Introduction

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive muscle wasting, and occurs in 1 out of 3,500 live male births. It is caused by mutations in the gene encoding the cytoskeletal protein dystrophin.^1,2,3 The absence of dystrophin results in compromised sarcolemmal integrity, rendering myofibers prone to contraction-induced injury. DMD is not amenable to treatment with existing medical options. Therefore, contemporary approaches are focused on symptom management.⁴

The milder and rarer allelic form of the disease, Becker muscular dystrophy, is frequently caused by mutations that result in the expression of an internally truncated dystrophin protein with reduced functionality. A review of these Becker muscular dystrophy mutations and the resultant genotype–phenotype relationships has helped delineate the essential regions and conformations necessary for dystrophin stability and function. These findings have been extended in studies of transgenic mice expressing modified dystrophin constructs, so as to elucidate the importance of the physical links provided at either end of the dystrophin protein.^1,5 At the cytoplasmic face of the sarcolemma, dystrophin plays a pivotal structural role in nucleating the assembly of a dynamic multi-protein complex, referred to as the dystrophin–glycoprotein complex (DGC). The absence of dystrophin results in a considerable reduction in DGC components,^5,6,7 and also produces membrane destabilization and membrane permeability defects, leading to increased build-up of intracellular calcium and consequent myofiber degeneration.⁸

Recombinant adeno-associated viral (rAAV) vectors are emerging as potential gene transfer vehicles in the treatment of neuromuscular disorders because of their ability to transduce the vast majority of the mouse striated musculature with a single administration. The additional advantages of these vectors include the existence of several muscle-tropic serotypes, and their inherent capacity for persistent transgene expression in postmitotic cells.^9,10,11 However, a potential complication of gene therapy in many DMD patients, particularly in those with genomic deletions, is a possibility that dystrophin expressed from exogenously administered transgenes could be perceived by the host as a "neoantigen," evoking destruction of transduced myofibers by immune effector cells. Indeed, previous transplantation studies in DMD patients have demonstrated that dystrophin can be recognized as nonself by the immune system leading to the generation of specific antibodies.^12,13,14

Utrophin, an autosomal paralog of dystrophin, was originally named for its ubiquitous expression and high degree of sequence similarity with dystrophin, particularly in regard to the critically important protein binding domains located near the amino and carboxy termini.^15,16 This observation raised the possibility of utrophin being used as a therapeutic replacement for dystrophin in DMD. Indeed, investigators have shown that utrophin can compensate for the lack of dystrophin in many circumstances.^{11,17,18,19,20,21} Nonetheless, the functionality of utrophins that are small enough to be delivered by rAAV vectors has not been investigated. We hypothesized that rAAV6-mediated delivery of a microutrophin (R4–R21/CT) to the mdx:utrn^-/- double-knockout mouse model would permit the recruitment of DGC component proteins, leading to improved membrane stabilization, enhancement of contractile performance, and a halt in the progression of the dystrophic phenotype. The mdx:utrn^-/- mouse model was chosen because it displays a highly dystrophic phenotype, in contrast with utrn^-/- mice that show almost no pathology, and dystrophin-deficient mdx mice in which extrasynaptic expression of utrophin contributes to a milder phenotype. Consequently, rAAV6/microutrophin was administered to 1-month-old mdx:utrn^-/- mice by tail vein injection. The most significant finding of this study was that microutrophin is highly functional in the mdx:utrn^-/- mouse model, suggesting that it could alter the course of DMD to one that more closely resembles a very mild Becker muscular dystrophy phenotype.

Results

Generation of an rAAV vector expressing microutrophin

An rAAV vector pseudotyped with serotype six capsids was used for gene transfer of a murine codon-optimized microutrophin under the transcriptional control of a minimal cytomegalovirus enhancer plus promoter, and was modeled on the lines of the previously described R4–R21/CT microdystrophin construct.22 This complementary DNA encodes the amino-terminal actin-binding domain, spectrin-like repeats 1–3, hinge 2, the final (22nd) spectrin-like repeat, and the dystroglycan-binding domain. The resulting microutrophin therefore lacks sequences encoding spectrin-like repeats 4 through 21 (Figure 1). Sequences encoding the carboxy-terminal domain within dystrophin have also been eliminated, because those regions have been shown to be nonessential in mice.^22,23 Production of the microutrophin protein was demonstrated by western analysis of a viral lysate preparation from 293D cells utilizing an antibody specific for the amino-terminus of utrophin (data not shown).

Figure 1.

Schematic view of the structural domains in dystrophin, utrophin, and microutrophin. Each of the three proteins contains an N-terminal actin-binding domain (ABD1). Within the central region of both dystrophin and utrophin there are a number of repeating units similar to the coiled-coil repeats of spectrin, and these are interspersed with four hinge regions (H1–H4). The central region of dystrophin uniquely contains a second actin-binding domain (ABD2) which is not found in utrophin (basically charged repeats are in white). The carboxy-terminal portions of both dystrophin and utrophin contain a cysteine-rich (CR) domain and a carboxy-terminal domain. Both domains contain numerous protein interaction motifs for components of the dystrophin–glycoprotein complex. NT, amino-terminus; R1–24, spectrin-like repeats 1–24; -DgBD, -dystroglycan-binding domain, composed of a WW domain (WW), two EF hand-like domains (EF) and a ZZ zinc finger domain (ZZ).⁵⁰

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Intravenous administration of rAAV6/CMV-microutrophin results in persistent expression of microutrophin in adult mdx:utrn^-/- mice

Four-week-old mdx:utrn^-/- mice were administered 3 10¹² vector genomes of rAAV6/CMV-microutrophin by tail vein injection. Cryosections of muscle were stained 4 months after vector administration with utrophin polyclonal antibodies that recognize the amino-terminus of the protein (Figure 2). The vast majority of the myofibers stained positive for microutrophin at the sarcolemma in all the muscles examined (gastrocnemius, quadriceps, tibialis anterior (TA), soleus, heart, and diaphragm). This sarcolemma localization was in sharp contrast to results in age-matched wild-type control mice in which myofiber utrophin staining was found only at the neuromuscular and myotendinous junctions, and in untreated 12-week-old mdx:utrn^-/- mice that do not express utrophin (Figures 2 and 3). In view of the natural function and localization of utrophin at the myotendinous junction, we performed immunostaining followed by confocal microscopy on longitudinal cryosections of gastrocnemius muscles from wild-type, mdx:utrn^-/- mice, and treated mdx:utrn^-/- mice (Figure 3). In mice that had received rAAV6/CMV-microutrophin, there was a qualitatively high degree of enrichment at the myotendinous junction, showing that microutrophin can localize to the myotendinous junction, as does full-length utrophin.

Figure 2.

Microutrophin properly localizes to the sarcolemma in striated muscle. Muscle cryosections from wild-type (wt), untreated mdx:utrn^-/- and treated mdx:utrn^-/- (t-mdx:utrn^-/-) mice were immunofluorescently stained with anti-utrophin polyclonal antibodies. Green fluorescence represents utrophin staining, red represents B2 laminin, blue represents nuclei staining, and blue arrows show utrophin staining (yellow) in wild-type mice. Bar = 100 m.

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Figure 3.

Microutrophin is enriched at the myotendinous junction in treated mdx:utrn^-/- mice. Immunofluorescent staining was performed on longitudinally cryosectioned gastrocnemius muscles using a NH₂-terminal utrophin antibody, and assessed using confocal microscopy. Representative photographs are shown of muscles from wild-type (wt), mdx:utrn^-/-, and t-mdx:utrn^-/- mice. The tendon abutting the myofiber is shown in the left portion of each panel and is denoted by an asterisk (^*). (Bar = 30 m).

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Microutrophin gene transfer alleviates the dystrophic phenotype in mdx:utrn^-/- mice

In an attempt to model therapeutic interventions in advanced cases of DMD, we assessed the properties of the hindlimb muscles of treated mdx:utrn^-/- mice. An analysis of muscle cryosections demonstrated that microutrophin recruits representative DGC components (-dystroglycan, -sarcoglycan, -dystrobrevin-2, and 1-syntrophin) to the sarcolemma, but it does not localize neuronal nitric oxide synthase (nNOS) (Figure 4). The absence of nNOS recruitment is consistent with results of studies using mini and microdystrophin constructs.^24,25

Figure 4.

Expression of microutrophin restores the dystrophin–glycoprotein complex, with the exception of nNOS, to the sarcolemma. Tibialis anterior muscles from wild-type (wt), mdx:utrn^-/-, and t-mdx:utrn^-/- mice were cryosectioned, and immunoflourescent staining for utrophin was performed. (-Dg, -dystroglycan; -Sg, -sarcoglycan; Db2, -dystrobrevin-2; 1Syn, 1-syntrophin; nNOS, neuronal nitric oxide synthase). (Bar = 100 m).

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In contrast to mdx mice, mdx:utrn^-/- mice experience a considerable reduction in body mass because of progressive muscle wasting that begins at about 6 weeks of age. By 12 weeks of age the average body mass of treated mdx:utrn^-/- mice was found to be approximately twice that of untreated mdx:utrn^-/- mice (Figure 5a). In addition, untreated mdx:utrn^-/- mice display kyphosis from the degenerative process of the disease, and also decreased ambulation. In contrast, age-matched treated mdx:utrn^-/- mice demonstrate minimal kyphosis and sufficient muscularity to participate actively on a voluntary running wheel (Supplementary Video S1). We have previously noted that rAAV6 vectors are extremely efficient at transducing cardiac muscle,^9,26 and treated mdx:utrn^-/- mice display complete transduction of the myocardium. Heart mass was likewise increased to levels more closely resembling those in wild-type mice (twice those in untreated mdx:utrn^-/- mice) (Figure 5e). In addition, treated mdx:utrn^-/- mice experienced an 60% reduction in serum creatine kinase levels (P < 0.001) when compared with untreated mdx:utrn^-/- mice (Figure 5f), consistent with a whole-body reduction in muscle degeneration.

Figure 5.

Expression of microutrophin compensates for the lack of utrophin and dystrophin. (a) Increase in body mass over time in mdx:utrn^-/- mice (N = 5) that received rAAV6/microutrophin, relative to untreated control wt and mdx:utrn^-/- mice (by week 14 all of the untreated mdx:utrn^-/- mice had died). (b) Ratio of tibialis anterior muscle mass to body mass (TA:BM). (c) t-mdx:utrn^-/- mice show increased musculature and minimal kyphosis and joint contractures when compared with (d) untreated mdx:utrn^-/- mice (not to scale) (e) t-mdx:utrn^-/- mice display increased heart mass when compared with control mdx:utrn^-/- (12 weeks) and wild-type (5 months) mice. (f) Serum creatine kinase levels are reduced by 60% in t-mdx:utrn^-/- mice. ^*P < 0.05 and ^***P < 0.001 when compared with control mdx:utrn^-/- mice; bars represent SEM.

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Histologically, mdx:utrn^-/- muscles display large inflammatory infiltrates and a high percentage of centrally nucleated myofibers which are often quite small in diameter, reflecting a continuum of the degeneration–regeneration process. Hematoxylin and eosin staining revealed a considerable reduction in inflammatory infiltrate (Figure 6a) and a dramatic reduction in the percentage of centrally nucleated myofibers (26.5%) when compared with untreated gender-matched mice of 14 weeks of age (84.2%) (Figure 6b). In treated mdx:utrn^-/- mice, the expression of microutrophin resulted in a shift in myofiber size, with considerably larger fibers being present in addition to a reduction in the small-fiber population (Figure 6c).

Figure 6.

Microutrophin improves muscle histopathology in mdx:utrn^-/- mice. (a) The upper panel shows immunoflourescent staining for utrophin (green), B2 laminin (red), and nuclei (blue). The lower panel shows adjacent sections stained with hemotoxylin and eosin. The green boxes indicate the same muscle fibers in the upper and lower panels. Blue arrows show utrophin staining localized at the neuromuscular junction in wild-type muscles. (Bar = 100 m). (b) Percentage of myofibers with central nucleation (N = 1,500 myofibers/cohort). (c) Box plots showing variance of the muscle fiber cross-sectional area (N = 800 myofibers/cohort). Boxes represent the middle quartiles from the 25th to the 75th percentiles, and the bar demonstrates the high and low values. ^***P < 0.001 when compared with control mdx:utrn^-/- mice, and bars represent SEM.

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Microutrophin increases force production in limb muscles of mdx:utrn^-/- mice

When compared with untreated mdx:utrn^-/- mice, there was a significant (N = 5; P < 0.001) increase in force-producing capacity in the TA muscles of treated mdx:utrn^-/- mice, reaching 93% of wild-type levels (Figure 7a). However, after normalizing the net force production to the cross-sectional area of the muscle (specific force), the benefit was less dramatic (P < 0.01), achieving 80% of the levels in wild-type muscles (Figure 7a). We also measured the resistance to eccentric-contraction-induced injury in the various cohorts. The protocol applied resulted in stressing the muscles with a series of 5% incremental stretches above the optimal length for force generation during in situ stimulated muscle contractions. Microutrophin-expressing muscles were protected from contraction-induced injury as were wild-type muscles, up to a 20% strain. For untreated mdx:utrn^-/- mice, this same level of strain resulted in a reduction of initial muscle contractile force to the extent of 46% (Figure 7c,d). There were significant differences between treated and untreated mdx:utrn^-/- mice (P < 0.001) at up to a 30% increase in optimal length (Figure 7c). No significant differences were found in respect of lengthening contractions within normal physiological levels of strain (i.e., <25%) when compared with wild-type control mice (Figure 7c,d). These results indicate that, in the mdx:utrn^-/- mouse model, the expression of microutrophin provides a significant functional benefit to dystrophic muscles even when treatment is initiated after the onset of histopathological degeneration.

Figure 7.

Intravenous administration of rAAV6/microutrophin to mdx:utrn^-/- mice results in increased muscle function. The TA muscles of untreated mdx:utrn^-/- mice exhibit (a) reduced force-producing capacity, (b) force generation per cross-sectional area, i.e., specific force, and (c,d) an increased susceptibility to eccentric contraction-induced injury. (c) The absolute force developed following strains of the indicated percentages beyond the length that is optimal for force development. (d) The percentage of force development relative to initial values prior to mechanical strain. By comparison, t-mdx:utrn^-/- mouse TA muscles exhibited significantly higher absolute force production (a) and force per cross-sectional area (b) (^**P < 0.01 and ^***P < 0.001 when compared to 3-month-old control mdx:utrn^-/- mice), and they also demonstrated markedly increased resistance to mechanical strain injury (c,d). When comparing wild-type mice with untreated mdx:utrn^-/- mice, no significant differences were found at lengthening contractions that are within normal physiological levels of strain (i.e., <30%). However, t-mdx:utrn^-/- mice showed a significant difference at up to 40% increase in optimal length in comparison with untreated mdx:utrn^-/- mice (c,d). (Bars represent SEM; ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).

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Discussion

Somatic gene transfer technology is one of a number of approaches that are being developed for the treatment of DMD. Numerous studies have demonstrated the potential of rAAV as a vector for gene transfer. rAAV has been shown to be not only a valuable research tool for the delivery of transgenes and gene-targeting cassettes such as those encoding shRNA²⁷ or zinc finger²⁸ technologies, but also an important reagent in numerous human clinical trials. Undoubtedly, there are issues of risk that should be considered, not only in respect of individual vectors but also as regards the inherent characteristics of specific disease processes. An understanding of the factors involved in immune activation following gene transfer is of great clinical significance, and much progress has been made in this area.²⁹ In many DMD patients there is little or no dystrophin expression, and in others the residual truncated protein produced in revertant myofibers lacks significant portions of the full amino acid sequence. Exogenous dystrophin production resulting from gene therapy protocols could, in theory, elicit a destructive cellular immune response against transduced muscle fibers. Using a therapeutic transgene based on a protein normally expressed in muscles of DMD patients, such as utrophin, could be an important approach to improving the efficiency and efficacy of gene therapy for DMD.

Utrophin is structurally similar to dystrophin, with the expression normally being restricted to the neuromuscular and myotendinous junctions within adult skeletal muscle. As a strategy to overcome the potential risk of an immune response to dystrophin, a microutrophin cassette was developed that can be packaged within rAAV vectors. The goal of this study was to characterize whether microutrophin could display functional efficacy in the mdx:utrn^-/- mouse model. In contrast to dystrophin-deficient mdx mice, whose mild skeletal muscle pathology is attributable, at least in part, to compensatory overexpression of utrophin at the sarcolemma,³⁰ mdx:utrn^-/- mice display an earlier onset of dystrophy, more extensive muscle wasting, significant weight loss, contractures of the joints, and a considerably shortened lifespan.^20,31 A number of investigators have previously shown that utrophin can compensate for the lack of dystrophin in many circumstances, including through adenoviral delivery or in transgenic mice.^{11,17,18,19,20,21} Further evidence for a compensatory role for utrophin derives from studies in the distal toe and extraocular muscles, which are largely spared from dystrophic degeneration, and where utrophin expression is normally high.^32,33,34 There is, therefore, a large body of data supporting the hypothesis that utrophin can functionally compensate for the absence of dystrophin in striated muscles. Indeed, upregulation of the utrophin gene has been actively pursued as a pharmacological target, as a potential therapy for DMD.^35,36 Here we demonstrate that intravenous injection of an rAAV6/microutrophin transgene can improve the morphology and function of muscles in the severely dystrophic mdx:utrn^-/- mouse model.

The DGC is distributed across the sarcolemma of striated muscle, and biochemical studies indicate that it provides a structural link between the extracellular matrix and the intracellular actin cytoskeleton.³⁷ Structurally, the DGC is thought to assemble in a dynamic manner in response to stress or contractile perturbation.³⁸ In the absence of dystrophin, there is extensive loss of sarcolemmal localization of DGC component proteins in both DMD patients and in mdx:utrn^-/- mice. As with dystrophin, utrophin interacts with this large oligomeric complex.³⁹ Previous experiments have shown the binding of dystrophin and utrophin to actin filaments to be noncompetitive, though with similar affinities.⁴⁰ Also, mutagenesis experiments have demonstrated that dystrophin and utrophin exhibit different modes of contact with the transmembrane protein -dystroglycan.⁴¹ Despite these differences, we observed that microutrophin readily recruits and localizes transmembrane and cytoplasmic components of the DGC (Figure 4). The level of staining for the transmembrane components appeared to be qualitatively higher than wild-type staining. This observation is consistent with results from transgenic studies using the larger mini-utrophin, where it was proposed that dystrophin (or utrophin) glycoprotein complexes are limited by the amount of dystrophin or utrophin present in the myofibers.²¹ However, nNOS localization to the sarcolemma was not restored by expression of microutrophin, consistent with previous reports showing that neither micro- nor minidystrophins could localize nNOS to the sarcolemma.^24,42

During muscle contraction, the integrity of the myotendinous junction is physiologically vital for the transmission of force from muscle to tendon. As the muscle transitions into tendon, the increasing proportion of connective tissue establishes a stiffness gradient, thereby preventing injury at the myotendinous junction. The high transverse stiffness of the muscle at the myotendinous junction and inextensibility of the central tendon combine to eliminate stress concentrations at the muscle–tendon interface, and may play an important role in preventing injury.⁴³ Consistent with the natural distribution and function of utrophin at the myotendinous junction, we found that microutrophin was highly enriched at this anatomical location (Figure 3). However, in contrast to the neuromuscular- and myotendinous junction–restricted localization of endogenous utrophin found in wild-type mice, the microutrophin delivered by rAAV was readily targeted to the sarcolemma. The uniform expression pattern of microutrophin likely reflects the use of the CMV promoter, which lacks N-boxes that can restrict transcription to nuclei adjacent to neuromuscular junctions and also lacks most of the untranslated regions that can influence mRNA trafficking and/or stability.^44,45,46 The extrasynaptic localization of microutrophin is vital in the consideration of a possible treatment for DMD.

In order to evaluate the benefit of treatment on a representative limb muscle, we chose to assess the morphological and functional properties of TA muscles in treated mdx:utrn^-/- mice. Delivery of microutrophin was found to result in a vast reduction in mononuclear cell infiltrates. We also observed the presence of myofibers of a more consistent, albeit larger, size, in addition to a reduction in the number of small-caliber fibers. This shift toward myofiber populations of larger size could be the result of an architectural specialization that may be influenced by activity patterns over the course of the treatment. In all the muscles that were tested, we found similar and significant reductions in the prevalence of regenerating myofibers in treated mdx:utrn^-/- mice when compared with untreated ones. A further encouraging finding from our study was a highly significant improvement in the contractile properties of the treated TA muscles (Figure 7). Previous studies of dystrophin-deficient mdx mice have shown that muscles from these animals display numerous centrally nucleated myofibers, ongoing degeneration/regeneration, a significant loss in force generating capacity, and a susceptibility to contraction-induced injury.²² Our results show that delivery of microutrophin to the muscles of mdx:utrn^-/- mice leads to a striking return to normalcy of all these parameters, showing that treated muscles are physiologically closer to wild-type muscles than those of mdx mice.²² Mice lacking only utrophin but not dystrophin display no signs of dystrophy or weakness. Our results with microutrophin are therefore in agreement with previous studies using mini- and full-length utrophin, namely, that utrophin constructs are able to compensate for the lack of both dystrophin and utrophin.^17,21

Taken together, our results show that the delivery of a highly truncated microutrophin cassette using rAAV6 vectors is able to achieve widespread and persistent transduction of the striated musculature in mdx:utrn^-/- mice. Microutrophin expression resulted in a significant amelioration of the histopathological features in the muscles of mdx:utrn^-/- mice and led to a nearly complete recovery of contractile properties. While we did not perform a detailed lifespan analysis, the data showed a significant extension of lifespan in treated mdx:utrn^-/- mice (Figure 5a, and data not shown). Overall, the phenotypic consequences of microutrophin delivery were broadly similar to those we previously observed following rAAV-mediated delivery of a structurally similar microdystrophin.⁴⁷ Both microutrophin and microdystrophin resulted in significant and similar reductions in centrally nucleated fibers, increased resistance to contraction-induced injury, decreased serum levels of creatine kinase, and a stoppage of ongoing myofiber necrosis.⁴⁷ In moving toward clinical applications of rAAV gene transfer, the potential immune responses of the host against both the viral capsid and the therapeutic protein encoded by the vector will need to be carefully considered. Given that utrophin is normally expressed in nearly all tissues including muscle, the results reported here suggest that gene therapy protocols for DMD could potentially benefit from the use of utrophin-based transgenes.

Materials and Methods

Microutrophin cloning and virus production. The microutrophin transgene (R4–R21/CT) was engineered to contain the amino-terminal actin-binding domain, the first three spectrin-like repeats followed by the second hinge, spectrin-like repeat 22, hinge 4, and the cysteine-rich domain. Twenty-five nucleotides of the adjacent 5' and 3' untranslated region were included, followed by the polyadenylation signal from the rabbit -globin gene at the carboxy terminus. The coding sequence of microutrophin was codon-optimized and synthesized (Blueheron Biotech, Bothell, WA) (GenBank accession no. EU293093). This synthesized microutrophin complementary DNA was excised from pENTR (Invitrogen, Carlsbad, CA) using Pme I, and subcloned into SnaB I-digested pARAP4.48 The CMV promoter obtained from pAAV-LacZ (Stratagene, La Jolla, CA) was excised by digestion with SpeI/BstBI digestion, and ligated into the Xho I/BstB I site of pAAV/microutrophin to create pAAV/CMV-microutrophin. Production of the rAAV6/microutrophin vector was carried out as described.⁴⁹

Animal experiments. Male and female wild-type C57Bl/6J mice (Jackson Laboratory, Bar Harbor, ME) and dystrophin/utrophin deficient (mdx:utrn^-/-) mice (N = 5) were derived as previously described.³¹ All the experiments involving animals were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Washington. Four-week-old dystrophic mdx:utrn^-/- mice received 3 10¹² vector genomes of rAAV6 vector in a single 300 l bolus intravenous injection through the tail vein. The mice were weighed every week and killed at 5 months of age for further evaluation.

Histological analyses. After functional analyses were complete, the mice were killed and necropsy was performed. Muscles were frozen in liquid nitrogen-cooled isopentane embedded in Tissue-Tek OCT medium (Sakura Finetek USA, Torrance, CA), and sectioned transversely in a cryostat at 10 m. For brightfield microscopy, sections were stained with hematoxylin and eosin and mounted with Permount (Fisher Scientific, Fairlong, NJ). For immunofluorescence studies, sections were blocked in 4% bovine serum albumin and 0.05% Tween-20 in 1 Dulbecco's phosphate buffered saline (PBS; Invitrogen GIBCO, Grand Island, NY). The sections were then washed three times in 1 PBS for 5 minutes. The slides were incubated for 60 minutes using rabbit anti-utrophin polyclonal antibody (1:400), (kindly provided by Stan Froehner, University of Washington) and rat anti-B2 laminin (1:800) (Sigma, St Louis, MO) in PBS containing 2% goat serum. The sections were then rinsed three times in PBS and incubated for 45 minutes with goat anti-rabbit-alexa-488 (1:1200) and goat anti-rat-alexa-540 (1:800) (Molecular Probes, Eugene, OR). The slides were mounted in antifade mounting media containing 4,6-diamidino-2-phenylindole (Vector Labs, Burlingame, CA). Fluorescent sections were imaged using a Nikon eclipse E1000 fluorescent microscope (Nikon, Melville, NY). Images were captured using a QIcam digital camera and processed using Qcapture Pro (Qimaging, British Columbia, Canada). Immunoflourescent detection of representative components of the DGC was performed on TA muscles with -Sg and -Dg monoclonal antibodies (1:100) (Novocastra, Newcastle, UK), using a mouse-on-mouse blocking kit (Novacastra, Newcastle, UK). The primary rabbit polyclonal antibodies used were -dystrobrevin-2, 1-syntrophin (1:200) (kindly provided by Stan Froehner, University of Washington), and nNOS (Sigma, St Louis, MO). For determining the extent of localization of microutrophin at myotendinous junctions, confocal microscopy was performed on longitudinal gastrocneminus cryosections with immunoflourescent staining of utrophin as described earlier, with the modification that 50% glycerol in PBS was used as mounting medium. Visualization was on a Zeiss 510 Meta microscopeusing 60 magnification. Images were taken with the same settings and were processed in an identical way. Limits were placed and maintained throughout image processing on the image "gain" so as to ensure that immunofluorescence saturation of the images was avoided.

Myofibers with centrally located nuclei were quantified from utrophin-positive myofibers in TA muscles by counting the fibers in a minimum of four random fields (800 myofibers/cohort). The mean numbers of centrally nucleated fibers in treated and untreated mice were compared using an unpaired Student's t-test. The myofiber cross-sectional area within the TA muscle (1,500 myofibers/cohort) was measured using Image J computer software (National Institutes of Health). Two days prior to being killed, the animals were bled retro-orbitally to determine serum creatine kinase levels, using a commercial kinetic kit (CK LiquiUV, Stanbio Laboratory, Boerne, TX).

Contractility assays. Sixteen weeks after vector administration, the mice were anesthetized with 2,2,2-tribromoethanol (Sigma, St Louis, MO) and assayed in situ (TA) for force generation and susceptibility to eccentric contraction-induced injury, using equipment and protocols described earlier.⁹ Control wild-type mice at 5 months of age and untreated mdx:utrn^-/- mice at 3 months of age (that is, not yet approaching the end of their life spans) were used as controls. The conditions established with this assay occur over a broad range of physiological operating conditions with the potential to produce injurious overload of the contractile apparatus. Briefly, through nerve stimulation we determined the maximum isometric force-producing capacity at optimal muscle fiber length. The TA muscle was then subjected to a series of progressively greater (5%) length changes under maximum stimulation (lengthening contractions) at twenty-second intervals. The impact of each lengthening contraction upon force production was recorded from the peak isometric force generated just prior to the initiation of the subsequent lengthening contraction. Cohorts from the lengthening contraction assay were analyzed using two-way ANOVA incorporating a Bonferroni posttest using the PRISM software.

Statistics. All results are expressed as mean s.e.m. unless otherwise stated. Differences identified between cohorts were determined using one-way ANOVA with a Student's t-test, with the exception of the contractility assays as mentioned earlier. All data analyses were performed using the PRISM software.

References

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Acknowledgments

We are grateful to Leonard Meuse for animal husbandry, Caitlyn Doremus for rAAV vector production assistance, and Miki Haraguchi for histopathological support. We thank Stan Froehner for providing the antibodies used in the study, and Marvin Adams of the University of Washington for his critical reading of this manuscript. We also thank Greg Martin at the Keck Imaging Center, University of Washington, for assistance with confocal microscopy, and Chamberlain laboratory members for critically reviewing the manuscript. This work was supported by grants from the National Institutes of Health (R37AR40864) and the Muscular Dystrophy Association (to J.S.C.). G.L.O. was supported by a National Institutes of Health National Research Service Award (T32 HL07828). P.G. was supported by a Development Grant from the Muscular Dystrophy Association.