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Genetic reduction of an extracellular matrix gene improves contractile function in dystrophic mdx mice

D.R. McCulloch,1 N.L. McRae,1 R. Kelly,1 L. Forgan2 and N. Stupka,1 1School of Medicine, Deakin University, Warun Ponds, VIC 3216, Australia and 2School of Life and Environmental Sciences, Deakin University, Warun Ponds, VIC 3216, Australia.

Versican is a chondroitin sulphate proteoglycan (CSPG) that serves as a transitional scaffold for extracellular matrix (ECM) deposition. Remodeling of versican by ADAMTS proteoglycanases regulates a plethora of cell behaviors associated with developmental morphogenesis. In skeletal muscle, expression of V0/V1 versican isoforms is increased during myogenesis and clearance of a versican rich pericellular matrix by ADAMTS5 and ADAMTS15 is necessary for efficient myoblast fusion (Stupka et al., 2013). Versican is also a mediator of cellular inflammatory processes, including macrophage polarization (Chang et al., 2012).

Duchenne muscular dystrophy (DMD) is a fatal hereditary disease caused by a mutation in the dystrophin gene rendering skeletal muscles highly susceptible to contraction-induced injury. In DMD, there is an inevitable expansion of the ECM and chronic inflammation leading to loss of contractile tissue. Versican protein levels are significantly elevated in skeletal muscle biopsies from patients with DMD, but absent in healthy, control muscle. In diaphragm and hindlimb muscles of dystrophin deficient mdx mice versican expression is also increased, with greater expression in the more severely affected diaphragm. Therefore, we hypothesised that the genetic reduction of versican will attenuate the pathology of dystrophic mdx mouse muscles.

All experiments were carried out with approval of Deakin University’s Animal Ethics Committee in accordance with NH&MRC guidelines. 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). The resultant F1 male dystrophic pups, mdx-hdf and mdx control littermates, were confirmed by genotyping and Q-RT-PCR. At 6 weeks of age, prior to contractile function testing, mouse body composition was assessed (ESF-005, EchoMRI). Then the mice were anaesthetized via i.p. injection of medetomidine (0.6 mg/kg), midazolam (5 mg/kg) and fentanyl (0.05 mg/kg) such that they were unresponsive to tactile stimuli. Fast twitch extensor digitorum longus (EDL) and the slow twitch soleus muscles were surgically excised, and muscle force production, endurance and recovery from fatigue were assessed in vitro (1300A Whole Mouse Test System, Aurora Scientific; N = 6). Following functional testing, anaesthetized mice were humanely euthanized by cervical dislocation and the heart and hindlimb muscles were collected for molecular and histological analysis.

Hindlimb muscles, specifically the fast twitch tibialis anterior muscle, of mdx-hdf mice had a ~60% reduction in versican (vcan) mRNA transcripts (P=0.04; independent t-test). Body weight, lean mass, fat mass, EDL and soleus muscle mass were similar in mdx-hdf mice and control mdx littermates. Twitch force (Pt), maximal force (Po) and specific force (sPo) were similar in EDL muscles from mdx-hdf mice and mdx littermates. In soleus muscles, Pt and sPo were also not different between mdx-hdf mice and control mdx littermates; however, a trend towards an increase in Po was observed in the mdx-hdf mice (P=0.054; independent t-test). To assess muscle endurance and force recovery, EDL and soleus muscles were stimulated submaximally at 60 Hz every 5 s for 4 min, and then again at 2 min, 5 min and 10 min post fatigue. During 4 min of contractile activity, fast twitch EDL muscles from mdx-hdf mice fatigued less than those from mdx littermates (P<0.001; main effect - general linear model ANOVA). Furthermore, EDL force recovery following fatigue was 50-70% greater in mdx-hdf mice (P=0.003; main effect - general linear model ANOVA).Whereas in soleus muscles of mdx-hdf mice, endurance and force recovery following fatigue were similar as compared to mdx littermates. Our data indicate that a genetic reduction of versican levels significantly improves muscle function on an mdx background. Follow up studies are ongoing to assess the effects of versican reduction on skeletal muscle fibre type and markers of muscle damage, fibrosis and regeneration.

Chang MY, Chan CK, Braun KR, Green PS, O'Brien KD, Chait A, Day AJ, Wight TN. (2012). Monocyte-to-macrophage differentiation: synthesis and secretion of a complex extracellular matrix. Journal of Biological Chemistry, 287, 14122-14135.

Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR. (1998). The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Developmental Biology, 202, 56-66.

Stupka N , Kintakas C, White JD, Fraser FW, Hanciu M, Aramaki-Hattori N, Martin S, Coles C, Collier F, Ward AC, Apte SS, McCulloch DR. (2013). Versican processing by a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats proteinases-5 and -15 facilitates myoblast fusion. Journal of Biological Chemistry, 88, 1907-1917.