(M-490) Fibrotic Fibro-adipogenic Progenitors Produce Altered Extracellular Matrix and Impair Myogenesis

Introduction:: Fibrosis is the pathological accumulation of extracellular matrix (ECM) components, impairing tissue function. In skeletal muscle, the main source of this excessive ECM deposition are fibro-adipogenic progenitors (FAPs). FAPs play a key role in muscle regeneration, activating after injury, depositing ECM to replace the damaged matrix, and releasing pro-myogenic signals to initiate regeneration. FAPs then undergo apoptosis, halting ECM deposition to prevent excess accumulation. However, in the context of chronic injury and fibrotic diseases, FAPs become resistant to apoptosis, progressively depositing ECM components leading to fibrosis. How fibrotic FAPs differ from regenerative FAPs in terms of ECM deposition and effect on muscle satellite cells (MuSCs) is not well understood. We hypothesized that FAPs isolated from mdx mice, a mouse model of skeletal muscle fibrosis, would have increased collagen deposition compared to wt FAPs. We also hypothesized that mdx FAPs would release soluble factors inhibiting myogenesis rather than promoting it.

Materials and Methods:: Both wt and mdx FAPs were isolated from the lower limb muscles. The muscles were dissected, mechanical and enzymatically digested, and both FAPs and muscle satellite cells (MuSCs) were isolated using magnetic-activated cell sorting. FAPs and MuSCs were cultured in growth media until confluent. 35mm glass bottom dishes were coated with 0.2% gelatin, 1% glutaraldehyde, and 1M ethanolamine prior to matrix deposition to improving anchoring of matrix to dish bottom. FAPs were plated on the coated 35mm dishes at a concentration of 2.5e5 cells/mL in 1 mL of matrix media (DMEM, 20% FBS, 1% Penn/Strep, 50μm ascorbic acid). Media was replaced every 24hrs with fresh ascorbic acid for 2 weeks straight to generate cell-derived matrices (CDMs). FAPs were then removed while keeping the matrix intact, using TritonX-100, NH₄OH, and DNAse. The cell-derived matrices were then imaged using second harmonic generation (SHG) imaging to visualize collagen deposition. MuSCs were then plated on the CDMs, in a transwell co-culture system with FAPs, and on plastic at a concentration of 5e5 cells/mL in differentiation media (DMEM, 5% HS, 1% Penn/Strep), and allowed to differentiate for 5 days. Cells were then fixed and stained with MF20 (MuSCs) or αSMA (FAPs), F-actin, and Hoescht. Cells were imaged using an inverted Leica DMi8. Images were captured with a dry 20X/0.40 objective using a Leica DFC9000 GTC camera and LAS X software. Images were analyzed using custom macros in FIJI: ImageJ.


Results, Conclusions, and Discussions:: The level of differentiation of MuSCs did not change significantly between wt CDMs and wt transwell co-culture experiments. However, there was a large amount of variability in the level of differentiation between samples that was strongly correlated to the amount of αSMA expression in FAPs (Fig. 1). This suggests that as FAPs activate into myofibroblasts and a more fibrotic phenotype, they produce ECM and soluble factors inhibit rather than promote myogenesis. Further experiments are currently being completed on the role of mdx CDMs on MuSC differentiation.

The matrices produced by both wt and mdx FAPs were imaged using SHG imaging to visualize collagen organization. CDMs derived from mdx FAPs had more collagen deposition and less aligned fibers compared to CDMs derived from wt FAPs. This suggests that FAPs in fibrotic muscles produce more ECM, leading to a progressively degenerative cycle of fibrosis. The increase in collagen and decrease alignment could be the reasons for decreased myogenesis on CDMs derived from fibrotic FAPs, as MuSCs have been show to preferentially differentiate on aligned substrates.

FAPs are considered a double-edged sword in skeletal muscle, with important roles in regeneration and homeostasis of the muscle. However, they are also the key drivers of fibrosis. This research highlights how the activation state of FAPs may play a key role in their regenerative versus fibrotic capacity. Inhibiting FAP activation into myofibroblasts may be a key therapeutic target for attenuating fibrosis without losing the pro-regenerative functions of FAPs.


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