AJP - Heart AJP: Lung Cellular and Molecular Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 289: H237-H242, 2005; doi:10.1152/ajpheart.01192.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (18)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shanmuganathan, S.
Right arrow Articles by Yellon, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shanmuganathan, S.
Right arrow Articles by Yellon, D. M.

Mitochondrial permeability transition pore as a target for cardioprotection in the human heart

Selvaraj Shanmuganathan,1 Derek J. Hausenloy,1 Michael R. Duchen,2 and Derek M. Yellon1

1Hatter Institute and Center for Cardiology, University College London Hospitals and Medical School, and 2Mitochondrial Biology Group, Department of Physiology, University College London, London, United Kingdom

Submitted 30 November 2004 ; accepted in final form 1 March 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
After an episode of myocardial ischemia, opening of the mitochondrial permeability transition pore (mPTP), at the onset of reperfusion, is a critical determinant of myocyte death. We investigated the role of the mPTP as a target for cardioprotection in the human heart. We subjected human atrial tissue, harvested from patients undergoing cardiac surgery, to a period of lethal hypoxia and investigated the effect of suppressing mPTP opening at the onset of reoxygenation. We found that suppressing mPTP opening at the onset of reoxygenation with known mPTP inhibitors cyclosporin A (CsA, 0.2 µmol/l) and sanglifehrin A (SfA, 1.0 µmol/l) 1) improved recovery of baseline contractile function from 29.4 ± 2.0% under control conditions to 48.7 ± 2.2% with CsA and 46.1 ± 2.3% with SfA (P < 0.01) and 2) improved cell survival from 62.8 ± 5.3% under hypoxic control conditions to 91.4 ± 4.1% with CsA and 87.2 ± 6.2% with SfA (P < 0.001). Furthermore, with a cell model in which oxidative stress was used to induce mPTP opening in human atrial myocytes, we demonstrated directly that CsA and SfA mediated their cardioprotective effects by inhibiting mPTP opening, as evidenced by an extension in the time required to induce mPTP opening from 116 ± 8 s under control conditions to 189 ± 10 s with CsA and 183 ± 12 s with SfA (P < 0.01). We report that suppressing mPTP opening at the onset of reoxygenation protects human myocardium against lethal hypoxia-reoxygenation injury. This suggests that, in the human heart, the mPTP is a viable target for cardioprotection.

hypoxia-reoxygenation; human cardiac muscle

THE OPENING OF THE MITOCHONDRIAL permeability transition pore (mPTP) is a critical determinant of cell death (10). Its opening causes mitochondrial swelling and uncoupling of mitochondrial oxidative phosphorylation, leading to both apoptotic and necrotic cell death (10). In the setting of myocardial ischemia and reperfusion injury, it has been demonstrated that the mPTP remains closed during ischemia and only opens at the onset of reperfusion, when the conditions of high mitochondrial Ca2+ concentration, Pi concentration, ATP depletion, and oxidative stress, factors that potentiate its opening, prevail (12, 19, 30, 33). Suppression of the opening of the mPTP at the onset of reperfusion therefore presents a powerful target for cardioprotection.

In this regard, inhibiting mPTP opening at the time of reperfusion has been demonstrated to be cytoprotective in various organ models including the isolated, perfused rat heart (18, 21, 22, 27), the isolated rat myocyte (23, 31), the isolated rat hepatocyte (3, 32, 33), and rat neuronal tissue (36, 38, 40). However, to the best of our knowledge, the role of the mPTP as a target for cardioprotection at the time of reperfusion has not been examined in human cardiac muscle.

To qualify suppression of opening of the mPTP as a viable strategy for cardioprotection in the clinical arena of myocardial ischemia-reperfusion injury, it is necessary to demonstrate a role for the mPTP as a target for cardioprotection in the human myocardium. In this regard, we investigated for the first time in human cardiac muscle the role of the mPTP as a target for cardioprotection. Using two different human models of hypoxia-reoxygenation injury, we examined whether suppressing mPTP opening at the onset of reoxygenation with the known pharmacological mPTP inhibitors cyclosporin A (CsA) and sanglifehrin A (SfA) offers any cardioprotection. Furthermore, with a cellular model in which oxidative stress is used to induce mPTP opening in human atrial myocytes, we investigated directly whether these pharmacological mPTP inhibitors actually exert their actions by suppressing mPTP opening.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. CsA (Sigma, Poole, UK) was dissolved in 50% ethanol and added to the buffer such that the final ethanol concentration was <0.005%. SfA (Novartis Pharma, Basel, Switzerland) was dissolved in DMSO (Sigma) and added to the buffer such that the final DMSO concentration was <0.01%. Tetramethylrhodamine methyl ester (TMRM; Molecular Probes Europe, Leiden, The Netherlands) was dissolved in DMSO. All other reagents were of standard analytical grade.

Human atrial trabecula model of hypoxia-reoxygenation. Experiments were performed on human atrial trabeculae, isolated from right atrial appendages, harvested from patients undergoing coronary artery bypass surgery. Prior ethical approval for this study was granted by the Ethics and Clinical Investigations Panel of the Middlesex Hospital, London, UK. Patients with a previous history of atrial arrhythmias, treatment with antiarrhythmic drugs, right ventricular failure, or diabetes mellitus were not included in the study.

Atrial trabeculae (of diameter ≤1 mm and length ≥2 mm) were isolated from the atrial appendage specimen, suspended horizontally in an organ bath, and superfused with modified Tyrode buffer comprising (in mM) 118.5 NaCl, 4.8 KCl, 24.8 NaHCO3, 1.2 KH2PO4, 1.44 MgSO4·7H2O, 1.8 CaCl2·2H2O, 10.0 glucose, and 10.0 pyruvic acid, oxygenated with a 95% O2-5% CO2 gas mixture, to maintain pH between 7.35 and 7.45, a partial pressure of O2 between 50 and 60 kPa, and a partial pressure of CO2 between 4.0 and 6.0 kPa. The temperature in the bath was maintained at 37°C with a heat exchanger (Techne Circulator C 85-A, Cambridge, UK). The developed contractile force of the atrial trabeculae was amplified and recorded with Powerlab/8sp (AD Instruments).

Atrial trabeculae were stimulated at 1 Hz and allowed to stabilize for 90 min. They were excluded if by the end of the stabilization period the maximal developed pressure, after stretching, was <1.0 g. They were then subjected to a period of simulated ischemia (SI), which comprised 90 min of perfusion with glucose-free hypoxic Tyrode buffer containing (in mM) 118.5 NaCl, 4.8 KCl, 24.8 NaHCO3, 1.2 KH2PO4, 1.44 MgSO4·7H2O, 1.8 CaCl2·2H2O, 7.0 choline chloride, and 10.0 pyruvic acid and pacing at 3 Hz. The hypoxic buffer was bubbled with 95% N2-5% CO2 to lower the partial pressure of O2 of the buffer in the organ bath to <7 kPa (pH 7.24–7.34). The atrial trabeculae were then subjected to simulated reperfusion comprising perfusion for 120 min with oxygenated Tyrode buffer and pacing at 1 Hz.

Atrial trabeculae were randomly assigned to the following treatment groups (see Fig. 1). In the control group (n = 6), atrial trabeculae were given either normal buffer or buffer containing the 0.005% ethanol or 0.02% DMSO vehicle controls for the first 30 min of the reoxygenation period. In the hypoxic preconditioning group (n = 6), immediately before the lethal 90-min period of hypoxia atrial trabeculae were subjected to 3 min of hypoxic substrate-free buffer and pacing at 3 Hz, followed by 7 min of reoxygenation with the normal oxygenated buffer and pacing at 1 Hz. This group was included as a positive control to verify that cardioprotection could be demonstrated in this atrial trabecula model of hypoxia-reoxygenation (39). In the CsA group (n = 6) and the SfA group (n = 6), after the lethal 90-min hypoxic period trabeculae were given either CsA (0.2 µmol/l) or SfA (1.0 µmol/l) for the first 30 min of reoxygenation, followed by a further 90 min of reoxygenation with normal buffer. These concentrations of CsA and SfA have been demonstrated to inhibit mPTP opening in the isolated, perfused rat heart (7, 18, 21, 22).



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1. Experimental protocols for human atrial trabecula model of hypoxia-reoxygenation. CsA, cyclosporin A; SfA sanglifehrin A.

 
At the end of the reoxygenation period, the contractile function expressed as a percentage of the baseline force of contraction was determined for each atrial trabecula. In addition, the width and length of each atrial trabecula were measured and all specimens were then weighed. The cross-sectional area was calculated from the measured diameter of the atrial trabecula.

Human atrial myocyte model of hypoxia-reoxygenation. Experiments were performed on atrial myocytes isolated from right atrial appendages harvested from patients undergoing coronary artery bypass surgery. Human atrial myocytes were isolated by enzymatic dissociation with both protease and collagenase digestion (20). Because of the fragility of human cardiomyocytes and the difficulty in isolating human myocytes, cell viability after isolation was in the order of 20–30%, which compares favorably with other studies in which human myocytes were used (20). After isolation, the cells were allowed to stabilize for 60 min in oxygenated medium calcium (MC) solution comprising (in mM) 120 NaCl, 5.4 KCl, 5 MgSO4, 5 pyruvate, 20 glucose, 20 taurine, 10 HEPES, and 0.05 Ca2+ (pH 7.4) at 37.0°C.

They were then subjected to lethal SI as follows, by replacing the oxygenated MC buffer with ischemic buffer containing (in mM) 137 NaCl, 12 KCl, 0.49 MgCl2, 0.9 CaCl2·H2O, 4 HEPES, and 20 Na-lactate (16) and incubating them at 37°C for 20 min in a hypoxic chamber (containing 20 g sodium dithionate) in an atmosphere of 0% O2-5% CO2 balanced with argon (BOC Gases). At the end of the SI period, the buffer was replaced with oxygenated MC buffer and incubated at 37.0°C for 30 min (to simulate reperfusion). Cell viability was assessed after 30 min of reoxygenation with light and fluorescent microscopy.

Isolated atrial myocytes were randomly assigned to the following treatment groups (see Fig. 2 for experimental protocol). In the time control group (n = 5), cells were left in normoxic conditions at 37.0°C for the duration of the experimental protocol to act as time controls. In the hypoxic control group, cells were subjected to 20 min of hypoxia followed by 30-min reoxygenation with either normal buffer (n = 6) or buffer containing the vehicle controls, either DMSO (n = 3) or ethanol (n = 3). In the CsA group (n = 6) and the SfA group (n = 6), cells were subjected to 20 min of hypoxia followed by 30-min reoxygenation with buffer containing either CsA (0.2 µM) or SfA (1.0 µM).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2. Experimental protocols for human atrial myocyte model of hypoxia-reoxygenation.

 
At the end of the 20-min reoxygenation period, the cells were incubated in the dark for 10 min with annexin V-Fluos (AV, 20 µM; Roche). Propidium iodide (PI, 3 nM; Sigma) was then added to the myocytes, and the samples were analyzed immediately with a fluorescent microscope. AV was shown previously to detect the early stages of apoptosis by binding to the phosphatidylserine residues, which are translocated to the external face of the cell membrane (1). Cellular necrosis was determined with PI, which binds to the nuclei of cells whose plasma membrane have become permeable (17).

For each treatment group, the numbers of rod-shaped, AV-stained, and PI-stained cells were counted in three randomly chosen fields by an operator blinded to the treatment and an average was taken. Results were then expressed as a percentage of the cells counted in the time control group and were assigned to three categories: 1) live cells (AV negative, PI negative, and rod-shaped), 2) apoptotic cells (AV positive, PI negative), and 3) necrotic cells (AV positive, PI positive).

Human atrial myocyte model for induction and detection of mPTP opening. Human myocytes were isolated as above and suspended in the MC solution. The cells were then seeded onto laminin-coated 25-mm-diameter round coverslips and incubated at 37.0°C for 50 min to allow the cells to adhere to the coverslips. Opening of the mPTP in adult rat myocytes was induced and detected with a well-characterized cellular model of oxidative stress (11, 13, 14, 23–26, 28).

Seeded human atrial myocytes were incubated with the fluorescent dye TMRM (3 µM) for 15 min at 37°C, washed, and visualized with confocal fluorescence microscopy as described below. TMRM, a lipophilic cation, accumulates selectively in mitochondria according to the mitochondrial membrane potential (15). Laser illumination of mitochondrial TMRM generates oxidative stress, used in this model to induce mPTP opening, that is detected by the loss of mitochondrial membrane potential, which in this model appears as an increase in TMRM fluorescence intensity. The relatively high concentration of TMRM in the mitochondria causes autoquenching of fluorescence, such that the fluorescence signal becomes a nonlinear function of dye concentration; therefore, mitochondrial depolarization results in the loss of dye into the cytosol, where the signal increases (4). After loading with TMRM, the cells were randomly assigned to the following treatment groups: 1) control group (n = 11 in total): incubation in MC medium in the presence or absence of the DMSO and ethanol vehicle controls; 2) CsA group (n = 10): incubation with CsA (0.2 µM) for 15 min at 37°C; and 3) SfA group (n = 10): incubation with SfA (1.0 µM) for 15 min at 37°C.

The coverslip with adherent myocytes was placed in a chamber and mounted on the stage of a Zeiss 510 CLSM confocal microscope equipped with x40 oil immersion, quartz objective lens (numerical aperture 1.3). The cells were illuminated with the 543-nm emission line of a HeNe laser. For all photosensitization experiments, all conditions of the confocal imaging system (laser power, confocal pinhole, optical slice, and detector sensitivity) were identical, to ensure comparability between experiments. The fluorescence of TMRM was collected with a 585-nm long-pass filter, and images were analyzed with Zeiss software (LSM 2.8).

Statistical analysis. All results are presented as group means ± SE. For comparison between more than two groups, factorial one-way ANOVA was used. Where a significant F-value was obtained, Fisher's protected least significance difference post hoc test was applied for between-group comparisons. Results were considered significant when P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Samples were obtained from 32 patients with stable ischemic heart disease (25 men and 7 women; age range 48–75 yr, mean age 67 yr). If two or three suitable trabeculae could be isolated from a single atrial appendage, each atrial trabeculae was allocated to one of three groups (3 sets of apparatus were used simultaneously). Ten atrial trabeculae were excluded because of poor baseline contractile function.

Human atrial trabecula model of hypoxia-reoxygenation. Baseline characteristics were similar in all groups (see Table 1). Figure 3 portrays the contractile function expressed as a percentage of the baseline contractile function measured at the end of the period of stabilization. In all treatment groups, SI resulted in a similar reduction in contractile function, which was evident within 15–30 min after the onset of SI, with a further reduction in contractile function occurring over the ensuing SI period. During reoxygenation in the control group, there was an improvement in contractile function to a maximum of 29.4 ± 2.0% of the baseline developed force by the end of the reoxygenation period (Fig. 3). Hypoxic preconditioning of the atrial trabeculae before the lethal hypoxia-reoxygenation injury resulted in a significant improvement in the force of contraction compared with the control group (29.4 ± 2.0% in control vs. 48.7 ± 4.3% with hypoxic preconditioning; P < 0.001; Fig. 3). Treatment with CsA and SfA for the first 30 min of the reoxygenation period resulted in a significant improvement in the force of contraction compared with the control group (29.4 ± 2.0% in control vs. 48.7 ± 2.2% with CsA and 46.1 ± 2.3% with SfA; P < 0.001; Fig. 3).


View this table:
[in this window]
[in a new window]
  		Table 1. Physical characteristics of human atrial trabecula study groups

 


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 3. Change in human atrial trabecula contractile function expressed as % of baseline contractile function at different time intervals (min) during simulated ischemia (SI) and reoxygenation (R). The presence of either CsA or SfA for the first 30 min of reoxygenation improved the recovery of contractile function to a similar extent as hypoxic preconditioning (HPC). Values are means ± SE; n ≥ 6/group. *P < 0.001.

 
Human atrial myocyte model of hypoxia-reoxygenation. After exposure of the atrial myocytes to hypoxia-reoxygenation, the numbers of live, apoptotic, and necrotic cells were determined and the numbers of live, apoptotic, and necrotic cells were then normalized to the percentage of cells in the time control group. In the hypoxic control group of atrial myocytes, exposure to 20 min of hypoxia and 30-min reoxygenation resulted in 62.8 ± 5.3% live cells, 126.3 ± 2.3% necrotic cells, and 86.3 ± 2.6% apoptotic cells (see Table 2). After exposure to 20 min of hypoxia, treatment with CsA and SfA for the 30-min reoxygenation period resulted in a significant improvement in the percentage of live cells [62.8 ± 5.3% in the hypoxic control vs. 91.4 ± 4.1% with CsA (P = 0.0006) and 87.2 ± 6.2% with SfA (P = 0.003); Table 2]. Treatment with CsA and SfA also resulted in a significant reduction in the percentage of necrotic cells compared with the hypoxic control group [126.3 ± 2.3% in the hypoxic control vs. 111.8 ± 3.8% with CsA (P = 0.01) and 109.8 ± 2.7% with SfA (P = 0.004) Table 2]. However, the percentage of apoptotic cells in all three treatment groups did not differ [86.3 ± 2.6% in the hypoxic control vs. 91.3 ± 4.1% with CsA (P = 0.42) and 94.7 ± 4.6% with SfA (P = 0.18); Table 2]. The presence of the vehicle controls DMSO or ethanol did not influence myocyte viability. Because of the fragility of the human myocytes, cell viability after atrial myocyte isolation was quite poor. Because of this, had the cell isolation been sorted for viable cells before the hypoxia-reoxygenation experiments, the treatment effect of the mPTP inhibitors might have been augmented.


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of inhibiting mPTP opening at time of reoxygenation on human atrial myocyte viability and apoptotic and necrotic components of cell death

 
Human atrial myocyte model for induction and detection of mPTP opening. Confocal fluorescence imaging of human atrial myocytes loaded with TMRM revealed mitochondria as fluorescent bands orientated with the longitudinal axis of the cell (see Fig. 4A). TMRM localizes selectively to the mitochondria according to the mitochondrial membrane potential. Figure 4 shows representative images extracted from a time sequence in which a human atrial myocyte was loaded with TMRM and subjected to laser-induced oxidative stress and demonstrates the sequential changes that take place in mitochondrial membrane potential over time. Laser illumination first induces global mitochondrial membrane depolarization, signifying mPTP opening, of the cell in the upper part of the image (Fig. 4B). Continued laser-induced oxidative stress results in the global mitochondrial membrane depolarization of the cell in the lower part of the image (Fig. 4C).



View larger version (49K):
[in this window]
[in a new window]
  		Fig. 4. Representative time series showing confocal fluorescence images of representative human atrial myocytes loaded with tetramethylrhodamine methyl ester (TMRM) and subjected to laser-induced oxidative stress over time. A: time 0 s: before oxidative stress. B: time 80 s: oxidative stress induces mitochondrial depolarization, represented by an increase in the intensity of fluorescence, signifying opening of the mitochondrial permeability transition pore (mPTP). C: time 95 s: with continued oxidative stress, global mitochondrial depolarization occurs, indicating opening of the mPTP.

 
The time taken to induce mPTP opening, indicated by global mitochondrial membrane depolarization, was 115.6 ± 7.8 s in the control group (Fig. 5). The presence of either DMSO or ethanol vehicle did not influence the time required to induce mPTP opening. The presence of CsA or SfA significantly prolonged the time taken to induce mPTP opening from 115.6 ± 7.8 s in control to 189.1 ± 10.2 s with CsA (P < 0.0001; Fig. 5) and 183 ± 12.2 s with SfA (P < 0.0001; Fig. 5), indicating that these drugs were able to delay oxidative stress-induced mPTP opening.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5. The effect of CsA and SfA hypoxic preconditioning on the time taken to induce global mitochondrial depolarization, indicating the opening of the mPTP, in TMRM-loaded human atrial myocytes. Compared with the control group, both CsA and SfA extended the time required to induce mPTP opening. The DMSO and ethanol vehicle controls did not affect the time taken to induce mPTP opening. Values are means ± SE; n ≥ 6/group. *P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have demonstrated for the first time with human cardiac muscle the role of the mPTP as a viable target for myocardial protection. Subjecting human atrial tissue to two different models of hypoxia-reoxygenation (to simulate hypoxia-reoxygenation injury), we found that pharmacologically inhibiting mPTP opening at the time of reoxygenation was cardioprotective. Using a cell model for inducing and detecting mPTP opening, we then demonstrated for the first time that the cardioprotective effects induced by CsA and SfA at the time of reoxygenation were mediated by the inhibition of mPTP opening.

The human atrial trabecula model has been demonstrated by our group (2, 5, 6, 37, 39) and others (8, 9) to be a reproducible and robust model of hypoxia-reoxygenation injury, with the percentage recovery of baseline contractile function a reproducible measure of protection. Using this model, we demonstrated that inhibition of mPTP opening for the first 30 min of reoxygenation was able to improve the recovery of contractile function to levels similar to those obtained by hypoxic preconditioning, implicating the mPTP as a critical determinant of hypoxia-reoxygenation injury in human myocardium.

In the next part of the study, we used a human atrial myocyte model of hypoxia-reoxygenation injury to demonstrate the protective effect of inhibiting mPTP opening at the time of reoxygenation on cell viability and on the mode of cell death. Interestingly, we found that inhibiting mPTP opening at the time of reoxygenation improved cell viability, with an attenuation in necrotic but not apoptotic cell death. Inhibition of mPTP opening has been demonstrated to protect against both necrotic and apoptotic cell death (10). However, in our human model of hypoxia-reoxygenation injury we were only able to demonstrate a reduction in the necrotic component of cell death. We can speculate that this may be due to the limited reoxygenation time. It may well be that if we had extended the reperfusion time a difference in the apoptotic component of cell death might have been observed between control and treatment groups. An alternative explanation could be that the opening of the mPTP only mediates necrotic and not apoptotic cell death, as suggested by a recent study that demonstrated that the overexpression of cyclophilin D (a component of the mPTP) promoted necrotic cell death but appeared to inhibit apoptotic cell death (29).

The opening of the mPTP has been demonstrated to be a critical determinant of cell death in many different animal models of ischemia-reperfusion injury, and to our knowledge this study is the first to demonstrate a role for the mPTP in the human heart. A previous study (35) demonstrated that CsA could protect slices of human atrial tissue from hypoxia-reoxygenation injury, but in that study CsA was given before the period of hypoxia and the mPTP was not investigated. In our study, we specifically administered two different mPTP inhibitors at the time of reoxygenation, the time period when the mPTP is believed to open. In addition, we demonstrated directly in the human atrial myocyte that these drugs exert their protective effect by inhibiting mPTP opening.

In this study, it was important to demonstrate that two different known mPTP inhibitors were cardioprotective in our human atrial models of hypoxia-reoxygenation injury, especially because CsA can also protect by inhibiting calcineurin. Importantly, the mPTP inhibitor SfA is a more specific inhibitor of mPTP as it does not inhibit calcineurin (7, 34).

In this study, the mPTP inhibitors were given at the time of reoxygenation, immediately after the period of hypoxia, to target the opening of the mPTP that has been demonstrated to occur during the first few minutes of reoxygenation/reperfusion (12, 19, 30, 33). The implications of these findings are of crucial importance in the clinical arena of myocardial protection, in which a cardioprotective strategy that can be applied during the reperfusion phase is far easier to implement than one applied before the index ischemic episode given the unpredictable onset of an acute myocardial infarction. Therefore, in the clinical settings of ischemia-reperfusion injury, such as after an acute myocardial infarction or at the time of cardiac surgery, intervening at the time of reperfusion offers a viable cardioprotective strategy that is under the control of the operator.

In conclusion, we show for the first time in the human muscle model that inhibiting the opening of the mPTP at the time of reoxygenation protects the human myocardium from lethal hypoxia-reoxygenation injury. Inhibition of the opening of mPTP may therefore provide a novel target for cardioprotection in the clinical settings of reperfusion.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We thank the Wellcome Trust for funding the confocal microscope and the British Heart Foundation for funding this research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. M. Yellon, Hatter Institute and Center for Cardiology, University College London Hospitals and Medical School, Grafton Way, London WC1E 6DB, UK (E-mail: hatter-institute{at}ucl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, and Willems GM. Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers. J Biol Chem 265: 4923–4928, 1990.[Abstract/Free Full Text]
  2. Bell SP, Sack MN, Patel A, Opie LH, and Yellon DM. Delta opioid receptor stimulation mimics ischemic preconditioning in human heart muscle. J Am Coll Cardiol 36: 2296–2302, 2000.[Abstract/Free Full Text]
  3. Broekemeier KM, Carpenter-Deyo L, Reed DJ, and Pfeiffer DR. Cyclosporin A protects hepatocytes subjected to high Ca2+ and oxidative stress. FEBS Lett 304: 192–194, 1992.[CrossRef][Web of Science][Medline]
  4. Bunting JR, Phan TV, Kamali E, and Dowben RM. Fluorescent cationic probes of mitochondria. Metrics and mechanism of interaction. Biophys J 56: 979–993, 1989.[Web of Science][Medline]
  5. Carr CS, Grover GJ, Pugsley WB, and Yellon DM. Comparison of the protective effects of a highly selective ATP-sensitive potassium channel opener and ischemic preconditioning in isolated human atrial muscle. Cardiovasc Drugs Ther 11: 473–478, 1997.[CrossRef][Web of Science][Medline]
  6. Carr CS, Hill RJ, Masamune H, Kennedy SP, Knight DR, Tracey WR, and Yellon DM. Evidence for a role for both the adenosine A1 and A3 receptors in protection of isolated human atrial muscle against simulated ischaemia. Cardiovasc Res 36: 52–59, 1997.[Abstract/Free Full Text]
  7. Clarke SJ, McStay GP, and 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 277: 34793–34799, 2002.[Abstract/Free Full Text]
  8. Cleveland JC Jr, Meldrum DR, Cain BS, Banerjee A, and Harken AH. Oral sulfonylurea hypoglycemic agents prevent ischemic preconditioning in human myocardium. Two paradoxes revisited. Circulation 96: 29–32, 1997.[Abstract/Free Full Text]
  9. Cleveland JC Jr, Meldrum DR, Rowland RT, Banerjee A, and Harken AH. Adenosine preconditioning of human myocardium is dependent upon the ATP-sensitive K+ channel. J Mol Cell Cardiol 29: 175–182, 1997.[CrossRef][Web of Science][Medline]
  10. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J 341: 233–249, 1999.
  11. De Giorgi F, Lartigue L, Bauer MK, Schubert A, Grimm S, Hanson GT, Remington SJ, Youle RJ, and Ichas F. The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J 16: 607–609, 2002.[Free Full Text]
  12. Di Lisa F, Menabo R, Canton M, Barile M, and 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 276: 2571–2575, 2001.[Abstract/Free Full Text]
  13. Duchen MR. Mitochondria and Ca2+ in cell physiology and pathophysiology. Cell Calcium 28: 339–348, 2000.[CrossRef][Web of Science][Medline]
  14. Duchen MR, Leyssens A, and Crompton M. Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes. J Cell Biol 142: 975–988, 1998.[Abstract/Free Full Text]
  15. Ehrenberg B, Montana V, Wei MD, Wuskell JP, and Loew LM. Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J 53: 785–794, 1988.[Web of Science][Medline]
  16. Esumi K, Nishida M, Shaw D, Smith TW, and Marsh JD. NADH measurements in adult rat myocytes during simulated ischemia. Am J Physiol Heart Circ Physiol 260: H1743–H1752, 1991.[Abstract/Free Full Text]
  17. Foglieni C, Meoni C, and Davalli AM. Fluorescent dyes for cell viability: an application on prefixed conditions. Histochem Cell Biol 115: 223–229, 2001.[Web of Science][Medline]
  18. Griffiths EJ and Halestrap AP. Protection by Cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts. J Mol Cell Cardiol 25: 1461–1469, 1993.[CrossRef][Web of Science][Medline]
  19. Griffiths EJ and Halestrap AP. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J 307: 93–98, 1995.
  20. Harding SE, Jones SM, O'Gara P, del Monte F, Vescovo G, and Poole-Wilson PA. Isolated ventricular myocytes from failing and non-failing human heart; the relation of age and clinical status of patients to isoproterenol response. J Mol Cell Cardiol 24: 549–564, 1992.[CrossRef][Web of Science][Medline]
  21. Hausenloy DJ, Duchen MR, and Yellon DM. Inhibiting mitochondrial permeability transition pore opening at reperfusion protects against ischaemia-reperfusion injury. Cardiovasc Res 60: 617–625, 2003.[Abstract/Free Full Text]
  22. Hausenloy DJ, Maddock HL, Baxter GF, and Yellon DM. Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning? Cardiovasc Res 55: 534–543, 2002.[Abstract/Free Full Text]
  23. Hausenloy DJ, Yellon DM, Mani-Babu S, and Duchen MR. Preconditioning protects by inhibiting the mitochondrial permeability transition. Am J Physiol Heart Circ Physiol 287: H841–H849, 2004.[Abstract/Free Full Text]
  24. Huser J and Blatter LA. Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore. Biochem J 343: 311–317, 1999.
  25. Ichas F, Jouaville LS, and Mazat JP. Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals. Cell 89: 1145–1153, 1997.[CrossRef][Web of Science][Medline]
  26. Jacobson J and Duchen MR. Mitochondrial oxidative stress and cell death in astrocytes—requirement for stored Ca2+ and sustained opening of the permeability transition pore. J Cell Sci 115: 1175–1188, 2002.[Abstract/Free Full Text]
  27. Javadov SA, Clarke S, Das M, Griffiths EJ, Lim KH, and Halestrap AP. Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart. J Physiol 549: 513–524, 2003.[Abstract/Free Full Text]
  28. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K, Antos CL, Olson EN, and Sollott SJ. Glycogen synthase kinase-3{beta} mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 113: 1535–1549, 2004.[CrossRef][Web of Science][Medline]
  29. Li Y, Johnson N, Capano M, Edwards M, and Crompton M. Cyclophilin-D promotes the mitochondrial permeability transition but has opposite effects on apoptosis and necrosis. Biochem J 383: 101–109, 2004.[CrossRef][Web of Science][Medline]
  30. Murata M, Akao M, O'Rourke B, and Marban E. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca2+ overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection. Circ Res 89: 891–898, 2001.[Abstract/Free Full Text]
  31. Nazareth W, Yafei N, and Crompton M. Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A. J Mol Cell Cardiol 23: 1351–1354, 1991.[CrossRef][Web of Science][Medline]
  32. Pastorino JG, Snyder JW, Serroni A, Hoek JB, and Farber JL. Cyclosporin and carnitine prevent the anoxic death of cultured hepatocytes by inhibiting the mitochondrial permeability transition. J Biol Chem 268: 13791–13798, 1993.[Abstract/Free Full Text]
  33. Qian T, Nieminen AL, Herman B, and Lemasters JJ. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am J Physiol Cell Physiol 273: C1783–C1792, 1997.[Abstract/Free Full Text]
  34. Sanglier JJ, Quesniaux V, Fehr T, Hofmann H, Mahnke M, Memmert K, Schuler W, Zenke G, Gschwind L, Maurer C, and Schilling W. 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) 52: 466–473, 1999.[Medline]
  35. Schneider A, Ad N, Izhar U, Khaliulin I, Borman JB, and Schwalb H. Protection of myocardium by cyclosporin A and insulin: in vitro simulated ischemia study in human myocardium. Ann Thorac Surg 76: 1240–1245, 2003.[Abstract/Free Full Text]
  36. Shiga Y, Onodera H, Matsuo Y, and Kogure K. Cyclosporin A protects against ischemia-reperfusion injury in the brain. Brain Res 595: 145–148, 1992.[CrossRef][Web of Science][Medline]
  37. Speechly-Dick ME, Grover GJ, and Yellon DM. Does ischemic preconditioning in the human involve protein kinase C and the ATP-dependent K+ channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ Res 77: 1030–1035, 1995.[Abstract/Free Full Text]
  38. Uchino H, Elmer E, Uchino K, Li PA, He QP, Smith ML, and Siesjo BK. Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat. Brain Res 812: 216–226, 1998.[CrossRef][Web of Science][Medline]
  39. Walker DM, Walker JM, Pugsley WB, Pattison CW, and Yellon DM. Preconditioning in isolated superfused human muscle. J Mol Cell Cardiol 27: 1349–1357, 1995.[CrossRef][Web of Science][Medline]
  40. Yoshimoto T, Uchino H, He QP, Li PA, and Siesjo BK. Cyclosporin A, but not FK506, prevents the downregulation of phosphorylated Akt after transient focal ischemia in the rat. Brain Res 899: 148–158, 2001.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
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]

Home page
 Am. J. Physiol. Renal Physiol.Home page
K. Devalaraja-Narashimha, A. M. Diener, and B. J. Padanilam
Cyclophilin D gene ablation protects mice from ischemic renal injury
Am J Physiol Renal Physiol, September 1, 2009; 297(3): F749 - F759.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
C. Fu, C. Wu, T. Liu, T. Ago, P. Zhai, J. Sadoshima, and H. Li
Elucidation of Thioredoxin Target Protein Networks in Mouse
Mol. Cell. Proteomics, July 1, 2009; 8(7): 1674 - 1687.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
L. Puente-Maestu, J. Perez-Parra, R. Godoy, N. Moreno, A. Tejedor, A. Torres, A. Lazaro, A. Ferreira, and A. Agusti
Abnormal Transition Pore Kinetics and Cytochrome C Release in Muscle Mitochondria of Patients with Chronic Obstructive Pulmonary Disease
Am. J. Respir. Cell Mol. Biol., June 1, 2009; 40(6): 746 - 750.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
E. N. Dedkova and L. A. Blatter
Characteristics and function of cardiac mitochondrial nitric oxide synthase
J. Physiol., February 15, 2009; 587(4): 851 - 872.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
R. G. Katare, M. Ando, Y. Kakinuma, M. Arikawa, T. Handa, F. Yamasaki, and T. Sato
Vagal nerve stimulation prevents reperfusion injury through inhibition of opening of mitochondrial permeability transition pore independent of the bradycardiac effect.
J. Thorac. Cardiovasc. Surg., January 1, 2009; 137(1): 223 - 231.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Thorac. Surg.Home page
B. G. Leshnower, S. Kanemoto, M. Matsubara, H. Sakamoto, R. Hinmon, J. H. Gorman III, and R. C. Gorman
Cyclosporine Preserves Mitochondrial Morphology After Myocardial Ischemia/Reperfusion Independent of Calcineurin Inhibition
Ann. Thorac. Surg., October 1, 2008; 86(4): 1286 - 1292.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
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]

Home page
 J. Pharmacol. Exp. Ther.Home page
F. N. Obame, C. Plin-Mercier, R. Assaly, R. Zini, J. L. Dubois-Rande, A. Berdeaux, and D. Morin
Cardioprotective Effect of Morphine and a Blocker of Glycogen Synthase Kinase 3{beta}, SB216763 [3-(2,4-Dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], via Inhibition of the Mitochondrial Permeability Transition Pore
J. Pharmacol. Exp. Ther., July 1, 2008; 326(1): 252 - 258.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
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]

Home page
 Cardiovasc ResHome page
M. Ruiz-Meana, A. Rodriguez-Sinovas, A. Cabestrero, K. Boengler, G. Heusch, and D. Garcia-Dorado
Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury
Cardiovasc Res, January 15, 2008; 77(2): 325 - 333.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
A. Jahangir, S. Sagar, and A. Terzic
Aging and cardioprotection
J Appl Physiol, December 1, 2007; 103(6): 2120 - 2128.
[Abstract] [Full Text] [PDF]

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

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. H. Kang, W. S. Park, N. Kim, J. B. Youm, M. Warda, J.-H. Ko, E. A Ko, and J. Han
Mitochondrial Ca2+-activated K+ channels more efficiently reduce mitochondrial Ca2+ overload in rat ventricular myocytes
Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H307 - H313.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
G. Kroemer, L. Galluzzi, and C. Brenner
Mitochondrial Membrane Permeabilization in Cell Death
Physiol Rev, January 1, 2007; 87(1): 99 - 163.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
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]

Home page
 SEMIN CARDIOTHORAC VASC ANESTHHome page
J. G. Bovill
Intravenous Anesthesia for the Patient with Left Ventricular Dysfunction
Seminars in Cardiothoracic and Vascular Anesthesia, March 1, 2006; 10(1): 43 - 48.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (18)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shanmuganathan, S.
Right arrow Articles by Yellon, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shanmuganathan, S.
Right arrow Articles by Yellon, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.