FAPs and Macrophages Team Up to Repair Aging Muscle After Injury
Spatial transcriptomics reveals how fibro-adipogenic progenitors signal macrophages via complement C3 to clear debris and drive elderly muscle repair.
Summary
Aging impairs skeletal muscle's ability to regenerate after injury, but the cellular mechanisms in humans remain poorly understood. Researchers used spatial RNA sequencing, single-cell transcriptomics, flow cytometry, and functional assays to map the regenerative landscape in elderly human muscle biopsies taken before and 2, 8, and 30 days after electrically induced injury. They identified fibro-adipogenic progenitors (FAPs) and macrophages as the central coordinators of repair, discovering that FAPs secrete complement factor C3 to enhance macrophage phagocytosis, survival, and metabolism. This FAP-to-macrophage signaling axis represents a previously uncharacterized communication pathway in human aged muscle and opens new avenues for therapeutic strategies to counter age-related regenerative decline.
Detailed Summary
Skeletal muscle makes up 30–50% of body mass and is essential for locomotion, metabolism, and immune modulation. Its regenerative capacity declines with age, contributing to sarcopenia and prolonged recovery from injury. Despite extensive rodent research, the cellular orchestration of regeneration in elderly human muscle has not been mapped with spatial and temporal resolution—until now.
Researchers recruited elderly subjects and induced standardized skeletal muscle injury in the vastus lateralis using electrically evoked eccentric contractions. Muscle biopsies were collected at baseline and 2, 8, and 30 days post-injury (dpi) from three individuals for Visium 10x Spatial Gene Expression profiling, capturing all human protein-coding genes across tissue sections. H&E staining confirmed substantial injury at 8 dpi and partial restoration by 30 dpi, with regenerating fibers showing central nuclei and smaller cross-sections. Median genes and UMI counts per spatial spot peaked at 8 dpi, reflecting intense transcriptional activity during the repair phase.
Unsupervised clustering identified ten spatial gene-expression clusters. Clusters 0–5, representing muscle fiber types, cytoskeletal function, proteasomal remodeling, and erythrocyte transcripts, declined after injury. Clusters 6, 7, and 8—enriched in extracellular matrix remodeling, immune response, and myofiber development genes, respectively—increased significantly post-injury and were most prominent at 8 dpi. Single-cell spatial deconvolution analysis mapped monocytes/macrophages and fibro-adipogenic progenitors (FAPs) as the dominant non-myogenic cell populations driving the regenerative response, with both populations expanding substantially at 8 dpi and partially resolving by 30 dpi.
Flow cytometry on nine additional subjects confirmed the increased abundance and activation of FAPs and macrophages during regeneration. Spatial correlation analysis showed FAPs and macrophages co-localizing within the same tissue niches at the injury site, and ligand-receptor interaction modeling identified complement factor C3 as the primary intercellular communication molecule. Immunostaining validated C3 protein expression in FAPs, and conditioned media experiments confirmed FAP secretion of C3. Functional assays then demonstrated that C3 significantly enhanced human monocyte phagocytic capacity, promoted monocyte survival, and shifted monocyte metabolic activity—all functions critical for clearing necrotic muscle debris and enabling tissue repair.
These findings establish a bidirectional communication loop: macrophages are known to regulate FAP survival and differentiation (via TNF-alpha and TGF-β), while FAPs now appear to actively recruit and potentiate macrophage function through C3 secretion. This axis may be impaired in aging, where complement dysregulation and chronic low-grade inflammation are well documented. The study provides the first spatially resolved, temporally mapped evidence of this FAP-macrophage interplay in elderly human muscle, offering mechanistic targets for interventions aimed at restoring regenerative capacity in older adults.
Key Findings
- FAPs and macrophages are the dominant non-myogenic cell types driving elderly human skeletal muscle regeneration, peaking at 8 days post-injury.
- FAPs secrete complement factor C3, which enhances macrophage phagocytosis, survival, and metabolic activity in functional assays.
- Spatial transcriptomics identified three injury-induced gene clusters enriched in ECM remodeling, immune response, and myofiber development.
- FAPs and macrophages co-localize at injury sites, confirmed by both spatial correlation analysis and flow cytometry in nine subjects.
- Tissue integrity was largely restored by 30 dpi, but regenerating fibers remained smaller with central nuclei, indicating incomplete recovery in aged muscle.
Methodology
Elderly human subjects underwent electrically induced eccentric muscle injury with biopsies at pre, 2, 8, and 30 days post-injury. Visium 10x Spatial Gene Expression profiling (n=3) was combined with single-cell deconvolution, flow cytometry (n=9), immunostaining, and functional monocyte assays to map cell types, spatial localization, and intercellular signaling.
Study Limitations
Spatial transcriptomics was performed in only three subjects, limiting statistical power for rare cell-type detection. The study used a non-physiological electrical injury model, which may not fully replicate natural aging-related muscle damage. Functional C3 assays were conducted in vitro with isolated monocytes, so in vivo relevance in the intact aged muscle niche requires further validation.
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