Background
The lack of dystrophin expression in Duchenne muscular dystrophy (DMD) induces muscle fibre and replacement by fibro-adipose tissue. Although the role of some growth factors in the process of fibrogenesis has been studied, pathways activated by PDGF-AA have not been described so far. Our
Conclusions
Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies.
Methods
Skeletal muscle fibro-adipogenic progenitor cells (FAPs) from three DMD treated with PDGF-AA at 50 ng/mL were analysed by quantitative mass spectrometry-based proteomics. Western-blot, immunofluorescence, and G-LISA were used to confirm the mass spectrometry
Results
Mass spectrometry revealed that RhoA pathway proteins were up-regulated in treated compared with non-treated DMD FAPs (n = 3, mean age = 8 ± 1.15 years old). Validation of proteomic data showed that Arhgef2 expression was significantly increased in DMD muscles compared with healthy controls by a 7.7-fold increase (n = 2, mean age = 8 ± 1.14 years old). In vitro studies showed that RhoA/ROCK2 pathway was significantly activated by PDGF-AA (n = 3, 1.88-fold increase, P < 0.01) and both C3-exoenzyme and fasudil blocked that activation (n = 3, P < 0.05 and P < 0.001, respectively). The activation of RhoA pathway by PDGF-AA promoted a significant increase in proliferation and migration of FAPs (n = 3, P < 0.001), while C3-exoenzyme and fasudil inhibited FAPs proliferation at 72 h and migration at 48 and 72 h (n = 3, P < 0.001). In vivo studies showed that fasudil improved muscle function (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, 1.76-fold increase, P < 0.013), and histological studies demonstrated a 23% reduction of collagen-I expression area (n = 5 non-treated dba/2J-mdx and n = 6 treated dba/2J-mdx, P < 0.01). Conclusions: Our results suggest that PDGF-AA promotes the activation of RhoA pathway in FAPs from DMD patients. This pathway could be involved in FAPs activation promoting its proliferation, migration, and actin reorganization, which represents the beginning of the fibrotic process. The inhibition of RhoA pathway could be considered as a potential therapeutic target for muscle fibrosis in patients with muscular dystrophies.