Identification of Collagen Producing Cells in a Model of Skeletal Muscle Fibrosis

Abstract

Skeletal muscle is a hierarchical composite tissue composed of myofibers embedded in extracellular matrix (ECM). When muscle is damaged or diseased, fibrosis often results. Skeletal muscle fibrosis is defined as an abnormal accumulation of ECM, specifically characterized by increased collagen content. However, the identity of collagen producing cells that contribute to fibrosis is unknown. PURPOSE: The purpose of this study was to create a transgenic mouse model to directly label the cells responsible for collagen deposition in muscle fibrosis. METHODS: A model of skeletal muscle fibrosis (nesprin-desmin double knockout [DKO] mouse) was crossed with a mouse line expressing GFP under the control of the collagen-α1 (I) promoter. This permitted direct identification of any cell that actively produces type I collagen based on green fluorescence. Fluorescence activated cell sorting was used to identify GFP+ cells in adult skeletal muscle. Additionally, antibodies were used against CD31, CD45, α-7 integrin, and Sca-1 to further divide the mononuclear cell population. GFP+ cells in wild-type (WT) (n=1) and DKO (n=4) mice were identified as either skeletal muscle progenitors (SMP) (CD31-, CD45-, Sca-1-, α-7 integrin+) or fibroadipogenic progenitors (FAP) (CD31-, CD45-, Sca-1+, α-7 integrin-). RESULTS: GFP+ SMP and FAP cells were identified in both WT and DKO skeletal muscle, with a small fraction of GFP+ cells falling in neither category. GFP+ cells in both genotypes were primarily FAP cells (~60% of GFP+ cells were also positive for Sca-1). Furthermore, 15-20% of GFP+ cells were SMPs in both genotypes. Importantly, a large increase in GFP+ cells/muscle mass was found in DKO mice (1506±58 GFP+ cells/mg muscle) compared with WT mice (657 GFP+ cells/mg muscle). CONCLUSION: These results demonstrate the heterogeneity of collagen I producing cells in both healthy and fibrotic muscle. Additionally, these data demonstrate that, in a model of muscle fibrosis, there a ~130% increase in collagen I producing cells compared with WT skeletal muscle. The factors that determine both the number and type of collagen I producing may permit development of antifibrotic therapies.