Fig 1: Targeting ET-1-mediated secretion of fibrotic FAPs improves myoblast fusion. (A) Principal component analysis (PCA) plot prepared following transcriptome analysis of FAPs from MCT (yellow), FibMCT (orange), and FibMOP (red) muscle biopsies. Each dot represents cells from one patient. (B) Quantification of ET-1 protein levels in unconcentrated conditioned medium from differentiated human myoblasts after 24, 48, and 72 h of differentiation, reanalyzed from a previous study. 26 (C) RT-qPCR quantification of EDNRb gene expression normalized to RPLP0 expression in FAPs from MCT, FibMCT, and FibMOP muscle biopsies (n = 5 biological replicates). (D) RT-qPCR quantification of Col7a1 gene expression normalized to RPLP0 expression in FAPs from MCT, FibMCT, and FibMOP muscle biopsies (n = 3 biological replicates). (E) FAPs from MCT, FibMCT, and FibMOP muscle biopsies were cultured in the presence of ET-1 (40 nM) or ET-1 and bosentan (10 µM) for 3 days in proliferation medium containing 1% FBS. Left: Immunofluorescence analysis of Phalloidin (red), Hoechst (blue) and collagen 7a1 (green) was performed. Right: Quantification of the percentage of COL7A1-positive cells (n = 3–4 biological replicates). (F) Quantification of the percentage of EdU incorporation after treatment of FAPs from MCT, FibMCT, and FibMOP muscle biopsies with ET-1 (40 nM) or ET-1 and bosentan (10 µM) for 3 days in proliferation medium containing 1% FBS (n = 3–4 biological replicates). (G) Myoblasts were cocultured alone or with FAPs from fibrotic muscles at a 70/30% ratio for 5 days in differentiation medium. Bosentan (10 µM) was added on days 0 and 3 of differentiation. Left: The fusion index was assessed by Desmin staining (n = 3 biological replicates). Right: Desmin immunostaining and Hoechst staining (scale bar = 100 µm). Data are presented as means ± SD, with P-values obtained by ordinary or RM one-way ANOVA test followed by Tukey's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).