystrophy focused on their possible role in oxidative damage to muscle, our advancing knowledge of free radical function in biological systems has shown that free radicals can play multiple and diverse regulatory roles through their functions as signaling molecules. Thus pathological modifications in free radical production in dystrophic muscle could yield defects in regulatory systems that may underlie some of the complex pathology of muscular dystrophy. However, the extent to which perturbations in free radicals contribute to misregulation of enzyme function or protein interactions that subsequently contribute to muscular dystrophy remains almost unexplored.

In the present review, we present current knowledge of the relationships between free radicals and muscular dystrophy, with an emphasis on dystrophinopathies (DMD in humans and the mdx mouse model of DMD). We present our assessment of data that address whether oxidative damage, especially to the muscle cell membrane structure, is an important pathogenic event in dystrophinopathies. In addition, regulatory roles played by free radicals in signaling pathways that may be disrupted in muscular dystrophy and thereby lead to specific functional defects are discussed. Finally, we address whether manipulations of free radicals may have therapeutic promise for the treatment of muscular dystrophy.


   DOES OXIDATIVE DAMAGE OF THE MUSCLE SARCOLEMMA CONTRIBUTE SIGNIFICANTLY TO MUSCULAR DYSTROPHY?
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Despite decades of investigation, there is no definitive proof that oxidative damage to the surface membrane of dystrophic muscle promotes muscular dystrophy. Most investigations of the role of oxidative damage in muscular dystrophy have relied on assays of oxidation products in the sera or muscles of DMD patients. In particular, some by-products of free radical damage to unsaturated fatty acids can react with thiobarbituric acid to form thiobarbituric acid-reactive substances (TBARS) that can be assayed in tissues as an index of oxidative damage. Other by-products of oxidative damage, such as pentane, can be detected in exhaled gases. The findings that TBARS are elevated in DMD muscle (44, 47) and that pentane is elevated in exhaled gases from DMD patients (36) provided early support for the hypothesis that oxidative stress promotes DMD pathology. Furthermore, muscles from DMD patients have elevated levels of products of lipid peroxidation (42), and they express elevated levels of enzymes that are responsive to oxidative stress (6, 47); both of these modifications show that oxidative stress is present in dystrophic muscle (Table 1). Nevertheless, whether the sarcolemma of dystrophic muscle is functionally impaired by free radical-mediated oxidative damage remains unknown. Indeed, only recently has direct proof been provided to show that the sarcolemma of DMD muscle fibers exhibits a response reflective of exposure to oxidative stress. Biophysical measurements to examine lipid distribution at the surface membrane of DMD fibers revealed that the antioxidants vitamin E and coenzyme Q were present at elevated concentrations, supporting the conclusion that the sarcolemma of DMD muscle is oxidatively stressed (83).


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  		Table 1. Changes in activity or concentrations of molecules associated with antioxidant functions in dystrophic or atrophying muscle

 
Although definitive data are not yet available to show that oxidative damage to the sarcolemma promotes dystrophinopathy, dystrophin-deficient muscle cells are clearly more susceptible to oxidative stress in vitro. In a series of investigations of the effects of oxidative stress on the viability of dystrophin-deficient muscle, Rando and colleagues (65) showed convincingly that dystrophin-deficient myotubes are killed more easily when exposed to oxidants. However, dystrophin-deficient myotubes were not more easily killed by other metabolic stresses (65), which indicated a selective sensitivity to free radical-mediated death in the absence of dystrophin. Further analyses addressed whether increased sensitivity to oxidative damage may also occur in dystrophic muscle in vivo by assaying primary cultures of myotubes that were derived from dystrophin-deficient mdx mice that expressed any of several transgenes for truncated dystrophin (22). A positive relationship was demonstrated between severity of the in vivo muscle pathology in each transgenic mdx line and the sensitivity to oxidative stress of myotubes in vitro that were derived from the specific lines (22). Collectively, these findings provide strong support that oxidative stress contributes to dystrophinopathy, although the mechanism of free radical-mediated cytolysis and the identity of key molecules that were affected by the oxidative stress remained unidentified. Nevertheless, controversy remains concerning whether oxidative stress is a necessary precondition for death of dystrophic muscle or is a consequence of pathology that does not advance the disease. On one hand, muscles collected from mdx mice before the onset of histologically discernible pathology showed elevated levels of the antioxidant enzymes catalase, glutathione peroxidase, superoxide dismutase (SOD)-1, and SOD-2, which indicated that oxidative stress may be an early, possibly pathogenic event that could be required for triggering necrosis of dystrophic muscle (21). On the other hand, muscles collected from mdx mice after the onset of pathology showed a poor relationship between the levels of expression of antioxidant enzymes and the degree of pathology. For example, antioxidant enzymes and products of lipid peroxidation were higher in mdx muscle than in controls, regardless of whether the muscles sampled were progressively necrotic (diaphragm), or in a stage of active regeneration (4-mo-old limb muscles) or spared the mdx pathology (extraocular muscles) (64). These observations supported the interpretation that oxidative stress may not be a determining pathogenic mechanism in mdx mice.

Perhaps the most parsimonious interpretation of these observations that relate oxidative stress to the pathology of dystrophin deficiency would be that oxidative stress has the potential to promote the pathology but is insufficient to cause muscular dystrophy in the absence of other perturbations of homeostasis. The necessity of interactions between perturbations in the pathogenesis of muscular dystrophy was proposed previously by Rando (67) as "the two-hit hypothesis" in which he stated that muscle ischemia followed by a second perturbation caused by defects in the dystrophin protein complex (DPC) caused muscle necrosis in dystrophinopathy. This hypothesis has received experimental support by findings that showed that application of mechanical stress to mdx muscle following a period of ischemia and reperfusion produced more sarcolemma damage than the sum of damages caused by ischemia-reperfusion or mechanical stress when applied to separate mice (24). While these findings show a potentially synergistic interaction between oxidative and mechanical stress in mdx muscle, whether they relate to stresses normally seen in dystrophic muscle is uncertain, because the period of ischemia involved application of a tourniquet to prevent blood flow to the affected muscle for 90 min.


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Elevations in oxidative stress in dystrophin-deficient muscles can be attributed to constitutive differences in the muscle, as well as to responses to the presence of muscle pathology and even behavioral changes in individuals with muscular dystrophy. The discovery that neuronal nitric oxide synthase (nNOS) is a member of the DPC that is lost in dystrophin deficiency provided the first link between dystrophin deficiency and inherent defects in free radical production in dystrophic muscle (9). Importantly, nNOS is not merely displaced from the cell membrane in dystrophin-deficient muscle; nNOS concentration is reduced to <20% of levels that occur in normal muscle, and nNOS mRNA is also dramatically decreased (14). As a consequence, the production of the free radical nitric oxide (NO) is tremendously reduced in dystrophin-deficient muscles (89). Thus any NO-related pathology in dystrophin-deficient muscle is a consequence of nNOS deficiency, not nNOS mislocalization. Because NO is a versatile and rapid reactant with other free radicals, these extreme disruptions of its production in muscle could cause major shifts in the redox environment in muscle. Furthermore, NO functions as a pluripotent signaling molecule in muscles and other tissues, and perturbations in its production could have broad effects on muscle homeostasis (76) (Fig. 1). Experimental evidence demonstrates the importance of nNOS deficiency in the pathology of mdx dystrophy. Expression of a muscle-specific nNOS transgene in dystrophic muscle prevented the majority of histologically discernible pathology in mdx mice, including reducing sarcolemmal lesions by ~80% (89).


Figure 1
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  		Fig. 1. Schematic representations of the systemic effects of dystrophin deficiency and some of the biological processes that are regulated by nitric oxide (NO). The broad similarities between functions that are NO mediated and those that are disrupted in Duchenne muscular dystrophy (DMD) and mdx dystrophy, in which there is a great reduction in muscle-derived NO, suggest the possibility that NO synthase (NOS) deficiency could contribute to multiple aspects of dystrophinopathy.

 
Muscle inflammation, a prominent feature of dystrophinopathy, provides an additional source for free radicals that can contribute to muscle pathology. Macrophages are the most common of the invasive leukocyte population, reaching concentrations as high as 105 macrophages/mm3 of muscle (89). Macrophages are capable of direct lysis of myotubes in vitro via free radical-mediated mechanisms (57), and macrophages from dystrophic muscle are highly cytolytic when present at concentrations that reflect their numbers in dystrophic muscle (89). Furthermore, depletion of macrophages from mdx muscle yields the same protective effect against mdx histopathology and fiber injury, as achieved by nNOS transgene expression (89). In addition, nNOS transgene expression prevents macrophage invasion into mdx muscle (89). Collectively, these observations support the hypothesis that much of the muscle fiber damage in mdx mice results from macrophage-derived free radicals and that muscle-derived NO protects against that damage by acting as an anti-inflammatory agent. Although the mechanism through which muscle-derived NO reduces inflammation and injury in mdx mice is unknown, several possible mechanisms have been identified in other systems. For example, NO can inhibit the expression of adhesion molecules that are necessary for leukocyte diapedesis (51) or inhibit the activity of enzymes that produce free radicals, such as superoxide and hypochlorous acid (1, 18), and it can induce apoptosis in leukocytes (3). NO can also scavenge other cytotoxic free radicals, which could further contribute to its protective role (54), although no differences in susceptibility to oxidative stress were observed in myotubes in vitro that were nNOS null mutants, wild type, or nNOS overexpressers (94).

Although normalizing NO production in dystrophin-deficient muscle can greatly reduce muscle pathology, recent findings show that there is not a simple relationship between increasing nNOS at the sarcolemma and the severity of dystrophinopathy. For example, mdx mice that express a transgene that encodes a minidystrophin ({Delta}H2-R19) or microdystrophin ({Delta}R4-R23) have a small increase in nNOS at the sarcolemma and display no muscle pathology (39). However, mdx mice that express a transgene encoding the dystrophin isoform Dp260 show higher levels of sarcolemma-associated nNOS but more muscle histopathology (87). Interestingly, mdx mice that expressed in their muscles a transgene that encodes Dp116, an isoform of dystrophin that is expressed in nonmuscle cells, showed little nNOS at the sarcolemma, although the rest of the DPC appeared to assemble normally (46). Despite the recovery of the DPC, except for nNOS, muscle pathology was worse in the DP116 transgenic mdx mice than in mdx mice without the transgene (46). These findings are consistent with a "two-hit hypothesis," because they may indicate that lack of nNOS exacerbates pathology only in the presence of other defects associated with dystrophin deficiency. This interpretation is consistent with the absence of muscle pathology in nNOS null mutant mice (20). However, the findings also emphasize that merely increasing nNOS expression and NO production may not always be sufficient to reduce pathology in dystrophin-deficient muscle. In vitro studies have shown that changes in NO concentration can either increase or decrease hydroxylation or oxidative modifications to reactants, depending on the stoichiometry of NO to other free radicals (54). This may become important in developing DMD therapeutics that cause changes in nNOS concentration, because increases in nNOS to nonphysiological levels may further promote muscle damage and cardiac arrhythmias (90).

Disruptions in free radical production also occur during reduced muscle use, which may further contribute to dystrophinopathies, although this possibility has been explored little. With progressive muscle weakness, DMD patients undergo greatly reduced physical activity that can produce large shifts in the expression and activity of enzymes that affect the production of free radicals or antioxidants. For example, nNOS activity and expression are both dramatically reduced in normal muscles that experience reduced use (80). Reduced muscle use also causes decreases in the activity of some antioxidant enzymes, such as catalase and glutathione peroxidase, at least in rodents (33, 49). However, reduced muscle use can result in a large increase in SOD-1 that is associated with increases in lipid peroxidation (49). Similar to changes observed during reduced use of healthy muscle, SOD-1 activity increases significantly in DMD and mdx muscles, although catalase and glutathione peroxidase have usually been reported at higher concentrations (21, 47, 64). Because SOD-1 converts superoxide to hydrogen peroxide that can cause lipid peroxidation, the increase in SOD-1 may have a pathogenic rather than protective role in dystrophinopathy and muscle disuse, despite its function as an antioxidant enzyme. Interestingly, this possibility is further supported by the severe, progressive muscular dystrophy that results from overexpression of SOD-1 in muscle, which yields muscle atrophy, elevated lipid peroxidation in muscles, and elevation of muscle cytosolic proteins in the serum (66). Furthermore, dystrophic mice that are null mutants for the extracellular matrix protein M-laminin (dy/dy mice) show significant elevations of glutathione peroxidase and catalase, but no increase in SOD-1, and a significant decrease in TBARS, indicating less lipid peroxidation (5). The comparison to dy/dy muscle may suggest useful insights into a mechanism of free radical-mediated damage in dystrophinopathy, because muscles with elevated SOD-1 (mdx, DMD, muscle atrophy) show increased lipid peroxidation, while in dy/dy muscle there is no increase in SOD-1 or in lipid peroxidation.


   DO PERTURBATIONS OF FREE RADICAL PRODUCTION CONTRIBUTE TO DYSTROPHINOPATHY BY DISRUPTION OF SPECIFIC SIGNALING OR METABOLIC PATHWAYS?
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NO and Regulation of Vasodilation in Dystrophin-Deficient Muscle

Although deficiencies in NO production by dystrophic muscle may contribute to disruptions in the redox environment of muscle that may then worsen muscle pathology, other observations suggest that changes in NO levels or other free radicals contribute to defects in specific signaling pathways. One of the first and a particularly exciting series of studies that illustrated this role for disrupted NO signaling focused on defects in vasodilation in mdx and DMD muscle. Thomas, Victor, and colleagues tested whether loss of muscle-derived NO yielded defects in vasodilation in mdx muscle, because endothelial cell-derived NO was a well-characterized activator of cGMP-mediating signaling that produces vasodilation (79). Their findings clearly established that NO derived from skeletal muscle could also produce vasodilation in vivo and showed that this regulatory function in mdx mice and DMD children was impaired (69, 79). Discovery of this novel role for muscle-derived NO raised the possibility that nNOS deficiency could lead to recurrent ischemia-reperfusion injury and provide an essential "first hit" (67) in promoting necrosis in dystrophin-deficient muscle. However, the conditions under which skeletal muscle-derived NO functioned in this signaling role were very specific. There were no significant differences in mean arterial pressure, femoral blood flow, or vascular conductance in resting muscle of mdx mice compared with wild-type mice (79). Similarly, these same hemodynamic parameters did not differ between wild-type or mdx mice during muscle contraction, which showed that muscle-derived NO does not play a significant role in regulating peripheral blood flow during muscle contraction. However, NO produced by contracting, wild-type muscle reversed the decrease in femoral blood flow velocity and vascular conductance caused by {alpha}-adrenergic stimulation, but this reversal did not occur in mdx. A similar defect was observed in nNOS null mutant mice. Thus release of NO from muscle that is caused by increased muscle contraction can oppose vasoconstriction caused by increased sympathetic outflow. However, questions remain to be answered. First, why does an increase in NO release from contracting muscle not have a vasodilatory effect in the absence of {alpha}-adrenergic stimulation? Furthermore, if muscle-derived NO plays a significant role in vasodilation only when there is an increase in {alpha}-adrenergic stimulation while muscle is actively contracting, is this defect sufficient to contribute to the pathology of DMD?

NO and Regulation of Synaptic Structure in Dystrophin-Deficient Muscle

More recent investigations have shown that deficiencies in NO production contribute to defects in synaptic structure in mdx mice. Neuromuscular junctions (NMJs) of mdx mice display disruptions in their architecture, changes in distribution of acetylcholine receptors (AChRs), and other postsynaptic membrane proteins and decreases in the concentration AChRs, which is associated with impaired synaptic transmission (12, 48, 52, 62, 82). Although these defects were originally attributed to loss of the structural role of dystrophin at the postsynaptic membrane, other findings indicated that this explanation was insufficient. For example, null mutations for other members of the DPC, such as {alpha}-dystrobrevin and {alpha}-syntrophin, cause defects in NMJ structure without causing reductions of dystrophin expression (2, 35, 41). However, the loss of other members of the DPC, such as {alpha}-sarcoglycan or {gamma}-sarcoglycan, does not cause disruptions of NMJ structure (23, 37). Intriguingly, all mutants of DPC members that lead to NMJ defects share the common characteristic that they are ligands for nNOS, or they are responsible for localizing nNOS ligands at the NMJ. The coincidence of the loss of direct or indirect binding partners of nNOS from the muscle cell membrane and the occurrence of defects in NMJ structure support the hypothesis that defects in nNOS localization or NO production may contribute to defects in NMJ structure. Indeed, expression of a muscle-specific nNOS transgene in mdx mice corrected structural defects at the NMJ, substantiating the role for NO-mediated signaling in regulating NMJ architecture (74).

Several investigations have provided strong support to show that NO effects on NMJ structure are mediated, at least in part, by cGMP-mediated signaling. NO can bind the heme group of guanylate cyclase, to increase enzyme activity and elevate cGMP, which in turn can activate protein kinase G. This portion of the signaling pathway is shared with the mechanism through which NO modulates vasorelaxation. Guanylate cyclase, PKG, and nNOS are all present at NMJs (15, 70), and clustering of AChRs at the postsynaptic membrane is significantly reduced, if nNOS activity is inhibited (45). Furthermore, application of NO donors or pharmacological activation of PKG or overexpression of guanylate cyclase or PKG increase AChR clustering (34, 45, 71).

Free Radicals and Fatigue in Dystrophin-Deficient Muscle

Increased susceptibility to muscle fatigue is a significant functional defect in DMD. For example, DMD boys who were tested between the ages of 8 and 10 yr maintained a contraction of the biceps brachii at 60–70% of maximum voluntary contraction (MVC) for only 10 s. In contrast, healthy boys could maintain contraction at 60–70% MVC for 45 s (31). Generally, similar findings have been reported in mdx mice. Tetanic stimulation of fast, glycolytic muscles for 280 ms every 5 s produced much more fatigue in mdx muscle than in age-matched C57, expressed as a decline in force production relative to pretetanic stimulation levels (92). These investigations that indicate an increased fatigability of dystrophin-deficient muscles appear at first view to be inconsistent with the finding of other investigators who have shown that dystrophic muscle is less fatigable than normal muscle (26, 72, 73). However, these latter studies have tested fatigability under conditions in which loss of force production was measured over longer periods of time (many minutes) at lower levels of force production (frequently ~20% MVC). The reduced fatigability of dystrophin-deficient muscles at relatively low levels of activation results in part from the increase in the concentration of slow-oxidative fibers in dystrophic muscle, which would reduce fatigability in endurance exercise.

Although the underlying defects that cause increased fatigability in dystrophin-deficient muscle have not been identified, several observations support the view that oxidative stress causes impaired ATP production during anaerobic function of dystrophic muscle. Some reports show tremendous reductions in muscle ATP concentration ([ATP]) in DMD; HPLC analysis of muscle [ATP] in 12 DMD children from 4 to 14 yr of age showed a 98% reduction in [ATP] compared with age-matched controls (10). Although part of this huge reduction in ATP in DMD muscle may result from the presence of noncontractile tissue in samples, a 50% reduction in muscle [ATP] has been demonstrated in isolated muscle fibers from patients at more advanced stages of the disease (6). A systematic analysis of changes of metabolic enzymes in DMD muscle showed that the activities of several enzymes involved in glycolysis were greatly reduced, especially in glycolytic type II fibers (16). However, GAPDH is the most functionally impaired glycolytic enzyme in DMD muscle, and its activity can be inhibited in vivo by oxidative stress. GAPDH contains a cysteine within its catalytic domain (Cys-149) that is highly susceptible to oxidative or nitrosative modification by hydrogen peroxide, hypochlorous acid, or NO, which results in inactivation of the enzyme (56, 75). Thus an attractive but untested hypothesis is that oxidative stress in dystrophin-deficient muscle, especially during exercise, leads to oxidation of the active site cysteine in GAPDH to reduce enzyme activity and slow ATP production.

Free Radicals and Cardiomyopathy in Muscular Dystrophy

Fibrosis of the heart of DMD patients contributes significantly to defects in cardiac function, which cause death in DMD. Most evidence indicates that the primary cause of cardiac involvement in DMD is the progressive accumulation of connective tissue that replaces cardiac myocytes and fibers of the Purkinje system (29). In addition to the fibrotic replacement of contractile tissue, the remaining myocytes in the DMD heart become encased in connective tissue, so that the number of gap junctions are greatly reduced, which may contribute further to conduction defects (28). Most fibrosis occurs in the posterolateral and lateral free walls of the heart, which become the foci of ventricular arrhythmias. As a consequence, many defects in cardiac function that are observed in DMD patients appear during systole and are consistent with the replacement of contractile and Purkinje fibers in the left ventricular myocardium by connective tissue (55).

Recent findings show that free radicals play an important role in cardiomyopathy in mdx mice. Cardiac muscle in mdx mice experiences a significant reduction in nNOS activity, similar to the reduction that occurs in skeletal muscle (7, 14). Also similar to mdx skeletal muscle, expression of an nNOS transgene in the myocardium prevents myocardial fibrosis and normalizes ventricular histology and improves cardiac function (90). Although the mechanism through which nNOS transgene expression improves mdx cardiomyopathy is unknown, the data suggest that reductions in myocardial fibrosis would produce the functional improvements that occurred in the mdx mouse hearts that expressed the nNOS transgene (90). Fibrosis is a consequence of oxidative stress in multiple tissues, including the heart (13, 58, 61), and elevations in the production of superoxide or its derivative hydrogen peroxide are able to activate numerous pathways leading to increased cardiac fibrosis (13). Given the ability of NO to inhibit NADPH oxidase (18), which generates superoxide that can promote cardiomyopathy (13), the normalization of myocardial nNOS-derived NO production may decrease the production of other profibrotic free radicals and thereby reduce cardiomyopathy in dystrophin-deficient hearts. However, an alternative possibility is that myocardial fibrosis in mdx muscle is secondary to inflammation, and the anti-inflammatory functions of NO thereby reduce mdx cardiac fibrosis. This latter possibility is supported by experimental observations that show a reduction of myocardial inflammation in mdx hearts that express an nNOS transgene (90).

Disruptions in NO production also contribute to cardiomyopathy in other muscular dystrophies. Null mutations in any one of several sarcoglycans, which are also members of the DPC, cause muscular dystrophy that involves dilated cardiomyopathy (19, 25, 37). {gamma}-Sarcoglycan and {delta}-sarcoglycan null mice have sites of elevated NO concentration in their myocardia, which are associated with areas of focal damage (40). Contrary to the vasodilatory role of NO in other systems, NO synthase (NOS) inhibition in sarcoglycan null mice produces a decrease in vasospasms that are associated with the cardiomyopathy (91). The underlying mechanism has not yet been identified.

Free Radicals and Regeneration in Dystrophin-Deficient Muscle

The progressive loss of muscle tissue in DMD patients and old mdx mice shows that the processes that drive injury and death of dystrophic muscle exceed the muscle's regenerative capacity. However, the factors that are most important in limiting the regenerative capacity of dystrophic muscle are not known, and there is controversy concerning whether the regenerative defect is primarily attributable to exhaustion of the normal replicative capacity of satellite cells or a pathological defect in the regenerative capacity that is caused indirectly by the dystrophin mutation. Again, differences in the constitutive properties of the dystrophic muscle compared with wild-type muscle, as well as responses of the muscle to the presence of pathology, may both contribute to the defects in regeneration.

Several observations indicate that the loss of constitutive expression of nNOS that is secondary to the dystrophin mutation may contribute significantly to the inability of dystrophic muscle to regenerate sufficiently. Release of NO from satellite cells in vitro stimulates the secretion of hepatocyte growth factor, which in turn accelerates the cell cycle, thereby providing a potential mechanism to enhance muscle growth, repair, or regeneration (78). Similarly, administration of NOS inhibitors to mice experiencing muscle crush injury reduced the number of proliferative cells in the injured muscle and slowed muscle repair (4). These observations have suggested that loss of nNOS expression in dystrophic muscle could impair regeneration, because there would be deficiencies in NO-stimulated satellite cell proliferation. However, satellite cell numbers in DMD muscle are two to three times greater than in wild-type muscle (43, 85, 88), which indicates that expansion of satellite cells in dystrophin-deficient muscle occurs in dystrophinopathy. Nevertheless, the rate of satellite cell expansion in DMD muscle may be insufficient to maintain muscle mass during rapidly advancing stages of the disease.

Loss of nNOS from dystrophin-deficient muscle could also disrupt normal differentiation of muscle that could then reduce the regenerative capacity of muscle. In particular, muscle-derived NO has been implicated in promoting myoblast fusion to form myotubes, which is a requisite step in the differentiation of muscle to form mature muscle fibers. Application of NO donors to myoblasts in vitro promotes their fusion (50), and NOS expression increases sharply at the time of myoblast fusion. Furthermore, the elevation of NOS expression coincides with an increase in cGMP in the muscle, which suggests that the NO effects on fusion may be mediated by guanylate cyclase and cGMP (50). A recent, interesting investigation (60) has further demonstrated that NO can stimulate myoblast fusion by upregulating follistatin through a process that is dependent on the activation of guanylate cyclase and production of cGMP. Although these findings suggest that the loss of nNOS from dystrophic muscle could contribute to defects in regeneration of dystrophic muscle, nNOS null mice and young mdx mice have normal, multinucleated muscle fibers, which indicates nNOS-derived NO is not essential for myoblast fusion in vivo. Whether satellite cell fusion in regenerative muscle has a greater dependence on NO-mediated processes than fusion in developing muscle has not been examined.


   DO ANTIOXIDANT-BASED THERAPEUTICS OFFER PROMISE FOR TREATMENT OF DYSTROPHINOPATHIES?
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The numerous features of the pathophysiology of DMD and mdx dystrophies that may be caused or exacerbated by perturbations in the production of free radicals suggest that treatments with antioxidants could have a therapeutic effect. Indeed, this possibility has been explored for decades. However, early clinical trials using vitamin E treatment of DMD patients yielded no clinical improvement (30, 86), although the failure of these attempts may be attributable to treatment of patients who were already far advanced in the disease. Similarly, treatment of DMD patients with selenium, a cofactor for glutathione peroxidase, produced no improvement in muscle function (32), and no significant functional improvements were observed in DMD patients who were treated with SOD (77).

Many reasons may underlie the failure of these antioxidant treatments to reduce the pathology of muscular dystrophy. First, perhaps oxidative stress is not an important factor in the pathophysiology of DMD. Alternatively, systemic treatments with antioxidants may be too broad and unregulated to provide clinical improvements. Because the stoichiometry of free radicals rather than their mere presence or absence is most important in determining whether the redox environment of cells is physiological or pathological (54), systemic treatments with high dosages of antioxidants may be insufficiently targeted or regulated to return specific tissues to a physiological redox environment. In addition, antioxidants can promote tissue injury as well as reduce tissue injury. For example, SOD is an antioxidant, but it functions to convert superoxide, a relatively noncytotoxic free radical, to form hydrogen peroxide, which is much more cytotoxic (38). Finally, antioxidants can have multiple functions, and disrupting their levels systemically may have unanticipated and detrimental effects. For example, vitamin E can modulate the expression of genes through signaling events that are independent of its antioxidant properties. Importantly, some of the genes that it can upregulate could reduce the pathology of DMD and mdx dystrophies by reducing fibrosis or inflammation (17, 93), although its downregulation of other genes could inhibit repair and regeneration of dystrophic muscle (84).


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Many experimental and clinical findings support the hypothesis that disruption in the production, distribution, or interactions of free radicals can promote and perhaps initiate much of the pathology of DMD and mdx dystrophy. However, much of the evidence that supports this hypothesis relies on correlations between the presence of pathology and the levels of markers of either oxidative damage or response to oxidative stress. Experimental interventions that substantiate this hypothesis generally consist of broad perturbations in the production of free radicals, after which the effect on pathology is assessed. Throughout these studies, changes in NO production appear to be of central importance in affecting the severity of the dystrophic pathology (Fig. 2). However, whether the protective effects of NO are mediated by its reactivity with other free radicals, by its activation with heme groups present on enzymes that regulate diverse metabolic processes, or by its more specific functions as a signaling molecule has been little explored. Because the complexity and unpredictability of free radical biology and chemistry in vivo will make it difficult to design therapies for the muscular dystrophies through systemic perturbations of free radicals or antioxidants, a more mechanistic knowledge of the roles of free radicals in dystrophinopathy is needed.


Figure 2
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  		Fig. 2. Schematic representation of the multiple routes through which decreases in muscle neuronal NOS and NO production may promote the pathology of dystrophin-deficient muscle. Central to the scheme (shaded boxes) is the decrease in neuronal NOS (nNOS) expression that occurs in dystrophin null mutant muscle that may be exacerbated by muscle disuse. Loss of normal NO production can contribute to pathology by disrupting normal signaling through cGMP-mediated pathways, by loss of NO regulation of enzymes involved in free radical production, and by disrupting NO scavenging of other free radicals that may serve cytotoxic or regulatory functions. CAT, catalase.

 

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The authors' laboratory received research support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR-47721 and AR-046855) and the Muscular Dystrophy Association, USA, during the time that this article was prepared.


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Address for reprint requests and other correspondence: J. G. Tidball, Dept. of Physiological Science, 5833 Life Science Bldg., Univ. of California, Los Angeles, CA 90095 (e-mail: jtidball{at}physci.ucla.edu)


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