Download Mitochondrial permeability transition pore as a target for - AJP

Am J Physiol Heart Circ Physiol 289: H237–H242, 2005;
doi:10.1152/ajpheart.01192.2004.
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
1
Hatter Institute and Center for Cardiology, University College London Hospitals and Medical School, and
Mitochondrial Biology Group, Department of Physiology, University College London, London, United Kingdom
2
Submitted 30 November 2004; accepted in final form 1 March 2005
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
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: [email protected]).
http://www.ajpheart.org
(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
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 ⱕ1 mm and length ⱖ2 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 䡠 7H2O, 1.8 CaCl2 䡠 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 contrac-
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.
0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society
H237
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 2, 2017
Shanmuganathan, Selvaraj, Derek J. Hausenloy, Michael R. Duchen,
and Derek M. Yellon. Mitochondrial permeability transition pore as target
for cardioprotection in the human heart. Am J Physiol Heart Circ Physiol
289: H237–H242, 2005; doi:10.1152/ajpheart.01192.2004.—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 ␮mol/l) and sanglifehrin A (SfA, 1.0 ␮mol/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 hypoxiareoxygenation injury. This suggests that, in the human heart, the
mPTP is a viable target for cardioprotection.
H238
MITOCHONDRIAL PERMEABILITY AND HUMAN HEART
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 䡠 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 ␮M) or SfA
(1.0 ␮M).
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 ␮M;
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).
Fig. 1. Experimental protocols for human atrial trabecula
model of hypoxia-reoxygenation. CsA, cyclosporin A; SfA
sanglifehrin A.
AJP-Heart Circ Physiol • VOL
289 • JULY 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 2, 2017
tile 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 䡠 7H2O, 1.8 CaCl2 䡠 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 ␮mol/l) or SfA
(1.0 ␮mol/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).
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)
MITOCHONDRIAL PERMEABILITY AND HUMAN HEART
H239
Fig. 2. Experimental protocols for human atrial myocyte model of
hypoxia-reoxygenation.
er’s protected least significance difference post hoc test was applied
for between-group comparisons. Results were considered significant
when P ⱕ 0.05.
RESULTS
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
Table 1. Physical characteristics of human atrial trabecula
study groups
Group
Control
CsA
SfA
Hypoxic
preconditioning
Length,
mm
Mass, mg
Area, mm2
Force, g
4.18⫾0.5
3.28⫾0.3
3.43⫾0.3
0.88⫾0.03
0.85⫾0.09
0.75⫾0.04
0.63⫾0.09
0.49⫾0.08
0.47⫾0.04
1.43⫾0.2
1.51⫾0.2
1.30⫾0.1
3.71⫾0.3
0.77⫾0.06
0.60⫾0.01
1.24⫾0.2
Data presented are means ⫾ SE. CsA, cyclosporin A; SfA, sanglifehrin A.
AJP-Heart Circ Physiol • VOL
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 ⱖ 6/group. *P ⬍ 0.001.
289 • JULY 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 2, 2017
Seeded human atrial myocytes were incubated with the fluorescent
dye TMRM (3 ␮M) 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
␮M) for 15 min at 37°C; and 3) SfA group (n ⫽ 10): incubation with
SfA (1.0 ␮M) 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 ⫻40 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, Fish-
H240
MITOCHONDRIAL PERMEABILITY AND HUMAN HEART
Table 2. Effect of inhibiting mPTP opening at time of
reoxygenation on human atrial myocyte viability and
apoptotic and necrotic components of cell death
Mean
SD
%
1.1
0.9
0.7
1.1
100
62.8⫾5.3
91.9⫾4.1*
87.2⫾6.2*
3.1
1.0
1.6
1.2
100
126.3⫾2.3
111.8⫾3.8*
109.8⫾2.7*
3.1
1.0
1.6
1.8
100
86.3⫾2.6
91.3⫾4.1
94.7⫾4.6
Live Cells
Control
Hypoxia
CsA
SfA
17.2
10.8
15.8
15.0
Control
Hypoxia
CsA
SfA
43.4
54.8
48.5
47.6
2.4
2.2
1.7
2.6
Necrotic Cells
6.9
2.5
4.0
2.9
Apoptotic Cells
Control
Hypoxia
CsA
SfA
39.6
34.2
36.2
37.5
7.0
2.5
4.0
4.5
The absolute numbers of cells and the numbers of cells in the 3 different
treatment groups, normalized and expressed as mean ⫾ SE % of the cells
counted in the time control group, are displayed. The presence of the mitochondrial permeability transition pore (mPTP) inhibitors (CsA and SfA) at the
time of reoxygenation 1) increases the number of live cells (annexin V
negative and propidium iodide negative) and 2) attenuates the number of
necrotic cells (annexin V positive and propidium iodide positive) but 3) has no
effect on the number of apoptotic cells (annexin V positive and propidium
iodide negative). *P ⬍ 0.01 compared with hypoxia treatment.
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 hypoxiareoxygenation 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).
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%
AJP-Heart Circ Physiol • VOL
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.
289 • JULY 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 2, 2017
SE
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.
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.
MITOCHONDRIAL PERMEABILITY AND HUMAN HEART
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).
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.
DISCUSSION
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 ⱖ 6/group. *P ⬍ 0.001.
AJP-Heart Circ Physiol • VOL
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 hypoxiareoxygenation 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
We thank the Wellcome Trust for funding the confocal microscope and the
British Heart Foundation for funding this research.
289 • JULY 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 2, 2017
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
H241
H242
MITOCHONDRIAL PERMEABILITY AND HUMAN HEART
REFERENCES
AJP-Heart Circ Physiol • VOL
289 • JULY 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.4 on May 2, 2017
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
13. Duchen MR. Mitochondria and Ca2⫹ in cell physiology and pathophysiology. Cell Calcium 28: 339 –348, 2000.
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.
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.
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.
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.
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.
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 nonfailing human heart; the relation of age and clinical status of patients to
isoproterenol response. J Mol Cell Cardiol 24: 549 –564, 1992.
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.
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.
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.
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.
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.
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.
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␤ mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest
113: 1535–1549, 2004.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.