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Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Ischemia-reperfusion injury
induces cell death, but the responsible mechanisms are not understood.
This study examined mitochondrial depolarization and cell death during
ischemia and reperfusion. Contracting cardiomyocytes were
subjected to 60-min ischemia followed by 3-h reperfusion.
Mitochondrial membrane potential (
m) was assessed
with tetramethylrhodamine methyl ester. During ischemia, m decreased to 24 ± 5.5% of baseline, but no
recovery was evident during reperfusion. Cell death assessed by Sytox
Green was minimal during ischemia but averaged 66 ± 7%
after 3-h reperfusion. Cyclosporin A, an inhibitor of mitochondrial
permeability transition, was not protective. However, pharmacological
antioxidants attenuated the fall in m during
ischemia and cell death after reperfusion and decreased lipid
peroxidation as assessed with C11-BODIPY. Cell death was also
attenuated when residual O2 was scavenged from the
perfusate, creating anoxic ischemia. These results suggested that reactive oxygen species (ROS) were important for the decrease in
m during ischemia. Finally,
143B-
0 osteosarcoma cells lacking a mitochondrial
electron transport chain failed to demonstrate a depletion of
m during ischemia and were significantly
protected against cell death during reperfusion. Collectively, these
studies identify a central role for mitochondrial ROS generation during
ischemia in the mitochondrial depolarization and subsequent
cell death induced by ischemia and reperfusion in this model.
reactive oxygen species; hypoxia; oxidants
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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REACTIVE OXYGEN SPECIES (ROS) have been implicated as participants in the myocardial damage induced by ischemia-reperfusion (I/R) (1, 4, 9, 17, 21, 25). Most studies have focused on the importance of oxidant stress generated during reperfusion, when a burst of ROS is generated after oxygen is reintroduced into the system after a prolonged period of ischemia (32, 39). However, growing evidence suggests that oxidant stress begins during ischemia before reperfusion. For example, in cardiomyocytes subjected to simulated I/R, we observed (32, 33) an increase in ROS generation during ischemia followed by a large burst of oxidant production during the first few minutes after reoxygenation. In that model, antioxidants were more protective when given throughout the experiment than when given only at reperfusion, which supports the idea that oxidants generated during the ischemic phase contribute to cell injury and are important determinants of cell survival and recovery of function (2, 35).
ROS generation cannot occur during ischemia unless some residual O2 is still present. Previous studies using cardiomyocytes revealed that trace levels of O2 are still detectable during simulated ischemia (PO2 = 5-7 mmHg). During ischemia, indexes of oxidant stress were attenuated by mitochondrial electron transport inhibitors, suggesting that the ROS are generated by mitochondria (2, 33, 34). Collectively, these observations support the notion that superoxide is generated during ischemia despite the conditions of low O2 concentration ([O2]) (11), and they suggest that these oxidants may play an important role in determining cell survival during I/R.
Although previous studies indicate that oxidants generated during
ischemia may contribute to cell damage, the specific mechanism by which these ROS disrupt cellular function is not known. The present
study sought to clarify the physiological consequences of oxidants
generated during ischemia before reperfusion. We hypothesized that oxidant stress generated at the mitochondria during
ischemia could contribute to a loss of mitochondrial membrane
potential (m), which in turn could contribute to the
overall cellular injury and survival.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell culture and perfusion system. Embryonic chick cardiomyocytes were prepared as previously described (35) and were grown on glass coverslips in a humidified incubator. Experiments were performed on spontaneously contracting cells at 3-5 days after isolation, under controlled O2-CO2 conditions at 37°C on an inverted microscope. A flow-through chamber was created by clamping a stainless steel spacer ring between two coverslips, allowing perfusion of the space between with buffered salt solutions (BSS; 0.5 ml/min) equilibrated with O2-CO2 gas mixtures in a water-jacketed column. Stainless steel tubing connecting the column to the chamber prevented diffusive entry of ambient O2 through the tubing wall.
I/R model. Cells were equilibrated for 30 min by superfusion with BSS (in mM: 120 NaCl, 18 NaHCO3, 4 KCl, 1 MgSO4, 0.8 NaH2PO4, 1.4 CaCl2, and 5.6 glucose; 5% CO2, pH 7.35). During simulated ischemia, cells were superfused with a variation of BSS containing 20 mM 2-deoxyglucose (2-DOG) to inhibit glycolysis, zero glucose, 8 mM K+, 5 mM lactate, and low [O2] and hypercarbia (pH = 6.8) obtained by bubbling with 80% N2-20% CO2. During hypoxic acidosis, cells were superfused with BSS bubbled with 80% N2-20% CO2. In other experiments, complete anoxia was achieved in the ischemic or hypoxic medium by adding EC-Oxyrase (10 µl/ml), an oxidase mixture that reduces O2 to H2O. Anoxia was confirmed with an optical phosphorescence quenching method using a porphryin probe in solution to measure PO2 within the perfusion chamber (Oxyspot) (24). After ischemia or hypoxia/anoxia (1 h), reperfusion was carried out with normoxic BSS (3 h). Tetramethylrhodamine methyl ester (TMRE) measurements were obtained during baseline, throughout ischemia, and during the first 90 min of reperfusion.
Mitochondrial membrane potential.
m was assessed with TMRE. This cationic dye enters
the cells and accumulates within mitochondria according to the Nernst equation (12). Cells were loaded with TMRE (100 nM) at
37°C for 45 min; studies were carried out in the continued presence of the dye (10 nM). Mean fluorescence intensity was measured every minute (excitation 535 nm, emission 610 nm) for a field of cells. Under
these nonquenching conditions, mitochondrial depolarization with FCCP
causes an immediate decrease in TMRE fluorescence, whereas oligomycin
causes an immediate increase (6). Fluorescence intensity is expressed as the percentage of initial brightness after background subtraction.
Cell viability.
Viability was measured in the same field of cells used to assess
m. Dead cells were identified with Sytox Green, a
membrane-impermeant dye that is excluded from cells when the plasma
membrane is intact. In dying cells with increased plasma membrane
permeability, nuclear fluorescence becomes apparent. To assess cell
death in a field of cells, a fluorescent image (×10 objective) was
acquired and the number of fluorescent nuclei was counted (Metamorph;
Universal Imaging). Digitonin (300 µM) was added to permeabilize all
cells in the field. Cell counts were then repeated, and the previous counts were normalized to that value.
Lipid peroxidation assay. Cardiomyocytes were loaded with C11-BODIPY (10 µM), and fluorescence was measured during baseline, ischemia, and reperfusion. Because of its lipophilic nature, this fluorophore localizes to cell membranes and can be used to assess oxidative stress in that environment (27). On oxidation, the fluorescence (excitation 480 nm, emission 525 nm) of C11-BODIPY increases.
Generation of respiration-deficient 0-cells.
To clarify the significance of mitochondrial ROS generation for
membrane damage and cell death during ischemia,
mitochondria-deficient cells (0-cells) were generated
from wild-type 143B osteosarcoma cells (American Type Culture
Collection) (22). The 0-cells were
generated by incubating rapidly dividing wild-type cells with ethidium
bromide (50 ng/ml), which inhibits replication of mitochondrial DNA
(8). The mitochondrial DNA encodes specific subunits that
are critical for electron transport, so 0-cells do not
possess a functional electron transport system and cannot generate ATP
or ROS in mitochondria. Cells were maintained in medium supplemented
with pyruvate (2 mM), uridine (50 µg/ml), and 5-bromouridine (0.015 mg/ml). Loss of mitochondrial DNA was confirmed by
semiquantitative PCR and by loss of cell viability when uridine
supplements to the media were withdrawn.
Reagents and analysis. Reagents were obtained from Sigma, and fluorophores were obtained from Molecular Probes. Replicate experiments were carried out with separate coverslips. Treatment and control group were matched by using cells isolated on the same day.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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To assess changes in m, contracting
cardiomyocytes on coverslips were loaded with TMRE, placed in a
flow-through chamber on an inverted microscope, and superfused with BSS
(37°C) equilibrated with normoxic (21% O2, 5%
CO2) gas. TMRE loading properties in cardiomyocytes, and
the nonquenching characteristics of this fluorophore in assessing
membrane potential under nonquenching conditions were reported
previously (6). Stable levels of fluorescence were
typically observed under baseline conditions (Fig.
1A). This fluorescence was
rapidly dissipated on addition of the protonophore FCCP, consistent
with the expected loss of m induced by this uncoupling agent.
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To assess the effects of ischemia on m, cells
loaded with TMRE were studied for 30 min under baseline normoxic
conditions followed by 60 min of simulated ischemia. During
ischemia, a progressive decrease in TMRE fluorescence was
observed, reaching 24.0 ± 5.5% of the initial intensity after
1 h. Minimal recovery of TMRE fluorescence was seen after return
to normoxia (reperfusion; Fig. 1B). Cell death within the
same field of cells was minimal (<5%) at the end of ischemia
but increased significantly during 3-h reperfusion (Fig.
1C). Inspection of TMRE fluorescence images revealed that the majority of cells lost all fluorescence during ischemia and some cells retained some fluorescence at an attenuated level. Reperfusion was not associated with significant recovery of
fluorescence in either case.
Mitochondrial depolarization could conceivably be caused by activation
of the mitochondrial permeability transition (MPT) pore, a
high-conductance putative channel in the inner mitochondrial membrane
(10). The opening of the MPT pore has been suggested to
contribute to the decrease in m during I/R injury
(18). Cyclosporin A inhibits the opening of this pore and
was therefore used to evaluate its contribution to membrane
depolarization and cell death during I/R. Cyclosporin A (0.2 µM) had
no significant effect on membrane depolarization during simulated
ischemia (Fig. 2A) and
had no significant effect on cell death after 3-h reperfusion (Fig.
2B). Additional studies using a higher concentration (0.5 µM) also failed to abolish the fall in TMRE fluorescence (data not
shown). These results suggest that opening of the MPT pore does not
contribute significantly to the depolarization and cell death in this
model.
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We previously found (33) evidence of mitochondrial oxidant
stress during ischemia, before the start of reperfusion. To
determine whether ROS generation during ischemia contributed to
the fall in m and subsequent cell death in this
model, pharmacological antioxidants were added to the perfusate
throughout the experiment and the effects on depolarization and cell
death were assessed (Table 1). The thiol
reductants 2-mercaptopropionyl glycine (2-MPG; 400 µM) and
pyrrolidine dithiocarbamate (PDTC, 10 µM) significantly attenuated the fall in m at the end of
ischemia, as did N-acetyl-L-cysteine (NAC; 0.5 mM). Likewise, the metal chelator 1,10-phenanthroline significantly attenuated the decrease in m during
ischemia. These antioxidant compounds also significantly
lessened cell death after 3-h reperfusion. Collectively, these studies
suggested that oxidant stress during ischemia contributes to
the fall in m.
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ROS generated from the mitochondrial electron transport chain can
induce cardiolipin oxidation within the inner mitochondrial membrane
(29). This oxidative damage could contribute to the observed membrane depolarization by compromising the integrity of the
inner membrane. To detect lipid peroxidation, cardiomyocytes loaded with the lipophilic probe C11-BODIPY were subjected to simulated
I/R. During ischemia, a significant increase in fluorescence was observed, consistent with an increase in the oxidation of this
fluorophore. If H2O2 is required for the
oxidative damage during ischemia, then lipid peroxidation
should be attenuated if SOD is inhibited. Accordingly, the Cu,Zn-SOD
inhibitor diethyldithiocarbamate (DDC; 1 mM) was used to attenuate SOD
activity. This caused a significant attenuation in C11-BODIPY
fluorescence (Fig. 3A). In
replicate experiments, the rate of increase in fluorescence during
ischemia (0.77 ± 0.19 arbitrary units (a.u./min);
n = 6) was significantly greater (P < 0.01) than during baseline (0.04 ± 0.09 a.u./min;
n = 6). Administration of DDC significantly
decreased the slope of this relationship (
0.43 ± 0.13, n = 3; P < 0.01), suggesting that
H2O2 contributes to lipid peroxidation. If free iron in the cell contributes to hydroxyl radical generation by the
Fenton reaction, then iron chelation should also attenuate lipid
peroxidation. Addition of the chelator 1,10-phenanthroline (10 µM)
during ischemia caused a significant decrease in the
fluorescence signal (Fig. 3B). In replicate experiments,
1,10-phenanthroline administration significantly decreased the rate of
fluorescence increase (0.63 ± 0.13 a.u./min, n = 3; P < 0.01) compared with ischemia. To
determine whether mitochondria are the source of these oxidants, the
electron transport inhibitor myxothiazol was added during
ischemia to inhibit ROS generation by complex III (30). Myxothiazol blocks electron transfer from ubiquinol
to the Rieske iron-sulfur center in complex III, thereby preventing the
generation of ubisemiquinone, which is a major source of superoxide generation. Myxothiazol (2 µM) significantly attenuated the rate of
increase in fluorescence during ischemia (0.026 ± 0.106 a.u./min, n = 3). Addition of DMSO, the solvent used in
myxothiazol experiments, produced no detectable effect.
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ROS generation during ischemia most likely begins with
univalent electron transfer to O2, thereby generating
superoxide. This process requires that some residual O2
must still be present to provide substrate for that reaction. In
previous studies (33) we found that low levels of
O2 were present during ischemia in this model. In
the present study, O2 tension within the flow-through chamber was assessed with a phosphorescence quenching technique previously shown to be accurate at low [O2] (24,
37). During simulated ischemia the O2
tension decreased progressively, reaching a value of ~7 mmHg within
~10 min (Fig. 4). Therefore, it is
possible that superoxide could be generated using residual
O2 that is present during ischemia.
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If residual O2 contributes to the generation of superoxide
during ischemia, and if these oxidants contribute to
mitochondrial depolarization and cell death, then significant
protection should ensue if residual O2 is scavenged from
the system during ischemia. To test this, an enzymatic
O2 scavenger was added to the perfusate to create anoxic
conditions during ischemia, thereby limiting the availability
of O2 as an electron acceptor. Anoxic conditions (PO2 = 0 mmHg) were created by adding
EC-Oxyrase to the ischemia buffer after it had been
equilibrated with 80% N2-20% CO2.
Measurements confirmed that this decreased the
PO2 within the chamber from ~7 to <0.1 mmHg
during ischemia. Heat inactivation of EC-Oxyrase (100°C for
15 min) abolished its O2-scavenging properties (data not
shown). During anoxic ischemia
(PO2 
0 mmHg), the decrease in
m was attenuated (43 ± 7% decrease; Fig.
5A) and
cell death after reperfusion was lessened (12 ± 6%;
P < 0.001) compared with standard hypoxic
ischemia (66 ± 7%; PO2 7 mmHg) (Fig. 5C). Heat treatment of EC-Oxyrase abolished
its protective effects on membrane potential and cell death (data not
shown). These findings suggested that residual O2
contributes to cell death and the irreversible decline in
m during I/R.
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During reperfusion after anoxic ischemia, TMRE fluorescence
increased progressively, indicating a restoration of
m (Fig. 5A). By contrast, minimal evidence
of recovery was seen during reperfusion after standard hypoxic
ischemia. This suggested that the mechanism responsible for
loss of m may be different between hypoxic and anoxic
ischemia. Normally, m reflects a balance between the rate of proton extrusion from the mitochondrial matrix (a
function of the rate of electron transport) and the rate at which
protons reenter the matrix (a function of ATP synthase activity and/or
ion leaks). During anoxia, electron flux should cease. Therefore,
m should decrease unless glycolytic ATP is available to maintain m through reverse operation of the ATP
synthase. During ischemia in our model, inhibition of
glycolysis by 2-DOG may have prevented reverse operation of the ATP
synthase. To explore the mechanism responsible for the fall in
m during anoxia, cells were subjected to anoxia under
the same acidic conditions used for ischemia (20%
CO2), except that glucose was added to the perfusate and
2-DOG was omitted to permit glycolysis to continue. During anoxic
acidosis, no decrease in m was observed (Fig.
5B) and minimal cell was evident after reperfusion (Fig.
5C), which suggested that m was maintained
during anoxia by reverse operation of the ATP synthase. To confirm that
reverse operation of the ATP synthase was responsible for sustaining
m during anoxia when glycolysis remained functional,
oligomycin (10 µM) was added to inhibit the ATP synthase during
anoxic acidosis. Under those conditions, m decreased
significantly (Fig. 5B). During reperfusion (21% O2, 5% CO2) without oligomycin, clear evidence
of mitochondrial repolarization was evident because proton pumping was
restored when electron transport resumed and the mitochondria membrane integrity was not compromised. The decrease in m
caused by anoxic acidosis with oligomycin was associated with
relatively low cell death (Fig. 5C). These findings indicate
that low levels of residual O2 during ischemia are
injurious because they contribute to an irreversible decline in
m. By contrast, the decline in m
caused by anoxia plus glucose deprivation is reversible and associated with minimal cell death.
Preliminary studies of standard hypoxic ischemia suggested that
a correlation may exist between the magnitude of the decrease in
m and subsequent cell death. To determine whether
such a dose-response relationship exists, we experimentally varied the severity of ischemia by adjusting the residual level of
O2 during ischemia without changing its duration (1 h). In these experiments the PO2 during
ischemia was increased from 7 mmHg (hypoxic ischemia) to ~15 mmHg. Subsequent cell death was measured after-3 h
reperfusion. As shown in Fig. 6, the
extent of cell death during reperfusion was significantly attenuated
when the fall in m during ischemia was less
severe. This suggested that the magnitude of mitochondrial depolarization during ischemia might contribute mechanistically to the cell death measured at the end of reperfusion.
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To determine the significance of mitochondrial ROS generation for the
depolarization and cell death during I/R, 0-cells were
loaded with TMRE and studied during simulated ischemia. Despite
a lack of electron transport, 0-cells maintain
m by ATP/ADP exchange via the adenine nucleotide translocator in the inner membrane (5). Wild-type 143B
osteosarcoma cells demonstrated a marked depletion of
m during ischemia that was qualitatively
similar to that seen in cardiomyocytes (Fig. 7). However, ischemia failed to
produce a similar depletion of TMRE fluorescence in
0-cells. Cell death in wild-type cells averaged
82.2 ± 9.9% vs. 28.7 ± 7.5% in the mitochondria-deficient
cells (P < 0.001). Thus the 0-cells
were protected against mitochondrial depolarization and subsequent cell
death compared with wild-type cells.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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These studies demonstrate that mitochondria undergo a significant
and irreversible decrease in potential during ischemia. The
degree of depolarization correlates with the extent of cell death
during reperfusion. However, mitochondrial depolarization by itself
does not cause cell death, because administration of anoxia plus
oligomycin caused a reversible mitochondrial depolarization without
causing significant cell death. Mitochondrial depolarization during
ischemia was triggered by ROS generated from the mitochondrial electron transport chain despite the low [O2] conditions.
These oxidants appear to initiate a cascade of lipid peroxidation that disrupts the integrity of the inner mitochondrial membrane, thereby preventing repolarization during reperfusion. Activation of the MPT
pore apparently did not contribute to this process, because attempts to
inhibit the activation of that pore failed to prevent depolarization or
cell death. By contrast, a variety of pharmacological antioxidant
compounds attenuated both the fall in m and
subsequent cell death. Furthermore, scavenging of residual
O2 during ischemia prevented the depletion of
m and significantly protected cells. Finally, 143B
cells lacking a mitochondrial electron transport chain failed to
demonstrate a depletion of m during ischemia and were significantly protected against cell death during reperfusion. Collectively these studies identify a central role for mitochondrial oxidant generation during ischemia in the irreversible
mitochondrial depolarization and subsequent cell death induced by
ischemia and reperfusion in this model (Fig.
8).
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Role of ROS during I/R.
ROS have long been associated with I/R injury. It is increasingly
evident that ROS play diverse roles in I/R, ranging from protective
effects at one extreme to damage-inducing effects at the other. For
example, low levels of oxidants appear to function as signaling agents
during the induction of ischemic preconditioning (13, 26,
31). The activation of preconditioning confers significant
protection against subsequent lethal ischemia. In contrast,
higher levels of oxidant stress are observed at the start of
reperfusion, when a transient burst of ROS generation is observed that
correlates with subsequent cell death (32). During
ischemia before reperfusion, ROS are generated by the electron transport chain of mitochondria (33, 34), although the
significance of these oxidants in cell injury is not fully understood.
The present study focused on the relationship between oxidant stress during ischemia, cell survival, and the fall in
m in a cardiomyocyte model.
Relationship between ischemic ROS and m.
m is normally maintained by proton pumping, which is
linked to the rate of electron transport. If the O2 tension
in the cell falls below a critical level of 1-4 mmHg,
m should decrease because electron transport becomes
limited by the availability of O2 at cytochrome oxidase. In
our study, large decreases in TMRE fluorescence were observed during
ischemia, indicating that a significant fall in
m must have occurred. However, the fall in
m could not be explained by a lack of O2,
because the O2 tension did not fall below the critical
level during the ischemic exposure (19). Moreover,
m failed to show significant recovery during
reperfusion, which suggests that the mitochondria sustained an
irreversible injury during the ischemic exposure. The extent of
cell death after 3-h reperfusion correlated significantly with the
extent of the loss in m, which suggests that the
mitochondrial injury sustained during ischemia could contribute
to cell death during reperfusion.
m during
ischemia. First, a variety of chemically dissimilar antioxidant
compounds given during ischemia were able to attenuate the fall
in m and to significantly lessen cell death after
reperfusion. Second, the scavenging of residual O2 during
anoxic ischemia abrogated both the fall in m
and later cell death. Both the enzymatic activity of EC-Oxyrase and its
protective effects were abolished by heat denaturation, which indicates
that the protection it provided was due to its O2
scavenging properties. The studies with anoxic ischemia
indicate that low residual levels of O2 during
ischemia are important for mitochondrial depolarization and
cell death because they act as a substrate for the generation of ROS.
Third, the results with C11-BODIPY indicate that lipid peroxidation
occurs during ischemia and that ROS originate from the
mitochondrial electron transport chain. Fourth, the
0-cells lacking an electron transport chain failed to
exhibit mitochondrial depolarization and were significantly protected
against cell death during reperfusion. We conclude that mitochondrial
ROS generation during ischemia contributes importantly to the
fall in m. This response is likely due to the
formation of lipid peroxides, which could undermine m
by destabilizing the inner membrane. A loss of membrane integrity could
also explain why m failed to recover when normal
O2 levels were restored during reperfusion.
This conclusion is consistent with the findings of Lesnefsky et al.
(23), who demonstrated that cardiolipin levels decrease during ischemia in subsarcolemmal mitochondria. Cardiolipin is a membrane phospholipid found in high abundance within mitochondria that interacts with electron transport proteins (14, 15)
and may also be important for maintaining the integrity of the inner membrane in terms of its ability to support the transmembrane potential. Mitochondrial oxidant generation during ischemia may explain the loss of cardiolipin, which could contribute to the mitochondrial dysfunction associated with I/R (28).
Alternatively, oxidants could contribute to mitochondrial damage
through direct oxidation of other lipids and proteins or by promoting
the opening of the MPT pore (3). Each of these mechanisms
could contribute to mitochondrial depolarization because of their
effects on electron transport and/or mitochondrial membrane integrity.
However, our findings suggest that the decrease in m
was not a result of MPT pore opening, because cyclosporin A treatment
had no significant effect on the decrease in membrane potential during
ischemia or the extent of cell death. This conclusion is
consistent with previous studies suggesting that MPT pore opening is
unlikely to occur under the low pH conditions of ischemia and
is more likely to occur after reperfusion (18, 20);
indeed, we observed a fall in m during
ischemia before reperfusion. However, other
investigators have used higher (10, 16) or lower
(38) concentrations of cyclosporin A to inhibit the
opening of that pore, so it is not clear whether protection would have
been observed at different concentrations or with other inhibitors of
the MPT pore.
The data suggest that H2O2 and hydroxyl
radicals, rather than superoxide, are responsible for the oxidative
damage to mitochondria. Normally, superoxide degradation by SOD is an
important step in preventing oxidant injury by that radical, so it is
surprising that SOD inhibition is protective during ischemia.
One explanation is that H2O2 may pose an
unusual threat to the cell under conditions of ischemia, when
release of iron from sites where it is normally chelated could
facilitate hydroxyl radical generation via the Fenton reaction.
Relative to hydroxyl radical, superoxide is far less reactive and may
be less injurious during relatively short periods of ischemia.
Relationship between mitochondrial depolarization and cell death.
We observed a significant correlation between the fall in TMRE
fluorescence and cell death. When the magnitude of the fall in
m was manipulated by adjusting the severity of the
ischemia, cell survival was found to be worse in experiments
where the fall in m was larger. Previous studies in
cardiomyocytes demonstrated that ROS generation tends to increase as
O2 tension is lowered from 35 to 7 mmHg (6).
We therefore suggest that milder ischemia was protective in the
present study because of the lesser oxidant stress it generated and the
associated decrease in oxidant damage to membranes. An oxidant-mediated
disruption of mitochondrial inner membrane integrity during
ischemia could conceivably lead to cell death by promoting
matrix swelling, release of cytochrome c to the cytosol, and
activation of the cell apoptotic machinery (36).
However, mitochondrial depolarization by itself was not lethal to these
cells, as evidenced by the minimal extent of death observed when
m was depleted with anoxic acidosis plus oligomycin. For a given degree of depolarization, it is conceivable that matrix swelling caused by lipid peroxidation and loss of membrane integrity is
more severe than when caused simply by electron transport inhibition. In the former case, reoxygenation would not promote recovery of m because the loss of membrane integrity would defeat
the effects of proton pumping. In the latter case, a restoration of
electron transport during reoxygenation would allow m
to recover.
7 mmHg) was not sufficient to induce significant cell death in this
study. Likewise, hypercapnic acidosis (20% CO2) was well
tolerated under normoxic conditions. However, when the two conditions
were combined, >60% cell death resulted. Acidosis may increase cell
death by causing the release of Fe2+ from intracellular
sites where it is normally chelated. The ROS released in response to
hypoxia alone appear to be well tolerated by cells (7).
However, in a setting where acidosis causes release of iron ions, these
ROS may lead to generation of hydroxyl radicals as a consequence of
Fenton interactions. The resulting loss of membrane integrity could
explain the observed decrease in m and subsequent
failure to recover during reoxygenation. This interpretation is
consistent with our observation that an iron chelator was most protective of m and cell viability. Although the
importance of the Fenton reaction in I/R injury is not new (1, 4,
9, 17, 21, 25), its potential involvement in cellular injury during ischemia before reperfusion has not been explored
previously, to our knowledge.
In summary, these studies reveal that ROS generated during
ischemia contribute to the irreversible loss of
m. The extent of this damage correlates with the
extent of cell death during reperfusion, and interventions that
minimize the oxidative stress during ischemia also attenuate
the loss of m. These findings therefore suggest that
oxidant generation during ischemia before reperfusion plays a
significant role in determining cell death during reperfusion.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grants HL-32646 and HL-35440. J. Levraut was supported by a grant from the Société de Réanimation de Langue Française.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. T. Schumacker, Dept. of Medicine MC6026, The Univ. of Chicago, 5841 South Maryland Ave., Chicago, IL 60637 (E-mail: pschumac{at}medicine.bsd.uchicago.edu).
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.
First published October 10, 2002;10.1152/ajpheart.00708.2002
Received 22 August 2002; accepted in final form 7 October 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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