Abstract Body: BACKGROUND: Chronic limb-threatening ischemia (CLTI), the most severe form of peripheral artery disease, is marked by ischemic rest pain or tissue loss, and increased risk of major adverse limb and cardiovascular events. Surgery is a pillar of the medical management of CLTI, yet amputation rates remain high after these procedures. Patients with CLTI have poor mitochondrial function and low O₂ availability which drive the accumulation of acetyl-CoA and lactate which are substrates for histone modifications. However, no studies have explored histone modifications in the CLTI limb. To test the hypothesis that mitochondrial dysfunction in CLTI drives epigenetic changes in ischemic muscle, we used a multi-faceted approach. METHODS: 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 again five days post-surgery. Additionally, 12-14-week-old C57BL/6J and BALB/cJ mice underwent femoral artery ligation (FAL) and hindlimb muscle was collected 24 hours and seven days post-FAL. Immunoblotting and histological analyses assessed epigenetic changes to quantify histone acetylation, lactylation, and methylation patterns in human and mouse ischemic muscle. RESULTS: In patients with CLTI, ischemic gastrocnemius muscle showed reduced H3K27ac compared to the quadriceps, accompanied by decreased H3K4me3 and H3K9me3 levels. Given strain-specific differences in mitochondrial function and ischemic regeneration following FAL, C57BL/6J and BALB/cJ mice were compared. At 24 hours post FAL, BALB/cJ mice showed significant reductions in H3K18ac (p<0.0001), H3K27ac (p=0.0173), and H3K18la (p=0.0021) in the ischemic limb relative to the control limb. By seven days post-FAL, both C57BL/6J and BALB/cJ mice demonstrated increased H3K18ac (p=0.0177; p=0.0001) and H3K27ac (p<0.0001; p=0.0023) in the ischemic limb, while methylation changes diverged, with decreased H3K4me3 (p=0.0001; p=0.0001) and elevated H3K9me3 (p=0.0006; p<0.0001) in the ischemic limb. CONCLUSION: Ischemia drives dynamic, time- and strain-dependent remodeling of histone acetylation, lactylation, and methylation in skeletal muscle. Together, these epigenetic shifts suggest that metabolic dysfunction contributes directly to chromatin regulation in CLTI, highlighting epigenetic pathways as potential targets to enhance muscle recovery following revascularization.