Fibro-Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

Orofacial and limb muscles differ in embryonic origin and regenerative capacity. Neuromuscular junction (NMJ) regeneration is critical for muscle restoration both histologically and functionally. The relative potential of orofacial and limb muscles to form postsynaptic apparatuses remains elusive. While the role of fibro-adipogenic progenitors (FAPs) in NMJ regeneration has been discussed in limb muscles, it remains unexplored in orofacial muscles.

NMJ regeneration was triggered by freeze injury in masseter (MAS) and tibialis anterior (TA) muscles and assessed using histological and functional tests. FAPs transplantation experiments and coculture with muscle stem cells (MuSCs) were performed to investigate their effects on postsynaptic apparatus formation. Transcriptome profiling of FAPs identified the key secretory molecule involved in NMJ regulation. The effect of this molecule was further investigated using in vitro gain- and loss-of-function assays, conditional knockout transgenic mice and pharmacological blockade.

Immunohistochemistry showed extensive fibrosis surrounded by regenerated myofibres in MAS, whereas no fibrosis but regenerated myofibres in TA. Restored myofibre calibre and resolved fibrosis in the regenerated lesion periphery are observed in both muscles, yet regenerated NMJs remained markedly below the intact level at 30 days post-injury (dpi) only in MAS (-52.1%, p < 0.001). Interestingly, transplantation of FAPs isolated from MAS reduced the number of postsynaptic acetylcholine receptors (AChRs) on regenerated myofibres in recipient TA muscle (-61.3%, p < 0.001). Conditioned medium of FAPs isolated from MAS at 7 dpi impaired AChR clustering on myotubes, decreasing the AChR/myotube area ratio (p < 0.001). RNA-seq analysis of 7 dpi MAS and TA FAPs identified myostatin (Mstn) as the key differentially expressed gene. Mstn transcripts in MAS FAPs were 1.7-fold higher than those in TA FAPs (p < 0.001). In vitro knockdown of Mstn in FAPs isolated from 7 dpi MAS reversed its negative effect on AChR clustering, as evidenced by a 4-fold increase in the AChR/myotube area ratio (p < 0.01). The number of nascent AChR clusters in injured MAS of FAP-specific Mstn knockout mice was higher than that of injured floxed controls (2.7-fold, p < 0.001). Pharmacological blockade of MSTN enhanced postsynaptic AChR neogenesis in MAS.

We demonstrated differential NMJ regeneration in MAS and TA muscle. Injury-activated MAS FAPs impede postsynaptic apparatus formation by secreting pathophysiological levels of MSTN. Lowering MSTN levels in injured MAS might enhance its regeneration through nerve-muscle signalling.