Poster U20 - The Biochemical Terrain of Tissue Regeneration After Volumetric Muscle Loss: Insights into Pathogenesis and Potential Therapeutic Targets

Introduction: Volumetric muscle loss (VML) is characterized by the substantial loss of skeletal muscle tissue, typically due to traumatic injury, surgical resection, or disease. VML can lead to persistent functional deficits, such as weakness, impaired mobility, and chronic pain, significantly impacting an individual's quality of life. Despite extensive research, the mechanisms underlying VML-induced muscle wasting and impaired regeneration remain incompletely understood.

Currently, treatment strategies for individuals with VML include surgical approaches, such as muscle flaps and grafts, as well as physical therapy and rehabilitation. However, these approaches have limited efficacy in restoring muscle volume and function, particularly in cases of severe VML. Approaching the chronic issue via the avenue of tissue engineering offers the promise of addressing the significant unmet clinical need in VML through the addition of supporting scaffolds and cellular intervention. VML pathophysiology involves a complex interplay of inflammation, fibrosis, and impaired muscle regeneration. Transcriptomics offers a powerful tool for unraveling the molecular intricacies of various physiological and pathological conditions, including VML. Here, we aim to provide an understanding of the biochemical landscape underlying VML, with a focus on elucidating key molecular pathways, gene expression signatures, and regulatory mechanisms involved in the progression and resolution of VML.

Materials and

Methods: Throughout a 21-day period, the regeneration and remodeling of skeletal muscle tissue in a partial thickness VML model was assessed. At day 0, 40% of the tibialis anterior muscle in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (8-week-old male) mice were surgically excised, harvesting of the tissues was done at day 3, day 7, day 14, and day 21. The tissues were divided into 3 sections to serve for transcriptomics, proteomics, and histological analysis. All histological samples were embedded in Optimal Cutting Temperature (OCT) media and frozen for cryosectioning.

Transcriptomic samples were placed in RNAlater and kept at -80C prior to RNA extraction. Homogenization of the tissues was done in Trizol, followed by two chloroform washes, allowing for a biphasic solution with the RNA component found at the upper section of the sample. The RNA is precipitated by the addition of isopropyl alcohol and any salts, phenols, or other impurities removed through the three subsequent ethanol washes. Purified RNA was then resuspended in RNAase-free water. Whole tissue RNA sequencing was done via Novogene. The tissue samples allocated for proteomic use were homogenized in a solution consisting of a combination of RIPA buffer and protease inhibitor, incubated on ice, and centrifuged to separate all superfluous tissue components. An inflammatory proteomic panel was done via RayBiotch.

Results, Conclusions, and Discussions: By examining the dynamic changes in gene expression patterns at the transcriptional level, we are offered insights into the molecular mechanisms driving VML pathogenesis and regeneration, allowing us to identify potential therapeutic targets. The transcription changes seen from day 3 onward demonstrate sustained upregulation of ECM organization, collagen synthesis, proliferation, differentiation, and pro-inflammatory genes. Downregulation at day 3, followed by normalization by day 21 of cellular respiration, fatty acid oxidation, and PPAR signaling pathway genes were also found. Proteomic analysis found significant increase of cell and ECM remodeling factors, which normalize by day 21. Those cytokines more closely related to immune response and vascularization show a steep initial decline at day 3, followed by a steady increase as time passes.

Overall, we find the transcriptomic landscape to show a continued increase in the synthesis of ECM and collagen with a decrease of the PPAR signaling pathway in the immediate recovery period of muscle regeneration. Whereas the protein panel demonstrates an offset of those changes seen in the transcriptomic analysis to changes seen in the inflammatory markers, showing a prolonged increase that does not completely return to base levels by day 21. Through this study, we were able to characterize the biochemical landscape of the regeneration process undergone by the skeletal muscle when subjected to VML, paving the way to uncovering a target mechanism to aid in a comprehensive solution for alternative treatments presented to individuals with VML.

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