Abstract Tu0081: In a murine model of PAD, genetic loss of the absent in melanoma 2 (AIM2) inflammasome promotes regional transcriptional activity favoring regeneration, cellular communication and management of oxidative stress

Introduction: We previously showed that the absent-in-melanoma 2 (AIM2) inflammasome is expressed in PAD myoblasts, reduces their ability to differentiate, and may cause myopathy. Here, using spatially resolved genetic profiling, we tested the hypothesis that beneficial effects of AIM2 inhibition in myoblasts can be detected in vivo , associated with genetic features favoring regeneration, and linked to greater cell-cell communication in muscle ischemia. Methods: Unilateral femoral artery ligation was performed on male 3mo C57Bl6/J (WT) and B6.129P2- Aim2 Gt(CsG445)Byg /J (AIM2 knockout [KO]) mice. Tibialis anterior/extensor digitorum longus ( TA/EDL ) muscle were procured for histology and spatial transcriptomics (ST; Visium 10X). Sequencing reads were processed with Space Ranger using the Visium mouse transcriptome. Expression data was processed with Seurat using Harmony for batch correction, and deconvolution identified cell-type colocalization based on a published scRNAseq reference. CellChat inferred cell-cell communication using the secreted signaling ligand-receptor pairs. Results: Recovering ischemic TA/EDL sections in KO had larger myofiber size (p<0.05) and capillary density (p<0.001) compared to WT mice (N=5 each/t-test). Uniform Manifold Approximation and Projection (UMAP) identified five clusters where Type IIX and IIB myofibers as well as fusing myocytes predominated (Fig.1A). Inflammatory cells were elevated in WT, while fusing myocytes and cell-cell interactions by number and strength were more numerous in KO mice (Fig.1B). Genes encoding myosin heavy chain 2 ( MYH2) , a myofiber differentiation marker, secreted protein acidic and rich in cysteine ( SPARC ), a mediator supporting regeneration, and the oxidoreductase thioredoxin 2 (TXN2) which protects against oxidative injury had significantly more expression in KO mice (Fig.1C) and were expressed in regions where emerging myocytes fused to established cells. Conclusion: This is one of the first applications of ST to understand regionally expressed genes at the peak of ischemic muscle recovery. We confirmed a protective effect of AIM2 inhibition on myocyte size, angiogenesis, differentiating myoblast colocalization with mature muscle cells, communication, and pro-regenerative differential gene expression in regions of fusing cells. Thus, inhibiting AIM2 may be a target for future therapeutics to improve muscle recovery in PAD.