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

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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.