GDF11PRO-Fc binds to both GDF11 and MSTN

To show that GDF11PRO-Fc can sequester the active mature GDF11 dimer via a direct interaction, we aimed to identify a protein-protein interaction between GDF11 and GDF11PRO-Fc. Additionally, due to the close structural homology of the GDF11 and MSTN mature dimers, we also wanted to determine whether GDF11PRO-Fc associates with MSTN. To identify these protein-protein interactions, we performed a set of pull-down assays (Fig. 1). The ligands rGDF11, rMSTN, and rActivin A were incubated with lysate fractions from HEK293 cells transfected with a plasmid encoding for GDF11PRO-Fc or MPRO-Fc at pH 7.4. Owing to the conjugated Fc fragments on the modified propeptides, fractions could be pulled down directly on a protein A/G agarose resin. As expected, GDF11PRO-Fc effectively pulled down both rGDF11 and rMSTN, indicating that GDF11PRO-Fc was capable of binding both ligands. In addition, MPRO-Fc successfully pulled down both rGDF11 and rMSTN as well. Both GDF11PRO-Fc and MPRO-Fc were not able to pull down the more distantly related TGF-β superfamily ligand rActivin A, indicating that ligand binding is specific (Fig. 1). No binding of GDF11PRO-Fc, MPRO-Fc, rGDF11, rMSTN, or rActivin A were observed on a control agarose resin without protein A/G. From these results, we conclude that GDF11PRO-Fc spontaneously associates with the mature GDF11 and MSTN dimers at physiological pH.

Fig. 1 GDF11PRO-Fc associates with GDF11 and MSTN. Protein-protein interactions between GDF11PRO-Fc or MPRO-Fc and rGDF11, rMSTN, or rActivin A were determined by a pull-down assay. GDF11PRO-Fc or MPRO-Fc was incubated with rGDF11, rMSTN, or rActivin A for 1 h at 4 °C. Fc-fused protein complexes were separated on a protein A/G-coated agarose resin and eluates were run on a 12% SDS-PAGE gel under reducing conditions and probed by western blot. Input control was 5% of the input material. WB western blot Full size image

GDF11PRO-Fc blocks GDF11/MSTN-induced atrophy in C2C12 myotubes

Addition of rGDF11 and rMSTN to differentiated myotubes has previously been shown to induce atrophy in murine C2C12 and primary human skeletal myotubes [16, 31]. Having determined that GDF11PRO-Fc binds GDF11 and MSTN, we next asked if GDF11PRO-Fc could prevent GDF11/MSTN-induced myotube atrophy in differentiated C2C12 myotubes. To achieve this, we packaged the DNA sequence encoding for GDF11PRO-Fc into the AAV6 capsid to generate the AAV6-GDF11PRO-Fc vector. In these experiments, the AAV6 vector was selected for its ability to efficiently and selectively transduce differentiated C2C12 myotubes, but not myoblasts [32, 33]. C2C12 myoblasts were cultured and differentiated for 5 days prior to treatment with AAV6-GDF11PRO-Fc or AAV6-EGFP (control vector) at a MOI of 105 (Fig. 2a). As expected, GFP expression was observable in C2C12 myotubes infected with AAV6-EGFP at 48–72 h post-infection, and quantification of internalized vector genome copies per diploid genome at 72 h post-infection indicated that vector transduction was adequately achieved (Fig. 2b and c).

Fig. 2 GDF11PRO-Fc blocks GDF11/MSTN-induced myotube atrophy in C2C12 cells. a Schematic detailing experimental timeline in C2C12 myotubes. AAV6-EGFP or AAV6-GDF11PRO-Fc was added to C2C12 myotubes at a MOI of 105 on day 5 post-differentiation and 100 ng/ml rGDF11 or rMSTN was added on day 7. Myotubes were stained and analyzed on day 10. b EGFP expression was evident at 48–72 h in C2C12 myotubes treated with AAV6-EGFP (MOI 10 [5]). Scale bars represents 50 μm. c Vector genome copy number per diploid genome in C2C12 myotubes 72 h after addition of AAV6-EGFP or AAV6-GDF11PRO-Fc (MOI 10 [5]). d Representative immunofluorescence images of C2C12 myotubes. C2C12 myotube membranes were visualized by staining with an anti-dystrophin antibody (red). Nuclei were stained with DAPI (blue). Inset shows a zoomed-in region. Scale bars represent 50 μm (main panel) and 25 μm (panel inset). e The fraction of nuclei incorporated into myotubes (differentiation index) was calculated and presented as a percentage of control. f Average myotube diameter relative to control and (g) distribution of diameter measurements. For myotube diameter measurements, each myotube was measured at three points along the length of the myotube and averaged. h Number of nuclei incorporated per myotube. A minimum of 50 myotubes were analyzed per experimental condition. i pSMAD2/3 relative to tSMAD2/3 was assessed by western blot. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control. Data represents results from three separate experiments. All error bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant; compared to AAV6-EGFP-treated control. †p < 0.05; ††p < 0.01; †††p < 0.001; compared to AAV6-EGFP + ligand-treated. pSMAD2/3: phosphorylated SMAD2/3; tSMAD2/3: total SMAD2/3 Full size image

Forty-eight hours after AAV treatment, rGDF11 or rMSTN was added to the media to a final concentration of 100 ng/ml. Seventy-two hours later, myotubes were fixed and stained for immunofluorescence analysis using an antibody recognizing dystrophin (rod 22/rod 23) to visualize myotube peripheral membranes and DAPI to stain myonuclei (Fig. 2d). A reduction in the differentiation index, which is defined as the proportion of myonuclei incorporated into myotubes, was observed in myotubes treated with rGDF11 (− 15%, p = 0.0008) or rMSTN (− 14%, p = 0.0013) relative to untreated control. However, this effect was blunted in GDF11PRO-Fc-expressing C2C12 myotubes treated with rGDF11 (− 1.9%; p = 0.0047, compared to rGDF11-treated control) or rMSTN (− 3.0%; p = 0.0040, compared to rMSTN-treated control; Fig. 2e). In addition, C2C12 myotubes that were treated with AAV6-GDF11PRO-Fc resisted rGDF11 or rMSTN-induced myotube atrophy. A substantial decrease in the average myotube diameter relative to control was detected in myotubes treated with rGDF11 (− 39%; 0.0002) or rMSTN (− 35%; p = 0.0003) compared to control. On the contrary, myotubes expressing GDF11PRO-Fc treated with rGDF11 (− 1.8%; p = 0.0005, compared to rGDF11-treated control) or rMSTN (− 5.3%; p = 0.0075, compared to rMSTN-treated control) were largely unaffected (Fig. 2f and g). In a separate experiment, GDF11PRO-Fc was not able to prevent rActivin A-induced myotube atrophy, which is in line with the observation that GDF11PRO-Fc does not bind activin A (Additional file 1: Figure S3).

Treatment with rGDF11 or rMSTN was also associated with a lower proportion of mature myotubes with higher numbers of myonuclei, which suggests an inhibition of myotube differentiation. Myotubes with more than 11 myonuclei comprised only 5.2% (p = 0.0215) and 7.2% (p = 0.0328) of myotubes analyzed in cells treated with rGDF11 and rMSTN, respectively. In comparison, myotubes with more than 11 nuclei made up 26% of myotubes analyzed in control myotubes. In myotubes expressing GDF11PRO-Fc that were treated with rGDF11 or rMSTN, the proportion of myotubes with more than 11 myonuclei was 22% (p = 0.0104, compared to rGDF11-treated control) and 19% (p = 0.0404, compared to rMSTN-treated control), respectively (Fig. 2h). Finally, in agreement with what would be expected following GDF11/MSTN signaling at ActRII, an increase in phosphorylated SMAD2/3 (pSMAD2/3) protein levels in myotubes treated with AAV6-EGFP and rGDF11 or rMSTN was observable on western blot 24 h after addition of ligand (Fig. 2i). This increase in pSMAD2/3 levels was absent in myotubes treated with AAV6-GDF11PRO-Fc, suggesting that GDF11PRO-Fc expression antagonizes GDF11/MSTN-induced activation of SMAD2/3 in differentiated myotubes. Overall, these results indicate that GDF11PRO-Fc was able to prevent rGDF11 and rMSTN-induced myotube atrophy and inhibition of differentiation in C2C12 cells.

Localized GDF11PRO-Fc expression induces skeletal muscle hypertrophy in adult mice

Previously, we have demonstrated that systemic AAV vector-mediated gene delivery of GDF11PRO-Fc into neonatal mice led to a significant increase in the rate of skeletal muscle growth [14]. However, it was unclear from this study whether or not GDF11PRO-Fc simply enhanced skeletal muscle growth or actually induced skeletal muscle hypertrophy. Furthermore, it was not possible to determine if the effects of GDF11PRO-Fc were mediated by local blockade of GDF11/MSTN in skeletal muscle or if the observed effects were due to systemic modulation of GDF11/MSTN. To address these questions, we evaluated the impact of intramuscular AAV9-GDF11PRO-Fc administration in adult C57BL/6J mice. In these studies, only the right-side hindlimb was injected, and the contralateral left-side hindlimb was used as a control.

Bodyweight increased slightly over time in mice treated with AAV9-GDF11PRO-Fc (+ 10% at 10 weeks; p = 0.0418; Fig. 3a). In addition, single-hindlimb grip strength normalized to body weight was significantly increased in both limbs tested individually, with the greater magnitude of effect seen in the treated right-side hindlimb (+ 37%; p = 0.0177), although a significant increase in normalized grip strength in the untreated left-side hindlimb (+ 18%; p = 0.0426) was also observed. However, this difference did not reach significance when both hindlimbs were tested simultaneously (+ 15%; p = 0.2375; Fig. 3b). Ten weeks after vector administration, the right-side hindlimb was visibly larger in mice treated with AAV9-GDF11PRO-Fc (Fig. 3c). In mice treated with AAV9-GDF11PRO-Fc, the wet tissue mass of the injected right-side tibialis anterior (+ 50%; p = 0.0046) and gastrocnemius (+ 37%; p = 0.0023) was significantly higher in comparison to vehicle-treated controls. There was no statistically significant difference in the wet tissue mass of the untreated left-side hindlimb muscles (Fig. 3d). Myofiber area analysis revealed an increase in the average myofiber cross-section area (+ 15%; p = 0.0078) in the AAV9-GDF11PRO-Fc-injected right-side gastrocnemius (Fig. 3e and f). MFD distribution analysis also revealed a shift toward larger myofibers in the right-side gastrocnemius of mice injected with AAV9-GDF11PRO-Fc (Fig. 3g), indicating that localized GDF11PRO-Fc expression induced muscle hypertrophy. In a separate cohort, we also assessed the impact of localized gene delivery of GDF11 by an intramuscular injection of AAV9-GDF11 into the right-side hindlimb, and significant muscle atrophy was observed 10 weeks post-treatment in the injected hindlimb (Additional file 1: Figure S4).

Fig. 3 GDF11PRO-Fc induces localized skeletal muscle hypertrophy after intramuscular gene delivery. 8-week-old C57BL/6J mice were treated with AAV9-GDF11PRO-Fc (n = 5) or vehicle (n = 5) via unilateral intramuscular injection into the right-side hindlimb. The contralateral left-side hindlimb was not treated. Mice were euthanized 10 weeks post-treatment. a Average bodyweight over time. b Hindlimb grip strength normalized to bodyweight at 10 weeks post-treatment. Shown are measurements from a single hindlimb and both hindlimbs. c Representative gross hindlimb musculature. The injected hindlimb is designated with a black arrow. d Wet tissue weight of tibialis anterior and gastrocnemius. e Representative immunofluorescence images of injected right-side gastrocnemius cross-sections stained with AlexaFluor-488-conjugated WGA to visualize myofibers. Scale bars represent 100 μm. f Average myofiber cross-section area in the injected right-side gastrocnemius. g Myofiber MFD distribution in the injected right-side gastrocnemius. A minimum of 500 myofibers were measured per mouse. h Western blot identification of GDF11PRO-Fc in tissue lysates from injected right-side gastrocnemius and liver samples of treated mice. GDF11PRO-Fc was not detectable in vehicle-treated mice. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control. All error bars represent mean ± SEM. *p < 0.05; **p < 0.01; n.s. not significant; compared to vehicle-treated control. TA tibialis anterior, Gas gastrocnemius Full size image

Western blot analysis indicated that the GDF11PRO-Fc protein was expressed in the AAV9-GDF11PRO-Fc-injected right-side gastrocnemius (58 kDa band; Fig. 3h). However, GDF11PRO-Fc was not detectable in the untreated left-side gastrocnemius at the same total protein amount loaded, suggesting that the majority of skeletal muscle GDF11PRO-Fc expression was localized to the injected right-side hindlimb (data not shown). GDF11PRO-Fc was also detectable in the liver by western blot indicating that some systemic exposure had occurred (Fig. 3h). This observation can most likely be attributed to vascular leakage of the AAV9 vector into systemic circulation from the site of injection [34]. From these data, we determine that GDF11PRO-Fc induces skeletal muscle hypertrophy in adult mice. Additionally, these effects are at least partially mediated by local blockade of GDF11/MSTN on skeletal muscle, probably via inhibition of ligand interactions with ActRII on skeletal muscle tissue.

Systemic GDF11PRO-Fc gene delivery increases skeletal muscle mass and strength in mdx mice

Next, we evaluated the impact of systemic GDF11PRO-Fc expression in mdx mice to determine if GDF11PRO-Fc could also induce hypertrophy and increase strength in dystrophic skeletal muscle. For the purposes of this trial, the GDF11PRO-Fc construct was modified with a mutation to render it impervious to endogenous BMP1/TLD-like metalloproteinase cleavage (GDF11PRO-Fc D122A) and increase factor persistence in systemic circulation [21]. For these experiments, both AAV9-GDF11PRO-Fc and AAV9-GDF11PRO-Fc D122A were evaluated to establish whether the mutated D122A transgene would lead to a more pronounced effect in vivo. To achieve systemic expression, mdx mice were treated with vector or vehicle by tail vein injection. Following intravenous delivery, the AAV9 vector transduces the mouse liver with high efficiency. Transduced hepatocytes can then express the transgene and secrete the protein product into systemic circulation [35].

Starting from 3 weeks post-injection, we observed a significant increase in body weight that persisted for the duration of the trial with AAV9-GDF11PRO-Fc (+ 7.7% at 12 weeks; p = 0.0094) and AAV9-GDF11PRO-Fc D122A (+ 11% at 12 weeks; p = 0.006) treatment (Fig. 4a). It should be noted that, due to the dystrophic pathology, mdx mice typically exhibit compensatory muscle hypertrophy, which may partially mask the increase in muscle mass induced by GDF11PRO-Fc expression. In regards to muscle function tests, treated mice displayed increased forelimb grip strength, even after normalizing to bodyweight. However, the difference in normalized forelimb grip strength only reached statistical significance in the AAV9-GDF11PRO-Fc D122A group. At 12 weeks post-treatment, the average normalized forelimb grip strength was increased by + 28% (p = 0.0873) and + 36% (p = 0.0248) in mice treated with AAV9-GDF11PRO-Fc and AAV9-GDF11PRO-Fc D122A, respectively (Fig. 4b). Rotarod performance was also improved by AAV9-GDF11PRO-Fc D122A treatment on average (+ 93% increase in rotarod latency time; p = 0.0127). Rotarod performance was also improved in the AAV9-GDF11PRO-Fc group, but this difference did not reach statistical significance (+ 44% increase in rotarod latency time; p = 0.2835; Fig. 4c). There was no difference observed in treadmill running time across any of the groups (Fig. 4d).

Fig. 4 GDF11PRO-Fc improves grip strength and rotarod performance in dystrophic mdx mice. 6-week-old mdx mice were treated with AAV9-GDF11PRO-Fc (n = 7), AAV9-GDF11PRO-Fc D122A (n = 7), or vehicle (n = 7) via tail vein injection. a Average bodyweight over time. b Forelimb grip strength normalized to bodyweight over time. c Best rotarod time-to-fall recorded from pre-treatment (baseline) and 12 weeks post-treatment. The best time from three attempts was used for analysis. d Total distance run on treadmill endurance test from pre-treatment (baseline) and 12 weeks post-treatment. All error bars represent mean ± SEM. *p < 0.05; **p < 0.01; n.s. not significant; compared to vehicle-treated control Full size image

At the end of the 12-week trial, muscle hypertrophy was grossly evident in the treated mice (Fig. 5a). Average wet tissue masses of the tibialis anterior, gastrocnemius, and quadriceps were increased by + 17% (p = 0.0472), + 20% (p = 0.0011), and + 14% (p = 0.0333), respectively, in the AAV9-GDF11PRO-Fc group. In the AAV9-GDF11PRO-Fc D122A group, these values were further increased to + 26% (p = 0.0030), + 31% (p = 0.0003), and + 23% (p = 0.0009) over vehicle-treated control for the change in average tibialis anterior mass, gastrocnemius mass, and quadriceps mass, respectively. In addition, the diaphragm wet tissue mass was increased by + 19% (p = 0.0162) and + 26% (p = 0.0014) in the AAV9-GDF11PRO-Fc and AAV9-GDF11PRO-Fc D122A groups, respectively. Interestingly, heart mass was not significantly changed by treatment. The average gastrocnemius myofiber cross-section area was higher in mice treated with AAV9-GDF11PRO-Fc (+ 23%; p = 0.0226) and AAV9-GDF11PRO-Fc D122A (+ 25%; p = 0.0170; Fig. 5c and d). In the MFD distribution analysis, MFD tended to peak around 20–30 μm across all the groups, which is likely due to the higher proportion of small regenerating myofibers in dystrophic muscle. However, treatment with AAV9-GDF11PRO-Fc and AAV9-GDF11PRO-Fc D122A did result in an increased proportion of myofibers with MFDs higher than 64 μm, suggesting that factor expression had induced myofiber hypertrophy (Fig. 5e) in skeletal muscle.

Fig. 5 GDF11PRO-Fc induces skeletal muscle hypertrophy in dystrophic mdx mice. 6-week-old mdx mice were treated with AAV9-GDF11PRO-Fc (n = 7), AAV9-GDF11PRO-Fc D122A (n = 7), or vehicle (n = 7) via tail vein injection. Mice were euthanized 12 weeks post-treatment. a Representative gross hindlimb musculature. b Wet tissue weight of limb muscles, diaphragm, and heart. c Representative immunofluorescence images of gastrocnemius cross-sections stained with AlexaFluor-488-conjugated WGA to visualize myofibers. Scale bars represent 100 μm. d Average myofiber cross-section area in the gastrocnemius. e Myofiber MFD distribution in the gastrocnemius. A minimum of 500 myofibers were measured per mouse. f Identification of GDF11PRO-Fc by western blot in liver tissue lysate and serum of mice treated with AAV9-GDF11PRO-Fc. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control in liver tissue lysates. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant; compared to vehicle-treated control Full size image

Western blot analysis confirmed protein expression of the transgenes in liver and serum. As expected, the band at 58 kDa corresponding to full-length GDF11PRO-Fc or GDF11PRO-Fc D122A was detectable in treated mice. Serum levels of the full-length propeptide were slightly higher in mice treated with AAV9-GDF11PRO-Fc D122A than in those treated with AAV9-GDF11PRO-Fc, suggesting that serum persistence of the active propeptide was increased by the D122A mutation (Fig. 5f).

Overall, these results indicate systemic expression of GDF11PRO-Fc and GDF11PRO-Fc D122A increases skeletal muscle mass in dystrophic mdx mice and that this skeletal muscle hypertrophy is associated with improvements in grip strength and motor coordination. However, treatment did not seem to affect muscle endurance based upon treadmill running performance. Additionally, the D122A mutant appeared to be slightly more effective at increasing muscle mass and function, presumably due to improved serum persistence.

Systemic GDF11PRO-Fc gene delivery does not reduce the dystrophic pathology in mdx mice

A number of studies have reported that blockade of TGF-β-ActRII-SMAD2/3 signaling, whether by MSTN inhibition or blockade of ActRII, may have therapeutic benefit in animal models of DMD [36,37,38,39,40,41,42,43,44,45]. To determine the impact of AAV9-GDF11PRO-Fc treatment on the dystrophic pathology, we performed histological analysis of limb muscles and the diaphragm.

Characteristic signs of the dystrophic pathology, including muscle necrosis, central nucleation, intramuscular collagen deposition, and presence of inflammatory infiltrates were evident across all the mdx groups (Fig. 6a). In the gastrocnemius, the fibrotic area percentage was reduced by − 39% (p = 0.0273) in mice treated with AAV9-GDF11PRO-Fc. Fibrosis in the gastrocnemius was also slightly decreased in the AAV9-GDF11PRO-Fc D122A group; however, due to high intersubject variability in limb muscle intramuscular fibrosis, this difference did not reach statistical significance (− 28%; p = 0.1230; Fig. 6b). There was also no significant difference observed in the proportion of centrally nucleated myofibers, suggesting that myofiber regeneration was actively occurring across all the groups (Fig. 6c). Serum CK, a marker of muscle breakdown, was also not significantly changed by treatment (Fig. 6d). In addition, localized deterioration of myofiber membrane integrity, as measured by anti-mouse IgG immunofluorescence staining, was evident across all the mdx groups. Unexpectedly, mdx mice treated with AAV9-GDF11PRO-Fc D122A exhibited a slight increase in IgG-positive area on average, but this difference did not reach statistical significance (+ 55%; p = 0.1729; Fig. 6e and f; Additional file 1: Figure S5). Obvious signs of myofiber necrosis, regeneration, central nucleation, and IgG penetrance were not apparent in the wild type C57BL/10J gastrocnemius, indicating that these markers are specific to the dystrophic pathology (Fig. 6a–f; Additional file 1: Figure S5) [46].

Fig. 6 GDF11PRO-Fc does not mitigate the dystrophic pathology in mdx mice. 6-week-old mdx mice were treated with AAV9-GDF11PRO-Fc (n = 7), AAV9-GDF11PRO-Fc D122A (n = 7) or vehicle (n = 7) via tail vein injection. Age-matched C57BL/10J (n = 5) mice were also included as a wild type control. Mice were euthanized 12 weeks post-treatment. a Representative HE and MTC staining from gastrocnemius cross-sections of C57BL/10J and mdx mice. Central nucleation is evident in all the mdx groups. Localized areas of myofiber necrosis and regeneration are marked with a black arrow. Fibrotic area is represented by the blue region on MTC staining. Scale bars represent 50 μm in HE sections and 100 μm in MTC sections. b Fibrotic area in the gastrocnemius expressed as a percentage of the total muscle cross-section area. c Percentage of myofibers exhibiting central nucleation. d Measurement of circulating levels of serum CK, a marker of muscle damage. e Representative immunofluorescence images of mouse IgG staining (red) in gastrocnemius tissue cross-sections. Positive staining for IgG indicates myofiber permeability to serum proteins and loss of membrane integrity. Sections were co-stained with WGA to visualize individual myofibers. Zoomed-in area is marked with a white box. Characteristic IgG-positive myofibers are depicted with a white arrow. Scale bars represent 100 μm. f The area of IgG-positive myofibers expressed as a percentage of the total muscle cross-section area. g Representative HE and MTC staining from diaphragm cross-sections of C57BL/10J and mdx mice. Extensive pathology is apparent in mdx but not C57BL/10J mice. Scale bars represent 100 μm. h Fibrotic area in the diaphragm expressed as a percentage of the total cross-section area. *p < 0.05; ***p < 0.001; ****p < 0.0001; n.s. not significant; compared to vehicle-treated mdx controls Full size image

In the diaphragm, H&E staining revealed extensive necrosis and intramuscular collagen deposition in both treated and untreated mdx groups (Fig. 6g). Similar to what was observed in the gastrocnemius muscle, treatment with AAV9-GDF11PRO-Fc or AAV9-GDF11PRO-Fc D122A produced a mild reduction in diaphragm intramuscular fibrosis overall. The average fibrotic area percentage in the diaphragm was reduced by 15% (p = 0.0328) and 17% (p = 0.019) with AAV9-GDF11PRO-Fc and AAV9-GDF11PRO-Fc D122A treatment, respectively (Fig. 6h). As expected, these pathological features were largely absent in the diaphragm of wild type C57BL/10J mice (Fig. 6g and h). From these data, we determine that GDF11PRO-Fc and GDF11PRO-Fc D122A are able to mildly inhibit intramuscular collagen deposition in the limb muscles and diaphragm over a 3-month period. However, treatment does not appear to halt the ongoing cycle of muscle degeneration and regeneration in the gastrocnemius or diaphragm of adult mdx mice.