Modulation of Oxidized Low-density Lipoprotein-Affected Macrophage Efferocytosis by Mitochondrial Calcium Uniporter in a Murine Model

Objective: Efferocytosis dysfunction contributes to the progression and rupture of atherosclerotic plaques. Efferocytosis is crucially modulated by intracytoplasmic Ca²⁺, and mitochondrial calcium uniporter (MCU) complex proteins serve as key channels for regulating Ca²⁺ concentration. Therefore, it was speculated that MCU may affect the development of atherosclerosis (AS) by regulating efferocytosis. In the present study, we aimed to investigate whether MCU could affect foam cell formation by regulating efferocytosis.

Methods: We stimulated primary macrophages (Møs) using oxidized low-density lipoprotein (ox-LDL) to mimic the atherosclerotic microenvironment and treated them with Ru360, an MCU-specific inhibitor, and UNC1062, an inhibitor of efferocytosis. Additionally, we conducted double staining to determine the Mø efferocytosis rate. We measured the expression of MCU complexes and efferocytosis-associated proteins using western blotting (WB) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, we separately detected the Ca²⁺ level in the cytoplasm and mitochondria (MT) using Fluo-4 AM and Rhod-2 methods. We separately determined the Reactive Oxygen Species (ROS) level in cytoplasm and MT using dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probing method and Mito-SOXTM superoxide indicator staining. Additionally, we conducted the enzyme-linked immunosorbent assay (ELISA) to detect the production of interleukin-6 (IL-6), interleukin-18 (IL-18), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α). Oil Red O staining was performed to measure cytoplasmic lipid levels.

Results: Ru360 attenuated ox-LDL-induced efferocytosis dysfunction, and attenuated the upregulation of MCU and MCUR1 induced by ox-LDL, and meanwhile attenuated the downregulation of MCUb induced by ox-LDL. Ru360 attenuated the decrease of intracytoplasmic Ca²⁺ concentration induced by ox- LDL, Ru360 also attenuated the ROS production induced by ox- LDL, attenuated the release of IL-6, IL-18, IL-1β, and TNF-α induced by ox- LDL, and attenuated the increase of intracytoplasmic lipid content induced by ox-LDL. UNC1062 attenuated the effects of Ru360 in reducing inflammatory cytokines and intracytoplasmic lipid content.

Conclusions: In this study, we found that MCU inhibition modulated intracytoplasmic Ca²⁺ concentration, improved impaired Mø efferocytosis, and reduced ROS generation. Macrophage efferocytosis removed apoptotic cells and prevented the release of inflammatory factor and foam cell formation, and this can be a potential new therapeutic target for alleviating atherosclerosis.

MCU; atherosclerosis; efferocytosis; macrophage; ox-LDL.