Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis

Abstract

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. 10: 173-183, 2009; Kjaer M. 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell.

High resolution three‐dimensional reconstruction of fibrotic skeletal muscle extracellular matrix

“…GFP expressing cells from aged wt ( n = 16) and aged des −/− ( n = 4) were sorted and counted by FACS analysis and normalized to muscle mass (Chapman et al . ). GFP + cells were significantly increased in aged des −/− muscle.…”

“…GFP expressing cells included fibroblasts, but also skeletal muscle progenitor cells and fibro/adipogenic progenitors (FAPs) (Chapman et al . ). Additionally, cells of haematopoietic and endothelial origin that exhibit fibrogenic plasticity and have low or no expression of CD45 and CD31, respectively, could be present in the pool of GFP + cells measured (Pessina et al .…”

“…The number of GFP + cells was significantly increased in des −/− muscle, indicating an ß90% increase in the number of collagen producing cells compared to wt ( Fig. 5B) (Chapman et al 2016).…”

“…FACS was used to determine the amount of collagen producing cells in wt ( n = 16) and des −/− ( n = 4) skeletal muscle (Chapman et al . ). Briefly, the tibialis anterior, quadriceps and gastrocnemius muscles were dissected and cleaned of all visible tendons and placed in a collagenase/dispase digestive solution (0.27% type I collagenase, 0.06 units ml −1 dispase II, 50 units ml −1 streptomycin, 50 units ml −1 penicillin) for 50 min at 37°C.…”

“…Identification of collagen I producing cells was achieved by breeding wt and des −/− mice with collagen I reporter mice expressing green fluorescent protein (GFP) under the control of the collagen α1(I) promoter (Yata et al 2003). FACS was used to determine the amount of collagen producing cells in wt (n = 16) and des −/− (n = 4) skeletal muscle (Chapman et al 2016). Briefly, the tibialis anterior, quadriceps and gastrocnemius muscles were dissected and cleaned of all visible tendons and placed in a collagenase/dispase digestive solution (0.27% type I collagenase, 0.06 units ml −1 dispase II, 50 units ml −1 streptomycin, 50 units ml −1 penicillin) for 50 min at 37°C.…”