THE EFFECTS OF CANNABIDIOL AND CANNABINOL ON C2C12 MYOBLAST PROLIFERATION AND DIFFERENTIATION

Abstract

Increasing interest has emerged in the field of nutrition and its role in promoting skeletal muscle growth. Recently, studies using both in vitro and in vivo models have suggested that cannabidiol – a constituent of Cannabis Sativa – can increase the growth and regenerative capacity of skeletal muscle stem cells. Other isolated compounds, such as cannabinol, have demonstrated anti-inflammatory effects in vivo. Due to the potential benefits of both compounds, our primary objective was to further elucidate the effects of cannabidiol and cannabinol on murine C2C12 myoblast proliferation and differentiation. We hypothesized that supplementation of cannabidiol and cannabinol would augment gene expression of myogenin, leading to enhanced myotube formation; as well as, induce greater gene expression of Myf5 and MyoD, accompanied by increased cell proliferation. In relation to skeletal muscle growth, myostatin and follistatin can substantially impact the regulation of hypertrophy; with down-regulation of myostatin being a potent stimulus for muscle growth, and follistatin being the antagonist to myostatin, we therefore examined if cannabidiol or cannabinol influenced these two proteins, as a possible rationale for increased myogenesis. In this study, cells were treated with either: (1) cannabidiol, (2) cannabinol, (3) or vehicle control (methanol). Cells were grown for 48 hrs in their respective media, the MTT assay was used to assess proliferation. Muscle differentiation experiments required cells to grow for seven days with media supplemented with the respective compound. The media was changed every 48 hrs. The extent of muscle differentiation was assessed via immunocytochemical and qPCR analysis. In preliminary experiments, cell proliferation was influenced by the duration of which cells were exposed to the compound and concentration of the compound within the media. It was noted that changing growth media and compound every 24 hrs augmented the proliferative response compared to leaving it on for 48 hrs for both cannabidiol and cannabinol (p<0.05). Furthermore, supplementing cells with cannabidiol at a 1 or 5 uM concentration resulted in considerable cell growth compared to vehicle control (p<0.0001). Cannabinol at 5 uM showed the same effect (p<0.0001). We also quantified the mRNA expression of genes involved in the myogenic regulatory pathway in proliferating and differentiating cells. Herein we report that using a 5 uM concentration of cannabidiol or cannabinol did not increase the expression of any of these genes in proliferating or differentiating cells. These findings help further characterize the effects of cannabidiol and cannabinol on the myogenic response.