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

缺血-再灌注（IR）损伤包括组织损伤和功能障碍，这是由氧合血供应的移除和随后的重新引入引起的。总的来说，这些疾病是迄今为止世界上死亡的主要原因，包括最普遍的心脏病发作造成的伤害。此外，在手术和器官移植的临床背景下，IR是不可避免的，并且对患者的预后有害。IR损伤的一个主要上游驱动因素是活性氧（ROS）的产生，它导致组织损伤和死亡。然而，由于活性氧产生的时间和分子来源尚未得到很好的表征，治疗策略主要依赖于抗氧化剂来抑制这些有害分子的产生。不幸的是，这种疗法在临床环境中被证明是无效的，很可能是由于体内ROS分子介质的不确定性。我们最近确定了体内心脏病发作和中风期间活性氧的一个重要分子来源：线粒体代谢物琥珀酸盐。重要的是，我们已经证明，当心脏病发作和中风期间组织缺氧时，琥珀酸盐大量积累，当氧气恢复时，积累的琥珀酸盐作为产生ROS的分子燃料。此外，我们发现琥珀酸盐对活性氧的补充是由于它与细胞中一种叫做线粒体复合体i的能量产生所必需的酶的相互作用。这些令人兴奋的发现为我们提供了对IR过程中活性氧起源的第一个分子理解。根据这里提出的研究计划，我们现在将应用这些知识来了解通过琥珀酸盐和复合物I控制体内ROS产生的分子机制，开发针对IR损伤的更有针对性的治疗策略，并开发评估琥珀酸盐途径的方法，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，现在可以在一次实验中量化活组织中的数百种代谢物，这种方法被称为代谢组学。这种代谢组学方法将使我们首次能够在体内追踪IRI事件期间心脏的代谢组学。我们的目标是确定在缺血期间发生的代谢物变化，这种变化可能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很有希望的主要候选分子：琥珀酸盐。在IR期间追踪的数百种代谢物中，琥珀酸盐是唯一发现在缺血组织中显著积累的线粒体代谢物。此外，再灌注仅5分钟后，积累的琥珀酸盐被代谢到接近静息水平。这种独特的积累和快速消耗模式，加上琥珀酸消耗驱动复合体I产生ROS的事实，使我们能够确定IRI过程中ROS的分子来源。我们现在的目标是确定驱动缺血琥珀酸盐积累的途径，并尝试使用药理学修改这些途径。这将使我们能够直接确定琥珀酸盐在再灌注时驱动ROS产生的作用，同时为IRI适应症提供新的药物靶点。与此同时，我们将研究再灌注时琥珀酸连接复合物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重要的见解。最后，我们将开发新的药物来直接控制体内琥珀酸水平或氧化，以制定合理的IRI病理治疗策略，防止IRI期间ROS的产生。

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.