Abstract

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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References

  1. 1.

    & Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol. Rev. 88, 581–609 (2008)

  2. 2.

    & Myocardial reperfusion injury. N. Engl. J. Med. 357, 1121–1135 (2007)

  3. 3.

    , & Cardioprotection by metabolic shut-down and gradual wake-up. J. Mol. Cell. Cardiol. 46, 804–810 (2009)

  4. 4.

    & Ischemia and reperfusion–from mechanism to translation. Nature Med. 17, 1391–1401 (2011)

  5. 5.

    et al. The innate immune response in reperfused myocardium. Cardiovasc. Res. 94, 276–283 (2012)

  6. 6.

    , & Hypoxanthine production by ischemic heart demonstrated by high pressure liquid chromatography of blood purine nucleosides and oxypurines. Clin. Chim. Acta 115, 73–84 (1981)

  7. 7.

    , & Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol. Rev. 58, 87–114 (2006)

  8. 8.

    et al. Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I. Nature Med. 19, 753–759 (2013)

  9. 9.

    , & Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc. Natl Acad. Sci. USA 84, 1404–1407 (1987)

  10. 10.

    et al. Succinate is an inflammatory signal that induces IL-1beta through HIF-1alpha. Nature 496, 238–242 (2013)

  11. 11.

    & A metabolic model of the mitochondrion and its use in modelling diseases of the tricarboxylic acid cycle. BMC Syst. Biol. 5, 102 (2011)

  12. 12.

    et al. The oxygen free radicals originating from mitochondrial complex I contribute to oxidative brain injury following hypoxia–ischemia in neonatal mice. J. Neurosci. 32, 3235–3244 (2012)

  13. 13.

    Metabolic responses to cardiac hypoxia. Increased production of succinate by rabbit papillary muscles. Circ. Res. 43, 808–815 (1978)

  14. 14.

    & Metabolic consequences of diving in animals and man. Science 187, 613–621 (1975)

  15. 15.

    , , , & The malate-aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast. Genes Dev. 22, 931–944 (2008)

  16. 16.

    , & Malate-aspartate shuttle, cytoplasmic NADH redox potential, and energetics in vascular smooth muscle. J. Mol. Cell. Cardiol. 30, 1571–1579 (1998)

  17. 17.

    , & Inborn errors of the purine nucleotide cycle: adenylosuccinase deficiency. J. Inherit. Metab. Dis. 20, 193–202 (1997)

  18. 18.

    et al. O2-sensing signal cascade: clamping of O2 respiration, reduced ATP utilization, and inducible fumarate respiration. Am. J. Physiol. 295, C29–C37 (2008)

  19. 19.

    & Studies on succinate dehydrogenase. I. spectral properties of the purified enzyme and formation of enzyme-competitive inhibitor complexes. Biochim. Biophys. Acta 92, 233–247 (1964)

  20. 20.

    Modulation of mitochondrial succinate dehydrogenase activity, mechanism and function. Mol. Cell. Biochem. 20, 41–60 (1978)

  21. 21.

    , , & Inhibition by (aminooxy)acetate of the malate-aspartate cycle in the isolated working guinea pig heart. Hoppe-Seyler's Z. Physiol. Chem. 361, 907–914 (1980)

  22. 22.

    , , , & Disruption of the purine nucleotide cycle by inhibition of adenylosuccinate lyase produces skeletal muscle dysfunction. J. Clin. Invest. 74, 1422–1427 (1984)

  23. 23.

    , & The production of reactive oxygen species by complex I. Biochem. Soc. Trans. 36, 976–980 (2008)

  24. 24.

    & The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc. Natl Acad. Sci. USA 103, 7607–7612 (2006)

  25. 25.

    & Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer. J. Biol. Chem. 286, 18056–18065 (2011)

  26. 26.

    How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13 (2009)

  27. 27.

    , & Assessing mitochondrial potential, calcium, and redox state in isolated mammalian cells using confocal microscopy. Methods Mol. Biol. 372, 421–430 (2007)

  28. 28.

    , , , & Physiological consequences of complex II inhibition for aging, disease, and the mKATP channel. Biochim. Biophys. Acta 1827, 598–611 (2013)

  29. 29.

    et al. Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of KATP channel activation. Cardiovasc. Res. 72, 313–321 (2006)

  30. 30.

    et al. G-Protein-coupled receptor 91 and succinate are key contributors in neonatal postcerebral hypoxia-ischemia recovery. Arterioscler. Thromb. Vasc. Biol. 34, 285–293 (2014)

  31. 31.

    et al. Cardioprotective effects of mineralocorticoid receptor antagonists at reperfusion. Eur. Heart J. 31, 1655–1662 (2010)

  32. 32.

    et al. Protection through postconditioning or a mitochondria-targeted S-nitrosothiol is unaffected by cardiomyocyte-selective ablation of protein kinase G. Basic Res. Cardiol. 108, 337 (2013)

  33. 33.

    , , & Lysine deacetylation in ischaemic preconditioning: the role of SIRT1. Cardiovasc. Res. 89, 643–649 (2011)

  34. 34.

    et al. Myocardial creatine levels do not influence response to acute oxidative stress in isolated perfused heart. PLoS One. 9, e109021 (2014)

  35. 35.

    et al. Positive impact of pre-stroke surgery on survival following transient focal ischemia in hypertensive rats. J. Neurosci. Methods 211, 305–308 (2012)

  36. 36.

    , , & Experimental studies of ischemic brain edema: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn. J. Stroke 8, 1–8 (1986)

  37. 37.

    et al. Functional assessments in mice and rats after focal stroke. Neuropharmacology 39, 806–816 (2000)

  38. 38.

    et al. Combined antiapoptotic and antioxidant approach to acute neuroprotection for stroke in hypertensive rats. J. Cereb. Blood Flow Metab. 33, 1215–1224 (2013)

  39. 39.

    et al. Quantitative assessment of early brain damage in a rat model of focal cerebral ischaemia. J. Neurol. Neurosurg. Psychiatry 50, 402–410 (1987)

  40. 40.

    et al. muma, An R Package for metabolomics univariate and multivariate statistical analysis. Curr. Metabol. 1, 180–189 (2013)

  41. 41.

    , & Statistical significance analysis of nuclear magnetic resonance-based metabonomics data. Anal. Biochem. 401, 134–143 (2010)

  42. 42.

    & Effects of NO on mitochondrial function in cardiomyocytes: Pathophysiological relevance. Cardiovasc. Res. 71, 10–21 (2006)

  43. 43.

    , & MitoMiner: a data warehouse for mitochondrial proteomics data. Nucleic Acids Res. 40, D1160–D1167 (2012)

  44. 44.

    , , , & BRENDA, AMENDA and FRENDA the enzyme information system: new content and tools in 2009. Nucleic Acids Res. 37, D588–D592 (2009)

  45. 45.

    et al. Computational prediction of human metabolic pathways from the complete human genome. Genome Biol. 6, R2 (2004)

  46. 46.

    , , , & Mitochondrial pH monitored by a new engineered green fluorescent protein mutant. J. Biol. Chem. 279, 11521–11529 (2004)

  47. 47.

    , & What is flux balance analysis? Nature Biotechnol. 28, 245–248 (2010)

  48. 48.

    et al. Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox. Nature Protocols 2, 727–738 (2007)

  49. 49.

    & Complex I is the major site of mitochondrial superoxide production by paraquat. J. Biol. Chem. 283, 1786–1798 (2008)

  50. 50.

    Citrate synthase. Methods Enzymol. 13, 3–11 (1969)

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Acknowledgements

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

Author notes

    • Edward T. Chouchani
    •  & Victoria R. Pell

    These authors contributed equally to this work.

Affiliations

  1. MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK

    • Edward T. Chouchani
    • , Ellen L. Robb
    • , Angela Logan
    • , Anthony C. Smith
    • , Filmon Eyassu
    • , Anna J. Dare
    • , Andrew M. James
    • , Sebastian Rogatti
    • , Alan J. Robinson
    •  & Michael P. Murphy
  2. Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0QQ, UK

    • Edward T. Chouchani
    • , Victoria R. Pell
    • , Chou-Hui Hu
    •  & Thomas Krieg
  3. MRC Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK

    • Edoardo Gaude
    • , Ana S. H. Costa
    •  & Christian Frezza
  4. King’s College London, British Heart Foundation Centre of Research Excellence, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK

    • Dunja Aksentijević
    •  & Michael J. Shattock
  5. Department of Cell and Developmental Biology and UCL Consortium for Mitochondrial Biology, University College London, Gower Street, London WC1E 6BT, UK

    • Stephanie Y. Sundier
    •  & Michael R. Duchen
  6. Department of Anesthesiology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642, USA

    • Sergiy M. Nadtochiy
    •  & Paul S. Brookes
  7. Institute of Cardiovascular & Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK

    • Emily N. J. Ord
    • , Rachel Shirley
    •  & Lorraine M. Work
  8. School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK

    • Richard C. Hartley
  9. Unit of Paediatric Surgery, UCL Institute of Child Health, London WC1N 1EH, UK

    • Simon Eaton
  10. Hatter Cardiovascular Institute, University College London, 67 Chenies Mews, London WC1E 6HX, UK

    • Sean M. Davidson
  11. University Department of Surgery and Cambridge NIHR Biomedical Research Centre, Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK

    • Kourosh Saeb-Parsy

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Contributions

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.

Competing interests

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.

Corresponding authors

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

Extended data

Extended data figures

  1. 1.

    Comparative analysis of metabolites significantly accumulated in ischaemic conditions.

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 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.

  6. 6.

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

  7. 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.

  8. 8.

    Predicted changes in pathways of succinate and oxidative phosphorylation metabolism during ischaemia and following reperfusion.

  9. 9.

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

  10. 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.

Supplementary information

Excel files

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    Supplementary Data

    This file contains Supplementary Table 1.

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    Supplementary Data

    This file contains Supplementary Table 2.

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    Supplementary Data

    This file contains Supplementary Table 3.

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    Supplementary data

    This file contains Supplementary Table 4.

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    Supplementary Data

    This file contains Supplementary Table 5.

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    Supplementary Data

    This file contains Supplementary Table 6.

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