Multiple Specific Cell Types Produce Type 1 Collagen During Skeletal Muscle Fibrosis
Authors: Chapman Mark , Mukund Kavitha , Subramaniam Shankar , Lieber Richard 
Published: November 2023

INTRODUCTIONAcute muscle damage and chronic pathologies such as muscular dystrophy and cerebral palsy frequently results in muscle fibrosis. Muscle fibrosis, defined as an abnormal level of extracellular matrix (ECM) protein deposition, results primarily from type I collagen. Although the identity of the cells that produce type 1 collagen is not known, it is often assumed to be fibroblasts. However, a rigorous understanding of cells causing fibrosis is vital to developing effective therapies. Our objective was to create a mouse model to label and identify the cells responsible for ECM deposition in muscle fibrosis.METHODSA model of muscle fibrosis (nesprin‐desmin double knockout [DKO] mouse [1]) was crossed with a mouse line expressing GFP under the control of the collagen‐α1 (I) promoter [2]. This permitted direct identification of collagen I producing cells based on green fluorescence. Fluorescence activated cell sorting was used to isolate GFP cells from adult muscle. Antibodies were used against CD31, CD45, α‐7 integrin, and Sca‐1 to subdivide the mononuclear cell population. GFP cells in wild‐type (WT) (n=16) and DKO (n=14) mice were identified as either muscle progenitors (SMP, CD31−, CD45−, Sca‐1−, α‐7 integrin+) or fibro/adipogenic progenitors (FAP, CD31−, CD45−, Sca‐1+, α‐7 integrin−). Remaining cells were considered to be primarily composed of fibroblasts (FB) since no specific FB marker is available and these are the most populous mononuclear cells. To characterize each cell population, RNA was isolated and RNAseq performed on Illumina Hi‐seq 2500 (rapid run mode).RESULTSThe GFP+ cell population in both genotypes was composed of ~60% FAP, ~20% of SMP and ~20% of FB cells. Additionally, a large increase in GFP+cells/muscle mass was found in DKO mice (1,506±58 GFP+ cells/mg muscle) compared to WT mice (657±81 GFP+ cells/mg muscle; p<0.001). This was clearly appreciated as increased fluorescence (Fig. 1). While the total number of GFP+ cells increased in DKO mice, the proportion of FAPs, SMPs, and FBs did not change, demonstrating that all three cell populations participated in the fibrotic response.Furthermore, we found a significant increase in the percentage of SMP cells positive for GFP in DKO (69.2±2.3%) versus WT mice (51.6±1.2%; p<0.0001). This was also found in FAP cells positive for GFP in DKO (83.6±2.1%) versus WT (71.9±1.4%; p<0.0001) muscle. The proportion of FBs positive for GFP was also increased in DKO (24.6±2.2%) versus WT (14.3±1.7%; p=0.0007) mice.RNAseq revealed differential expression of type collagen I expression in the three cell types. The highest collagen I expression was in FBs, followed by FAPs, and finally, SMPs (Fig. 2). Differences between cell types demonstrated a distinct role of each cell population in the production of muscle ECM (Fig. 2).DISCUSSIONThese results demonstrate the heterogeneity of collagen I producing cells in healthy and fibrotic muscle. Additionally, these data demonstrate that there is a 130% increase in collagen I producing cells compared with WT in this model. Furthermore, our data demonstrate that all three GFP+ cell populations show a significant increase in numbers, while the GFP+ cell composition generally remains the same between WT and DKO muscle. We now know that three distinct populations of collagen I producing cells all increase their number while their ECM gene expression remains roughly constant.Support or Funding InformationResearch reported here was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Numbers AR061303 & T32AR0607 and by the National Institute of Child Health and Human Development under Award Number HD050837. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We also acknowledge the National Science Foundation for an NSF‐Graduate Research Fellowship to MAC.