Cardiovascular Research

Cardiovascular Research 2003 60(3):617-625; doi:10.1016/j.cardiores.2003.09.025
© 2003 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Hausenloy, D. J Articles by Yellon, D. M Search for Related Content PubMed PubMed Citation Articles by Hausenloy, D. J Articles by Yellon, D. M Social Bookmarking          What's this?

Copyright © 2003, European Society of Cardiology

Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia–reperfusion injury

Derek J Hausenloy^a, Michael R Duchen^b and Derek M Yellon^*,a

^aThe Hatter Institute and Centre for Cardiology, University College London Hospitals and Medical School, Grafton Way, London, WC1E 6DB, UK
^bThe Mitochondrial Biology Group, Department of Physiology, University College London, London, WC1E 6BT, UK

*Corresponding author. Tel.: +44-207-380-9776; fax: +44-207-388-5095. Email address: hatter-institute{at}ucl.ac.uk

Received 6 January 2003; accepted 10 September 2003

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: The opening of the mitochondrial permeability transition pore (mPTP) in the first few minutes of post-ischaemic reperfusion is a critical determinant of reperfusion-induced cell death. We hypothesised that the novel immunosuppressant, sanglifehrin-A (SFA), given at the time of reperfusion, protects the myocardium from ischaemia–reperfusion injury, by suppressing mPTP opening. Methods: Isolated perfused rat hearts were subjected to 35 min ischaemia/120 min reperfusion, and were treated with (1) SFA (1.0 µM) or (2) DMSO vehicle for the first 15 min of reperfusion or (3) SFA (1.0 µM) after the first 15 min of reperfusion. We examined the effect of SFA on mPTP opening directly, using a myocyte model of oxidative stress. Laser illumination of adult rat myocytes loaded with the fluorophore, TMRM, generates oxidative stress, which induces mPTP opening (represented by mitochondrial membrane depolarisation) followed by rigour contracture. Results: In the isolated perfused heart model, SFA, given during the first 15 min of post-ischaemic reperfusion, reduced the infarct-risk volume ratio from 43.9±2.5% in the control group to 23.8±4.2% with SFA (p = 0.001). However, when SFA was given after the first 15 min of reperfusion, there was no change in infarct size (43.8±5.7% with SFA vs. 43.9±2.5% in control; p = NS), suggesting that SFA has to be present during the first 15 min of reperfusion to induce protection. In the isolated adult myocyte model, SFA was shown to inhibit mPTP opening in the setting of oxidative stress, represented by an increase in the ROS threshold required to induce: mitochondrial membrane depolarisation (from 269±21 to 777±100 s; p<0.001) and rigour contracture (from 613±14 to 1329±129 s; p<0.001). Conclusions: Inhibiting mPTP opening during the first few minutes of reperfusion, using sanglifehrin-A, limits infarct size and protects myocytes from oxidative stress.

KEYWORDS Infarction; Ischaemia; Membrane permeability; Membrane potential; Reperfusion

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Reperfusion is a pre-requisite for salvaging viable myocardium from an episode of prolonged myocardial ischaemia. Paradoxically, the act of reperfusion is not without risk, as it can in itself, induce further myocyte death—a phenomenon termed lethal reperfusion-induced injury [1]. The concept of reperfusion-induced injury as an independent determinant of cell death is unresolved, with some studies suggesting that reperfusion only exacerbates the cellular injury sustained during the ischaemic period [2,3]. Other studies, on the other hand, indicate that the oxidative stress and abrupt metabolic changes that accompany reperfusion can themselves initiate cellular injury in the absence of ischaemia [4,5]. The most convincing evidence for the existence of reperfusion-induced injury as a distinct entity is provided by the observation that myocyte survival can be influenced by interventions applied solely at the time of post-ischaemic reperfusion, implying that crucial events which occur at time of reperfusion can be modified.

In this regard, a critical determinant of cell death in the setting of ischaemia–reperfusion injury is the opening of the mitochondrial permeability transition pore (mPTP) [6]. By opening in the first few minutes of reperfusion, the mPTP mediates reperfusion-induced myocyte death [7,8]. The mPTP is a large conductance pore of the inner mitochondrial membrane, which on opening renders the otherwise impermeable inner membrane, freely permeable to solutes up to 1500 Da in size [6,9]. This results in swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane, leading to the translocation of cytochrome C and other pro-apoptotic factors into the cytosol, which initiate the apoptotic death pathway [10]. Furthermore, the opening of the mPTP uncouples mitochondrial oxidative phosphorylation, resulting in the collapse of the mitochondrial membrane potential, which leads to cellular death by necrosis if the mPTP opening is prolonged and ATP levels are depleted [6].

In the setting of ischaemia–reperfusion injury, the mPTP is believed to open in the first few minutes of reperfusion, when conditions that increase its opening probability prevail; these include a high mitochondrial calcium and inorganic phosphate load, ATP depletion, oxidant stress and a corrected matrix pH [7,8,11]. We and others have shown that inhibiting its opening at the time of reperfusion using the immunosuppressant cyclosporin-A (CsA) protects the myocardium from ischaemia–reperfusion-induced injury [12,13]. Although CsA is a potent inhibitor of mPTP opening, it also has other actions [14], which include inhibiting the phosphatase, calcineurin [15].

Recently, the novel immunosuppressant, sanglifehrin-A (SFA), has been shown to also act as a potent inhibitor of mPTP opening [16]. This drug has been demonstrated to be a more potent inhibitor of mPTP opening than CsA and, in addition, SFA does not inhibit calcineurin [16,17]. When administered prior to the lethal ischaemic period in the isolated perfused heart, SFA was shown to improve recovery of LV function and reduce LDH release [16]. However, its action at the crucial time of reperfusion alone, when the mPTP is believed to open, has never been studied.

As such, the aim of this study was to investigate the effect of SFA at reperfusion, in the isolated perfused rat heart using infarct size as the end-point of injury, to ascertain its ability to protect the myocardium against the consequences of lethal reperfusion-induced injury. Furthermore, in order to demonstrate the mechanism by which SFA may induce protection, we used a myocyte model for detecting mPTP opening in isolated adult rat ventricular myocytes subjected to oxidative stress.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1. Animals
Male Sprague–Dawley rats (n = 23, 300±50 g body weight) were used. All animals were obtained from Charles River UK (Margate, UK) and received humane care in accordance with The Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (The Stationery Office, London, UK). The investigation conforms with the Guide for the Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2. Isolated heart perfusion
Rats were anaesthetised with sodium pentobarbital (55 mg/kg intraperitoneally) and given heparin sodium (300 IU). Hearts were rapidly excised and placed in ice-cold buffer and mounted on a constant pressure (100 mm Hg) Langendorff-perfusion apparatus. They were perfused retrogradely with modified Krebs–Henseleit bicarbonate buffer containing (in mM): NaCl 118.5, NaHCO₃ 25.0, KCl 4.8, MgSO₄ 1.2, KH₂PO₄ 1.2, CaCl₂ 1.7 and glucose 12.0. All solutions were gassed with 95% O₂/5% CO₂ with pH maintained between 7.35 and 7.45 at 37 °C. Temperature was continuously monitored by a thermo-probe inserted into the pulmonary artery and maintained between 36.5 and 37.5 °C. A latex, fluid-filled, isovolumic balloon was introduced into the left ventricle through the left atrial appendage and inflated to give a pre-load of 8–10 mm Hg. Left ventricular developed pressure, heart rate and coronary flow were measured at regular intervals. A surgical needle was passed under the left main coronary artery, and the ends of the suture were passed through a pipette tip to form a snare. Tightening the snare induced regional ischaemia and releasing the ends of the suture initiated reperfusion [13].

2.3. Treatment protocols for infarct study
The experimental protocols for the infarct studies are presented in Fig. 1. All hearts received 35 min regional ischaemia and 120 min reperfusion. SFA (Novartis Pharma, Basel) was dissolved in dimethyl sulphoxide (DMSO) and added to the Krebs–Henseleit buffer such that the final DMSO concentration was less than 0.01%. The hearts were randomly assigned to one of the following treatment groups: (1) control hearts were perfused with 0.01% DMSO (n = 6) for the first 15 min of reperfusion; (2) hearts (n = 6) were perfused with SFA (1.0 µM) for the first 15 min of reperfusion. This concentration of SFA has been shown to give the most potent inhibition of mPTP opening [3,16]. Hearts (n = 5) were perfused with SFA (1.0 µM) after the first 15 min of reperfusion. This group was included to determine whether SFA would have any effect if given after the crucial first 15 min of reperfusion, the time period during which the mPTP has been demonstrated to open [7,8].

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Treatment protocols for the infarct studies. TTC—triphenyltetrazolium chloride.

 
2.4. Infarct size measurement
At the end of the 120 min reperfusion period, the snare was pulled tight and the heart was slowly perfused with saline solution containing 0.25% Evans blue dye, to delineate the non-ischaemic zone of the myocardium as a dark blue area. After 1–4 h at –20 °C, the hearts were sliced into 2-mm-thick transverse sections and incubated in triphenyltetrazolium chloride solution (1% in phosphate buffer, pH 7.4) at 37 °C for 10–15 min. The tissue slices were then fixed in 10% formalin. In the risk zone, the viable tissue was stained red and the infarcted tissue appeared pale. The slices were drawn onto acetate sheets and with the use of a computerised planimetry package (Summa Sketch III, Summagraphics, Seymour, CT, USA); the percentage of infarcted tissue within the volume of myocardium at risk was calculated [13].

2.5. Preparation of adult rat myocytes
Adult rat myocytes were isolated by collagenase perfusion using a previously described method with modifications [18]. Briefly, following anaesthesia with sodium pentobarbital (55 mg/kg intraperitoneally) and the administration of heparin sodium (300 IU), hearts were rapidly excised and placed in ice-cold buffer, and mounted on a non-recirculating perfusion apparatus. All solutions used were based on a modified calcium-free Krebs–Ringers–Hepes (KRH) buffer (in mM) NaCl 116.0, KCl 5.4, MgSO₄ 0.4, HEPES 20.0, Na₂HPO₄ 0.9, glucose 5.6 (pH 7.4). The perfusate was bubbled with 100% O₂ and maintained at 37 °C. Hearts were first perfused at 14 ml/min with KRH buffer containing 1 mg/ml BSA and 3.3 µM EGTA. After 5 min, the hearts were switched to KRH buffer containing 0.75 mg/ml collagenase (Worthington type II) and 25 µM calcium for 10–15 min. They were finally perfused with KRH buffer containing 50 µM calcium for 5 min. Following perfusion, the hearts were removed from the perfusion apparatus and the atria trimmed away. The ventricles were minced and underwent several more digestions with collagenase. The cells were then filtered through a nylon mesh and washed with restoration buffer: KRH buffer+10 mg/ml BSA, 0.5 mM Na pyruvate, 5.0 mM taurine, 2.0 mM carnitine, 1.0 mM creatine and 75 µM calcium. Next, the calcium concentration was gradually increased to 1.25 mM. After isolation, the cells were seeded onto sterilised laminin-coated 25 mm diameter round cover-slips and incubated overnight at 37 °C in an atmosphere of 95% air/5% CO₂ in M-199 medium (M7653, Sigma) containing 10% foetal calf serum and 1% penicillin–streptomycin (Sigma). The next day, the cells were washed and kept in restoration buffer for the duration of the experiments.

2.6. Model for detecting mPTP opening in the intact cell
In order to open the mPTP routinely and predictably, we have used controlled intra-mitochondrial oxidative stress and have used mitochondrial membrane depolarisation and the rigor contracture that follows the resultant ATP depletion as endpoints. The fluorescent dye TMRM was used: (a) to report mitochondrial membrane potential, with global mitochondrial depolarisation signalling mPTP opening, and (b) to act as a photosensitising agent to generate the oxidative stress on illumination required to induce mPTP opening followed by rigour contracture [19–24]. Laser-induced oxidative stress was continued until mPTP opening had been provoked, and then continued until rigour contracture took place. This model represents a widely reported and reliable way to reproducibly induce the loss of mitochondrial potential, which has been unequivocally identified as a reflection of mPTP opening [19–24]. TMRM, a lipophilic cation, accumulates selectively into mitochondria according to the mitochondrial membrane potential. On laser-induced photo-sensitisation, TMRM generates reactive oxygen species (ROS) from within the mitochondria, which provoke mPTP opening [19–22,24]. As the oxidative stress generated at reperfusion also involves excess production of radical species from mitochondria, this should represent a useful model for reoxygenation-induced cell injury. Cells were incubated with TMRM (3 µM) for 15 min at 37 °C followed by washing. Opening of the mPTP results in mitochondrial membrane depolarisation, which is detected in this model as an increase in TMRM fluorescence intensity. The relatively high concentration of TMRM in the mitochondria causes auto-quenching of fluorescence, so that the fluorescence signal becomes a non-linear function of dye concentration; therefore, mitochondrial depolarisation results in loss of dye into the cytosol where the signal increases [25].

Cells were assigned to: (1) control-incubation with 0.01% DMSO vehicle control for 20 min (n = 12); (2) incubation with sanglifehrin-A (1.0 µM, n = 12) for 20 min. We measured the time taken to the beginning of global mitochondrial depolarisation (represented by an increase in cytosolic TMRM fluorescence and indicates mPTP opening) and the time taken to the beginning of irreversible cell shortening (rigor contracture, signalling ATP depletion).

2.7. Confocal imaging and analysis
The cover-slip with adherent myocytes was placed in a chamber and mounted on the stage of a Zeiss 510 CLSM confocal microscope equipped with x 40 oil immersion, quartz objective lens (NA 1.3). The cells were illuminated using the 543-nm emission line of a HeNe laser. For all photo-sensitisation experiments, all conditions of the confocal imaging system (laser power, confocal pinhole—set to give an optical slice of 1 µm—pixel dwell time and detector sensitivity) were identical to ensure comparability between experiments. The fluorescence of TMRM was collected using a 585-nm long pass filter. Images were analysed using the Zeiss software (LSM 2.8).

2.8. Statistical analysis
All values are expressed as mean±S.E.M. Infarct size and times taken to induce global mitochondrial depolarisation and irreversible cell shortening were analysed by one-way ANOVA and Fisher's protected least significant difference test for multiple comparisons. Rate pressure product and coronary flow were analysed by a two-way ANOVA for repeated measures. Differences were considered significant when p<0.05.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1. Isolated perfused rat heart model
Baseline data relating to cardiac function (assessed by the rate-pressure-product, RPP) and coronary flow before the lethal ischaemic period, were similar in the experimental groups (see Table 1). During regional ischaemia, coronary flow and RPP decreased to a similar extent in both groups. An increase in cardiac function and coronary flow upon reperfusion was indicative of successful re-flow. Perfusion with SFA for the first 15 min of reperfusion had no immediate effect on cardiac function and coronary flow compared to control. However, at the end of the reperfusion period, hearts treated with SFA for the first 15 min of reperfusion had significantly better cardiac function (RPP of 17.5±2.4 mm Hg/min with SFA vs. 8.3±2.3 mm Hg/min in control; p<0.005) and coronary flow (9.2±0.9 ml/min with SFA vs. 5.3±0.3 ml/min in control; p<0.05; Table 1). In hearts treated with SFA for the first 15 min of reperfusion, the % recovery of baseline cardiac function after 120 min of reperfusion was 60.6% with SFA vs. 29.6% in control hearts (p<0.005). However, in hearts perfused with SFA given after the first 15 min of reperfusion, compared to control, there was no difference in either cardiac function (RPP of 11.5±0.6 mm Hg/min with SFA given after the first 15 min of reperfusion vs. 8.3±2.3 mm Hg/min in control; p = NS) or coronary flow (6.6±0.8 ml/min with SFA given after the first 15 min of reperfusion vs. 5.3±0.3 ml/min in control; p = NS; Table 1) at the end of the 120 min of reperfusion.

View this table: [in this window] [in a new window]   Table 1 Haemodynamic variables throughout the ischaemia–reperfusion protocol

 
The risk zone volumes were similar in the experimental groups (0.411±0.017 cm³ in control vs. 0.485±0.046 cm³ with SFA given for the first 15 min of reperfusion vs. 0.505±0.046 cm³ with SFA given after the first 15 min of reperfusion; p = NS). Infarct size was represented as the percentage of tetrazolium-negative tissue in the ischaemic risk zone. The presence of SFA for the first 15 min of reperfusion significantly reduced infarct size compared with control hearts (23.8±4.2% with sanglifehrin-A vs. 43.9±2.5% in control; p<0.01; Fig. 2). However, when SFA was omitted for the first 15 min of reperfusion and only given after 15 min of reperfusion had elapsed, there was no influence on infarct size (43.8±5.7% with SFA given after the first 15 min of reperfusion vs. 43.9±2.5% in control; p = NS; Fig. 2), which suggests that SFA needs to be present during the first 15 min of reperfusion to exert its cardio-protective effect.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Sanglifehrin-A given during the first 15 min of reperfusion reduces the infarct-risk volume ratio, but not when given after the first 15 min of reperfusion (values are mean±S.E.M.; n = 5–6 per group; *p = 0.001).

 
3.2. Model for detecting mPTP opening in intact cells
Confocal imaging of TMRM-loaded adult ventricular myocytes revealed bands of intracellular fluorescence throughout the cell. These bands of fluorescence correspond to mitochondria, and their identity is confirmed by their co-localisation with mitochondrial NADH autofluorescence (Fig. 3).

View larger version (96K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Confocal image of representative myocyte showing co-localisation of (A) TMRM fluorescence and (B) NADH fluorescence within the mitochondria.

 
Fig. 4i–v shows a representative TMRM-loaded myocyte, subjected to oxidative stress, and demonstrates the sequential changes in mitochondrial membrane potential. With initial oxidative stress, one observes, depolarisation of individual mitochondria (which appear as areas devoid of TMRM fluorescence, see arrow in Fig. 4ii). Of interest, with continued oxidative stress, global mitochondrial membrane depolarisation, which reflects mPTP opening, begins as a wave of increased cytosolic TMRM fluorescence at one end of the cell (see arrow in Fig. 4iii) and progresses across the full length of the cell. The cause of this wave of global mitochondrial depolarisation is not known but this phenomenon may reflect a self-propagating wave of mPTP opening in mitochondria, with calcium released from one mitochondria on mPTP opening, initiating mPTP opening in the adjacent mitochondria and so on, [26,27] leading to global mitochondrial depolarisation of the whole cell (Fig. 4iv). As oxidative stress continues, rigour contracture occurs, signifying ATP depletion (Fig. 4v). Therefore, using this model of oxidative stress, we measured the time taken to induce the initiation of global mitochondrial membrane depolarisation (which represents mPTP opening) and the time taken to induce the initiation of rigor contracture (representing ATP depletion).

View larger version (48K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Confocal image of representative adult ventricular myocyte loaded with TMRM showing the sequential changes on exposure to oxidative stress: (i) the cell prior to oxidative stress showing TMRM localised to the mitochondria as bands of increased red-orange fluorescence; (ii) with continued oxidative stress areas devoid of fluorescence appear (see arrow), which precede (iii) a wave of global mitochondrial membrane depolarisation (appearing as increased cytosolic fluorescence, signalling mPTP opening) that begins at one end of the cell and progresses across the full length of the cell, until the whole cell is globally depolarised; (iv) and finally rigour contracture ensues following ATP depletion (v).

 
The time required to induce mPTP opening (indicated by the beginning of global mitochondrial membrane depolarisation) in control cells was 269.1±21.7 s and the time taken to induce the beginning of rigour contracture was 613.5±14.2 s (Fig. 5). The presence of SFA significantly prolonged the time taken to induce both mPTP opening and rigour contracture to 777.4±100.7 and 1329.0±129.0 s (p<0.001), respectively (Fig. 5). Sanglifehrin-A was therefore shown to inhibit mPTP opening (as shown by a delay in the time taken to induce global mitochondrial membrane depolarisation) and protect against rigour contracture in the setting of oxidative stress.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Sanglifehrin-A prolongs the time taken to induce mPTP opening and the time taken to induce rigour contracture in TMRM-loaded myocytes subjected to oxidative stress (values are mean±S.E.M.; n = 12 per group; *p<0.001).

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
In the isolated perfused heart model, we show for the first time that inhibiting mPTP opening in the first few minutes of reperfusion, following a lethal ischaemic period, using sanglifehrin-A, protects the myocardium, as shown by a reduction in infarct size and improved recovery of baseline cardiac function. Furthermore, in the isolated adult rat myocyte subjected to oxidative stress, we confirm that sanglifehrin-A protects myocytes by inhibiting mPTP opening.

Importantly, in the isolated perfused heart model, we demonstrated protection when SFA was administered during the first 15 min of reperfusion only, to cover the crucial time period when the mPTP would be expected to open. Furthermore, we demonstrated that targeting the first 15 min of reperfusion is crucial to its protective effect, since administering SFA after the first 15 min of reperfusion induced no cardio-protection. This last finding suggests that SFA has to be present during the first 15 min of reperfusion to exert it protective effect, which corresponds to the time-period during which mPTP opening has been demonstrated to occur [7,8]. Although SFA has been investigated in the setting of ischaemia–reperfusion injury, the drug was administered prior to index global ischaemia and for a further 10 min of reperfusion [16]. Furthermore, in that study, the end-points of post-ischaemic recovery of LV function and LDH release were used. As such, it is difficult to interpret whether this agent was only having its effect during the reperfusion phase, given that the drug was administered prior to the lethal ischaemic episode.

The current proposed model of the mPTP is believed to comprise a physical interaction between three core components: the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane, the adenine nucleotide translocase (ANT) of the inner mitochondrial membrane and cyclophilin D in the mitochondrial matrix [6]. Through its cis–trans isomerase action, it is believed that cyclophilin D induces a conformational change in the ANT from its normal translocating configuration to a pore-forming configuration [28]. The mechanism by which SFA inhibits mPTP opening has been shown to differ from that of CsA. The latter appears to inhibit binding of mitochondrial cyclophilin D to the ANT [29], whereas SFA inhibits the peptidyl-prolyl cis–trans isomerase activity of cyclophilin D, thereby preventing the conformational change of the ANT to the pore-forming configuration required to manifest as mPTP opening [16]. Furthermore, in contrast to CsA, the drug SFA is a more specific inhibitor of mPTP opening as it does not inhibit calcineurin, which is important as calcineurin inhibition can in itself result in cardio-protection [16,17].

The contribution of the mPTP to ischaemia–reperfusion injury was first proposed in 1987, when it was found that factors, which increase the opening probability of the mPTP, prevailed in the setting of ischaemia–reperfusion injury [30]. These factors included, a rise in tissue Ca²⁺ and iP levels, and depletion of ATP levels during the ischaemic period, followed by a burst of oxidant stress and further influx of calcium at time of reperfusion [30,31]. In agreement with the crucial role of the mPTP in ischaemia–reperfusion injury, inhibiting its opening, using the drug cyclosporin-A, has been shown to protect against necrosis in myocytes exposed to anoxia-reoxygenation [32,33], and protect the myocardium from ischaemia–reperfusion injury in the isolated perfused heart [12,13].

The actual time when mPTP opening occurs in the setting of ischaemia–reperfusion has been characterised, and studies have shown that the mPTP remains closed during the ischaemic period and only opens in the first few minutes of reperfusion [7,8,34]. It has been suggested that the mPTP remains closed during the ischaemic period, because its opening is inhibited by the lactate-induced acidic conditions. It has previously been shown that mPTP opening is inhibited at pH values below 7 [11,35,36]. At reperfusion, there is a further influx of calcium into the mitochondria, a burst of oxidant stress and rapid correction of the acidosis as the lactate is washed away. All these factors contribute to increasing the opening probability of the mPTP in the early minutes of reperfusion following a lethal ischaemic period.

The first few minutes of reperfusion following a lethal ischaemic insult, therefore, represent a ‘window of opportunity’ for interventions directed to attenuating reperfusion-induced cell death, via inhibition of mPTP opening. This study and others have shown that pharmacologically inhibiting the mPTP opening that normally occurs at reperfusion protects the myocardium from ischaemia–reperfusion injury in the laboratory setting [12,13]. Further research is needed to apply this approach to the clinical settings of reperfusion, such as during thrombolysis post-myocardial infarction, during coronary angioplasty and during coronary artery bypass surgery, since effective inhibition of mPTP opening in the first few minutes of reperfusion could provide further protection against ischaemia–reperfusion injury.

	Acknowledgements

 
Dr. Derek Hausenloy is supported by a British Heart Foundation Clinical PhD Studentship (FS/02017). We are grateful to Novartis Pharma (Basel) for providing us with sanglifehrin-A.

	Notes

 
Time for primary review 24 days

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

1. Braunwald E., Kloner R.A. Myocardial reperfusion: a double-edged sword? J. Clin. Invest. (1985) 76(5):1713–1719.[Web of Science][Medline]
2. Hearse D.J. Reperfusion-induced injury: a possible role for oxidant stress and its manipulation. Cardiovasc. Drugs Ther. (1991) 5(Suppl. 2):225–235.[CrossRef][Web of Science][Medline]
3. Kloner R.A. Does reperfusion injury exist in humans? J. Am. Coll. Cardiol. (1993) 21(2):537–545.[Abstract]
4. Hearse D.J. Reperfusion of the ischemic myocardium. J. Mol. Cell Cardiol. (1977) 9(8):605–616.[Web of Science][Medline]
5. Vanden Hoek T.L., Shao Z., Li C., Zak R., Schumacker P.T., Becker L.B. Reperfusion injury on cardiac myocytes after simulated ischemia. Am. J. Physiol. (1996) 270(4 Pt. 2):H1334–H1341.[Web of Science][Medline]
6. Crompton M., Virji S., Doyle V., Johnson N., Ward J.M. The mitochondrial permeability transition pore. Biochem. Soc. Symp. (1999) 66:167–179.[Medline]
7. Griffiths E.J., Halestrap A.P. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem. J. (1995) 307(Pt. 1):93–98.[Web of Science][Medline]
8. Di Lisa F., Menabo R., Canton M., Barile M., Bernardi P. Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart. J. Biol. Chem. (2001) 276(4):2571–2575.[Abstract/Free Full Text]
9. Hunter D.R., Haworth R.A. The Ca2+-induced membrane transition in mitochondria. I. The protective mechanisms. Arch. Biochem. Biophys. (1979) 195(2):453–459.[CrossRef][Web of Science][Medline]
10. Kroemer G., Dallaporta B., Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu. Rev. Physiol. (1998) 60:619–642.[CrossRef][Web of Science][Medline]
11. Qian T., Nieminen A.L., Herman B., Lemasters J.J. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am. J. Physiol. (1997) 273(6 Pt. 1):C1783–C1792.[Web of Science][Medline]
12. Griffiths E.J., Halestrap A.P. Protection by Cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts. J. Mol. Cell. Cardiol. (1993) 25(12):1461–1469.[CrossRef][Web of Science][Medline]
13. Hausenloy D.J., Maddock H.L., Baxter G.F., Yellon D.M. Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning? Cardiovasc. Res. (2002) 55(3):534–543.[Abstract/Free Full Text]
14. 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(1):357–360.[Web of Science][Medline]
15. Petronilli V., Nicolli A., Costantini P., Colonna R., Bernardi P. Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A. Biochim. Biophys. Acta (1994) 1187(2):255–259.[Medline]
16. Clarke S.J., McStay G.P., Halestrap A.P. 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(38):34793–34799.[Abstract/Free Full Text]
17. Sanglier J.J., Quesniaux V., Fehr T., Hofmann H., Mahnke M., Memmert K., et al. Sanglifehrins A, B, C and D, novel cyclophilin-binding compounds isolated from Streptomyces sp. A92-308110. I. Taxonomy, fermentation, isolation and biological activity. J. Antibiot. (Tokyo) (1999) 52(5):466–473.[Medline]
18. Maddock H.L., Mocanu M.M., Yellon D.M. Adenosine A(3) receptor activation protects the myocardium from reperfusion/reoxygenation injury. Am. J. Physiol, Heart Circ. Physiol. (2002) 283(4):H1307–H1313.[Abstract/Free Full Text]
19. Huser J., Blatter L.A. Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore. Biochem. J. (1999) 343(Pt. 2):311–317.[CrossRef][Web of Science][Medline]
20. Huser J., Rechenmacher C.E., Blatter L.A. Imaging the permeability pore transition in single mitochondria. Biophys. J. (1998) 74(4):2129–2137.[Web of Science][Medline]
21. Duchen M.R., Leyssens A., Crompton M. Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes. J. Cell Biol. (1998) 142(4):975–988.[Abstract/Free Full Text]
22. Duchen M.R. Mitochondria and Ca(2+)in cell physiology and pathophysiology. Cell Calcium. (2000) 28(5–6):339–348.[CrossRef][Web of Science][Medline]
23. Jacobson J., Duchen M.R. Mitochondrial oxidative stress and cell death in astrocytes—requirement for stored Ca2+ and sustained opening of the permeability transition pore. J. Cell Sci. (2002) 115(Pt. 6):1175–1188.[Abstract/Free Full Text]
24. De Giorgi F., Lartigue L., Bauer M.K., Schubert A., Grimm S., Hanson G.T., et al. The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J. (2002) 16(6):607–609.[Free Full Text]
25. Bunting J.R., Phan T.V., Kamali E., Dowben R.M. Fluorescent cationic probes of mitochondria. Metrics and mechanism of interaction. Biophys. J. (1989) 56(5):979–993.[Web of Science][Medline]
26. Riley W.W. Jr., Pfeiffer D.R. Relationships between Ca2+ release, Ca2+ cycling, and Ca2+-mediated permeability changes in mitochondria. J. Biol. Chem. (1985) 260(23):12416–12425.[Abstract/Free Full Text]
27. Ichas F., Jouaville L.S., Mazat J.P. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell (1997) 89(7):1145–1153.[CrossRef][Web of Science][Medline]
28. Woodfield K., Ruck A., Brdiczka D., Halestrap A.P. 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(Pt. 2):287–290.[Web of Science][Medline]
29. Halestrap A.P., Davidson A.M. Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin 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(1):153–160.[Web of Science][Medline]
30. 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(3):915–918.[Web of Science][Medline]
31. 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(2):489–501.[Web of Science][Medline]
32. Nazareth W., Yafei N., Crompton M. Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A. J. Mol. Cell Cardiol. (1991) 23(12):1351–1354.[CrossRef][Web of Science][Medline]
33. Griffiths E.J., Ocampo C.J., Savage J.S., Stern M.D., Silverman H.S. Protective effects of low and high doses of cyclosporin A against reoxygenation injury in isolated rat cardiomyocytes are associated with differential effects on mitochondrial calcium levels. Cell Calcium (2000) 27(2):87–95.[CrossRef][Web of Science][Medline]
34. Murata M., Akao M., O'Rourke B., Marban E. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca(2+) overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ. Res. (2001) 89(10):891–898.[Abstract/Free Full Text]
35. Haworth R.A., Hunter D.R. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch. Biochem. Biophys. (1979) 195(2):460–467.[CrossRef][Web of Science][Medline]
36. Halestrap A.P. 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(Pt. 3):715–719.[Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				S. Javadov, M. Karmazyn, and N. Escobales Mitochondrial Permeability Transition Pore Opening as a Promising Therapeutic Target in Cardiac Diseases J. Pharmacol. Exp. Ther., September 1, 2009; 330(3): 670 - 678. [Abstract] [Full Text] [PDF]

				J. Inserte, J. A. Barrabes, V. Hernando, and D. Garcia-Dorado Orphan targets for reperfusion injury Cardiovasc Res, July 15, 2009; 83(2): 169 - 178. [Abstract] [Full Text] [PDF]

				A. Granfeldt, D. J. Lefer, and J. Vinten-Johansen Protective ischaemia in patients: preconditioning and postconditioning Cardiovasc Res, July 15, 2009; 83(2): 234 - 246. [Abstract] [Full Text] [PDF]

				P. S. Pagel and J. G. Krolikowski Transient Metabolic Alkalosis During Early Reperfusion Abolishes Helium Preconditioning Against Myocardial Infarction: Restoration of Cardioprotection by Cyclosporin A in Rabbits Anesth. Analg., April 1, 2009; 108(4): 1076 - 1082. [Abstract] [Full Text] [PDF]

				Z. Zhong, V. K. Ramshesh, H. Rehman, R. T. Currin, V. Sridharan, T. P. Theruvath, I. Kim, G. L. Wright, and J. J. Lemasters Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion Am J Physiol Gastrointest Liver Physiol, October 1, 2008; 295(4): G823 - G832. [Abstract] [Full Text] [PDF]

				H. Kobayashi, S. Minatoguchi, S. Yasuda, N. Bao, I. Kawamura, M. Iwasa, T. Yamaki, S. Sumi, Y. Misao, H. Ushikoshi, et al. Post-infarct treatment with an erythropoietin-gelatin hydrogel drug delivery system for cardiac repair Cardiovasc Res, September 1, 2008; 79(4): 611 - 620. [Abstract] [Full Text] [PDF]

				D. J. Hausenloy and D. M. Yellon Time to Take Myocardial Reperfusion Injury Seriously N. Engl. J. Med., July 31, 2008; 359(5): 518 - 520. [Full Text] [PDF]

				L. Gomez, M. Paillard, H. Thibault, G. Derumeaux, and M. Ovize Inhibition of GSK3{beta} by Postconditioning Is Required to Prevent Opening of the Mitochondrial Permeability Transition Pore During Reperfusion Circulation, May 27, 2008; 117(21): 2761 - 2768. [Abstract] [Full Text] [PDF]

				S. J. Clarke, I. Khaliulin, M. Das, J. E. Parker, K. J. Heesom, and A. P. Halestrap 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., May 9, 2008; 102(9): 1082 - 1090. [Abstract] [Full Text] [PDF]

				E. Murphy and C. Steenbergen Mechanisms Underlying Acute Protection From Cardiac Ischemia-Reperfusion Injury Physiol Rev, April 1, 2008; 88(2): 581 - 609. [Abstract] [Full Text] [PDF]

				A. B. Gustafsson and R. A. Gottlieb Heart mitochondria: gates of life and death Cardiovasc Res, January 15, 2008; 77(2): 334 - 343. [Abstract] [Full Text] [PDF]

				D. M. Yellon and D. J. Hausenloy Myocardial Reperfusion Injury N. Engl. J. Med., September 13, 2007; 357(11): 1121 - 1135. [Full Text] [PDF]

				C. P. Baines The mitochondrial permeability transition pore as a target of cardioprotective signaling Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H903 - H904. [Full Text] [PDF]

				S. Y. Lim, S. M. Davidson, D. J. Hausenloy, and D. M. Yellon Preconditioning and postconditioning: The essential role of the mitochondrial permeability transition pore Cardiovasc Res, August 1, 2007; 75(3): 530 - 535. [Abstract] [Full Text] [PDF]

				M. T. Dirksen, G. J. Laarman, M. L. Simoons, and D. J.G.M. Duncker Reperfusion injury in humans: A review of clinical trials on reperfusion injury inhibitory strategies Cardiovasc Res, June 1, 2007; 74(3): 343 - 355. [Abstract] [Full Text] [PDF]

				M. V. Cohen, X.-M. Yang, and J. M. Downey The pH Hypothesis of Postconditioning: Staccato Reperfusion Reintroduces Oxygen and Perpetuates Myocardial Acidosis Circulation, April 10, 2007; 115(14): 1895 - 1903. [Abstract] [Full Text] [PDF]

				S. Venkatapuram, C. Wang, J. G. Krolikowski, D. Weihrauch, J. R. Kersten, D. C. Warltier, P. F. Pratt Jr, and P. S. Pagel Inhibition of Apoptotic Protein p53 Lowers the Threshold of Isoflurane-Induced Cardioprotection During Early Reperfusion in Rabbits Anesth. Analg., December 1, 2006; 103(6): 1400 - 1405. [Abstract] [Full Text] [PDF]

				J. C. Bopassa, D. Vandroux, M. Ovize, and R. Ferrera Controlled reperfusion after hypothermic heart preservation inhibits mitochondrial permeability transition-pore opening and enhances functional recovery Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2265 - H2271. [Abstract] [Full Text] [PDF]

				C. Wang, D. Weihrauch, D. A. Schwabe, M. Bienengraeber, D. C. Warltier, J. R. Kersten, P. F. Pratt Jr, and P. S. Pagel Extracellular signal-regulated kinases trigger isoflurane preconditioning concomitant with upregulation of hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression in rats. Anesth. Analg., August 1, 2006; 103(2): 281 - 8, table of contents. [Abstract] [Full Text] [PDF]

				M. Nishihara, T. Miura, T. Miki, J. Sakamoto, M. Tanno, H. Kobayashi, Y. Ikeda, K. Ohori, A. Takahashi, and K. Shimamoto Erythropoietin affords additional cardioprotection to preconditioned hearts by enhanced phosphorylation of glycogen synthase kinase-3beta Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H748 - H755. [Abstract] [Full Text] [PDF]

				S.-S. Park, H. Zhao, Y. Jang, R. A. Mueller, and Z. Xu N6-(3-Iodobenzyl)-adenosine-5'-N-methylcarboxamide Confers Cardioprotection at Reperfusion by Inhibiting Mitochondrial Permeability Transition Pore Opening via Glycogen Synthase Kinase 3beta J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 124 - 131. [Abstract] [Full Text] [PDF]

				T. Takeda, M. Akao, M. Matsumoto-Ida, M. Kato, H. Takenaka, Y. Kihara, T. Kume, A. Akaike, and T. Kita Serofendic Acid, a Novel Substance Extracted From Fetal Calf Serum, Protects Against Oxidative Stress in Neonatal Rat Cardiac Myocytes J. Am. Coll. Cardiol., May 2, 2006; 47(9): 1882 - 1890. [Abstract] [Full Text] [PDF]

				C. Wang, D. A. Neff, J. G. Krolikowski, D. Weihrauch, M. Bienengraeber, D. C. Warltier, J. R. Kersten, and P. S. Pagel The influence of B-cell lymphoma 2 protein, an antiapoptotic regulator of mitochondrial permeability transition, on isoflurane-induced and ischemic postconditioning in rabbits. Anesth. Analg., May 1, 2006; 102(5): 1355 - 1360. [Abstract] [Full Text] [PDF]

				D. V. Cuong, N. Kim, J. B. Youm, H. Joo, M. Warda, J.-W. Lee, W. S. Park, T. Kim, S. Kang, H. Kim, et al. Nitric oxide-cGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K+ channels in rat hearts Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1808 - H1817. [Abstract] [Full Text] [PDF]

				J.-S. Kim, Y. Jin, and J. J. Lemasters Reactive oxygen species, but not Ca2+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H2024 - H2034. [Abstract] [Full Text] [PDF]

				E. R. Gross and G. J. Gross Ligand triggers of classical preconditioning and postconditioning Cardiovasc Res, May 1, 2006; 70(2): 212 - 221. [Abstract] [Full Text] [PDF]

				S. Lecour, N. Suleman, G. A. Deuchar, S. Somers, L. Lacerda, B. Huisamen, and L. H. Opie Pharmacological Preconditioning With Tumor Necrosis Factor-{alpha} Activates Signal Transducer and Activator of Transcription-3 at Reperfusion Without Involving Classic Prosurvival Kinases (Akt and Extracellular Signal-Regulated Kinase) Circulation, December 20, 2005; 112(25): 3911 - 3918. [Abstract] [Full Text] [PDF]

				J. G. Krolikowski, M. Bienengraeber, D. Weihrauch, D. C. Warltier, J. R. Kersten, and P. S. Pagel Inhibition of Mitochondrial Permeability Transition Enhances Isoflurane-Induced Cardioprotection During Early Reperfusion: The Role of Mitochondrial KATP Channels Anesth. Analg., December 1, 2005; 101(6): 1590 - 1596. [Abstract] [Full Text] [PDF]

				A. Tsang, D. J. Hausenloy, M. M. Mocanu, R. D. Carr, and D. M. Yellon Preconditioning the Diabetic Heart: The Importance of Akt Phosphorylation Diabetes, August 1, 2005; 54(8): 2360 - 2364. [Abstract] [Full Text] [PDF]

				A. Tsang, D. J. Hausenloy, and D. M. Yellon Myocardial postconditioning: reperfusion injury revisited Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H2 - H7. [Full Text] [PDF]

				S. Shanmuganathan, D. J. Hausenloy, M. R. Duchen, and D. M. Yellon Mitochondrial permeability transition pore as a target for cardioprotection in the human heart Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H237 - H242. [Abstract] [Full Text] [PDF]

				J. C. Bopassa, P. Michel, O. Gateau-Roesch, M. Ovize, and R. Ferrera Low-pressure reperfusion alters mitochondrial permeability transition Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2750 - H2755. [Abstract] [Full Text] [PDF]

				H.-Y. Sun, N.-P. Wang, F. Kerendi, M. Halkos, H. Kin, R. A. Guyton, J. Vinten-Johansen, and Z.-Q. Zhao Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1900 - H1908. [Abstract] [Full Text] [PDF]

				G. Wang, D. A. Liem, T. M. Vondriska, H. M. Honda, P. Korge, D. M. Pantaleon, X. Qiao, Y. Wang, J. N. Weiss, and P. Ping Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1290 - H1295. [Abstract] [Full Text] [PDF]

				R. Schulz A new paradigm: cross talk of protein kinases during reperfusion saves life! Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H1 - H2. [Full Text] [PDF]

				A. Tsang, D. J. Hausenloy, M. M. Mocanu, and D. M. Yellon Postconditioning: A Form of "Modified Reperfusion" Protects the Myocardium by Activating the Phosphatidylinositol 3-Kinase-Akt Pathway Circ. Res., August 6, 2004; 95(3): 230 - 232. [Abstract] [Full Text] [PDF]

				M. Ruiz-Meana, P. Pina, D. Garcia-Dorado, A. Rodriguez-Sinovas, I. Barba, E. Miro-Casas, M. Mirabet, and J. Soler-Soler Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition J. Physiol., August 1, 2004; 558(3): 873 - 882. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Hausenloy, D. J Articles by Yellon, D. M Search for Related Content PubMed PubMed Citation Articles by Hausenloy, D. J Articles by Yellon, D. M Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics