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The consequences of the genetic reduction of the extracellular protein versican on hindlimb muscle function and structure depend on muscle fibre type and age in dystrophic mdx mice

N.L. McRae, L.G. Forgan, A. Addinsall, D.R. McCulloch and N. Stupka, School of Medicine, Deakin University, 75 Pigdons Road, Waurn Ponds, VIC 3216, Australia.

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and ineffective repair. Accumulation of extracellular matrix (ECM) is a consequence of the disease and exacerbates the pathology. Versican, a transitional ECM proteoglycan, is upregulated in fibrotic lesions of dystrophic muscles. Versican is remodelled by A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) versicanases to generate the bioactive DPEAAE versikine fragment. In cultured C2C12 muscle cells, versican expression is down-regulated by glucocorticoids, the only treatment with clinical efficacy in DMD. Thus, we hypothesized that the genetic reduction of versican would ameliorate the pathology of dystrophic hindlimb muscles from mdx mice. Hence, female mdx mice were crossed with male mice heterozygous for a transgene insertional mutation in the versican gene (hdf – heart defect mice (Mjaatvedt et al., 1998)). All experiments were carried out in accordance with NHMRC guidelines with approval from the Animal Ethics Committee at Deakin University (A79-2011; G06-2015). At 6 or 26 weeks of age, F1 mdx and mdx-hdf male pups were anesthetized via intraperitoneal injection of medetomidine (0.6 mg/kg), midazolam (5 mg/kg) and fentanyl (0.05 mg/kg) until unresponsive to tactile stimuli. Fast twitch extensor digitorum longus (EDL), and slow twitch soleus muscles were surgically excised, and then muscle strength, fatiguability and recovery from fatigue were assessed in vitro (1300A Whole Mouse Test System, Aurora Scientific). Mice were euthanized by cervical dislocation and tissues collected for molecular and histological analysis.

To confirm the genetic reduction of versican, immunohistochemical staining and specific antibodies for versican (anti-GAGβ domain; Merk Millipore) and versikine (versican V0, V1 neo antibody; ThermoFisher Scientific) were used. Versican and versikine protein levels were greater in dystrophic hindlimb muscle at 6 versus 26 weeks of age. Versikine, but not versican, protein levels were decreased 51% (P=0.01) in EDL muscles of mdx-hdf mice. Whereas, in soleus muscles of 6 week old mdx-hdf mice, versican expression was decreased by 91% (P=0.004) with similar versikine expression. By 26 weeks of age, this decrease in versican expression was no longer apparent.

Bodyweight, EDL and soleus muscle mass were similar in mdx-hdf and mdx mice. The genetic reduction of versican had no effect on EDL muscle strength normalized to muscle size (sPo) at 6 and 26 weeks of age. Following 4 min of intermittent, submaximal (60 Hz) stimulation, EDL muscles from 6 week old mdx-hdf mice fatigued less (P<0.001; main effect GLM ANOVA) and recovered force better (P<0.001; main effect GLM ANOVA) than those from mdx littermates. By 26 weeks, this improvement in EDL muscle endurance was lost. Soleus muscles from 6 week old mdx-hdf mice were stronger than those from mdx mice, as indicated by an upward shift of the force (sPo) frequency curve (P=0.02; main effect GLM ANOVA). However by 26 weeks, soleus muscles from mdx-hdf mice produced less force (P<0.001; main effect GLM ANOVA). Fatigability was reduced in soleus muscles from mdx-hdf mice at 6 (P<0.03, main effect GLM ANOVA) and 26 weeks (P<0.02, main effect GLM ANOVA), with no change in force recovery.

The genetic reduction of versican had distinct effects on muscle pathology depending on fibre type and age. At 6 weeks, EDL and soleus muscles from mdx-hdf mice had fewer muscle fibres/mm2 of tissue (P=0.01 and P=0.04, respectively, t-test). In soleus muscles, this was associated with a trend for an increase in fibre size (P=0.07), which may contribute to the increase in muscle strength. Whilst at 26 weeks, the number of muscle fibres/mm2 was similar in EDL and soleus muscles from mdx-hdf and mdx mice. Muscle fibre cross-sectional area was reduced in soleus, but not EDL, muscles from mdx-hdf mice. Only in soleus muscles, was an increase in mRNA transcript abundance of genes associated with ECM synthesis and remodelling observed; including, TGF-β1 (P=0.02), collagen a1 (P=0.05) and a trend in ADAMTS5 (P=0.07). All together, these changes may account for the decrease in soleus muscle strength in mdx-hdf mice at 26 weeks of age.

Overall, our data demonstrate that the genetic reduction of versican has distinct effects on muscle structure and function depending on the muscle fibre type and age, specifically postnatal growth. These factors need to be considered when developing preclinical strategies to target fibrosis and ECM remodelling in dystrophic muscles.

Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR. (1998). Dev Biol 202, 56-66.