reperfusion injury.

YJMCC-07878; No. of pages: 13; 4C: 4, 7, 8 Journal of Molecular and Cellular Cardiology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc

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Review article

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Andrew P. Halestrap ⁎, Andrew P. Richardson

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School of Biochemistry and Bristol CardioVascular, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK

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Article history: Received 1 August 2014 Received in revised form 21 August 2014 Accepted 24 August 2014 Available online xxxx

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Keywords: Adenine nucleotide translocase Contact sites Cyclophilin D Cyclosporin A FoF1 ATP synthase Ischaemia/reperfusion injury Hexokinase Phosphate carrier Oxidative stress Preconditioning

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The properties of the MPTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Historical perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The open pore is a non-selective channel — evidence and consequences . . . . . . 2.3. MPTP opening is triggered by elevated matrix [Ca2 +] . . . . . . . . . . . . . . 2.4. MPTP opening is modulated by additional factors that change its sensitivity to [Ca2 +] 2.5. Low conductance states and transient opening of the MPTP . . . . . . . . . . . . The molecular identity of the MPTP . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The role of cyclophilin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The role of the adenine nucleotide translocase . . . . . . . . . . . . . . . . . . 3.3. The role of the phosphate carrier . . . . . . . . . . . . . . . . . . . . . . . .

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The mitochondrial permeability transition pore (MPTP) is a non-specific pore that opens in the inner mitochondrial membrane (IMM) when matrix [Ca2+] is high, especially when accompanied by oxidative stress, high [Pi] and adenine nucleotide depletion. Such conditions occur during ischaemia and subsequent reperfusion, when MPTP opening is known to occur and cause irreversible damage to the heart. Matrix cyclophilin D facilitates MPTP opening and is the target of its inhibition by cyclosporin A that is cardioprotective. Less certainty exists over the composition of the pore itself, with structural and/or regulatory roles proposed for the adenine nucleotide translocase, the phosphate carrier and the FoF1 ATP synthase. Here we critically review the supporting data for the role of each and suggest that they may interact with each other through their bound cardiolipin to form the ATP synthasome. We propose that under conditions favouring MPTP opening, calcium-triggered conformational changes in these proteins may perturb the interface between them generating the pore. Proteins associated with the outer mitochondrial membrane (OMM), such as members of the Bcl-2 family and hexokinase (HK), whilst not directly involved in pore formation, may regulate MPTP opening through interactions between OMM and IMM proteins at “contact sites”. Recent evidence suggests that cardioprotective protocols such as preconditioning inhibit MPTP opening at reperfusion by preventing the loss of mitochondrial bound HK2 that stabilises these contact sites. Contact site breakage both sensitises the MPTP to [Ca2+] and facilitates cytochrome c loss from the intermembrane space leading to greater ROS production and further MPTP opening. This article is part of a Special Issue entitled ‘Mitochondria’. © 2014 Published by Elsevier Ltd.

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The mitochondrial permeability transition: A current perspective on its identity and role in ischaemia/reperfusion injury

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Abbreviations: Akt, also known as protein kinase B; ANT, adenine nucleotide translocase; Bad, Bcl-2-associated death promoter; Bak, Bcl-2 homologous antagonist/killer; Bax, Bcl-2-like protein 4; Bcl2, B cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; Bid, BH3 interacting-domain death agonist; CAT, carboxyatractyloside; CK, creatine kinase; CsA, cyclosporin A; CyP-D, cyclophilin D; G-6-P, glucose-6-phosphate; GSK3β, glycogen synthase kinase 3β; HK, hexokinase; IF1, ATP synthase inhibitor factor 1; IMM, inner mitochondrial membrane; IP, ischaemic preconditioning; I/R, ischaemia reperfusion; MCF, mitochondrial carrier family; MPTP, mitochondrial permeability transition pore; NHE, sodium/proton exchanger; OMM, outer mitochondrial membrane; OPA-1, optic atrophy 1; PCr, phosphocreatine; PiC, phosphate carrier; PKA, protein kinase A; PKCε, protein kinase Cε; pmf, proton motive force; PPIase, peptidylprolyl isomerase; ROS, reactive oxygen species; SR, sarcoplasmic reticulum; T0, time of ischaemic rigour start; TAT-HK2, cell permeable peptide of HK2 binding domain; tBid, truncated BID; TP, temperature preconditioning; TSPO, translocator protein of the outer membrane; VDAC, voltage-dependent anion channel; ρ0, mitochondrial DNA-depleted cells. ⁎ Corresponding author. Tel.: +44 117 3312118; fax: +44 117 3312168. E-mail address: [email protected] (A.P. Halestrap).

http://dx.doi.org/10.1016/j.yjmcc.2014.08.018 0022-2828/© 2014 Published by Elsevier Ltd.

Please cite this article as: Halestrap AP, Richardson AP, The mitochondrial permeability transition: A current perspective on its identity and role in ischaemia/reperfusion injury, J Mol Cell Cardiol (2014), http://dx.doi.org/10.1016/j.yjmcc.2014.08.018

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3.4. The FoF1 ATP synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Could the MPTP involve an interaction between the FoF1 ATP synthase, ANT and PiC? . . . . . . . . . . . . . 3.6. Outer membrane proteins are not directly involved in MPTP formation but may regulate its opening . . . . . . 4. The role of the MPTP in ischaemia/reperfusion injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Inhibiting the MPTP is cardioprotective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Other cardioprotective protocols are mediated by inhibition of MPTP opening . . . . . . . . . . . . . . . . . 5.2. Attenuation of oxidative stress provides a link between preconditioning protocols and inhibition of MPTP opening 5.3. Cytochrome c loss during ischaemia is regulated by mitochondrial hexokinase 2 binding . . . . . . . . . . . . 5.4. The role of contact sites in HK2-mediated modulation of MPTP opening and cardioprotection . . . . . . . . . 6. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Measurement of the permeability properties of mitochondria that have undergone the permeability transition demonstrated that the MPTP is a non-specific pore with a diameter of about 2.3 nm [7,15]. Further evidence for this came from patch-clamp studies that identified the presence of a megachannel within the IMM whose opening and electrophysiological properties matched those predicted for the MPTP [16,17]. However, it is important to note that such megachannel behaviour may also reflect other molecular entities [18,19]. This becomes important when the role of specific proteins in the formation of the MPTP is investigated using electrophysiological techniques as discussed in Section 3. Opening of the MPTP makes the IMM freely permeable to protons and hence uncouples mitochondria as noted above; but it also allows all small molecular weight metabolites, cofactors and ions to equilibrate between the mitochondrial matrix and the cytosol. This includes [Ca2+] and there is evidence that this provides a mechanism for releasing excessive accumulated calcium from the mitochondrial matrix in some (patho)physiological situations [20,21]. This is discussed further in Section 2.4. MPTP opening also induces swelling of mitochondria. This occurs because small molecular weight metabolites and cofactors equilibrate across the IMM leaving proteins within the matrix to exert a colloidal osmotic pressure [1,2]. In vitro, in the absence of extramitochondrial proteins to provide an osmotic support, this causes extensive swelling of the matrix accompanied by cristae unfolding and rupture of the outer mitochondrial membrane (OMM). Such swelling is accompanied by a decrease in light scattering which is often used to monitor the progress of MPTP opening [22,23]. In vivo, swelling is still observed but it is of lesser magnitude because of the colloidal osmotic support provided by proteins in the cytosol. However, it may still be sufficient to cause rupture of the outer membrane and release of intermembrane proteins such as cytochrome c that can induce apoptosis [24,25].

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The major role of mitochondria in the heart is the provision of ATP by oxidative phosphorylation to drive the contractile cycle and maintain ionic homeostasis. Oxidative phosphorylation requires the permeability barrier of the inner mitochondrial membrane (IMM) to be maintained. However, mammalian mitochondria contain a latent non-specific pore within their inner membrane, known as the mitochondrial permeability transition pore (MPTP). Opening of the MPTP not only prevents mitochondria from synthesising ATP by oxidative phosphorylation, but also allows reversal of the FoF1 ATP synthase causing hydrolysis of the ATP produced by glycolysis or any remaining “healthy” mitochondria [1]. If this occurs for any length of time, cells become depleted of ATP and will eventually die by necrosis. In essence, MPTP opening converts mitochondria from ATP providers that energise the cell to agents of cell death, akin to the caring Dr Jekyll turning into the murderous Mr Hyde [2]. It is now widely accepted that the MPTP plays a major role in determining the extent of injury the heart suffers during reperfusion after a prolonged period of ischaemia; such ischaemia/reperfusion injury (I/R) is reflected in the size of the necrotic area or infarct (see [2–4]). In this article we will first review what is currently known about the mechanism and molecular identity of the MPTP, paying particular attention to significant new developments since our previous review in this journal [1]. We will then briefly summarise the evidence that MPTP opening is a key event in I/R injury and finally review how inhibiting MPTP opening during reperfusion is cardioprotective.

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It has been known for more than sixty years that mitochondria become leaky, uncoupled and massively swollen if they are exposed to high calcium concentrations, especially in the presence of phosphate and when accompanied by oxidative stress (see [2,5]). This phenomenon became known as the permeability transition and was originally thought to reflect activation of endogenous phospholipase A2 leading to phospholipid breakdown within the IMM [6]. However, seminal studies in the late seventies by Haworth and Hunter [7,8] revealed that the permeability transition involved the opening of a non-specific channel permeable to any molecule b 1.5 kDa. This was subsequently confirmed by Crompton [9] who demonstrated that the pore could be specifically blocked by sub-micromolar concentrations of cyclosporin A (CsA) [10]. This critical observation was rapidly confirmed by others [11,12] and provided the first clue as to the identity of one component of the MPTP, cyclophilin-D. Since then several laboratories in addition to ours, most notably those of Crompton, Bernardi and Molkentin, have been involved in characterising the properties of the MPTP and its molecular identity (see [1,5,13,14]).

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A matrix-facing Ca2+ binding site is essential to trigger MPTP opening, and any factor that influences calcium loading, such as modulators of Ca2+ import and efflux pathways, will affect MPTP opening, whilst chelation of matrix calcium causes rapid closure [7,15,26–29]. Interestingly, unlike most mitochondrial Ca2+-sensitive processes such as the Ca2+-activated dehydrogenases, Sr2+ cannot substitute for Ca2+ as a trigger for MPTP opening. Indeed, the Ca2+ trigger site can be inhibited by Sr2+ and other divalent cations such as Mg2+ [7,30,31], and also by H+ which accounts for the potent inhibition of MPTP opening by low pH [8,32,33]. There is an additional divalent cation regulatory site on the MPTP that faces the cytosolic side of the inner membrane and inhibits MPTP opening. This has a broader specificity than the Ca2 +trigger site, and inhibition is observed with many divalent cations including Ca2+ and Mg2+ [34].

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Electrophysiological studies suggested that the MPTP may rapidly switch between fully open, fully closed and intermediate sub-states [16,17]. Such partially open states would provide a mechanism that allows rapid passage of protons (to produce uncoupling) or calcium (to promote rapid calcium release) without total equilibration of small molecular weight metabolites and thus without swelling of mitochondria [42,43]. However, it is uncertain whether such intermediate conductance states exist under physiological conditions. Kinetic measurements of sucrose permeation into mitochondria provide no evidence for intermediate states but rather suggest that the MPTP can rapidly oscillate between fully open and closed states [15]. Furthermore, following MPTP opening, isolated mitochondria are either “normal” or massively swollen, without any intermediate states (see [44]). Within a population of isolated mitochondria, individual mitochondria appear to possess different sensitivities to MPTP opening perhaps reflecting their age and consequent exposure to oxidative stress [1]. The situation for mitochondria in vivo may be more complex since a modest trigger for MPTP could induce transient MPTP opening in only a few mitochondria at any one time with depolarisation and loss of accumulated [Ca2+]. Such transient opening can be monitored in isolated cells as a slow CsA-sensitive permeation of large molecules such as calcein without an observable drop in membrane potential across the whole mitochondrial population [22,45]. It has been proposed that transient MPTP opening is important in releasing the excessively large amounts of mitochondrial calcium that can accumulate in conditions associated with prolonged increased intracellular [Ca2 +] such as pressure overload [21,46]. CyP-D deficient mice would be unable to utilize this rapid calcium release pathway and this could explain the observed perturbation of their pyruvate, citric acid cycle and branched chain amino acid metabolism [47] that may in turn account for the greater cardiac hypertrophy, fibrosis, and reduction in myocardial function observed in these mice in response to pressure overload stimulation [48].

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Although the exact molecular identity of the MPTP is yet to be determined, several proteins have been implicated in its formation and regulation. Here we will critically review the evidence for the involvement of each of these proteins, focussing especially on recent papers proposing a role for the FoF1 ATP synthase [49–52]. For a more detailed account of the evidence for the role of the adenine nucleotide translocase (ANT)

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Studies in this laboratory first identified the target for CsA to be a small matrix peptidyl-prolyl cis–trans isomerase (PPIase) [12,56]. We subsequently identified this PPIase as cyclophilin D (CyP-D) [57] and demonstrated that it facilitates MPTP opening by enhancing its calcium sensitivity [58]. Extensive work in many laboratories, including our own, has confirmed these observations and identified other inhibitors of the PPIase activity of CyP-D including CsA analogues and sanglifehrin A (SfA) [36]. We demonstrated that CyP-D binds to mitochondrial membranes and sub-mitochondrial particles in a CsA-sensitive manner and that binding is increased by the chaotropic agent, KSCN, by oxidative stress induced by diamide or t-butylhydroperoxide and by the vicinal thiol reagent phenylarsine oxide (PAO). All of these treatments potently activate the MPTP [58–60]. Subsequently three major IMM proteins have been shown to bind CyP-D: the ANT [61,62], the PiC [37,63] and the oligomycin sensitivity conferring protein (OSCP) of the FoF1 ATP synthase [64]. However, CyP-D binding to only the PiC and ANT is increased by PAO and oxidative stress [37,60]. Furthermore, treatment with carboxyatractyloside (CAT), which binds specifically to the ANT and enhances MPTP opening, also increases CyP-D binding to the PiC [37]. Another important observation is that CyP-D binding to the PiC and ANT is inhibited by CsA whereas SfA increases CyP-D binding to these proteins despite their similar behaviour against the PPIase activity of CyP-D [36,60]. Another difference between CsA and Sfa is that the CsA concentration dependence of MPTP inhibition is linear whereas that of SfA is sigmoidal with the lowest SfA concentrations providing little or no inhibition [36]. This is illustrated in Fig. 1. One possible explanation for these data may come from the observation that SfA binding to cyclophilin A, a very close relative of CyP-D, causes it to dimerise, which is not the case for CsA [65]. We have confirmed that this is also the case for CyP-D (Fig. 1). Thus it would seem that, unlike CsA, SfA inhibits MPTP opening by inhibiting the PPIase activity of CyP-D without dissociating it from its target protein. Indeed it may actually enhance binding by promoting dimerisation [36] as illustrated in Fig. 1. However, when only 1 SfA molecule is bound to the dimer, the other subunit may remain active to facilitate MPTP opening thus accounting for the sigmoidal shape of the inhibition profile. Definitive proof that CyP-D plays a key role in MPTP opening came from the use of mitochondria from CyP-D knockout mice [66–68]. MPTP opening in these mitochondria is insensitive to CsA and shows a greatly reduced sensitivity to [Ca2+] that matches that of control mitochondria in the presence of CsA. These data are consistent with the PPIase activity of CyP-D facilitating a conformational change in a membrane protein that forms the transmembrane pore. However, it is important to note that even in the absence of CyP-D (or the presence of CsA) the MPTP can still open in response to a strong enough stimulus such as higher [Ca2+] and oxidative stress [38,66–69]. It has been proposed that the ability of CyP-D to facilitate MPTP opening is attenuated by nitrosylation of cysteine 203 [70], a modification that has been observed in the perfused mouse heart [71], and its mutation to serine impairs the ability of CyP-D to facilitate MPTP opening [70]. This led to the proposal that oxidation of Cys203 may play a role

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There are many factors that can activate or inhibit MPTP opening and some of these have played a major role in identifying candidate proteins involved in the formation and regulation of the MPTP as outlined below (Section 3). They usually act by changing the calcium sensitivity of the MPTP as has been reviewed in detail elsewhere [1,23,35]. Thus, oxidative stress and phosphate enhance the calcium sensitivity of the MPTP whilst adenine nucleotides (ATP or ADP), cyclosporin A and sanglifehrin A (SfA) decrease its sensitivity [28,36–39]. This means that MPTP opening can be induced without an increase in matrix [Ca2+] if one of the other regulatory parameters changes appropriately. As discussed in Section 4, this is the case during reperfusion of the heart following a period of ischaemia when there is oxidative stress, adenine nucleotide depletion and elevated phosphate concentrations [2]. Membrane potential may also regulate MPTP opening independently of Ca2+ uptake, and mitochondria loaded with insufficient Ca2+ to induce MPTP opening on its own can be triggered to undergo the permeability transition by addition of uncoupler [27,40]. These data have been interpreted as showing the MPTP to be a voltage regulated pore [41] although the mechanisms involved are uncertain [38,41].

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and phosphate carrier (PiC) the reader is referred to our previous review in this journal [1], whilst different perspectives on the molecular composition of the MPTP may be found in other recent reviews [5,21,53,54]. However, it should be noted that He and Lemasters have proposed that there may not be a unique protein responsible for pore formation. Rather, they suggest that the MPTP could be formed from aggregates of denatured membrane proteins, and that these are normally stabilised by a chaperone, but form a pore when acted upon by CyP-D in the presence of calcium [55]. In this hypothesis, although any membrane protein might be capable of forming the pore, the ANT, PiC and FoF1 ATP synthase could play a dominant role because of their high expression levels.

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Many studies from this and other laboratories have implicated the ANT in MPTP opening (see [1,35]), and several members of the Bcl-2 family. Furthermore, some viral proteins that regulate apoptosis and necrosis have been reported to bind to the ANT and modulate MPTP activity [35]. We originally proposed that Ca2+ triggers a conformational change in some ANT molecules to form the transmembrane components of the pore and that this was facilitated by the PPIase activity of CyP-D [12]. Indeed, the action of many known activators and inhibitors of the MPTP, such as oxidative stress, thiol reagents, carboxyatractyloside (CAT) and membrane potential can be explained by their interaction with the ANT [38]. Of particular significance, phenylarsine oxide, a vicinal thiol reagent which cross-links Cys160 with Cys257 of the ANT, is a potent MPTP inducer as is eosin 5maleimide which modifies only Cys160. Both act to prevent the inhibition of pore opening by adenine nucleotides [60] which is consistent with the known role of Cys160 in the binding and translocation of adenine nucleotides by the ANT [35]. Furthermore, the sensitivity of MPTP opening to inhibition by ADP, ATP and a range of other purine nucleotides matches their ability to act as substrates of the ANT [38]. In addition, prior exposure of mitochondria to the established MPTP inhibitors ubiquinone-0 (UQ0) or Ro 68–3400 [75] prevented both activation of MPTP opening by PAO and ANT binding to immobilised PAO [37]. If the ANT is a critical component of the MPTP it would be expected to bind CyP-D in a CsA sensitive manner and this was confirmed in both this laboratory and that of Crompton [61,62]. It was later reported that CyP-D binding to the ANT is increased by PAO and oxidative stress induced by diamide [60], although subsequent studies suggested that the antibody used to detect ANT binding to CyP-D also recognises the

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Strong evidence that the phosphate carrier (PiC) may be a component of the MPTP was provided by this laboratory [37]. As noted in Section 3.2 above, PAO is a potent activator of the MPTP and we used immobilised PAO to establish which IMM proteins could bind in a manner consistent with their involvement in MPTP formation. Using detergent-solubilised IMMs from beef heart mitochondria, we showed that only a few proteins bound to PAO and of these only the binding of the ANT and PiC could be totally prevented by prior exposure to the MPTP inhibitors UQ0 and Ro 68–3400 [37]. No subunits of the FoF1 ATP synthase were detected in the bound fraction. Interestingly, in yeast mitochondria, the adenine nucleotide carrier (AAC2) associates with the PiC, but not components of the FoF1 ATP synthase, in a supercomplex that is dependent on the presence of cardiolipin [83]. This phospholipid has been implicated in the regulation of the MPTP [76,84,85] as discussed further below. These data led us to investigate

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phosphate carrier [37] (see Section 3.3 below). Others have shown that when reconstituted into proteoliposomes, the purified ANT can form Ca2 +-activated high conductance channels that are modulated by ligands of the ANT and by CyP-D [76–78]. However, despite this strong evidence in support of an important role, it fell out of favour as a major component of the MPTP when pore opening was demonstrated in liver mitochondria from ANT1/ANT2 knockout mice [79]. Nevertheless, it should be emphasised that the sensitivity of MPTP opening to [Ca2+] in these mitochondria was greatly reduced and no longer sensitive to ANT ligands such as CAT and ADP [79]. Moreover, in QGY7703 cells, knockdown of ANT3 (a major ANT isoform in these cells) protects against mitochondrial depolarisation and apoptosis induced by the cytotoxic alkaloid, camptothecin (CPT), suggestive of reduced MPTP opening [80]. These data confirm a major role for the ANT in facilitating MPTP opening in this (patho)physiological setting even if it is not an essential structural component. It should also be noted that the mouse liver mitochondria employed from ANT1/ANT2 knockout mice will still contain a small amount of ANT4 which has been detected by proteomic analysis [81]. Indeed, this probably accounts for the normal liver function of these mice which would not be expected if ANT was totally absent [82]. Nevertheless, it would seem unlikely that ANT4 could account for the adenine nucleotide-insensitive MPTP opening observed in these mitochondria and the PiC and the FoF1 ATP synthase have been identified as potential alternative candidates for this role. The evidence for each is considered below.

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in the activation of the MPTP by oxidative stress and that its nitrosylation might be protective. However, the extent of nitrosylation of this residue in vivo remains ill-defined and direct evidence that it is modulated in response to cardioprotective regimes such as ischaemic precondition (IP) is lacking [72]. CyP-D has also been reported to be a target for phosphorylation by glycogen synthase kinase-3-β (GSK-3β) whose inhibition in cancer cells protects them from MPTP opening [73]. Since inhibition of GSK-3β has been implicated in the mechanism by which IP inhibits MPTP opening in reperfusion injury, this would be an attractive regulatory mechanism. However, we were unable to find any evidence for translocation of GSK-3β into mitochondria in response to IP, or any phosphorylation of CyP-D [74].

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Fig. 1. SfA causes dimerisation of CyP-D and shows a different concentration dependence of MPTP inhibition. The left panel confirms earlier data [36] that inhibition of the MPTP by SfA is distinct from that of CsA despite them both exhibiting similar Ki values (2 nM) for inhibition of the PPIase activity of recombinant CyP-D [36]. Data are presented as means ± S.E.M. of 3 separate liver mitochondrial preparations. The right panel compares the gel filtration profile of CyP-D in the presence of CsA or SfA and reveals that the latter encourages dimer formation as reported for CyP-A [65].


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Recently, evidence has been presented in support of a role for the Fo F1 ATP synthase in MPTP formation, although there are competing hypotheses as to how this may be achieved [5,54]. Using Blue Native gel electrophoresis and co-immunoprecipitation experiments, Bernardi's group had previously shown that CyP-D associates with the FoF1-ATP synthase from bovine heart mitochondria, and that this association is reduced by Bz-423, a benzodiazepine that binds to the FoF1-ATP synthase and also induces MPTP opening [50,64]. Cross-linking studies with 3,3-dithiobis-propionimidate suggested an involvement of the OSCP and b and d subunits of the lateral stalk of the ATP synthase in

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CyP-D binding. Phosphate was found to increase CyP-D binding and decrease the ATPase activity of the FoF1 ATP synthase slightly, whereas CsA had the reverse effects. However, it should be noted that the K0.5 of CsA for this effect was about 1 μM compared to the value of 2 nM for its inhibition of the MPTP and the PPIase activity of CyP-D [56]. A similar effect of CsA and CyP-D knockout on FoF1-ATP synthase activity was subsequently confirmed by Chinopoulos et al. [100]. In their more recent experiments, Bernardi and colleagues presented indirect evidence that the activity state of the ATP synthase modulates the sensitivity of the MPTP to [Ca2+] in intact mitochondria [50]. More direct evidence that the FoF1-ATP synthase might form the MPTP was obtained by eluting the dimeric fraction of the protein from Blue Native gels and reconstituting it into proteoliposomes. Electrophysiological techniques demonstrated that the reconstituted dimers, but not monomers, exhibited Ca2+-activated channels with a conductance similar to the MPTP [50]. However, high levels (0.3 mM) Ca2+ were required and opening was not observed unless Bz-423 was also added. A range of sub-conductance states was detected and the presence of Mg2+, ADP or the non-hydrolysable γ-imino ATP significantly reduces the conductance. No data were presented on the effects of CsA or cyclophilin on channel activity and, rather surprisingly, when the OSCP, the proposed binding partner for CyP-D, was knocked down by siRNA MPTP the opening was actually sensitised to [Ca2+] rather than attenuated as might be expected. From their data, the authors concluded that dimers of the FoF1-ATP synthase are responsible for the formation of the MPTP [50] and they subsequently demonstrated that a Ca2+-activated IMM pore could also be demonstrated in yeast mitochondria which was strongly attenuated in mutants lacking the ε and γ subunits of the ATPase that are required for dimer formation [51]. Benardi has speculated that an interface between the dimers involving subunits associated with the c-ring in the membrane such as a, e, f, g and A6L may move to produce the pore [5]. Since the ATP synthase binds ATP, ADP and Pi it is suggested that this is how these metabolites affect MPTP opening, although it is unclear why Pi should activate and ATP and ADP inhibit. It was further suggested that Ca2+ binds to the Mg2+ binding site involved in catalysis but no consideration is given as to why Sr2 +, which normally mimics Ca2+, is unable to activate the pore. Nor is it apparent how the binding of CyPD facilitates MPTP opening or how thiol reagents and oxidative stress activate the MPTP and prevent inhibition by adenine nucleotides. This should be contrasted with the evidence for the involvement of the ANT and PiC presented above (Sections 3.2 and 3.3) where sites for these reagents and others such as CAT, UQ0 and Ro 68–3400 have been defined, and where knockout experiments do modulate MPTP activity. Furthermore, reconstitution of the ANT produces a Ca-activated pore with properties similar to the MPTP that is modulated by adenine nucleotides, bongkrekic acid, CyP-D and oxidative stress. The group of Pinton has also raised concerns over the data of Bernardi and colleagues [54]. Most notably they note that previous work from Bernardi's group had demonstrated MPTP opening in ρ0 cells, which lack the mitochondrial DNA encoding the α and A6L subunits of the ATP synthase and so prevents proper assembly of the protein complex. Furthermore, dimerization of ATP synthase is promoted by binding of the ATPase inhibitor protein F1, yet IF1 has been implicated in protecting mitochondria from MPTP opening and enhancing cell survival under ischaemic conditions [101,102]. In addition, yeast mitochondria expressing mutants of the dimer-specific subunits e and g, which destabilise dimeric and oligomeric FoF1 ATP synthase supracomplexes, exhibit a decreased mitochondrial membrane potential which is the opposite of what would be predicted if dimers of FoF1 ATP synthase formed the MPTP [103]. Nevertheless, Pinton and colleagues still advocate of role for the ATP synthase in MPTP formation, but they present data to implicate the c subunits [49]. In the mature protein complex, the c subunits form a ring in the inner membrane and it would not be hard to envisage how this might produce a pore when dissociated from the rest of the ATP synthase. In HeLa cells they demonstrated that siRNA knockdown of the c subunit

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whether the PiC might be a key component of the MPTP. It is an attractive candidate since Pi had long been recognised as a potent activator of MPTP opening [9,12,28,86]. We demonstrated that the ability of N-ethylmaleimide to inhibit MPTP opening [60,87] correlated with its ability to inhibit PiC-mediated phosphate transport and a similar correlation was found for the inhibition of both processes by UQ0 and Ro 68– 3400 [37]. We also showed that the PiC binds CyP-D in a CsA sensitive manner and that binding increases in response to factors that enhance the sensitivity of MPTP opening to [Ca2+], such as oxidative stress and CAT [37]. Others have confirmed the binding of CyP-D to PiC and shown that the binding site on CyP-D involves residues 70–110 [63]. Bernardi and colleagues have reported that the ability of phosphate to activate MPTP opening is dependent on CyP-D and that when CyP-D is absent or inhibited by CsA, Pi actually inhibits MPTP opening. Indeed, in their experiments genetic or pharmacological (CsA) ablation of CyPD was unable to inhibit MPTP opening in the absence of Pi [39,88]. However, extensive data from our own laboratory [38,56,89] and others [90] have consistently demonstrated potent CsA inhibition of MPTP opening in the absence of Pi under a range of different conditions. Despite the strong circumstantial evidence for a role of the PiC in MPTP formation, neither partial knockdown nor over-expression of the PiC had any measurable effect on MPTP opening [63,89] suggesting that if the PiC is involved, its expression level does not usually limit MPTP opening. Nevertheless, in HeLa cells, where apoptosis induced by staurosporine is mediated by MPTP opening [91], PiC knockdown has been reported to reduce their sensitivity to staurosporine, whilst PiC over-expression induced apoptosis [92]. Similarly, hearts from mice with cardiac-specific PiC knockout showed some resistance to reperfusion injury whilst mitochondria isolated from them were less sensitive to MPTP opening [93]. Conversely, hearts from mice fed a nonobesogenic high-fat diet show increased PiC expression (two-fold) without significant changes in ANT or CyP-D expression, and these hearts are more susceptible to reperfusion injury [94]. Taken together, these data imply that either the PiC, like the ANT, plays only a regulatory role in MPTP opening, or that under appropriate conditions it can form the MPTP as can other members of the mitochondrial carrier family (MCF). Since the ANT and the PiC are the most highly expressed members of the MCF they would be responsible for the majority of pores. We have provided preliminary evidence for the latter using proteoliposomes reconstituted from a purified ANT preparation that was subsequently shown to contain the PiC [37,95]. It may also be significant that both these proteins require bound annular cardiolipin for their stability and function [96]. This might explain how agents that interact with cardiolipin such as 10-N-nonyl acridine orange and doxorubicin [97] can sensitise MPTP opening to [Ca2+] [98]. Indeed, it has been suggested that it is the cardiolipin associated with the ANT that may be the site at which Ca2+ binds to activate MPTP opening [76,84]. This has some attraction since binding of Sr2 + to large unilamellar cardiolipin vesicles has quite distinct effects on their phase behaviour to those of Ca2+ [99] which may provide an explanation for the unusual ability of the MPTP trigger site to discriminate between the two very similar cations [7,30]. Furthermore, peroxidation of cardiolipin sensitises mitochondria to MPTP opening [85].

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Overall, we would suggest that the evidence for a role of the FoF1 ATP synthase in MPTP formation remains tentative and is certainly not stronger than that for the PiC or ANT discussed in Sections 3.2 and 3.3 above. Nevertheless, it would certainly be unwise to rule out a role for the FoF1 ATP synthase, especially since the PiC and ANT have been shown to associate closely with the FoF1 ATP synthase in the ATP synthasome [106]. Indeed, we have previously suggested that all these components may interact/cooperate in some way to form the MPTP [1] and this conclusion was also reached by others commenting on the role of the FoF1 ATP synthase in MPTP formation [14]. Additional support for this hypothesis comes from the use of Blue Native Gel electrophoresis which confirmed that the ATP synthase associates with the ANT and PiC in digitonin solubilised bovine and yeast IMMs [83,107]. Furthermore, in yeast immunogold electron microscopy has revealed that the proteins show a similar dynamic subcompartmentalisation in relation to cristae structure [108]. Clearly, it cannot be the native conformation of these proteins that forms the MPTP and we have proposed that under conditions favouring MPTP formation, a small fraction of these proteins adopt a conformation that forms the pore, perhaps at the interface between two or more of the proteins. A scheme illustrating how the different components may interact is presented in Fig. 2.

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3.6. Outer membrane proteins are not directly involved in MPTP formation 589 but may regulate its opening 590

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The MPTP is located in the IMM and does not require the OMM to function since its opening can be demonstrated in mitoplasts (mitochondria with the outer membrane removed) [109] and submitochondrial particles [110]. The early suggestion that OMM components such as VDAC and the peripheral benzodiazepine receptor, now known as TSPO (TranSlocator Protein of the Outer membrane) are also involved in MPTP formation was largely based on conjecture rather than strong experimental evidence [1,5]. Indeed, it has now been demonstrated that MPTP formation can be demonstrated in mitochondria from mice in which all isoforms of VDAC [111] or the TSPO [112] have been knocked out. Nevertheless, proteins that bind to the outer membrane such as hexokinase and members of the Bcl-2 family can modulate MPTP opening (see [1,113]). Although Bcl-2 (anti-apoptotic) overexpression does not appear to inhibit MPTP opening in liver mitochondria [114] Bax and Bak do appear to enhance opening since their knock out inhibits MPTP-induced mitochondrial swelling and cell death [115,116]. Interestingly, oligomerisation-deficient Bax can act to restore MPTP sensitivity and cell death suggesting that its interaction with the MPTP is distinct from its role in oligomerisation-dependent permeabilisation of the OMM to cytochrome c. Another protein that binds to the OMM and attenuates MPTP opening is hexokinase 2 (HK2) that is particularly highly expressed in tumour cells whose mitochondria are very resistant to MPTP opening [113,117,118]. In recent years, several groups, including our own, have provided evidence that binding of HK2 to the outer mitochondrial membrane (OMM) also plays an important role in cardioprotection [119,120]. How these effects of proteins binding to the OMM influence MPTP opening in the IMM remains uncertain but recent data has focussed on a role for contact sites between the OMM and IMM which allow proteins in both membrane compartments to interact [121]. Furthermore, purification of a membrane fraction rich in contact sites reveals that they contain proteins of the Bcl2 family as well as VDAC, ANT and TSPO [121,122] all of which have been implicated in the regulation of the MPTP as discussed above. Also associated with these contact sites is creatine kinase-1 which resides in the intermembrane space between the IMM and OMM. Interestingly, its knockout greatly sensitises mitochondria to MPTP opening [123] and makes hearts more sensitive to I/R injury [124]. Thus we propose that stabilisation of contact sites inhibits MPTP

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reduced MPTP opening in response to ionomycin or hydrogen peroxide. Conversely, over-expression of the MYC-tagged c subunit enhanced MPTP opening in response to these stimuli [49]. However, without further experimentation it is not possible to conclude with confidence that these effects reflect a direct involvement of the c subunit in MPTP formation rather than being an indirect consequence of reduced ATP production by the ATP synthase (knockdown) or a misfolded protein response (over-expression). The authors also reported that cellular ATP levels were not reduced by knockdown of the c subunit, but were enhanced by overexpression that also induced mitochondrial depolarisation. They argue that normal HeLa cells are largely glycolytic for their energy metabolism but when provided with more c subunit they can increase mitochondrial oxidative phosphorylation which reduces the membrane potential. However, no data are provided to support this unlikely scenario. Furthermore, the antibody used by the authors only detects the uncleaved immature form of the c subunit (MWt 14.2 kDa) rather than the mature form (7.6 kDa) which makes the significance of their overexpression and knockdown data difficult to assess. In a very recent paper Alavian et al. have provided further evidence for a role of the c subunit in MPTP formation [52]. They confirm the observations of Bonora et al. [54] that knocking down the c-subunit with siRNA reduces MPTP opening and cell death in response to ionomycin and oxidative stress to a similar extent as CsA. They also demonstrated the presence of a multiconductance channel in excised patches from proteoliposomes containing the purified c subunit. The majority of channels conducted at ~100 pS but a few showed high conductance in the range of 1.5–2 nS, which is similar to values described for the Ca2+-activated megachannel detected in the IMM of isolated mitochondria that is CsA-sensitive and thought to represent the MPTP [104,105]. However, the channels were insensitive to Ca2+ and CsA and could only be inhibited by ATP, ADP and AMP at much higher (~1 mM) concentrations than required to inhibit opening of the megachannel/MPTP which is insensitive to AMP [8,38]. The authors argue that other parts of the FoF1 ATP synthase are required for appropriate regulation by these factors and evidence for this was provided by using purified monomeric FoF1 ATP synthase reconstituted into proteoliposomes where some infrequent channel activity was observed that was increased by addition of recombinant CyP-D and further by 100 μM Ca2+ in a CsA (5 μM) sensitive manner. In this context it should be noted that Bernardi and colleagues only demonstrated channel activity when the dimeric FoF1 ATP synthase was reconstituted [50]. Nevertheless, in patches from sub-mitochondrial vesicles (SMVs) enriched in F1Fo ATP synthase or IMMs, Alavian et al. [52] were able to demonstrate robust Ca2 + and CsA sensitive channel activity which was prevented by urea treatment to remove extramembrane components including the OSCP, β subunit and bound CyP-D. This treatment did not prevent inhibition of channel activity by 1 mM ATP, as was also observed for the purified c subunit. From these data Alavian et al. [52] propose that components of the F1 domain of the ATP synthase modulate channel activity of the c subunits thus explaining why only the whole FoF1 ATP synthase with CyP-D bound can be regulated by Ca2+, ADP and CsA. They suggest that binding of Ca2+ and CyP-D to the F1 component of the ATP synthase loosens its association with the c subunit ring causing it to expand and become a high conductance channel. Using fluorescent probes they presented evidence consistent with the subunits moving apart under conditions that favour pore opening and provided some further evidence for this hypothesis by adding back different purified subunits of the ATP synthase to the reconstituted c subunits. These experiments show that the presence of the β-subunit largely inhibits channel activity whilst other sub-units were without effect. Furthermore, changing glycines to valines in the transmembrane domains of the c subunit greatly enhanced channel activity as might be predicted if the c subunits could not pack so tightly [52]. However, these data with purified modified c subunits are hard to interpret because the expressed protein ran at 15 kDa on SDS-PAGE suggesting that it was not properly processed into the mature c subunit.

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opening as is illustrated schematically in Fig. 2 and this will be considered more fully below in the context of the regulation of MPTP opening in I/R (Section 5.4).

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4. The role of the MPTP in ischaemia/reperfusion injury

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It is now widely accepted that mitochondrial dysfunction, and particularly MPTP opening, plays a major role in determining the extent of injury the heart suffers during reperfusion after a prolonged period of ischaemia. This has been well reviewed elsewhere [2,4,21,125] and only a brief summary will be given here. First, the conditions that occur following ischaemia and reperfusion are exactly those that would induce MPTP opening. In particular, the heart experiences calcium overload, high [Pi] and low adenine nucleotide concentrations during ischaemia. These prime the MPTP for opening when reperfusion causes oxidative stress and accumulation of calcium by the reenergised mitochondria. Direct measurement of MPTP opening in the perfused heart using [3H]-deoxyglucose entrapment confirmed that the pore remains closed during ischaemia but opens early in reperfusion [69,126]. This occurs as the pH returns to normal from the low values of ischaemia which inhibit the MPTP opening [32,127]. The importance of MPTP opening in mediating I/R injury was confirmed by demonstrating that inhibition of MPTP opening by CsA is cardioprotective. This was first shown in this laboratory using the Langendorff-perfused rat heart [128] and protection was demonstrated in an isolated cardiac myocyte model of I/R by others [129]. Similar data have subsequently been obtained in many laboratories (see [2]). The target of this inhibition by CsA was confirmed to be CyP-D through the use of sanglifehrin A [36] and CsA analogues such as NIM811 and Debio-025 that inhibit MPTP opening without off-target effects mediated through calcineurin inhibition [130,131]. Further confirmation was provided by showing that hearts of CyP-D knockout mice are greatly protected against I/R injury [67,68]. Very recently a class of substituted cinnamic anilides has been discovered that acts as novel and potent MPTP inhibitors that also provide potent cardioprotection in a rabbit model of acute myocardial infarction [132]. Furthermore, it has now been established that many other well-documented cardioprotective

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regimes mediate their effects, at least in part, through inhibition of MPTP opening. Fig. 3 presents a scheme summarising the role of the MPTP in I/R and how we propose that cardioprotection is mediated through a range of pathways that attenuate its opening. This is discussed in detail below (Section 5). Despite the strong evidence in favour of a central role for the MPTP in I/R injury discussed above, a recent study by Finkel, Murphy and colleagues [133] might be considered to call this into question. The authors employed a gene-trap deletion of the mitochondrial calcium uniporter (MCU) which they confirmed prevented mitochondrial calcium accumulation and MPTP opening. Nevertheless, MPTP-dependent cell death induced by a range of toxins (hydrogen peroxide, tunicamycin, C2-ceramide, thapsigargin and doxorubicin) was not prevented; nor was I/R injury in the perfused heart, although the latter became insensitive to protection by CsA [133]. However, these observations are quite compatible with the central role of the MPTP in I/R since in isolated myocytes subject to simulated ischaemia/reperfusion, the amount of damage to the cells correlates with the [Ca2+] within the matrix at the end of the ischaemic period rather than on reoxygenation [134,135] and similar rises in matrix [Ca2+] are also observed in the whole ischaemic heart [136]. The dominant entry pathway for calcium into the mitochondria after prolonged ischaemia is unlikely to be MCU since the mitochondria will be largely depolarized and unable to accumulate calcium through this channel. Rather, calcium is more likely to enter in exchange for sodium and protons through the operation of the sodium calcium exchanger and sodium proton antiporter [137]. It will also be argued below (Section 5) that in addition to elevated matrix [Ca2 +], other events occurring during ischaemia, such as decreased oxidative stress, dissociation of bound hexokinase and contact site breakage, play major roles in stimulating MPTP opening on reperfusion, and are the targets of cardioprotection by ischaemic preconditioning. The data of Finkel, Murphy and colleagues [133] are entirely consistent with this proposal. It is less clear why I/R injury in the MCU knockout hearts should be insensitive to protection by CsA [133]. However, it is well established that MPTP opening is only facilitated by CyP-D and can occur in its absence or in the presence of CsA if the stimulus is sufficient (e.g. high levels of matrix [Ca2 +], oxidative stress and adenine

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Fig. 2. A schematic model of the MPTP that integrates current data on the role of CyP-D, ANT, PiC and FoF1 ATP synthase and the role of OMM proteins regulating MPTP activity through contact sites. The cartoon of the ATP synthase is based on the structure published in [198]. The detailed nature of the ATP synthase interaction with the PiC and ANT is not established but is implied by the existence of ATP synthasome [106] which is discussed more fully in the text. It is proposed that an additional level of MPTP regulation is provided by the contact sites between the IMM and OMM as discussed in the text. The proteins associated with the contact sites shown in the figure are those that copurify in fractions enriched in contact sites, but this does not require all of them to exert regulatory effects on the MPTP. Indeed mitochondria from VDAC knockout mice show normal MPTP function [111]. The presence of VDAC in contact sites may be more important for their role in the creatine phosphate shuttle as discussed more fully in [141].


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Fig. 3. A scheme illustrating how MPTP opening is triggered in I/R injury and how cardioprotective regimes may attenuate this. Pale blue and red boxes/arrows refer to events occurring in ischaemia and reperfusion respectively whilst other coloured boxes with dashed arrows illustrate the proposed locus of action of different cardioprotective regimes. Plus and minus symbols refer to activation and inhibition respectively. Further details may be found in the text.

nucleotide depletion) [38,66–69]. Thus if MCU knockout has additional effects on one of these parameters it could explain why CsA is no longer cardioprotective. 5. Inhibiting the MPTP is cardioprotective

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As noted above, inhibitors of MPTP opening such as CsA and SfA provide protection from reperfusion injury in a variety of experimental models including those relevant to the clinical setting. Thus, using the Langendorff perfused rat heart, Hausenloy and colleagues demonstrated that reduction of infarct size is observed when CsA is added during the first 15 min of reperfusion [138]. Protection has also been observed using in vivo mouse and rabbit models of reperfusion injury [130,131] although, surprisingly, in the in vivo rat model CsA alone did not show protection [139]. Proof of principle clinical trials have demonstrated that CsA may improve recovery of patients undergoing angioplasty following myocardial infarction [140,141] and a large Phase 3 clinical trial is underway [142].

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5.1. Other cardioprotective protocols are mediated by inhibition of MPTP opening

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Inhibition of the MPTP also underlies other cardioprotective regimes that attenuate matrix calcium overload and oxidative stress or maintain a low pH during the early phase of reperfusion [2,3,143,144]. Thus inhibitors of the plasma membrane sodium proton antiporter (NHE1) such as cariporide are cardioprotective and inhibit MPTP opening during reperfusion [145]. Reperfusion at low pH during the first phase of reperfusion has a similar effect [146] and may, at least in part, underlie cardioprotection by post-conditioning [147,148] which is known to inhibit MPTP opening [149]. Mitochondria-targeted scavengers of reactive oxygen such as mitoQ [150] and Szeto–Schiller peptides [151,152] are cardioprotective in some models of I/R injury and free radical scavenging may also underlie the cardioprotection offered by pyruvate [127] and the anaesthetic propofol [153,154]. The cardioprotective effect of the former is especially profound, probably because it not only acts as a ROS scavenger but also maintains a more acid pH during ischaemia and reperfusion [127,155,156].

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One of the most potent cardioprotective strategies, ischaemic preconditioning (IP), has also been shown to exert its effects through inhibition of MPTP opening [157,158]. A key question is how this inhibition is mediated. A wide range of different signalling pathways have been implicated and suggested mitochondrial targets include the putative mitochondrial KATP (mitoKATP) channels, components of the MPTP itself such as CyP-D in the matrix and VDAC in the OMM or HK2 bound to it. However, there is no firm consensus and the reader is referred elsewhere to review the evidence on which these claims are made [2,159–163]. Work from our own laboratory has identified an even more potent cardioprotective regime, temperature preconditioning (TP), in which the heart is subject to brief hypothermic episodes (26 °C) prior to normothermic ischaemia and this is also mediated through an inhibition of MPTP opening [164].

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5.2. Attenuation of oxidative stress provides a link between preconditioning 749 protocols and inhibition of MPTP opening 750 Studies in this laboratory on the mechanisms by which IP inhibited MPTP opening revealed that no inhibition could be detected immediately after the IP stimulus; rather inhibition developed as ischaemia progressed. This was seen as an increased sensitivity of MPTP opening to [Ca2+] in ischaemia that was attenuated by IP [74]. No phosphorylation of any component of the MPTP was detected that might be responsible for this effect. However, we were able to show that sensitisation of the MPTP during ischaemia was associated with an increase in mitochondrial protein carbonylation, a surrogate marker for oxidative stress which is known to sensitise MPTP opening [74]. Furthermore, TP showed an even greater reduction in end-ischaemic protein carbonylation and MPTP sensitisation than IP, and this was associated with greater cardioprotection [164]. This led us to suggest that a common mechanism by which preconditioning protocols inhibit MPTP opening, and thus mediate cardioprotection, is through attenuating oxidative stress. We subsequently explored how this decrease in oxidative stress might be mediated. We confirmed that cytochrome c is lost from mitochondria during ischaemia [165] as reported by others [166,167], and that this is prevented by IP [119]. We further demonstrated that cytochrome c loss causes increased ROS production following ischaemia through two mechanisms. First, oxidised cytochrome c acts as a


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In apoptosis, cytochrome c release involves a specific permeabilisation of the OMM mediated by pro-apoptotic members of the Bcl-2 family such as Bax, Bak and Bad, whose actions are opposed by the anti-apoptotic members of the family such as Bcl-2 and Bcl-xL [168]. In addition, as noted in Section 3.4 above, these proteins are also able to modulate MPTP function by a mechanism yet to be elucidated [115,116]. Furthermore, knockout of both Bax and Bak does protect hearts from I/R injury to the same extent as CyP-D knockout, implying that they play a role in regulating MPTP opening [115]. However, the mitochondrial content of these proteins was found to be unchanged following 30 min ischaemia [165,169] although we did detect a decrease in the anti-apoptotic protein Bcl-xL [165]. The significance of this decrease is unclear since it was not prevented by IP despite the mitochondria being protected from MPTP opening [119]. Thus it seems unlikely that IP attenuates cytochrome c release through changing the expression levels of members of the Bcl-2 family, although this does not discount a change in their disposition such as might occur through changes in contact sites (see Section 5.4 below). One protein whose mitochondrial content did decrease following ischaemia was hexokinase 2 (HK2) [165] and this loss was prevented by IP [119]. It has been known for some time that binding of HK2 to the OMM decreases apoptosis mediated by members of the Bcl-2 family, and that mitochondria of tumour cells exhibit both extensive HK2 binding and a resistance to opening of the MPTP. Furthermore, tumour cells are very resistant to apoptotic stimuli including to agents that induce MPTP opening [113,117,118]. A role of HK2 binding to mitochondria in cardioprotection was first proposed by Zuurbier et al. who demonstrated that the amount of HK2 bound to mitochondria increased following IP or morphine treatment of hearts, without any change in its phosphorylation or in HK1 binding [170,171]. We have subsequently confirmed and extended these studies as we describe below. For a more detailed consideration of the role HK2 in cardioprotection and the mechanisms involved the reader is referred to two very recent reviews [120,141]. Our own studies confirmed that 30 min global ischaemia caused a substantial loss of mitochondrial HK2, but not HK1, which was largely prevented by IP [119]. The importance of mitochondrial HK2 in cardioprotection received further support from experiments by Zuurbier et al. in which a cell permeable peptide (TAT-HK2) was used to dissociate HK2 from mitochondria without affecting baseline cardiac function, yet it dramatically increased ischaemia–reperfusion injury when perfused prior to ischaemia as well as preventing the protective effects of IP [169]. Similar studies in this laboratory confirmed that TAT-HK2 peptide increases I/R injury, but suggested that rather than directly dissociating HK2 from myocyte mitochondria, the peptide was having an indirect effect on heart function through vasoconstriction of the coronary circulation and consequent hypoxia [172]. Although Zuurbier and colleagues have challenged these conclusions [173] we sought other means to modulate mitochondrial HK2 binding that could be used to investigate its role in cardioprotection [119]. We found that we could alter mitochondrial HK2 binding by changing cytosolic [G-6-P] using metabolic interventions that target glycogen metabolism and glycolysis (see below). A very strong negative correlation was

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5.3. Cytochrome c loss during ischaemia is regulated by mitochondrial hexokinase 2 binding

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found between the amount of HK2 bound to mitochondria at the end of ischaemia and the infarct size after 2 h reperfusion [119]. We showed that the mechanism by which ischaemia causes dissociation of HK2 is through the combination of increased [G-6-P] and decreased pH that occurs during ischaemia rather than through changes in phosphorylation of HK2 or VDAC, its proposed mitochondrial binding partner [74]. IP decreases the glycogen content of the heart prior to ischaemia and thus decreases glycolysis during ischaemia with a reduction in [G-6-P] and lactic acid accumulation [174]. The latter means that the drop in pH during ischaemia is less as has been observed by many groups (see [119]). In IP hearts, the lower [G-6-P] and higher pH at the end of ischaemia can explain the decrease in HK2 dissociation. Indeed, we found that these effects could be enhanced or attenuated by treatments that decreased or increased respectively pre-ischaemic glycogen content and rates of glycolysis during ischaemia [119]. Since a range of signalling pathways can affect both glycogen metabolism and glycolysis, this may provide some insight into why such a variety of signalling pathways have been implicated in preconditioning [143,159, 161]. However, there may be additional mechanisms that affect HK2 binding involving the Akt and GSK3 signalling pathway whose activation during reperfusion is known to be cardioprotective [143,161,175]. Indeed, in tumour cells, Akt activation enhances HK2 binding to mitochondria and Akt inhibition causes HK2 dissociation probably via inhibition of GSK-3β [176,177]. Potential relevant targets for phosphorylation by Akt include VDAC1 and HK2 itself [178,179], although we found no evidence that IP modulates either in the rat heart [74].

5.4. The role of contact sites in HK2-mediated modulation of MPTP opening 862 and cardioprotection 863

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scavenger of superoxide produced by the external-facing ubiquinone site of complex 3 and its loss leads to greater release of superoxide into the IMS. Second, loss of cytochrome c also causes electrons to build up in complex 1 and this enhances superoxide production into the matrix [165]. The ability of IP to prevent cytochrome c loss during ischaemia [119] provides an attractive explanation as to how IP reduces oxidative stress and thus MPTP opening. However, it raises the question as to the mechanisms of cytochrome c loss during ischaemia and how IP attenuates this.

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Mitochondrial HK2 binding makes cells more resistant to apoptosis induced by pro-apoptotic members of the Bcl-2 family such as Bax and tBid [180–182]. Furthermore, tumour cells with high levels of mitochondrial HK2 are very resistant to the action of cytotoxic agents that enhance MPTP opening [113,117,118] and this can be overcome when dissociation of HK2 is induced by activation of GSK-3β or inhibition of Akt [177]. These data suggest that HK2 binding to the OMM both reduces its permeability to cytochrome c and makes the mitochondria less vulnerable to MPTP opening. This could be explained if bound mitochondrial HK2 interacted directly with members of the Bcl-2 family, perhaps in association with VDAC, to prevent them from forming an active permeability pathway for cytochrome c in the OMM, whilst at the same time modulating MPTP opening directly. This would require an interaction between the IMM and OMM such as is known to occur at “contact sites” [183,184]. This is illustrated in Fig. 2. Contact sites are thought to be held in place by a complex of proteins comprising VDAC, the adenine nucleotide translocase (ANT) and HK or, when present, creatine kinase (CK) which co-purify together with members of the Bcl-2 family during sub-fractionation of mitochondrial membranes [122,184]. Contact sites were originally thought to function primarily to enhance the transfer of ATP from the matrix to the cytosol either directly or via creatine phosphate [183]. Only subsequently were they implicated in the regulation of OMM permeability and the MPTP [121]. However, previous work in this laboratory had demonstrated that breakage of contact sites in liver mitochondria led to both cytochrome c release and sensitisation of the MPTP to [Ca2+] [185]. Since the ANT has been shown to play an important role in MPTP formation and/or regulation (see Section 3.2), this provides a potential link by which ligands or proteins that associate with the OMM could, via stabilising or breaking contact sites, modulate MPTP opening. This would also explain how binding of HK2 to the OMM, which is known to stabilise contact sites [186,187], attenuates both MPTP opening and cytochrome c release. Furthermore, knockout of creatine kinase, which also stabilises contact sites [188], makes hearts more sensitive to I/R injury [124]. Interestingly, mitochondria from the livers of mice genetically modified to express mitochondrial creatine kinase (which is normally


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The central role of MPTP opening in causing reperfusion injury appears well established as is the cardioprotection offered by its inhibition and pharmacological interventions targeting CyP-D have been proven effective in a wide range of models [2,21,194,195] including a small proof of principle clinical trial [140]. Nevertheless, the effects are modest and not observed in all species [139,194]. One reason for this may be that CyP-D only facilitates MPTP opening which can occur in its absence when the stimulus is sufficient. Development of new drugs that target the pore forming component of the MPTP would be preferable, but uncertainties over the molecular identity of the MPTP (Section 3) make this difficult. Clearly, a major priority must be to clarify the molecular identity of the proteins that make up the MPTP and how they interact. Since the “front runners”, the ANT, PiC and FoF1 ATP synthase, all have essential roles in oxidative phosphorylation, it is possible that knock down or knockout experiments will not be a suitable approach to provide unequivocal evidence for their role. This is especially true if the pore is formed from a novel conformation of one or more of these proteins or at an interface between them as we have suggested (Section 3.5). Some additional clues may emerge through the use of substituted cinnamic anilides recently discovered to act as novel and potent MPTP inhibitors that work independently of CyP-D [132]. Elucidating the binding partner of these drugs should identify at least one component of MPTP with certainty, but the “Holy Grail” is the reconstitution of the MPTP from its constituent proteins to produce a pore with properties that closely match those determined in mitochondria. An alternative to direct pharmacological inhibition of the MPTP will be to modulate its activity indirectly by mimicking the effects of cardioprotective regimes such as post-conditioning, IP and TP. IP and TP are both thought to act via decreasing the oxidative stress experienced by mitochondria [74,164] and thus mitochondria-targeted scavengers of ROS provide another approach to cardioprotection as discussed in Section 5.1. So too may targeting of HK2 binding to mitochondria. In this laboratory we have demonstrated that TP can be mimicked by consecutive activation of PKA and PKC prior to the ischaemic episode and this intervention, like IP, decreases the pre-ischaemic glycogen content of the heart suggesting that it probably maintains HK2

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We would like to thank the many colleagues who have contributed to the research we have presented in this article that was performed in our own laboratory over many years. We are also extremely grateful for continuous funding of our research by the British Heart Foundation.

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References

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[1] Halestrap AP. What is the mitochondrial permeability transition pore? J Mol Cell Cardiol 2009;46:821–31. [2] Halestrap AP. A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection. Biochem Soc Trans 2010;38:841–60. [3] Halestrap AP, Pasdois P. The role of the mitochondrial permeability transition pore in heart disease. Biochim Biophys Acta 2009;1787:1402–15. [4] Hausenloy DJ, Ong SB, Yellon DM. The mitochondrial permeability transition pore as a target for preconditioning and postconditioning. Basic Res Cardiol 2009;104: 189–202. [5] Bernardi P. The mitochondrial permeability transition pore: a mystery solved? Front Physiol 2013;4:95. [6] Gunter TE, Pfeiffer DR. Mechanisms by which mitochondria transport calcium. Am J Physiol 1990;258:C755–86. [7] Haworth RA, Hunter DR. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch Biochem Biophys 1979;195:460–7. [8] Hunter DR, Haworth RA. The Ca2+-induced membrane transition in mitochondria. I. The protective mechanisms. Arch Biochem Biophys 1979;195:453–9. [9] Al-Nasser I, Crompton M. The reversible Ca2+-induced permeabilisation of rat liver mitochondria. Biochem J 1986;239:19–29. [10] Crompton M, Ellinger H, Costi A. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J 1988;255:357–60. [11] Broekemeier KM, Dempsey ME, Pfeiffer DR. Cyclosporin-A Is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J Biol Chem 1989;264:7826–30. [12] Halestrap AP, Davidson AM. Inhibition of Ca2+-induced large amplitude swelling of liver and heart mitochondria by Cyclosporin A is probably caused by the inhibitor binding to mitochondrial matrix peptidyl-prolyl cis–trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochem J 1990;268:153–60. [13] Crompton M, Virji S, Doyle V, Johnson N, Ward JM. The mitochondrial permeability transition pore. Biochem Soc Symp 1999;66:167–79. [14] Karch J, Molkentin JD. Identifying the components of the elusive mitochondrial permeability transition pore. Proc Natl Acad Sci U S A 2014;111:10396–7. [15] Crompton M, Costi A. A heart mitochondrial Ca-2+-dependent pore of possible relevance to re-perfusion-induced injury — evidence that ADP facilitates pore interconversion between the closed and open states. Biochem J 1990;266:33–9. [16] Szabo I, Zoratti M. The giant channel of the inner mitochondrial membrane is inhibited by Cyclosporin-A. J Biol Chem 1991;266:3376–9. [17] Zoratti M, Szabo I. Electrophysiology of the inner mitochondrial membrane. J Bioenerg Biomembr 1994;26:543–53. [18] Lohret TA, Murphy RC, Drgon T, Kinnally KW. Activity of the mitochondrial multiple conductance channel is independent of the adenine nucleotide translocator. J Biol Chem 1996;271:4846–9. [19] Lohret TA, Jensen RE, Kinnally KW. Tim23, a protein import component of the mitochondrial inner membrane, is required for normal activity of the multiple conductance channel, MCC. J Cell Biol 1997;137:377–86. [20] Bernardi P, Petronilli V. The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J Bioenerg Biomembr 1996;28(2):131–8. [21] Elrod JW, Molkentin JD. Physiologic functions of cyclophilin D and the mitochondrial permeability transition pore. Circ J 2013;77:1111–22. [22] Bernardi P, Scorrano L, Colonna R, Petronilli V, DiLisa F. Mitochondria and cell death — mechanistic aspects and methodological issues. Eur J Biochem 1999; 264:687–701. [23] Halestrap AP, McStay GP, Clarke SJ. The permeability transition pore complex: another view. Biochimie 2002;84:153–66.

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binding to mitochondria [196,197]. Furthermore, maximal protection can be observed using sequential treatment with the β-agonist, isoproterenol, followed by adenosine, both at concentrations already routinely used in the clinic, and importantly the protection is seen in both healthy and failing hearts [197]. This intervention looks especially promising for translation into the clinic for use in cardiac surgery. Whether it will also be effective when drugs are added just prior to reperfusion of an occluded vessel, as required in the treatment of myocardial infarction by PCI remains to be established.

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absent) demonstrated a 3-fold increase in the number of contact sites as observed by transmission electron microscopy [188], and were more resistant to MPTP opening by calcium [189]. Modulation of contact sites may also provide the mechanism through which ligands of peripheral membrane benzodiazepine receptor (also known as TSPO) act indirectly to inhibit MPTP opening and induce cardioprotection [190]. If HK2 binding to mitochondria exerts its cardioprotective effects by stabilising contact sites it would be predicted that, following ischaemia, mitochondria would exhibit fewer contact sites than before ischaemia and that this loss would be attenuated in IP hearts. Indirect evidence for this comes from the observation that the phosphocreatine shuttle, which uses contact sites to transport ATP generated within the mitochondria to the cytosol as phosphocreatine (PCr) [121,191], is impaired following ischaemia [192] but less so following IP [193]. Indeed, time courses of the restoration of normal diastolic function and the recovery of PCr and ATP levels over the first 90 s of reperfusion following ischaemia are also consistent with contact site breakage during ischaemia and its prevention by protocols such as IP that reduces dissociation of mitochondrial HK2 [119]. Furthermore, in saponin-permeabilised heart muscle fibres we were able to mimic the ischaemia-induced perturbation of the creatine shuttle by incubating them with G-6-P at pH 6.3 to dissociate HK2 from the mitochondria [119]. However, direct demonstration of contact site breakage in situ and its modulation by HK2 binding will require analysis of the inner and outer membrane interactions and cristae morphology of mitochondria from control and preconditioned hearts before and after ischaemia using electron microscopy with 3D tomography.

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[54] Bonora M, Wieckowski MR, Chinopoulos C, et al. Molecular mechanisms of cell death: central implication of ATP synthase in mitochondrial permeability transition. Oncogene 2014 Epub ahead of print PMID: 24727893. [55] 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. [56] Griffiths EJ, Halestrap AP. Further evidence that cyclosporin-A protects mitochondria from calcium overload by inhibiting a matrix peptidyl-prolyl cis–trans isomerase — implications for the immunosuppressive and toxic effects of cyclosporin. Biochem J 1991;274:611–4. [57] Connern CP, Halestrap AP. Purification and N-terminal sequencing of peptidylprolyl cis–trans-isomerase from rat liver mitochondrial matrix reveals the existence of a distinct mitochondrial cyclophilin. Biochem J 1992;284:381–5. [58] Connern CP, Halestrap AP. Chaotropic agents and increased matrix volume enhance binding of mitochondrial cyclophilin to the inner mitochondrial membrane and sensitize the mitochondrial permeability transition to [Ca2+]. Biochemistry 1996;35:8172–80. [59] Connern CP, Halestrap AP. Recruitment of mitochondrial cyclophilin to the mitochondrial inner membrane under conditions of oxidative stress that enhance the opening of a calcium-sensitive non-specific channel. Biochem J 1994;302:321–4. [60] 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;367:541–8. [61] Crompton M, Virji S, Ward JM. Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore. Eur J Biochem 1998;258:729–35. [62] Woodfield K, Ruck A, Brdiczka D, Halestrap AP. Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition. Biochem J 1998; 336:287–90. [63] Gutierrez-Aguilar M, Douglas DL, Gibson AK, Domeier TL, Molkentin JD, Baines CP. Genetic manipulation of the cardiac mitochondrial phosphate carrier does not affect permeability transition. J Mol Cell Cardiol 2014;72:316–25. [64] Giorgio V, Bisetto E, Soriano ME, et al. Cyclophilin D modulates mitochondrial F0F1ATP synthase by interacting with the lateral stalk of the complex. J Biol Chem 2009; 284:33982–8. [65] Kallen J, Sedrani R, Zenke G, Wagner J. Structure of human cyclophilin A in complex with the novel immunosuppressant sanglifehrin A at 1.6 angstrom resolution. J Biol Chem 2005;280:21965–71. [66] Basso E, Fante L, Fowlkes J, Petronilli V, Forte MA, Bernardi P. Properties of the permeability transition pore in mitochondria devoid of cyclophilin D. J Biol Chem 2005;280:18558–61. [67] Nakagawa T, Shimizu S, Watanabe T, et al. Cyclophilin D-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death. Nature 2005;434:652–8. [68] Baines CP, Kaiser RA, Purcell NH, et al. Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 2005;434:658–62. [69] Griffiths EJ, Halestrap AP. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J 1995;307:93–8. [70] Nguyen TT, Stevens MV, Kohr M, Steenbergen C, Sack MN, Murphy E. Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore. J Biol Chem 2011;286:40184–92. [71] Kohr MJ, Aponte AM, Sun J, et al. Characterization of potential S-nitrosylation sites in the myocardium. Am J Physiol 2011;300:H1327–35. [72] Kohr MJ, Sun J, Aponte A, et al. Simultaneous measurement of protein oxidation and S-nitrosylation during preconditioning and ischemia/reperfusion injury with resin-assisted capture. Circ Res 2011;108:418–26. [73] Rasola A, Sciacovelli M, Chiara F, Pantic B, Brusilow WS, Bernardi P. Activation of mitochondrial ERK protects cancer cells from death through inhibition of the permeability transition. Proc Natl Acad Sci U S A 2010;107:726–31. [74] Clarke SJ, Khaliulin I, Das M, Parker JE, Heesom KJ, Halestrap AP. Inhibition of mitochondrial permeability transition pore opening by ischemic preconditioning is probably mediated by reduction of oxidative stress rather than mitochondrial protein phosphorylation. Circ Res 2008;102:1082–90. [75] Walter L, Miyoshi H, Leverve X, Bernardi P, Fontaine E. Regulation of the mitochondrial permeability transition pore by ubiquinone analogs. A progress report. Free Radic Res 2002;36:405–12. [76] Brustovetsky N, Becker A, Klingenberg M, Bamberg E. Electrical currents associated with nucleotide transport by the reconstituted mitochondrial ADP/ATP carrier. Proc Natl Acad Sci U S A 1996;93:664–8. [77] Ruck A, Dolder M, Wallimann T, Brdiczka D. Reconstituted adenine nucleotide translocase forms a channel for small molecules comparable to the mitochondrial permeability transition pore. FEBS Lett 1998;426:97–101. [78] Brustovetsky N, Tropschug M, Heimpel S, Heidkamper D, Klingenberg M. A large Ca2+-dependent channel formed by recombinant ADP/ATP carrier from Neurospora crassa resembles the mitochondrial permeability transition pore. Biochemistry 2002;41:11804–11. [79] Kokoszka JE, Waymire KG, Levy SE, et al. The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 2004;427:461–5. [80] Hu Z, Guo X, Yu Q, et al. Down-regulation of adenine nucleotide translocase 3 and its role in camptothecin-induced apoptosis in human hepatoma QGY7703 cells. FEBS Lett 2009;583:383–8. [81] Da Cruz S, Xenarios I, Langridge J, Vilbois F, Parone PA, Martinou JC. Proteomic analysis of the mouse liver mitochondrial inner membrane. J Biol Chem 2003;278: 41566–71. [82] Halestrap AP. Dual role for the ADP/ATP translocator? Nature 2004;430:U1.

E

T

[24] Martinou JC, Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001;2:63–7. [25] Halestrap AP. Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans 2006;34:232–7. [26] Crompton M, Costi A, Hayat L. Evidence for the presence of a reversible Ca2+dependent pore activated by oxidative stress in heart mitochondria. Biochem J 1987;245:915–8. [27] Petronilli V, Cola C, Bernardi P. Modulation of the mitochondrial Cyclosporin A-sensitive permeability transition pore. 2. The minimal requirements for pore induction underscore a key role for transmembrane electrical potential, matrix pH, and matrix Ca2+. J Biol Chem 1993;268:1011–6. [28] Crompton M, Costi A. Kinetic evidence for a heart mitochondrial pore activated by Ca2+, inorganic phosphate and oxidative stress. A potential mechanism for mitochondrial dysfunction during cellular Ca2+ overload. Eur J Biochem 1988;178: 489–501. [29] Gunter TE, Buntinas L, Sparagna G, Eliseev R, Gunter K. Mitochondrial calcium transport: mechanisms and functions. Cell Calcium 2000;28:285–96. [30] Bernardi P, Vassanelli S, Veronese P, Colonna R, Szabo I, Zoratti M. Modulation of the mitochondrial permeability transition pore — effect of protons and divalent cations. J Biol Chem 1992;267:2934–9. [31] Novgorodov SA, Gudz TI, Brierley GP, Pfeiffer DR. Magnesium ion modulates the sensitivity of the mitochondrial permeability transition pore to cyclosporin A and ADP. Arch Biochem Biophys 1994;311:219–28. [32] Halestrap AP. Calcium-dependent opening of a non-specific pore in the mitochondrial inner membrane is inhibited at pH Values below 7 — implications for the protective effect of low pH against chemical and hypoxic cell damage. Biochem J 1991; 278:715–9. [33] Szabo I, Bernardi P, Zoratti M. Modulation of the mitochondrial megachannel by divalent cations and protons. J Biol Chem 1992;267:2940–6. [34] Bernardi P, Veronese P, Petronilli V. Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore. 1. Evidence for 2 separate Me2+ binding sites with opposing effects on the pore open probability. J Biol Chem 1993;268: 1005–10. [35] Halestrap AP, Brenner C. The adenine nucleotide translocase: a central component of the mitochondrial permeability transition pore and key player in cell death. Curr Med Chem 2003;10:1507–25. [36] Clarke SJ, McStay GP, Halestrap AP. Sanglifehrin A acts as a potent inhibitor of the mitochondrial permeability transition and reperfusion injury of the heart by binding to cyclophilin-D at a different site from cyclosporin A. J Biol Chem 2002;277: 34793–9. [37] Leung AWC, Varanyuwatana P, Halestrap AP. The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition. J Biol Chem 2008;283:26312–23. [38] Halestrap AP, Woodfield KY, Connern CP. 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; 272:3346–54. [39] Di Lisa F, Bernardi P. A CaPful of mechanisms regulating the mitochondrial permeability transition. J Mol Cell Cardiol 2009;46:775–80. [40] Bernardi P. Modulation of the mitochondrial cyclosporin-A-sensitive permeability transition pore by the proton electrochemical gradient — evidence that the pore can be opened by membrane depolarization. J Biol Chem 1992;267:8834–9. [41] Scorrano L, Petronilli V, Bernardi P. On the voltage dependence of the mitochondrial permeability transition pore — a critical appraisal. J Biol Chem 1997;272: 12295–9. [42] Ichas F, Jouaville LS, Mazat JP. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 1997;89:1145–53. [43] Ichas F, Mazat JP. From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to highconductance state. Biochim Biophys Acta 1998;1366:33–50. [44] Bernardi P. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 1999;79:1127–55. [45] Petronilli V, Miotto G, Canton M, et al. 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;76:725–34. [46] Korge P, Yang L, Yang JH, Wang Y, Qu Z, Weiss JN. Protective role of transient pore openings in calcium handling by cardiac mitochondria. J Biol Chem 2011;286: 34851–7. [47] Menazza S, Wong R, Nguyen T, Wang G, Gucek M, Murphy E. CypD(−/−) hearts have altered levels of proteins involved in Krebs cycle, branch chain amino acid degradation and pyruvate metabolism. J Mol Cell Cardiol 2013;56:81–90. [48] Elrod JW, Wong R, Mishra S, et al. Cyclophilin D controls mitochondrial poredependent Ca(2+) exchange, metabolic flexibility, and propensity for heart failure in mice. J Clin Invest 2010;120:3680–7. [49] Bonora M, Bononi A, De Marchi E, et al. Role of the c subunit of the Fo ATP synthase in mitochondrial permeability transition. Cell Cycle 2013;12:674–83. [50] Giorgio V, von Stockum S, Antoniel M, et al. Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci U S A 2013;110:5887–92. [51] Carraro M, Giorgio V, Sileikyte J, et al. Channel formation by yeast F-ATP synthase and the role of dimerization in the mitochondrial permeability transition. J Biol Chem 2014;289:15980–5. [52] Alavian KN, Beutner G, Lazrove E, et al. An uncoupling channel within the c-subunit ring of the F1Fo ATP synthase is the mitochondrial permeability transition pore. Proc Natl Acad Sci U S A 2014;111:10580–5. [53] Baines CP. The mitochondrial permeability transition pore and ischemia–reperfusion injury. Basic Res Cardiol 2009;104:181–8.

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benzodiazepine receptor (TSPO). J Biol Chem 2014 Epub ahead of print PMID: 24692541. [113] Pastorino JG, Hoek JB. Hexokinase II: the integration of energy metabolism and control of apoptosis. Curr Med Chem 2003;10:1535–51. [114] Yang JC, Kahn A, Cortopassi G. Bcl-2 does not inhibit the permeability transition pore in mouse liver mitochondria. Toxicology 2000;151:65–72. [115] Whelan RS, Konstantinidis K, Wei AC, et al. Bax regulates primary necrosis through mitochondrial dynamics. Proc Natl Acad Sci U S A 2012;109:6566–71. [116] Karch J, Kwong JQ, Burr AR, et al. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice. Elife 2013;2:e00772. [117] Mathupala SP, Ko YH, Pedersen PL. Hexokinase-2 bound to mitochondria: cancer's stygian link to the “Warburg Effect” and a pivotal target for effective therapy. Semin Cancer Biol 2009;19:17–24. [118] Robey RB, Hay N. Mitochondrial hexokinases: guardians of the mitochondria. Cell Cycle 2005;4:654–8. [119] Pasdois P, Parker JE, Halestrap AP. Extent of mitochondrial hexokinase II dissociation during ischemia correlates with mitochondrial cytochrome c release, reactive oxygen species production, and infarct size on reperfusion. J Am Heart Assoc 2012;2:e005645. [120] Nederlof R, Eerbeek O, Hollmann MW, Southworth R, Zuurbier CJ. Targeting hexokinase II to mitochondria to modulate energy metabolism and reduce ischaemia– reperfusion injury in heart. Br J Pharmacol 2014;171:2067–79. [121] Brdiczka DG, Zorov DB, Sheu SS. Mitochondrial contact sites: their role in energy metabolism and apoptosis. Biochim Biophys Acta 2006;1762:148–63. [122] Beutner G, Ruck A, Riede B, Brdiczka D. Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases. Biochim Biophys Acta 1998;1368:7–18. [123] Datler C, Pazarentzos E, Mahul-Mellier AL, et al. CKMT1 regulates the mitochondrial permeability transition pore in a process that provides evidence for alternative forms of the complex. J Cell Sci 2014;127:1816–28. [124] Spindler M, Meyer K, Stromer H, et al. Creatine kinase-deficient hearts exhibit increased susceptibility to ischemia–reperfusion injury and impaired calcium homeostasis. Am J Physiol 2004;287:H1039–45. [125] Di Lisa F, Carpi A, Giorgio V, Bernardi P. The mitochondrial permeability transition pore and cyclophilin D in cardioprotection. Biochim Biophys Acta 1813;2011: 1316–22. [126] Halestrap AP, Connern CP, Griffiths EJ, Kerr PM. Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury. Mol Cell Biochem 1997;174:167–72. [127] Kerr PM, Suleiman MS, Halestrap AP. Reversal of permeability transition during recovery of hearts from ischemia and its enhancement by pyruvate. Am J Physiol 1999;276:H496–502. [128] Griffiths EJ, Halestrap AP. Protection by cyclosporin A of ischemia reperfusion-induced damage in isolated rat hearts. J Mol Cell Cardiol 1993; 25:1461–9. [129] Duchen MR, McGuinness O, Brown LA, Crompton M. On the involvement of a cyclosporin-A sensitive mitochondrial pore in myocardial reperfusion Injury. Cardiovasc Res 1993;27:1790–4. [130] Argaud L, Gateau-Roesch O, Muntean D, et al. Specific inhibition of the mitochondrial permeability transition prevents lethal reperfusion injury. J Mol Cell Cardiol 2005;38:367–74. [131] Gomez L, Thibault H, Gharib A, et al. Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice. Am J Physiol 2007;293:H1654–61. [132] Fancelli D, Abate A, Amici R, et al. Cinnamic anilides as new mitochondrial permeability transition pore inhibitors endowed with ischemia–reperfusion injury protective effect in vivo. J Med Chem 2014;57:5333–47. [133] Pan X, Liu J, Nguyen T, et al. The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. Nat Cell Biol 2013; 15:1464–72. [134] Silverman HS, Stern MD. Ionic basis of ischaemic cardiac injury: insights from cellular studies. Cardiovasc Res 1994;28:581–97. [135] Allen SP, Darleyusmar VM, Mccormack JG, Stone D. Changes in mitochondrial matrix free calcium in perfused rat hearts subjected to hypoxia reoxygenation. J Mol Cell Cardiol 1993;25:949–58. [136] Varadarajan SG, An J, Novalija E, Smart SC, Stowe DF. Changes in [Na(+)](i), compartmental [Ca(2+)], and NADH with dysfunction after global ischemia in intact hearts. Am J Physiol 2001;280:H280–93. [137] Griffiths EJ. Mitochondrial calcium transport in the heart: physiological and pathological roles. J Mol Cell Cardiol 2009;46:789–803. [138] Hausenloy DJ, Duchen MR, Yellon DM. Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia–reperfusion injury. Cardiovasc Res 2003;60:617–25. [139] De Paulis D, Chiari P, Teixeira G, et al. Cyclosporine A at reperfusion fails to reduce infarct size in the in vivo rat heart. Basic Res Cardiol 2013;108:379. [140] Piot C, Croisille P, Staat P, et al. Effect of cyclosporine on reperfusion injury in acute myocardial infarction. N Engl J Med 2008;359:473–81. [141] Halestrap AP, Pereira GC, Pasdois P. The role of hexokinase in cardioprotection — mechanism and potential for translation. Br J Pharmacol 2014 Invited review; accepted subject to minor revision. [142] Kloner RA. Current state of clinical translation of cardioprotective agents for acute myocardial infarction. Circ Res 2013;113:451–63. [143] Murphy E, Steenbergen C. Mechanisms underlying acute protection from cardiac ischemia–reperfusion injury. Physiol Rev 2008;88:581–609.

N

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O

R

R

E

C

T

[83] Claypool SM, Oktay Y, Boontheung P, Loo JA, Koehler CM. Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane. J Cell Biol 2008;182:937–50. [84] Pestana CR, Silva CH, Pardo-Andreu GL, et al. Ca(2+) binding to c-state of adenine nucleotide translocase (ANT)-surrounding cardiolipins enhances (ANT)-Cys(56) relative mobility: a computational-based mitochondrial permeability transition study. Biochim Biophys Acta 2009;1787:176–82. [85] Petrosillo G, Casanova G, Matera M, Ruggiero FM, Paradies G. Interaction of peroxidized cardiolipin with rat-heart mitochondrial membranes: induction of permeability transition and cytochrome c release. FEBS Lett 2006;580:6311–6. [86] Chalmers S, Nicholls DG. The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria. J Biol Chem 2003;278: 19062–70. [87] Chernyak BV, Bernardi P. The mitochondrial permeability transition pore is modulated by oxidative agents through both pyridine nucleotides and glutathione at two separate sites. Eur J Biochem 1996;238:623–30. [88] Basso E, Petronilli V, Forte MA, Bernardi P. Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation. J Biol Chem 2008;283:26307–11. [89] Varanyuwatana P, Halestrap AP. The roles of phosphate and the mitochondrial phosphate carrier in the mechanism of the permeability transition. Mitochondrion 2012;12:120–5. [90] McGee AM, Baines CP. Phosphate is not an absolute requirement for the inhibitory effects of cyclosporin A or cyclophilin D deletion on mitochondrial permeability transition. Biochem J 2012;443:185–91. [91] Tafani M, Minchenko DA, Serroni A, Farber JL. Induction of the mitochondrial permeability transition mediates the killing of HeLa cells by staurosporine. Cancer Res 2001;61:2459–66. [92] Alcala S, Klee M, Fernandez J, Fleischer A, Pimentel-Muinos FX. A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release. Oncogene 2008;27:44–54. [93] Kwong JQ, Davis J, Baines CP, et al. Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy. Cell Death Differ 2014;21:1209–17. [94] Littlejohns B, Pasdois P, Duggan S, et al. Hearts from mice fed a non-obesogenic high-fat diet exhibit changes in their oxidative state, calcium and mitochondria in parallel with increased susceptibility to reperfusion injury. PLoS One 2014;9: e100579. [95] Halestrap AP, Doran E, Gillespie JP, OToole A. Mitochondria and cell death. Biochem Soc Trans 2000;28:170–7. [96] Kadenbach B, Mende P, Kolbe HV, Stipani I, Palmieri F. The mitochondrial phosphate carrier has an essential requirement for cardiolipin. FEBS Lett 1982;139: 109–12. [97] Chavez E, Zazueta C, Garcia N, Martinez-Abundis E, Pavon N, Hernandez-Esquivel L. Titration of cardiolipin by either 10-N-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition. J Bioenerg Biomembr 2008;40:77–84. [98] Cheneval D, Muller M, Carafoli E. The mitochondrial phosphate carrier reconstituted in liposomes is inhibited by doxorubicin. FEBS Lett 1983;159:123–6. [99] Ortiz A, Killian JA, Verkleij AJ, Wilschut J. Membrane fusion and the lamellar-toinverted-hexagonal phase transition in cardiolipin vesicle systems induced by divalent cations. Biophys J 1999;77:2003–14. [100] Chinopoulos C, Konrad C, Kiss G, et al. Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels. FEBS J 2011;278: 1112–25. [101] Campanella M, Parker N, Tan CH, Hall AM, Duchen MR. IF(1): setting the pace of the F(1)F(o)-ATP synthase. Trends Biochem Sci 2009;34:343–50. [102] Campanella M, Casswell E, Chong S, et al. Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, IF1. Cell Metab 2008;8:13–25. [103] Bornhovd C, Vogel F, Neupert W, Reichert AS. Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes. J Biol Chem 2006;281:13990–8. [104] Szabo I, Zoratti M. The mitochondrial megachannel is the permeability transition pore. J Bioenerg Biomembr 1992;24:111–7. [105] Zoratti M, De Marchi U, Biasutto L, Szabo I. Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore. FEBS Lett 2010; 584:1997–2004. [106] Chen C, Ko Y, Delannoy M, Ludtke SJ, Chiu W, Pedersen PL. Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP. J Biol Chem 2004; 279:31761–8. [107] Wittig I, Schagger H. Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes. Biochim Biophys Acta 2009;1787:672–80. [108] Vogel F, Bornhovd C, Neupert W, Reichert AS. Dynamic subcompartmentalization of the mitochondrial inner membrane. J Cell Biol 2006;175:237–47. [109] Sileikyte J, Petronilli V, Zulian A, et al. Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor). J Biol Chem 2011;286:1046–53. [110] de Macedo DV, da Costa C, Pereira-Da-Silva L. The permeability transition pore opening in intact mitochondria and submitochondrial particles. Comp Biochem Physiol B Biochem Mol Biol 1997;118:209–16. [111] 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;9:550–5. [112] Sileikyte J, Blachly-Dyson E, Sewell R, et al. Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral

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[173] Nederlof R, Xie C, Eerbeek O, et al. Pathophysiological consequences of TAT-HKII peptide administration are independent of impaired vascular function and ensuing ischemia. Circ Res 2013;112:e8-13. [174] Cross HR, Opie LH, Radda GK, Clarke K. Is a high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved. Circ Res 1996;78: 482–91. [175] Hausenloy DJ, Yellon DM. Survival kinases in ischemic preconditioning and postconditioning. Cardiovasc Res 2006;70:240–53. [176] Neary CL, Pastorino JG. Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells. J Cell Physiol 2013;228:1943–8. [177] Pastorino JG, Hoek JB, Shulga N. Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltagedependent anion channel and potentiates chemotherapy-induced cytotoxicity. Cancer Res 2005;65:10545–54. [178] Miyamoto S, Murphy AN, Brown JH. Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II. Cell Death Differ 2008;15:521–9. [179] Roberts DJ, Tan-Sah VP, Smith JM, Miyamoto S. Akt phosphorylates HK-II at Thr473 and increases mitochondrial HK-II association to protect cardiomyocytes. J Biol Chem 2013;288:23798–806. [180] Gottlob K, Majewski N, Kennedy S, Kandel E, Robey RB, Hay N. Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev 2001;15:1406–18. [181] Pastorino JG, Shulga N, Hoek JB. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis. J Biol Chem 2002;277:7610–8. [182] Majewski N, Nogueira V, Bhaskar P, et al. Hexokinase–mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak. Mol Cell 2004;16:819–30. [183] Brdiczka D. Contact sites between mitochondrial envelope membranes — structure and function in energy-transfer and protein-transfer. Biochim Biophys Acta 1991; 1071:291–312. [184] Knoll G, Brdiczka D. Changes in freeze-fractured mitochondrial membranes correlated to their energetic state. Dynamic interactions of the boundary membranes. Biochim Biophys Acta 1983;733:102–10. [185] Doran E, Halestrap AP. Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites. Biochem J 2000;348:343–50. [186] Beutner G, Ruck A, Riede B, Brdiczka D. Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases. Biochim Biophys Acta Biomembr 1998;1368(1):7–18. [187] Kottke M, Adams V, Wallimann T, Nalam VK, Brdiczka D. Location and regulation of octameric mitochondrial creatine kinase in the contact sites. Biochim Biophys Acta 1991;1061:215–25. [188] Speer O, Back N, Buerklen T, et al. Octameric mitochondrial creatine kinase induces and stabilizes contact sites between the inner and outer membrane. Biochem J 2005;385:445–50. [189] Dolder M, Walzel B, Speer O, Schlattner U, Wallimann T. Inhibition of the mitochondrial permeability transition by creatine kinase substrates — requirement for microcompartmentation. J Biol Chem 2003;278:17760–6. [190] Schaller S, Paradis S, Ngoh GA, et al. TRO40303, a new cardioprotective compound, inhibits mitochondrial permeability transition. J Pharmacol Exp Ther 2010;333: 696–706. [191] Dolder M, Wendt S, Wallimann T. Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage. Biol Signals Recept 2001;10: 93–111. [192] Rauch U, Schulze K, Witzenbichler B, Schultheiss HP. Alteration of the cytosolic–mitochondrial distribution of high-energy phosphates during global myocardial ischemia may contribute to early contractile failure. Circ Res 1994;75:760–9. [193] Laclau MN, Boudina S, Thambo JB, et al. Cardioprotection by ischemic preconditioning preserves mitochondrial function and functional coupling between adenine nucleotide translocase and creatine kinase. J Mol Cell Cardiol 2001;33:947–56. [194] Gomez L, Li B, Mewton N, et al. Inhibition of mitochondrial permeability transition pore opening: translation to patients. Cardiovasc Res 2009;83:226–33. [195] Hausenloy DJ, Yellon DM. Myocardial ischemia–reperfusion injury: a neglected therapeutic target. J Clin Invest 2013;123:92–100. [196] Khaliulin I, Parker JE, Halestrap AP. Consecutive pharmacological activation of PKA and PKC mimics the potent cardioprotection of temperature preconditioning. Cardiovasc Res 2010;88:324–33. [197] Khaliulin I, Halestrap A, Bryant S, Dudley D, James A, Suleiman M-S. Clinicallyrelevant consecutive treatment with isoproterenol and adenosine protects the failing heart against ischaemia and reperfusion. J Transl Med 2014;12:139. [198] Baker LA, Watt IN, Runswick MJ, Walker JE, Rubinstein JL. Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM. Proc Natl Acad Sci U S A 2012;109:11675–80.

E

T

[144] Javadov S, Karmazyn M, Escobales N. Mitochondrial permeability transition pore opening as a promising therapeutic target in cardiac diseases. J Pharmacol Exp Ther 2009;330:670–8. [145] Javadov S, Choi A, Rajapurohitam V, Zeidan A, Basnakian AG, Karmazyn M. NHE-1 inhibition-induced cardioprotection against ischaemia/reperfusion is associated with attenuation of the mitochondrial permeability transition. Cardiovasc Res 2008;77:416–24. [146] Kitakaze M, Takashima S, Funaya H, et al. Temporary acidosis during reperfusion limits myocardial infarct size in dogs. Am J Physiol 1997;272:H2071–8. [147] Fujita M, Asanuma H, Hirata A, et al. Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning. Am J Physiol 2007;292:H2004–8. [148] Cohen MV, Yang XM, Downey JM. The pH hypothesis of postconditioning: staccato reperfusion reintroduces oxygen and perpetuates myocardial acidosis. Circulation 2007;115:1895–903. [149] GateauRoesch O, Argaud L, Ovize M. Mitochondrial permeability transition pore and postconditioning. Cardiovasc Res 2006;70:264–73. [150] Adlam VJ, Harrison JC, Porteous CM, et al. Targeting an antioxidant to mitochondria decreases cardiac ischemia–reperfusion injury. FASEB J 2005;19:1088–95. [151] Zhao KS, Zhao GM, Wu DL, et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 2004;279:34682–90. [152] Szeto HH. Mitochondria-targeted cytoprotective peptides for ischemia–reperfusion injury. Antioxid Redox Signal 2008;10:601–19. [153] Javadov SA, Lim KHH, Kerr PM, Suleiman MS, Angelini GD, Halestrap AP. Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res 2000;45:360–9. [154] Lim KHH, Halestrap AP, Angelini GD, Suleiman MS. Propofol is cardioprotective in a clinically relevant model of normothermic blood cardioplegic arrest and cardiopulmonary bypass. Exp Biol Med 2005;230:413–20. [155] Deboer LWV, Bekx PA, Han LH, Steinke L. Pyruvate enhances recovery of rat hearts after ischemia and reperfusion by preventing free radical generation. Am J Physiol 1993;265:H1571–6. [156] Mallet RT, Sun J, Knott EM, Sharma AB, Olivencia-Yurvati AH. Metabolic cardioprotection by pyruvate: recent progress. Exp Biol Med 2005;230:435–43. [157] Argaud L, GateauRoesch O, Chalabreysse L, et al. Preconditioning delays Ca2+induced mitochondrial permeability transition. Cardiovasc Res 2004;61:115–22. [158] Hausenloy DJ, Yellon DM, Mani-Babu S, Duchen MR. Preconditioning protects by inhibiting the mitochondrial permeability transition. Am J Physiol 2004;287: H841–9. [159] Halestrap AP, Clarke SJ, Khaliulin I. The role of mitochondria in protection of the heart by preconditioning. Biochim Biophys Acta 2007;1767:1007–31. [160] Murphy E, Steenbergen C. Preconditioning: the mitochondrial connection. Annu Rev Physiol 2007;69:51–67. [161] Yang X, Cohen MV, Downey JM. Mechanism of cardioprotection by early ischemic preconditioning. Cardiovasc Drugs Ther 2010;24:225–34. [162] Garlid KD, Halestrap AP. The mitochondrial K(ATP) channel—fact or fiction? J Mol Cell Cardiol 2012;52:578–83. [163] Hausenloy DJ. Cardioprotection techniques: preconditioning, postconditioning and remote conditioning (basic science). Curr Pharm Des 2013;19:4544–63. [164] Khaliulin I, Clarke SJ, Lin H, Parker J, Suleiman M-S, Halestrap AP. Temperature preconditioning of isolated rat hearts — a potent cardioprotective mechanism involving a reduction in oxidative stress and inhibition of the mitochondrial permeability transition pore. J Physiol 2007;581:1147–61. [165] Pasdois P, Parker JC, Griffiths EJ, Halestrap AP. The role of oxidized cytochrome c in regulating mitochondrial reactive oxygen species production and its perturbation in ischaemia. Biochem J 2011;436:493–505. [166] Borutaite V, Budriunaite A, Morkuniene R, Brown GC. Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia. Biochim Biophys Acta 2001;1537:101–9. [167] Lesnefsky EJ, Tandler B, Ye JA, Slabe TJ, Turkaly J, Hoppel CL. Myocardial ischemia decreases oxidative phosphorylation through cytochrome oxidase in subsarcolemmal mitochondria. Am J Physiol 1997;273:H1544–54. [168] Tait SW, Green DR. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol 2010;11:621–32. [169] Smeele KM, Southworth R, Wu R, et al. Disruption of hexokinase II-mitochondrial binding blocks ischemic preconditioning and causes rapid cardiac necrosis. Circ Res 2011;108:1165–9. [170] Gurel E, Smeele KM, Eerbeek O, et al. Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia–reperfusion. J Appl Physiol 2009;106:1909–16. [171] Zuurbier CJ, Eerbeek O, Meijer AJ. Ischemic preconditioning, insulin, and morphine all cause hexokinase redistribution. Am J Physiol 2005;289:H496–9. [172] Pasdois P, Parker JE, Griffiths EJ, Halestrap AP. Hexokinase II and reperfusion injury TAT-HK2 peptide impairs vascular function in Langendorff-perfused rat hearts. Circ Res 2013;112:e3–7.

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