Prostaglandin E2 Controls Skeletal Muscle Damage via The Activation of Anti-Apoptotic Birc-3

Introduction: Prostaglandin E2 (PGE2) is a signaling molecule that has been shown to play a role in protecting skeletal muscle and enhancing regeneration1. PGE2 has an anti-inflammatory role, and it might act through inhibition of muscle cell death “anti-apoptotic” genes, however this mechanism has not been validated experimentally. Previous studies indicated that Barium Chloride (BaCl2) can induce apoptosis in various cell types including muscle cells2. Thus, we hypothesized that by establishing an in-vitro muscle injury model using BaCl2 could be a potential approach to study the underlying molecular mechanisms and screen potential therapeutics for muscle regeneration. The goal of the current study was specific to test the role of PGE2 on muscle regeneration after damage by BaCl2. Methods: In-vitro chemical muscle damage using BaCl2 model was developed and optimized. Studies using C2C12 and mouse primary muscle cells were performed. Live/dead cell imaging was used to monitor cell’s apoptosis. Fusion index (FI) calculation was used to quantify the differentiation studies and muscle damage. Live dynamic microscopy was used to quantify cell’s migration and muscle regeneration using a wound-healing test. Pathway finder RT-PCR array was used to uncover the activated pathways. All experiments were tested with n=4 and in triplicates. Statistical analysis using T-Test and ANOVA were performed. Results: In-vitro results indicated the BaCl2 chemical injury induce myoblast cell apoptosis as indicated by significant cell death after 6 hrs of BaCl2 treatment. Also, BaCl2 induced muscle damage via breakdown of multi-nucleated myotubes as indicated by the significant decrease in FI ( p=0.001). BaCl2 significantly decreased muscle cell’s regeneration; while PGE2 attenuated this effect and significantly increased the rate of cell's migration. RT-PCR Gene arrays results indicated that BaCl2 treatment upregulates the BCl-2 gene, which is involved in programmed cell death “apoptosis.” Treatment with 50nM of PGE2 significantly upregulated the anti-apoptotic gene Birc-3 (~7.5-fold upregulation) compared to normal control. Conclusion: BaCl2 induces cell apoptosis leading to muscle degeneration through the upregulation of BCl-2 gene, while PGE2 normalizes these deleterious effects after BaCl2 through the upregulation of Birc-3 leading to muscle regeneration. This study establishes and validates a new in-vitro model of BaCl2 induces muscle damage. The data suggests that BaCl2 induces damage via upregulation of apoptosis, while PGE2 exerts anti-apoptotic effects via Birc-3. References: 1- Mo, Chenglin; et al. Recent patents on biotechnology vol. 6,3 (2012): 223-9. doi: 10.2174/1872208311206030223 . 2- Morton, Aaron; et al. Skeletal Muscle 9, 27 (2019). https://doi.org/10.1186/s13395-019-0213-2 . KA, LM, JH, LB, and MB were supported by National Institutes of Health Grants: NIA 2-PO1AG039355, NIA R01AG056504; National Institute of Diabetes and Digestive and Kidney Diseases R01DK119066 to M.B.; and National Institutes of Neurological Disorders and Stroke (NINDS) 2-R01NS105621 to M.B. The authors are thankful for the generous support from the George W. and Hazel M. Jay Research Endowments, and the UTA College of Nursing and Health Center of Research and Scholarship. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.