Succinate accumulation induces pyroptosis and mitochondrial damage via the inhibition of ATP5F1D in HUVECs.

Atherosclerosis, a chronic inflammatory disorder, is pathophysiologically linked to endothelial cell (EC) pyroptosis. This study aims to elucidate the mechanisms by which succinate exacerbates EC pyroptosis through mitochondrial damage. Serum samples are collected from patients with coronary heart disease (CHD) and healthy controls (HCs), and the levels of succinate, interleukin (IL)-6, and IL-18 are quantified. To establish a succinate accumulation model, human umbilical vein endothelial cells (HUVECs) are treated with diethyl butyl malonate (DEBM), followed by analysis of inflammatory cytokines. The expression of pyroptosis-related proteins is assessed via western blot analysis. Morphological changes in pyroptotic vesicles and membrane pores are examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The mitochondrial membrane potential and reactive oxygen species (ROS) levels are evaluated via a JC-1 kit and MitoSOX, respectively. RNA sequencing (RNA-seq) is performed to identify potential target genes and regulatory pathways. To investigate the functional role of ATP5F1D, small interfering RNAs (siRNAs) are used to knockdown , while lentiviral vectors are used to overexpress ATP5F1D in HUVECs. The results reveal significantly elevated levels of succinate, IL-6, and IL-18 in both CHD patients and DEBM-treated HUVECs. Succinate accumulation induced by DEBM triggers pyroptosis and mitochondrial damage in HUVECs, as evidenced by the upregulation of pyroptosis-related proteins and the impairment of mitochondrial structure and function. RNA sequencing analysis identifies ATP5F1D as a key downstream target of succinate accumulation. Downregulation of ATP5F1D promotes pyroptosis and mitochondrial injury in HUVECs, whereas restoration of ATP5F1D expression effectively mitigates these detrimental effects. Succinate-induced downregulation of ATP5F1D drives mitochondrial dysfunction and pyroptosis in HUVECs.