PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of cardiovascresLink to Publisher's site
 
Cardiovasc Res. 2009 May 1; 82(2): 333–340.
Published online 2008 December 2. doi:  10.1093/cvr/cvn323
PMCID: PMC2675927

Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection

Abstract

Aims

Both mitochondria and nitric oxide (NO) contribute to cardioprotection by ischaemic preconditioning (IPC). IPC causes mild uncoupling of mitochondria via uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT), and mild uncoupling per se is cardioprotective. Although electrophilic lipids are known to activate mitochondrial uncoupling, the role of such species in IPC-induced uncoupling and cardioprotection is unclear. We hypothesized that endogenous formation of NO-derived electrophilic lipids (nitroalkenes such as nitro-linoleate, LNO2) during IPC may stimulate mitochondrial uncoupling via post-translational modification of UCPs and ANT, thus affording cardioprotection.

Methods

Hearts from male Sprague-Dawley rats were Langendorff-perfused and subjected to IPC. Nitroalkene formation was measured by HPLC-ESI-MS/MS. The effects of exogenous LNO2 and biotin-tagged LNO2 on isolated heart mitochondria and cardiomyocytes were also investigated.

Results

Nitroalkenes including LNO2 were endogenously generated in mitochondria of IPC hearts. Synthetic LNO2 (<1 µM) activated mild uncoupling, an effect blocked by UCP and ANT inhibitors. LNO2 (<1 µM) also protected cardiomyocytes against simulated ischaemia–reperfusion injury. Biotinylated LNO2 covalently modified ANT thiols and possibly UCP-2. No effects of LNO2 were attributable to NO release, cGMP signalling, mitochondrial KATP channels, or protective kinase signalling.

Conclusion

Components of a novel signalling pathway are inferred, wherein nitroalkenes formed by IPC-stimulated nitration reactions may induce mild mitochondrial uncoupling via post-translational modification of ANT and UCP-2, subsequently conferring resistance to ischaemia–reperfusion injury.

Keywords: Nitric oxide, Mitochondria, Ischaemia, Reperfusion, Preconditioning, Nitroalkenes

1. Introduction

Cardiac ischaemic preconditioning (IPC) is an endogenous protective mechanism, in which short cycles of non-lethal ischaemia–reperfusion (IR) elicit protection from subsequent prolonged IR injury.1 The mechanisms underlying IPC-mediated cardioprotection are debated, but a consensus has emerged that nitric oxide (NO) and mitochondria play essential roles.26 Despite this consensus, links between NO signalling and effector mechanisms at the mitochondrial level remain elusive. For example, while NO signalling via cGMP-dependent protein kinase (PKG) can phosphorylate several mitochondrial targets of relevance to IPC,79 the importance of PKG-independent effects of NO on mitochondria is less clear.4 Similarly, mild uncoupling of mitochondria is an important IPC-induced protective event,10,11 but its potential upstream regulation by NO is unclear. The aim of this study was to elucidate novel mechanisms linking NO and mitochondria in IPC.

One unexplored aspect of NO signalling in IPC is the nitration of unsaturated fatty acids to yield electrophilic nitroalkene derivatives (e.g. nitro-linoleate and nitro-oleate, LNO2 and OA-NO2, respectively).12 Although the biochemical mechanisms of lipid nitration are not fully elucidated,13 nitroalkenes are found endogenously in humans14 and can mediate pluripotent cell signalling effects.15 These effects may be mediated by electrophilic reaction of nitroalkenes with protein thiols, to form covalent ‘nitroalkylation’ adducts.16 Notably, conditions during IPC could favour nitroalkene generation from the abundance of polyunsaturated fatty acids in mitochondrial membranes.17 These conditions include elevated NO, 5,6 transient reactive oxygen species (ROS) generation,18,19 acidic pH,13 and the activation of both lipoxygenases and mitochondrial phospholipase A2.20,21 In addition, both peroxynitrite and electrophilic lipids can activate mitochondrial uncoupling,22,23 and mild uncoupling itself is cardioprotective.11,24 Combining these observations we hypothesized that nitroalkenes may be formed in mitochondria during IPC, and may nitroalkylate mitochondrial proteins thereby activating mild uncoupling, leading to cardioprotection against IR injury.

2. Methods

Detailed experimental procedures are given in the Supplementary material online. All chemicals were of the highest grade available from Sigma (St Louis, MO, USA) unless otherwise stated. LNO2 and biotinylated LNO2 (Bt-LNO2) were synthesized, purified, quantified, and stored as previously,14,25,26 with all procedures performed under subdued light. Non-nitrated linoleic acid (LA) served as a control throughout.

Male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA), 200–250 g body mass, were housed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (US National Institutes of Health Publication No. 85–23, revised 1996). All procedures were also approved by the University of Rochester Committee on Animal Resources (UCAR, protocols 2003-111 and 2007-087). Hearts were perfused as previously,10 and subjected to either (i) normoxic perfusion, (ii) IPC, (iii) IPC plus the NO synthase inhibitor l-nitro-arginine methyl ester (l-NAME, 100 µM), (iv) ischaemia, or (v) IPC plus ischaemia.

Heart mitochondria were isolated and protein determined as previously.10 Mitochondrial respiration and uncoupling were measured as previously.10 Optional additions to incubations were: 1–5 µM LNO2, 1 mM guanosine diphosphate (GDP), or 50 µM Genipin (Wako, Richmond, VA, USA)27 to inhibit uncoupling proteins (UCPs) or UCP-2, respectively; 5 µM carboxyatractyloside (CATr, Calbiochem, San Diego, CA, USA) to inhibit adenine nucleotide translocase (ANT); 20 µM ethanethiol (E-SH) to reverse thiol modifications; 30 µM 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO, Axxora, San Diego, CA, USA) to scavenge NO; or 1 mM 2,6-di-tert-butyl-4-methyl-phenol (BHT), a lipid-soluble antioxidant. Mitochondrial permeability transition (PT) pore opening was measured as previously.28

Adult rat ventricular cardiomyocytes were isolated and state 4 respiration plus mitochondrial membrane potential (Δψm) were measured as previously.29 Cells were subjected to simulated ischaemia–reperfusion (SIR) injury as previously,29 with cell viability measured by Trypan blue exclusion. This model eliminates the potentially confounding vascular effects of LNO2.15 Briefly, SIR comprised 1 h hypoxia in glucose-free buffer at pH 6.5, then 30 min reoxygenation in glucose-replete buffer at pH 7.4. The following were optionally present: LNO2 (0.25–1 µM), LA (0.5–1 µM), the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ, 10 µM), the mitochondrial KATP channel antagonists 5-hydroxydecanoate (5-HD, 300 µM) or glybenclamide (2 µM), the extracellular signal regulated kinase (ERK) inhibitor UO126 (10 µM), or the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin (100 nM).

Biotinylated proteins were immunoprecipitated from Bt-LNO2-treated mitochondria or cardiomyocytes using neutravidin-agarose as previously.10 Immunoprecipitated samples or whole extracts were western blotted as previously,10 using antibodies against biotin, ANT, or UCP-2.

Lipids were extracted from 5 mg mitochondrial protein as previously.30 A synthetic [13C18]-LNO2 internal standard was added to correct for extraction losses. Nitroalkenes were detected by HPLC electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) as previously.14 Biologically derived nitroalkenes were identified by retention time, precursor ion mass, MS/MS fragmentation pattern, and thiol reactivity,14 and were quantified relative to the internal standard.

All experiments were performed three to eight times, each n representing an independent mitochondrial, cell, or heart preparation (separate animal). Significance between groups was established by ANOVA.

3. Results

In the current study, we investigated whether fatty acid nitration could occur in cardiac mitochondria during IPC. Mitochondrial lipid extracts from perfused hearts were analysed by HPLC-ESI-MS/MS using a multiple reaction monitoring (MRM) transition of 324/46 in negative ion mode. The chromatograms (Figure 1A) revealed that mitochondria from IPC hearts contained elevated levels of an LNO2 nitroalkene derivative, with positional isomers eluting at identical times to a synthetic LNO2 nitroalkene standard. Concomitant product ion analysis (Figure 1B) revealed a fragmentation pattern for the IPC-derived nitroalkene consistent with the previously reported LNO2 structure.14 Quantitation of LNO2 via internal standard revealed its concentration in IPC mitochondria to be 619 ± 137 fmol/mg mitochondrial protein (Figure 1C). The NOS inhibitor l-NAME attenuated the IPC-induced increase in mitochondrial LNO2 by ~60% (P = 0.06 vs. IPC), although it is not known if the l-NAME insensitive fraction of LNO2 is due to incomplete NOS inhibition or represents LNO2 generation from other reactive nitrogen species (RNSs) such as NO2. Ischaemia alone generated a miniscule amount of LNO2, but notably in mitochondria from hearts subjected to IPC plus ischaemia, LNO2 levels dropped to ~16% of those seen in IPC alone, suggesting rapid LNO2 degradation. Mitochondria also contained OA-NO2, but its levels did not change in IPC (215 ± 74 vs. 245 ± 41 fmol/mg protein in control vs. IPC, respectively). In addition nitroalkenes were detected in other subcellular compartments (data not shown). Due to space restrictions the current study focuses on mitochondrial IPC samples, and a more complete characterization of cardiac nitroalkenes during IPC and IR, including their metabolism by mitochondrial β-oxidation, is anticipated to be the subject of a subsequent manuscript.

Figure 1

Endogenous LNO2 formation in mitochondria during IPC. (A) Lipid extracts were prepared from mitochondria isolated from control and IPC-treated hearts, and analysed by HPLC ESI-MS/MS in MRM mode using m/z 324/46 transition to identify LNO2. Blank solvent ...

Next, the potential for exogenous LNO2 to protect against SIR injury was tested in isolated cardiomyocytes. Figure 2A shows that LNO2 significantly improved post-SIR cardiomyocyte viability, with maximal protection at 0.5 µM LNO2. Non-nitrated LA was without effect, and the mito-KATP channel antagonists 5-HD or glybenclamide31 did not reverse the effect of LNO2, suggesting no role for this channel in LNO2-mediated protection. Notably in this system, 5-HD did block protection by the mito-KATP channel agonist diazoxide, indicating appropriate 5-HD efficacy (not shown). In addition, the sGC inhibitor ODQ,47 the ERK inhibitor UO-126,32 and the PI3K inhibitor wortmannin32 did not affect LNO2-mediated protection, indicating no role for classical NO/cGMP/PKG signalling, or ERK/PI3K signalling. Furthermore, post-SIR mitochondrial function (intracellular Δψm) correlated well with cell viability and benefited from LNO2 treatment (Figure 2B).

Figure 2

LNO2 protects cardiomyocytes from SIR injury and stimulates myocyte respiration. (A) Post-SIR cell viability. Cardiomyocytes were subjected to SIR injury in the presence of indicated concentrations of LNO2 or LA, added 20 min before ischaemia. Where indicated, ...

We next investigated the mechanism of LNO2-mediated protection and hypothesized that LNO2, like other electrophilic lipids,23 may uncouple mitochondria, which itself is known to be cardioprotective.10,11,24 Consistent with this hypothesis, Figure 2C shows that LNO2 stimulated cellular state 4 respiration (a surrogate marker for uncoupling), while LA was without effect. Such respiratory stimulation could be due to uncoupling, or an acceleration of oxidative-phosphorylation,10 therefore, we next assayed the direct effects of LNO2 on uncoupling in isolated mitochondria. Titration curves of state 4 respiration vs. Δψm (Figure 3) showed that 1 µM LNO2 stimulated uncoupling, as indicated by a left shift of the curve (a more H+ permeable membrane necessitates faster respiratory chain activity to maintain a given Δψm10), while LA was without effect. Figure 3B and C shows that LNO2-induced uncoupling was inhibited by the ANT inhibitor CATr and the UCP inhibitor GDP. Neither inhibitor affected baseline function in the absence of LNO2 (Figure 3D).

Figure 3

LNO2 stimulates mitochondrial uncoupling, in a manner sensitive to inhibitors of ANT and UCPs. Isolated mitochondrial uncoupling (H+ leak) was determined as previously.10 The upper-right point in each curve represents state 4 respiration, with the remaining ...

Toxicity studies (Figure 3E) revealed that LNO2 >10 µM both inhibited respiration and dropped ΔΨm. This respiratory inhibition was not due to complex I inhibition (result not shown), as has been shown for other electrophilic lipids.33 Furthermore, similar to other electrophilic lipids,34 LNO2 >10 µM induced large-scale mitochondrial swelling indicative of PT pore opening (Figure 4). This effect was insensitive to the PT pore inhibitor cyclosporin A (CsA), indicating a possible role for the ‘unregulated’ PT pore resulting from membrane protein aggregation.35 PT pore opening was not induced by 5 µM LNO2 (Figure 4), suggesting this was not the mechanism of uncoupling induced by 1 µM LNO2 (Figure 3).

Figure 4

Induction of unregulated PT pore opening at high [LNO2] (A) Typical PT pore swelling traces28 are shown. LNO2 or LA was added at the arrow. Where indicated, the PT pore inhibitor cyclosporin A (CsA, 2 µM) was present from the beginning of the ...

Experiments to elucidate the mechanism of LNO2-induced uncoupling (Figure 5) employed oligomycin-clamped state 4 mitochondrial respiration as a surrogate marker for uncoupling.10 LNO2-induced uncoupling was not inhibited by BHT or c-PTIO, respectively, indicating no role for secondary lipid oxidation or NO released from LNO2 36. However, LNO2-induced uncoupling was sensitive to the UCP-2 inhibitor genipin27 and was also reversed by E-SH suggesting protein thiol modification as a possible mechanism.16 Neither BHT, GDP, genipin, nor E-SH affected baseline state 4 respiration.

Figure 5

The effect of various reagents on LNO2-induced uncoupling. Oligomycin-clamped state 4 mitochondrial respiration was used as a surrogate for uncoupling (see Methods). Indicated concentrations of reagents were added prior to LNO2, except E-SH, which was ...

To define mitochondrial targets of LNO2, biotin-tagged LNO226 was employed. Importantly, Bt-LNO2 induced mitochondrial uncoupling in the same GDP- and CATr-sensitive manner as native LNO2 (Figure 6A, B). Following Bt-LNO2 addition to mitochondria, biotinylated proteins were immunoprecipitated and western blotted with anti-biotin or anti-ANT antibodies. Figure 6C (upper panel) shows that Bt-LNO2 adducted several mitochondrial proteins, including a prominent band at ~32 kDa which was identified as ANT (6C, lower panel). Furthermore, Bt-LNO2 labelled several proteins including ANT in intact cardiomyocytes (Figure 6D), thus indicating that Bt-LNO2, similar to other electrophilic lipids, can enter cells and target mitochondria.37 Full characterization of the nitroalkene-reactive proteome is anticipated to be the subject of subsequent studies. Nevertheless, as detailed in the Supplementary material online, several other mitochondrial and non-mitochondrial proteins were also identified as nitroalkylation targets.

Figure 6

LNO2 nitroalkylates ANT and UCP-2. (A) Bt-LNO2 induced uncoupling similar to LNO2 (c.f. Figure 3), and (B) this effect of Bt-LNO2 was sensitive to GDP and CATr. Data are means ± SEM, n = 4. (C) Biotin labelled proteins were immunoprecipitated ...

As shown in Figure 6E, two conformations of the ANT can be enforced by different inhibitors. Furthermore, in the CATr-induced c-conformation, a redox-sensitive thiol (C57) is inaccessible, whereas this thiol is exposed in the bongkrekic acid (BKA)-induced m-conformation.38 Support for this thiol as a potential target of LNO2 is provided by observations that CATr and E-SH inhibited Bt-LNO2 ANT modification, whereas BKA did not (Figure 6C). These results are consistent with the ability of both CATr and E-SH to inhibit LNO2-induced uncoupling (Figures 3B and 55). c-PTIO was without effect on ANT modification, indicating no role for NO release. To quantify ANT modification, the fraction of ANT immunoprecipitated (i.e. ANT in pellet vs. supernatant) was examined. Figure 6F shows a pellet/supernatant pair from a biotin immunoprecipitation, western blotted for ANT. Densitometry on several such blots revealed that upon Bt-LNO2 treatment (1 µM) 48.2 ± 4.1% of ANT disappeared from the supernatant and appeared in the pellet.

Since LNO2-induced H+ leak was also sensitive to UCP-2 inhibitors (Figures 3C and 55), nitroalkylation of UCP-2 was also investigated. However, biotin-immunoprecipitation studies similar to those performed for ANT revealed no UCP-2 pull-down (data not shown). Subsequently, mitochondria were treated with native LNO2 in the presence or absence of the UCP inhibitor GDP, followed by western blotting for UCP-2. Figure 6G shows that UCP-2 progressively disappeared from the blot with increasing LNO2 doses, and this disappearance was attenuated by GDP. We hypothesized this may be due to nitroalkylation increasing the hydrophobicity of UCP-2 and preventing its SDS-PAGE migration. Supplementary material online, Figure S1D provides support for this hypothesis; blotting the entire gel including stacker plus comb revealed that high-LNO2 treatment resulted in aggregation of UCP-2 immunoreactivity at the stacker/separating gel interface.

4. Discussion

The major findings of this study are: (i) Electrophilic nitroalkene derivatives are formed endogenously in mitochondria during IPC; (ii) Exogenous LNO2 protects cardiomyocytes from SIR injury; (iii) LNO2 stimulates mitochondrial uncoupling, via ANT and UCP-2 dependent mechanisms; (iv) LNO2 nitroalkylates ANT and possibly UCP-2.

Central roles in IPC signalling are played by mitochondria,24 NO,46 and ROS,18,19,24. However, the relationships between these key players are not fully understood. One unexplored mechanism for the interaction of ROS, NO, and mitochondria in IPC may be the generation of electrophilic lipids such as nitroalkenes. Oxidative lipid derivatives are known to be cardioprotective3941 and can induce mitochondrial uncoupling.23 Furthermore, mild mitochondrial uncoupling itself is cardioprotective.10,11,24 Therefore, the current data together with these previous findings suggest that electrophilic nitroalkenes may be endogenously generated in mitochondria during IPC and may induce mitochondrial uncoupling, thereby contributing to cardioprotection.

Regarding the contribution of this pathway to the overall cardioprotective effects of IPC, if a mitochondrial volume of 0.65 µl/mg protein is assumed,10 the amount of LNO2 in IPC mitochondria (Figure 1C) translates to an intra-mitochondrial concentration of 0.95 µM, which is the same level of exogenous LNO2 (1 µM) that stimulated uncoupling in isolated mitochondria (Figure 3). Thus, the mitochondrial concentration of LNO2 generated in IPC is theoretically capable of inducing uncoupling.

Considering the mechanism of acyl chain nitration in IPC, the finding that l-NAME only partially inhibited IPC-induced LNO2 formation (assuming efficient NOS inhibition by l-NAME in this system) suggests that non-NOS sources of RNS may be involved. In this regard, an observed ~40% increase in mitochondrial NO2 levels during IPC (Nadtochiy and Brookes, unpublished) suggests NO2 may contribute to lipid nitration under the acidic conditions of IPC. The overall role of NOS in IPC is somewhat controversial,42 since in vivo studies suggest NOS is essential for IPC,43 whereas in vitro studies have found that l-NAME does not block IPC.44,45 The mechanism by which nitro fatty acids are liberated from membranes may involve PLA2, which is known to liberate linoleate and arachidonate, but not oleate, during IPC.46 This is consistent with our finding that mitochondrial OA-NO2 levels did not change in IPC, indicating some specificity in fatty-acid liberation.

Regardless the mechanism of nitroalkene formation or liberation in IPC, exogenous LNO2 was protective in a cardiomyocyte model of SIR injury. The signalling pathways by which nitroalkenes elicit protection could include PPARγ activation4749 and subsequent HO-1 up-regulation.50 However, such gene transcription effects unlikely account for the immediate short-term (20 min) effects of LNO2 observed herein. Thus, we chose to focus on the short-term direct effects of LNO2 on mitochondrial function as a potential mechanism of protection and demonstrated that LNO2 induces mild mitochondrial uncoupling.

Since RNSs are known to have a number of other effects on mitochondria including the modulation of many proteins implicated in IPC,4 control experiments were performed to exclude the involvement of NO released from LNO2, sGC, ERK, PI3K, or mKATP channels in LNO2-mediated protection. Furthermore, even though the mKATP channel has been proposed to uncouple mitochondria,18,31,51 the magnitude of mKATP flux is insufficient to account for IPC-induced uncoupling,10 and the current lack of a molecular identity for mKATP precludes its identification as an LNO2 target.

Having established that LNO2 uncoupled mitochondria, we next investigated the mechanism of uncoupling. One possibility is that cycling of protonated/deprotonated nitro-fatty-acids across the membrane, as observed for nitro-aromatics (e.g. dinitrophenol),52 could uncouple mitochondria. However, such uncoupling should not occur with Bt-LNO2 in which the carboxylic acid group is blocked by biotin. The data in Figure 6A and B thus precludes this mechanism. Rather, based on evidence from biotin-tagged LNO2 (Figure 6), the likely mechanism of LNO2-induced uncoupling is the nitroalkylation of ANT and UCP-2. Although the exact mechanism of H+ transport by these proteins is unknown,52 we speculate that nitroalkylation of cysteine residues may result in structural/conformation changes that cause uncoupling.

Mild mitochondrial uncoupling is thought to be cardioprotective via inhibition of mitochondrial Ca2+ overload24 or ROS generation,53 although we consider it unlikely54 that a direct role for UCPs in mitochondrial Ca2+ transport55 exists. Regarding ROS, it has been shown that uncoupling-induced cardioprotection is blocked by antioxidants, suggesting a role for ROS downstream of uncoupling.24 These apparently inconsistent findings are reconciled by the paradigm that low levels of ROS generated during IPC (prior to index ischaemia) may activate signalling processes that inhibit large- scale ROS generation during subsequent IR injury.10,19

In summary, the current work has elucidated components of a potential signalling pathway, in which nitroalkenes are generated in mitochondria during IPC and activate mitochondrial uncoupling via nitroalkylation of proteins such as ANT. These studies advance our understanding of the biological roles of nitroalkenes and also suggest that nitroalkenes may be useful cardioprotective pharmacologic agents. It is also possible that some of the cardioprotective benefits of NO256 or mitochondrially targeted NO donors29 may involve nitroalkene generation, and that some cardioprotective benefits of the ‘Mediterranean diet’ may be due to intra-gastric nitroalkene generation.57

Funding

This work was supported by grants from the US National Institutes of Health (RO1 HL071158 to P.S.B. and HL58115 and HL64937 to B.A.F.) and the American Diabetes Association (ADA 7-06-JF-06 to P.R.S.B).

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: BAF acknowledges financial interest in Complexa, Inc.

[Supplementary Data]

References

1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124–1136. [PubMed]
2. Garcia-Dorado D, Rodriguez-Sinovas A, Ruiz-Meana M, Inserte J, Agullo L, Cabestrero A. The end-effectors of preconditioning protection against myocardial cell death secondary to ischemia–reperfusion. Cardiovasc Res. 2006;70:274–285. [PubMed]
3. Murphy E, Steenbergen C. Preconditioning: the mitochondrial connection. Annu Rev Physiol. 2007;69:51–67. [PubMed]
4. Burwell LS, Brookes PS. Mitochondria as a target for the cardioprotective effects of nitric oxide in ischemia–reperfusion injury. Antioxid Redox Signal. 2008;10:579–600. [PubMed]
5. Cohen MV, Yang XM, Downey JM. Nitric oxide is a preconditioning mimetic and cardioprotectant and is the basis of many available infarct-sparing strategies. Cardiovasc Res. 2006;70:231–239. [PubMed]
6. Jones SP, Bolli R. The ubiquitous role of nitric oxide in cardioprotection. J Mol Cell Cardiol. 2006;40:16–23. [PubMed]
7. Costa AD, Garlid KD, West IC, Lincoln TM, Downey JM, Cohen MV, et al. Protein kinase G transmits the cardioprotective signal from cytosol to mitochondria. Circ Res. 2005;97:329–336. [PubMed]
8. Kim JS, Ohshima S, Pediaditakis P, Lemasters JJ. Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH- dependent cell death after reperfusion. Free Radic Biol Med. 2004;37:1943–1950. [PubMed]
9. Wang G, Liem DA, Vondriska TM, Honda HM, Korge P, Pantaleon DM, et al. Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition. Am J Physiol Heart Circ Physiol. 2005;288:H1290–H1295. [PubMed]
10. Nadtochiy SM, Tompkins AJ, Brookes PS. Different mechanisms of mitochondrial proton leak in ischaemia/reperfusion injury and preconditioning: implications for pathology and cardioprotection. Biochem J. 2006;395:611–618. [PubMed]
11. McLeod CJ, Aziz A, Hoyt RF, Jr, McCoy JP, Jr, Sack MN. Uncoupling proteins 2 and 3 function in concert to augment tolerance to cardiac ischemia. J Biol Chem. 2005;280:33470–33476. [PubMed]
12. Kalyanaraman B. Nitrated lipids: a class of cell-signaling molecules. Proc Natl Acad Sci USA. 2004;101:11527–11528. [PubMed]
13. O'Donnell VB, Eiserich JP, Chumley PH, Jablonsky MJ, Krishna NR, Kirk M, et al. Nitration of unsaturated fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and nitronium ion. Chem Res Toxicol. 1999;12:83–92. [PubMed]
14. Baker PR, Schopfer FJ, Sweeney S, Freeman BA. Red cell membrane and plasma linoleic acid nitration products: synthesis, clinical identification, and quantitation. Proc Natl Acad Sci USA. 2004;101:11577–11582. [PubMed]
15. Freeman BA, Baker PR, Schopfer FJ, Woodcock SR, Napolitano A, d'Ischia M. Nitro-fatty acid formation and signaling. J Biol Chem. 2008;283:15515–15519. [PubMed]
16. Batthyany C, Schopfer FJ, Baker PR, Duran R, Baker LM, Huang Y, et al. Reversible post-translational modification of proteins by nitrated fatty acids in vivo. J Biol Chem. 2006;281:20450–20463. [PMC free article] [PubMed]
17. Daum G. Lipids of mitochondria. Biochim Biophys Acta. 1985;822:1–42. [PubMed]
18. Facundo HT, Carreira RS, de Paula JG, Santos CC, Ferranti R, Laurindo FR, et al. Ischemic preconditioning requires increases in reactive oxygen release independent of mitochondrial K+ channel activity. Free Radic Biol Med. 2006;40:469–479. [PubMed]
19. Vanden Hoek TL, Becker LB, Shao Z, Li C, Schumacker PT. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J Biol Chem. 1998;273:18092–18098. [PubMed]
20. Murphy E, Glasgow W, Fralix T, Steenbergen C. Role of lipoxygenase metabolites in ischemic preconditioning. Circ Res. 1995;76:457–467. [PubMed]
21. Williams SD, Gottlieb RA. Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective. Biochem J. 2002;362:23–32. [PubMed]
22. Brookes PS, Land JM, Clark JB, Heales SJ. Peroxynitrite and brain mitochondria: evidence for increased proton leak. J Neurochem. 1998;70:2195–2202. [PubMed]
23. Echtay KS, Esteves TC, Pakay JL, Jekabsons MB, Lambert AJ, Portero-Otin M, et al. A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling. EMBO J. 2003;22:4103–4110. [PubMed]
24. Brennan JP, Southworth R, Medina RA, Davidson SM, Duchen MR, Shattock MJ. Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of KATP channel activation. Cardiovasc Res. 2006;72:313–321. [PubMed]
25. Lim DG, Sweeney S, Bloodsworth A, White CR, Chumley PH, Krishna NR, et al. Nitrolinoleate, a nitric oxide-derived mediator of cell function: synthesis, characterization, and vasomotor activity. Proc Natl Acad Sci USA. 2002;99:15941–15946. [PubMed]
26. Cui T, Schopfer FJ, Zhang J, Chen K, Ichikawa T, Baker PR, et al. Nitrated fatty acids: Endogenous anti-inflammatory signaling mediators. J Biol Chem. 2006;281:35686–35698. [PMC free article] [PubMed]
27. Zhang CY, Parton LE, Ye CP, Krauss S, Shen R, Lin CT, et al. Genipin inhibits UCP2-mediated proton leak and acutely reverses obesity- and high glucose-induced beta cell dysfunction in isolated pancreatic islets. Cell Metab. 2006;3:417–427. [PubMed]
28. Brookes PS, Salinas EP, Darley-Usmar K, Eiserich JP, Freeman BA, Darley-Usmar VM, et al. Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release. J Biol Chem. 2000;275:20474–20479. [PubMed]
29. Nadtochiy SM, Burwell LS, Brookes PS. Cardioprotection and mitochondrial S-nitrosation: Effects of S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) in cardiac ischemia–reperfusion injury. J Mol Cell Cardiol. 2007;42:812–825. [PMC free article] [PubMed]
30. Brookes PS, Rolfe DF, Brand MD. The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: comparison with isolated mitochondria. J Membr Biol. 1997;155:167–174. [PubMed]
31. Gross GJ, Fryer RM. Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning. Circ Res. 1999;84:973–979. [PubMed]
32. Lim SY, Davidson SM, Paramanathan AJ, Smith CC, Yellon DM, Hausenloy DJ. The novel adipocytokine visfatin exerts direct cardioprotective effects. J Cell Mol Med. 2008;12:1395–1403. [PMC free article] [PubMed]
33. Martinez B, Perez-Castillo A, Santos A. The mitochondrial respiratory complex I is a target for 15-deoxy-delta12,14-prostaglandin J2 action. J Lipid Res. 2005;46:736–743. [PubMed]
34. Landar A, Shiva S, Levonen AL, Oh JY, Zaragoza C, Johnson MS, et al. Induction of the permeability transition and cytochrome c release by 15-deoxy-delta12,14-prostaglandin J2 in mitochondria. Biochem J. 2006;394:185–195. [PubMed]
35. He L, Lemasters JJ. Regulated and unregulated mitochondrial permeability transition pores: a new paradigm of pore structure and function? FEBS Lett. 2002;512:1–7. [PubMed]
36. Schopfer FJ, Baker PR, Giles G, Chumley P, Batthyany C, Crawford J, et al. Fatty acid transduction of nitric oxide signaling. Nitrolinoleic acid is a hydrophobically stabilized nitric oxide donor. J Biol Chem. 2005;280:19289–19297. [PubMed]
37. Landar A, Zmijewski JW, Dickinson DA, Le GC, Johnson MS, Milne GL, et al. Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species. Am J Physiol Heart Circ Physiol. 2006;290:H1777–H1787. [PubMed]
38. McStay GP, Clarke SJ, Halestrap AP. Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. Biochem J. 2002;367:541–548. [PubMed]
39. Nithipatikom K, Moore JM, Isbell MA, Falck JR, Gross GJ. Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury. Am J Physiol Heart Circ Physiol. 2006;291:H537–H542. [PubMed]
40. Nowak G, Grant DF, Moran JH. Linoleic acid epoxide promotes the maintenance of mitochondrial function and active Na+ transport following hypoxia. Toxicol Lett. 2004;147:161–175. [PubMed]
41. Karliner JS. Mechanisms of cardioprotection by lysophospholipids. J Cell Biochem. 2004;92:1095–1103. [PubMed]
42. Ferdinandy P, Schulz R. Nitric oxide, superoxide, and peroxynitrite in myocardial ischaemia–reperfusion injury and preconditioning. Br J Pharmacol. 2003;138:532–543. [PMC free article] [PubMed]
43. Vegh A, Szekeres L, Parratt J. Preconditioning of the ischaemic myocardium; involvement of the L-arginine nitric oxide pathway. Br J Pharmacol. 1992;107:648–652. [PMC free article] [PubMed]
44. Weselcouch EO, Baird AJ, Sleph P, Grover GJ. Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts. Am J Physiol. 1995;268:H242–H249. [PubMed]
45. Nakano A, Liu GS, Heusch G, Downey JM, Cohen MV. Exogenous nitric oxide can trigger a preconditioned state through a free radical mechanism, but endogenous nitric oxide is not a trigger of classical ischemic preconditioning. J Mol Cell Cardiol. 2000;32:1159–1167. [PubMed]
46. Starkopf J, Andreasen TV, Bugge E, Ytrehus K. Lipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in ischaemic preconditioning of rat heart. Cardiovasc Res. 1998;37:66–75. [PubMed]
47. Baker PR, Lin Y, Schopfer FJ, Woodcock SR, Groeger AL, Batthyany C, et al. Fatty acid transduction of nitric oxide signaling: multiple nitrated unsaturated fatty acid derivatives exist in human blood and urine and serve as endogenous peroxisome proliferator-activated receptor ligands. J Biol Chem. 2005;280:42464–42475. [PMC free article] [PubMed]
48. Schopfer FJ, Lin Y, Baker PR, Cui T, Garcia-Barrio M, Zhang J, et al. Nitrolinoleic acid: an endogenous peroxisome proliferator-activated receptor gamma ligand. Proc Natl Acad Sci USA. 2005;102:2340–2345. [PubMed]
49. Gonon AT, Bulhak A, Labruto F, Sjoquist PO, Pernow J. Cardioprotection mediated by rosiglitazone, a peroxisome proliferator-activated receptor gamma ligand, in relation to nitric oxide. Basic Res Cardiol. 2007;102:80–89. [PubMed]
50. Liu X, Pachori AS, Ward CA, Davis JP, Gnecchi M, Kong D, et al. Heme oxygenase-1 (HO-1) inhibits postmyocardial infarct remodeling and restores ventricular function. FASEB J. 2006;20:207–216. [PubMed]
51. Liu Y, Sato T, O'Rourke B, Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation. 1998;97:2463–2469. [PubMed]
52. Jezek P, Zackova M, Ruzicka M, Skobisova E, Jaburek M. Mitochondrial uncoupling proteins–facts and fantasies. Physiol Res. 2004;53(Suppl. 1):S199–S211. [PubMed]
53. Cannon B, Shabalina IG, Kramarova TV, Petrovic N, Nedergaard J. Uncoupling proteins: a role in protection against reactive oxygen species–or not? Biochim Biophys Acta. 2006;1757:449–458. [PubMed]
54. Brookes PS, Parker N, Buckingham JA, Vidal-Puig A, Halestrap AP, Gunter TE, et al. UCPs - unlikely calcium porters. Nat Cell Biol. 2008;10:1235–1237. [PMC free article] [PubMed]
55. Trenker M, Malli R, Fertschai I, Levak-Frank S, Graier WF. Uncoupling proteins 2 and 3 are fundamental for mitochondrial Ca2+ uniport. Nat Cell Biol. 2007;9:445–452. [PubMed]
56. Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, et al. Cytoprotective effects of nitrite during in vivo ischemia–reperfusion of the heart and liver. J Clin Invest. 2005;115:1232–1240. [PMC free article] [PubMed]
57. Napolitano A, Panzella L, Savarese M, Sacchi R, Giudicianni I, Paolillo L, et al. Acid-induced structural modifications of unsaturated fatty acids and phenolic olive oil constituents by nitrite ions: a chemical assessment. Chem Res Toxicol. 2004;17:1329–1337. [PubMed]

Articles from Cardiovascular Research are provided here courtesy of Oxford University Press