Fig 2: Recombinant MSTN suppressed AChR clustering and miR‐206 expression. (a) AChR and MyHC staining of MuSCs cultures treated with or without recombinant MSTN (100 ng/mL) and follistatin (500 ng/mL). Scale bar = 50μm. (b) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. (c) The levels of miR‐206 expression in MuSCs cultures. Mature myotubes differentiated from MAS‐derived MuSCs were treated with control, agrin alone, agrin plus MSTN or agrin plus MSTN and follistatin. (d) AChR and MyHC staining of myotubes transfected with miR‐206 mimics or negative control (NC) prior to treatment with agrin and MSTN. Scale bar = 50 μm. (e) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: MSTN knockdown reversed the negative effect of MAS FAPs on in vitro AChR clustering. (a) MSTN protein and mRNA level in 7 dpi MAS FAPs transfected with negative control (pGPU6/GFP/Neo‐NC) or knockdown vectors (pGPU6/GFP/Neo‐shMSTN). (b) MSTN protein and mRNA level in 7 dpi TA FAPs transfected with negative control (pcDNA3.1(+)) or overexpression vectors (pcDNA3.1(+)‐MSTN). (c) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster counts normalized to myotube area (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN KD FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (d) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster density (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN OE FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (e,f) Transcript levels of Chrna1, Chrne, Chrng, Musk and Rapsn in myotubes treated with CM from NC or KD FAPs, and NC or OE FAPs. KD, knock down; OE, overexpression. The data are shown as mean ± SD. Unpaired Student's t test was used in (a–d). Two‐way ANOVA followed by Sidak post hoc test was used in (e,f). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: FAP‐specific MSTN knockout and pharmacological blockade of MSTN enhanced NMJ regeneration in MAS muscle. (a) AChR immunostaining of transverse sections from intact, PBS‐treated, or ACE‐031‐treated mice. Graphs in the right panel show the quantification of AChR counts per myofibre in MAS (upper) and TA (lower). n = 3 mice/group. (b) Masseter bite force in intact, PBS‐treated and ACE‐031‐treated mice at 30 dpi (upper). Hindlimb grip time assessed in intact, PBS‐treated and ACE‐031‐treated mice at 30 dpi (lower). n = 3 mice/group. (c) Schematic showing the generation of FAPs‐specific MSTN knockout mice (MSTN cKO). (d) AChR labelling of transverse sections of MAS or TA muscles of MSTN cKO mice (MSTNfl/fl; PdgfraCre/+) and control mice (MSTNfl/fl; Pdgfra+/+). Graphs in the right panel show the quantification of AChR counts per myofibre in MAS (upper) and TA (lower). n = 6 mice/group. (e) Masseter bite force (upper) and hindlimb grip time (lower) were measured in intact and injured MSTN cKO mice and controls. n = 3 mice/group. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: MSTN was the paracrine factor that distinguishes MAS‐derived FAPs from TA‐derived FAPs. (a) AChR and MyHC staining of MuSC single culture or cocultures with conditioned medium (CM) of MAS‐ or TA‐isolated FAPs. Scale bar = 50 μm. (b) Graphs show (from left to right) the quantification of myotube area, average AChR cluster size, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 6. (c) Venn diagram depicting the overlap among DEGs between 7 dpi MAS‐ and TA‐derived FAPs (orange), NMJ‐regulating genes (green) and genes encoding secreted proteins (blue). (d) Mstn expression levels were quantified by qPCR. n = 3. (e) Myostatin levels were quantified by ELISA in CM from 7 dpi MAS‐ or TA‐derived FAPs. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used in (b). Unpaired Student's t test was used in (d,e). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.