Abstract Body (Do not enter title and authors here): Background: Endothelial cells (ECs) utilize molecular mechanosensors that are critical for sensing disturbed flow (DF) to promote atherosclerosis. Copper (Cu), an essential micronutrient, is increased in human atherosclerotic plaques, while Cu chelation inhibits atherosclerosis in mice. “Cuproptosis” is a recently recognized form of programmed cell death which is driven by Cu-dependent mitochondrial protein aggregation and mitochondrial dysfunction; however, its role in atherosclerosis remains unknown.
Results: Here we show that the Cu uptake transporter CTR1, which mainly localizes in the plasma membrane and caveolae/lipid rafts (C/LR), functions as a novel mechanosensor for DF to promote cuproptosis and atherosclerosis. Using X-ray fluorescence and ICP-MS analysis, we found that Cu, but not Fe and Zn levels, were increased in DF-exposed EC layers in the aortic arch of high fat diet-induced atherosclerotic (ApoE-KO) mice (2.3-fold) and cultured human aortic ECs (1.73-fold). Notably, DF vs. laminar flow (LF) increased mitochondrial Cu (1.6-fold) in ECs and induced mitochondrial dysfunction and cell death. These responses were rescued by mitochondrial-targeted Cu-depleting nanoparticle (mitoCDN) or CTR1siRNA. Functionally, the partial carotid ligation model revealed that EC-Ctr1 KO mice (by 70%) or mitoCDN (i.v.)(by 58%) reduced DF-induced atherosclerotic lesions in ApoE-KO mice, suggesting that DF-induced mitochondrial Cu promotes atherosclerosis through CTR1. Importantly, DF induced cuproptosis-related responses (increased DLAT aggregation, reduced DLAT lipoylation (47%) and Fe-S cluster proteins vs. LF), thereby increasing proteotoxic stress and decreasing mitochondrial respiration. All of these DF-induced responses were blocked by mitoCDN or CTR1siRNA. To address how DF increased mitochondrial Cu via CTR1, we performed subcellular fractionation and colocalization analysis and found that DF promoted mitochondrial Cu transporter SLC25A3 translocation to the plasma membrane (C/LR)(10.2-fold) and its association with CTR1 (2.8-fold), which were blocked by the disruption of C/LR using methyl-beta cyclodextrin. Furthermore, CTR1siRNA prevented DF-induced SLC25A3 translocation to plasma membrane.
Conclusion: Endothelial CTR1 functions as a novel DF “mechanosensor” to increase mitochondrial Cu levels via recruiting mitochondrial SLC25A3 to plasma membrane C/LR where it binds to CTR1, leading to cuproptosis, which contributes to atherosclerosis.