Mitochondria-mediated mechanisms of ferroptosis in response to cardiac ischemia-reperfusion injury

摘要 冠心病是世界范围内死亡和发病率的主要原因，其原因是 心肌梗死(MI)/缺血再灌注损伤(IRI)的不利影响。心肌梗死/再灌注损伤的机制 不完全清楚，因此，没有有效的治疗策略；目前的治疗方法 减轻心脏损伤的方法主要集中在恢复冠脉血流灌注，而不是限制 再灌注损伤。多种形式的细胞死亡发生在MI/IRI后的不同阶段，这取决于 疾病的严重程度。这个项目将阐明最近发现的一种铁的调节机制- 心脏IRI中的依赖性程序性细胞死亡，铁性下垂。铁下垂是由过度的氧合作用引起的。 伴有硒蛋白谷胱甘肽过氧化物酶能力不足的磷脂(Pls) 4(Gpx4)，以消除氧化的PL，特别是磷脂酰乙醇胺(PEox)，代价是 还原型谷胱甘肽(GSH)我们最近鉴定和量化了前铁上性PEOX物种在 用最新技术研究心肌细胞对RSL3(Gpx4抑制剂、铁下垂诱导剂)的反应。在……里面 此外，我们的初步研究显示，铁上链PEOX物种在线粒体中积累。 从暴露于全球IRI的心脏以及从RSL3挑战的心肌细胞分离。我们 提示线粒体通过两种主要的GSH途径参与MI/IRI诱导的铁链信号转导。 依赖机制：i)IR诱导ETC和TCA循环产生线粒体ROS耗尽GSH， 因此，使GSH/Gpx4系统失活，以及ii)由于抑制 由于谷氨酸和半胱氨酸水平较低，线粒体中的谷氨酰胺分解作用会抑制GSH的合成。 因此，该项目将通过研究线粒体在心脏IRI中的作用来探索一种新的范式。 铁性下垂。我们的中心假设是线粒体参与了由心脏IRI引起的铁性下垂。 通过降低Gpx4活性和无法控制前铁眼性PEOX物种的积累。 此外，我们还提出，主要细胞死亡机制在MI/IRI后依赖期间有不同的影响 关于MI/IRI后的严重程度和持续时间。因此，我们将评估特定的生物标志物以区分 细胞凋亡、铁性下垂、坏死性下垂和下垂在MI/IRI后的作用。两种方法将是 通过i)特异性地抑制mtROS的产生/GSH耗竭，以及ii) 通过一种有效的还原剂硫辛酸/二氢硫辛酸(LA/DHL)补充GSH池。的具体目标 该项目将(I)确定在IRI期间铁性下垂对心功能不全的相对贡献，(Ii) 探索线粒体介导的心肌细胞铁链信号的分子机制，以及(Iii) 检查新的铁下垂抑制剂对心脏IRI的有效性。该项目将建立 与其他主要细胞死亡机制相比，铁下垂在MI/IRI诱导的细胞死亡中的作用 心功能障碍，并发现新的线粒体介导的铁上链信号机制，将 引领治疗心脏IRI的创新治疗策略。

ABSTRACT Coronary heart disease is the leading cause of mortality and morbidity worldwide that occurs due to the detrimental effects of myocardial infarction (MI)/ischemia-reperfusion injury (IRI). Mechanisms of MI/IRI are not completely clear, and hence, there are no effective therapeutic strategies; current therapeutic approaches to mitigate heart damage mostly focus on restoring coronary perfusion but not limiting reperfusion injury. Multiple forms of cell death occur at different stages of post-MI/IRI depending on the severity of the disease. This project will elucidate the regulatory mechanisms of a recently discovered iron- dependent programmed cell death, ferroptosis, in cardiac IRI. Ferroptosis arises via excessive oxygenation of phospholipids (PLs) accompanied by the insufficient capacity of a selenoprotein glutathione peroxidase 4 (GPX4) to eliminate oxidized PLs, particularly, phosphatidylethanolamine (PEox) at the expense of reduced glutathione (GSH). We have recently identified and quantified pro-ferroptotic PEox species in response to RSL3 (GPX4 inhibitor, ferroptosis inducer) in cardiomyocytes by a state-of-art technique. In addition, our preliminary studies revealed accumulation of ferroptotic PEox species in mitochondria isolated from hearts exposed to global IRI as well as from cardiomyocytes challenged with RSL3. We propose that mitochondria participate in MI/IRI-induced ferroptotic signaling through two major GSH- dependent mechanisms: i) IR-induced mitochondrial ROS generation by ETC and TCA cycle deplete GSH, and hence, inactivate the GSH/GPX4 system, and ii) glutamate deficiency due to inhibition of glutaminolysis in mitochondria inhibits GSH synthesis as a result of low glutamate and cysteine levels. Thus, this project will explore a novel paradigm by investigating the role of mitochondria in cardiac IRI ferroptosis. Our central hypothesis is that mitochondria are engaged in ferroptosis induced by cardiac IRI through diminishing GPX4 activity and inability to control the accumulation of pro-ferroptotic PEox species. Also, we propose that major cell death mechanisms have different impacts during post-MI/IRI depending on the severity and duration of post-MI/IRI. Hence, we will evaluate specific biomarkers to distinguish the contributions of apoptosis, ferroptosis, necroptosis, and pyroptosis to post-MI/IRI. Two approaches will be employed to mitigate cardiac IRI via i) specific suppression of mtROS production/GSH depletion, and ii) replenishment of the GSH pool by a potent reductant lipoic/dihydrolipoic acid (LA/DHLA). Specific Aims of the project will (i) determine comparative contributions of ferroptosis to cardiac dysfunction during IRI, (ii) explore molecular mechanisms of mitochondria-mediated ferroptotic signaling in cardiac cells, and (iii) examine the effectiveness of novel inhibitors of ferroptosis against cardiac IRI. The project will establish the contribution of ferroptosis, in comparison with other major cell death mechanisms, to MI/IRI-induced cardiac dysfunction, and discover new mitochondria-mediated mechanisms of ferroptotic signaling that will lead to innovative therapeutic strategies for the treatment of cardiac IRI.