Poster 155: Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes that Treat Rotator Cuff Muscle Degeneration in Mice and are Identified in Humans by Single-Cell RNA Sequencing
Authors: Liu Xuhui , Garcia Steven , Kim Hubert , Raffai Robert , Feeley Brian , Davies Michael 
Published: July 2022

Objectives: Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white- and beige adipose tissue (WAT; BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in a variety of injury models. The purpose of this study was to characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their pro-myogenic effect is mediated via exosomes, and analyze human FAPs for an analogous pro-myogenic, exosome-rich subpopulation. Given their non-myogenic differentiation capacity, it was hypothesized that FAP-BAT exerts a pro-myogenic effect on the injured muscle environment through the secretion of exosomes. Methods: FAPs from UCP1-reporter mice were isolated via FACS and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1-), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNAseq was performed on UCP1-, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ (Exo-FB) and UCP1- (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and MEFs in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat WT C57B/L6J mice that had undergone massive rotator cuff tear. At 6 weeks mice were sacrificed and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing (scRNAseq) was then performed on FAPs isolated from human rotator cuff muscle that were treated with mirabegron or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans. Results: Flow cytometry analysis of sorted UCP1-reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with three distinct transcriptomic profiles characterized with bulk RNAseq. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers (Table 1). UCP1- cells were marked by fibrogenic markers and UCP1+ cells were characterized differential enrichment of WAT markers (Table 1). Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, with a myotube fusion index of 33.3 ± 6.3% compared to 7.1 ± 3.2% with Exo-nFB (p <0.001) and 8.8 ± 3.3% with exosome-free media (p = 0.0014), while treatment of MEFs resulted in transdifferentiation into myotubes (Fig. 1). Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and FI compared to treatment with Exo-nFB or PBS (Fig. 2). ScRNAseq of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with differential expression of mitochondrial genes, certain BAT markers, and exosome surface markers noted within one of the subpopulations (Fig. 3). Conclusions: FAP-BAT cells comprise a subpopulation of FAPs with upregulated mitochondrial gene expression and exosome production that mediates promyogenic effects in vitro and in vivo, and are present as a transcriptomically similar subpopulation of FAPs in humans. [Table: see text][Figure: see text][Figure: see text]