Abstract 4371070: Mechanistic Insight into Disturbed Flow–Induced Mitochondrial Copper Overload and Cuproptosis in Atherosclerosis

Background: Disturbed flow (D-flow), a pro-atherogenic hemodynamic force, promotes endothelial cell (EC) dysfunction and atherosclerosis through poorly understood mechanisms. Copper (Cu) is essential micronutrient regulated by specific transporters; however, excessive intracellular Cu, particularly within mitochondria, can trigger cuproptosis, a newly characterized form of Cu-dependent cell death. Our preliminary studies indicate that D-flow increases mitochondrial Cu levels in human ECs. This study aims to define how D-flow drives mitochondrial Cu accumulation and promotes cuproptosis and atherosclerosis. Results: Using synchrotron-based X-ray fluorescence microscopy, ICP-MS, and Cu-sensitive fluorescent probes, we found that D-flow significantly elevates Cu accumulation (but not Fe or Zn) in atheroprone arterial regions (2.3-fold) and in human ECs exposed to D-flow (5.8-fold), accompanied by increased expression of the Cu importer CTR1. D-flow also leads to a 1.6-fold increase in mitochondrial Cu, inducing classic features of cuproptosis, such as DLAT aggregation, Fe-S cluster protein loss, disrupted oxidative phosphorylation, and EC death. Mechanistically, D-flow stimulates the translocation of mitochondria harboring the Cu transporter SLC25A3 to plasma membrane caveolae/lipid rafts (C/LR), where SLC25A3 interacts with CTR1 (2.8-fold) in a caveolin-1-dependent manner. Focusing on mitochondrial trafficking, we found that silencing GTPases Miro1/2, key regulators of mitochondrial movement, prevented SLC25A3 translocation to C/LR and its association with CTR1. Disruption of components within this pathway including CTR1, Miro1/2, SLC25A3, or lipid rafts, prevented mitochondrial Cu overload and cuproptosis responses. Notably, endothelial-specific Ctr1 knockout or treatment with a mitochondria-targeted Cu chelator (mitoCDN) restored mitochondrial function, reduced EC death, and decreased atherosclerotic lesion formation in a partial carotid ligation model. Conclusion: Our findings uncover a novel mechanism by which D-flow induces mitochondrial Cu overload through SLC25A3 translocation mediated by Miro1/2 to C/LR, where it associates with CTR1. This axis drives endothelial cuproptosis and promotes atherosclerosis, revealing new therapeutic targets for vascular disease.