Endocannabinoids and Sphingosine 1-Phosphate: a crosstalk between two lipid systems in the regulation of myoblasts metabolism and differentiation

Skeletal muscle regeneration after trauma or during myopathies depends on myogenic differentiation process that involves cell cycle arrest and fusion of myoblasts into myofibers. Among different signals, sphingosine 1-phosphate (S1P) and endocannabinoids affect this process. S1P is a bioactive sphingolipid that exerts a plethora of biological functions mainly via the engagement of five specific G protein-coupled receptors, S1P1-5. Specifically, S1P2 receptor and the selective S1P transporter Spinster homolog 2 are known to be involved in myogenic differentiation induced by the sphingolipid1. Endocannabinoid system regulates myogenic differentiation since Cannabinoid receptor 1 (CB1) antagonism was shown to enhance the expression of myogenic markers in human satellite cells2. This study, performed in collaboration with the University of Teramo, uncovers a functional interplay between the two bioactive lipid systems in C2C12 murine myoblasts. S1P, indeed, increased TRPV1 expression and counteracted the effect on the mitochondrial membrane potential of methanandamide (mAEA), a stable analogue of the endocannabinoid anandamide. On this basis, the effect of mAEA on myogenic differentiation was investigated, following the expression of myogenic markers by Real-Time PCR, western blot and confocal immunofluorescence analysis. The involvement of TRPV1 in myogenesis was demonstrated using pharmacological approaches and RNA interference. Noteworthy, our results indicate a dose dependent anti-myogenic action of mAEA, that is worsened by TRPV1 antagonism or downregulation and reverted by CB1 antagonist. During myogenic differentiation TRPV1 mRNA levels increase up to 130 folds in 4 day-differentiated myotubes compared to control myoblasts. Noteworthy, both S1P and TRPV1 agonist capsaicin, appear to exert a positive effect on myogenic differentiation which is attenuated by TRPV1 antagonism and downregulation. These data highlight the importance of TRPV1 action on the myogenic differentiation process and suggest opposite roles of eCBs receptors in myogenic differentiation: CB1 is anti-myogenic while TRPV1 exerts a promyogenic effect. Moreover, S1P is known to induce the release of Ca2+ in the cytoplasm. Since TRPV1 is a Ca2+ channel and given its upregulation upon S1P treatment, we investigated if S1P signalling could rely on TRPV1 for the Ca2+ increase in C2C12 myoblast. The data did not show an involvement of TRPV1 in the S1P-induced Ca2+ increase since it was not reverted by TRPV1 specific antagonism. Further studies will clarify the interaction between S1P and eCBs signalling pathways in myogenesis, possibly leading to the identification of new therapeutic targets for muscle disorders characterized by impaired myogenic differentiation. References: 1. Cencetti. et al. Sphingosine 1-phosphate lyase blockade elicits myogenic differentiation of murine myoblasts acting via Spns2/S1P2 receptor axis. BBA Molecular and cell biology of lipids. 1865(9), 158759 (2020) 2. Iannotti, F. A. et al. Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy. Nat. Commun. 9, (2018).