Pre-clinical developments in neuromuscular disorders

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin gene and characterized by progressive skeletal muscle weakness. The lack of dystrophin expression induces sarcolemma instability that leads to muscle necrosis and replacement of muscle tissue by fibro-adipose tissue. Although some growth factors have been widely studied in the process of fibrogenesis, the pathways activated by PDGF-AA in muscular dystrophies have not been described so far. We report here the effects of PDGF-AA on the fibrotic process in muscle dystrophies by performing a proteomic study in FAPs isolated from DMD patients and treated with PDGF-AA. Mass spectrometry revealed that proteins of the RhoA pathway were upregulated in PDGF-AA treated FAPs compared to non-treated. In vitro studies showed that RhoA/ROCK2 pathway is activated by PDGF-AA and induces the activation of FAPs in vitro, which includes increased actin reorganization, cell proliferation and migration. The inhibition of RhoA/ROCK signalling pathway by two inhibitors, C3-exoenzyme and fasudil, attenuated the effects of PDGF-AA. The blockade effects of RhoA/ROCK pathway were also analysed in vivo by treating the dba/2J-mdx murine model of DMD with fasudil. Grip strength test showed an improvement in the muscle function and histological studies demonstrated a significant reduction of the fibrotic area present in quadriceps. Fasudil also induced a decreased number of macrophages together with a reduction of macrophage polarization toward M2-like phenotype. The results of the study suggest that RhoA signalling is increased in DMD patients and that PDGF-AA activates this pathway is DMD FAPs. This pathway induces FAPs activation which is essential for the development of fibrosis in muscular dystrophies. Our results indicate that the inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies.