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Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS



Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS)1,2,3,4,5. Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion1,3. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

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Figure 1: Comparative metabolomics identifies succinate as a potential mitochondrial metabolite that drives reperfusion ROS production.
Figure 2: Reverse SDH activity drives ischaemic succinate accumulation by the reduction of fumarate.
Figure 3: Ischaemic succinate levels control ROS production in adult primary cardiomyocytes and in the heart in vivo.
Figure 4: NADH and AMP sensing pathways drive ischaemic succinate accumulation to control reperfusion pathologies in vivo through mitochondrial ROS production.


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Supported by the Medical Research Council (UK) and by grants from Canadian Institutes of Health Research and the Gates Cambridge Trust (E.T.C.) and the British Heart Foundation (T.K., V.R.P., L.M.W.). We thank J. Hirst and G. C. Brown for discussions.

Author information

Authors and Affiliations



E.T.C. designed research, carried out biochemical experiments, analysed data from in vivo experiments and co-wrote the paper. T.K., V.R.P. and C.-H.H. designed and carried out the ex vivo and in vivo experiments. C.F. and E.G. designed and carried out mass spectrometry and metabolomics analyses, with A.S.H.C. assisting. D.A. and M.J.S. designed and carried out ex vivo perfused heart experiments. S.Y.S., S.M.D., M.R.D., S.M.N., E.L.R. and P.S.B. designed and carried out cell experiments. L.M.W., E.N.J.O. and R.S. designed and carried out brain experiments. A.J.D., S.R. and K.S.-P. designed and carried out kidney experiments. A.L. and R.C.H. carried out ROS analyses. S.E. carried out analyses. A.M.J. helped with data interpretation. A.C.S., A.J.R. and F.E. designed and performed bioinformatic analyses. E.T.C., T.K., C.F. and M.P.M. directed the research and co-wrote the paper, with assistance from all other authors.

Corresponding authors

Correspondence to Christian Frezza, Thomas Krieg or Michael P. Murphy.

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M.P.M., E.T.C., L.M.W., C.F. and T.K. have applied for a patent on some of the work described here.

Extended data figures and tables

Extended Data Figure 1 Comparative analysis of metabolites significantly accumulated in ischaemic conditions.

a, Various rat and mouse tissues exposed to sufficient periods of ischaemia to prime for reperfusion ROS production were subjected to targeted LC–MS metabolomics analysis and comparison of metabolites that accumulated significantly when compared to normoxic levels. After this, metabolites were scored according to the prevalence of their accumulation across five ischaemic tissue conditions. B, brain; H, whole heart ischaemia ex vivo; HL, left anterior descending coronary artery ischaemia in vivo; K, kidney, L, liver. b, Determination of linearity of the relationship between LC–MS metabolite peak intensity and concentration for CAC and related metabolites. c, Quality control determination of coefficient of variation for LC–MS quantification of CAC and related metabolites.

Extended Data Figure 2 Time course of succinate levels in the in vivo heart during ischaemia and reperfusion and potential metabolic inputs for succinate.

a, Time course of succinate levels during myocardial ischaemia and reperfusion for the in vivo heart (5 min and 15 min ischaemia n = 4; 30 min ischaemia n = 9; 5 min reperfused n = 5). b, Summary of the three potential metabolic inputs for succinate-directed ischaemic flux. To understand the metabolic pathways that could contribute to succinate production under ischaemia, an updated version of the iAS253 model of cardiac metabolism11 was used to simulate ischaemia using flux balance analysis. The model showed three possible mechanisms for producing succinate: from α-ketoglutarate produced by the CAC, derived from glycolysis, fatty acid oxidation, and glutaminolysis (grey box), from succinic semialdehyde produced from the GABA shunt (blue box), and from fumarate produced from the malate-aspartate shuttle and purine nucleotide cycle (red box) via the reversal of SDH. Data are mean ± s.e.m. of at least four biological replicates.

Extended Data Figure 3 Metabolic labelling of CAC and proximal metabolites by 13C-glucose in the ischaemic and normoxic myocardium.

Proportional isotopic labelling profile of CAC and proximal metabolites during normoxic and ischaemic myocardial perfusion. Mouse hearts were perfused with 11 mM [U-13C]glucose (+6 labelled) for 10 min followed by either 30 min no flow ischaemia or 30 min normoxic perfusion followed by snap-freezing and LC–MS metabolomic analysis (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test). Data are mean ± s.e.m. of at least four biological replicates.

Extended Data Figure 4 Metabolic labelling of CAC and proximal metabolites by 13C-palmitate in the ischaemic and normoxic myocardium.

a, Mouse hearts were perfused with 0.3 mM [U-13C]palmitate (+16 labelled) for 10 min resulting in a significant proportion of the endogenous palmitate pool being +16 labelled. Following this, hearts were subjected to either 30 min ischaemia or continued normoxic respiration with 13C-palmitate followed by snap-freezing and metabolomic analysis. b, Isotopic flux from palmitate to CAC and proximal metabolites following normoxic and ischaemic myocardial respiration. The isotopic profile for each metabolite is expressed as a proportion of the total pool (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test). Data are shown as the mean ± s.e.m. of at least four biological replicates.

Extended Data Figure 5 Metabolic labelling of CAC and proximal metabolites by 13C-glutamine in the ischaemic and normoxic myocardium, and measurement of the effect of inhibition of GABA transaminase on succinate accumulation in the ischaemic myocardium.

a, Mouse hearts were perfused with 4 mM [U-13C]glutamine (+5 labelled) for 10 min followed by either 30 min no flow ischaemia or 30 min normoxic respiration followed by snap freezing and metabolomic analysis. The isotopic profile for each metabolite is expressed as a proportion of the total pool (n = 4). b, Furthermore, flux to α-ketoglutarate was determined relative to the proportion of the +5 glutamine pool in the heart (n = 4). c, d, Perfused mouse hearts were subjected to 30 min no flow ischaemia ± continuous infusion of vigabatrin (vig; 100, 300 and 700 μM) 10 min before ischaemia. Heart tissue was snap frozen and GABA (c) and succinate (d) abundance quantified relative to normoxic levels by LC–MS (n = 4; ischaemia n = 5). *P < 0.05 (two-tailed Student’s t-test). Data are mean ± s.e.m. of at least four biological replicates.

Extended Data Figure 6 Unabridged metabolic model identifying pathways that can become activated by tissue ischaemia to drive succinate accumulation.

To identify the metabolic pathways that could contribute to succinate production under ischaemia, we simulated these conditions using flux balance analysis in conjunction with an expanded version of the iAS253 mitochondrial model of central cardiac metabolism. The major pathways contributing to succinate accumulation (bold red lines) were via fumarate feeding into the reverse activity of SDH. This was produced by the PNC and the MAS, which consumed glucose and aspartate, and also led to significant production of lactate and alanine. Lesser sources of succinate (thin red lines) included glycolysis and glutaminolysis but this was relatively minor as this route was constrained by the overproduction of NADH. In addition, a small amount of fumarate was generated by pyruvate carboxylase activity. The GABA shunt did not contribute (black dashed line).

Extended Data Figure 7 Effects of dimethyl malonate and dimethyl succinate treatment of cells and in vivo on intracellular accumulation of malonate and succinate, and respiration and comparison of 13C-labelled ischaemic metabolite fluxes to succinate relative to isotopic donor pools.

a, Intravenous infusion of dimethyl malonate in vivo results in accumulation of malonate in the ischaemic myocardium (n = 4). b, c, C2C12 cells were incubated with: no additions, glucose, 5 mM dimethyl succinate, 5 mM dimethyl malonate, or 5 mM dimethyl malonate and 5 mM dimethyl succinate. Cellular oxygen consumption rate due to ATP synthesis (b) and maximal rates (c) in the presence of p-triflouromethoxyphenylhydrazone (FCCP) were determined using a Seahorse XF96 analyser (n = 4). d, Mouse hearts were perfused with 13C-glucose (+6 labelled), 13C-glutamine (+5 labelled), 13C-aspartate (+1 labelled), or 13C-palmitate (+16 labelled) for 10 min followed by 30 min no-flow ischaemia or 30 min normoxic respiration, followed by snap-freezing and metabolomic analysis. To compare the relative magnitude of metabolite flux from each carbon source, 13C incorporation to succinate during normoxia and ischaemia was determined relative to the proportion of the total pool of the relevant infused 13C donor. 13C incorporation into succinate was considered in terms of the proportion of the +4 isotope in the entire succinate pool for 13C-glucose, 13C-glutamine and 13C-palmitate infusions; and the proportion of the +1 isotope in the entire pool for the 13C-aspartate infusion (n = 4). *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test for pairwise comparisons, and one-way ANOVA followed by Bonferroni’s test for multiple comparisons). Data are mean ± s.e.m. of at least four biological replicates.

Extended Data Figure 8 Predicted changes in pathways of succinate and oxidative phosphorylation metabolism during ischaemia and following reperfusion.

To determine possible changes in succinate metabolism during ischaemia, reperfusion and normoxia, cardiac metabolism was simulated in these conditions using an expanded version of the iAS253 model with flux balance analysis. a, The simulations predicted that under ischaemia, SDH ran in reverse by using ubiquinol produced by complex I to reduce fumarate to succinate, thereby acting as a terminal electron acceptor instead of oxygen. Fumarate was produced from the PNC and reversal of the CAC. Flux through the rest of the respiratory chain was diminished and AMP was produced from ADP owing to insufficient ATP production. b, With oxygen restored SDH metabolised excess succinate. A delay in regenerating AMP to ADP, as typified in the first minute of reperfusion, limited the flux through ATP-synthase. This in turn prevented complex III consuming all the ubiquinol generated by SDH, as the membrane became hyperpolarized. The excess flux of ubiquinol and protons forced complex I to run in reverse, which would generate ROS by RET. c, Once the flux of succinate was reduced to normal levels, as in the transition from late reperfusion to normoxia, the fluxes through the respiratory chain and citric acid cycle returned to normal.

Extended Data Figure 9 Tracking DHE oxidation, NAD(P)H reduction state, and mitochondrial membrane potential in primary cardiomyocytes during in situ IR.

a, Inhibition of mitochondrial complex I RET reduces DHE oxidation on reperfusion (n = 6; rotenone n = 4). b, c, Effect of manipulation of ischaemic succinate levels on NAD(P)H oxidation during early reperfusion (n = 3). Primary rat cardiomyocytes were subjected to 40 min ischaemia followed by reoxygenation and NAD(P)H reduction state was tracked throughout the experiment by measurement of NAD(P)H autofluorescence. Ischaemic buffer contained no additions, 4 mM dimethyl malonate, or 4 mM dimethyl succinate. Average (b) and representative (c) traces from each condition are shown. The highlighted window in c indicates the period of the experiment expanded in detail in b. d, Effect of inhibition of ischaemic succinate accumulation on mitochondrial membrane potential following late ischaemia (left) and early reperfusion (right). e, f, Primary rat cardiomyocytes were subjected to 40 min ischaemia and reoxygenation and mitochondrial membrane potential was tracked throughout the experiment by measurement of TMRM fluorescence. Ischaemic buffer contained either no additions or 4 mM dimethyl malonate. e, TMRM signal throughout the entire experiment. f, TMRM signal during the transition from ischaemia to reoxygenation (n = 3). *P < 0.05 (two-tailed Student's t-test and one-way ANOVA). Data are mean ± s.e.m. of at least three biological replicates. Replicates represent separate experiments on independent cell preparations.

Extended Data Figure 10 Quantification of CAC intermediates in the heart following infusion of dimethyl succinate and in the brain after infusion of dimethyl malonate, and extended summary cytoprotection and neurological scores of rats subjected to tMCAO IR in vivo ± dimethyl malonate infusion.

a, Effect of intravenous infusion of dimethyl succinate on CAC metabolite abundance in the ischaemic and non-ischaemic myocardium (normoxia and peripheral heart tissue plus dimethyl succinate n = 3; ischaemia plus dimethyl succinate n = 4; α- ketoglutarate and aconitate in peripheral heart tissue n = 2). b, Profile of mitochondrial CAC metabolite levels after tMCAO ischaemia ± dimethyl malonate (n = 4). c, Representative images of cross-sections from rat brains after undergoing tMCAO in vivo ± treatment with dimethyl malonate. Brains were treated with haematoxylin and eosin to delineate infarcted tissue. d, Locomotor and sensorimotor assessment of rats by quantification of average number of footfalls after tCMAO ± dimethyl malonate (control n = 6; dimethyl malonate n = 4). *P < 0.05, **P < 0.01 (two-tailed Student’s t-test for pairwise comparisons, and one-way (a, b) or two-way (d) ANOVA for multiple comparisons). Data are mean ± s.e.m. of at least three biological replicates, unless otherwise stated.

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Chouchani, E., Pell, V., Gaude, E. et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515, 431–435 (2014).

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