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. 2009 Jun 30;106(26):10764-9.
doi: 10.1073/pnas.0903250106. Epub 2009 Jun 15.

A Mitochondria-Targeted S-nitrosothiol Modulates Respiration, Nitrosates Thiols, and Protects Against Ischemia-Reperfusion Injury

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A Mitochondria-Targeted S-nitrosothiol Modulates Respiration, Nitrosates Thiols, and Protects Against Ischemia-Reperfusion Injury

Tracy A Prime et al. Proc Natl Acad Sci U S A. .
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Abstract

Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.

Conflict of interest statement

Conflict of interest statement: M.P.M. and R.A.J.S. have submitted a patent on the technology described in this manuscript.

Figures

Fig. 1.
Fig. 1.
Rationale for developing mitochondria-targeted S-nitrosothiols (MitoSNOs). (A) The Nernst equation dictates that lipophilic TPP cations should be accumulated approximately 10-fold for every 60 mV of membrane potential (Δψ), with values of the plasma Δψ (≈70 mV) and mitochondrial Δψ (≈140 mV) for the heart leading to approximately 3,000-fold concentration of MitoSNO1 within mitochondria. In the oxidizing extracellular environment, MitoSNO1 should be relatively stable. In contrast, glutathione (GSH) within mitochondria should release NO from MitoSNO1, mainly by by transnitrosation to GSNO followed by reduction to release NO by mechanisms that are currently uncertain. The high concentration of NO generated will compete with O2 for the active site of cytochrome c oxidase, particularly when the O2 concentration is low, and slow respiration while elevating the cytosolic concentration of O2. MitoSNO1 will also S-nitrosate mitochondrial thiol proteins by transferring a nitrosonium group (NO+) to protein thiolates, modifying their function and may lead to protection during ischemia-reperfusion injury. (B) The structure and synthesis of MitoSNO1 by the S-nitrosation of the free thiol precursor MitoNAP.
Fig. 2.
Fig. 2.
Stability of MitoSNO1 and its decomposition to produce NO. (A) Stability of MitoSNO1 (5 μM) in KCl buffer measured by RP-HPLC. The identities of the RP-HPLC peaks for MitoNAP and the disulfide dimer of MitoNAP obtained at 21 h were confirmed by electrospray mass spectrometry (ESMS). (B) Stability of MitoSNO1 (50 μM) and SNAP (50 μM) in KCl buffer measured by nitrite accumulation. Data are means of triplicate determinations. (C) Release of NO from MitoSNO1 (5 μM) or SNAP (5 μM) on reaction with GSH measured using an NO electrode. Where indicated GSH (200 μM) or oxyhemoglobin (OxyHb, 5 μM) was added. (D) Reduction of MitoSNO1 (5 μM) to MitoNAP by GSH (500 μM) analyzed by RP-HPLC.
Fig. 3.
Fig. 3.
Membrane potential-dependent MitoSNO1 mitochondrial uptake and NO generation. (A–D) Mitochondria were incubated with rotenone and electrodes were used to measure the concentrations of O2 and NO simultaneously. Where indicated succinate (10 mM), OxyHb (5 μM), or FCCP (500 nM) were added. (A and B) Two milligrams mitochondrial protein/mL and 20 μM MitoSNO1 (A) or SNAP (B) were used. (C and D) One-half milligram mitochondrial protein/mL and 5 μM MitoSNO1 were used.
Fig. 4.
Fig. 4.
Effect of MitoSNO1 on respiration in cells. (A–D) Jurkat cells were incubated with 5 μM MitoSNO1 (A), MitoNAP (B), SNAP (C), or MitoSNO1 and FCCP (500 nM) (D). Where indicated 5 μM OxyHb was added. Sections of the O2 electrode trace where OxyHb was added have been expanded 4-fold in the vertical dimension. The dashed line indicates anaerobiosis. (E) Effect of MitoSNO1 on O2 bioavailability in hypoxia. HeLa cells were incubated at 1% O2 concentration with 5% CO2 and the balance N2 for 60 min before the indicated treatments. Cellular pO2 values, measured by fluorescence quenching oximetry and expressed as mean ± SD, are shown. (**, P < 0.001 compared to hypoxia alone and to MitoNAP treatments by ANOVA).
Fig. 5.
Fig. 5.
S-nitrosation of mitochondrial thiols by MitoSNO1. (A) Time course of S-nitrosation of mitochondrial proteins by MitoSNO1. Mitochondria were incubated with rotenone and succinate in the presence of 5 μM MitoSNO1. Data are means ± range of duplicate samples. (B) S-nitrosation of mitochondrial proteins by different concentrations of MitoSNO1 and SNAP. Mitochondria were incubated withMitoSNO1 or SNAP for 2.5 min ± 500 nM FCCP. For the +NEM sample, 10 mM NEM was added followed 1 min later by 5 μM MitoSNO1 and then incubated for a further 2.5 min. Data are means ± range of duplicate samples. (C) S-nitrosation of cellular protein thiols by MitoSNO1. C2C12 cells were preincubated for 5 min with either 20 μM FCCP or DMSO carrier, then 5 μM MitoSNO1 or SNAP was added and incubated for 5 min. Data are means ± range of duplicate determinations. (D) S-nitrosation of a mitochondria-enriched cell subfraction. C2C12 cells were incubated as in (C), and the cells were then processed to isolate a mitochondria-enriched fraction. Data are mean ± SEM of triplicate determinations, or mean ± range of duplicate determinations (+FCCP). (E and F) Visualisation of S-nitrosated mitochondrial proteins. Liver (E) or heart (F) mitochondria were incubated with rotenone and succinate in the presence of no additions, 10 μM MitoSNO1 or 500 μM diamide for 5 min. S-nitrosated thiol proteins were then selectively tagged with Cy3 maleimide and separated by SDS PAGE gel. The Cy3 fluorescence (SNO) and coomassie staining are shown.
Fig. 6.
Fig. 6.
Inhibition and S-nitrosation of complex I by MitoSNO1. (A) Effect of MitoSNO1 on mitochondrial respiration. Heart mitochondria were incubated in an oxygen electrode with 5 mM glutamate and 5 mM malate, or succinate, and incubated with 10 μM MitoSNO1, 10 μM MitoNAP or carrier for 2–3 min, then 250 μM adenosine diphosphate (ADP) was added and the rate of respiration measured. Respiration +MitoSNO1 is expressed as a percentage of that with MitoNAP, and are means ± SEM from 3 mitochondrial preparations; *P < 0.05 by Student's paired t test. (B) Effect of MitoSNO1 on respiration by mitochondrial membranes. Bovine heart mitochondrial membranes were incubated in an O2 electrode with 75 μM MitoSNO1 or MitoNAP, or ethanol carrier for 5 min ± rotenone, then 1 mM NADH or 10 mM succinate was added and respiration measured. Data are expressed as respiration as a percentage of the appropriate controls and are means ± range of 2 separate experiments. (C) S-nitrosation of complex I by MitoSNO1. Bovine heart mitochondrial membranes were incubated with 75 μM MitoSNO1 or carrier for 5 min. Then protein SNOs were labeled by maleimide-Cy3 and respiratory complexes were separated by BN-PAGE and scanned for Cy3 fluorescence. The locations of respiratory complexes I, III, and V were determined by immunoblotting. (D) Effect of GSH on S-nitrosation of complex I by MitoSNO1. Bovine heart mitochondrial membranes were incubated and processed as in (C) except that after incubation ± MitoSNO1 the membranes were incubated ± 1 mM GSH for 15 min.
Fig. 7.
Fig. 7.
Vasodilatory and tissue protective effects of MitoSNO1. (A) Dose-response curves for the effect of MitoSNO1 and related compounds on blood vessel relaxation. Data are means ± SEM, n = 8 (MitoSNO1) or n = 4 (decomposed MitoSNO1 and SNAP). (B) EC50 for the effect of MitoSNO1 and SNAP on blood vessel relaxation from (A) above. ***, P < 0.001 by Student's unpaired t test. (C and D) MitoSNO1 protects against I/R injury in Langendorff-perfused mouse hearts. Hearts were subjected to 25 min of ischemia followed by 1 h of reperfusion. MitoSNO1 or MitoNAP (100 nM final), or vehicle carrier, was added during reperfusion. Hearts were then stained with 2,3,5-triphenyltetrazolium chloride (TTC) to visualize infarct. (C) Protection of heart function by MitoSNO1 during I/R injury. Data show rate pressure product (RRP). The arrow indicates where infusion with MitoSNO1 or MitoNAP was initiated. Data are means ± SEM, n = 6–7. (D) Decreased cardiac infarct size following infusion with MitoSNO1. Upper panel shows typical heart staining. Pale white staining is necrotic infarct, while live tissue stains deep red. Lower panel shows quantitation of infarct size versus area at risk. Data are means ± SEM, n = 6–7. *, P < 0.05 versus vehicle control group; #, P < 0.05 versus vehicle control group (ANOVA).

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