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Human Molecular Genetics Advance Access originally published online on December 17, 2003
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Human Molecular Genetics, 2004, Vol. 13, No. 4 379-388
DOI: 10.1093/hmg/ddh037

Stimulation of calcineurin signaling attenuates the dystrophic pathology in mdx mice

Joe V. Chakkalakal1, Mary-Ann Harrison2, Salvatore Carbonetto3, Eva Chin4, Robin N. Michel2 and Bernard J. Jasmin1,5,*

1Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, Ontario Canada K1H 8M5, 2Neuromuscular Research Laboratory, Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada P3E 2C6, 3Centre for Neuroscience Research, Montreal General Hospital, McGill University, Montreal, Quebec, Canada H3G 1A4, 4Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT 06340 and 5Ottawa Health Research Institute, Molecular Medicine Program, Ottawa Hospital, General Campus, Ottawa, Ontario, Canada K1H 8L6

Received September 25, 2003; Revised November 25, 2003; Accepted December 3, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Utrophin has been studied extensively in recent years in an effort to find a cure for Duchenne muscular dystrophy. In this context, we previously showed that mice expressing enhanced muscle calcineurin activity (CnA*) displayed elevated levels of utrophin around their sarcolemma. In the present study, we therefore crossed CnA* mice with mdx mice to determine the suitability of elevating calcineurin activity in preventing the dystrophic pathology. Muscles from mdx/CnA* displayed increased nuclear localization of NFATc1 and a fiber type shift towards a slower phenotype. Measurements of utrophin levels in mdx/CnA* muscles revealed an ~2-fold induction in utrophin expression. Consistent with this induction, we also observed that members of the dystrophin-associated protein (DAP) complex were present at the sarcolemma of mdx/CnA* mouse muscle. This restoration of the utrophin–DAP complex was accompanied by significant reductions in the extent of central nucleation and fiber size variability. Importantly, assessment of myofiber sarcolemmal damage, as monitored by the intracellular presence of IgM and albumin as well as by Evans blue uptake in vivo, revealed a net amelioration of membrane integrity. Finally, immunofluorescence experiments using Mac-1 antibodies showed a reduction in the number of infiltrating immune cells in muscles from mdx/CnA* mice. These results show that elevated calcineurin activity attenuates the dystrophic pathology and thus provides an effective target for pharmacological intervention.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Over the last 15 years, a large number of laboratories have focused their efforts on trying to develop an effective therapy for Duchenne muscular dystrophy (DMD). Although several therapeutic strategies have been explored including gene transfer and cell therapy (1–4), there is currently no cure for this disease. One proposed strategy aimed at alleviating the debilitating symptoms of DMD involves the up-regulation of the autosomal homolog of dystrophin referred to as utrophin (5–7). In skeletal muscle, utrophin is known to accumulate preferentially at the post-synaptic membrane of the neuromuscular junction, although a low level of expression in extra-synaptic compartments can also be detected in slow myofibers (8,9). Studies using either transgenic mouse models or adenoviral-based gene transfer have shown that increasing utrophin levels can rescue muscle fibers by decreasing the extent of the dystrophic pathology in both canine and mouse models of DMD (10–12). The demonstration in these studies that utrophin can functionally compensate for the lack of dystrophin shows the importance of defining the mechanisms regulating utrophin in skeletal muscle. Elucidation of these mechanisms will identify specific molecular targets for designing pharmacological interventions aimed at increasing the abundance of utrophin throughout muscle fibers.

Within the last few years, several groups have shown that expression of utrophin can vary according to the state of differentiation and innervation of muscle cells (6). In this context, it has been shown that expression of utrophin is regulated at different levels since it appears to involve the contribution of transcriptional events (reviewed in 6,7) as well as post-transcriptional mechanisms (8,13–15). Recently, we reported that slower, high oxidative, muscle fibers contained significantly more utrophin in comparison to faster, more glycolytic counterparts (8,16, see also 9). Using a series of complementary approaches, we were also able to demonstrate that the increased expression of utrophin in slower, high oxidative, fibers was due to greater levels of the utrophin A isoform and involved the calcineurin/NFAT signaling cascade acting on the utrophin A promoter (16). Specifically, we showed in these recent studies that constitutively activated calcineurin and NFATc1 could directly transactivate the utrophin A promoter in myogenic cells in culture and in vivo (16).

Given these findings, we decided to examine whether stimulation of the calcineurin pathway could attenuate the dystrophic pathology in mdx mice. To this end, we crossed mice that expressed an activated form of calcineurin (CnA*) in their skeletal muscle with mdx mice. We then determined the levels of utrophin expression in these transgenic mdx mice and examined several indices that reflect the extent of the muscle pathology. Our results demonstrate that utrophin A expression is indeed under the control of calcineurin signaling and that increased utrophin A expression achieved under these conditions, can have substantial beneficial effects on dystrophic muscle fibers in vivo. These findings have significant implications in assessing the suitability of calcineurin activation as a therapeutic avenue for DMD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stimulation of the Calcineurin Pathway in mdx mouse muscle
Stimulation of calcineurin activity is known to result in the dephosphorylation of NFAT transcription factors leading to their translocation to nuclei where they can increase transcription of target genes (17,18). The CnA* mice used to generate mdx/CnA* mice have been shown previously to display increased levels of activated calcineurin mRNA and calcineurin activity (19). We thus initially performed a series of immunofluorescence experiments to ascertain that muscles from mdx/CnA* mice contain a higher number of NFAT-positive nuclei compared to muscles obtained from mdx mice. As shown in Figure 1A, staining for NFATc1 in mdx tissue appeared diffuse and cytoplasmic but greater than background labeling. In contrast, staining of NFATc1 in mdx/CnA* muscles displayed a preferential accumulation in myonuclei with a reduction in the diffuse cytoplasmic staining pattern seen in mdx tissue. This is expected given that expression of the transgene is under the control of the muscle-specific creatine kinase promoter. In these experiments, controls performed by omitting the primary antibody resulted in an absence of labeling (data not shown). Comparison of the average number of NFATc1-positive nuclei revealed a >2-fold increase in the number of myonuclei stained for NFATc1 in mdx/CnA* mice (Fig. 1B). These findings not only confirm the genotype of mdx/CnA* mice, but further our previous contentions (19,20) that expression of this transgene results in a functional stimulation of the calcineurin signaling pathway.



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  		Figure 1. Enhanced localization of NFATc1 in myonuclei of mdx/CnA* mice. Shown are representative photomicrographs of cross sections from mdx and mdx/CnA* muscles double labeled to detect NFATc1 and myonuclei with DAPI. * denotes NFATc1 labeling and corresponding nuclei (A). Quantification of the number of nuclei stained for NFATc1 shows that mdx/CnA* muscles contain considerably more positive nuclei in 20x cross-sectional views (B). The asterisk denotes significant difference from mdx (P<0.05), n=4 animals per group, three or four 20x sections from each animal (B). Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 
Since calcineurin signaling has been shown to be involved in controlling the slow oxidative myofiber program and, ultimately, the transcriptional activity of slower isoforms of myosin heavy chain (MyHC) II genes (17,18,20–23), we next sought to determine whether a fiber type shift occurred in mdx/CnA* muscles. Using isoform-specific antibodies in immunocytochemical experiments, we observed an increase in the percentage of MyHC IIa fibers which was accompanied by a parallel reduction in the percentage of MyHC IIb fibers in mdx/CnA* muscles (Fig. 2). These results demonstrate that muscle-specific expression of an activated calcineurin transgene in an mdx background is capable of inducing a shift towards a slower muscle phenotype.



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  		Figure 2. mdx/CnA* mice display shifts in fiber type towards a slower phenotype. Shown are representative photomicrographs of cross sections from mdx and mdx/CnA* muscles processed to detect MyHC IIb or MyHC IIa by immunocytochemsitry (A). Quantification revealed a significant induction in MyHC IIa expressing fibers and a reduction in MyHC IIb (B). The asterisk denotes significant difference from mdx (P<0.05), n=4 animals per group. Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 
Changes in utrophin expression in mdx/CnA* mouse muscle
Recently, we showed that levels of utrophin A in myofibers are positively correlated with the expression of slower myosin heavy chain isoforms (16). In the present study, immunoblotting experiments using muscle proteins revealed an ~2-fold induction in utrophin levels in muscles from mdx/CnA* versus mdx mice (Fig. 3A). In agreement with the immunoblot data, immunofluorescence experiments using three different antibodies showed an increased expression of utrophin (Figs 3B, 5A and B). These experiments further revealed that the increase in extra synaptic utrophin A occurred at the level of the sarcolemma and led to a more homogeneous pattern of utrophin expression amongst myofibers. RT–PCR analysis using RNA extracted from muscles isolated from mdx/CnA* mice revealed an ~2-fold induction (P<0.05) in utrophin transcripts (Fig. 4A). Using isoform-specific primers, we also demonstrated that this increase was accompanied by a corresponding induction in utrophin A mRNA with utrophin B transcripts remaining largely unchanged (Fig. 4).



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  		Figure 3. Increase in utrophin and utrophin A protein levels in mdx/CnA* muscle fibers. (A) Representative immunoblot for utrophin. Note the increased levels of utrophin in mdx/CnA* muscles. Immunofluorescence analyses demonstrated that muscles from mdx/CnA* mice express high levels of both utrophin and utrophin A at the sarcolemma of each individual fiber (B). The asterisk denotes significant difference from mdx (P<0.05), n=3 animals per group. Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 


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  		Figure 5. Increase in sarcolemmal expression of dystrophin associated proteins. Shown are representative photomicrographs of cross sections from muscles from mdx and mdx/CnA* animals processed to detect utrophin (A, B), ß-dystroglycan (C, D), syntrophin (E, F) and nNOS (G, H). Immunofluorescence analyses demonstrated that muscles from mdx/CnA* mice express high sarcolemmal levels of utrophin, ß-dystroglycan, syntrophin and neuronal nitric oxide synthase.

 


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  		Figure 4. Increase in utrophin and utrophin A mRNA expression in mdx/CnA* muscle fibers. (A) Examples of ethidium bromide stained gels showing PCR products from muscles from mdx and mdx/CnA* for total utrophin, utrophin A and utrophin B. (B) Note the increased levels of utrophin and utrophin A mdx/CnA* without any change in utrophin B levels. * denotes significant difference (P<0.05), n=6–8 animals per group. Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 
The induction in utrophin expression in mdx/CnA* mouse muscle prompted us to examine the localization of several components of the dystrophin-associated protein complex to determine whether they also accumulated at the sarcolemma of individual myofibers. As shown in Figure 5, immunofluorescence experiments demonstrated a greater presence of ß-dystroglycan, syntrophin and nNOS at the sarcolemma of mdx/CnA* muscles.

Improvement in morphological features of mdx/CnA* mouse muscle
The induction of utrophin and the restoration of dystrophin-associated proteins has been shown by others to have significant beneficial effects in mdx mouse muscles (see Introduction). To determine whether stimulation of the calcineurin signaling pathway could result in an attenuation of the dystrophic pathology, we examined several morphological parameters in muscles from mdx/CnA* mice. Hematoxylin and eosin staining of muscle sections revealed a healthier appearance of mdx/CnA* muscle fibers in comparison to mdx counterparts (Fig. 6A). An assessment of the percentage of central nucleation seen in mdx/CnA* muscles showed an ~20% reduction of this parameter (Fig. 6B), in agreement with other studies that used different paradigms to improve the dystrophic phenotype (11,24,25). Importantly, the reduction in the percentage of central-nucleated fibers reflects the enhanced survival of myofibers in mdx/CnA* mouse muscle (26).



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  		Figure 6. Reduction in central nucleation and fiber size variability in mdx/CnA* muscle fibers. Shown are representative photomicrographs of cross sections from mdx and mdx/CnA* muscles processed for hematoxylin and eosin staining (A). Myofibers from mdx/CnA* animals display a reduction in central nucleation (B). Similarly, mdx/CnA* myofibers displayed a reduction in fiber size variability (C). The asterisk denotes significant difference from mdx (P<0.05), n=4 animals per group. Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 
Among the several abnormal morphological indices, dystrophic muscle fibers are known to display greater variations in their cross-sectional area (27). Assessment of fiber size variability as determined by the averaged standard deviation of the cross sectional areas of myofibers, showed greater homogeneity in mdx/CnA* muscles (Fig. 6A and C). This correction in the morphological features of muscles from mdx/CnA* mice is similar to those reported by others using different paradigms and correlates favorably with the functional improvements seen in dystrophic muscle (24).

Improved sarcolemmal integrity of mdx/CnA* myofibers
We also determined the percentage of fibers staining positively for IgM (mass of 900 kDa) and albumin (mass of 65 kDa) taken as indices of membrane stability. Normally, these serum components are extracellular but sarcolemmal disruption, caused by the dystrophic pathology, confers membrane leakiness, thereby allowing serum proteins to penetrate myofibers (5,28). As shown in Figure 7, immunofluorescence experiments showed that approximately 10% of the fibers in muscles from 4- to 7-week-old mdx mice stained for IgM. Related studies using different paradigms have found a similar percentage of damaged fibers in mdx muscles (10,24,29). Transgenic expression of CnA* led to a significant reduction (P<0.05) in the percentage of IgM-positive fibers. Additional studies performed on older mice revealed that this beneficial effect was still present in 14-week-old mice. Similar results were also obtained in experiments using cytoplasmic labeling of albumin as a marker of membrane disruption (data not shown).



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  		Figure 7. Reduction in membrane lesions in mdx/CnA* mice. Cross sections from 4–7- and 14-week-old muscles demonstrate a reduction in the occurrence of cytoplasmic staining for IgM (A). Assesment of the percentage of fibers stained for IgM revealed an ~2-fold reduction in mdx/CnA* in comparison to age-matched mdx mice (B). The asterisk denotes significant difference from mdx (P<0.05), n=3–4 animals per group. Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 
To further examine the beneficial effects of utrophin up-regulation on the integrity of the sarcolemma in mdx/CnA* muscles, we injected mice with the small dye Evans blue and monitored the uptake of the dye in myofibers using fluorescence microscopy as described previously (28). As shown in Figure 8, there was a significant reduction in the levels of Evans blue uptake in myofibers of mdx/CnA* mice compared with mdx counterparts. In agreement with our data showing an improvement in membrane integrity in muscles from mdx/CnA* mice, serum levels of creatine kinase activity was also reduced by ~50% in these animals (data not shown).



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  		Figure 8. Reduction in the levels of Evans blue staining in skeletal muscles from mdx/CnA* mice. Cross sectional views of Evans blue dye-positive regions of both medial gastrocnemius (MG) and hamstring (Ham) muscles from mice intravenously injected demonstrate levels of dye infiltration. Assessment of Evans blue dye staining in damaged regions of mdx/CnA* hamstring and medial gastrocnemius muscles revealed an approximately 2- and 3-fold reductions, respectively, in comparison to age matched mdx mice. The asterisk denotes significant difference from mdx (P<0.05), n=4 animals per group. Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 
Attenuation of the inflammatory response in mdx/CnA* mouse muscle
Several recent studies demonstrate that the dystrophic process in mdx mouse muscle is accompanied by a marked inflammatory response (30,31). Therefore, in a last set of experiments, we determined whether muscles from mdx/CnA* mice contained fewer immune cells as identified by immunofluorescence experiments using Mac-1 antibodies which recognize several cells involved in the immune response. As shown in Figure 9, we noted a significant reduction in the levels of Mac-1 immunolabeling in muscles from mdx/CnA* mice. In these experiments, we observed similar results with different muscles including the hamstring, gastrocnemius and diaphragm (data not shown).



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  		Figure 9. Reduction in the levels of infiltrating immune cells in mdx/CnA* mice. Muscle cross sections demonstrate a reduction in the occurrence of Mac-1 staining, a marker for invading cells involved in the inflammatory response (A). Assessment of the levels of Mac-1 positive regions revealed an ~2-fold reduction in mdx/CnA* in comparison to age-matched mdx mice (B). The asterisk denotes significant difference from mdx (P<0.05), n=3–4 animals per group. Means±SEM are shown. Statistical analysis was conducted using Student's t-tests.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have previously shown utrophin levels to be positively correlated with the expression of slower MyHC isoforms and to be regulated, at least partially, by calcineurin/NFAT signaling (16). In the present study, we significantly extend these initial observations by showing that transgenic expression of an activated form of calcineurin in an mdx background results in an increase in the number of NFATc1-positive myonuclei. In turn, enhanced calcineurin signaling in an mdx background increases the expression of MyHC IIa and utrophin A (16,22,23). Within the last few years, numerous studies have shown the remarkable beneficial effects of increased levels of utrophin at the sarcolemma of mdx mice (5,10,11,32,33). Coherent with these earlier reports, we used a battery of different markers to show significant improvements in several morphological features normally associated with the dystrophic process. Taken together, the results of the present study clearly show the benefits of calcineurin activation in attenuating muscular dystrophy in mdx mice.

Normally, activation of calcineurin/NFAT gene expression involves sustained increases in the intracellular levels of calcium in a pattern similar to that seen in slower contracting muscles (34–36). Therefore, treatments designed to enhance calcineurin activity could potentially involve either the direct stimulation of calcineurin activity or sustained elevations in intracellular calcium levels. Although still controversial, abnormal calcium handling has been postulated to be an important factor resulting in the progression of the dystrophic pathology (37). Such disease progression via impaired calcium kinetics may occur as a result of alterations in the normal activation of the calcineurin/NFATc1 signaling pathway. In this context, altered calcium levels can also result in the activation of other transcriptional networks including those involving the stress-activated MAPK JNK1 pathway (38). Interestingly, the activation of this pathway has been shown to be associated with the progression of the dystrophic pathology and the higher JNK1 activity seen in dystrophic muscles can result in increased interactions with NFATc1, thereby displacing it from the nucleus (39). Therefore, imbalances in calcium homeostasis in dystrophic muscles may result in the activation of pathways that antagonize calcineurin/NFAT signaling resulting in impaired gene expression eventually triggering muscle necrosis/apoptosis.

Although it appears likely, based on our previous (16) and current findings, that NFATc1 is directly involved in the transcriptional regulation of utrophin A, it is not possible at this point to completely rule out the contribution of other transcription factors. For instance, MEF2 and SRF are factors known to be capable of responding to various nerve-dependent calcium-regulated signals and have been shown to be implicated in the expression of fiber type-specific genes and cytokine production in lymphocytes together with NFAT (19,40,41). Given that mdx myofibers have elevated levels of intracellular calcium (37), it is conceivable that MEF 2 and SRF or other, yet to be identified, calcium-regulated transcription factors, act alone or in concert with NFATc1 to stimulate the expression of MyHC IIa and utrophin A in mdx/CnA* muscles.

Pharmacological strategies enhancing calcineurin activity in combination with other proposed therapies could thus provide an effective treatment for counteracting the devastating symptoms caused by dystrophin deficiency. In our system, the ability of enhanced calcineurin activity to potentiate endogenous utrophin levels coupled with the potential to attenuate the inflammatory response may promote survival of dystrophin-deficient fibers. In this context, calcineurin/NFAT signaling has been shown to regulate the expression of IL-4 in myogenic cells which promotes the fusion of myoblasts with myotubes (42). This apparent paradox, whereby enhanced calcineurin activity results in a reduction in the number of infiltrating immune cells in muscle (this study) while stimulating cytokine production (42), may be reconciled if we consider the selectivity of NFAT factors in preferentially regulating the expression of specific cytokines (43). Alternatively, it appears likely that the role of IL-4 in promoting myoblast fusion may be most relevant during the early stages of myogenesis in our mdx/CnA* mice, whereas in mature muscles from these mice, the induction of utrophin A and of the dystrophin-associated proteins preserves the integrity of existing fibers and hence results in an attenuation of the inflammatory response. It will be interesting in future studies to examine whether myoblast fusion proceeds normally in these mice and whether cytokine production is affected by enhanced calcineurin activation. In any case, the ability of calcineurin to both elevate utrophin expression and attenuate the immune response in mdx muscles is relevant particularly towards the design of future therapies involving the use of the immunosuppressants and calcineurin inhibitors, cyclosporine A and FK506, that could have deleterious effects on DMD patients by decreasing expression of utrophin in their muscles (16).

Through these studies (8,16), we have effectively shown an association between utrophin A expression with both calcineurin signaling and the slower oxidative myogenic program that can serve as the basis for designing a therapeutic strategy for the treatment of DMD. This is particularly relevant considering that fast MyHC IIb fibers are preferentially affected in DMD patients (44). In the present study, we show beneficial effects stemming from a fiber type shift towards a slower more oxidative phenotype in dystrophin-deficient muscles. Interestingly, such a shift in fiber type towards a slower more oxidative phenotype has previously been shown to result in corrections of the dystrophic pathology. Specifically, mdx mice with ADR (arrested development of righting response) mutations resulting in myotonia and enhanced muscle activity, show reductions in various markers of the dystrophic pathology with a corresponding shift in muscle fiber type to a more oxidative phenotype (45,46). Based on the foregoing discussion, it is tempting to speculate that the improved phenotype of ADR-mdx mice is a result of increased utrophin expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of CnA* Tg/mdx mice
Transgenic mice expressing a constitutively active form of calcineurin have been characterized (19). Female mdx mice were paired with male CnA* transgenic mice, resulting in male dystrophic pups. Male pups that expressed the transgene were identified through PCR screening of genomic DNA extracted from tail tissue. The absence of dystrophin from these mice was confirmed by immunolabeling of muscle sections using dystrophin antibodies (Santa-Cruz Biotech). Male pups that did not incorporate the CnA* transgene were used as dystrophic controls.

Immunofluorescence
Detection of sarcolemmal utrophin and utrophin A were conducted as described (16). Briefly, cross sections were incubated with H-300 polyclonal antibodies recognizing the C-terminus of utrophin (Santa-Cruz Biotech), NCL-DRP2 (Novocastra) recognizing the N-terminus of utrophin or with previously characterized antibodies generated in our laboratory specific for residues in the N-termini of utrophin A (16). Detection of sarcolemmal levels of dystrophin-associated proteins was done using the following antibodies: 1351 recognizing syntrophin, anti-nNOS (Zymed) and ß-dystroglycan.

The ß-dystroglycan antibody was made in rabbits to a synthetic peptide encompassing the last 15 amino acids. Antiserum was subsequently purified on a peptide affinity column. Western blotting using microsomes from rat muscle or brain homogenates, or following immunoprecipitation from Triton X-100 extracts and fractionation by SDS–PAGE, reveals a specific band of 43 kDa. Affinity chromatography with this antibody followed by electrophoresis and mass spectrometry yields an unambiguous match with the sequence of rat ß-dystroglycan.

Assessment of NFATc1 nuclear localization was performed in a similar manner using polyclonal NFATc1 antibodies (Santa-Cruz Biotech). The number of myonuclei positively staining for NFATc1 was counted in three or four 20x cross sectional views of myofibers from the midbelly of muscles from three or four animals per group. These values were then averaged and compared between samples. Sections were viewed with a Zeiss Axioskop-2 microscope with 10x and 20x plan-neofluor objectives and imaged using a digital camera (DVK). The images were captured and analyzed with Northern Eclipse software and Adobe Photoshop.

Western blot analysis
Protein extraction and quantitation was conducted as described in detail elsewhere (47). Briefly, skeletal muscles were harvested and crushed in extraction buffer (pH 6.8) consisting of 5 mM Tris, 10% SDS, 0.2 M DTT, 1 mM EDTA, and protease inhibitor (Roche) subsequently boiled and centrifuged for 10 min at 10 000g. Supernatant was separated with one part used for determination of protein concentration using a BCA protein assay (Pierce) and the other for electrophoresis analysis. Total protein for each sample (100 µg) was diluted in a loading dye consisting of Tris, 10% SDS, 2-mercaptoethanol and bromophenol blue and then separated on a 6% SDS–PAGE gel with 5% stacking at 75V for 6 h. The gels were then transferred onto nitrocellulose membranes (BioRad) overnight at 4°C. Membranes were then stained with ponceau red to ensure equal loading of protein sample. Blots were incubated with the NCL-DRP2 primary antibody, washed thoroughly and incubated with peroxidase-labeled, anti-mouse secondary antibodies (Jackson Laboratories). The presence of utrophin was detected using ECL reagents (Perkin-Elmer). The utrophin bands were visualized and their intensity determined using image analysis software (Kodak Image Analysis Software).

RNA extraction and quantitative RT–PCR
Total RNA was extracted using TriPure (Boehringer Mannheim) as recommended by the manufacturer. Quantitative RT–PCR was carried out to determine the relative abundance of total utrophin (both A and B together), utrophin A and utrophin B using primers that selectively amplify total utrophin, utrophin A, utrophin B and S12 rRNA (8,16). Cycling conditions were optimized for all targets. In all these assays, negative controls consisted of RT mixtures in which total RNA was replaced with RNase-free water. PCR products were first visualized on 1% agarose gels containing ethidium bromide (Sigma-Aldrich). For quantitative measurements, PCR products were separated and visualized on agarose gels containing the fluorescent dye Vistra Green (Amersham). The labeling intensity of the PCR product which is linearly related to the abundance of cDNAs, was quantified using a Storm PhosphorImager (Molecular Dynamics). Values obtained for utrophin, utrophin A and utrophin B were standardized relative to the amount of S12 rRNA present in the same sample. For all quantitative measurements, PCR experiments were performed during the linear range of amplification as described and shown in detail in our previous work (48,49).

Muscle fiber typing
Muscle cross-sections were incubated for 1 h with primary antibodies recognizing either MyHC IIa or IIb (16), washed with PBS and incubated with affinity purified goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (Jackson Laboratories Inc.). Following several washes with PBS, the sections were incubated for 10 min with diaminobenzidene (DAB) media made up of 3'3-diaminobenzidine, deionised water, 2x buffer (0.2 M Tris–HCl pH 7.6) and 1% hydrogen peroxide (Sigma-Aldrich). The sections were subsequently washed with running tap water, dehydrated using alcohol solutions, cleared with xylene and mounted with permount. Using light microscopy, the percentage of fibers staining for MyHC IIa or IIb were determined from three or four 20x cross sectional views of myofibers from the midbelly of muscles from three or four animals per group. The percentage for both MyHC isoforms were then averaged and compared between samples.

Assessment of muscle fiber size and central nucleation
Cross-sections of muscles were stained with hematoxylin and eosin, dehydrated through a series of alcohol solutions, cleared with xylene and mounted using permount (Fisher Scientific). The extent of regeneration occurring in muscles was determined by comparing the averaged percentage of central nuclei between samples. Variability of muscle fiber size was determined because an increase in this value has been shown to be a pathological feature of mdx muscles (24,27). Cross sectional areas for each individual fiber were measured using Northern Eclipse Software. Variability in fiber size was determined by averaging the standard deviations from three or four 20x cross sectional views of myofibers from the midbelly of muscles from three or four animals per group and comparing this value between samples.

Assessment of muscle membrane damage
Sarcolemmal integrity was assessed by evaluating the levels of cytoplasmic staining within muscle fibers for the normally membrane impermeable markers IgM and albumin (28). Muscle cross-sections were incubated for 1 h with fluorescein-conjugated IgM anti-mouse secondary antibodies (Sigma-Aldrich), or with and anti-albumin antibody subsequently revealed in this case with an AlexaFluor 594 secondary antibody (Molecular Probes). Sections were viewed using fluorescent microscopy to identify injured fibers. The percentage of injured fibers was then determined from three or four 10x cross sectional views of myofibers from the midbelly of muscles from three or four animals per group. The percentage of damaged fibers was then averaged and compared between samples.

Evans blue uptake and staining
Evans blue dye injections were performed as described elsewhere (28). Briefly, 50 µl/10 mg of b.w. of Evans blue dye was injected intravenously. Six to 12 h later, muscles were isolated and frozen in melting isopentane. Prior to observing the sections under the microscope, muscle sections were incubated in ice-cold acetone for 10 min, washed three times for 10 min with PBS and mounted with Vectashield mounting medium (Vector Laboratories). The presence of Evan blue dye in myofibers was observed under fluorescence microscopy and the intensity level was determined using northern Eclipse software by converting images to 8 bit gray scale and determining the total and average gray intensity taken as a measure of Evans blue dye fluorescence in the entire area of each 10x cross section. The average gray intensity was then compared between groups with n=4 animals per group. Three 10x cross sections per animal was used to obtain average intensities.

Assessment of inflammation
The extent of inflammation was determined by monitoring the presence of Mac-1 immunoreactivity using a commercially available antibody (BD Biosciences Pharmigen). Levels of Mac-1 staining were determined using northern eclipse software by converting images to 8 bit gray scale and determining the total and average gray intensity taken as a measure of Mac-1 labeling in the entire area of each 10x cross-section. The average gray intensity was then compared between groups (n=3–4 animals per group) using three 10x cross sections per animal.


   ACKNOWLEDGEMENTS
 
We thank John Lunde for technical assistance, Dr R.S. Williams for assistance with generating CnA* mice, C. Blomme and L. Brosseau for breeding and maintaining these lines, N. Paquette and N. Polasek for help with the Evans blue dye experiments, and Dr S.C. Froehner for the syntrophin antibody. This work was supported by grants from the Canadian Institutes of Health Research (CIHR to B.J.J.), the CIHR/Muscular Dystrophy Association of Canada/Amyotrophic Lateral Sclerosis Society Partnership (to R.N.M.), the Muscular Dystrophy Association (to B.J.J.), L'Association Francaise contre les Myopathies (to B.J.J.), and the Natural Sciences and Engineering Research Council of Canada (to R.N.M.). J.V.C. is supported by a studentship from the CIHR. B.J.J. is a CIHR Investigator.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. Tel: +1 6135625800, ext. 8383; Fax: +1 6135625636; Email: jasmin{at}uottawa.ca


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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