Editorial Views  |   February 2008
Mito-controversies: Mitochondrial Permeability Transition Pore and Myocardial Reperfusion Injury
Author Affiliations & Notes
  • Hemal H. Patel, Ph.D.
  • Yasuo M. Tsutsumi, M.D., Ph.D.
  • David M. Roth, Ph.D., M.D.
  • *Department of Anesthesiology, University of California, San Diego, California. . †Department of Anesthesiology, University of California, San Diego, and Veterans Affairs San Diego Healthcare System, San Diego, California.
Article Information
Editorial Views / Cardiovascular Anesthesia
Editorial Views   |   February 2008
Mito-controversies: Mitochondrial Permeability Transition Pore and Myocardial Reperfusion Injury
Anesthesiology 2 2008, Vol.108, 182-184. doi:10.1097/01.anes.0000299750.34472.93
Anesthesiology 2 2008, Vol.108, 182-184. doi:10.1097/01.anes.0000299750.34472.93
POSTCONDITIONING, repetitive ischemia applied before reperfusion, protects against ischemia–reperfusion injury.1 Of the therapies proposed for protecting ischemic myocardium, postconditioning offers a significant clinical advantage; it obviates predicting when someone will have an ischemic attack. As such, mechanisms involved in postconditioning are of significant interest. In this issue of Anesthesiology, Jang et al.  2 demonstrate that ischemic postconditioning in the heart involves activation of δ-opioid receptors. Morphine, a mixed opioid agonist, produced postconditioning that was abolished by a δ-opioid receptor antagonist or pharmacologic opening (via  atractyloside) of the mitochondrial permeability transition pore (mPTP). The authors showed that morphine exposure in isolated cardiac myocytes produced nitric oxide and attenuated hydrogen peroxide oxidant stress–induced loss of the mitochondrial membrane potential (ΔΨm). The attenuation of ΔΨmproduced by morphine was sensitive to δ-opioid antagonism, a nonselective nitric oxide synthase inhibitor and an inhibitor of protein kinase G. The authors concluded that (1) postconditioning protects the heart by targeting the mPTP via  activation of δ-opioid receptors and (2) the ability of δ opioids to activate the nitric oxide–cyclic guanosine monophosphate–protein kinase G pathway may account for the effect of postconditioning on the mPTP. The authors are to be complimented for their original work regarding a role for δ-opioid signaling on the mPTP.
Mitochondria, a source of cellular adenosine triphosphate, are increasingly being implicated in cell survival and death signaling. mPTP opening leads to an increase in mitochondrial membrane permeability to small molecules and plays an integral role in regulating cytoprotection. The mPTP, a putative high conductance channel on the inner mitochondrial membrane, is thought to be the final end effector in cardiac myocyte protection and therefore an important therapeutic target for cardiac protective strategies.3,4 The molecular composition of the mPTP is controversial. The pore putatively is composed of the adenine nucleotide translocase on the inner mitochondrial membrane, voltage-dependent anion channel on the outer membrane, and cyclophilin D in the mitochondrial matrix. These components exist as individual entities that assemble into a complex in response to stress to form the mPTP.3 A benzodiazepine receptor, hexokinase, and creatine kinase also have been proposed as regulators of the pore. The role of adenine nucleotide translocase and voltage-dependent anion channel in forming the pore recently has been questioned. The adenine nucleotide translocase may act as a regulator of the mPTP pore, but not as a pore-forming unit of the complex.5 In addition, voltage-dependent anion channel knockout mice (voltage-dependent anion channels 1, 2, 3, 1/3, and 1/2/3) show stress-induced mPTP opening indistinguishable from wild-type mitochondria, questioning whether the voltage-dependent anion channel is an essential component of the pore.6 Cyclophilin D seems to be the only essential component of the mPTP described thus far.7 The authors treat the mPTP as one entity, not a multiprotein complex. It is unclear whether cardiac protective agents work to inhibit the opening of a preformed mPTP complex, inhibit a particular subunit of the complex, or inhibit the assembly or organization of the complex. Recent work showed that increased phosphorylation of glycogen synthase kinase-3β in a model of protection reduces the affinity of the adenine nucleotide translocase for cyclophilin D, suggesting that assembly of the complex is targeted by protective signals to limit mPTP opening.8 
A limitation of the current study deals with the indirect means by which mPTP opening was assessed. Tetramethylrhodamine ethyl ester, a dye used to measure ΔΨm, was used to infer mPTP opening. Although the literature largely agrees that mPTP opening can be inferred by loss of ΔΨm, they are not one in the same. A loss in ΔΨmcan be caused by factors other than mPTP opening (e.g.  , increased adenosine triphosphate demand). To circumvent this limitation, calcein acetoxymethylester–loaded cells in the presence of Co2+can be used.9 Calcein is loaded into cells and taken up by mitochondria. Residual calcein is quenched by Co2+. If a stress is induced to open the mPTP, calcein is released and fluorescence is lost; this is reversed by addition of cyclosporine A. Dual loading of cells with tetramethylrhodamine ethyl ester and calcein can measure both ΔΨmand mPTP opening simultaneously.10 In the whole heart, mPTP opening can be assessed by a method devised by Halestrap et al.  11,12 in which radioactive 2-deoxyglucose (3H-DOG) is loaded into cells and accumulates as a phosphate. Functioning mitochondria exclude the 3H-DOG, and opening of the mPTP allows accumulation of 3H-DOG in mitochondria, which can be assessed by isolating mitochondria in the presence of EGTA to trap mitochondrial 3H-DOG during isolation.
The authors also used atractyloside, a pharmacologic mPTP opener, to block ischemic and opioid postconditioning, leading to the conclusion that opioids impact mPTP opening to affect postconditioning. Because mPTP opening is proposed to be an end effector of protection, interventions that open the mPTP pharmacologically would be expected to abrogate cardiac protection induced by any form of preconditioning or postconditioning (i.e.  , ischemic or pharmacologic). Atractyloside would not implicate a role for protective agents in the modulation of mPTP. Therefore, use of atractyloside does not address the role of mPTP in protection, but shows that mPTP opening is responsible for tissue injury. In addition, it has been reported that atractyloside not only opens mPTP but also inhibits adenosine diphosphate transport by inhibition of adenine nucleotide translocase,13 therefore limiting oxidative phosphorylation. As such, it would be difficult to differentiate the effects of atractyloside on mPTP opening versus  loss of energy production as causative factors in attenuated protection. Therefore, to assess whether protective agents use mPTP as a downstream mechanism, future studies should directly test whether these agents impact mPTP opening in response to stress.
The role of nitric oxide and/or reactive oxygen species in postconditioning events is intriguing, especially with respect to their impact on the mPTP. We and others have shown a role for reactive oxygen species in triggering postconditioning induced by ischemia, isoflurane, and the δ-opioid agonist SNC-121.14,15 There is evidence that reactive oxygen species may impact downstream mediators of protection (e.g.  , mitochondrial adenosine triphosphate–sensitive potassium channels)16; however, the nature of the reactive species generated and the role in inducing protection is under debate. During severe ischemia–reperfusion injury, overproduction of reactive oxygen species and mitochondrial Ca2+overload produce mPTP opening, ΔΨmdepolarization, inhibition of adenosine triphosphate production, mitochondrial swelling, additional reactive oxygen species production, and further Ca2+accumulation, all of which initiate mitochondrial dysfunction. However, reactive oxygen species at low concentrations can initiate signaling cascades that preserve mitochondrial integrity and myocardium during ischemia and reperfusion. The finding by Jang et al.  2 that morphine generates nitric oxide and that changes in ΔΨmare sensitive to nitric oxide synthase inhibition may provide a potential mechanism of action for opioids in ischemic postconditioning and implicate nitric oxide as the triggering reactive species. Low levels of endogenous nitric oxide and low concentrations of nitric oxide donors (<1 μm) protect mitochondria via  suppression of mPTP opening, whereas high concentrations of nitric oxide (>5 μm) can produce mPTP opening and cytochrome c  release.17 The fact that nitric oxide could be detected by fluorescence microscopy (a relatively insensitive technique) begs the question: Was this a small or large concentration of nitric oxide, and what does it mean to downstream protection? The dilemma seems to be that if a reactive species is easily detectable, the level likely is high and potentially injurious, whereas if it is low, it may be a beneficial trigger to cytoprotective signaling but evade detection.
The current study by Jang et al.  2 has focused welcomed attention on a possible role for δ-opioids in the modulation of the mPTP. Future studies will need to focus on the effects of modulators of preconditioning and postconditioning directly on the mPTP at the cellular and molecular levels. Resolution of these mito-controversies will add important information to our understanding of anesthetics as cytoprotective drugs and potential therapeutics for ischemia–reperfusion injury.
*Department of Anesthesiology, University of California, San Diego, California. . †Department of Anesthesiology, University of California, San Diego, and Veterans Affairs San Diego Healthcare System, San Diego, California.
Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten-Johansen J: Inhibition of myocardial injury by ischemic postconditioning during reperfusion: Comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003; 285:H579–88Zhao, ZQ Corvera, JS Halkos, ME Kerendi, F Wang, NP Guyton, RA Vinten-Johansen, J
Jang Y, Xi J, Wang H, Mueller RA, Norfleet EA, Xu Z: Postconditioning prevents reperfusion injury by activating δ-opioid receptors. Anesthesiology 2008; 108:243–50Jang, Y Xi, J Wang, H Mueller, RA Norfleet, EA Xu, Z
Javadov S, Karmazyn M: Mitochondrial permeability transition pore opening as an endpoint to initiate cell death and as a putative target for cardioprotection. Cell Physiol Biochem 2007; 20:1–22Javadov, S Karmazyn, M
Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, Sollott SJ: Glycogen synthase kinase-3β mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 2004; 113:1535–49Juhaszova, M Zorov, DB Kim, SH Pepe, S Fu, Q Fishbein, KW Ziman, BD Wang, S Ytrehus, K Antos, CL Olson, EN Sollott, SJ
Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J, Jones DP, MacGregor GR, Wallace DC: The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 2004; 427:461–5Kokoszka, JE Waymire, KG Levy, SE Sligh, JE Cai, J Jones, DP MacGregor, GR Wallace, DC
Baines CP, Kaiser RA, Sheiko T, Craigen WJ, Molkentin JD: Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol 2007; 9:550–5Baines, CP Kaiser, RA Sheiko, T Craigen, WJ Molkentin, JD
Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA, Brunskill EW, Sayen MR, Gottlieb RA, Dorn GW, Robbins J, Molkentin JD: Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 2005; 434:658–62Baines, CP Kaiser, RA Purcell, NH Blair, NS Osinska, H Hambleton, MA Brunskill, EW Sayen, MR Gottlieb, RA Dorn, GW Robbins, J Molkentin, JD
Nishihara M, Miura T, Miki T, Tanno M, Yano T, Naitoh K, Ohori K, Hotta H, Terashima Y, Shimamoto K: Modulation of the mitochondrial permeability transition pore complex in GSK-3β-mediated myocardial protection. J Mol Cell Cardiol 2007; 43:564–70Nishihara, M Miura, T Miki, T Tanno, M Yano, T Naitoh, K Ohori, K Hotta, H Terashima, Y Shimamoto, K
Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F: Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 1999; 76:725–34Petronilli, V Miotto, G Canton, M Brini, M Colonna, R Bernardi, P Di Lisa, F
Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ: Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 1995; 307(pt 1):99–106Nieminen, AL Saylor, AK Tesfai, SA Herman, B Lemasters, JJ
Griffiths EJ, Halestrap AP: Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J 1995; 307(pt 1):93–8Griffiths, EJ Halestrap, AP
Halestrap AP, Clarke SJ, Javadov SA: Mitochondrial permeability transition pore opening during myocardial reperfusion: A target for cardioprotection. Cardiovasc Res 2004; 61:372–85Halestrap, AP Clarke, SJ Javadov, SA
Gateau-Roesch O, Argaud L, Ovize M: Mitochondrial permeability transition pore and postconditioning. Cardiovasc Res 2006; 70:264–73Gateau-Roesch, O Argaud, L Ovize, M
Penna C, Rastaldo R, Mancardi D, Raimondo S, Cappello S, Gattullo D, Losano G, Pagliaro P: Post-conditioning induced cardioprotection requires signaling through a redox-sensitive mechanism, mitochondrial ATP-sensitive K+channel and protein kinase C activation. Basic Res Cardiol 2006; 101:180–9Penna, C Rastaldo, R Mancardi, D Raimondo, S Cappello, S Gattullo, D Losano, G Pagliaro, P
Tsutsumi YM, Yokoyama T, Horikawa Y, Roth DM, Patel HH: Reactive oxygen species trigger ischemic and pharmacological postconditioning: In vivo  and in vitro  characterization. Life Sci 2007; 81:1223–7Tsutsumi, YM Yokoyama, T Horikawa, Y Roth, DM Patel, HH
Jiang MT, Nakae Y, Ljubkovic M, Kwok WM, Stowe DF, Bosnjak ZJ: Isoflurane activates human cardiac mitochondrial adenosine triphosphate-sensitive K+ channels reconstituted in lipid bilayers. Anesth Analg 2007; 105:926–32Jiang, MT Nakae, Y Ljubkovic, M Kwok, WM Stowe, DF Bosnjak, ZJ
Brookes PS, Salinas EP, Darley-Usmar K, Eiserich JP, Freeman BA, Darley-Usmar VM, Anderson PG: Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome c release. J Biol Chem 2000; 275:20474–9Brookes, PS Salinas, EP Darley-Usmar, K Eiserich, JP Freeman, BA Darley-Usmar, VM Anderson, PG