In vivo characterisation and manipulation of succinate-dependent free radical injury during ischaemia-reperfusion

缺血再灌注过程中琥珀酸依赖性自由基损伤的体内表征和处理

• 批准号: MR/P000320/1
• 负责人: Thomas Krieg
• 金额: $ 62.87万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FP000320%2F1
• 关键词: vivo; characterisation; manipulation; succinate; dependent

Ischaemia-reperfusion (IR) injury comprises tissue damage and dysfunction caused by the removal and subsequent reintroduction of the oxygenated blood supply. Collectively, these pathologies are by far the leading cause of death in the world, including most prevalently the injuries from heart attack. Additionally, in the clinical context of surgery and organ transplantation, IR is unavoidable and detrimental to patient outcomes. A major upstream driver of IR damage is the production of reactive oxygen species (ROS), which lead to tissue damage and death. However, as the timing and molecular sources of ROS production were not well characterised, therapeutic strategies have mostly relied on antioxidants to quench these damaging molecules once they have been produced. Unfortunately, such therapies have proved ineffective in the clinical setting, most likely due to uncertainty about the molecular mediators of ROS in vivo. We have recently defined an essential molecular source of ROS during heart attack and stroke in vivo: the mitochondrial metabolite succinate. Importantly, we have demonstrated that succinate accumulates substantially when tissues are deprived of oxygen during heart attack and stroke, and when oxygen is restored this accumulated succinate acts as a molecular fuel for producing ROS. Moreover, we have discovered that this fuelling of ROS by succinate is due to its interaction with an enzyme essential for energy production in our cells called mitochondrial complex I.These exciting findings provide us with a first molecular understanding of the origins of ROS during IR. With the research program proposed here, we will now apply this knowledge to understand the molecular mechanisms that control ROS production in vivo through succinate and complex I, to develop better-targeted therapeutic strategies against IR injury, and develop methods to assess the succinate pathways to better diagnose outcomes of IR injury in various settings.To address these questions, we will use state-of-the-art technologies to investigate the metabolic state of living tissue. Using mass spectrometric methods, it is now possible to quantify hundreds of metabolites from living tissue in a single experiment, an approach termed metabolomics.This metabolomics method will enable us for the first time to track the metabolome of the heart during IRI events in vivo. Our goal is to identify metabolite shifts that occur during ischaemia that can result in interactions with complex I at reperfusion to generate ROS. Our preliminary analyses have already yielded a promising lead candidate molecule: succinate. Of the hundreds of metabolites tracked during IR, succinate was the only mitochondrial metabolite found to accumulate significantly in ischaemic tissue. Furthermore, following only 5 minutes of reperfusion, this accumulated succinate was metabolized to near resting levels. This unique pattern of accumulation and rapid consumption, combined with the fact that succinate consumption drives ROS production at complex I, enabled us to determine the molecular source of ROS during IRI.We will now aim to identify the pathway(s) that drive ischaemic succinate accumulation and attempt to modify those pathways using pharmacological. This will allow us to determine directly the role played by succinate in driving ROS production at reperfusion, while providing novel drug targets for IRI indications. In parallel, we will look to determine the mechanism of succinate-linked complex I free radical production at reperfusion that will provide essential insight into the metabolic parameters that drive ROS production in vivo. Finally, we will develop new drugs designed to directly manipulate succinate levels or oxidation in vivo in order to develop rational therapeutic strategies for IRI pathologies preventing the ROS production during IRI.

缺血再灌注（IR）损伤包括由氧合血液供应的去除和随后的再引入引起的组织损伤和功能障碍。总的来说，这些病理是迄今为止世界上主要的死亡原因，包括最常见的心脏病发作造成的伤害。此外，在外科手术和器官移植的临床背景下，IR是不可避免的，并且对患者结果有害。IR损伤的主要上游驱动因素是活性氧（ROS）的产生，其导致组织损伤和死亡。然而，由于ROS产生的时间和分子来源没有得到很好的表征，因此治疗策略主要依赖于抗氧化剂来在这些破坏性分子产生后淬灭它们。不幸的是，这种疗法已被证明在临床环境中无效，最有可能是由于体内ROS的分子介质的不确定性。我们最近定义了一个重要的分子来源的活性氧在心脏病发作和中风在体内：线粒体代谢产物琥珀酸。重要的是，我们已经证明，当组织在心脏病发作和中风期间缺氧时，琥珀酸会大量积累，当氧气恢复时，这种积累的琥珀酸会作为产生ROS的分子燃料。此外，我们发现琥珀酸对ROS的这种燃料是由于它与我们细胞中能量产生所必需的酶（称为线粒体复合物I）的相互作用。这些令人兴奋的发现为我们提供了IR期间ROS起源的第一个分子理解。我们现在将应用这些知识来了解通过琥珀酸和复合物I控制体内活性氧产生的分子机制，为了开发针对IR损伤的更有针对性的治疗策略，并开发评估琥珀酸途径的方法，以更好地诊断各种情况下IR损伤的结果。为了解决这些问题，我们将使用最先进的技术来研究活组织的代谢状态。使用质谱方法，现在可以在一个实验中定量来自活组织的数百种代谢物，这种方法称为代谢组学。这种代谢组学方法将使我们能够首次在体内追踪IRI事件期间心脏的代谢组。我们的目标是确定在缺血期间发生的代谢物变化，其可以导致在再灌注时与复合物I相互作用以产生ROS。我们的初步分析已经产生了一个有希望的主要候选分子：琥珀酸。在IR期间追踪的数百种代谢物中，琥珀酸是发现在缺血组织中显著累积的唯一线粒体代谢物。此外，再灌注仅5分钟后，这种积累的琥珀酸被代谢到接近静息水平。这种独特的积累和快速消耗模式，结合琥珀酸消耗驱动复合物I的ROS产生的事实，使我们能够确定IRI期间ROS的分子来源。我们现在的目标是确定驱动缺血性琥珀酸积累的途径，并尝试使用药理学来修饰这些途径。这将使我们能够直接确定琥珀酸在再灌注时驱动ROS产生中所起的作用，同时为IRI适应症提供新的药物靶点。与此同时，我们将着眼于确定再灌注时琥珀酸连接的复合物I自由基产生的机制，这将为体内驱动ROS产生的代谢参数提供必要的见解。最后，我们将开发新的药物，旨在直接操纵体内琥珀酸水平或氧化，以制定合理的治疗策略，IRI病理，防止ROS的生产过程中IRI。

项目成果

ND3 Cys39 in complex I is exposed during mitochondrial respiration.

• DOI: 10.1016/j.chembiol.2021.10.010
• 发表时间: 2022-04-21
• 期刊: CELL CHEMICAL BIOLOGY
• 影响因子: 8.6
• 作者: Burger, Nils;James, Andrew M.;Mulvey, John F.;Hoogewijs, Kurt;Ding, Shujing;Fearnley, Ian M.;Loureiro-Lopez, Marta;Norman, Abigail A., I;Arndt, Sabine;Mottahedin, Amin;Sauchanka, Olga;Hartley, Richard C.;Krieg, Thomas;Murphy, Michael P.
• 通讯作者: Murphy, Michael P.

Selective mitochondrial superoxide generation in vivo is cardioprotective through hormesis

体内选择性线粒体超氧化物生成通过毒物兴奋效应具有心脏保护作用

• DOI: 10.17863/cam.36909
• 发表时间: 2019
• 作者: Antonucci S

Ester Prodrugs of Malonate with Enhanced Intracellular Delivery Protect Against Cardiac Ischemia-Reperfusion Injury In Vivo.

丙二酸酯的酯前药具有增强的细胞内递送能力，可预防体内心脏缺血再灌注损伤。

• DOI: 10.17863/cam.55714
• 发表时间: 2020
• 作者: Prag H

Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

• DOI: 10.1074/jbc.m117.798744
• 发表时间: 2017-09-01
• 期刊: The Journal of biological chemistry
• 作者: Chouchani ET;James AM;Methner C;Pell VR;Prime TA;Erickson BK;Forkink M;Lau GY;Bright TP;Menger KE;Fearnley IM;Krieg T;Murphy MP
• 通讯作者: Murphy MP

Ischemia-Selective Cardioprotection by Malonate for Ischemia/Reperfusion Injury.

丙二酸对缺血/再灌注损伤的缺血选择性心脏保护作用。

• DOI: 10.17863/cam.88285
• 发表时间: 2022
• 作者: Prag H