Chantal A. Coles

Versican Processing by a Disintegrin-like and Metalloproteinase Domain with Thrombospondin-1 Repeats Proteinases-5 and -15 Facilitates Myoblast Fusion

Background: Skeletal muscle fiber formation requires myoblast cell-cell membrane contact and fusion. Results: A versican-rich pericellular matrix surrounding myoblasts is proteolytically cleared by ADAMTS versicanases facilitating myoblast contact and fusion. Conclusion: Versican processing by ADAMTS versicanases contribute to muscle fiber formation. Significance: Targeting versican remodeling could enhance the regenerative capacity of muscle by improving muscle fiber fusion during regeneration. Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.

Alterations in the expression of leukemia inhibitory factor following exercise: comparisons between wild-type and mdx muscles.

Background: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine, belonging to the interleukin-6 family of cytokines, that has been suggested to have positive effects on myogenesis following injury and to minimise dystrophic pathology in mdx mice. Previous reports have suggested that Lif mRNA is up-regulated in the limb and diaphragm muscles of mdx mice, in human cases of dystrophy and acutely following exercise. This study examined expression of Lif mRNA in the quadriceps muscles of mdx and wild-type mice that were either sedentary or allowed to exercise voluntarily for two weeks. Results: Exercise caused a decrease in Lif mRNA expression in wild-type muscle, but this was not the case in mdx muscle. Lif mRNA levels in sedentary mdx mice were similar to those in exercised wild type muscles, and in mdx mice there was no further decrease in levels following exercise. Similar down-regulation of Lif mRNA was observed in the tibialis anterior and diaphragm muscles of mdx mice at three and six weeks of age respectively, compared with wild-type controls. Transcripts for the LIF receptor (Lifr) were also down-regulated in these mdx muscles, suggesting LIF activity may be minimised in dystrophic muscle. However fluorescent immunohistochemical labeling of LIF did not correlate with transcript expression data, as LIF immunoreactivity could not be detected in wild-type muscle, where mRNA expression was high, but was present in dystrophic muscle where mRNA expression was low. This study also described the translocation of membrane proteins, including LIFR, to the nuclei of syncytial muscle cells during differentiation and fusion. In addition this study demonstrates that survival of donor myoblasts injected into dystrophic muscle was enhanced by co-administration of recombinant LIF. Conclusions: This study provides new evidence to support a role for LIF in normal muscle biology in response to exercise. Although expression levels of Lif transcript in mdx muscles were not consistent with previous studies, the detection of LIF protein in mdx muscle but not wild-type muscle supports a role for LIF in dystrophy. This study also provides evidence of the differential localisation of the LIFR, and the potential for anti-inflammatory actions of LIF that promote survival of transplanted myoblasts in dystrophic muscle. *corresponding author: Jason White, Muscular Dystrophy Research Group, Murdoch Childrens Research Institute; email: jasondw@unimelb.edu.au

A disintegrin and metalloprotease-12 is type I myofiber specific in Bos taurus and Bos indicus cattle.

A disintegrin and metalloproteinase-12 (ADAM12) is involved in the regulation of myogenesis and adipogenesis and is of interest as a potential target to manipulate skeletal muscle development and intramuscular fat (IMF) deposition in cattle to increase beef yield and improve meat quality. The longissimus thoracis muscle (LM) and semitendinosus muscle (STM) from 5 Bos taurus (Angus) and 5 Bos indicus (Brahman) cattle were collected for histological and ADAM12 gene and protein expression analysis. Myofiber typing was used to determine if ADAM12 expression patterns related to differences in muscling and IMF deposition, which are influenced by proportions of the different myofiber types. The STM was found to contain a higher proportion of glycolytic myofibers than the LM, which contained a greater proportion of oxidative myofibers (myofiber ratio of glycolytic to more oxidative types in LM and STM of 1.1 and 3.5, respectively; P < 0.05). ADAM12 gene expression, fluorescent immunohistochemical staining for ADAM12, and image analysis found ADAM12 to be greater in the LM (P < 0.05). Regression analysis found a strong, positive relationship for the distribution of ADAM12 against the proportion of type I myofibers (P < 0.05, r(2) = 0.86). These findings suggest ADAM12 is upregulated in muscles with more slow-oxidative myofibres, such as the LM, and is linked to type I myofibers in cattle. ADAM12 may be important in the regulation and maintenance slow myofibers in the LM muscle.

Proliferation Rates of Bovine Primary Muscle Cells Relate to Liveweight and Carcase Weight in Cattle

Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5–20 hours in culture) in vitro (P < 0.05). The proliferation rates of myoblasts during early stages of culture in vitro were also found to be positively related to the liveweight and carcase weight of cattle (P < 0.05). Gene expression of MYF5 was also found to be significantly down-regulated in WagyuX compared with Angus cattle (P < 0.05). These findings suggest that early events during myogenesis are important for determining liveweight and caracase weights in cattle.

Ablation of Grb10 Specifically in Muscle Impacts Muscle Size and Glucose Metabolism in Mice

Grb10 is an adaptor-type signaling protein most highly expressed in tissues involved in insulin action and glucose metabolism, such as muscle, pancreas, and adipose. Germline deletion of Grb10 in mice creates a phenotype with larger muscles and improved glucose homeostasis. However, it has not been determined whether Grb10 ablation specifically in muscle is sufficient to induce hypermuscularity or affect whole body glucose metabolism. In this study we generated muscle-specific Grb10-deficient mice (Grb10-mKO) by crossing Grb10flox/flox mice with mice expressing Cre recombinase under control of the human α-skeletal actin promoter. One-year-old Grb10-mKO mice had enlarged muscles, with greater cross-sectional area of fibers compared with wild-type (WT) mice. This degree of hypermuscularity did not affect whole body glucose homeostasis under basal conditions. However, hyperinsulinemic/euglycemic clamp studies revealed that Grb10-mKO mice had greater glucose uptake into muscles compared with WT mice. Insulin signaling was increased at the level of phospho-Akt in muscle of Grb10-mKO mice compared with WT mice, consistent with a role of Grb10 as a modulator of proximal insulin receptor signaling. We conclude that ablation of Grb10 in muscle is sufficient to affect muscle size and metabolism, supporting an important role for this protein in growth and metabolic pathways.

Low dose resveratrol promotes hypertrophy in wildtype skeletal muscle and reduces damage in skeletal muscle of exercised mdx mice.

Duchenne muscular dystrophy (DMD) is a progressive and fatal neuromuscular disorder for which there is no treatment. Therapies to restore dystrophin deficiency are not ready for clinical use and long-term efficiency is yet to be established. Therefore, there is a need to develop alternative strategies to treat DMD. Resveratrol is a nutraceutical with anti-inflammatory properties and previous studies have shown that high doses can benefit mdx mice. We treated 4-week-old mdx and wildtype mice with low-dose resveratrol (5mg/kg bodyweight/day) for 15 weeks. A voluntary exercise protocol was added to test if low dose resveratrol could reduce exercise-induced damage. We showed that resveratrol promoted skeletal muscle hypertrophy in the wildtype mice. There was no change in markers of pathology in the mdx mice; however, the low-dose resveratrol reduced exercised induced damage. Gene expression of immune cell markers such as CD86, CD163 and PCNA was reduced; however signalling targets associated with resveratrol’s mechanism of action of action including SIRT1 and NF-κB were unchanged. In conclusion, low-dose resveratrol was not effective in reducing disease pathology; however, its ability to promote hypertrophy in wildtype skeletal muscle could have direct applications to the livestock industry or in sports medicine.

Benfotiamine reduces pathology and improves muscle function in mdx mice.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal neuromuscular disease which arises from mutations in the dystrophin gene (DMD) that result in the absence or severe reduction of the cytoskeletal protein dystrophin. In addition to the primary dystrophin defect, secondary processes such as inflammation, calcium influx, dysregulated autophagy and fibrosis exacerbate dystrophic pathology and thus increase disease progression. While therapies to restore dystrophin deficiency are being developed, strategies which target these secondary processes could be of benefit to patients. Benfotiamine is a lipid soluble precursor to thiamine that can reduce secondary processes such as inflammation and oxidative stress in diabetic patients. As such we tested it in the mdx mouse model of DMD and found that benfotiamine reduced multiple markers of dystrophic pathology and improved grip strength. In addition, members of the utrophin and dystrophin glycoprotein complexes were significantly increased at the sarcolemma which could improve cell adhesion. We also demonstrated that benfotiamine treatment lowered the expression of macrophage markers and pro-inflammatory cytokines suggesting that benfotiamine is reducing dystrophic pathology by acting on inflammatory processes.

Nutraceuticals and Their Potential to Treat Duchenne Muscular Dystrophy: Separating the Credible from the Conjecture

In recent years, complementary and alternative medicine has become increasingly popular. This trend has not escaped the Duchenne Muscular Dystrophy community with one study showing that 80% of caregivers have provided their Duchenne patients with complementary and alternative medicine in conjunction with their traditional treatments. These statistics are concerning given that many supplements are taken based on purely “anecdotal” evidence. Many nutraceuticals are thought to have anti-inflammatory or anti-oxidant effects. Given that dystrophic pathology is exacerbated by inflammation and oxidative stress these nutraceuticals could have some therapeutic benefit for Duchenne Muscular Dystrophy (DMD). This review gathers and evaluates the peer-reviewed scientific studies that have used nutraceuticals in clinical or pre-clinical trials for DMD and thus separates the credible from the conjecture.