The involvement of oxidative stress in determining the severity and progress of pathological processes in dystrophin-deficient muscles.

In both forms of muscular dystrophy, the severe Duchenne's muscular dystrophy (DMD) with lifespan shortened to about 20 years and the milder Becker dystrophy (BDM) with normal lifespan, the gene defect is located at chromosome locus Xp21. The location is the same in the experimental model of DMD in the mdx mice. As the result of the gene defect a protein called dystrophin is either not synthesized, or is produced in traces. Although the structure of this protein is rather well established there are still many controversies about the dystrophin function. The most accepted suggestion supposes that it stabilizes sarcolemma in the course of the contraction-relaxation cycle. Solving the problem of dystrophin function is a prerequisite for introduction of an effective therapy. Among the different factors which might be responsible for the appearance and progress of dystrophic changes in muscles there is an excessive action of oxidative stress. In this review data indicating the influence of oxidative stress on the severity of the pathologic processes in dystrophy are discussed. Several pieces of data indicating the action of oxidative damage to different macromolecules in DMD/BDM are presented. Special attention is devoted to the degree of oxidative damage to muscle proteins, the activity of neuronal nitric oxide synthase (nNOS) and their involvement in defining the severity of the dystrophic processes. It is indicated that the severity of the morbid process is related to the degree of oxidative damage to muscle proteins and the decrease of the nNOS activity in muscles. Estimation of the degree of the destructive action of oxidative stress in muscular dystrophy may be a useful marker facilitating introduction of an effective antioxidant therapy and regulation of nNOS activity.

Duchenne's dystrophy (DMD) is a severe genetically determined disease.The patients are immobilized at the age of about 10 yrs and their lifespan is shortened to about 20 yrs.Becker dystrophy (BDM) is a milder form of dystrophy.The first clinical symptoms appear at the age of about 10 years, the patients are usually not immobilized and the lifespan is nearly normal.The mdx mice, an experimental model of DMD, is clinically almost asymptomatic.The gene is located at chromosome locus Xp21.Its product called dystrophin is located at the cytoplasmic face of the sarcolemma (Zubrzycka-Gaarn et al., 1988).In DMD, BDM and the mdx mice dystrophin is either absent, or appears in traces.The structure of this protein has been extensively studied for the last 15 years (Fig. 1).It is known already that several proteins, localized mainly in the membrane, collaborate with dystrophin.Spe-

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I. Niebrój-Dobosz and I. Hausmanowa-Petrusewicz mdx diaphragm and also sparing of some muscles in DMD/BDM is among the most fundamental questions which remain to be answered.The answer can provide important clues regarding the disease etiology and also therapeutic measures.
The function of dystrophin in the muscle is still a mystery.It has been suggested that: 1) dystrophin supports sarcolemma against mechanical stress and stabilizes it in course of the contraction-relaxation cycle (Koenig et al., 1988), 2) dystrophin takes part in the regulation of intracellular calcium and the further cascade of calcium-related events (Franco & Lansman, 1990), 3) dystrophin works in force and signal transduction (Gee et al., 1998), 4) dystrophin influences the aggregation of neurotransmiĴer receptors (Kong & Anderson, 1999), 5) dystrophin prevents excessive generation of reactive oxygen free radical species (Brown, 1995).
All these mechanisms are in no way exclusive and may interact with one another to a significant degree.

OXIDATIVE STRESS IN DYSTROPHINOPATHIES
Recent studies strongly support the notion of the importance of oxidative stress in dystrophinopathies.Free radical injury to sarcolemma may contribute to changes of its integrity (Murphy & Kehrer, 1989).There is increasing evidence that the degenerative processes in dystrophic muscles may be due to oxidative stress.An imbalance of free radicals synthesis and the antioxidant capacity may contribute to the necrotic process (Mendell et al., 1971).The significance and precise extent of the oxidative stress contribution is, however, poorly understood.
Free radicals are produced in the course of different physiological processes.Their action should be limited by several defense mechanisms (Woodford & Whitehead, 1998).Free radicals are known to be responsible for chemical and molecular damage of DNA, nucleotides, proteins, lipids, carbohydrates and cell membrane structure (Slater, 1984).

OXIDATIVE STATUS OF MUSCLE PROTEINS IN DYSTROPHINOPATHIES
In addressing the notion that the severity of muscle changes is mediated by reactive free radical species (ROS) it is of value to determine the formation of carbonyl derivatives of amino-acid residues Oxidative stress in dystrophy in proteins.Their content indicates the proteins oxidative status.Their quantification by testing the reaction of these groups with carbonyl-specific reagents is considered to be the most sensitive and reliable method for determining the oxidative modification to proteins and the free radical-induced protein damage (Levine et al., 1994).Proteins of different structure, function or intracellular localization exhibit various susceptibility to oxidative damage (Haycock et al., 1996).
In nearly all DMD cases increased protein carbonyl values are found (Haycock et al., 1996;Niebroj-Dobosz et al., 2002).In BMD and LGMD this is a rare finding.In mdx mice the carbonyl level is normal both in the hind limb and in the diaphragm muscles (Niebroj-Dobosz et al., 2002).The most heavily oxidized proteins in DMD appear to be actin (43 kDa), desmin (57 kDa) and an unidentified 34 kDa protein, and less oxidized proteins appear at 125 and 83 kDa (Niebroj-Dobosz et al., 2002).In BDM generally the same paĴern is present, although it is less expressed.In some LGMD cases increased oxidation of proteins between 83 and 34 kDa is also observed.In a previously published paper (Haycock et al., 1998) the only oxidized protein in DMD and BDM was a 125 kDa protein.Although in the mdx mice the carbonyl content both in the hind limb muscle and the diaphragm is normal, an 83 kDa protein is more heavily oxidized.This protein has been identified as α-actinin (Niebroj-Dobosz et al., 2002).

THE CONTRIBUTION OF NEURONAL NITRIC OXIDE SYNTHASE (nNOS) TO OXIDATIVE DAMAGE OF PROTEINS AND ENHANCEMENT OF DEGENERATIVE PROCESSES OF MUSCLE FIBRES
nNOS is a member of the dystrophin-glycoprotein complex and is implicated in several vital functions including regulation of the homeostasis of reactive free radical species including NO which protects the muscles against oxidative injury and may function as an antioxidant and takes part in mediating the signaling function of dystrophin and related proteins (Wink et al., 1993;1995).Measurements of nNOS/NO may be helpful in answering the question whether there is a correlation between its activity and the severity of the dystrophic process.
The protective action of nNOS is reduced in dystrophinopathies (Brenman et al., 1996;Chang et al., 1996;Bredt, 1999;Niebroj-Dobosz et al., 2002).In Duchenne's dystrophy nNOS (Niebroj-Dobosz et al., 2001) appears to be either drastically reduced or even absent.In very advanced stages of this disease only endomysial tissue reacts with nNOS antibodies.In Becker dystrophy (Niebroj-Dobosz et al., 2002) nNOS is either decreased or normal, in limb-girdle dystrophy staining of nNOS appears in the cytoplasm.In mdx mouse muscles nNOS reactivity is observed on the surface of the fiber and from day 30 of life clusters of nNOS are present (Niebroj-Dobosz et al., 2001).When immuno-detected in Western bloĴing, nNOS appears either reduced or absent in DMD, decreased or normal in BMD and LGMD, and normal or slightly decreased in mdx mice (Niebroj-Dobosz et al., 2001) Altered activity of nNOS, apart from its influence on the homeostasis of ROS, is implicated also in the abnormalities in blood flow during exercise in dystrophic patients.Contraction of dystrophic muscles may not properly stimulate a level of NO production (Gucuyener et al., 2000;Kasai et al., 2004) sufficient for relieving vasoconstriction.This results in local muscle ischemia because of inadequate blood flow to the actively contracting muscles.The sustained vascular constriction in Duchenne dystrophy may explain the focal necroses in dystrophin-deficient muscles.At least part of muscle degeneration in DMD may result from the reduced production of nNOS/NO as it may lead to impaired regulation of vasocontrictor response (Crosbie, 2001).
The oxidative damage of macromolecules possibly appears as a result of insufficient antioxidant defense and abnormal nNOS/NO, which correlates with the severity of the dystrophic process.

STRATEGY OF MUSCULAR DYSTROPHY TREATMENT BASED ON PREVENTION OF OXIDATIVE STRESS ACTION AND IMPROVEMENT OF nNOS ACTIVITY
No effective treatment of muscular dystrophies is known yet.Prevention of ROS damage by providing different antioxidants could have positive clinical effect.Administration of a set of naturally occurring antioxidants and manipulation of the nNOS activity and NO level by exercise.(Tidball et al., 1998) plus physiotherapy could improve the quality of life and decrease the severity of the disease.

CONCLUSIONS
1.In dystrophinopathies myofibers are subject to intra-/extracellular oxidative stress.The oxidized macromolecules are more susceptible to degradation.
2. Oxidative stress is a potential pathogenetic factor which may determine the severity of pathological changes in dystrophic muscle.
3. Oxidative stress in dystrophinopathies interacts with sarcolemmal weakening, inappropriate calcium influx, aberrant cell signaling and recurrent muscle ischemia.
5. Lowering the oxidative stress by increasing the antioxidant capacity through application of anti-oxidants and corrections of the nNOS activity/NO level by daily long-term physiotherapy and exercise, opens a new strategy of treatment which may modify the disease process and slow down its progress.