Abstract 20523: Immune Responsive Gene 1-Itaconate Induces Mitochondrial Dysfunction to Impair Ischemic Muscle Revascularization and Perfusion Recovery

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Abstract

Introduction: IRG1 processes citrate to itaconate in mitochondrial Krebs cycle. We have recently shown that IRG1 expression inversely correlates with perfusion recovery in preclinical PAD model (hind limb ischemia (HLI)) and IRG1 overexpression or itaconate decreased endothelial (EC) angiogenic potential and induced an M1 like macrophage (MΦ) phenotype. Based on these data, we hypothesize that increased IRG1-itaconate levels induce mitochondrial dysfunction (defined as any abnormalities in mitochondrial ATP generation, membrane potential, reactive oxygen species (ROS), metabolite synthesis, mitophagy and/or biogenesis) in ischemic muscle (IM) to impair perfusion recovery

Results: qPCR showed a significant increase in IRG1 expression in both human and mouse IM as well as in hypoxia serum starved (HSS) EC (EOMA), skeletal muscle cells (C2C12) and MΦs (Raw264.7) vs. controls in vitro. Flow cytometry showed a significant increase of IRG1 expression in IM mitochondria vs. non IM. LC-MS/MS showed a significant increase in itaconate levels in HSS EC, C2C12 and MΦs vs. controls. IRG1 overexpression or itaconate significantly decreased mitochondrial potential and increased cellular ROS in EC, C2C12 and MΦs under normal conditions, whereas IRG1 inhibition in HSS EC, C2C12 and MΦs reversed these processes vs. respective controls. Immunoblot of LC3 (mitophagy gene) in IRG1 overexpressing EC, C2C12 and MΦs showed a significant decrease in LC3-II:LC3-I levels without caspase-3 activation vs. controls. In vivo, IRG1 overexpression significantly decreased EC and MΦ mitochondrial potential and increased cellular ROS resulting in increased M1 like MΦs (CD80+), decreased M2 like MΦs (CD206+), decreased angiogenesis (2X), increased necrosis and impaired perfusion by day 7 and 14 post HLI (d14: Con Pld 74±3 vs. IRG1 Pld 50±6). IRG1 inhibition significantly induced angiogenesis (2.5X), decreased necrosis and enhanced perfusion recovery by day 7 and 14 post HLI (d14: Con pld 48±3vs. IRG1 KO Pld 66±2). Statistical analysis by GraphPad Prism7, P<0.05 considered significant

Conclusion: Our data show that IRG1-itaconate induces mitochondrial dysfunction and impairs mitophagy in EC and MΦs to induce cell phenotypes that impair ischemic muscle revascularization

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