© 2000 American Heart Association, Inc.
Cellular Biology |
Preconditioning in Cardiomyocytes Protects by Attenuating Oxidant Stress at Reperfusion
From the Section of Emergency Medicine, Department of Medicine, The University of Chicago, Chicago, Ill.
Correspondence to Paul T. Schumacker, PhD, Department of Medicine, MC6026, The University of Chicago, 5841 S Maryland Ave, Chicago, IL 60637. E-mail pschumac{at}medicine.bsd.uchicago.edu
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Abstract |
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Abstract—Cardiomyocyte death after ischemia/reperfusion correlates with oxidant stress, and antioxidants confer protection in that model. Preconditioning (PC) with hypoxia or adenosine also confers protection, leading us to hypothesize that PC protects by attenuating oxidant generation during subsequent ischemia/reperfusion. Chick cardiomyocytes were preconditioned with 10 minutes of hypoxia or adenosine (100 µmol/L), followed by 1 hour of simulated ischemia and 3 hours of reperfusion. Adenosine PC decreased cell death from 50±3% to 18±4% and enhanced the return of contractions during reperfusion, as observed previously with hypoxic PC. A transient burst of dichlorofluorescein (sensitive to H2O2) oxidation that was significantly attenuated by PC initiated by hypoxia or adenosine was seen at reperfusion. The protein kinase C (PKC) inhibitor Go-6976 and the mitochondrial ATP-sensitive K+ (KATP) channel inhibitor 5-hydroxydecanoate each abolished protection and abrogated the PC-induced attenuation of reperfusion oxidant stress. By contrast, when given only at reperfusion, the K+ channel opener pinacidil or the antioxidants 2-mercaptopropionylglycine and 1,10-phenanthroline decreased oxidant stress at reperfusion and improved survival and return of contractions. Thus, PC protection is associated with an attenuation of the oxidant burst at reperfusion, regardless of the method by which PC is triggered. Loss of PC protection associated with PKC inhibition or KATP channel inhibitors is associated with a restoration of that oxidant stress. These results suggest a mechanism for PC protection and reveal a functional link between PKC activation and KATP channel activation in that pathway.
Key Words: reperfusion • protein kinase C • hydrogen peroxide • KATP channels • reactive oxygen species
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Introduction |
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Myocardial preconditioning was first described as a phenomenon whereby brief episodes of nonlethal ischemia confer protection against subsequent ischemia/reperfusion (I/R) injury in the heart.1 Since then, work directed at understanding the factors that initiate and mediate preconditioning2 has identified multiple triggers, including transient hypoxia3 4 and adenosine.5 6 Despite the description of numerous preconditioning triggers, the process by which preconditioning protects the heart from subsequent ischemia and reperfusion injury is unclear. Activation of protein kinase C (PKC) and phosphorylation of ATP-sensitive K+ (KATP) channels have been implicated as common participants in this protective pathway, but the specific mechanism of protection is not known.7 8
Regarding the injury against which preconditioning protects, studies have linked I/R injury with the generation of reactive oxygen species (ROS), especially within the first few minutes of reoxygenation.9 Moreover, the development of cellular injury in these models correlates with the degree of oxidant stress (for review, see Reference 10 ). We hypothesized that preconditioning protects by attenuating the generation of oxidants at reperfusion. If so, then preconditioning should be associated with an attenuation of that oxidant stress regardless of the method used to trigger preconditioning protection. Furthermore, given that PKC inhibition blocks the protection associated with preconditioning, this should also abolish the attenuation of oxidant stress at reperfusion. Finally, given that activators of KATP channels confer preconditioning-like protection but that inhibitors abolish protection, we reasoned that KATP channel activation or inhibition should attenuate or augment oxidant generation at reoxygenation in accordance with this model.
Because it is technically difficult to detect transient changes in intracellular oxidant generation in intact organs,11 we studied cardiomyocytes that permit the use of intracellular dyes to assess oxidant generation during simulated ischemia and during reoxygenation.12 Previous studies demonstrated that cultured cardiomyocytes exhibit preconditioning3 4 13 and exhibit increased oxidant stress14 during I/R,15 rendering them suitable for addressing the hypothesis.
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Materials and Methods |
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Cardiac Cell Culture
Ventricular embryonic chick cardiomyocytes were prepared by using a method described previously.16 Cells were studied at days 3 to 5, when viability exceeded 99%. Cells were studied in a flow-through chamber as previously described.16 The perfusate consisted of balanced salt solution (with PO2 149 mm Hg, PCO2 36 mm Hg, pH 7.4, and [K+] 4.0 mEq/L) containing glucose (5.6 mmol/L). To simulate ischemia, the perfusate consisted of balanced salt solution without glucose containing 2-deoxyglucose (20 mmol/L) to inhibit glycolysis and [K+] of 8.0 mEq/L. This solution was bubbled with 80% N2/20% CO2 to produce a PO2 of
4 mm Hg, a
PCO2 of 144 mm Hg, and a final
pH of 6.8. Hypoxic media used for preconditioning consisted of balanced
salt solution with no glucose bubbled with 95%
N2/5% CO2. Reperfusion was
with normal perfusate with glucose. Hypoxia
(PO2 15 mm Hg) and simulated
ischemia (4 mm Hg) were verified by use of an optical
phosphorescence quenching technique (Oxyspot, Medical Systems
Inc)17 to confirm the
PO2 in contact with the cells.
Video/Fluorescent Microscopy
Fluorescent images were obtained by using a cooled
slow-scanning personal computer–controlled camera (Hamamatsu).
Propidium iodide (PI, 5 µmol/L; Molecular Probes) was used to
assess viability, as previously described.18 Cells were
permeabilized with digitonin (300 µmol/L) at the
end of the experiment to maximize PI staining. Vigorous cell
contractile motion was always observed at the start of each study.
Within minutes after the start of ischemia, visible contractile
motion ceased in every experiment. During reperfusion, some contractile
motion returned in some experiments. Return of contraction was assessed
at 3 hours into reperfusion.
Measurement of ROS Generation
Intracellular oxidant stress was monitored as previously
described4 14 by using the probe
2',7'-dichlorofluorescin diacetate (DCFH-DA, 5
µmol/L; Molecular Probes). Present in the media throughout the
experiment (5 µmol/L), this dye enters the cells and is cleaved
by esterases, yielding nonfluorescent
2',7'-dichlorofluorescin (DCFH). Intracellular oxidants can
lead to DCFH oxidation, yielding the fluorescent compound
dichlorofluorescein (DCF).
Preconditioning Protocols
To simulate I/R, cardiomyocytes were exposed to 1
hour of simultaneous hypoxia, hypercarbic acidosis,
hyperkalemia, and substrate deprivation,
followed by 3 hours of reperfusion. This results in significant cell
death, particularly during reperfusion, which appears related to
oxidant damage.15 16 For preconditioning before I/R,
cardiomyocytes were exposed to 10 minutes of
hypoxia (PO2 15 mm Hg)
without glucose and then 10 minutes of normoxic recovery, followed by
I/R. As a second preconditioning protocol, adenosine (100
µmol/L, Sigma Chemical Co) was used in lieu of hypoxia. Cell
viability, contraction, and oxidant generation were measured during
subsequent I/R.
Data Analysis
For each experiment, a single field of 500 cells was
observed. Treatment and control groups were matched by using cells
isolated on the same day. Additional coverslips were used for replicate
experiments.
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Results |
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Effects of Preconditioning on Cell Death and Contraction
In this model of simulated I/R, the majority of cell death develops during reperfusion.16 In cells preconditioned with adenosine (Figure 1
), the
pattern and extent of cell death were similar to those previously
reported for hypoxia-preconditioned cells.4 After
ischemia but before reperfusion, cell death was minimal and
averaged <2% in all groups. Cell death after 3 hours of reperfusion
in adenosine-preconditioned cells (18±4%, n=6) was similar to
that seen after preconditioning with hypoxia (16±2%, n=5). In
cells preconditioned by either hypoxia or adenosine,
cell death was less than that found in
nonpreconditioned control cells (50±3%, n=10;
P<0.001). After 3 hours of reperfusion, visible contractile
activity returned in all preconditioned cells (11 of 11) compared with
nonpreconditioned cells (0 of 10). Thus,
preconditioning with adenosine or hypoxia conferred
significant protection from cell death and contractile dysfunction.
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Effects of Preconditioning on Reperfusion Oxidant Generation
via PKC
If the mechanism of preconditioning protection involves an
attenuation of oxidant stress at reperfusion, then induction of
preconditioning by either hypoxia or adenosine should
attenuate the oxidant burst at reperfusion. Figure 1
shows the effect of adenosine preconditioning on DCF
fluorescence during baseline and I/R compared with no
preconditioning. In the absence of preconditioning, control cells
demonstrated an increase in DCFH oxidation to a peak value of
2.2±0.1 at 10 minutes of reperfusion (n=3), whereas
adenosine-preconditioned cells increased to 1.6±0.1 (n=3,
P=0.02). Figure 2 shows the
effect of hypoxic preconditioning on intracellular oxidant generation
during I/R, as assessed by the oxidation of DCFH. As seen in
adenosine preconditioning, a transient burst of DCF
fluorescence was noted in nonpreconditioned
cells at the start of reperfusion (1.4±0.1 to 4.0±0.3 by 10 minutes
of reperfusion, n=6). This was significantly attenuated in
hypoxia-preconditioned cells (1.4±0.1 to 2.6±0.3, n=9;
P=0.01).
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PKC inhibition has been shown to block preconditioning protection in
multiple studies. If preconditioning protects by attenuating oxidant
stress at reperfusion, then PKC inhibition should abrogate this
attenuation. In adenosine preconditioning, PKC inhibition with
Go-6976, a selective PKC inhibitor,19
abrogated the attenuation of the reperfusion oxidant burst, causing
DCFH oxidation to increase in preconditioned cells to 2.2±0.1 by 10
minutes of reperfusion (n=3), similar to
nonpreconditioned cells (Figure 1). In hypoxic
preconditioning, Go-6976 caused DCFH oxidation to increase from
1.5±0.1 at the end of ischemia to 4.1±0.3 at 10 minutes of
reperfusion (n=3) (P=NS versus
nonpreconditioned controls, Figure 2). Go-6976
also abrogated preconditioning protection, with 46±6% cell death at
0.01 to 0.1 µmol/L (n=7) and no return of contractions compared
with preconditioned cells (P<0.001, Figure 2).
Exposure to Go-6976 (0.1 µmol/L) for 4 hours under normoxia had
no effect on viability or contraction (data not shown). In the absence
of preconditioning, cells subjected to ischemia in the presence
of Go-6976 (0.05 µmol/L) showed no exacerbation of cell death
(n=3, Figure 2). The less specific PKC inhibitors
Ro-31-8220 (0.01 µmol/L, n=3) and chelerythrine (2
µmol/L, n=4) also abolished preconditioning protection in this system
and were not associated with increased cell death during prolonged
normoxia or during the standard I/R protocol (data not shown). These
results confirm that preconditioning requires PKC
activation20 and indicate that the attenuation of
oxidant production during reperfusion involves PKC.
Role of the KATP Channel in Reperfusion
Oxidants
Studies have shown that KATP channel openers
can induce preconditioning-like protection, whereas
inhibitors abolish protection in the heart. If
preconditioning protects cells by attenuating the oxidant burst at
reperfusion, then activators and inhibitors of
that channel should modify the magnitude of the oxidant burst at
reperfusion in accordance with our model. The compound
5-hydroxydecanoate (5-HD) inhibits mitochondrial
KATP channels21 and abolishes
protection in chick cardiomyocytes.22 The
attenuation of the reperfusion oxidant burst after hypoxic
preconditioning was abolished by 5-HD (n=3, Figure 3). Likewise, cell death after 3 hours
reperfusion in hypoxia-preconditioned cells increased from
16±2% to 50±8% (P=0.04) when 5-HD was added during the
preconditioning (n=4, Figure 3).
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The KATP channel opener pinacidil induces
preconditioning-like protection in the chick cardiomyocyte
system.22 In the present study, pinacidil (10
µmol/L) that was added for 1 hour at the start of reperfusion
abrogated oxidant generation at reperfusion and reduced cell death
(Figure 4). In addition, this
attenuation in oxidant generation and cell death could be reversed with
the addition of 5-HD. Compared with cell death in
nonpreconditioned control cells (50±3%), cells
treated with pinacidil exhibited 30±2% cell death, with 3 of 4 groups
exhibiting return of contraction during reperfusion (versus 0 of 10 in
control cells). The addition of 5-HD before and during pinacidil
treatment at reperfusion reversed this attenuation in cell death,
resulting in 46±8.4% cell death and 0 of 4 groups exhibiting any
return of contraction.
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Preconditioning and Antioxidant Protection
If protection conferred by preconditioning is mediated by an
attenuation of oxidant stress at reperfusion, then similar protection
should be obtained when antioxidants are given only at reperfusion. The
antioxidants 1,10-phenanthroline and 2-mercaptopropionylglycine were
previously found to attenuate oxidant stress in this
model.14 Figure 5 shows the
percent reduction in cell death achieved with anti-oxidants compared
with other interventions in the present study. When
1,10-phenanthroline (10 µmol/L) plus 2-mercaptopropionylglycine
(400 µmol/L) was given only at reperfusion, cell death decreased
to a similar extent as seen with preconditioning. Return of contractile
motion was observed in 4 of 4 studies using antioxidants compared with
0 of 10 control studies. Addition of the PKC inhibitor
Go-6976 or the KATP channel inhibitor
5-HD largely abrogated the protection conferred by hypoxic
preconditioning. In nonpreconditioned cells, addition
of the KATP channel opener pinacidil reduced cell
death by 37±6% only at reperfusion.
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Discussion |
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Ischemic preconditioning was initially described in the heart,1 but isolated cardiomyocytes demonstrate similar protection from I/R after brief exposure to adenosine, hypoxia, or ischemia,3 4 13 23 suggesting that the same mechanisms act in both systems. In our model, preconditioning conferred a reproducible degree of protection against cell death, and it improved recovery of contractile function during reperfusion. Using DCFH to assess ROS generation, we detected a transient burst of oxidant stress at the start of reoxygenation, which was consistent with previous findings in the intact heart.9 Preconditioning induced by hypoxia, adenosine, or KATP channel activation attenuated this oxidant burst. By contrast, inhibition of PKC or inhibition of mitochondrial KATP channels abolished the protection and restored oxidant generation at reperfusion to a level indistinguishable from the control level. The level of protection seen with preconditioning was similar to that seen with antioxidants given only at reperfusion. These findings suggest that preconditioning can protect cells from I/R by attenuating oxidant generation at reperfusion, through a process involving activation of a PKC-dependent signaling pathway and KATP channels.
Cell viability was assessed by using PI, which showed a progressive increase in staining during 3 hours of reperfusion yet only a minimal increase during ischemia. Previous studies in cardiomyocytes have shown minimal cell death during 1 hour of ischemia but accelerated cell death during 3 hours of reperfusion.15 Our present results link the transient oxidant burst at reperfusion to the loss in cell viability, yet PI staining continued to increase during reperfusion even though the transient oxidant burst had long since subsided. This may reflect a delayed response of PI, which provides a measure of sarcolemma permeability, to a lethal oxidant stress occurring at the start of reperfusion. Alternatively, a burst of oxidant stress at reperfusion could activate a sequence of events that later results in the increase in cell permeability. Could the PI measurements reflect a delayed response to a lethal insult incurred during ischemia? We argue against this, on the basis of the observation that cell death was minimal after 4 hours of continuous ischemia, when cells were studied without reperfusion.16
Preconditioning Protection and Attenuation of Oxidant
Stress
Our findings are consistent with reports suggesting that
preconditioning decreases oxidant stress, but our findings are novel in
suggesting that this process occurs during the "early window" of
protection. In rat cardiomyocytes, Zhai et
al24 and Zhou et al25 found decreases in
superoxide levels and increases in magnesium superoxide dismutase
(magnesium SOD) activity by 24 hours after preconditioning. They
suggested that preconditioning may activate existing SOD during
the early window of protection and confer protection during the late
phase by causing the expression of additional SOD. However, Turrens et
al26 found no increase in the activities of SOD, catalase,
or glutathione peroxidase during the early window of protection.
Another report has suggested that mitochondria isolated from
preconditioned hearts generates less superoxide than those isolated
from nonpreconditioned hearts.27 Those
results suggest that preconditioning could attenuate oxidant generation
by mitochondria. However, controversy exists regarding the source of
the ROS generated at reperfusion. Yabe et al28 found that
preconditioning preserved mitochondrial function in rat hearts, raising
the possibility that preconditioning may attenuate ROS generation and
thereby lessen mitochondrial injury by oxidants produced elsewhere in
the cell. Indeed, studies suggest that oxidants originate from a
nonmitochondrial source at reperfusion.29 30 In the
present study, antioxidants administered at reperfusion were as
effective as preconditioning in protecting cells. These findings
suggest that ROS generation at reperfusion is responsible for the cell
injury and that preconditioning protects by attenuating that oxidant
stress. Because the cardiomyocyte system used in these
studies does not contain neutrophils, endothelial
cells, or other potential sources of oxidants, the present study
also demonstrates that the cardiomyocytes themselves can
act as a source of lethal oxidant stress.
Although some studies in the intact heart have shown a beneficial effect of antioxidants given only at reperfusion,31 other studies have failed to detect a beneficial effect unless they are given before reperfusion (as reviewed by Opie32 ). To the extent that a rapid burst of ROS generation at reperfusion is an important determinant of myocardial injury, antioxidants administered at reperfusion will be protective only if they reach their targets before significant oxidant stress has occurred. Any delay in the delivery of the drug to the site of oxidant generation will likely degrade the extent of protection by allowing unchecked oxidant stress to begin. The consistent protection afforded by antioxidants in our cardiomyocyte model may reflect the ability of such a system to delivery the drugs simultaneously with oxygen at the start of reperfusion.
Role of KATP Channels in Reducing Reperfusion
Oxidant Injury
The KATP channel may function as an effector
of preconditioning, although the mechanism by which it confers
protection is unknown.33 34 35 Antagonists of
the channel, such as glibenclamide and 5-HD, block ischemic
preconditioning protection in the heart, and KATP
channel openers mimic the beneficial effect of
preconditioning.36 37 Some studies indicate that
KATP channel activators are
protective only when given during ischemia (for review, see
References 38 and 39 ), whereas other
studies suggest that it can be effective when given just before
reperfusion.40 Because reperfusion oxidant stress occurs
rapidly after reperfusion begins, any delay in intracellular access
could degrade the extent of protection. We found that pinacidil was
protective when administered at the start of reperfusion, which may be
explained by a more rapid access of the drug to the cells in a
flow-through system. The ability of pinacidil to confer protection when
given at the start of reperfusion points to a possible connection
between that channel and the system responsible for ROS generation. To
the extent that the KATP channel is the effector
of preconditioning, our data suggest that the channel modulates oxidant
generation at the start of reperfusion. The inhibitor 5-HD
is selective for the mitochondrial KATP
channel,21 and mitochondria have the potential to generate
ROS.41 This might suggest that KATP
channels function by modulating mitochondrial superoxide generation,
yet other evidence points away from mitochondria as the source of
oxidants generated at reperfusion.29 30 We speculate that
the mitochondrial KATP channel may influence
oxidant generation by affecting the supply of NAD(P)H from the
mitochondrial matrix dehydrogenases to a nonmitochondrial oxidase
system. However, additional work is required to test this
hypothesis.
Interestingly, in the present study, the extent of protection conferred by pinacidil was somewhat less than that conferred by preconditioning, yet it appeared to be as effective as preconditioning at attenuating the reoxygenation ROS burst. One explanation is that preconditioning protects cells by a second mechanism that is independent of the KATP channel activation. Alternatively, it is possible that pinacidil exhibits nonspecific detrimental effects that undermine the extent of protection otherwise afforded by KATP channel activation. However, the finding that 5-HD abolished preconditioning strongly implicates the KATP channel as a significant effector for preconditioning protection with pinacidil in this system.
Role of PKC Signaling
PKC has been implicated as a mediator of preconditioning in
different models.7 42 43 44 45 However, the specific targets of
PKC in preconditioning have not been fully established. In
ventricular myocytes, Hu et al46 found that
PKC activation reduced the sensitivity of sarcoplasmic
KATP channels to ATP, an effect that would tend
to promote channel opening at a given [ATP]. Light et
al47 showed that PKC activation increased the open
probability of the KATP channel in
cardiomyocyte membrane patches. Speechly-Dick et
al35 found that preconditioning of human
trabeculae induced by PKC activation could be blocked by
inhibition of the KATP channel. Sato et
al21 have suggested that activated PKC acts on
mitochondrial KATP channels. Collectively, these
findings support a model in which the PKC targets include the
KATP channel(s). Our findings confirm the linkage
between PKC activation and KATP channel
activation in preconditioning and extend previous work by linking this
pathway to a modulation of oxidant generation at
reoxygenation. This is based on the observation that
the effects of preconditioning on the reoxygenation
oxidant burst were ablated when either PKC activation or
KATP channel activation was inhibited.
Go-6976 has been characterized as selective for Ca2+-dependent isoforms of PKC,19 yet other studies have implicated Ca2+-independent isoforms in the preconditioning of adult hearts.48 The particular isoforms involved in preconditioning of chick cardiomyocytes have not been characterized, so it is possible that this difference is due to the different source of our cardiomyocytes.
Role of Oxidants in the Induction of Preconditioning
ROS have been implicated as second messengers in the activation of
preconditioning. Exogenous oxidants induce preconditioning in intact
hearts,49 and antioxidants block the beneficial effects of
preconditioning.50 51 We previously reported that
increased ROS are generated by mitochondria during hypoxic
preconditioning. According to that model, low levels of mitochondrial
ROS function as signaling agents required for the activation of
protection.4 Antioxidants administered only during hypoxic
preconditioning attenuated the transient ROS signal and abolished the
protective effect. In the present study, evidence of an oxidant
signal was again observed during hypoxic preconditioning (see
Figure 2A, minute 40). However, compared with magnitude
of the ROS burst at reperfusion, the magnitude of this signal is
small.
Regarding the induction phase, no oxidant signal was observed during adenosine preconditioning, suggesting the existence of alternative pathways that do not involve oxidants. It is also possible that adenosine triggers preconditioning by acting downstream from the oxidant-dependent step. We speculate that mitochondrial oxidants generated during hypoxic preconditioning lead to the activation of PKC, which subsequently acts on the KATP channel. Evidence suggests that oxidants can activate PKC,52 whereas adenosine may activate PKC by a receptor-mediated pathway, possibly involving diacylglycerol, that bypasses mitochondria. In either case, KATP channel activation appears to attenuate the oxidant burst seen in reperfusion, which appears to be responsible for the cell death later in reperfusion. These findings present a dual role for ROS in preconditioning: during induction (when low levels of oxidants generated by mitochondria activate PKC and open KATP channels) and during the protection phase (when the subsequent oxidant burst at reperfusion is attenuated).
Regarding the mechanism of reversal of preconditioning protection by
5-HD, it is noteworthy that 5-HD had no effect on the oxidant
generation during hypoxia (see Figure 3A, minute 40).
Thus, abrogation was not due to attenuation of mitochondrial oxidant
generation during hypoxia, an intervention previously
demonstrated with antioxidants to abrogate preconditioning
protection.4 The result also suggests that oxidants
generated during hypoxic preconditioning may originate from a source
different from the oxidant burst after I/R, because interventions
affecting the KATP channel appear to alter the
oxidants generated at reperfusion but not during preconditioning.
Further work is needed to identify the oxidant source at
reperfusion.
Timing of Preconditioning Protection: Ischemia Versus
Reperfusion
The shorter the duration of myocardial ischemia, the less
is the degree of ischemic injury. Classically, it has been
thought that most injury occurs during ischemia, yet a growing
number of studies have shown that interventions at the start of
reperfusion may improve recovery.53 It is difficult to
know whether the protection conferred by preconditioning of intact
hearts is mediated by its effects on events occurring during
ischemia or whether preconditioning affects events during the
first minutes of reperfusion. Because reperfusion injury may occur
within seconds and because some amount of reperfusion of
ischemic tissue is necessary to quantify the extent of necrosis
in intact hearts when dyes are used,9 54 this issue has
been difficult to address in the intact heart. In vitro models have the
potential to shed light on this controversy by providing a continuous
assessment of viability in the same cells during ischemia and
reperfusion phases. To the extent that our findings in
cardiomyocytes apply to the intact heart, these data
suggest that interventions such as a K+ channel
agonists or antioxidants applied at reperfusion have the potential to
protect against the consequences of ischemia but that the rapid
generation of oxidants on reperfusion leads to a narrow window of
opportunity for therapy.
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-32646, HL-35440, HL-03779, and HL-03459.
Received July 1, 1999; accepted January 3, 2000.
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Z. Yao, B. C. McPherson, H. Liu, Z. Shao, C. Li, Y. Qin, T. L. Vanden Hoek, L. B. Becker, and P. T. Schumacker Signal transduction of flumazenil-induced preconditioning in myocytes Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1249 - H1255. [Abstract] [Full Text] [PDF]
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 C. Ozcan, E. L. Holmuhamedov, A. Jahangir, and A. Terzic Diazoxide protects mitochondria from anoxic injury: Implications for myopreservation J. Thorac. Cardiovasc. Surg., February 1, 2001; 121(2): 0298 - 306. [Abstract] [Full Text] [PDF]
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B. O'Rourke Pathophysiological and protective roles of mitochondrial ion channels J. Physiol., November 15, 2000; 529(1): 23 - 36. [Abstract] [Full Text] [PDF]
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B. O'Rourke Myocardial KATP Channels in Preconditioning Circ. Res., November 10, 2000; 87(10): 845 - 855. [Abstract] [Full Text] [PDF]
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G. J. Gross and R. M. Fryer Mitochondrial KATP Channels : Triggers or Distal Effectors of Ischemic or Pharmacological Preconditioning? Circ. Res., September 15, 2000; 87(6): 431 - 433. [Full Text] [PDF]
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R. M. Mentzer Jr Ischemic preconditioning: how close are we to therapeutic implementation? Ann. Thorac. Surg., August 1, 2000; 70(2): 356 - 357. [Full Text] [PDF]
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R. A. Forbes, C. Steenbergen, and E. Murphy Diazoxide-Induced Cardioprotection Requires Signaling Through a Redox-Sensitive Mechanism Circ. Res., April 27, 2001; 88(8): 802 - 809. [Abstract] [Full Text] [PDF]
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D. X. Zhang, Y.-F. Chen, W. B. Campbell, A.-P. Zou, G. J. Gross, and P.-L. Li Characteristics and Superoxide-Induced Activation of Reconstituted Myocardial Mitochondrial ATP-Sensitive Potassium Channels Circ. Res., December 7, 2001; 89(12): 1177 - 1183. [Abstract] [Full Text] [PDF]
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