SERCA2 dysfunction drives vascular calcification via coupling with TSPO-MCU at mitochondria-associated endoplasmic reticulum membranes

Vascular calcification arises from osteogenic transdifferentiation of vascular smooth muscle cells (VSMC) driven by disordered calcium signaling. Sarcoplasmic/ER calcium ATPase 2 (SERCA2) maintains intracellular calcium homeostasis. Irreversible oxidation of SERCA2 Cys⁶⁷⁴ (C674) indicates SERCA2 dysfunction. The impact of SERCA2 dysfunction on vascular calcification remains poorly understood. Here, we demonstrate that oxidized SERCA2 C674, a SERCA2 dysfunction marker, was elevated in the calcified arteries of mice and chronic kidney disease (CKD) patients. Heterozygous SERCA2 Cys674Ser knock-in (SKI) mice, a SERCA2 dysfunction model, exhibited aggravated aortic calcification in cholecalciferol (VitD3)-overloaded and 5/6 nephrectomy-induced CKD mice. SERCA2 dysfunction exacerbates calcification, mitochondrial impairment and calcium overload in cultured VSMC. Mechanistically, SERCA2 dysfunction increased mitochondria-associated endoplasmic reticulum membrane (MAM) formation. Furthermore, outer-mitochondrial translocator protein (TSPO) was identified to facilitate SERCA2 coupling to the IP3R1-Grp75-VDAC1-MCU complex at MAM. TSPO knockdown alleviated SERCA2 dysfunction-induced MAM formation and vascular calcification in SKI mice and cultured SKI VSMC, while TSPO overexpression aggravated these effects. Inhibition of the downstream mitochondrial calcium uniporter (MCU) reduced mitochondrial calcium overload and thus alleviated the pro-calcific effects of TSPO in SKI VSMC. In conclusion, SERCA2 dysfunction promotes TSPO-dependent MAM formation, which couples SERCA2 to the IP3R1-Grp75-VDAC1-MCU complex, causing mitochondrial impairment and calcium overload. This study uncovers the pivotal role of SERCA2 during vascular calcification, delineates the novel components of the MAM complex, and highlights potential therapeutic targets of vascular calcification.