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Cited by 619Cardiac Ischemia and ReperfusionMitochondrial Function and PathologyNitric Oxide and Endothelin Effects

Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I

Open access
Chouchani, Edward T, Methner, Carmen, Nadtochiy, Sergiy M, Logan, Angela, Pell, Victoria R, Ding, Shujing, James, Andrew M, Cochemé, Helena M +10 more·2013·Nature Medicine
DOI 10.1038/nm.3212PMC PMC4019998
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Abstract

Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.

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Claims the paper makes

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  • Findingown data

    S-nitrosation of the other three cysteines was unaffected by changes in complex I activity.

    “Conversely, S-nitrosation of the other three cysteines was unaffected by changes in complex I activity (Fig. 2d and Supplementary Figs. 8–10).”
    Quote in the paperSubstantial claim
  • Findingown data

    MitoSNO reproducibly S-nitrosated three complex I subunits: the 75 kDa, B8 and ND3 subunits.

    “MitoSNO reproducibly S-nitrosated three subunits: the 75 kDa, B8 and ND3 subunits (Fig. 2b,c, Supplementary Fig. 7b and Supplementary Table 2).”
    Quote in the paperSubstantial claim
  • Findingown data

    MitoSNO treatment did not inhibit complex I in normoxic myocardium in vivo.

    “Notably, MitoSNO treatment did not inhibit complex I in the normoxic myocardium in vivo (Fig. 1g)”
    Quote in the paperSubstantial claim
  • Findingown data

    In mice after 30 min LAD ischemia and 120 min reperfusion, MitoSNO given at reperfusion significantly reduced infarct size.

    “MitoSNO administered at reperfusion significantly decreased infarct size (Fig. 1b and Supplementary Fig. 2).”
    Quote in the paperSubstantial claim
  • Findingown data

    Four cysteines were S-nitrosated by MitoSNO: two on the 75 kDa subunit and one each on the ND3 and B8 subunits.

    “we identified four cysteines that were S-nitrosated by MitoSNO: two on the 75 kDa subunit and one each on the ND3 and B8 subunits (Fig. 2c,d and Supplementary Table 3).”
    Quote in the paperSubstantial claim
  • May combine two claims. 33
  • Classified as probably not a substantial claim. 19
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Neither SNAP nor MitoNAP reduced infarct size in the same mouse ischemia-reperfusion model.

    “Neither SNAP nor the MitoSNO-targeting moiety (MitoNAP)11 had this effect (Fig. 1b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Without MitoSNO, complex I was unaffected after 5 min reperfusion but its activity decreased by about 50% during 30 min reperfusion and could not be restored ex vivo by reductants.

    “In contrast, in the absence of MitoSNO treatment in vivo, complex I was not affected after 5 min of reperfusion, but its activity then decreased by ~50% during 30 min of reperfusion and could not be restored ex vivo by reductants”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In intact ischemic hearts perfused ex vivo in Langendorff, MitoSNO produced substantial (~40%) S-nitrosation of ND3 that was absent in normoxic hearts; S-nitrosation of 75 kDa cysteines was similar under normoxia and ischemia.

    “Within the intact, ischemic heart perfused ex vivo in a Langendorff system, there was substantial (~40%) S-nitrosation of ND3 by MitoSNO that was not present in normoxic hearts; in contrast, the amount of S-nitrosation of the 75 kDa subunit cysteines was similar under normoxia and ischemia (Fig. 3a, Supplementary Table 4 and Supplementary Fig. 11).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MitoSNO given at reperfusion blocked the post-reperfusion rise in mitochondrial hydrogen peroxide, whereas SNAP did not.

    “MitoSNO administered at reperfusion blocked this rise in the amount of hydrogen peroxide, whereas administration of SNAP did not (Fig. 1c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mice after 30 min LAD ischemia and 5 min reperfusion, MitoSNO injected systemically before reperfusion caused S-nitrosation selectively in heart mitochondrial proteins; an equal dose of SNAP instead modified only nonmitochondrial proteins in myocardium and plasma.

    “This led to the selective S-nitrosation of mitochondrial proteins in the reperfused heart (Fig. 1a), as visualized by labeling with a red fluorescent probe15–17. In contrast, equal doses of the untargeted precursor S-nitrosating compound S-nitroso-N-acetylpenicillamine (SNAP) S-nitrosated only nonmitochondrial myocardial and plasma proteins (Fig. 1a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Intravenous MitoSNO in mice caused S-nitrosation of the ND3 cysteine in vivo during reperfusion of the ischemic heart, but not in normoxic hearts.

    “Intravenous injection of MitoSNO into mice also resulted in S-nitrosation of the ND3 cysteine in vivo during reperfusion of the ischemic heart, whereas MitoSNO injection did not have this effect in normoxic hearts (Fig. 3b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mice after 30 min LAD occlusion and 5 min reperfusion, complex I activity was lower in heart mitochondria from MitoSNO-treated mice than untreated mice, and this decrease was normalized by copper/ascorbate.

    “Complex I activity was lower in heart mitochondria from MitoSNO-treated mice compared to untreated mice, and this decreased activity was normalized by selective S-nitrosothiol reduction using copper and ascorbate15–17 (Fig. 1g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In isolated rat heart mitochondria, MitoSNO extensively S-nitrosated ND3 Cys39 (>50%) during anoxia but negligibly under normoxia.

    “Moreover, ND3 Cys39 was extensively S-nitrosated (>50%) by MitoSNO in isolated rat heart mitochondria during anoxia but was negligibly modified under normoxic conditions (Fig. 2e,f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MitoSNO protected only when administered during reperfusion; injection 10 min after reperfusion was ineffective.

    “MitoSNO was protective only during reperfusion: injection 10 min after reperfusion was ineffective (Supplementary Fig. 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In vivo, mitochondrial hydrogen peroxide remained low during ischemia and increased only after reperfusion.

    “Using MitoB we found that the amount of mitochondrial hydrogen peroxide remained low during ischemia and increased only after reperfusion in vivo (Fig. 1c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The post-reperfusion rise in mitochondrial hydrogen peroxide was associated with increased protein carbonylation, necrotic and apoptotic cell death, and MitoSNO prevented all these effects.

    “The rise in the amount of mitochondrial hydrogen peroxide was associated with increased protein carbonylation (Fig. 1d), a marker of oxidative damage, as well as necrotic (Fig. 1b) and apoptotic (Fig. 1e) cell death, and these effects were all prevented by MitoSNO.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MitoSNO protected control cardiomyocytes from ischemia-reperfusion injury, but NDUFAF1-targeted RNAi completely abolished this protection.

    “MitoSNO protected control cardiomyocytes treated with a scrambled RNAi construct from ischemia-reperfusion injury, but decreasing complex I activity by NDUFAF1-targeted RNAi completely abolished MitoSNO protection (Fig. 4a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Copper and ascorbate treatment restored complex I activity, indicating that the inhibition was due to S-nitrosation of ND3 Cys39.

    “Copper and ascorbate treatment restored complex I activity, indicating that the inhibition of activity was due to S-nitrosation of ND3 Cys39 (Fig. 3d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Exposure of isolated bovine heart mitochondrial membranes to MitoSNO under high complex I activity led to S-nitrosation of 75 kDa and B8 cysteines but not ND3, and did not affect complex I activity.

    “Exposure of isolated bovine heart mitochondrial membranes to MitoSNO under conditions in which complex I is in a high activity state led to S-nitrosation of the 75 kDa and B8 subunit cysteines but not of the ND3 subunit, and this exposure did not affect complex I activity (Fig. 3d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NDUFAF1-targeted RNAi decreased NDUFAF1 protein amounts by 80% and lowered complex I activity while leaving complex II amount unaffected.

    “We selectively knocked down expression of the complex I assembly factor NDUFAF1 (ref. 36) by RNAi, which decreased protein amounts by 80% (Supplementary Fig. 16a–c) and lowered complex I activity (Supplementary Fig. 16d) while leaving the amount of complex II unaffected (Supplementary Fig. 16a,e).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] Mimaki, M. et al. Understanding mitochondrial complex I assembly in health and disease. Biochim. Biophys. Acta 1817, 851–862 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    The model shows Cys39 is at the hinge region of complex I facing the mitochondrial matrix, adjacent to the half-channel element of one proposed proton translocation pathway and close to the ubiquinone binding pocket.

    “Our model shows that Cys39 is located at the hinge region of complex I facing the mitochondrial matrix. Furthermore, Cys39 is adjacent to the half-channel element of one of the four proposed proton translocation pathways of complex I and is close to the ubiquinone binding pocket, where ubiquinone receives electrons from the N2 iron-sulfur center (Fig. 3g)34.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Baradaran, R., Berrisford, J.M., Minhas, G.S. & Sazanov, L.A. Crystal structure of the entire respiratory complex I. Nature 494, 443–448 (2013). source

      Not checked in the free run.

    • Rests on an assumption: The prokaryotic complex I structures used for modeling are sufficiently homologous to mammalian complex I to locate ND3 Cys39 accurately.

  • May combine two claims.

    Findingown data

    Nitrite infusion during prolonged ischemia caused substantial S-nitrosation of complex I ND3 Cys39, which was negligibly modified by nitrite under normoxia.

    “Nitrite infusion during prolonged ischemia resulted in substantial S-nitrosation of this cysteine, which was negligibly modified by nitrite under normoxic conditions (Fig. 4b,c and Supplementary Table 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Incubation with MitoSNO under low complex I activity led to S-nitrosation of ND3 Cys39 and the other three cysteines and inhibited complex I activity.

    “In contrast, incubation with MitoSNO under conditions in which complex I is in a low activity state led to S-nitrosation of ND3 Cys39, as well as of the other three cysteines, and inhibited complex I activity (Fig. 3d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    MitoSNO cardioprotection acts through mitochondrial S-nitrosation, which inhibits mitochondrial hydrogen peroxide production at reperfusion.

    “Together these data indicate that cardioprotection by MitoSNO acts through mitochondrial S-nitrosation, which inhibits mitochondrial hydrogen peroxide production at reperfusion.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Complex I was the only S-nitrosation target identified in the proteomic analysis whose activity was affected by this modification, making it a probable node for cardioprotection.

    “As complex I was the only S-nitrosation target identified in our proteomic analysis whose activity was affected by this modification, we consider it to constitute a probable node for cardioprotection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    MitoSNO cardioprotection is not due to nonmitochondrial cyclic GMP-protein kinase G pathways, because MitoSNO protected mice with cardiomyocyte protein kinase G deficiency.

    “MitoSNO has been reported to be protective in mice with cardiomyocyte deficiency of protein kinase G18, indicating that the cardio-protection conferred by MitoSNO is not due to nonmitochondrial pathways that operate through cyclic GMP–protein kinase G signaling3,6”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [18] Methner, C., Lukowski, R., Hofman, F., Murphy, M.P. & Krieg, T. 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). source

      Not checked in the free run.

    • cites [3] Lundberg, J.O., Weitzberg, E. & Gladwin, M.T. The nitrate-nitrite–nitric oxide pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156–167 (2008). source

      Not checked in the free run.

    • cites [6] Costa, A.D. et al. Protein kinase G transmits the cardioprotective signal from cytosol to mitochondria. Circ. Res. 97, 329–336 (2005). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Prior work showed that complex I is inhibited by various nitric oxide donors in vitro and that this inhibition is associated with S-nitrosation.

    “previous work has shown that complex I is inhibited by various nitric oxide donors in vitro and that this inhibition is associated with S-nitrosation2,10,11,21–24.”
    Quote in the paperSubstantial claim0 of 7 sources checked
    • cites [2] Shiva, S. et al. Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer. J. Exp. Med. 204, 2089–2102 (2007). source

      Not checked in the free run.

    • cites [10] Sun, J., Morgan, M., Shen, R.F., Steenbergen, C. & Murphy, E. Preconditioning results in S-nitrosylation of proteins involved in regulation of mitochondrial energetics and calcium transport. Circ. Res. 101, 1155–1163 (2007). source

      Not checked in the free run.

    • cites [11] Prime, T.A. et al. A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury. Proc. Natl. Acad. Sci. USA 106, 10764–10769 (2009). source

      Not checked in the free run.

    • cites [21] Clementi, E., Brown, G.C., Feelisch, M. & Moncada, S. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc. Natl. Acad. Sci. USA 95, 7631–7636 (1998). source

      Not checked in the free run.

    • cites [22] Burwell, L.S., Nadtochiy, S.M., Tompkins, A.J., Young, S. & Brookes, P.S. Direct evidence for S-nitrosation of mitochondrial complex I. Biochem. J. 394, 627–634 (2006). source

      Not checked in the free run.

    • cites [23] Dahm, C.C., Moore, K. & Murphy, M.P. Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite. J. Biol. Chem. 281, 10056–10065 (2006). source

      Not checked in the free run.

    • cites [24] Galkin, A. & Moncada, S. S-nitrosation of mitochondrial complex I depends on its structural conformation. J. Biol. Chem. 282, 37448–37453 (2007). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Complex I ND3 Cys39 is the crucial functional S-nitrosation target, and other MitoSNO interactions with mitochondria do not contribute to protection.

    “These data suggest that complex I ND3 Cys39 is the crucial functional S-nitrosation target and that other interactions of MitoSNO with mitochondria do not contribute to protection.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    This mechanism probably contributes to cardioprotection by untargeted nitric oxide donors and alterations to nitric oxide metabolism in general, although nonmitochondrial pathways may also contribute.

    “This mechanism probably contributes to cardioprotection by untargeted nitric oxide donors and alterations to nitric oxide metabolism in general, although nonmitochondrial pathways may also contribute.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Infarct size was assessed after 120 min reperfusion by triphenyltetrazolium chloride staining and expressed as a percentage of the risk zone as described.

    “We assessed infarct size after 120 min of reperfusion using triphenyltetrazolium chloride staining and expressed it as a percentage of the risk zone as described recently40.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [40] Nadtochiy, S.M., Redman, E., Rahman, I. & Brookes, P.S. Lysine deacetylation in ischaemic preconditioning: the role of SIRT1. Cardiovasc. Res. 89, 643–649 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    The study used an established open-chest in situ mouse heart model of myocardial infarction.

    “We used an open-chest, in situ mouse (C57BL/6) heart model as recently described41.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Schmidt, K. et al. Cardioprotective effects of mineralocorticoid receptor antagonists at reperfusion. Eur. Heart J. 31, 1655–1662 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Nitrite protects from ischemia-reperfusion injury in vivo and is thought to act as an S-nitrosating agent in ischemic myocardium.

    “The nitrite (NO2−) anion protects from ischemia-reperfusion injury in vivo1–3,37 and is thought to act as an S-nitrosating agent in the ischemic myocardium2.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [1] Duranski, M.R. et al. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J. Clin. Invest. 115, 1232–1240 (2005). source

      Not checked in the free run.

    • cites [2] Shiva, S. et al. Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer. J. Exp. Med. 204, 2089–2102 (2007). source

      Not checked in the free run.

    • cites [3] Lundberg, J.O., Weitzberg, E. & Gladwin, M.T. The nitrate-nitrite–nitric oxide pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 7, 156–167 (2008). source

      Not checked in the free run.

    • cites [37] Shiva, S. & Gladwin, M.T. Nitrite mediates cytoprotection after ischemia/reperfusion by modulating mitochondrial function. Basic Res. Cardiol. 104, 113–119 (2009). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Because ND3 S-nitrosation occurs only after anaerobic incubation, MitoSNO decreases ROS production by S-nitrosating the ND3 subunit.

    “As the S-nitrosation of the ND3 subunit only occurs after anaerobic incubation, these findings suggest that MitoSNO decreases ROS production by S-nitrosating the ND3 subunit.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

    Study design and conductown data

    The SNO-DIGE experiment used a biological variance analysis design with four biological replicates per experiment, comparing each treatment to a pooled standard.

    “To assess differences in protein fluorescence using two-dimensional DIGE, we used an experimental design that included biological variance analysis (BVA)12. In each DIGE experiment, we used four biological replicates, where a biological replicate is an independently prepared heart tissue subjected to the CyDye sample preparations described above.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Chouchani, E.T. et al. Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation. Biochem. J. 430, 49–59 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Fluorescent signal intensities from the gels were kept within the linear acquisition range by adjusting the photomultiplier tube.

    “We scanned all gels at 100- to 200-μm pixel size, and the photomultiplier tube was set to ensure a maximum pixel intensity within the linear acquisition range for the region of the gel being analyzed (most commonly the sub–20 kDa region of gels containing the complex I ND3 subunit) and thereby ensuring that fluorescent signal intensities were in the linear range.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Samples were shielded from light throughout experiments to preserve S-nitrosothiols.

    “All samples were shielded from light throughout the experiment to preserve S-nitrosothiols.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Reperfusion rapidly reactivates complex I and generates hydrogen peroxide, which causes oxidative damage leading to cell death underlying ischemia-reperfusion injury.

    “Reperfusion of the ischemic tissue rapidly reactivates complex I and generates hydrogen peroxide, which causes oxidative damage that leads to the cell death underlying ischemia-reperfusion injury.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All animal experiments were carried out in accordance with UK Home Office and NIH guidelines and approved under Project License PPL 80/2393.

    “All animal experiments described here were carried out in accordance with the UK Home Office Guide on the Operation of Animal (Scientific Procedures) Act of 1986 and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication 85–23, revised 1996). All procedures were approved under the Project License number PPL 80/2393.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    ND3 is encoded by mitochondrial DNA, its cysteine cannot be replaced genetically, and there are no known natural mutations or polymorphisms in MT-ND3.

    “As the ND3 subunit is encoded by mitochondrial DNA, its cysteine cannot be replaced genetically, and there are no known natural mutations or polymorphisms in the gene encoding this subunit (MT-ND3)31.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Bridges, H.R., Birrell, J.A. & Hirst, J. The mitochondrial-encoded subunits of respiratory complex I (NADH:ubiquinone oxidoreductase): identifying residues important in mechanism and disease. Biochem. Soc. Trans. 39, 799–806 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Together the data suggest that ischemia exposes ND3 Cys39 for S-nitrosation and that this modification specifically mediates reversible inhibition of complex I in vivo.

    “Together these data suggest that ischemia exposes ND3 Cys39 for S-nitrosation and that this modification specifically mediates the reversible inhibition of complex I in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    S-nitrosation of ND3 Cys39 does not disrupt complex I function by affecting NADH interaction with the flavin mononucleotide site, and S-nitrosation of other residues in complex I does not affect its activity.

    “This result indicates that S-nitrosation of ND3 Cys39 does not disrupt complex I function by affecting the interaction of NADH with the flavin mononucleotide site and that the S-nitrosation of other residues in complex I does not affect its activity.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Unreacted thiols in mitochondrial samples were quenched with 100 mM NEM before differential labeling of S-nitrosothiols.

    “We incubated mitochondria or mitochondrial membranes from both the in vitro and in vivo treatment groups in 100 mM NEM to quench unreacted thiols.”
    • Also stated in: § Differential labeling of respiratory complex I S-nitrosothiols · plain
      “Mitochondria from mice treated in vivo with an S-nitrosating agent were isolated as described above in buffer supplemented with 100 mM NEM before differential labeling.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    NEM was included in isolation buffers when protein S-nitrosothiols were assessed in mitochondria from in vivo treatments.

    “When protein S-nitrosothiols were to be assessed in mitochondria isolated from hearts that had been subjected to in vivo treatments, 100 mM NEM was included in the isolation buffers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    When S-nitrosating species are present during reperfusion, exposed ND3 cysteine becomes S-nitrosated, locking complex I in low activity and decreasing ROS production.

    “When S-nitrosating species are present during reperfusion, the exposed ND3 cysteine becomes S-nitrosated, locking complex I in a low activity state and decreasing ROS production during the reperfusion phase.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Pelleted samples were washed with Chelex 100-treated PBS to remove metal ions before labeling.

    “Pelleted samples were washed three times in Chelex 100–treated PBS before resuspension in 100 μl of the same buffer containing 1 mM ascorbate and 10 μM CuSO4”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Error bars represent the standard error of the mean from at least three replicates unless otherwise stated.

    “Error bars represent the s.e.m. from at least three replicates unless otherwise stated.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Tissue samples were spiked with deuterated internal standards for quantification of MitoB and MitoP.

    “For analysis, we spiked the tissues with deuterated internal standards, extracted the MitoB and its product MitoP from the reaction with hydrogen peroxide and determined the amounts of MitoB and MitoP present in the tissue by liquid chromatography and tandem mass spectrometry as described previously20.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Cochemé, H.M. et al. Measurement of H2O2 within living Drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix. Cell Metab. 13, 340–350 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    MitoSNO stability was assessed before use by its absorbance at 340 nm.

    “MitoSNO stability was assessed before use from its absorbance at 340 nm.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    A constant final concentration of nitrite was maintained in the heart during ischemia.

    “NO2− and control saline infusions began 15 min before ischemia and continued throughout the ischemic phase so that a constant final concentration of NO2− was maintained in the heart.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Mitochondrial hydrogen peroxide flux in vivo was measured using the MitoB mass spectrometric probe as previously described.

    “We measured mitochondrial hydrogen peroxide in vivo using the MitoB mass spectro-metric probe as described previously20.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Cochemé, H.M. et al. Measurement of H2O2 within living Drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix. Cell Metab. 13, 340–350 (2011). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    The nitrite treatment protocol used was previously established to elicit cardioprotection.

    “We treated mouse hearts with nitrite using previously established protocols known to elicit cardioprotection38.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] Webb, A. et al. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc. Natl. Acad. Sci. USA 101, 13683–13688 (2004). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Complex I activity rates were normalized to citrate synthase activity to control for mitochondrial content.

    “In parallel, we determined the citrate synthase activity of each sample46. We then normalized the complex I rate to the citrate synthase rate and expressed it as a percentage of the appropriate control.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] Srere, P.A. Citrate synthase. Methods Enzymol. 13, 3–10 (1969). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Nitrite and saline solutions given to ischemic hearts were deoxygenated before administration.

    “When administering NO2− or saline solutions to ischemic hearts, we deoxygenated the solutions during preparation by bubbling with 95% N2 and 5% CO2.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cells were protected from light during hypoxia and reperfusion.

    “We protected cells from light during hypoxia (2 h) and reperfusion (1.5 h) and measured cell death using a lactate dehydrogenase (LDH) release assay (Cytotoxicity Detection, Roche Applied Science) according to the manufacturer's instructions.”
    Quote in the paperNot a substantial claim
49 of 49

  • The paper relies on this without support.

    Before this study, the existence and physiological importance of S-nitrosation of complex I in vivo had not been demonstrated.

    “However, to our knowledge, the existence and physiological importance of S-nitrosation of complex I in vivo has not previously been demonstrated.”

  • The paper relies on this without support.

    The conservation of ND3 Cys39 across metazoans implies that this residue has a physiological role.

    “Cys39 in ND3 is conserved throughout metazoans, consistent with a physiological role for this residue.”

  • The paper relies on this without support.

    The prokaryotic complex I structures used for modeling are sufficiently homologous to mammalian complex I to locate ND3 Cys39 accurately.

    “We modeled the location of ND3 Cys39 using structures of prokaryotic complex I (refs. 32–34). This modeling led to an orthologous mammalian core structure containing ND3 Cys39”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • MitoSNO protection is dependent on transnitrosating a critical cysteine in the active site of complex I.

    “Mechanistically, this protection is dependent on MitoSNO transnitrosating a critical cysteine in the active site of complex I.”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [170]

  • S-nitrosation of Cys39 in the ND3 subunit of complex I inhibits its catalytic activity and attenuates reverse electron transport and ROS production.

    “This S-nitrosation of Cys39 in the ND3 subunit of complex I inhibits its catalytic activity and, hence, attenuates reverse electron transport and ROS production (170).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [170]

  • MitoSNO, a mitochondrially targeted S-nitrosothiol, significantly attenuates infarct size after MI in rodent models.

    “For example, MitoSNO (FIGURE 14), a mitochondrially targeted S-nitrosothiol, was shown in rodent models to significantly attenuate infarct size after MI (170).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [170]

  • Mitochondria-selective S-nitrosating agent studies show that Complex I nitrosation protects during ischemia/reperfusion by reducing oxidative damage and tissue necrosis.

    “Murphy and coworkers used a mitochondria-selective S-nitrosating agent to demonstrate that nitrosation of Complex I serves a protective role during ischemia/reperfusion by reducing oxidative damage and tissue necrosis [212, 213].”

    From Signaling and stress: The redox landscape in NOS2 biology (2015) · cites this paper as [213]

Cites

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Related

Authors
Chouchani, E. T., Methner, C., Nadtochiy, S. M., Logan, A., Pell, V. R., Ding, S., James, A. M., Cochemé, H. M., Reinhold, J., Lilley, K. S., Partridge, L., Fearnley, I. M., Robinson, A. J., Hartley, R. C., Smith, R. A. J., Krieg, T., Brookes, P. S., & Murphy, M. P.
Affiliations
  • MRC Mitochondrial Biology Unit (GB)
  • Max Planck Society (DE)
Corresponding author
Murphy, Michael P
ORCID
Venue
Nature Medicine
Publisher
Springer Science and Business Media LLC
Published
2013-05-26
Volume
19
Issue
6
Pages
753-759
ISSN
  • 1078-8956 (ISSN-L)
  • 1546-170X
DOI
10.1038/nm.3212
PMC
PMC4019998
OpenAlex
W1994270731
Semantic Scholar
97e1eb3beeba3f16e5a83fa5a94d80446f1bb0b7
Cited by
619
References
46
Citations by year
2026: 19 · 2025: 38 · 2024: 39 · 2023: 31 · 2022: 42 · 2021: 51 · 2020: 59 · 2019: 37 · 2018: 60 · 2017: 50 · 2016: 60 · 2015: 54 · 2014: 58 · 2013: 20
FWCI
47.86
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Medicine (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • ENLIGHTEN (Jurnal Bimbingan dan Konseling Islam) (submittedVersion)
  • UCL Discovery (University College London) (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
  • Spiral (Imperial College London) (submittedVersion)
  • UEA Digital Repository (University of East Anglia) (submittedVersion)
Funders
  • Wellcome Trust (098565)
  • British Heart Foundation (PG/12/42/29655)
  • Canadian Institutes of Health Research
  • Biotechnology and Biological Sciences Research Council (BB/I012923/1, BB/I012826/1, BB/I012923)
  • National Heart, Lung, and Blood Institute (R01HL071158)
Topics
  • Health Sciences › Medicine › Pathology and Forensic Medicine › Cardiac Ischemia and Reperfusion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
Keywords
S-nitrosylation, mitochondrial complex I, nitric oxide donors, myocardial ischemia-reperfusion injury, cardioprotection, reactive oxygen species, oxidative damage, mitochondrial respiratory chain, myocardial infarction, stroke
MeSH terms
Animals, Cysteine, Male, Mice, Inbred C57BL, Mitochondria, Heart, Myocardial Reperfusion Injury, Nitrosation, Reactive Oxygen Species, Protein Subunits, Mitochondrial Proteins, Electron Transport Complex I, Mice, Rats

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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