Abstract Body: Introduction: Skeletal muscle fibers contain hundreds of myonuclei normally located at the cell periphery. However, lower extremity muscles of people with peripheral artery disease (PAD) frequently have myonuclei positioned away from the cell periphery. The significance of non-peripheral myonuclei in PAD remains unclear. While traditionally considered a sign of muscle regeneration, recent data suggest more centrally-located myonuclei may not always indicate repair. This study used spatial transcriptomics to compare gene expression of non-peripheral vs. peripheral myonclei in PAD muscle cross-sections. Methods: Visium (10x Genomics) was applied to gastrocnemius biopsies of three male patients with PAD for whole transcriptome spatial analysis. IHC was used to detect dystrophin (fiber borders) and nuclei were stained (DAPI) prior to library preparation and sequencing. Following spatial mapping, a pipeline for resolution enhancement was applied to classify barcoded sub-spots as containing a non-peripheral or peripheral myonucleus based on its location relative to the muscle fiber border. DESeq2 was used for paired-design differential expression analysis to test the effect of nuclear position status while controlling for sample-to-sample variation. The Wald test assessed statistical significance, with p-values adjusted for multiple testing using the BH method. Results: Of 2,794 myonuclei analyzed, 2,515 (90%) were peripherally located and 279 (10%) were not peripherally located. Compared to peripherally located myonuclei, 74 genes were significantly overexpressed in myonuclei that were not peripheral (p_adj<0.05). Genes overexpressed in non-peripheral myonuclei included those associated with ‘sarcomere disorganization’ (p_adj=5.5x10^-4) and ‘ischemia’ (p_adj=0.003) pathways. Overexpression of several inflammatory S100 protein-encoding genes, S100A4 (fold-change [FC]=2.10, p_adj=0.01), S100A6 (FC=1.64, p_adj=0.002), S100A13 (FC=1.53, p_adj=0.001), and S100A16 (FC=1.53, p_adj=0.005), and Ankyrin Repeat Domain 1 (ANKRD1, FC=2.33, p_adj=1.13x10^-7), were common in non-peripheral myonuclei, compared to peripheral myonuclei. Conclusions: Compared to peripherally located myonuclei, non-peripheral myonuclei were characterized by gene expression associated with inflammation, stress sensing, and pathological remodeling. Based on their expression profiles, these myonuclei do not reflect regenerating fibers and may contribute to further damage and increased muscle pathology in PAD.