Single Cell Transcriptomics Identifies Immunomodulation in Fibro‐Adipose Progenitor Cells and Changes to Macrophage Dynamics Following Mechanotherapy in Aged Rats Recovering From Disuse Muscle Atrophy

Abstract

Age-related changes to the muscle extracellular matrix (ECM) are a potential barrier to muscle recovery from atrophy. The involvement of individual cell types in age-related ECM remodeling is currently unknown. The purpose of this study was to determine whether mechanotherapy can affect ECM-related gene expression in cells responsible for ECM remodeling in aged rats recovering from atrophy using single cell RNA sequencing (scRNA-seq). We hypothesized that (1) there would be reduced ECM-related gene expression in cell populations responsible for ECM remodeling in aged compared to adult rats and (2) mechanotherapy would promote ECM-related gene expression in cell populations responsible for ECM regulation. Adult (10 month) and aged (30 month) male F344/BN rats were randomly assigned to weight bearing controls (WB; N=1), or to 14-days of hindlimb unloading followed by 7-days of recovery with (RE+M; N=1) or without (RE; N=1) mechanotherapy intervention. Mechanotherapy consisted of 30 minutes of cyclic compression, a massage mimetic, to the right gastrocnemius muscle at 4.5 N, 0.5 Hz, every other day for a total of 4 bouts during recovery. Rats were euthanized and the right gastrocnemius was harvested for immediate digestion in collagenase, filtered for cellular debris and labeled with propidium iodide. Live cells were isolated using fluorescence activated cell sorting, partitioned using the 10X Genomics Chromium Controller, and sequenced by Novogene. Following quality control assessment and filtering, a total of 51,582 cells across 6 groups were used for targeted analyses using Partek Flow. Uniform manifold approximation and projection identified fibro-adipose progenitor cells (FAPs) as the predominant cell population responsible for ECM remodeling. Subcluster annotation showed an immunomodulatory population of FAPs present only in aged RE rats, reflected by a 4-fold higher expression of Nfkb1 compared to adult RE rats (p<0.0001). Importantly, there were no differences in Nfkb1 expression between adult or aged WB rats. ECM-related gene expression in FAPs was lower in aged as evidenced by 4-fold lower Col1a1 compared with adult RE rats (p<0.0001). Furthermore, a subpopulation of macrophages annotated as “M1” was present only in aged RE rats. Mechanotherapy in aged rats associated with lower FAP Nfkb1 (p<0.0001), higher Col1a1 expression (p<0.0001) and elimination of the “M1” annotated macrophage subcluster. Results from this study suggest that mechanotherapy influences ECM remodeling by reprogramming the inflammatory response in aged rats recovering from atrophy, to levels similar to younger adult. Further, findings from this study identify a novel role for immunomodulation in skeletal muscle FAPs of aged rats recovering from disuse.