Abstract 15616: Single Cell Transcriptomic Profiling Identifies Ferroptosis and Oxidative Stress as Potential Drivers of Muscle Dysfunction in Pad

Introduction: Peripheral artery disease (PAD) affects >8 million people nationwide and is disabling. PAD-associated myopathy causes weakness yet is understudied. Unbiased transcriptomic profiling of muscle satellite cells (MuSC) which drive recovery may elucidate the etiology of ischemic myopathic change. Hypothesis: MuSC in PAD differentially express injury associated pathways that may be novel targets for intervention to reduce ischemic myopathy. Methods: Muscle tissue biopsies were obtained during lower extremity amputation or bypass in PAD/ischemic (N=4; ABI<0.6) and perfused conditions (N=4). CD56+ magnetic assisted cell sorting (MACS)-purified MuSCs were processed for single-cell RNA sequencing using the 10X Genomics platform. Unsupervised clustering and differential gene expression were performed. Western blot (WB) informed by transcriptomics confirmed pathway activation in perfused, ischemic and PAD(-) cells. Results: Among 25,298 analyzed ischemic and perfused cells, a unique cluster of MuSC transcriptionally programmed for apoptosis was detected under chronically ischemic PAD but not control conditions. Furthermore, quiescent cells with renewal capacity were lower in MuSC from ischemic muscle (17.8% vs. 26.3%, P =0.03) while pathologic myofibroblasts were almost exclusively seen in PAD (Fig.1A-B). Genes associated with iron metabolism (FTL, FTH), ferroptosis regulation (GSS), oxidative stress (HMOX) and the damage associated molecular pattern (DAMP) HMGB1 were upregulated in ischemic MuSC shown in violin plots (Fig.1C). By WB, CISD1 protein which regulates ferroptosis was elevated in PAD (p <0.03Fig.1C). Conclusions: The data implicate oxidative damage and iron-mediated programmed cell death as potential mechanisms driving muscle dysfunction from chronic ischemia. Targeting ferroptosis and oxidative stress represent novel ways to improve outcomes by reducing myopathy in PAD.