A Selective and Cell-Permeable Mitochondrial Calcium Uniporter (MCU) Inhibitor Preserves Mitochondrial Bioenergetics after Hypoxia/Reoxygenation Injury

Mitochondrial Ca2+ (mCa2+) uptake mediated by the mitochondrial calcium uniporter (MCU) plays a critical role in signal transduction, bioenergetics, and cell death, and its dysregulation is linked to several human diseases. In this study, we report a new ruthenium complex Ru265 that is cell-permeable, minimally toxic, and highly potent with respect to MCU inhibition. Cells treated with Ru265 show inhibited MCU activity without any effect on cytosolic Ca2+ dynamics and mitochondrial membrane potential (ΔΨm). Dose-dependent studies reveal that Ru265 is more potent than the currently employed MCU inhibitor Ru360. Site-directed mutagenesis of Cys97 in the N-terminal domain of human MCU ablates the inhibitory activity of Ru265, suggesting that this matrix-residing domain is its target site. Additionally, Ru265 prevented hypoxia/reoxygenation injury and subsequent mitochondrial dysfunction, demonstrating that this new inhibitor is a valuable tool for studying the functional role of the MCU in intact biological models.由线粒体钙单向转运体（mitochondrial calcium uniporter, MCU）介导的线粒体钙离子（mCa2+）摄取，在信号转导、生物能量代谢与细胞死亡过程中发挥关键作用，其调控异常与多种人类疾病存在密切关联。本研究报道了一种新型钌配合物Ru265，该化合物具备细胞可通透性、低毒性，且对MCU具有高效抑制活性。经Ru265处理的细胞其MCU活性受到抑制，但对胞质钙离子动态变化及线粒体膜电位（ΔΨm）无显著影响。剂量依赖性实验结果显示，Ru265的抑制效力优于当前广泛使用的MCU抑制剂Ru360。对人MCU N端结构域内Cys97位点的定点诱变可消除Ru265的抑制活性，提示该基质驻留结构域是其作用靶点。此外，Ru265可有效阻断缺氧/复氧损伤及后续的线粒体功能障碍，证明这一新型抑制剂是探究MCU在完整生物模型中功能角色的极具价值的研究工具。