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氧合血供的去除和重新引入所引起的功能障碍。总的来说，这些疾病是迄今为止世界上最主要的死亡原因，其中最常见的是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，对患者的预后不利。IR损伤的一个主要上游驱动因素是活性氧物种(ROS)的产生，它会导致组织损伤和死亡。然而，由于ROS产生的时间和分子来源尚未得到很好的描述，治疗策略主要依赖于抗氧化剂在这些有害分子产生后将其熄灭。不幸的是，这种疗法在临床上被证明是无效的，很可能是由于体内ROS分子介体的不确定性。我们最近在体内确定了心脏病发作和中风期间ROS的一个重要的分子来源：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，琥珀酸大量积累，当氧气恢复时，积累的琥珀酸充当产生ROS的分子燃料。此外，我们还发现，琥珀酸对ROS的补充是由于它与一种名为线粒体复合体I的酶的相互作用，这种酶是我们细胞中产生能量所必需的。这些令人兴奋的发现为我们提供了第一个关于IR期间ROS起源的分子理解。随着这里提出的研究计划，我们现在将应用这些知识来了解体内通过琥珀酸和复合体I控制ROS产生的分子机制，开发更有针对性的针对IR损伤的治疗策略，并开发评估琥珀酸途径的方法，以更好地诊断不同环境下的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。使用质谱学方法，现在可以在一次实验中量化数百种来自活组织的代谢物，这种方法被称为代谢物。这种代谢组学方法将使我们第一次能够在活体IRI事件期间跟踪心脏的代谢组。我们的目标是确定在缺血期间发生的代谢物变化，这些变化可以导致在再灌注时与复合体I相互作用而产生ROS。我们的初步分析已经产生了一种有希望的候选先导分子：丁二酸酯。在IR过程中跟踪的数百种代谢物中，琥珀酸是唯一在缺血组织中发现显著积累的线粒体代谢物。此外，仅在再灌流5分钟后，积累的琥珀酸就被代谢到接近静止的水平。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体I产生ROS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推动缺血性琥珀酸积累的途径(S)，并试图利用药理学来修改这些途径。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。同时，我们将寻求确定琥珀酸连接的复合体I在再灌流时产生自由基的机制，这将为体内驱动ROS产生的代谢参数提供必要的洞察。最后，我们将开发新的药物，旨在直接操纵琥珀酸水平或体内氧化，以开发合理的治疗策略，以防止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.