Abstract Body: BACKGROUND: Chronic limb-threatening ischemia (CLTI), the most severe form of peripheral artery disease (PAD), is marked by ischemic rest pain or tissue loss, affecting 1 in 10 PAD patients, and increasing risks of limb amputation, cardiovascular events, and mortality. Surgical revascularization is a pillar of the medical management of CLTI, yet a thorough understanding of how revascularization impacts limb muscle is lacking. To begin to fill this knowledge gap, we performed temporal single-nuclei multi-omic sequencing (snRNA-seq and snATAC-seq) on calf muscle obtained from CLTI patients before and after revascularization. METHODS: Four patients with CLTI (Rutherford category 4 or 5) undergoing open revascularization (vein bypass) were enrolled. Biopsies were taken from gastrocnemius (ischemic) and quadriceps (non-ischemic) muscles pre-surgery, and gastrocnemius biopsies were collected on post-operative day (POD) 1, 5, and 28, with a final quadriceps biopsy on POD28. Nuclei were isolated from 20 samples and 10x Genomics multi-omics was performed. Bioinformatic analysis assessed gene expression and chromatin accessibility. RESULTS: 34,533 high quality nuclei for each snRNA-seq and snATAC-seq dataset were captured after filtering and quality control. SnRNA-seq clustering revealed eleven distinct cell populations. SnATAC-seq datasets were clustered and cell populations were labeled according to the corresponding snRNA-seq 10x barcode cell type. Temporal shifts in cellular abundance were detected in the data, notably an increase in the relative abundance of macrophages between POD0 (8.9%), POD1 (22.7%) and POD5 (36.6%), followed by a decrease at POD28 (31.8%). Type I myonuclei underwent a decrease in relative abundance between POD0 (26.2%) and POD5 (17.9%), with an increase at POD28 (26.8%). Comparatively, satellite cells saw a gradual increase in relative abundance from POD0 (11.1%) through POD1 (18.7%), POD5 (30.5%), to POD28 (39.8%). Differentially expressed gene analysis of myonuclei populations uncovered downregulation of genes (p<0.05) involved in oxidative phosphorylation, adaptive immune response, and ribosome assembly. CONCLUSION: Single-nuclei multi-omics reveals transcriptional and epigenetic changes during recovery after revascularization, highlighting shifts in cellular composition and gene regulation in CLTI. These provide insights into muscle remodeling, offering potential biomarkers or therapeutic targets to optimize limb recovery and function.