Fibro-Adipogenic Progenitors require autocrine IGF-I in homeostatic and regenerating skeletal muscle

Background

Muscle regeneration refers to restoration of muscle mass lost as a result of muscle injury. Like any other biological process, this process is highly dynamic in nature involving a complex network of interactions between muscle resident and non-muscle resident cell populations.

Fibro-adipogenic progenitors (FAPs) are muscle resident mesenchymal stromal cells which provide a regeneration conducive niche to muscle stem cells (MuSCs), which give rise to new muscle fibers upon injury (1). In healthy muscle, FAPs aid muscle regeneration by controlling their proliferation and by deposition of regeneration-specific extracellular matrix (ECM) (1). However, under pathological conditions, FAPs can differentiate into fibroblasts—leading to fibrosis—or adipocytes—leading to deposition of intramuscular fat, thereby limiting the muscle regeneration capacity (2,3).

One of the key factors secreted by FAPs is Insulin growth factor 1 (IGF-1), which is of high interest due to its role in facilitating skeletal muscle regeneration and glucose regulation (4,5). While FAPs secrete IGF-1, the majority of the IGF-1 found in muscle is secreted from muscle fiber, hence most of the studies so far focused on the contribution of IGF-1 from muscle fibers on skeletal muscle regeneration.

The current study investigated the role of IGF-1 secreted exclusively by FAPs on skeletal muscle regeneration, more specifically on how it governs FAPs function.

Study Model/Injury Model

1. Barium Chloride (BaCl₂) induced acute muscle injury in FAP-Igf1-Deficient (FID) mice to study fibrotic muscle injury.
2. Glycerol induced muscle injury in FAP-Igf1-Deficient (FID) mice to study adipogenic muscle injury.

 Key Findings

1. Regenerating muscles from FID mice (male and female) showed a marked reduction in relative muscle mass and fiber size at 7 days post injury (7dpi) compared to control mice (Igf1^fl/fl). Immunofluorescence staining revealed a decrease in number of MuSCs (Pax7⁺ ) at 5dpi, while macrophages (CD68⁺) were persistently present in FID mice at 14dpi and 28dpi as compared to Igf1^fl/fl control mice, where macrophage cell numbers peaked at 7dpi. Overall, these findings suggest that the depletion of FAPs-secreted IGF-1 results in impaired muscle regeneration.
2. Immunostaining the muscle sections for PDGFRα⁺(marker for FAPs) showed that FAPs fail to expand at 3, 5 and 7dpi, as compared to control muscle section, where FAPs exhibited a transient expansion at 5dpi. Moreover, FAP proliferation was impaired in the absence of FAP IGF-1 in vivo and in vitro, which was confirmed by staining the sections and isolated primary FAPs from injured muscle with Ki67 (a marker for cell cycle).
3. Bulk-RNA sequencing analysis of FAPs isolated from 3dpi FID muscle showed a downregulation of ECM related genes (Fibronectin, Collagens etc.) and cell proliferation related genes. On the other hand, genes associated with immune response particularly inflammation were upregulated in FID FAPs as compared to control.
4. Quantitative analysis of ECM content using Pico-Sirius Red staining at 3 and 5dpi revealed FID muscles exhibit less ECM deposition as compared to control injured muscle. This shows that IGF-1 from FAPs is required for ECM deposition during regeneration in vivo. To assess the adipogenic potential of FAPs from FID mice, glycerol injury was performed. Immunofluorescence staining for adipocytes (Perilipin⁺ cells) showed that FID muscles had significantly lower adipocyte accumulation at 21dpi compared to control. Taken together these findings suggest that loss of FAP IGF-1 results in reduced ECM deposition and adipocyte differentiation following muscle injury.
5. Lastly, to understand the global effect of IGF deletion in FAPs, single cell RNA sequencing was performed from control (Igf1^fl/fl) and FID homeostatic muscles. scRNA sequencing revealed that FAPs from FID mice adopt a senescent phenotype marked by elevated inflammatory signaling and reduced expression of cell proliferation markers. Moreover, FAPs isolated from FID mice and cultured in vitro, showed enlarged cytoplasm, a flattened cell shape and multinucleation, which are typical hallmarks of senescent cells.

What I like about the preprint?

Overall, I find this study really interesting as it provides a preliminary insight into the potential roles of autocrine IGF-1 in controlling FAPs cellular state. Understanding the exact mechanism by which IGF-1 pushes FAPs into senescence can help delineate the mechanisms involved in FAPs senescence in aging muscle. I chose to highlight this preprint as it relates very closely to my current research project investigating how post- muscle injury FAPs adopt a pre- fibrogenic cell state upon knockdown of a transcription factor Osr1 (Odd Skipped Related 1). Therefore, this study serves as a perfect addition to my literature review for understanding the mechanisms by which FAPs govern their cellular state.

Questions for the authors

1. At 3, 5 and 7dpi, FAP numbers are consistently lower in FID injured mice due to lesser expansion of FAPs which can be explained by lower proliferation (low Ki67+ cells and proliferation related genes downregulated as revealed by Bulk-RNA seq). However, apoptosis is also a major pathway by which cell expansion is regulated during muscle regeneration i.e., FAP numbers are controlled by TNF-alpha secretion from pro-inflammatory macrophages (M1). Does the loss of autocrine IGf-1 trigger FAPs apoptosis hence leads to the lower cell numbers? or are these low numbers entirely due to proliferation defects?

2. Single-cell RNA sequencing analysis in homeostatic muscles showed that FAPs and fibroblasts are the biggest contributor of IGF-1, while there are negligible amounts detected from the myonuclei and satellite cells (Figure 7B, C). At the protein level, Figure 1I shows a slight decrease in total IGF-1 content in FID muscle compared to control, which also hints at FAPs secreting lower amounts of IGF-1 as compared to other cell types. Moreover, previous studies point out that 75% of the local IGF-1 pool is produced by muscle fibers. So, taken together, are FAPs the major source of IGF-1 in muscles or is it the myonuclei? Or can it be both depending on the state (homeostatic or injured)?

References

• Wosczyna, M. N., & Rando, T. A. (2018). A muscle stem cell support group: coordinated cellular responses in muscle regeneration. Developmental cell, 46(2), 135-143.
• Lemos DR, Babaeijandaghi F, Low M, Chang CK, Lee ST, Fiore D, Zhang RH, 916 Natarajan A, Nedospasov SA, Rossi FM (2015) Nilotinib reduces muscle fibrosis in chronic muscle injury by promoting TNF-mediated apoptosis of fibro/adipogenic Nat Med 21: 786-794.
• Uezumi A, Fukada S, Yamamoto N, Takeda S, Tsuchida K (2010) Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol 12: 143-152.
• Musaro A, McCullagh K, Paul A, Houghton L, Dobrowolny G, Molinaro M, Barton ER, Sweeney HL, Rosenthal N (2001) Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet 27: 195-200.
• Vassilakos G, Lei H, Yang Y, Puglise J, Matheny M, Durzynska J, Ozery M, Bennett K, Spradlin R, Bonanno H et al (2019) Deletion of muscle IGF-I transiently impairs growth and progressively disrupts glucose homeostasis in male mice. FASEB J 33: 181-194.

Tags: fibroadipogenic progenitors, igf-i, senescence, skeletal muscle regeneration

doi: https://doi.org/10.1242/prelights.41784

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