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. 2013 Jun;19(6):753-9.
doi: 10.1038/nm.3212. Epub 2013 May 26.

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

Edward T Chouchani  1 , Carmen Methner, Sergiy M Nadtochiy, Angela Logan, Victoria R Pell, Shujing Ding, Andrew M James, Helena M Cochemé, Johannes Reinhold, Kathryn S Lilley, Linda Partridge, Ian M Fearnley, Alan J Robinson, Richard C Hartley, Robin A J Smith, Thomas Krieg, Paul S Brookes, Michael P Murphy
Affiliations

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

Edward T Chouchani et al. Nat Med. 2013 Jun.

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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Figures

Figure 1
Figure 1
S-nitrosation of mitochondrial proteins is required for S-nitrosation–mediated protection from cardiac ischemia-reperfusion injury. (a) Protein S-nitrosation in mitochondrial and cytosolic fractions of the myocardium, as well as in blood plasma, of mice after tail-vein injection of MitoSNO or SNAP just before reperfusion. Top, S-nitrosated proteins, as detected by selective reduction of protein S-nitrosothiols in the presence of a red fluorescent maleimide. Bottom, protein loading of the scanned gels (top) assessed by Coomassie staining. SNO, S-nitrosation. (b) Top, quantification of myocardial infarct size after tail-vein injection of MitoSNO, SNAP or MitoNAP. Each open circle represents data from a single mouse, and filled circles represent the mean values of all mice for a particular condition. Bottom, representative images of cross-sections from mouse hearts treated as indicated above. Infarcted tissue is white, the rest of the area at risk is red, and nonrisk tissue is dark blue. n = 7 mice per group. (c) Mitochondrial hydrogen peroxide measured in vivo by the selective oxidation of the mitochondria-targeted mass spectrometric probe MitoB to its product MitoP during myocardial ischemia-reperfusion. MitoSNO, SNAP or MitoNAP was injected at reperfusion, and control hearts were collected from mice without intervention. n = 3–6 mice per group. Control, 60 min of normoxic perfusion; I 30, 30-min ischemia; R 30, 30-min reperfusion. (d,e) Assessment of protein oxidative damage (as assessed by protein carbonyl formation, d) and apoptosis (as assessed by caspase-3 and cleaved caspase-3 amounts, e) after myocardial ischemia-reperfusion (IR) with or without mitochondrial S-nitrosation by MitoSNO. The representative western blots in e show the amounts of caspase-3 and cleaved caspase-3, along with the amounts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. n = 3 mice per group. (f) BN-PAGE analysis of heart mitochondria identifying S-nitrosated respiratory complexes in mice injected with MitoSNO during ischemia-reperfusion injury followed by S-nitrosothiol labeling. CI–CV indicate the locations of oxidative phosphorylation complexes I–V. (g) Complex I activity in vivo at baseline and after ischemia and reperfusion for 5 min (IR 5 min) or 30 min (IR 30 min) with or without MitoSNO injection. Following mitochondrial isolation from hearts, the S-nitrosothiol-selective reductant Cu/Asc, or the general thiol reductant DTT, were added where indicated in vitro to test for the reversibility of complex I inhibition. Complex I activity was normalized to citrate synthase activity. Cu/Asc, copper and ascorbate; DTT, dithiothreitol. n = 3 mice per group. *P < 0.05, **P < 0.01 determined by one-way analysis of variance (ANOVA). Data (c–e,g) are shown as the mean ± s.e.m. of at least three replicates.
Figure 2
Figure 2
Respiratory complex I ND3 Cys39 S-nitrosation is dependent on low complex I activity. (a) Resolution of the subunits of complex I by diagonal SDS-PAGE after treatment of bovine heart mitochondrial membranes with or without MitoSNO and processing to label S-nitrosothiols with a red fluorescent tag. Red spots, S-nitrosated subunits; yellow spots, subunits containing cysteine thiols that are occluded by other modifications. The red spot at ~37 kDa was not reproducible. Pr-SNO, protein S-nitrosothiol; Pr-SX, occluded cysteine thiol. (b) Scheme for the identification of peptides using mass spectrometry after ratiometric labeling of complex I cysteine residues with light NEM (d0-NEM (unmodified), blue) or heavy NEM (d5-NEM (S-nitrosated), red) followed by isolation of complex I by BN-PAGE electrophoresis, tryptic digestion and LC-MS. (c) Summary of complex I cysteines sensitive to S-nitrosation by MitoSNO. In the d0 column, `+' indicates that the NEM-modified cysteine residue could be detected by mass spectrometry. In the d5 column, `+' indicates that the cysteine was S-nitrosated by MitoSNO, and `−' indicates that it was not. Cys, cysteine. (d) The percentage S-nitrosation of sensitive complex I cysteines in mitochondrial membranes that underwent this modification after incubation with MitoSNO with or without NADH. n = 3 individual experiments on bovine heart mitochondrial membranes. (e) Representative mass spectroscopic traces of unmodified and S-nitrosated ND3 Cys39 peptides indicating their relative intensities from rat heart mitochondria incubated with or without O2 and MitoSNO. (f) Percentage of ND3 Cys39 in rat heart mitochondria that was S-nitrosated after incubation with or without O2 and MitoSNO. n = 3 individual experiments on rat heart mitochondria per group. *P < 0.05, **P < 0.01, ***P < 0.001 determined by one-way ANOVA. Data in d and f are shown as the mean ± s.e.m. of three replicates.
Figure 3
Figure 3
ND3 Cys39 S-nitrosation mediates inhibition of complex I activity and ROS production. (a) The percentage S-nitrosation of sensitive complex I cysteines within the intact heart during normoxia and ischemia with or without MitoSNO treatment. n = 4–6 ex vivo hearts per group. (b) Quantification of the extent of ND3 S-nitrosation (top) and the ND3 subunit- complex I was isolated by BN-PAGE, and complex I subunits were resolved by SDS-PAGE. Data are shown as the means ± range. n = 2 hearts per group. (c) Left, time course showing the amount of S-nitrosated ND3 subunit after MitoSNO treatment in vivo after LAD ischemia and MitoSNO treatment of isolated mitochondria in vitro during anoxia. The inset shows the log10 of these data plotted against the time to calculate the half-life of ND3 S-nitrosation from the slope. Right, the ND3 subunit-containing region of representative fluorescent-scanned gels. The half-life of the ND3 S-nitrosothiol signal both in vivo and in vitro was determined by setting the fluorescence at 5 min to 100% and measuring the relative decrease in this signal at subsequent time points. The half-life values (in minutes) generated by this analysis for in vitro and in vivo conditions are shown next to the gels. – indicates non–fluorescently labeled sample. n = 3 experiments (in vitro), and n = 3 mice (in vivo). (d) The activity of complex I in bovine heart mitochondrial membranes with no modifications compared to the effects of S-nitrosating the 75 kDa and B8 subunits only (75 kDa/B8 SNO) by incubation with MitoSNO during high complex I activity and of S-nitrosating the ND3, 75 kDa and B8 subunits (ND3 SNO) by incubation with MitoSNO during high complex I activity generated by anoxia. n = 3–4 experiments per condition. (e) Rat heart mitochondria at baseline and after being subjected to anoxia and reoxygenation (AR) with and without MitoSNO and monitored for hydrogen peroxide production. n = 3 experiments. (f) A model of the core structure of mammalian complex I generated using orthologous bovine sequences modeled on the crystal structure of the entire complex I from Thermus thermophilus. Structural modeling places Cys39 near the matrix-facing ND3 loop region of mammalian complex I (blue, ND3; green, other modeled complex I subunits; gray, entire core complex I). FMN, flavin mononucleotide. (g) Expanded modeled structure from f indicating the functional components of complex I close to ND3 Cys39. ND3 Cys39 (yellow sphere) maps to the interface of the ubiquinone binding site (Q site, orange surface) and the mitochondrial matrix surface of the complex, as well as the half-channel element of one of the proposed proton translocation pathways within complex I (charged residues within the channel are shown in magenta, and an approximate proton translocation path is highlighted with a blue arrow). The N2 iron-sulfur cluster that transfers electrons to bound ubiquinone is also shown. *P < 0.05, **P < 0.01 determined by one-way ANOVA. Data (a–e) are shown as the mean ± s.e.m. of at least three replicates.
Figure 4
Figure 4
S-nitrosation of ND3 Cys39 underlies protection from ischemia-reperfusion injury by mitochondrial S-nitrosation in vivo and represents a general mechanism for cardioprotection. (a) Cell death of rat heart–derived H9C2 cells subjected to ischemia-reperfusion injury with or without MitoSNO or NDUFAF1-targeted RNAi. n = 8 experiments per group. (b) S-nitrosation of complex I ND3 Cys39 in the intact heart followed by exposure to nitrite during ischemia as determined by fluorescent labeling of the entire complex after its resolution by BN-PAGE (top gel). In a separate experiment, after the isolation of complex I by BN-PAGE, the constituent subunits of complex I were resolved, allowing for direct assessment of the S-nitrosation state of ND3 (middle gel). Loading of complex I was assessed by western blotting of the 75 kDa subunit (bottom gel). Bottom, quantification of ND3 Cys39 S-nitrosation signals relative to those of complex I loading as determined by western blotting. n = 4 hearts per group. (c) A representative mass spectrum of the Cys39-containing tryptic peptide of ND3 indicating S-nitrosation of ND3 Cys39 after exposure of the heart to nitrite during ischemia by labeling of the ND3 S-nitrosothiol with d5-NEM. (d) A model showing how S-nitrosation of ND3 Cys39 protects from ischemia-reperfusion injury. During normal complex I activity, ND3 Cys39 is occluded. The low activity of complex I after ischemia causes this cysteine to become exposed. Reperfusion of the ischemic tissue rapidly reactivates complex I and generates superoxide and hydrogen peroxide, causing oxidative damage and cell death. When MitoSNO, or other S-nitrosating species, are present during reperfusion, the exposed ND3 cysteine becomes S-nitrosated, temporarily holding complex I in a low activity state and decreasing ROS production. The S-nitrosothiol is subsequently reduced by glutathione and thioredoxin, gradually reactivating complex I. SH, unmodified ND3 Cys39; Q, ubiquinone. *P < 0.05, ***P < 0.001 determined by one-way ANOVA. Data (a,b) are shown as the mean ± s.e.m.
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References

    1. Duranski MR, et al. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J. Clin. Invest. 2005;115:1232–1240. - PMC - PubMed
    1. Shiva S, et al. Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer. J. Exp. Med. 2007;204:2089–2102. - PMC - PubMed
    1. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite–nitric oxide pathway in physiology and therapeutics. Nat. Rev. Drug Discov. 2008;7:156–167. - PubMed
    1. Methner C, Schmidt K, Cohen MV, Downey JM, Krieg T. Both A2a and A2b adenosine receptors at reperfusion are necessary to reduce infarct size in mouse hearts. Am. J. Physiol. Heart Circ. Physiol. 2010;299:H1262–H1264. - PMC - PubMed
    1. Cohen MV, Yang XM, Liu YP, Solenkova NV, Downey JM. Cardioprotective PKG-independent NO signaling at reperfusion. Am. J. Physiol. Heart Circ. Physiol. 2010;299:H2028–H2036. - PMC - PubMed

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