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

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.

This research will investigate the essential role of succinate driven ROS production and injury during IR. To do so, complementary research aims will:

(1) determine the in vivo mechanisms that control succinate-driven ROS production
(2) elucidate the generality of the succinate-ROS mechanism in established cardioprotective interventions
(3) develop targeted inhibitors for the manipulation of the succinate-ROS pathway in vivo, and
(4) establish succinate as a novel diagnostic marker of IR.

1. Succinate driven ROS during IR occurs by a process of reverse electron transport (RET) at mitochondrial complex I, but the bioenergetic parameters required for this process to occur in vivo are unclear. We will use pharmacological tools to manipulate mitochondrial parameters and characterize the RET mechanism in vivo for the first time.

2. We will apply our recently developed tools for comparative metabolomics to examine the consequences on metabolic flux of established cardioprotective regimes that have uncharacterized modes of action. We hypothesise that manipulation of succinate-ROS mechanism provides a unifying explanation of various cardioprotective strategies. Testing this hypothesis will provide critical mechanistic insight and determine the extent to which manipulation of ischaemic succinate accumulation is a universal predictor of IR damage.

3. Having established that competitive inhibition of succinate dehydrogenase mediated succinate accumulation is a promising strategy to protect against the upstream damage of IR injury, we will now develop and test targeted inhibitors of the pathways that control succinate driven ROS production.

4. Using a combination of in vivo models of IR injury with established and variable levels of consequent pathophysiology, we will develop a mass spectrometry based screen of mitochondrial metabolites from circulating fluids as a predictive diagnostic for the extent or IR injury.

This work will make advances at the intersection of mitochondrial biology, metabolism, and cardiovascular research, and therefore will benefit a significant body of researchers. The most immediate benefit will be to the very active pre-clinical and clinical research programs in the UK and abroad dedicated to devising therapeutic strategies to mitigate acute and chronic injury resulting from ischaemia-reperfusion (IR) events, which are leading killers in the UK. Our findings here, building on our recent high impact work, will seek to manipulate and characterise the recently established molecular source of reactive oxygen species (ROS) damage that is known to drive morbidity and mortality due to IR.

Myocardial infarction and resulting heart failure, stroke, and organ transplantation, are prominent examples of leading causes of morbidity and mortality due to reperfusion injury for which there are no specific therapies in routine clinical practice. So, establishing the mechanisms that control succinate driven ROS and IR damage in the myocardium will be informative for researchers developing therapies against all IR pathologies.
Furthermore, our development of inhibitors of succinate accumulation and oxidation pathways on a rational basis will provide for a new class of therapies targeted towards the upstream events that underlie damage during IR. Our team is well suited to the development of such a class of therapies, with extensive experience in bridging pre-clinical and clinical research in the UK. So, in the next decade we anticipate the translational potential of this work will provide an economic and healthcare advantage in the UK.

More broadly, this work will inform a longstanding intellectual and public health issue, that is the role of oxidative stress in age related disease and dysfunction generally. For over half a century there has been a strengthening correlation between ROS damage and age related disease. However, the mechanisms and role of ROS in cancer, diabetes, obesity, and ageing remain elusive. While our identification of molecular sources of ROS will be in the context of myocardial IR, this research will provide a mechanistic framework that those studying these varied diseases can target using similar manipulations as those detailed in our proposal. We believe strongly that in the coming years this will encourage a more nuanced view of the role of ROS in age related disease and dysfunction. Such a view will allow for a refinement by researchers of strategies seeking to manipulate and understand the role of ROS in health and disease.