09. Single-cell And Spatiotemporal Transcriptomics Of Volumetric Muscle Loss Highlights Discoidin Domain Receptor 2 Expression In Fibroadipogenic Progenitors
Year: 2024
Type: article
Abstract: Purpose: Muscle has the remarkable ability to regenerate small defects, however, when the defect is large, the injured tissue results in fibrosis. Fibrotic volumetric muscle loss (VML) injuries are painful and result in dysfunctional muscle tissue characterized by dense collagen deposition. We discovered that mesenchymal progenitor cells (MPCs) regulate collagen extracellular matrix (ECM) organization after tendon injury using discoidin domain receptor 2 (DDR2). The current study aims to identify the role of DDR2 expression by fibroadipogenic progenitor cells (FAPs) after VML injury. Methods: A previously published single-cell RNA sequencing (scRNA-seq) of uninjured, regenerative 2mm, and fibrotic 3mm quadricep VML defects were analyzed (GSE163376). Analyses were performed using Seurat v4 (Hao et al., 2022). Uninjured and fibrotic injury samples were isolated for analysis. Pdgfra+ clusters were isolated and clustered for further analysis. Differential gene expression analyses were performed utilizing negative binomials. Clusters were identified by marker gene expression. Published Visium spatial transcriptomics (GSE205707) analyses of fibrotic 2mm tibialis anterior VML defects and uninjured muscle were used. VML defect zones were previously labeled (Larouche et al. 2023). Novel spatial distance analyses were performed by marking the defect region in ImageJ, matching pixel regions to spots on Visium sequencing spots, performing distance analysis from the defect to each Visium spot, and scaling the distance value to a score of 0 to 1. Monocle2 (Trapnell et al., 2014) was used to determine genes changing over distance by replacing the pseudo-time values with distance score per spot. Results: ScRNA-seq analyses of previously labeled Pdgfra+ FAPs isolated from a dataset of uninjured and fibrotic VML defect tissue showed 6 mesenchymal lineage clusters, defined by marker gene expression (A-B). All clusters were Ddr2+ (C). Visium spatial transcriptomics of VML injuries 7 and 14 days after injury showed high transcription of type 1 collagen (Col1a1) within the defect region (D). Interestingly, Pdgfra and Ddr2 expression was upregulated in the defect region compared to transition and intact muscle zones (E). Pdgfra/Ddr2 spots were found at higher proportions within the defect zone compared to transition and intact muscle zones (F). This suggests that Pdgfra+/Ddr2+ FAPs populate in the injury site and may be contributing to fibrotic collagen deposition following injury. Distance analysis was used to score spots based on distance from the defect region and determine trends of gene expression over distance (G). Col1a1 was found to decrease as distance score increased (H) compared to Desmin, which increased with increasing score (I), as expected. To determine the spatial trends in FAPs, Pdgfra and Ddr2 expression was visualized with respect to distance, which were both found to decrease as distance increased (J-K). Conclusion: These findings suggest that Ddr2-expressing FAPs are within VML defects compared to intact muscle and may be contributing to the fibrotic tissue after injury. DDR2 may serve as a therapeutic target in the prevention of post-traumatic muscle fibrosis.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,195
OpenAlex ID: W4395029463