(E-185) Maresin-1 Repletion Improves Muscle Regeneration After Volumetric Muscle Loss

Introduction:: Volumetric muscle loss (VML) is the frank loss of a large mass of skeletal muscle, which leads to persistent inflammation, severe fibrosis, and reduced muscle function [1]. Existing therapeutic strategies have shown minimal success in recovering proper muscle function and structure [2], in part due to limited knowledge of the molecular and cellular basis of regenerative dysfunction following VML. Our goal is to enhance our understanding of the VML response in order to identify potential therapeutic avenues to reduce fibrosis and restore muscle function after this injury. Recently, we used metabolipidomic analysis to elucidate differences between healing and non-healing VML injuries in order to identify potential determinants of successful regeneration. After finding a lower relative abundance of pro-resolving lipid mediators in non-healing injuries, we proposed the exogenous administration of one pro-resolving mediator, Maresin-1, as a potential pro-regenerative therapeutic strategy. Herein, we used immunohistochemical staining and cell-based in vitro assays to evaluate the efficacy and understand the mechanism of Maresin-1 as a therapeutic to stimulate regeneration post-VML. Our results demonstrate Maresin-1’s ability to reduce pathological fibrosis, attenuate exacerbated inflammation, and stimulate muscle regeneration following VML.

Materials and Methods:: In vivo studies were performed by administering VML injuries to young adult mice (3-4 months) as 2mm full-thickness punch biopsy defects in the tibialis anterior (TA) muscle. After VML, mice were treated with Maresin-1 and 0.1% ethanol in saline (vehicle) in opposite limbs via intramuscular injection 1, 3, and 5 days post injury (dpi). Tissues were removed from the mice at 7 dpi and cross-sectionally cryosectioned. Picrosirius red staining and immunohistochemical staining for CD68 and MYH3 was performed. Resulting images of Picrosirius red, CD68, and MYH3 stains were processed using Cellpose, LabelsToROIs, and/or MATLAB to quantify collagen content, macrophage quantity, and myofiber cross sectional area.



To understand the effect of Maresin-1 on muscle satellite cells (MuSCs) directly, MuSCs were isolated from mouse tissue via magnetic-activated cell-sorting (MACS). Cells were treated with media containing Maresin-1 (10nM) and a proliferation marker, 5-ethynyl-2’deoxyuridine (EdU). After EdU staining, cells were imaged and MATLAB was used to quantify the percentage of EdU+ cells.


To interrogate the mechanism of Maresin-1’s effect on MuSCs, the expression of Lgr6 was assessed in MuSCs, as Maresin-1 selectively binds to the Leucine-rich repeat containing G protein-coupled receptor 6 (LGR6) [3]. This was done through RT-qPCR using MuSCs in three different states—quiescent, proliferating, and differentiated. The role of this receptor in MuSCs was then assessed by performing an Lgr6 knockdown with Dsi-RNA using cells isolated from Pax7CreERT2-Rosa26Tcf/Lef-LSL-H2B-GFP reporter mice, which express GFP in MuSCs in response to canonical Wnt signaling, then immunostaining for GFP to assess Wnt signaling.

Results, Conclusions, and Discussions:: Analysis of the Picrosirius red stains of treated and untreated limbs revealed that exogenous Maresin-1 administration reduced collagen content and fibrosis (Figure 1). With excessive fibrosis being a defining characteristic of a non-healing VML response, this result was an initial indication of Maresin-1’s therapeutic efficacy. Reduced fibrosis suggested underlying attenuation of the immune response, which was confirmed in our analysis of the CD68 immunohistochemical stains. This quantification showed a lower CD68+ percentage of the defect area, which serves as a proxy for macrophage quantity in the region (Figure 2). Furthermore, the in vivo results indicated that Maresin-1 was able to not only suppress fibrosis and inflammation, but could also stimulate myogenesis. This was evidenced by the MYH3 immunohistochemistry, which revealed that MYH3+ (regenerating) myofibers tended to be larger in Maresin-1-treated tissues, with size evaluated by fiber cross sectional area and minimum Feret diameter (Figure 3).

Acknowledgements (Optional): :

References (Optional): : [1] Greising, S. M., Rivera, J. C., Goldman, S. M., Watts, A., Aguilar, C. A., & Corona, B. T. (2017). Unwavering Pathobiology of Volumetric Muscle Loss Injury. Scientific reports, 7(1), 13179. https://doi.org/10.1038/s41598-017-13306-2

[2] Greising, S. M., Corona, B. T., McGann, C., Frankum, J. K., & Warren, G. L. (2019). Therapeutic Approaches for Volumetric Muscle Loss Injury: A Systematic Review and Meta-Analysis. Tissue engineering. Part B, Reviews, 25(6), 510–525. https://doi.org/10.1089/ten.TEB.2019.0207

[3] Chiang, N., Libreros, S., Norris, P. C., de la Rosa, X., & Serhan, C. N. (2019). Maresin 1 activates LGR6 receptor promoting phagocyte immunoresolvent functions. The Journal of clinical investigation, 129(12), 5294–5311. https://doi.org/10.1172/JCI129448

[4] Liu, S. L., Zhou, Y. M., Tang, D. B., Zhou, N., Zheng, W. W., Tang, Z. H., Duan, C. W., Zheng, L., & Chen, J. (2019). LGR6 promotes osteogenesis by activating the Wnt/β-catenin signaling pathway. Biochemical and biophysical research communications, 519(1), 1–7. https://doi.org/10.1016/j.bbrc.2019.08.122

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