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Does Normal Nitric Oxide Synthase Prevent Pathologic MuscleChanges in Dystrophin Deficiency?

Irena Niebroj-Dobosz(1, 2), Anna Fidziaska(2), Zofia Glinka(1) and Irena Hausma-nowa-Petrusewicz(2)

(1) Department of Neurology, Medical University, and (2) Neuromuscular Unit,Medical Research Center, Polish Academy of Sciences, Warsaw, Poland

AbstractNeuronal nitric-oxide synthase (nNOS) is a member of the dystrophin-associated proteins, re-gulates homeostasis of reactive free radical species and may contribute to oxidative damage toproteins in muscle diseases. To test the hypothesis that nNOS activity may be involved in spa-ring from muscle pathology in dystrophin deficient muscles we examined nNOS immunoreac-tivity in muscles from Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy(BMD). The results were compared to nNOS in dystrophin positive limb-girdle dystrophy(LGMD) patients. Similar studies in dystrophin deficient hind limb muscles and diaphragm ofclinically almost asymptomatic mdx mice were performed. In the DMD patients nNOS appea-red to be either drastically reduced, or absent. In BMD and LGMD it was decreased, or nor-mal. In mdx mice muscles no changes in nNOS immunoreactivity were present. In the immu-nocytochemical examination in DMD nNOS was either not stained, or the staining was obser-ved in the surrounding connective tissue. In BMD nNOS staining was decreased or absent, inLGMD it appeared in the muscle cell cytoplasm. In mdx mice muscles nNOS reactivity wasobserved on the surface of the muscle fiber, starting from 30 days of age of the animals clustersof nNOS positive cells were observed.It is suggested that the decrease of nNOS content in dystrophinopathies is contributing to oxi-dative damage to muscle proteins, which enhances the degeneration of the muscle fibers.Normal nNOS may be one of factors, which prevent pathological muscle changes in mdxmice muscles. Regulations of the activity of this enzyme may be one of the possible strate-gies in dystrophy treatment.Key words: dystrophinopathies, mdx mice, nNitric oxide synthase, oxidative damage.

Basic Appl Myol 11 (2): 105-110, 2001

nNitric oxide synthase (nNOS), originally purifiedfrom neuronal tissues, has an important level of expres-sion in skeletal muscles [27]. It appeared to be associ-ated with sarcolemma [20]. It is enriched at myotendi-nous and neuromuscular junctions [41] and binds todystrophin directly/undirectly via (α’-syntrophin [6, 7,15, 17, 38]. It is suggested that nNOS is not attached tosarcolemma in Duchenne dystrophy (DMD) [6, 9, 16,19]. As the consequence of the dystrophin-glycoproteincomplex disruption nNOS is downregulated in the cyto-sol of the muscle cell [6, 16, 17, 19]. nNOS is also ab-sent in Becker dystrophy (BMD) [9, 10, 16] and themdx mice muscles [5, 6, 9].

As nNOS is implicated in several vital functions in themuscle cell it is of importance to answer the question,whether its deficiency may be responsible, at least

partly, for the severe and widespread pathologic changesin the muscles of DMD patients.

Material and Methods

Twenty five patients with DMD (4 to 18 yrs of age),10 patients with BMD (aged 8 to 20 yrs of age), 11 pa-tients with LGMD (9 to 44 years of age) and 20 aged-matched healthy controls were examined. The diagnosisof the diseases was established by clinical, genetic,electromyographic, histologic, ultrastructural, immuno-cytochemical, biochemical and immunochemical exami-nations. Diagnostic muscle biopsies were obtained afterinformed consent of the patients or their families.

Male mutant C57BL/1O mice (14 days to 12 monthsof age) and control age-matched normal mice ofC57BL/6J strain were examined. The animals (5 in eachgroup) were fed a standard laboratory diet. At 14, 30 90

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and 365 days of age the extensor digitorum longus(EDL) and diaphragm muscles for immunohistochemicalexamination, and gastrocnemius and diaphragm for im-munochemical examination were taken.

Cryostat frozen sections (8 mm) of the muscles werestained according to the standard techniques. For immu-nocytochemical studies monoclonal anti-nitric oxidaseantibodies (Sigma), diluted 1:3000, were used. Immu-nostaining: an indirect immunofluorescence method withTRITC immunoglobulins was applied.

For Western blotting examinations human and animalsmuscles were frozen quickly in dry ice and preserved at-72°C until used. After thawing all procedures wereconducted at +4°C. The samples were weighed, mincedand homogenized in 50 mM phosphate buffer (pH 7.5)with added protease inhibitors (5 mg leupeptin, 5 mgantipain, 5mg pepstatin and 170 mg PMSF in 10 ml ofDMSO. 100 ml of the mixture per 10 ml of the buffer

was used). The concentration of the proteins in the ho-mogenized samples was estimated by the method of Pe-terson [31]. An aliquot equivalent to 100 µg of proteinswas treated with a Laemmli sample buffer. The SDS-polyacrylamide gel electrophoresis in MiniProtean II 4-15% gradient gels (BioRad) in the system of Lae-mmli[21] in MiniProtean II Electrophoresis Cell was devel-oped. The separated proteins were blotted on nitrocel-lulose membranes 0.2 µm (LKB) in 25 µM Tris-HCl,192 mM glycine and 20% methanol buffer (pH 8.3) us-ing Mini Trans-Blot Electrophoretic Transfer Cell(BioRad). The blots were incubated with monoclonalanti-nitric synthase antibodies (Sigma), diluted 1:3000,goat anti-mouse-HRP secondary antibodies, and strepta-vidin HRP (Opti-4CN detection kit - BioRad). The ni-trocellulose sheets were further processed in a GelDoc1000/2000 system (BioRad), Multi An-alyst/PC version1.1 and Mitsubishi Video Printer P91.

Figure 1. Quadriceps femoris muscle. (a) Dystrophin-positive muscle fibers in healthy human muscle. (b) nNOS-positivefibers in healthy human muscle. (c) nNOS-negative muscle fibers in DMD. (d) nNOS-decorated endomysial connec-tive tissue in a patient with advanced stage of DMD. x 448

Figure 2. Quadriceps femoris muscle. (a) Early stage of BMD - faint nNOS stained muscle fibers. (b) Advanced stage ofBMD - in a small proportion of fibers nNOS is partially stained. (c) LGMD - nNOS stained in the muscle cellcytoplasm. x 448

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Analyses of variance, comparison of means and thedegree of significance were performed. A single valuewas defined to be significant when it was over the nor-mal mean ± (SDx2) using the Student’s test and theWilkoxon’s test. A value over P > 0.05 was consideredas non-significant.

ResultsIn the immunocytochemical studies (ICH) in control

human muscle immunoreactivity of nNOS was presentin the sarcolemma and closely corresponded to dystro-phin. In early stages of DMD no immunoreactivity ofnNOS was observed, in more advanced DMD cases theendomysial connective tissue reacted with the anti-

nNOS antibodies (Fig. 1). In early stages of BMD veryweak nNOS staining in the sarcolemma was present, inmore advanced BMD cases nNOS immunoreactivitywas more variable. In some fibers positive immu-nostaining appeared on the muscle cell surface, butsome fibers were nNOS negative. In LGMD nNOS wasstained in the muscle cell cytoplasm (Fig. 2). In normalmice nNOS was localized at the surface of the musclefiber. The fluorescence was more distinct in EDL, ascompared to the diaphragm (Fig. 3, Fig. 4). In mdx micenNOS reactivity was present on the surface of the EDLand diaphragm fibers. Starting from 30 days of age anincreasing number of cell clusters with intensively

Figure 3. EDL mice muscle. (a) nNOS-positive normal mice muscle. (b) Mdx mice, 1 month of age - nNOS stained in fewmuscle fibers. (c) Mdx mice, 3 months of age nNOS stained fibers form small clusters. (d) Mdx mice, 12 months ofage - nNOS stained fibers form large clusters. x 224

Figure 4. Diaphragm mice muscle. (a) nNOS is weakly stained in normal mice diaphragm. (b) Mdx mice, 3 months ofage - nNOS is stained in few muscle fibers. (c) Mdx mice, 12 months of age - nNOS is stained in the endomysialconnective tissue. x 224

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stained sarcoplasm was present (Fig. 3, Fig. 4). In thediaphragm some immunoreactivity in the endomysialconnective tissue was also observed (Fig. 4).

In the Western blotting analysis (WB) a nNOS immu-noreactive protein at 156 kDa was detected. Howeverboth in human as well in mice muscles there were sev-eral other immune reactive bands (190 kDa, 170 kDa,109 kDa, 84 kDa, 45 kDa, 33 kDa and 18 kDa). InDMD muscles the nNOS protein band, was either absentor decreased. In BMD and LGMD nNOS was moder-ately decreased, or normal (Fig. 5, Table 1). In normalhind limb muscles of non-mature mice (14 days of age)nNOS reactivity was lower than in adult animals, innormal diaphragm the nNOS reactivity was comparablebetween 14 days and 1 yr of age. Normal nNOS immu-noreactivity was present in the mdx hind limb musclesand diaphragm (Fig. 5, Table 1).

DiscussionnNOS is one of the most regulated enzymes in biol-

ogy. It is responsible for the synthesis of the molecularmessenger nitric oxide (NO), which is implicated as amodulator of skeletal muscle contractility and exercise-induced glucose uptake, mitochondrial respiration, car-bohydrate metabolism, neuromuscular transmission,muscle development, and blood flow to skeletal muscles[5, 23, 39]. Both the synthesis of NO and the nNOS sta-bility are finely regulated by Ca2+/calmodulin interac-tions [4], and transient changes in intracellular Ca2+

[28].Increases in [Ca2+

i] are required for binding of calmo-dulin to nNOS and consequently for nNOS activationand its synthesis [28, 32]. nNOS participates also inregulations of the free radical species homeostasis. Aprogressive increase of nNOS is observed in mice [9]and rats during aging [8], which may indicate a physio-logical role for NO during the aging process.

Several speculations indicate that decreased nNOSmay underlie some aspects of the pathophysiology ofdystrophic muscles. nNOS decrease is observed in dys-trophin-deficient DMD, BMD muscles [5, 6, 9, 16, 19,41] and mdx mice hind limb muscles [6, 9]. Downregu-lation of nNOS is occurring also in dystrophin-positivedenervated muscles, neurogenic diseases, metabolic andinflammatory myopathies [16, 33]. nNOS mislocalizedto muscle cell cytosol in human dystrophy and in mdxmice is also indicated [11, 12]. It is suggested that cyto-solic nNOS could compensate the loss of this enzyme inthe sarcolemma [6, 19]. According to other opinions,however, accumulation of nNOS in the cytosol is harm-ful for the muscle cell, as it may enhance the toxicity ofNO or superoxide and contribute to the myofiber necro-sis [18].

In all these studies immunocytochemical techniquesare used. No immunochemical examination and itsquantitation of the nNOS immunoreactive band of 150kDa is presented, yet. It should be stressed that ICH re-sults should not be directly compared to those of the

WB analyses. In the latter the 156 kDa protein is sepa-rated from the other immunoreactive bands, which is notpossible using the ICH techniques. The question re-mains, whether the muscle cell cytosol immunoreactivityis connected with the mislocalized nNOS, and with deg-radation products of nNOS, or other proteins with epi-topes reacting with the anti-nNOS antibodies.

The presented results generally confirm the previousreports of nNOS in DMD and BMD. LGMD should ac-complish the list of the diseases with nNOS changes inthe muscles. No changes of nNOS, however, using theWB technique, in hind limb mdx muscles were present.In the mdx diaphragm nNOS was well preserved, too.We do suppose that the observed immunoreactivity inICH in the connective tissue and cytosol in human dys-trophies and mdx mice muscles represents immunoreac-tive material not represented by nNOS.

Figure 5. Western blotting of nNOS in human and micemuscles. The position of nNOS is indicated bythe arrow.

Table 1. nNitric oxide synthase immunoreactivity in humanand mdx mice dystrophinopathy.

n Area of the tracingat 156 kDa

mm X 10-4 OD

Duchenne dystrophy 25 12.0 ± 11.6* (25)Becker dystrophy 8 26.1 ± 13.9* (6)Limb-girdle dystrophy 11 40.1 ± 25.8 (7)Healthy controls 20 66.9 ± 13.3

Mdx mice - hind limb muscles 20 56.5 ± 14.6Mdx mice - diaphragm 20 121.8 ± 35.3Normal mice - hind limb muscles 20 60.8 ± 20.9Normal mice - diaphragm 20 154.3 ± 64,0

Values are means ± SD. * Significant at P < 0.001. Thenumber of patients with significantly decreased nNOS val-ues (over the mean ± SDx2) is presented in parentheses.

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As nNOS mediates several functions in the normalmuscles loss of this enzyme may underlie some aspectsof the pathology in diseased muscles, DMD included.The fact that the most evident nNOS changes were pres-ent in dystrophin-deficient DMD/BMD, also in somecases of dystrophin-positive LGMD muscles, but not inthe dystrophin-deficient mdx mice muscles, indicatesthat nNOS decrease is not the consequence of dystro-phin deficiency per se. The question remains, what isresponsible for the decrease of nNOS in course of mus-cular dystrophies. One of the possibilities is that nNOS,a protein highly sensitive to calpains, is the subject ofthe calpains action. The activity of calpains is known tobe increased in DMD, at least. This may be one of themechanisms, which contribute to nNOS changes inDMD [22]. Another mechanism may be connected withthe calmodulin-[Ca2+]-nNOS interplay. nNOS activity ischanged by transient changes of [Ca2+], which altersnNOS binding to calmodulin, and consequently inducesits activation and synthesis [28, 32]. Both calmodulin[25, 29, 30], and [Ca2+] [2, 3, 14, 26, 35, 36] are knownto be increased in DMD. This mechanism may contrib-ute to abnormal nNOS activity in DMD.

The question is also, whether nNOS attributes tosparing from dystrophic pathology in dystrophin-deficient but almost clinically asymptomatic mdx mice.This possibility has to be taken into account, but othermechanism(s) in sparing the muscles from dystrophicpathology than is the normal concentration and locationof nNOS have to be thought over [40].

Although loss of sarcolemmal nNOS does not seem tobe the consequence of dystrophy, the absence/decrease ofthis enzyme in the muscles may contribute to the oxidativedamage to muscle proteins and their increased suscepti-bility to degeneration. As nNOS is known to increase fol-lowing exercise [1, 34] and NO regulates blood flow inskeletal muscle [24], playing a major role in muscle toler-ance to endurance exercise, the old trials of physiotherapyand exercise in dystrophinopathies should be reminded[13, 37]. Strengthening exercises have rather a harmfuleffect in DMD, but resisted exercises could have someadvantage in management of these patients. The expectedexercise induced increase of nNOS activity may improveblood flow and induce a favorable effect on glucose andenergy supply to the dystrophic muscle cell.

Address correspondence to:Irena Niebroj-Dobosz, NW, PhD, Department of Neurol-

ogy, Medical University, 1a Banacha Str., 02-097 Warsaw,Poland, tel. +48 (22) 659 7505, fax +48 (22) 668 8512.

AcknowledgementThe study was supported by a grant of State Commit-

tee for Scientific Research (No 0497/P05/98/15).

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