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.

Introduction Although skeletal muscle has a remarkable regenerative capacity, certain traumatic injuries are beyond the normal physiologic repair and require extensive regenerative therapy. Musculoskeletal injury (MSI) afflicts more than 2.3 billion people globally and ~30 million Americans leading to nearly one‐trillion dollars in medical care costs (~ 6% of our GDP). In the tissue engineering field, muscle regeneration to overcome the compromised regeneration after traumatic injuries has emerged. Tissue engineering introduces novel in‐vitro models that simulate in‐vivo models that aid in reducing/eliminating the use of animal models, allow rapid screening of potential therapies and clinical interventions, reduce the associated cost and complexity, and are broadly available for research laboratories. Following our previous results showing that PGE2 and WNT3a enhance muscle cell differentiation, this study intended to validate in‐vitro muscle damage model induced by barium chloride (BaCl2) and screen PGE2 and WNT3a as potential molecules for muscle regeneration after injuries. Materials and Methods C2C12 skeletal muscle cells under proliferation were firstly used to determine the concentration of BaCl2 for this study, then differentiation studies were performed to test chemical damage in muscle and regeneration under PGE2 and WNT3a treatment. In the first part of study, C2C12 cells were seeded in 12 well‐plates and cultured in growth medium (GM) with different concentrations of BaCl2 for 24 and 48 hr. and their effects on cell viability were monitored. After adjusting the BaCl2 concentration, C2C12 cells were seeded in 12 well‐plates and cultured in GM till 70% confluency, then allowed to differentiate in differentiation medium (DM) for 48 hr. before damage introduced using OPTI‐MEM containing BaCl2 for 6 hours, followed by treatment with condition media (DM + PEG2, WNT3a) for 48 hrs. Cells were finally stained with live/dead assay kit and imaged directly, or RNA was extracted for our custom‐built muscle specific PCR array. Results and Conclusion Our preliminary data indicated that 25 mM BaCl2 significantly (p<0.0007) decreased the cells viability and increased the number of dead cells as observed at 24 and 48 hr. under proliferative condition. Post‐treatment with 50nM PGE2 or 10ng/ml WNT3a significantly enhanced the regeneration as indicated by the significant increase in the percentage area of live cells (myotubes/myoblast) and significant decrease in the number of dead cells. Results of the custom‐built muscle specific PCR array indicated that 6 and 3 genes were differentially expressed after WNT3a and PGE2 treatment, respectively, compared to the BaCl2 group. Several of detected genes (e.g., Tnnc2, Actc1, Il6, Myh1, Nppa, Itpr1, etc.) are main contributors to the contractile machinery, while others are associated with inflammation and the extracellular matrix. This study concluded that BaCl2 affects both mononuclear and multinuclear C2C12 cells and PGE2 and Wnt3a could enhance the regeneration after the chemi...

Osteoarthritis (OA) is one of the most common causes of disability in aged people, and it is defined as a degenerative arthropathy, characterized by the disruption in joint tissue. The synovium plays a vital role in maintaining the health of the joint by supplying the nutrients to the surrounding tissues and the lubrication for joint movement. While it is well known that all the joint tissues are communicating and working together to provide a functioning joint, most studies on OA have been focused on bone and cartilage but much less about synovium have been reported. The purpose of this review was to investigate the current literature focused on RNA sequencing (RNAseq) of osteoarthritic synovial tissues to further understand the dynamic transcriptome changes occurring in this pivotal joint tissue. A total of 3 electronic databases (PubMed, CINHAL Complete, and Academic Complete) were systematically searched following PRISMA guidelines. The following criteria was used for inclusion: English language, free full text, between the period 2011–2022, size of sample (n &gt; 10), study design being either retrospective or prospective, and RNAseq data of synovial tissue from OA subjects. From the initial search, 174 articles, 5 met all of our criteria and were selected for this review. The RNAseq analysis revealed several differentially expressed genes (DEGs) in synovial tissue. These genes are related to the inflammatory pathway and regulation of the extracellular matrix. The MMP family, particularly MMP13 was identified by three of the studies, indicating its important role in OA. IL6, a key contributor in the inflammation pathway, was also identified in 3 studies. There was a total of 8 DEGs, MMP13, MMP1, MMP2, APOD, IL6, TNFAIP6, FCER1G, and IGF1 that overlapped in 4 out of the 5 studies. One study focused on microbial RNA in the synovial tissue found that the microbes were differentially expressed in OA subjects too. These differentially expressed microbes have also been linked to the inflammatory pathway. Further investigation with more clinical gene profiling in synovial tissue of OA subjects is required to reveal the causation and progression, as well as aid in the development of new treatments.

Skeletal muscles play key roles in maintaining the homeostasis of the musculoskeletal system (MSK). The current models of osteoarthritis (OA) focus on the direct injury to the joint and have not tested whether direct muscle damage can lead to OA. This study’s objective was to investigate this possibility. We hypothesize that following muscle damage there will be periarticular muscle weakness causing joint damage. During muscle regeneration there will be a release of lipid signaling mediators into the circulation. The combination of biochemical and biomechanical modifications will alter the joint resulting in evidence of the onset of OA.This study was approved by the UTA IACUC. A total of 48 skeletally mature, young (28±1 weeks) C57BL/6 mice were used. The mice were blindly randomized into 4 groups: Baseline-Control, 4-Day post injection, 7-Day, and 1-month. Muscle damage was induced by intra-muscular (IM) injection of 50 μL of 1.2% Barium Chloride (BaCl2). The contralateral limb served as the sham control. Physical strength was accessed through grip strength. Locomotor activity was assessed using a force plate actimeter. Targeted lipidomics on serum was performed [1]. Paraffin embedded muscle was transversely cut at 7-μm thick and stained with H&E. The tibia was paraffin embedded, and 7-μm thick sections of the medial tibial plateau was stained with Safranin O/Fast Green. Before fixation of the tibia, bone and cartilage was accessed using Raman spectroscopy, and subchondral bone measured in μCT. Statistical analysis was conducted using ANOVA.The muscle damage in the TA showed a significant initial decrease in all limb grip strength (MD -54.62 p<.001 4-days post injury to baseline control). Strength significantly recovered over time (4-Day to 1-Month all limb grip MD 32.49 p<.001). Muscle regeneration was confirmed by our histological imaging of the TA which showed similar results to previous findings [2]. Serum lipidomics revealed a significant immune response with elevated concentration of Prostaglandin E2 (MD 2.752 p<.05), and lipid mediator derivatives of the EPA and DHA signaling pathway. The subchondral cortical shell showed significant increase in BV/TV by one month. Raman spectroscopy showed alterations in the cartilage consistent with our preliminary results in the histology. The articular cartilage showed a gradual fibrillation, and at 1-month post injury showed surface erosion and cartilage matrix loss consistent with the development of OA. While skeletal muscle has a remarkable ability to recover from injury, the resulting damage has prolonged effects on the joint. These finding suggest that a direct damage to the TA muscle could provide a new model in OA research, which are highly significant in the process of understanding the mechanism of OA. References: Wang, Z., et al (2017) Ana Chimica doi: 10.1016/j.aca.2017.07.024 ; Hardy, D., et al (2016) PLOS One doi: 10.1371/journal.pone.0147198 . LM, KA, 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.

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