Z20 - Investigating a Novel Pro-regenerative Therapeutic to Treat Volumetric Muscle Loss

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

VM

Victoreya McKissick

undergraduate researcher
University of Oregon, United States

Co-Author(s)

FP

Frank Pittman

PhD Candidate
University of Oregon
Eugene, Oregon, United States

Primary Investigator(s)

NW

Nick J. Willet, PhD

Associate Professor
University of Oregon, United States

Introduction: : Volumetric muscle loss (VML) is characterized by the permanent loss of substantial amounts of skeletal muscle mass, leading to chronic pain, lasting disability, and an overall reduction in the quality of life. This is typically the result of sustained trauma from motor vehicle accidents or military combat, neuromuscular diseases, or extensive surgeries. The current standard of clinical treatment is surgical repair using autologous muscle and skin grafts in efforts to salvage the affected limb. However, these treatments fail to address chronic inflammation, which can lead to extensive fibrosis, impaired muscle fiber regeneration, and impede overall functional recovery. Following a VML injury, the persistent inflammatory response can lead to persistent high levels of a profibrotic cytokine known as transforming growth factor beta (TGF-𝛽). Previous research has shown that high levels of TGF-𝛽 can promote fibrotic scarring and directly impair myogenesis.1 Additionally, we have shown that treatment with specialized pro-resolving mediators (SPMs), specifically AT-RvD1, at the cellular level mitigates the anti-myogenic effects of TGF-𝛽 in vitro.2 However, the translational potential of SPM-based therapeutics is limited due to their instability in vivo, complex chemical synthesis, and extremely high cost. Synthetic agonists of SPM receptors have the potential to mitigate the current limitations of SPM-based therapeutics. Thus, our objective was to investigate a synthetic agonist of SPM receptors, BMS-986235 (BMS), to elicit a pro-regenerative recovery outcome like that of previously tested SPMs. We hypothesized that the treatment of cells with the BMS compound in the presence of high TGF-𝛽 levels would improve myogenesis.

Materials and

Methods: : BMS Dose Response- c2c12s, an immortalized murine myoblast cell line, were cultured in differentiation media in vitro and a logarithmic dose of 0.01 nM to 100 nM BMS was assessed under profibrotic conditions created using 2.5 ng/mL TGF-𝛽. There was a sample size of three wells per group. The cells were fixed with 4% paraformaldehyde after 96 hrs of treatment. Immunofluorescence staining of myosin heavy chain II (MHC-II) and DAPI was carried out to quantify an outcome of myogenesis. Five images were taken per well and nuclear fusion index was quantified in ImageJ to determine the extent of myoblast fusion into myotubes. The equation for fusion index is: (# Myonuclei) / (Total # Nuclei). A One-way ANOVA with Tukey’s multiple comparisons post-hoc test was conducted to determine if there was any significant difference across all groups. The alpha level was set to 0.05.

Results, Conclusions, and Discussions:: Fusion index was compared between groups to assess the effects of varying BMS doses, on myotube formation, in the presence of TGF-𝛽. We detected a significant increase in fusion index when comparing the 0.01 nM BMS + 2.5 ng/mL TGF-𝛽 group to the 2.5 ng/mL TGF-𝛽 negative control. We also detected a significant increase in fusion index when comparing the 1 nM BMS + 2.5 ng/mL TGF-𝛽 group to the 2.5 ng/mL TGF-𝛽 negative control. The increase in fusion index in a BMS treated group compared to TGF-𝛽 alone is more statistically significant for 0.01 nM BMS rather than 1 nM. There was also a significant increase in fusion index at the 0.01 nM BMS treatment group compared to the 0.1 nM, 10 nM, 100 nM, and differentiation media only groups. These results suggest that lower doses of BMS might be more effective at recovering myotube formation under profibrotic conditions. However, we cannot yet form a definitive conclusion from these results because we did not detect a significant difference between our positive (differentiation media only) and negative (TGF-𝛽 only) controls, which is contradictory to what has been consistently reported in the literature. We are in the process of re-establishing our controls in a forthcoming, fully powered study with the goal of gaining a more accurate measure of the effects of BMS on myogenesis in vitro. BMS is a promising small molecule that has the potential to reduce fibrosis, resolve inflammation, and improve tissue regeneration. Successful therapeutic outcomes can shift a patient’s trajectory from unavoidable muscle loss to actual functional regeneration.

Acknowledgements and/or References (Optional): : 1 J.A. Larouche et. al, PNAS (April 2022)

2 T.C. Turner et. al, bioRxiv (September 2024)