| Human Molecular Genetics | Pages |
mdx muscle pathology is independent of nNOS perturbation
Introduction
Results And Discussion
Absence of nNOS correlates with mdx phenotype
Genetic removal of nNOS from mdx mice
mdx:nNOS-/- mice maintain membrane damage
mdx:nNOS-/- muscle maintains dystrophic morphology
Materials And Methods
Mice
Antibodies
Immunofluorescence
Generation and genotyping of dystrophin-nNOS double mutant mice
Northern blot analysis
Pyruvate kinase assay
Evans blue assay
Histology
Acknowledgements
References
mdx muscle pathology is independent of nNOS perturbation
In skeletal muscle, neuronal nitric oxide synthase (nNOS) is anchored to the sarcolemma via the dystrophin-glycoprotein complex. When dystrophin is absent, as in Duchenne muscular dystrophy patients and in mdx mice, nNOS is mislocalized to the interior of the muscle fiber where it continues to produce nitric oxide. This has led to the hypothesis that free radical toxicity from mislocalized nNOS may contribute to mdx muscle pathology. To test this hypothesis directly, we generated mice devoid of both nNOS and dystrophin. Overall, the nNOS-dystrophin null mice maintained the dystrophic characteristics of mdx mice. We evaluated the mice for several features of the dystrophic phenotype, including membrane damage and muscle morphology. Removal of nNOS did not alter the extent of sarcolemma damage, which is a hallmark of the dystrophic phenotype. Furthermore, muscle from nNOS-dystrophin null mice maintain the histological features of mdx pathology. Our results demonstrate that relocalization of nNOS to the cytosol does not contribute significantly to mdx pathogenesis.
INTRODUCTION
Muscular dystrophy represents a group of skeletal muscle diseases that are characterized primarily by progressive muscle wasting and weakness. In the case of Duchenne muscular dystrophy (DMD) and the murine model (mdx mouse) for this disease, mutations in the dystrophin gene result in the absence of dystrophin and loss of the dystrophin-glycoprotein complex (DGC) (1-4). This oligomeric complex spans the sarcolemma and links the intracellular actin cytoskeleton to the extracellular matrix (5). Exactly how absence of the DGC leads to muscle cell necrosis is not clearly understood, but several lines of evidence suggest that oxidative stress may play an important role (6). The possibility that an imbalance of free radicals contributes to the necrotic process in muscular dystrophy was proposed in 1971 by Mendell et al. (7), who noted the similarity between oxidative lesions of ischemic muscle and some of the pathologic features of muscular dystrophy. The free radical damage hypothesis was strengthened by experiments which showed elevated levels of free radical-scavenging enzymes in dystrophic muscle, suggesting that expression of these proteins was induced as a result of oxidative stress (8).
Recently, several laboratories have shown that the free radical-producing enzyme neuronal nitric oxide synthase (nNOS; type I) is anchored to the sarcolemma by the DGC (9,10). The PDZ domain of nNOS interacts directly with syntrophin, which is associated with dystrophin (10). In the absence of dystrophin, nNOS is mislocalized to the cytosol where it maintains some of its enzymatic activity (9,11), namely conversion of arginine to citrulline and nitric oxide. Nitric oxide is a highly reactive molecule that potentially can modify lipids, proteins and DNA through a variety of mechanisms (12-16). These observations have led to the hypothesis that free radicals from mislocalized nNOS may contribute to mdx muscle pathology (6,9). Regulation of nNOS activity, which normally occurs via Ca2+-calmodulin (17-19), may be altered by elevated levels of intracellular Ca2+ present in mdx muscle (17-19). The possible aberrant activation of nNOS activity might also contribute to inappropriate free radical production.
Using mice expressing various dystrophin transgenes, we found a correlation between the mdx phenotype and the absence of nNOS from the sarcolemma, supporting the hypothesis that mislocalization of nNOS may contribute to the dystrophic phenotype. To test this hypothesis directly, we genetically removed nNOS from mdx mice and examined the phenotype of these nNOS-dystrophin null mice. We found that the nNOS-dystrophin double knockout mice maintained the dystrophic phenotype, suggesting that relocalization of nNOS to the cytosol does not significantly contribute to mdx pathogenesis.
Table 1.
| Dystrophina | Deleted region in transgene | Muscle morphology | nNOS | DGCb |
| Wild-type | no deletion | normal | + | + |
| mdx | premature stop | dystrophic | - | - |
| [Delta]3-7 ([Delta]abd)c | actin-binding domain | quasi-normal (specific force of muscle intermediate between wt and mdx) | +/- | + |
| [Delta]17-48 (becker)d | rod domain | quasi-normal (slightly elevated central nucleation) | + | + |
| [Delta]1-62 (Dp71)e | N-terminus and rod domain | dystrophic | - | + |
| [Delta]64-67f | Cys-rich domain | dystrophic | - | - |
| [Delta]68-70f | C-terminus | dystrophic | - | - |
| [Delta]71-74 ([Delta]330)f,g | syntrophin-binding domain | normal | + | + |
| [Delta]75-78f | C-terminus | normal | + | + |
RESULTS AND DISCUSSION
Absence of nNOS correlates with mdx phenotype
In order first to explore the role of nNOS in mdx pathology, we examined nNOS expression in a collection of seven dystrophin transgenic mice. The transgenes, which individually were expressed on an mdx background, encode truncated dystrophin products (20-26). Muscle from the transgenic mice was evaluated previously for its morphology and for the ability of the transgene to restore sarcolemma localization of the DGC (Table 1).
We detected nNOS expression in skeletal muscle cross-sections by immunofluorescence staining with nNOS antibodies for each of the transgenic mice. These results, summarized in Table 1, suggest a correlation between the mdx phenotype and the absence of nNOS from the sarcolemma. We found sarcolemma staining of nNOS in muscle from the [Delta]3-7 (26) (deletion of the actin-binding domain), [Delta]17-48 (24) (becker), [Delta]71-74 (23,25) (formerly referred to as the [Delta]330) and [Delta]75-78 (25) mice, which have normal or quasi-normal muscle morphology (Fig. 1). However, nNOS staining was not detected in muscle from the [Delta]1-62 (21) (Dp71), [Delta]64-67 (25) and [Delta]68-70 (25) mice, which maintain the dystrophic, mdx phenotype. Interestingly, expression of the Dp71 transgene restores the DGC complex to the sarcolemma, but is not able to rescue the mdx morphology (21,22). Chao et al. found similar results with the Dp71 mouse (27); however, our results differ concerning the [Delta]17-48 mouse. The differences observed between the two studies are likely to be due to mouse to mouse variations in the level of [Delta]17-48 transgene expression, which has been shown to affect the ability of this specific transgene to rescue the mdx phenotype (24). Taken together, these data support the hypothesis put forward by Bredt et al. (9) that free radicals produced from mislocalized nNOS may contribute to the pathogenesis of muscular dystrophy.
Figure 1. Immunohistochemical analysis of nNOS in muscle from dystrophin transgenic mice. Skeletal muscle cryosections from wild-type, mdx, [Delta]3-7 (deletion of the actin-binding domain), [Delta]17-48 (`becker'), [Delta]1-62 (Dp71), [Delta]64-67, [Delta]68-70, [Delta]71-74 (formerly referred to as [Delta]330) and [Delta]75-78 mice were stained with an affinity-purified rabbit polyclonal antibody against the N-terminal domain of nNOS. The dystrophin transgenes are depicted schematically on the right of each panel. Skeletal muscle which maintains the mdx morphology does not have sarcolemma staining of nNOS. Bar indicates 100 µm. 
Genetic removal of nNOS from mdx mice
To test directly whether misregulated or mislocalized nNOS activity contributes to the pathogenesis of muscular dystrophy, we genetically removed nNOS from mdx mice. This was accomplished by mating nNOS-/- males (28) with mdx females, as depicted in Figure 2. a. The mdx phenotype is inherited as an X-linked recessive trait, which stems from a premature stop codon in the dystrophin gene, leading to complete absence of dystrophin (29-31). The nNOS-/- mice were created by a targeted removal of exon 1, which deletes the first 200 amino acids of the protein encoding the PDZ domain. Muscle from nNOS mutant mice specifically lacks the alpha and mu isoforms of nNOS (nNOS[alpha] and nNOSµ), which are the only isoforms that bind to the DGC (10). Beta and gamma isoforms of nNOS (nNOS[beta] and nNOS[gamma]) are not affected by the targeted disruption of this gene (10). However, nNOS[beta] is not expressed in muscle, and nNOS[gamma], which has nearly negligible activity (3% catalytic activity compared with nNOS[alpha] and nNOSµ), is cytosolic and does not bind to the DGC (10). a
Figure 2. Generation of dystrophin-nNOS double mutant mice. (a) Breeding scheme to produce mdx mice devoid of nNOS. nNOS single mutant males (nNOS-/- dyst+/Y) were bred with mdx females (nNOS+/+ dyst-/-) to produce heterozygote F1 offspring. F1 generation mice were intercrossed to produce F2 generation mice with a one in eight prospect for obtaining mice with an nNOS and dystrophin double mutant genotype (nNOS-/- dyst-/-, female or nNOS-/- dyst-/Y, male). (b) nNOS-dystrophin genotyping by PCR amplifications. Upper panel: for nNOS genotyping, separate amplifications were performed using neo-specific (neo PCR) or nNOS-specific (nNOS PCR) primers. The neo gene was used in the targeted disruption of the nNOS gene (28) and therefore serves as a marker for the nNOS mutant allele. PCR amplifications were performed using genomic DNA from wild-type (lane 1), nNOS mutant (lane 2), nNOS heterozygous (lane 3) and mdx:nNOS-/- mutant (lane 4) mice. Lane 5 is a no-DNA template control. The arrowhead marks the 500 bp product that is expected for both theneo and nNOS PCR reactions. Heterozygous females (nNOS-/- dyst-/-) can be distinguished by this PCR-based method. Note the presence of PCR products for both neo and nNOS in lanes 3 as well as PCR products for mdx and wild-type dystrophin alleles in lanes 3[prime]. Lower panel: offspring were genotyped for the mdx allele using PCR analysis based on the mdx amplification-resistant mutation system (mdx-ARMS) (41). For each DNA sample, two sets of PCRs are utilized. One PCR set utilizes a reverse oligonucleotide primer specific for the wild-type allele (WT PCR) and therefore only allows amplification of the wild-type allele. The second PCR reaction utilizes a reverse oligonucleotide primer specific for the mdx allele (MDX PCR) and therefore only amplifies the mdx allele. The same forward primer is used in each reaction. The PCR product for each amplification is 105 bp. Genotyping was performed for wild-type (lane 1[prime]), mdx (lane 2[prime]), mdx heterozygous (lane 3[prime]) and mdx:nNOS-/- (lane 4[prime]) genomic DNA. Lane 5[prime] is a no DNA template control. (c)Analysis of nNOS expression in mouse skeletal muscle.Northern blots of total RNA (20 µg/lane) from wild-type, mdx, nNOS-/- and mdx:nNOS-/- skeletal muscle were hybridized with 32P-labeled nNOS probe (left panel). As a control for loading, the same blot was probed with [beta]-actin (right panel). Molecular size standards (in kilobases) are indicated on the left of each panel. In our study, the dystrophin-nNOS mutant (mdx:nNOS-/-) mice were identified by PCR genotyping (Fig. 2b) and Southern analysis (data not shown). These data were confirmed by northern analysis showing that nNOS transcript was present in mdx mice, but completely absent from nNOS-/- mutant and mdx:nNOS-/- mutant mice (Fig. 2c). Western blot analysis of total muscle homogenates and skeletal muscle membranes confirmed that nNOS is mislocalized in mdx mice, as previously shown (9,10), and that the nNOS-/- and mdx:nNOS-/- mice lacked nNOS at the protein level (data not shown). We have tested the mdx:nNOS-/- mice for several characteristics of the mdx phenotype and, overall, we found that the mdx:nNOS-/- mice maintained these mdx features. The mdx:nNOS-/- mice were examined up to 12 months of age, and we found no quantitative differences compared with mdx mice, suggesting that the disease pathogenesis was not altered by nNOS removal. 
mdx:nNOS-/- mice maintain membrane damage
Sarcolemma damage is one of the early hallmarks of mdx pathology (32-34). The possibility that free radicals from mislocalized nNOS contribute to sarcolemma damage is consistent with the observations of Rando (35), Ragusa (36) and their colleagues, who recently reported elevated levels of lipid peroxidation in muscles of mdx mice. This disruption in membrane integrity permits unregulated exchange of molecules between the circulating blood serum and the muscle fiber. We evaluated membrane damage in the nNOS-dystrophin mutant mice by employing two different assays. First, permeability of the muscle fiber was analyzed by evaluating leakage of muscle-specific pyruvate kinase (PK) in the blood serum (Fig. 3a). Wild-type and nNOS mutant mice have low serum levels of PK activity. mdx mice, on the other hand, exhibit high serum levels of PK activity. The PK activities in the serum of mdx:nNOS-/- mice were similar to that of mdx mice, indicating that membrane damage persisted despite the removal of nNOS.a
Figure 3. Serum pyruvate kinase and Evans blue assays detect sarcolemma damage. (a) Measurement of pyruvate kinase released from the muscle fiber into the circulating blood. Pyruvate kinase activities were evaluated in the blood serum from wild-type, mdx, nNOS-/- and mdx:nNOS-/- mice. Error bars indicate the deviation between six sets of experiments (duplicate measurements for three separate mice). (b) Evans blue assay for infiltration of blood serum proteins into the muscle fiber. Intravenous injection of the Evans blue tracer can be detected in fibers with damaged plasma membranes. Transverse cross-sections of quadriceps muscle were observed with a fluorescence confocal microscope equipped with green activation filters. Evans blue-positive fibers are denoted by red staining. Bar, 100 µm. To examine leakage into the muscle fiber, we employed an Evans blue tracer assay (37,38). This intravenously injected fluorescent dye accumulates in the interior of damaged muscle fibers presumably by entering through unrepaired tears in the membrane. Work from several groups has demonstrated that skeletal muscle fibers from mdx mice exhibit intracellular Evans blue staining (37,38). With the Evans blue injections, we demonstrated that extensive membrane damage was present in quadriceps muscle from both mdx and mdx:nNOS-/- mutant mice (Fig. 3b). We have also examined the diaphragm muscle from these mice and found that Evans blue accumulated to the same extent in mdx and mdx:nNOS-/- mice. These data are consistent with the pyruvate kinase measurements. 
mdx:nNOS-/- muscle maintains dystrophic morphology
Additional features of mdx muscle pathology include necrosis, variation in fiber size and increased fiber degeneration/regeneration with centrally located nuclei. Hematoxylin and eosin (H&E) staining of quadriceps muscle cross-sections clearly showed the presence of these pathological features in age-matched mdx:nNOS-/- mice, indicating that these characteristics of the mdx phenotype were independent of nNOS (Fig. 4a). H&E staining of the diaphragm muscle showed that the mdx:nNOS-/- mice maintained the hallmark features of the mdx morphology (data not shown). Quantitation of central nucleation for both muscle types demonstrated that the mdx:nNOS-/- mice had undergone levels of regeneration identical to the mdx mice (Fig. 4b). We have examined the mdx:nNOS-/- mice up to 12 months of age and found no quantitative differences compared with mdx mice, suggesting that the disease pathogenesis is not altered by nNOS removal.
Figure 4. Skeletal muscle morphology of wild-type, mdx, nNOS-/- and mdx:nNOS-/- mice. (a) H&E staining of quadriceps sections shows that the mdx:nNOS-/- mutant mice maintain the characteristics of mdx pathology, including variations in fiber size, centrally located nuclei and phagocytic infiltration. Identical morphological features were found in diaphragm muscle (not shown). (b) Quantitation of central nucleation for both quadriceps and diaphragm muscles. Central nucleation is represented as the percentage of total nucleated fibers with centrally located nuclei. nNOS is anchored to the sarcolemma by its association with the DGC. In addition to dystrophin, the DGC is composed of the sarcoglycans, syntrophins and dystroglycan (1-5). Sarcospan (25 kDa) is the most recently identified member of the DGC, and is unique in that it is predicted to have four transmembrane-spanning domains (43). Although nNOS is not an integral component of this complex, it is linked to dystrophin via attachment to the PDZ domain of sytrophin (9,10). Interestingly, syntrophin has been shown to bind to the voltage-gated sodium channels in skeletal and cardiac muscle, suggesting that nNOS may regulate these channels in vivo (44). Our results suggest that redirection of nNOS from the sarcolemma to the cytoplasm in mdx muscle is not likely to be critical for the oxidative damage and other necrotic processes observed in muscular dystrophy. Although we cannot rule out that cytosolic nNOS may contribute partially to these events, its contribution must be minimal, given the extent of damage observed in the mdx:nNOS-/- mice. Furthermore, knowledge of the prerequisite factors necessary for restoring normal muscle morphology is imperative for designing strategies to alleviate the dystrophic phenotype. Our results support the idea that removing cytosolic nNOS from mdx muscle is not essential for correcting the mdx phenotype. It is possible, though, that loss of nNOS function at the sarcolemma contributes to the muscular dystrophy pathogenesis. For instance, several reports have documented that nitric oxide produced by nNOS can provide protection from more toxic free radicals (16). Perhaps loss of this protective function is the basis for elevated oxidative stress in dystrophic muscle. Defining the functional interactions between nNOS and the DGC will provide insight into the role of nNOS at the sarcolemma. 
MATERIALS AND METHODS
Mice
Wild-type (C57BL/10) and mdx (C57BL/10ScSn) mice, obtained from Jackson Laboratories, were maintained at the University of Iowa Animal Care Unit in accordance with animal usage guidelines. mdx mice were maintained as a breeder colony of mdx males and homozygous mdx females. nNOS single mutant mice (C57BL/6) were obtained from the colony produced by Huang et al. (28). The targeted disruption of the nNOS gene in these mice was accomplished previously by replacement of exon 1 with the neomycin resistance gene (neo) through homologous recombination (28). The dystrophin transgenic mice have been described previously (21,24-26,39)
Antibodies
Anti-nNOS antibodies were generated by intramuscular and subcutaneous injection of New Zealand white rabbits (Knapp Creek Farms) with 500 µg of an nNOS (rat amino acids 1-200)-glutathione S-transferase (GST) fusion protein in an emulsion of Freund's complete adjuvant (Sigma). Rabbits 200 and 201 were boosted 2 weeks later with an s.c. injection of nNOS-GST fusion protein (1 mg) in TBS (20 mM Tris-HCl, 100 mM NaCl, pH 7.5). Blood serum was collected from the rabbits 1 week following the boost and tested for the presence of anti-nNOS antibodies. The serum was cleared of anti-GST antibodies with Immobilon-P (Millipore) strips containing GST and subsequently affinity-purified using nNOS-GST Immobilon-P strips. C-terminal dystrophin antibodies were affinity- purified from goat 20 (immunized with the DGC complex) (40) using dystrophin (mouse C-terminal 315 amino acids)-GST Immobilon-P strips.
Immunofluorescence
Transverse cryosections (7 µm thick) from mouse quadriceps were fixed with 4% freshly depolymerized paraformaldehyde for 10 min at room temperature. Muscle sections were washed briefly with TBS and incubated with affinity-purified nNOS antibodies (1:10 dilution; rabbit 200) for 24 h.
Generation and genotyping of dystrophin-nNOS double mutant mice
nNOS single mutant males were bred with mdx females in the breeding strategy outlined in Figure 2. a. Heterozygous F1 offspring were mated to produce F2 generation mice. Identification of the dystrophin-nNOS mutant mice was performed by PCR genotyping of genomic DNA prepared from mouse tail snips (41). DNA from wild-type, mdx and nNOS single mutant mice were used for controls.
mdx genotyping. An amplification-resistant mutation system (ARMS) assay was used to screen for the mdx point mutation as previously described (41). Briefly, two separate PCR reactions were performed. One reaction (MDX PCR) utilizes oligonucleotide primers which only allow amplification of the mdx allele, and the second PCR reaction (WT PCR) amplifies only the wild-type allele (41). Both PCR products are 105 bp. nNOS genotyping. PCR amplification of nNOS exon 1 (which is disrupted in the nNOS mutant mice) was performed with mouse tail DNA using either nNOS- or neo-specific oligonucleotides. PCR primers for the neo and nNOS amplifications were as follows: 5[prime]-TGGAGAGGCTATTCGGCTATGAC-3[prime] (neo, sense); 5[prime]-CACCATGATATTCGGCAAGCAC-3[prime] (neo, antisense); 5[prime]-atctcagatctgatccgaggag-3[prime] (nNOS, sense); 5[prime]-CTTTCATCTCTGCTTTGGCTGG-3[prime] (nNOS, antisense). PCR cycling conditions were 94°C, 45 s; 55°C, 45 s; 72°C, 45 s for 35 cycles in a buffer consisting of 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3. The PCR product for both reactions was 500 bp. The genotyping was confirmed by Southern analysis (28) (data not shown).Northern blot analysis
Total RNA from skeletal muscle of wild-type, mdx, nNOS-/- and mdx:nNOS-/- mice was isolated using RNAzol (Tel-Test, Friendswood, TX) according to the manufacturer's directions. The RNA (20 µg/lane) was fractionated by electrophoresis through a 1.25% agarose, 5% formaldehyde gel and overnight transfer to Biosbrane membranes by capillary action. Northern blot filters were hybridized with a radiolabeled cDNA probe encoding the first N-terminal 200 residues of rat nNOS. Hybridization was carried out using standard methods, and blots were exposed for autoradiography.
Pyruvate kinase assay
The activities of muscle-specific PK isozyme found in the blood serum were measured as previously documented (42). Blood was collected from the retroorbital sinus of 4- to 12-week-old mice and the serum was stored at -80°C prior to measurements. Data shown in all figures are from 8-week-old mice.
Evans blue assay
Evans blue dye (10 mg/ml) dissolved in phosphate-buffered saline (10 mM phosphate buffer, 150 mM NaCl, pH 7.4) was sterile filtered using a 0.2 µm pore filter. The Evans blue was injected into the tail vein of the mouse (0.1 ml/10 g body weight). The mice were euthanized 5-8 h after injection. Quadriceps muscle was prepared and observed under a Bio-Rad MRC-600 laser scanning confocal microscope as described previously (37,38). Digitized images were captured under identical conditions.
Histology
Transverse cross-sections (10 µm) of quadriceps and diaphragm were fixed for 10 min at room temperature with 10% freshly depolymerized paraformaldehyde. Muscle sections were washed briefly with TBS and stained with H&E. Sections were visualized with a Leitz diaplan light microscope, and digitized images were captured under identical conditions. To quantitate central nucleation, histological sections were photographed, and the percentage of centrally nucleated myofibers was determined by dividing the number of myofibers containing one or more centrally located nuclei by the total number of nucleated fibers. Myofibers with no nuclei in the plane were not counted. On average, 1200 myofibers were counted for both quadriceps and diaphragm.
ACKNOWLEDGEMENTS
R.H.C. is supported by the Robert G. Sampson postdoctoral research fellowship from the Muscular Dystrophy Association. V.S. is supported by a grant from Deutsche Forschungsgemeinschaft (DFG). This research was supported by grants from the US Public Health Service (NS33277 and NS01578 to T.M.D.; NS33142 to V.L.D.), the Muscular Dystrophy Association (K.P.C. and V.L.D.) and the National Institutes of Health (AR40864 to J.S.C.). K.P.C. is an Investigator of the Howard Hughes Medical Institute.
REFERENCES
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