Systemic microdystrophin gene delivery improves skeletal muscle structure and function in old dystrophic mdx mice

Abstract

Restoring dystrophin expression in the muscles of patients with Duchenne muscular dystrophy (DMD) may halt or reverse the degenerative wasting and weakness that causes premature death. However, the therapeutic efficacy of an intervention may be limited by the extent of disease progression prior to treatment. In the present study, we considered the potential for ameliorating pathology in a mouse model of advanced-state muscular dystrophy via systemic administration of recombinant adeno-associated viral vectors (rAAV6) encoding a microdystrophin expression construct. Treatment of 20 month-old mdx mice restored body-wide expression of a dystrophin-based protein in striated musculature. In treated old mice, dystrophin expression as a consequence of treatment was associated with improved hindlimb and respiratory muscle morphology and function concomitant with reduced muscle fiber degeneration. The findings demonstrate that an established dystrophic state remains amenable to improvement with appropriate intervention, and by some measures, may even achieve similar benefits as observed with intervention early in disease progression. The capacity to ameliorate pathology in an animal model of an advanced-state muscular dystrophy suggests that interventions ultimately proven to exert a therapeutic effect in young patients may offer benefit to patients with advanced conditions of progressive muscular dystrophy.

INTRODUCTION

Duchenne muscular dystrophy (DMD) is caused by mutation of the gene encoding the 427 kDa dystrophin protein, an integral component of the dystrophin-glycoprotein complex (DGC) that links the cytoskeleton and cellular membrane in skeletal and cardiac muscle cells.^1–3 Disruption of the DGC as a consequence of dystrophin deficiency increases the susceptibility of myofibers to damage with contraction, and perturbs signaling processes implicated in cellular homeostasis.^4–6 Consequent cell degeneration with intramuscular fibrotic and adipose accumulation gradually depletes patients’ functional muscle mass, and ultimately culminates in premature death attributed to either respiratory or cardiac failure.^{3, 7}

Restoration of dystrophin expression in the muscles of patients may be achievable by administering engineered constructs intended to function in place of the defective endogenous gene.⁸ Expression of miniaturized genes encoding for truncated “microdystrophin” proteins has been demonstrated to restore organization of the cytoskeleton-DGC interaction in muscle fibers, and prevent the onset of pathology in transgenic dystrophic mice.^{9, 10} Furthermore, systemic administration of microdystrophin constructs to young mice with a severe form of muscular dystrophy using recombinant viral vectors can considerably reduce deterioration of muscle function, and extend lifespan.^{11, 12} The results demonstrate that timely administration of an intervention that restores dystrophin expression can slow or halt the development of dystrophic symptoms in mice. However, DMD is a condition of progressive deterioration, and some aspects of pathology associated with recurring cycles of degeneration and regeneration may not be amenable to complete reversal or resolution with treatment.^{3, 13–15} Consequently, it has not been established to what extent the severity of the disease state at the time of intervention may limit the potential to halt or reverse disease progression with treatment. As patients frequently present with features of advanced pathology even when diagnosed in early childhood,^{3, 7} it is essential to determine whether the efficacy of potential treatments may depend on the progression of disease severity, and therefore the age of the patient at intervention.

While dystrophic mice do not exactly reproduce all the disease attributes observed in DMD patients, they remain very useful for advancing our understanding of muscular dystrophy and for assessing the potential of promising interventions. In the present study, we tested the null hypothesis that old dystrophic mdx mice receiving a microdystrophin gene-based intervention proven to be highly efficacious in young dystrophic mice, would derive reduced (if any) therapeutic benefit, because of considerable disease progression prior to experimental treatment. In contrast to untreated old dystrophic mice, we observed that treated animals exhibited evidence of improved skeletal muscle morphology and function, consistent with reduced muscle fiber degeneration after treatment.

RESULTS

Configuration of our existing microdystrophin expression construct to incorporate a ubiquitous CMV promoter achieves robust transgene expression in the heart and diaphragm musculature, as well as in the axial and appendicular skeletal muscles of young adult mice, when delivered via recombinant pseudotype-6 adeno-associated viral vectors.^{11, 12} Having verified the expression potential of the modified vector construct in vivo in young adult mice, we administered a bolus dose of ~1×10¹³ rAAV6:CMV-microdystrophin vectors genomes to 20 month-old mdx mice via tail vein injection (see Methods).

Dystrophic mdx mice typically exhibit an increased body mass compared with wildtype animals during adolescence and early adulthood, but demonstrate a loss of mass relative to wildtype animals into late adulthood, consistent with advancing disease state^{14, 16, 17} Upon examination at ~24 months of age (corresponding to ~4 months after treatment of the experimental cohort), old untreated dystrophic mice exhibited a reduced body mass compared with age-matched wildtype mice (p = 0.057, Fig. 1a), caused by reductions in both lean and fat mass (Figs. 1b,c). Blood analyses confirmed that untreated dystrophic mice also presented dramatically elevated serum creatine kinase activity compared with wildtype mice (Fig. 1d), suggestive of ongoing muscle fiber degeneration at this advanced age. In contrast to the untreated dystrophic mice, the cohort that had received a systemic intervention of rAAV6:microdystrophin ~4 months prior to analyses exhibited a 15% increased body mass as a product of an increased lean body mass specifically (Figs. 1a,b), and a reduction in serum CK levels accounting for ~90% of the discrepancy between the values of untreated dystrophic and wildtype cohorts. Inspection of numerous muscles from mice in each cohort confirmed that untreated mdx mice did not exhibit the uniform expression of dystrophin in cardiac and skeletal musculature body-wide that is typical of non-dystrophic muscle (Fig. 1e). However, mdx mice that received treatment exhibited widespread dystrophin immunoreactivity of an intensity and typical uniformity (in most muscles and within individual transduced muscle fibers) that was difficult to distinguish from that of the full-length protein in the muscles of wildtype mice (Fig. 1e). These findings demonstrate that the advanced age and dystrophic state of 24 month old dystrophic mdx mice does not present a significant impediment to systemic transduction of the striated musculature via the intravascular administration of rAAV6:microdystrophin.

Figure 1. Systemic microdystrophin gene delivery improves whole-body indices of disease state as a consequence of restoring dystrophin expression in the striated musculature of old dystrophic mice.

Untreated 24 month old dystrophic mdx mice (mdx, grey columns) presented with reduced body mass (p = 0.057) compared with age-matched wildtype mice (WT, black columns) (a), which was a product of reductions in lean (b) and fat (c) mass. By comparison, treated mdx mice (Tmdx, red columns) that were analyzed 4 months after receiving ~1×10¹³ vg of rAAV6:microdystrophin via tail vein injection at 20 months of age exhibited an increased body mass and lean mass compared with untreated dystrophic mice. Untreated dystrophic mice demonstrated dramatically elevated serum levels of creatine kinase compared with wildtype mice (d), while treated animals demonstrated CK levels more than 90% reduced compared the untreated cohort. Cryosections of striated muscles (e, also Figs. 2a,4a) examined for dystrophin expression (green) demonstrate that the muscles of untreated dystrophic animals lack the uniform protein expression that is observed in wildtype mice, and which is readily restored as a consequence of treatment with systemic administration of rAAV6:microdystrophin. * p < 0.05 for mdx vs. wildtype. ** p < 0.05 for Tmdx vs. mdx. Scale bar 100μm.

Having established systemic gene transfer is achievable in old mdx mice, we sought to determine whether the systemic microdystrophin gene transfer late in life could ameliorate functional and structural symptoms of advanced dystrophinopathy in the limb and respiratory musculature of these mice, as a model for the potential to improve the locomotory and respiratory musculature of dystrophic patients. As predicted, the tibialis anterior hindlimb muscles of untreated old mdx mice exhibited marked evidence of myofiber turnover as a consequence of dystrophin deficiency (Figs. 2a,b), including increased prevalence of small, immature myotubes (Fig. 2c), and myofibers with centrally-located nuclei (typically indicative of incomplete/ongoing muscle fiber remodeling during regeneration,^{3, 9} Fig 2d). In contrast, the majority of the myofibers in the TA muscles of treated mdx mice demonstrated robust microdystrophin expression (Figs. 2a,b). In treated muscles, dystrophin expression was associated with reduced numbers of degenerating (Fig. 2a) and centrally-nucleated (Fig. 2d) muscle fibers, indicating reduced muscle fiber turnover. The improved morphological characteristics of the TA muscles of treated mice correlated with a trend (p = 0.089) towards increased TA muscle mass compared with that of untreated mice (Fig. 2e). A body-wide effect of this nature as a consequence of treatment would account for the generalized increase in lean mass reported for the treated animals compared with the untreated dystrophic cohort (Fig. 1b).

Figure 2. Systemic microdystrophin gene delivery improves the morphological properties of the limb muscles of old dystrophic mice.

Cryosections of tibialis anterior hindlimb muscles (a) from untreated dystrophic mice (mdx) lack the uniform dystrophin expression that is observed in wildtype mice, and instead demonstrate substantial evidence of myofiber degeneration and remodeling. In contrast, dystrophin expression is restored to the considerable majority of myofibers in the TA muscles of treated mice (b). Expression of microdystrophin did not appear to significantly reduce the proportion of unusually small myofibers (c) in the limb muscles of treated dystrophic animals (Tmdx dys+ve, yellow line, and in (d), yellow column), although the incidence of centrally-located myonuclei (a measure of myofiber remodeling and incomplete regeneration) was reduced in the transduced muscle fibers of treated mouse TA muscles compared with in the muscles of untreated dystrophic mice (d). The TA muscles of treated animals displayed a trend (p = 0.089) towards an increased mass compared with the muscles of untreated dystrophic animals (e), which even surpassed that of muscles obtained from age-matched wildtype mice. * p < 0.05 for mdx vs. wildtype. ** p < 0.05 for Tmdx vs. mdx. Scale bar 100μm.

Deleterious changes in muscle structure typically reflect impaired muscle functionality. The compromised morphological characteristics of the TA muscles of untreated mdx mice were associated with a reduced capacity of these muscles to generate force under maximal activation (Figs. 3a,b), and to maintain force production after exposure to potentially injurious strain during contraction (Figs. 3c,d), compared with the muscles of non-dystrophic wildtype mice. As with previous studies that positively correlate improvements in the aforementioned morphological attributes with improved muscle function, we established that the TA muscles of treated mdx mice exhibited considerably increased (~30%) maximum force producing capacity (accounting for more than two-thirds of the deficit between dystrophic and widltype mice, Fig. 3a) and also a 12% improvement in force normalized for cross sectional area, compared with the muscles of untreated mice. The enhanced force-producing capacity of the muscles of treated mice also conferred improvements in force output after application of strain during maximal contraction (Figs. 3c,d), but did not significantly protect muscles from injury in a manner resembling the capabilities of muscles from wildtype mice. These data demonstrate that the intervention still achieves significant improvements in the functional performance of hindlimb muscles in mice that have experienced progressive dystrophy for nearly 20 months prior to intervention.

Figure 3. Systemic microdystrophin gene delivery improves the functional properties of the limb muscles of old dystrophic mice.

The tibialis anterior muscles of untreated dystrophic mdx mice (mdx, grey) displayed reduced absolute (a) and normalized (b) maximal force-producing capacity compared with the muscles of age-matched non-dystrophic mice (WT, black). In contrast, the muscles of mice systemically treated with rAAV6:microdystrophin (Tmdx, red) demonstrated a significantly greater capacity to generate force (a) compared with that of the muscles of untreated dystrophic mice. The absence of dystrophin in the muscles of untreated dystrophic animals contributes to an increased susceptibility to contraction-induced injury when repeatedly subjected to strain of 20% (c) or 40% (d) beyond optimum length during maximal contraction. LC1, LC2 and LC3 denote force output after 1, 2, and 3 “lengthening contractions” of this nature. The muscles of dystrophic mice that received rAAV6:microdystrophin 4 months prior to evaluation exhibit improved force output compared with the muscles of untreated animals as a consequence of greater initial capacity (c,d top panels) though offered little in the way of improved relative resistance to contraction-induced injury (c,d bottom panels). * p < 0.05 for mdx vs. wildtype. ** p < 0.05 for Tmdx vs. mdx.

Respiratory muscle function is a key predictor of mortality in dystrophic patients. Additionally, it has been established that the diaphragm musculature of the dystrophic mdx mouse presents a more severely affected phenotype (with regards to structural degeneration and loss of functional output) compared with other muscles, suggesting it to be the preferred murine muscle in which to model the human disease state.^18–20 Therefore, we chose to study the effects of the intervention upon structural and functional attributes of the diaphragm muscle from mice in each cohort. As in limb muscles, the diaphragm musculature of untreated dystrophic mice typically exhibited fewer than 1% dystrophin-positive myofibers (Figs. 4a,b), and an associated increase in the prevalence of small and centrally-nucleated regenerating myofibers (Figs. 4c,d). Also, the diaphragm musculature of untreated dystrophic mice appeared to contain fewer muscle fibers than observed in the muscles of wildtype mice (mean number of myofibers through the cross sectional thickness of analysed diaphragm segments, wildtype vs mdx; 12.7 ± 0.3 vs 5.6 ± 0.5, mean ± sem, n=5). Analyses performed 4 months after administration of rAAV6:microdystrophin identified persistent dystrophin expression in ~50% of diaphragm muscle fibers – notably less than in the limb muscles of the treated mice. However the expression of microdystrophin in the diaphragm musculature of treated mdx mice considerably reduced the prevalence of small myotubes, and also dramatically reduced the prevalence of centrally-nucleated muscle fibers (Figs. 4c,d). Additionally, there was an increased number of muscle fibers in the cross sections of the muscles of treated mice compared with the diaphragm musculature of untreated mice (mean number of myofibers through the cross sectional thickness of analysed diaphragm segments, mdx vs Tmdx; 5.6 ± 0.5 vs 10.3 ± 0.7, mean ± sem, n=5). These findings indicate that considerable muscle fiber loss occurs in the diaphragm musculature of mdx mice beyond 20 months of age, but that this process can be slowed or halted with the administration of rAAV6:microdystrophin. These data affirm that substantial morphological improvements remain attainable in the advanced state of the old dystrophic mdx mouse model.

Figure 4. Systemic microdystrophin gene delivery improves the morphological and functional properties of the respiratory musculature of old dystrophic mice.

Old dystrophic mice that received a systemic administration of rAAV6:microdystrophin 4 months prior to evaluation demonstrated considerably improved morphology of the diaphragm musculature compared with that of muscles from untreated dystrophic mice (a) despite sustaining less than complete restoration of dystrophin expression (Tmdx, red, b). The diaphragm musculature of untreated dystrophic mice (mdx, grey) was comprised of an increased proportion of small myofibers (c), and muscle cells with centrally-located nuclei (d) compared with the musculature of age-matched non-dystrophic mice (WT, black). By comparison, the dystrophin-postive myofibers within the muscles of treated animals (Tmdx dys+ve, yellow) were on average larger, and considerably less frequently centrally nucleated, corresponding to reduced incidence of myofiber turnover. Treatment had little effect on restoring the depressed normalized force-producing capacity observed as a consequence of dystrophic pathology in the diaphragm musculature (e), but dramatically enhanced the resistance of muscles to mild (20% strain, f) and severe (40% strain, g) contraction-induced injury, compared with the muscles of untreated dystrophic mice. * p < 0.05 for mdx vs. wildtype. ** p < 0.05 for Tmdx vs. mdx. Scale bar 400μm.

Regarding contractile properties of the respiratory musculature, the diaphragm muscles of untreated dystrophic mice generated little more than 25% of the force per cross sectional area of muscles from wildtype mice (Fig. 4e), and exhibited notably impaired contractile capacity subsequent to strain challenge, compared with wildtype mice (Fig. 4f). The diaphragm muscles of treated mice did not demonstrate any significant improvement regarding force normalized for muscle cross sectional area compared with the muscles of untreated animals (Fig. 4e). However, treatment considerably improved the muscles’ resistance to contraction-induced injury (Fig. 4f). Specifically, the muscles of treated mice maintained force output (as a percentage of initial performance) when subject to increasingly severe strain during contraction that was not different from the values of muscles from wildtype animals. These data demonstrate that the intervention proved highly capable of achieving desirable functional improvements in the respiratory musculature of animals of a highly advanced dystrophic state. The findings also show that individual muscles can respond differently to the same intervention (i.e. systemic administration of rAAV6:microdystrophin), and as has been reported elsewhere with transgenic animals,⁹ the more severe disease state in the diaphragm¹⁸ appears notably responsive to interventions (subject to successful delivery) compared with typically less severely affected limb muscles. The cause of the differential pathology and response to intervention observed in specific muscles has not been completely elucidated, but may be influenced several factors including the patterns of mechanical loading imparted on different muscles.

DISCUSSION

The most important finding of this study is that so-called “late stage” intervention with systemically-administered rAAV6:microdystrophin can still elicit physiologically relevant improvements in muscle structure and function in the aged mdx mouse, despite the challenges of intervening upon a considerably advanced dystrophic condition. Most studies that have considered the effects of an intervention as a means to halt or reverse the pathology of severe muscular dystrophy have typically been administered to young mice, which better model the dystrophic state in newborn or very young patients.^{9–11, 21–29} By comparison, we have chosen to test this promising intervention in the most time-advanced murine model of severe muscular dystrophy that we can generate, the old mdx mouse.^{14, 15, 17, 30} Consistent with the literature, we observed that untreated old dystrophic mice exhibited signs of advanced dystrophy that are not observed in younger animals, such as reduced body mass and muscle mass compared with age-matched non-dystrophic animals caused by ongoing disease. ^{15, 30, 31} We acknowledge that mdx mice do not perfectly reproduce the severity of pathology exhibited by DMD patients. However, aged mdx mice represent the most advanced dystrophic condition that can be generated as a consequence of dystrophin deficiency alone in a small mammal model. The disease features confirm that the benefits we observed with treatment were achieved in a model that presents with aspects of disease severity that are not associated with younger dystrophic mdx mice, and are more in keeping with the pathology of the human condition. These observations support the use of the old mdx mouse (and especially the diaphragm musculature) as a model of pathology associated with muscular dystrophy.

Our findings determined that the advanced dystrophic state observed of these animals did not appear to present a significant impediment to the systemic dissemination and subsequent transduction of striated musculature using rAAV6 vectors. Ascertaining feasibility of transduction as shown here is an important consideration, as the accumulation of non-muscle (i.e fibrotic and adipose) tissue with progressive dystrophy could conceivably present significant physical barriers to the delivery and uptake of vectors by remaining muscle fibers. Most studies examining interventions for DMD have relied upon local, intramuscular administration to assess safety and efficacy in a specific muscle. Patient trials utilizing a similar mode of delivery would not only require an excessive number of injections to achieve similarly successful transduction of a single muscle (much less multiple muscles), but would likely encounter significant challenges in realizing practical dissemination of vector on account of obstructive intramuscular non-muscle tissue that accumulates with disease progression.^{3, 7} As the microvascular network is closely associated with every striated muscle cell to provide for metabolic, thermal and gas exchange, more recent methodological developments have been employed to administer interventions via the intravascular route to facilitate body-wide treatment and circumvent the impediments to intramuscular diffusion that are presented by non muscle tissue. Here too, it has not been conclusively established that the accumulation of adipose and fibrotic material with the development of dystrophy, and vascular remodeling with advancing age does not impede systemic delivery of vectors to striated muscle. Our data establish convincingly that the striated musculature of the old mdx mouse remains readily amenable to systemic transduction with rAAV6 vectors consistent with that observed in young animals, and suggests that host age (at least in mice) is not necessarily a limiting factor in achieving gene transfer to treat the symptoms of a form of muscular dystrophy.

We observed that systemic treatment of old mdx mice with rAAV6:microdystrophin attained body-wide expression of a therapeutic protein for at least 4 months, which is also a key demonstration that the transcriptional mechanisms that operate in the musculature of these animals remain sufficiently operable to sustain a level of transgene expression that can exert beneficial effects upon phenotype. We envisage that the ultimate configurations of clinical vectors will utilize expression cassettes with tissue-specific promoter elements. However such designs will need to be verified for functionality in appropriate models of advanced disease state, as it is recognized that patterns of gene expression vary between muscles, as well as with disease and with advancing age.^32–34 Therefore, promoter elements may experience reduced functionality in some instances where the disease state is more advanced, or patients are older. Our data confirm that expression of microdystrophin at a level which is comparable to that observed in treated young-adult mice can be sustained in the musculature of old, severely dystrophic mdx mice.

Our data establish that the body-wide expression of microdystrophin observed in aged mdx mice as a consequence of treatment with a systemic rAAV6:microdystrophin injection can enhance the structural and functional attributes of both respiratory and limb musculature. The degree of benefit attained in treated animals differed between muscles, which may hold important implications for prospective human efficacy trials. As previously mentioned, many previous animal studies have tended to focus on specific limb muscles, which may allow for straightforward treatment and evaluation. However sufficient evidence exists to show that the hallmarks of disease vary between muscles in mice, and that diaphragm musculature in particular may actually be the tissue of choice for modeling human dystrophy.^{18, 19} Why specific muscles exhibit different degrees of pathology and response to interventions is not clear, but our findings clearly demonstrate that both axial and appendicular musculature of old mdx mice can benefit from the systemic administration of rAAV6:microdystrophin despite the advanced pathological state. The ability to achieve notable phenotypic improvement in the muscles of old mdx mice via the methods used indicate that the prospect of rAAV-mediated microdystrophin administration as a treatment for advanced dystrophy merits ongoing study.

The findings of this study are especially important because clinical trials of interventions intended to treat DMD and other serious forms of muscular dystrophy will frequently need to treat the symptoms of patients who are of legal consenting age or at least in their teen years, and therefore already of advanced disease state. Promising data on the efficacy of interventions which are acquired from studies of young mouse models need to be supported with data establishing the validity of the interventions in models with considerably advanced disease progression to indicate the scope for benefit in prospective patient populations. Our data presented here expand upon the rigorous work performed in young mouse models, and help to extend our understanding of the scope for therapy in the more advanced condition of the old mdx mouse. Given the encouraging findings described here, we propose that further studies should consider whether the disease state progresses similarly and remains amenable to treatment in canine models of DMD.^{35, 36} Dogs live considerably longer than mice, and may therefore serve as a comparatively better model for the progression of the human condition beyond two years of life (as is the practical limit in mice), and in which to study the persistence of the transgene expression following intervention. Also, some groups feel that the canine model exhibits a more severe phenotype than the mouse, which may better model the human condition. Some aspects of interpretation may remain challenging owing to quadrupedal posture placing different patterns of loading and recruitment on specific muscle groups in dogs compared with humans. However, canine studies may also help to determine if the mechanisms of systemic gene delivery via rAAV6 vectors are affected with aging and disease progression in larger mammals contrary to that observed in mice.

In summary, within the lifespan limitations of the mouse model-based studies (which remain an imperfect model of the human state), we believe that the findings provide support for “proof of concept” that aspects of an advanced human condition may benefit from interventions intended to enhance muscle function and performance, despite the progression of disease state symptoms. These findings represent a valuable addition to the body of knowledge concerning therapeutic strategies for preventing and/or treating the symptoms of severe muscular dystrophies such as DMD.

METHODS

Construct cloning and Vector Production

Recombinant AAV genomes containing an expression cassette for microdystrophin (ΔR4-23/ΔCT) and serotype 2 inverted terminal repeats were generated by standard cloning techniques.⁹ Eag I fragments of the coding sequence for microdystrophin were blunted with T4 DNA polymerase and cloned into the plasmid ARAP4 ³⁷ after Hind III digestion and blunting of the vector plasmid. The CMV promoter was introduced into this construct via ligation of a Mlu I-blunted/Sac II fragment of pCMV-MCS (Stratagene, La Jolla, CA) into the Xba I-blunted/Sac II portion of the plasmid. The resulting construct was co-transfected with the pDGM6 packaging plasmid into HEK293 cells to generate recombinant AAV vectors comprising serotype 6 capsids that were harvested, enriched, and quantitated as described previously.³⁸ Enriched (up to ~ 1×10¹⁴ vg/ml) preparations of rAAV6 vector were stored in phosphate buffered saline (PBS) at −80°C are previously reported³⁸ until thawed for use.

Manipulation of experimental animals

All experimental manipulation of young adult (12 wk) or old (19.5 mo) male wild type C57Bl/10J, or dystrophic, C57Bl/10ScSn-Dmdmdx/J mice (Jackson Labs, Barr Harbor, ME) was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Washington. Individual mice (from cohorts containing 6 animals) were administered a 300 μL bolus injection (corresponding to ~15% of the circulating blood volume, a readily tolerable dose) via the tail vein comprising physiological Ringer’s solution containing ~ 3×10¹³ vg of rAAV6 vector, 10 μg recombinant human VEGF-165 (R&D Systems, Minneapolis, MN), 0.008% mouse serum albumin (Sigma, St. Louis, MO), and 2 IU sodium heparin as employed previously.¹¹ All testing after this step was performed in a blinded manner when possible.

Determination of body composition

Whole body composition analysis was performed using a quantitative magnetic resonance method (EchoMRI, Echo Medical Systems, Houston, TX) as described previously.^{39, 40}

Functional analyses

Eighteen weeks post-injection, mice were anesthetized with 2,2,2-tribromoethanol (Sigma) and assayed in situ (tibialis anterior) and in vitro (diaphragm) for force generation and protection from contraction-induced injury using methods and hardware described peviously.^{16, 17, 21} To test the susceptibility of muscles to strain-mediated injury of varying intensity, individual muscle preparations (a minimum of 4–5 per cohort) were forcibly lengthened 20% or 40% beyond optimum length whilst maximally contracted a total of three times at 30 second intervals. After functional testing, animals were sacrificed and tissues were rapidly excised and processed for histology as below.

Tissue processing and analysis

Histological, and immunochemical processing of muscle cryosections was completed as described previously.¹² Immunofluorescent detection utilized primary antibodies against the N-terminal region of dystrophin (raised in rabbit, used at 1:600), laminin B2 (raised in rat, Chemicon, Temecula, CA, used at 1:600) and the commercial Alexa 488-labeled goat-anti-rabbit and Alexa 594-lableled goat-anti-rat secondary antibodies (Molecular Probes, Eugene, OR, used at 1:1200). Sections were coverslipped with DAPI supplemented hard-set mountant (Vector Laboratories, Burlingame, CA). Images were obtained and digitally merged (Nikon E-1000 microscope, Japan, supported with Spot software) to assess dystrophin expression, myonuclear position, and myofiber morphology (Image Pro Plus software suite, Media Cybernetics). Cell diameter was measured by tracing the perimeter of individual muscle fibers via on-screen image assessment, and calculating the minimum diameter passing through the centroid of each object of interest. An average of 100 muscle fibers was sampled spanning the diameter of individual tibialis anterior muscle sections or the full thickness of diaphragm sections. The mean number of muscle fibers spanning transverse sections of diaphragm muscle was determined by counting the number of fibers underlying a 4 pixel line drawn across the widest portion of a minimum of six separate fields from each diaphragm muscle. Serum CK levels were assayed with a kinetic kit (StanBio Laboratory, Boerne, TX) using blood obtained from the animals at the experimental endpoint. All data are presented as mean ± SEM (typically n=5 each cohort) unless otherwise stated. Reported differences between cohorts are statistically significant at p < 0.05 according to Student’s t-test analyses unless specifically stated otherwise.