Cell-permeable Peptide Antioxidants Targeted to Inner Mitochondrial Membrane inhibit Mitochondrial Swelling, Oxidative Cell Death, and Reperfusion Injury

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress. Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N2A cells (EC50 in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca2+ in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress. The mitochondrial respiratory chain on the inner mitochondrial membrane (IMM) 1The abbreviations used are: IMM, inner mitochondrial membrane; ROS, reactive oxygen species; 3NP, 3-nitropropionic acid; OMM, outer mitochondrial membrane; MPT, mitochondrial permeability transition; tBHP, t-butylhydroperoxide; Dmt, 2′,6′-dimethyltyrosine; LDL, low density lipoprotein; FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; CLSM, confocal laser scanning microscopy; TMRM, tetramethylrhodamine methyl ester. is a major intracellular source of reactive oxygen species (ROS). ROS cause nonspecific damage to lipids, proteins, and DNA, leading to alteration or loss of cellular function. Mitochondria are continuously exposed to ROS and accumulate oxidative damage more rapidly than the rest of the cell, especially because ROS are highly reactive and shortlived (1Kowaltowski A.J. Vercesi A.E. Free Radic. Biol. Med. 1999; 26: 463-471Crossref PubMed Scopus (706) Google Scholar). Many studies have associated mitochondrial dysfunction caused by ROS with both necrotic and apoptotic cell death (2Zamzami N. Hirsch T. Dallaporta B. Petit P.X. Kroemer G. J. Bioenerg. Biomembr. 1997; 29: 185-193Crossref PubMed Scopus (304) Google Scholar). The rate of mitochondrial ROS production can be altered by several physiological or pathological conditions. Inhibitors of the respiratory chain such as 3-nitropropionic acid (3NP), an irreversible inhibitor of the complex II enzyme succinate dehydrogenase, tend to increase ROS production (3Beal M.F. Ferrante R.J. Henshaw R. Matthews R.T. Chan P.H. Kowall N.W. Epstein C.J. Schulz J.B. J. Neurochem. 1995; 65: 919-922Crossref PubMed Scopus (157) Google Scholar, 4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar). The inhibition of this complex seems to be related to neuronal death similar to those occurring in Huntington's disease (6Beal M.F. Brouillet E. Jenkins B.G. Ferrante R.J. Kowall N.W. Miller J.M. Storey E. Srivastava R. Rosen B.R. Hyman B.T. J. Neurosci. 1993; 13: 4181-4192Crossref PubMed Google Scholar), and antioxidants can attenuate the neurochemical changes and some behavioral disturbances caused by 3NP in animals (5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar, 7Matthews R.T. Yang L. Browne S. Baik M. Beal M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8892-8897Crossref PubMed Scopus (515) Google Scholar). Mitochondrial Ca2+ is another powerful signal for ROS production. Calcium is taken up into mitochondria via a uniporter in the IMM, and elevation of mitochondrial Ca2+ and ROS production is thought to play an important part in cell death associated with ischemia-reperfusion as well as 3NP (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar). Increasing evidence suggests that ROS play a key role in promoting cytochrome c release from the mitochondria (8Petrosillo G. Ruggiero F.M. Pistolese M. Paradies G. FEBS Lett. 2001; 509: 435-438Crossref PubMed Scopus (204) Google Scholar, 9Nishimura G. Proske R.J. Doyama H. Higuchi M. FEBS Lett. 2001; 505: 399-404Crossref PubMed Scopus (39) Google Scholar, 10Petrosillo G. Ruggiero F.M. Paradies G. FASEB J. 2003; 17: 2202-2208Crossref PubMed Scopus (307) Google Scholar, 11Galindo M.F. Jordan J. Gonzalez-Garcia C. Cena V. Br. J. Pharmacol. 2003; 139: 797-804Crossref PubMed Scopus (68) Google Scholar), and cytochrome c in the cytoplasm triggers activation of the caspase cascade that ultimately leads to apoptosis (12Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4463) Google Scholar, 13Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6239) Google Scholar). The mechanism underlying ROS-mediated cytochrome c release from mitochondria is still not fully understood. Cytochrome c is normally bound to the IMM by an association with cardiolipin (14Nicholls P. Biochim. Biophys. Acta. 1974; 346: 261-310Crossref PubMed Scopus (178) Google Scholar). It is now believed that cytochrome c release from mitochondria proceeds by a two-step process: dissociation of cytochrome c from cardiolipin in the IMM, followed by release of cytochrome c through the outer mitochondrial membrane (OMM) (15Ott M. Robertson J.D. Gogvadze V. Zhivotovsky B. Orrenius S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1259-1263Crossref PubMed Scopus (792) Google Scholar). Cardiolipin is rich in unsaturated fatty acids, and peroxidation of cardiolipin induces the dissociation of cytochrome c from mitochondria into the cytosol (16Shidoji Y. Hayashi K. Komura S. Ohishi N. Yagi K. Biochem. Biophys. Res. Commun. 1999; 264: 343-347Crossref PubMed Scopus (222) Google Scholar). However, the mechanism by which cytochrome c is released through the OMM is not clear. One mechanism may involve ROS-induced promotion of Ca2+-dependent mitochondrial permeability transition (MPT), with swelling of the mitochondrial matrix and rupture of the OMM (17Byrne A.M. Lemasters J.J. Nieminen A.L. Hepatology. 1999; 29: 1523-1531Crossref PubMed Scopus (121) Google Scholar, 18Kowaltowski A.J. Castilho R.F. Vercesi A.E. FEBS Lett. 2001; 495: 12-15Crossref PubMed Scopus (714) Google Scholar). ROS may promote MPT by causing oxidation of thiol groups on the adenine nucleotide translocator (19Costantini P. Belzacq A.S. Vieira H.L. Larochette N. de Pablo M.A. Zamzami N. Susin S.A. Brenner C. Kroemer G. Oncogene. 2000; 19: 307-314Crossref PubMed Scopus (261) Google Scholar, 20Vieira H.L. Belzacq A.S. Haouzi D. Bernassola F. Cohen I. Jacotot E. Ferri K.F. El Hamel C. Bartle L.M. Melino G. Brenner C. Goldmacher V. Kroemer G. Oncogene. 2001; 20: 4305-4316Crossref PubMed Scopus (228) Google Scholar, 21Kanno T. Sato E.E. Muranaka S. Fujita H. Fujiwara T. Utsumi T. Inoue M. Utsumi K. Free Radic. Res. 2004; 38: 27-35Crossref PubMed Scopus (114) Google Scholar). This mechanism seems likely in 3NP toxicity and ischemia-reperfusion injury, where increased intracellular Ca2+ and ROS are both present (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar, 22Crompton M. Andreeva L. Basic Res. Cardiol. 1993; 88: 513-523Crossref PubMed Scopus (80) Google Scholar, 23Paradies G. Petrosillo G. Pistolese M. Di Venosa N. Serena D. Ruggiero F.M. Free Radic. Biol. Med. 1999; 27: 42-50Crossref PubMed Scopus (205) Google Scholar). However, there is also evidence showing that cytochrome c can be released through the OMM in an MPT-independent manner (24Jurgensmeier J.M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1373) Google Scholar, 25Doran E. Halestrap A.P. Biochem. 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These peptide antioxidants are cell-permeable and are very intracellular ROS and cell death caused by the t-butylhydroperoxide We have used these IMM-targeted antioxidants to investigate the role of ROS in mitochondrial dysfunction in cells exposed to 3NP. investigate the by which these peptide antioxidants mitochondrial we used isolated mitochondria to to prevent MPT and cytochrome c release caused by Ca2+ and 3NP. In addition, because ROS have been implicated in contractile dysfunction associated with reperfusion of ischemic hearts, we the of these peptide antioxidants in myocardial stunning in an ex vivo heart model. to that the of these peptide antioxidants are caused by to ROS, we a peptide that properties. results that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT and rupture of the These results also a major role for ROS in mitochondrial dysfunction and reperfusion and demonstrate the of these peptide antioxidants in ischemia-reperfusion and and peptides are with alternating aromatic and basic amino and as I. S. G. C. J. Med. Chem. 2000; PubMed Scopus Google Scholar, X. J. Pharmacol. 2003; PubMed Scopus Google Scholar). acid in of the in for mitochondrial and cellular studies I. C. B. 2003; PubMed Scopus Google Scholar). from of of in of peptides to Y. H. M.A. Biochim. Biophys. 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J. 1999; 276: Google and of the intracellular of can be to be than of used to cellular and intracellular by The confocal of into cells in the cytoplasm of the peptide from the The mitochondrial as by The of and suggests of to mitochondria cellular Mitochondrial of of peptides isolated mitochondria. of isolated mitochondria to in of the signal that the of by mitochondria not an of the we mitochondria with and in the mitochondrial of by mitochondria with by in the mitochondrial and in the mitochondrial of S.A. M. Halestrap A.P. J. 2002; PubMed Scopus Google Scholar), can be that in mitochondria. of mitochondria with reduced or by and that of this peptide into the mitochondrial matrix in a mitochondria with for and the mitochondrial to of in the membrane of both IMM and OMM of the mitochondrial with to the OMM to that of the in the and These results that the peptides are to the IMM. ROS and by that peptides are to neuronal N2A cells treated with tBHP or for and then with or or for with tBHP in increase in intracellular ROS and in cell of these cells with or reduced intracellular ROS and increased cell and with in the 3NP-induced Mitochondrial in demonstrate that in ROS can mitochondrial we the of on mitochondrial depolarization caused by treatment of cells with 3NP. cells treated with 3NP in the or of and mitochondrial by confocal In cells the mitochondria are clearly as the In cells treated with 3NP the In treatment with mitochondrial depolarization caused by 3NP ROS by mitochondria then used to the of by these peptide reduced generation of in isolated mitochondria results with In to ROS and to inhibit ROS production induced by which not have on or induced ROS production by isolated mitochondria not of on Mitochondrial in of or in mitochondria did not mitochondrial function. mitochondria with or did not rate of oxygen or not Mitochondrial membrane as by TMRM, also not by or In the of caused and Ca2+ in loss of mitochondrial of MPT MPT in of isolated mitochondria with or of Ca2+ in depolarization of the mitochondria without the of MPT and of mitochondria with also the of MPT induced by 3NP However, which mitochondrial ROS production, on or 3NP-induced MPT not Mitochondrial in of isolated mitochondria with inhibited swelling as by in nm Mitochondrial swelling also inhibited by not by Ca2+ in the of cytochrome c from the mitochondrial to the The release of mitochondrial cytochrome c inhibited by and not in the of the isolated heart of is associated with loss of contractile force D. Y. J. 2002; PubMed Scopus Google Scholar). of contractile force with of the heart with the peptides or improved contractile force of reperfusion with In not to prevent the contractile dysfunction from providing for a major role for ROS in reperfusion These peptides and are the antioxidants that target and concentrate in the IMM, scavenging of ROS the of production. these peptide we to that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP. studies with isolated mitochondria also that ROS mediate cytochrome c release via MPT and rupture of the ROS production, these peptide antioxidants to prevent mitochondrial depolarization in cells exposed to 3NP. these peptide antioxidants to prevent myocardial stunning associated with reperfusion in the ischemic heart in an ex vivo model. The of which not have to prevent mitochondrial swelling or reperfusion that the of these peptides are via The structural motif of these peptides centers on alternating aromatic and basic amino The of peptides can be to the can which can be followed by to M. I. S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. K.F. Biochem. Pharmacol. PubMed Scopus Google Scholar), or scavenging by H. D. Biochem. Biophys. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). peptides have been to B. C. Mol. Pharmacol. 2002; PubMed Scopus Google Scholar, M. L. M.A. Biochem. Pharmacol. 2001; PubMed Scopus Google Scholar). We have that of the as in Dmt, structural to both have the of the amino acid on the scavenging of the of in with in loss of the peptides are and cell in a manner K. G. J. 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Bernassola F. Cohen I. Jacotot E. Ferri K.F. El Hamel C. Bartle L.M. Melino G. Brenner C. Goldmacher V. Kroemer G. Oncogene. 2001; 20: 4305-4316Crossref PubMed Scopus (228) Google Scholar, 21Kanno T. Sato E.E. Muranaka S. Fujita H. Fujiwara T. Utsumi T. Inoue M. Utsumi K. Free Radic. Res. 2004; 38: 27-35Crossref PubMed Scopus (114) Google Scholar). The of peptides to prevent MPT ROS and oxidative damage on mitochondria C. X. E. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). ROS production also a role in cell death caused by 3NP, an irreversible inhibitor of complex II of the respiratory The production of ROS seems to be from elevation of mitochondrial which can then to MPT, mitochondrial and cell death (4Lee W.T. Yin H.S. Shen Y.Z. Neuroscience. 2002; 112: 707-716Crossref PubMed Scopus (45) Google Scholar, 5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google Scholar). an inhibitor of MPT, can prevent mitochondrial depolarization and neuronal death caused by 3NP N. J.M. J. Neurosci. 2000; 20: PubMed Google Scholar). We to that peptides can prevent mitochondrial depolarization in cells treated with 3NP, and studies in isolated mitochondria that can inhibit MPT induced by 3NP. It that some 3NP-induced behavioral in by and with (5Rosenstock T.R. Carvalho A.C. Jurkiewicz A. Frussa-Filho R. Smaili S.S. J. Neurochem. 2004; 88: 1220-1228Crossref PubMed Scopus (105) Google this of that to be 3NP and is related to the of the highly E. Reactive oxygen species and MPT have been to play a major role in reperfusion M. Andreeva L. Basic Res. Cardiol. 1993; 88: 513-523Crossref PubMed Scopus (80) Google Scholar, Pharmacol. 2004; PubMed Scopus Google Scholar). reperfusion there is of contractile as myocardial stunning. However, the of oxygen to the treatment of the been because of the of antioxidants to cell B. D. N. E. PubMed Scopus Google Scholar). results the myocardial stunning. and to prevent stunning in the ex vivo heart reperfusion In which to the ischemic heart reperfusion stunning. We that can prevent myocardial stunning reperfusion D. Y. J. 2002; PubMed Scopus Google Scholar), with as the mechanism of The of to prevent stunning been in in M. K. and H. H. and may have myocardial and are reperfusion In we have cell-permeable peptide antioxidants that target the of ROS generation and mitochondrial function. results demonstrate that ROS play a major role in mitochondrial dysfunction induced by tBHP, and 3NP. These peptides may be beneficial in the treatment of aging and diseases associated with oxidative damage such as ischemia-reperfusion and We and for We for with the confocal and and for with the mitochondria by of of The N2A cells by of of The studies in the the and the of