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• W2064096645 abstract "Electrophilic nitrated lipids (nitroalkenes) are emerging as an important class of protective cardiovascular signaling molecules. Although species such as nitro-linoleate (LNO2) and nitro-oleate can confer acute protection against cardiac ischemic injury, their mechanism of action is unclear. Mild uncoupling of mitochondria is known to be cardioprotective, and adenine nucleotide translocase 1 (ANT1) is a key mediator of mitochondrial uncoupling. ANT1 also contains redox-sensitive cysteines that may be targets for modification by nitroalkenes. Therefore, in this study we tested the hypothesis that nitroalkenes directly modify ANT1 and that nitroalkene-mediated cardioprotection requires ANT1. Using biotin-tagged LNO2 infused into intact perfused hearts, we obtained mass spectrometric (MALDI-TOF-TOF) evidence for direct modification (nitroalkylation) of ANT1 on cysteine 57. Furthermore, in a cell model of ischemia-reperfusion injury, siRNA knockdown of ANT1 inhibited the cardioprotective effect of LNO2. Although the molecular mechanism linking ANT1-Cys57 nitroalkylation and uncoupling is not yet known, these data suggest that ANT1-mediated uncoupling may be a mechanism for nitroalkene-induced cardioprotection. Electrophilic nitrated lipids (nitroalkenes) are emerging as an important class of protective cardiovascular signaling molecules. Although species such as nitro-linoleate (LNO2) and nitro-oleate can confer acute protection against cardiac ischemic injury, their mechanism of action is unclear. Mild uncoupling of mitochondria is known to be cardioprotective, and adenine nucleotide translocase 1 (ANT1) is a key mediator of mitochondrial uncoupling. ANT1 also contains redox-sensitive cysteines that may be targets for modification by nitroalkenes. Therefore, in this study we tested the hypothesis that nitroalkenes directly modify ANT1 and that nitroalkene-mediated cardioprotection requires ANT1. Using biotin-tagged LNO2 infused into intact perfused hearts, we obtained mass spectrometric (MALDI-TOF-TOF) evidence for direct modification (nitroalkylation) of ANT1 on cysteine 57. Furthermore, in a cell model of ischemia-reperfusion injury, siRNA knockdown of ANT1 inhibited the cardioprotective effect of LNO2. Although the molecular mechanism linking ANT1-Cys57 nitroalkylation and uncoupling is not yet known, these data suggest that ANT1-mediated uncoupling may be a mechanism for nitroalkene-induced cardioprotection. IntroductionMitochondria play a central role in cardiac ischemia-reperfusion (IR) 3The abbreviations used are: IRischemia-reperfusionLAlinoleic acidOAoleic acidLNO2nitro-linoleateBt-LNO2biotinylated LNO2OANO2nitro-oleateANT1adenine nucleotide translocase 1IPCischemic preconditioningPT porepermeability transition poreTMREtetramethylrhodamine ethylCtrlcontrolCHCAα-cyano-4-hydroxycinnaminic acid. injury (1Halestrap A.P. Mitochondria and reperfusion injury of the heart-a holey death but not beyond salvation.J. Bioenerg. Biomembr. 2009; 41: 113-121Crossref PubMed Scopus (95) Google Scholar). IR severely impairs mitochondrial function, causing inhibition of Ox-Phos (2Chen Q. Moghaddas S. Hoppel C.L. Lesnefsky E.J. Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria.Am. J. Physiol. Cell Physiol. 2008; 294: C460-C466Crossref PubMed Scopus (242) Google Scholar), enhanced ROS generation, and Ca2+ dysregulation (3Brookes P.S. Yoon Y. Robotham J.L. Anders M.W. Sheu S.S. Calcium, ATP, and ROS. A mitochondrial love-hate triangle.Am. J. Physiol. Cell Physiol. 2004; 287: C817-C833Crossref PubMed Scopus (1877) Google Scholar), which in combination with high inorganic phosphate and low ATP promote opening of the mitochondrial permeability transition (PT) pore (4Di Lisa F. Bernardi P. A CaPful of mechanisms regulating the mitochondrial permeability transition.J. Mol. Cell. Cardiol. 2009; 46: 775-780Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The latter is a key factor in IR-induced cell death (5Halestrap A.P. A pore way to die. The role of mitochondria in reperfusion injury and cardioprotection.Biochem. Soc. Trans. 2010; 38: 841-860Crossref PubMed Scopus (243) Google Scholar). The composition of the PT pore is still a matter of debate, and whereas a role for cyclophilin D is well defined (6Baines C.P. Kaiser R.A. Purcell N.H. Blair N.S. Osinska H. Hambleton M.A. Brunskill E.W. Sayen M.R. Gottlieb R.A. Dorn G.W. Robbins J. Molkentin J.D. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death.Nature. 2005; 434: 658-662Crossref PubMed Scopus (1808) Google Scholar), opposing data exist regarding involvement of the adenine nucleotide translocase (ANT) (7Brustovetsky N. Klingenberg M. Mitochondrial ADP/ATP carrier can be reversibly converted into a large channel by Ca2+.Biochemistry. 1996; 35: 8483-8488Crossref PubMed Scopus (399) Google Scholar, 8Kokoszka J.E. Waymire K.G. Levy S.E. Sligh J.E. Cai J. Jones D.P. MacGregor G.R. Wallace D.C. The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore.Nature. 2004; 427: 461-465Crossref PubMed Scopus (891) Google Scholar). In addition to its potential role in the PT pore and cell death regulation (7Brustovetsky N. Klingenberg M. Mitochondrial ADP/ATP carrier can be reversibly converted into a large channel by Ca2+.Biochemistry. 1996; 35: 8483-8488Crossref PubMed Scopus (399) Google Scholar, 9Baines C.P. Molkentin J.D. Adenine nucleotide translocase-1 induces cardiomyocyte death through up-regulation of the pro-apoptotic protein Bax.J. Mol. Cell. Cardiol. 2009; 46: 969-977Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 10Klingenberg M. The ADP and ATP transport in mitochondria and its carrier.Biochim. Biophys. Acta. 2008; 1778: 1978-2021Crossref PubMed Scopus (439) Google Scholar), ANT plays essential roles in mitochondrial bioenergetics (10Klingenberg M. The ADP and ATP transport in mitochondria and its carrier.Biochim. Biophys. Acta. 2008; 1778: 1978-2021Crossref PubMed Scopus (439) Google Scholar) and ANT1 (the major cardiac isoform (11Levy S.E. Chen Y.S. Graham B.H. Wallace D.C. Expression and sequence analysis of the mouse adenine nucleotide translocase 1 and 2 genes.Gene. 2000; 254: 57-66Crossref PubMed Scopus (78) Google Scholar)) is known to mediate a basal proton leak in mitochondria (12Brand M.D. Pakay J.L. Ocloo A. Kokoszka J. Wallace D.C. Brookes P.S. Cornwall E.J. The basal proton conductance of mitochondria depends on adenine nucleotide translocase content.Biochem. J. 2005; 392: 353-362Crossref PubMed Scopus (274) Google Scholar, 13Cadenas S. Buckingham J.A. St-Pierre J. Dickinson K. Jones R.B. Brand M.D. AMP decreases the efficiency of skeletal-muscle mitochondria.Biochem. J. 2000; 351: 307-311Crossref PubMed Scopus (50) Google Scholar). Mild increases in mitochondrial proton leak (uncoupling) can confer protection against IR injury (14Brennan J.P. Southworth R. Medina R.A. Davidson S.M. Duchen M.R. Shattock M.J. Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of KATP channel activation.Cardiovasc. Res. 2006; 72: 313-321Crossref PubMed Scopus (178) Google Scholar, 15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar, 16Quarrie R. Cramer B.M. Lee D.S. Steinbaugh G.E. Erdahl W. Pfeiffer D.R. Zweier J.L. Crestanello J.A. Ischemic preconditioning decreases mitochondrial proton leak and reactive oxygen species production in the post-ischemic heart.J. Surg. Res. 2011; 165: 5-14Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and this raises the possibility that ANT may play a role in cardioprotection.Nitroalkenes, such as nitro-linoleic acid (LNO2) and nitro-oleic acid (OANO2), are electrophiles that trigger a variety of intracellular signaling events (17Groeger A.L. Freeman B.A. Signaling actions of electrophiles. Anti-inflammatory therapeutic candidates.Mol. Interv. 2010; 10: 39-50Crossref PubMed Scopus (52) Google Scholar, 18Baker P.R. Schopfer F.J. O'Donnell V.B. Freeman B.A. Convergence of nitric oxide and lipid signaling. Anti-inflammatory nitro-fatty acids.Free Radic. Biol. Med. 2009; 46: 989-1003Crossref PubMed Scopus (104) Google Scholar). The mechanisms of endogenous nitroalkene formation are poorly understood, but several factors present in mitochondria are thought to be important (19Koenitzer J.R. Freeman B.A. Redox signaling in inflammation. Interactions of endogenous electrophiles and mitochondria in cardiovascular disease.Ann. N.Y. Acad. Sci. 2010; 1203: 45-52Crossref PubMed Scopus (70) Google Scholar). This includes a large mitochondrial pool of unsaturated membrane lipids (20Daum G. Lipids of mitochondria.Biochim. Biophys. Acta. 1985; 822: 1-42Crossref PubMed Scopus (701) Google Scholar), a ready availability of nitrite (21Rodriguez J. Maloney R.E. Rassaf T. Bryan N.S. Feelisch M. Chemical nature of nitric oxide storage forms in rat vascular tissue.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 336-341Crossref PubMed Scopus (187) Google Scholar), and elevated levels of reactive oxygen/nitrogen species (22Brookes P.S. Levonen A.L. Shiva S. Sarti P. Darley-Usmar V.M. Mitochondria. Regulators of signal transduction by reactive oxygen and nitrogen species.Free Radic. Biol. Med. 2002; 33: 755-764Crossref PubMed Scopus (252) Google Scholar). The conditions encountered during ischemia, including acidosis, may promote endogenous nitroalkene formation (23O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Krishna N.R. Kirk M. Barnes S. Darley-Usmar V.M. Freeman B.A. 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: 83-92Crossref PubMed Scopus (244) Google Scholar, 24Rudolph V. Rudolph T.K. Schopfer F.J. Bonacci G. Woodcock S.R. Cole M.P. Baker P.R. Ramani R. Freeman B.A. Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischemia and reperfusion.Cardiovasc. Res. 2010; 85: 155-166Crossref PubMed Scopus (149) Google Scholar). However, far from being pathologic in nature, nitroalkenes are thought to play an important role in the endogenous cardioprotective paradigm of ischemic preconditioning (IPC) (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar). Formation of LNO2 has been shown in mitochondria isolated from perfused hearts subjected to IPC (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar). Furthermore, prior administration of synthetic nitroalkenes prevented IR injury in both in vivo hearts and isolated cardiomyocytes (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar, 24Rudolph V. Rudolph T.K. Schopfer F.J. Bonacci G. Woodcock S.R. Cole M.P. Baker P.R. Ramani R. Freeman B.A. Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischemia and reperfusion.Cardiovasc. Res. 2010; 85: 155-166Crossref PubMed Scopus (149) Google Scholar).One mechanism of nitroalkene signaling is the nitroalkylation of nucleophilic protein residues such as cysteines (25Batthyany C. Schopfer F.J. Baker P.R. Durán R. Baker L.M. Huang Y. Cerveñansky C. Branchaud B.P. Freeman B.A. Reversible post-translational modification of proteins by nitrated fatty acids in vivo.J. Biol. Chem. 2006; 281: 20450-20463Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). We previously obtained immunoblot data strongly supportive of ANT1 nitroalkylation in isolated mitochondria and cardiomyocytes. This was accompanied by an increase in mitochondrial proton leak and by potent cytoprotection against simulated IR injury (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar). ANT1 is an attractive candidate for cardioprotective nitroalkene signaling, as it contains a number of redox active cysteines (26Lin T.K. Hughes G. Muratovska A. Blaikie F.H. Brookes P.S. Darley-Usmar V. Smith R.A. Murphy M.P. Specific modification of mitochondrial protein thiols in response to oxidative stress. A proteomics approach.J. Biol. Chem. 2002; 277: 17048-17056Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) that have been shown to play critical roles in adenine nucleotide binding (27Halestrap A.P. Woodfield K.Y. Connern C.P. Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase.J. Biol. Chem. 1997; 272: 3346-3354Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar) and opening of the PT pore (28McStay G.P. Clarke S.J. Halestrap A.P. 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: 541-548Crossref PubMed Scopus (307) Google Scholar, 29Vyssokikh M.Y. Katz A. Rueck A. Wuensch C. Dörner A. Zorov D.B. Brdiczka D. Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.Biochem. J. 2001; 358: 349-358Crossref PubMed Scopus (83) Google Scholar). Notably, however, the role of these cysteines in regulating proton conductance by ANT1 is unknown. Furthermore, a direct role for ANT1 in the acute cardioprotective effect of nitroalkenes has not been established. Therefore, the goal of this study was to determine the requirement for ANT1 in nitroalkene-mediated cardioprotection.RESULTSAn acute cardioprotective effect of nitroalkenes was demonstrated in adult cardiomyocytes (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar). Furthermore, a long term in vivo treatment with nitroalkenes in mice conferred protection against IR injury. However, the ability of nitroalkenes to afford acute protection at the isolated heart level has not been investigated. Fig. 1 demonstrates that nitroalkenes significantly improved cardiac contractile function (rate × pressure product) during reperfusion and drastically decreased infarct size compared with vehicle treated controls. Both LNO2 and OANO2 provided equal magnitudes of protection, and regular non-nitrated LA and OA were not protective, indicating that the small doses used herein (100 nm) were not sufficient to provide any cardioprotective effect of free fatty acids, as has been observed in other studies (34Al-Khalifa A. Maddaford T.G. Chahine M.N. Austria J.A. Edel A.L. Richard M.N. Ander B.P. Gavel N. Kopilas M. Ganguly R. Ganguly P.K. Pierce G.N. Effect of dietary hempseed intake on cardiac ischemia-reperfusion injury.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 292: R1198-R1203Crossref PubMed Scopus (32) Google Scholar, 35Leprán I. Szekeres L. Effect of dietary sunflower seed oil on the severity of reperfusion-induced arrhythmias in anesthetized rats.J. Cardiovasc. Pharmacol. 1992; 19: 40-44Crossref PubMed Scopus (22) Google Scholar). Together these data support the cardioprotective efficacy of nitroalkenes against IR injury.The time-frame for this effect (15 min. infusion) indicates that the mechanism of protection was most likely via protein nitroalkylation rather than at the level of transcriptional activity. To investigate protein modifications by LNO2 in the perfused heart, Bt-LNO2 was synthesized. Bt-LNO2 was administered in the same manner as LNO2 and exhibited the same degree of protection against IR injury (Figs. 1, A and C). These data indicate that the addition of the biotin group did not interfere with the bioactivity of LNO2, suggesting that the active portion of the LNO2 molecule is not the biotin-blocked carboxyl group.Based on our previous studies (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar), we focused next on the nitroalkene modification of ANT1. After Bt-LNO2 delivery to perfused hearts, homogenates were subjected to immunoprecipitation with either anti-ANT1 antibodies or neutravidin to capture ANT1 and other biotin-tagged proteins followed by Western blotting. Fig. 2 shows that Bt-LNO2 adducted several proteins, including ANT1, suggesting that nitroalkenes can cross the vascular and plasma membranes and enter mitochondria. This is in agreement with our previous results (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar) where Bt-LNO2 modified ANT1 in both isolated mitochondria and intact adult cardiomyocytes. Although the modification site was not elucidated, we demonstrated that pretreatment with the ANT inhibitor carboxyatractyloside abrogated Bt-LNO2 modification (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar). Because carboxyatractyloside is known to block the accessibility of a cysteine residue in ANT (Cys57), we therefore speculated this may be a site of LNO2 modification.FIGURE 2ANT1 modification by Bt-LNO2. A, ANT1 was immunoprecipitated from whole heart homogenates (Ctrl or treated with 100 nm Bt-LNO2) using anti-ANT1 antibodies (see “Experimental Procedures”). The first two lanes are inputs (i.e. intact homogenate), and the next two lanes are immunoprecipitated (IP) pellets. Upper and lower panels correspond to immunoblots (WB) for biotin and ANT1, respectively. At least two independent experiments were performed for each condition. B, biotinylated proteins were immunoprecipitated from whole heart homogenates using neutravidin. The left panel represents immunoblot for ANT1, and the right panel is the corresponding Coomassie Blue (CB)-stained gel. Four bands at >250, 80, 55, and 32 kDa (highlighted by dotted lines) were analyzed by mass spectrometry (MALDI-TOF) and are identified as labeled in the figure. Mass spectrometry of the gel band at 32 kDa corresponding to ANT1 is shown in supplemental Fig. S1B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To check if any of the four known cysteines in ANT were modified in perfused heart after Bt-LNO2 treatment, cardiac homogenates were immunoprecipitated using neutravidin beads. The immunoprecipitate pellet was separated on duplicate SDS-PAGE gels. One gel was transferred and probed for ANT1 to verify successful nitroalkylation and pulldown of the Bt-LNO2-ANT1 complex (Fig. 2B, left panel). The second gel was stained (Fig. 2B, right panel), and the prominent bands at >250, 80, 55, and 32 kDa were excised and subjected to mass spectrometric identification. The >250-kDa band could not be identified and is likely a nonspecific protein aggregate. The band at 80 kDa was the α-subunit of propionyl CoA-carboxylase, a biotin-containing enzyme that often contaminates biotin detection systems. The 55-kDa band was the α-subunit of ATP synthase (see “Discussion”).The 32-kDa band was identified as ANT1, with 80% sequence coverage, including all four cysteine residues (supplemental Fig. S1). The confidence of identification was above >98 for at least 5 peptides. Further MS/MS analysis (Fig. 3) revealed that a peptide at m/z 1438.8 containing Cys57 was modified by Bt-LNO2, as indicated by a mass shift of 564 Da corresponding to the molecular mass of Bt-LNO2 (supplemental Fig. S2). These data are the first identification of a nitroalkylation site in ANT1.FIGURE 3Detection of Bt-LNO2-Cys adduct site in ANT1. The MS/MS spectrum of 1438.8 m/z ion was obtained in positive reflector mode fitted with peptide 53GIIDC*VVR60 from a ANT1 mouse sequence (gi:148747424) modified at Cys57 by Bt-LNO2. Colored numbers represent masses found in MS/MS with mass error less than 0.3 ppm. Collision of biotinylated nitro-lipid-modified peptides during MS/MS produces multiple products of nitrolipid side-chain breakage, which PEAKS software cannot take into account. We performed manual assignment of the four major peaks on the MS/MS; 1136.931 corresponds to the y5-H2O ion, 1393.184 corresponds to decarboxylated metastable ion, 1279.124 corresponds to metastable ion with water and biotin ring loss, 1311.005 corresponds to y7 ion with C5H10 neutral loss from nitro-lipid side chain. Supplemental Table S1 shows the full list of b and y ions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Although supraphysiological doses of electrophiles have previously been shown to modify mitochondrial proteins (36Chavez J.D. Wu J. Bisson W. Maier C.S. Site-specific proteomic analysis of lipoxidation adducts in cardiac mitochondria reveals chemical diversity of 2-alkenal adduction.J. Proteomics. 2011; 74: 2417-2429Crossref PubMed Scopus (56) Google Scholar, 37Wong H.L. Liebler D.C. Mitochondrial protein targets of thiol-reactive electrophiles.Chem. Res. Toxicol. 2008; 21: 796-804Crossref PubMed Scopus (68) Google Scholar) and modification of ANT cysteines has been shown using various thiol reagents (38Girón-Calle J. Zwizinski C.W. Schmid H.H. Peroxidative damage to cardiac mitochondria. II. Immunological analysis of modified adenine nucleotide translocase.Arch. Biochem. Biophys. 1994; 315: 1-7Crossref PubMed Scopus (35) Google Scholar), to the best of our knowledge this is the first ever in situ identification of a cysteine modification in ANT by a low dose (100 nm) of a physiologically relevant (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar, 39Baker P.R. Schopfer F.J. Sweeney S. Freeman B.A. Red cell membrane and plasma linoleic acid nitration products. Synthesis, clinical identification, and quantitation.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11577-11582Crossref PubMed Scopus (177) Google Scholar) electrophile, delivered to an intact tissue. Notably, the levels of LNO2 found in mitochondria in preconditioned hearts are in the micromolar range, suggesting they may be capable of nitro-alkylating ANT in situ, although preliminary studies in this area 4S. M. Nadtochiy and P. S. Brookes, unpublished information. have so far failed to detect nitroalkylation of ANT in hearts exposed to IPC.To investigate if ANT1 was required for LNO2-mediated protection, we employed an in vitro model of IR injury. ANT1 was knocked down by siRNA in the H9c2 cardiomyocyte cell line. Fig. 4A demonstrates that 72 h post-transfection, ANT1 levels were 35% of control. The levels of another mitochondrial protein (70-kDa subunit of respiratory complex II) did not change, and a scrambled siRNA control was without effect.FIGURE 4ANT1 knock down abolishes effects of LNO2 in H9c2 cells. A, knockdown of ANT1 in H9c2 cells is shown. 72 h post-transfection cells (Ctrl and ANT1 siRNA) were harvested for immunoblotting analysis using anti-ANT1, anti-70 kDa complex II, and anti-actin antibodies. A blot image representative of four independent experiments is shown. Densitometry of ANT1, 70-kDa complex II (CX II), and actin bands was performed using Scion Image™ software. Relative ANT1 expression (in siRNA cells versus controls) is shown below the blot (means ± S.E., n = 4). B, is shown LDH release upon simulated IR injury. Transfected cells were subjected to simulated IR (see the “Experimental Procedures”). LDH release (cell death) is expressed as a percentage of total LDH (upon lysis with Triton X-100). Data are the means ± S.E., n = 7. *, p < 0.05 versus Ctrl siRNA; #, p < 0.05 versus Ctrl siRNA + LNO2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To verify the metabolic effects of ANT1 knockdown in cells, we employed extracellular flux analysis (40Gerencser A.A. Neilson A. Choi S.W. Edman U. Yadava N. Oh R.J. Ferrick D.A. Nicholls D.G. Brand M.D. Quantitative microplate-based respirometry with correction for oxygen diffusion.Anal. Chem. 2009; 81: 6868-6878Crossref PubMed Scopus (243) Google Scholar) and TMRE spectrofluorimetry (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar). ANT1 knockdown caused an ∼30% decrease in basal respiration rate (124 ± 20 pmol/min in ANT1 siRNA versus 177 ± 26 pmol/min in Ctrl siRNA, n = 7, p = 0.001), likely due to the known bioenergetic role of ANT1. To assess mitochondrial uncoupling (proton leak), respiration in the presence of oligomycin (enforced state 4) was used as a surrogate marker, as previously described (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar, 41Bosetti F. Baracca A. Lenaz G. Solaini G. Increased state 4 mitochondrial respiration and swelling in early post-ischemic reperfusion of rat heart.FEBS Lett. 2004; 563: 161-164Crossref PubMed Scopus (46) Google Scholar, 42Muscari C. Bonafè F. Gamberini C. Giordano E. Lenaz G. Caldarera C.M. Ischemic preconditioning preserves proton leakage from mitochondrial membranes but not oxidative phosphorylation during heart reperfusion.Cell Biochem. Funct. 2006; 24: 511-518Crossref PubMed Scopus (7) Google Scholar). ANT1 knockdown caused a significant decrease in state 4 respiration (37 ± 5 pmol/min in ANT1 siRNA versus 51 ± 6 pmol/min in Ctrl siRNA, n = 7, p = 0.001), consistent with a role for ANT1 in basal proton leak (12Brand M.D. Pakay J.L. Ocloo A. Kokoszka J. Wallace D.C. Brookes P.S. Cornwall E.J. The basal proton conductance of mitochondria depends on adenine nucleotide translocase content.Biochem. J. 2005; 392: 353-362Crossref PubMed Scopus (274) Google Scholar). Furthermore, although the addition of LNO2 caused a small but significant increase in state 4 respiration in control cells (51 ± 6 pmol/min → 61 ± 9 pmol/min, n = 7, p = 0.02), LNO2 was without effect in ANT1 siRNA cells (37 ± 5 pmol/min → 41 ± 6 pmol/min, n = 7, p = 0.185) (supplemental Fig. S3). To further characterize proton leak, mitochondrial membrane potential was also measured in H9c2 cells using TMRE, normalizing the signal to MitoTracker Green. Consistent with the respiration data, in oligomycin-enforced state 4, LNO2 addition caused a significant decrease in Δψ in control cells (TMRE/MitoTracker Green signal 1439 ± 124 → 900 ± 80, p = 0.021) but not in ANT1 knock-down cells (TMRE/MitoTracker Green signal 1456 ± 371 → 1017 ± 216, p = 0.365). Together, these data indicate that the ability of LNO2 to induce mitochondrial proton leak is partly dependent on ANT1.Finally, because a mild induction of mitochondrial proton leak can elicit cardioprotection (14Brennan J.P. Southworth R. Medina R.A. Davidson S.M. Duchen M.R. Shattock M.J. Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of KATP channel activation.Cardiovasc. Res. 2006; 72: 313-321Crossref PubMed Scopus (178) Google Scholar, 43Teshima Y. Akao M. Jones S.P. Marbán E. Uncoupling protein-2 overexpression inhibits mitochondrial death pathway in cardiomyocytes.Circ. Res. 2003; 93: 192-200Crossref PubMed Scopus (262) Google Scholar) and we previously suggested this may be a mechanism of LNO2-induced protection (15Nadtochiy S.M. Baker P.R. Freeman B.A. Brookes P.S. Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection.Cardiovasc. Res. 2009; 82: 333-340Crossref PubMed Scopus (104) Google Scholar), we sought to investigate whether knocking down ANT1 would also block LNO2 protection. Fig. 4B shows that pretreatment with LNO2 resulted i" @default.
• W2064096645 created "2016-06-24" @default.
• W2064096645 creator A5008948496 @default.
• W2064096645 creator A5014989179 @default.
• W2064096645 creator A5016644607 @default.
• W2064096645 creator A5060599255 @default.
• W2064096645 creator A5062302454 @default.
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• W2064096645 date "2012-01-01" @default.
• W2064096645 modified "2023-09-26" @default.
• W2064096645 title "Nitroalkenes Confer Acute Cardioprotection via Adenine Nucleotide Translocase 1" @default.
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