Department of Cardiology (G.I., Y.K., J.E., A.A., K. Shirakawa, H.M., H.K., T.H., Shinichi Goto, S.K., Y.I., K. Sugai, K.D., M.I., M. Sano), Keio University School of Medicine, Tokyo, Japan.
Institute for Integrated Sports Medicine (Y.K., K. Sato), Keio University School of Medicine, Tokyo, Japan.
Circ Res. 2023 Oct 27;133(10):861-876. doi: 10.1161/CIRCRESAHA.123.323517. Epub 2023 Oct 11.
The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis.
In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain.
Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury.
Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.
心肌细胞的膜成分富含多不饱和脂肪酸，容易氧化。因此，维持细胞功能需要一个有效的基于谷胱甘肽的脂质氧化还原系统。然而，缺血再灌注（IR）过程中氧化还原系统的破坏、氧化脂质的产生以及随之而来的细胞死亡（铁死亡）之间的关系仍不清楚。我们研究了 IR 过程中与铁死亡相关的谷胱甘肽介导的还原系统破坏的机制，并开发了抑制铁死亡的干预策略。
通过微透析和组织代谢组学分析研究了 IR 过程中细胞内外代谢物水平的体内波动。使用液相色谱高分辨率质谱法评估氧化的磷脂酰胆碱。通过三苯基四唑氯/伊文思蓝染色评估 IR 后的危险区域。
代谢组学分析结合微透析显示，在缺血期间和再灌注后，谷胱甘肽从缺血区域大量释放到细胞外空间。在体外心肌细胞模型的缺氧再灌注过程中，也观察到谷胱甘肽向细胞外空间释放，同时细胞内谷胱甘肽浓度降低。化学抑制或谷胱甘肽转运体（主要是多药耐药蛋白 1 [MRP1]）的基因抑制可防止这种细胞外谷胱甘肽释放。MRP1 抑制剂的治疗降低了细胞内活性氧水平和脂质过氧化，从而抑制细胞死亡。随后在 IR 后对体内氧化磷脂的评估表明，铁死亡参与其中，因为在再灌注后 12 小时缺血区域中多种氧化的磷脂酰胆碱水平显著升高。MRP1 转运体的抑制也减轻了体内谷胱甘肽的耗竭，并显著减少了氧化的磷脂酰胆碱的产生。MRP1 抑制剂的给药显著减轻了 IR 损伤后的梗死面积。
谷胱甘肽在 IR 过程中持续释放，主要以 MRP1 依赖的方式释放，并诱导铁死亡。抑制谷胱甘肽释放可减轻铁死亡并减少 IR 后的心肌梗死面积。
