Delayed ischemic preconditioning is mediated by opening
of ATP-sensitive potassium channels in the rabbit heart
NELSON L. BERNARDO,1 MICHAEL D’ANGELO,1 SHINJI OKUBO,2
ARCHI JOY,1 AND RAKESH C. KUKREJA1
1Division of Cardiology, Department of Medicine, Medical College of Virginia, Virginia
Commonwealth University, Richmond, Virginia 23298; and 2Department of Cardiology,
Kanazawa Medical University, Daigaku, Uchinada, Kahoku, Ishikawa 920-0293, Japan
action potential; myocardial infarction; ischemia-reperfusion
injury; mitochondrial and sarcolemmal adenosine 58-triphosphate-sensitive potassium channel; protein kinase C
of ischemia and reperfusion
protect the heart against subsequent sustained ischemia and reperfusion (35). This phenomenon, known as
ischemic preconditioning (PC) is an inherent capability
of the myocardium to protect itself from ischemic
damage. The initial protective effect of preconditioning
is transient, disappearing within 2–3 h (50). Recent
studies showed that cardioprotection from PC reappears 24 h after the initial stimulus. This phenomenon
REPEATED BRIEF EPISODES
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is known as ‘‘delayed preconditioning’’ or ‘‘second window of preconditioning’’ (27, 30). Delayed preconditioning can also be produced by heat shock (12) and free
radicals (56) and with pharmacological agents including monophosphoryl lipid A (MLA) (54) or A1-adenosine
agonist 2-chloro-N6-cyclopentyladenosine (4, 7). Understanding the mechanism(s) underlying the second window of preconditioning may provide a better insight
into the nature of myocardial adaptation to ischemia
and open new therapeutic avenues capable of modifying the outcome after myocardial ischemic episodes.
Opening of the ATP-sensitive potassium (KATP ) channel has been shown to be one of the potential mechanisms in early preconditioning (17, 18, 43, 48) and
delayed preconditioning induced by heat shock (20, 41)
as well as pharmacological agents (15, 24, 32). However, it is not known whether opening of the KATP
channel also mediates the delayed protective effect of
ischemic preconditioning in rabbit. The protection afforded by this channel has been attributed to an
increase in the outward potassium current followed by
shortening of the action potential duration (APD). This,
in turn, may spare ATP, thereby allowing less entry of
calcium into the myocyte through the voltage-sensitive
calcium channels. Decreased intracellular calcium overload then reduces the ischemic injury, and therefore
enhances the preservation of myocytes. Within 1–3 min
of acute coronary occlusion, there is a pronounced
shortening in APD secondary to activation of the KATP
channel (11) although the APD shortening is not related to the extent of cardiac protection. It has been
proposed that the lack of such an association could be
caused by the opening of the mitochondrial KATP channel rather than the sarcolemmal channel (16). In
support of this hypothesis, a potent opener of the
mitochondrial KATP channel, diazoxide, was shown to
induce a significant cardioprotective effect in the isolated, perfused heart (16) and in ventricular myocytes
(29, 47). The protective effect of diazoxide was blocked
by 5-hydroxydecanoate (5-HD), a specific blocker of the
mitochondrial KATP channel (16, 29, 47).
The purpose of the present investigation was to
determine whether the KATP channel also plays a role in
the development of delayed phase of ischemic preconditioning. A second goal was to test the hypothesis if the
epicardial APD shortening, which occurs because of the
opening of the sarcolemmal KATP channel, is suppressed
by glibenclamide (the blocker of sarcolemmal and mitochondrial KATP channels) but not by 5-HD (29, 47). Our
results show that the delayed effect of ischemic preconditioning as well as epicardial APD shortening was
0363-6135/99 $5.00 Copyright r 1999 the American Physiological Society
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Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on October 1, 2016
Bernardo, Nelson L., Michael D’Angelo, Shinji Okubo,
Archi Joy, and Rakesh C. Kukreja. Delayed ischemic
preconditioning is mediated by opening of ATP-sensitive
potassium channels in the rabbit heart. Am. J. Physiol. 276
(Heart Circ. Physiol. 45): H1323–H1330, 1999.—Cardioprotection from preconditioning reappears 24 h after the initial
stimulus. This phenomenon is called the second window of
protection (SWOP). We hypothesized that opening of the
ATP-sensitive potassium (KATP ) channel mediates the protective effect of SWOP. Rabbits were preconditioned (PC) with
four cycles of 5-min regional ischemia each followed by 10 min
of reperfusion. Twenty-four hours later, the animals were
subjected to sustained ischemia for 30 min followed by 180
min of reperfusion (I/R). Glibenclamide (Glib, 0.3 mg/kg ip) or
5-hydroxydecanoate (5-HD, 5 mg/kg iv) was used to block the
KATP channel function. Infarct size was reduced from 41.2 ⫾
2.6% in sham-operated rabbits to 11.6 ⫾ 1.0% in PC rabbits, a
71% reduction (n ⫽ 11, P ⬍ 0.01). Treatment with Glib or
5-HD before I/R increased the infarct size to 43.4 ⫾ 2.6 and
37.8 ⫾ 1.9%, respectively (P ⬍ 0.01 vs. PC group, n ⫽
12/group). Sham animals treated with either Glib or 5-HD
had an infarct size of 39.0 ⫾ 3.4 and 37.8 ⫾ 1.5%, respectively,
which was not different from control (40.0 ⫾ 3.8%) or sham
(41.2 ⫾ 2.6%) I/R hearts. Monophasic action potential duration (APD) at 50% repolarization significantly shortened by
28.7, 26.6, and 23.3% in sham animals during 10, 20, and 30
min of ischemia. However, no further augmentation in the
shortening of APD was observed in PC hearts. Glib and 5-HD
significantly suppressed ischemia-induced epicardial APD
shortening, suggesting that 5-HD may not be a selective
blocker of the mitochondrial KATP channel in vivo. We conclude that SWOP is mediated by a KATP channel-sensitive
mechanism that may have occurred because of the opening of
the sarcolemmal KATP channel in vivo.
H1324
KATP CHANNEL IN DELAYED ISCHEMIC PRECONDITIONING
equally suppressed by glibenclamide and 5-HD. These
data raise the question of the specificity of 5-HD in
blocking the mitochondrial KATP channel in vivo. To our
knowledge, this is the first report investigating the
KATP channel as the mediator of delayed ischemic
preconditioning in the rabbit myocardium.
METHODS
APD (%) ⫽
APD50 or 90 ⫺ APD50 or 90 (before occlusion)
APD50 or 90 (before occlusion)
⫻ 100
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Animal care. New Zealand White rabbits (males; 2.5–3.3
kg) were used for this study. The care and use of the animals
was conducted in accordance with the guidelines of the
Committee on Animals of Virginia Commonwealth University
and the National Institutes of Health (NIH) Guide for the
Care and Use of Laboratory Animals [DHHS Publication No.
(NIH) 80–23, Revised 1985].
Drugs and chemicals. 5-HD was purchased from Research
Biochemicals. Glibenclamide, Evans blue dye, and triphenyltetrazolium chloride (TTC) were obtained from Sigma Chemical (St. Louis, MO). The vehicle for glibenclamide was 40%
propylene glycol and 10% ethanol in distilled water. All other
chemicals were of analytical reagent quality.
Surgical preparation. The animals were anesthetized with
an intramuscular injection of ketamine HCl (35 mg/kg) and
xylazine (5 mg/kg). Further injections of ketamine-xylazine
were given as needed throughout the surgical procedure. The
animals were intubated orotracheally and ventilated on a
positive-pressure ventilator. The tidal volume was set at ⬃15
ml, and the respiratory rate was adjusted to 30–40 cycles/
min. Ventilator setting and PO2 were adjusted as needed to
maintain the blood gas parameters within the physiological
range. The prominent ear artery and a marginal ear vein
were cannulated with a Jelco 24-gauge striped radiopaque
intravenous catheter for blood sampling and measurement of
arterial blood pressure. The cannulation of the vein allowed
for continuous infusion of 0.9% NaCl solution. Electrocardiographic leads were attached to subcutaneous electrodes to
monitor heart rhythm.
Day 1: Ischemic preconditioning protocol. All surgical procedures were performed under sterile conditions. The chest was
opened by a left thoracotomy in the fourth intercostal space,
and the pericardium was opened to expose the heart. A 5-0
silk suture with an atraumatic needle was then passed
around the left anterior descending artery (LAD) midway
between the atrioventricular groove and the apex of the
heart. The animal was then heparinized with 500 U of
heparin sodium to prevent thrombosis. The ends of the suture
were passed through a small, hollow plastic tube, which was
tapered at one end. This produced a snare, which could
occlude the artery with minimal mechanical injury to the
epicardium. The snare was pulled and then fixed in place
with a hemostat, thus inducing regional ischemia. Myocardial ischemia was confirmed visually in situ by regional
cyanosis, electrocardiograph S-T elevation/depression or T
wave inversion, hypokinetic movement of the myocardium,
and relative hypotension. Five minutes later, the clamp was
released, and the heart was allowed to reperfuse for 10 min.
Release of the snare for reperfusion was readily confirmed by
hyperemia over the surface of the previously ischemiccyanotic segment. The heart was subjected to three additional
5-min episodes of ischemia, each followed by 10 min of
reperfusion. After the completion of the preconditioning protocol, the incision was closed in layers and the chest was
evacuated of air. The animals were observed during recovery
until fully conscious and then extubated. After surgery, the
animals received intramuscular doses of analgesia (buprenor-
phine 0.02 mg/kg) and antibiotic (penicillin 200,000 U/kg).
They were then returned to their cages and allowed free
access to food and water. In the sham operation, the rabbits
underwent the same surgical preparation as the preconditioned animals, minus the induction of ischemia by pulling
ends of the suture. Instead, the chest remained open for 50
min. These animals were also allowed to recover for
24 h before sustained ischemia.
Day 2: Myocardial infarction protocol. Sham-operated and
preconditioned rabbits were reanesthetized with doses of
ketamine and xylazine identical to those used during the
preconditioning protocol. Body temperature was maintained
at 37°C with the use of a heating pad. The neck was
subsequently opened with a ventral midline incision, and a
tracheotomy was performed. The animals were intubated and
ventilated according to the parameters of the previous day.
The jugular vein was cannulated with a polyethylene catheter for infusion of saline solution and drugs. The carotid
artery was also cannulated with a polyethylene catheter for
blood sampling and pressure monitoring. Electrocardiographic leads were reattached to the limbs of the animal. The
chest was reopened, and another hollow plastic tube was
placed around the already-existing suture. The animal was
then heparinized with 500 U of heparin sodium. The snare
was clamped once again, and the LAD was occluded. After 30
min of ischemia the ligature was released, and the heart was
allowed to reperfuse for 180 min. The thoracic cavity was
covered with a plastic film to minimize heat loss.
Infarct size assessment. At the end of the infarction protocol, the ligature around the coronary artery was retightened
and ⬃4–5 ml of 10% Evans blue dye was injected as a bolus
into the jugular vein until the eyes turned blue. The animal
was immediately killed, and the heart was removed and
frozen. The frozen heart was then cut into six to eight
transverse slices of equal thickness from the apex to the base.
The area at risk was determined by negative staining with
Evans blue. The slices were then incubated in a 1% TTC
solution in isotonic pH 7.4 phosphate buffer at 37°C for 20
min. Tetrazolium reacts with NADH in the presence of
dehydrogenase enzymes, causing viable tissue to stain a deep
red color. The slices were subsequently fixed in a 10%
Formalin solution. Red-stained viable tissue was easily distinguished from the infarcted pale, unstained necrotic tissue.
The areas of infarcted tissue, the risk zone, and the whole left
ventricle (LV) were determined by digital planimetry with
computer morphometry using a Bioquant imaging software.
The area for each region was averaged from the slices. Infarct
size was expressed both as a percentage of the total LV and as
a percentage of the ischemic risk area.
Epicardial APD. The activity of the ventricular KATP channel was assessed during ischemia by placing an epicardial
probe (MAP electrode, EP Technologies, Sunnyvale, CA) in
the center of the ischemic region. The electrode was placed
with a constant pressure to the perceived center of the
ischemic zone. The APD at 50% and 90% repolarization
(APD50 and APD90, respectively) was determined during
preischemia and after every 10 min of LAD occlusion. The
APD was accepted only if it fulfilled the following criteria: 1)
constant configuration and stable resting membrane potential and 2) stable amplitude of phase 2 ⬎10 mV during control
recording. The percent APD change was defined as
KATP CHANNEL IN DELAYED ISCHEMIC PRECONDITIONING
between different time points within the same group or
between different groups were performed using a t-test.
Statistical differences with a P value of ⬍0.05 were considered significant.
RESULTS
Exclusions and mortality. A total of 96 rabbits were
entered into the present study. A summary of the
number of animals in each group and the reasons for
exclusion is shown in Table 1.
Blood pH and gases. The pH was maintained between 7.20 and 7.50. The PCO2 was maintained between
20 and 50 and the PO2 between 65 and 150. The HCO3
was sustained between 15.0 and 28.0, and the O2
saturation was consistently kept above 95%. An exception to these ranges was in one animal of group V that
was respirated with 100% O2 during the reperfusion.
Systemic hemodynamics. Heart rate and MAP remained reasonably stable throughout the reperfusion
period, although these parameters dropped gradually
at most of the data points in all groups (Table 2). Except
for a few time points, the mean values were not
significantly different between the groups and at any
time point within the groups.
Infarct size. Infarct size (in % of risk area) was not
significantly different between the control and shamoperated animals (40.0 ⫾ 3.8% vs. 41.2 ⫾ 2.6%, P ⬎
0.05, n ⫽ 9–11/group; Fig. 2). Preconditioning resulted
in a significant decrease in infarct size from 41.2 ⫾
2.6% of the area at risk in the sham-operated hearts to
11.6 ⫾ 1.0% of the area at risk, a 71% reduction from
sham-operated hearts (P ⬍ 0.01, n ⫽ 11). Treatment of
preconditioned rabbits with glibenclamide or 5-HD
before ischemia-reperfusion resulted in a significant
increase in the infarct size to 43.4 ⫾ 2.6 and 37.8 ⫾
1.9%, respectively (P ⬍ 0.01 from preconditioned group,
n ⫽ 12/group). Also, nonpreconditioned control rabbits
treated with either glibenclamide or 5-HD had an
Fig. 1. Experimental protocol. Glib, glibenclamide; 5-HD, 5-hydroxydecanoate; PC, preconditioning.
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Study protocol. Rabbits were randomly assigned to one of
the seven groups, and all animals were subjected to 30 min of
sustained ischemia followed by 180 min of reperfusion. In
addition, five to eight animals from each group were used for
measurement of APD. The experimental protocol is illustrated in Fig. 1. In group I (n ⫽ 13), rabbits received no
treatment on day 1 of the experiment. In group II (n ⫽ 11),
nonpreconditioned control rabbits were treated with glibenclamide (0.3 mg/kg ip) 30 min before sustained ischemia and
reperfusion on day 2. In group III (n ⫽ 11), nonpreconditioned
control rabbits were treated with 5-HD (5 mg/kg iv) 15 min
before sustained ischemia and reperfusion on day 2. In group
IV (sham operated, n ⫽ 9), the chest was opened and a suture
was placed around the coronary artery on day 1 of the
experiment. However, the artery was not occluded and thus
the animals were not preconditioned. In group V (n ⫽ 11), the
animals received preconditioning with four 5-min coronary
artery occlusions each followed by a 10-min reperfusion
period on day 1 of the experiment. In group VI (n ⫽ 12),
preconditioned animals received glibenclamide (0.3 mg/kg ip)
30 min before sustained ischemia and reperfusion on day 2.
In group VII (n ⫽ 12), preconditioned animals were treated
with 5-HD (5 mg/kg iv) 15 min before sustained ischemia and
reperfusion on day 2.
Blood pH and gases. Arterial blood gases and pH were
measured seven times for groups IV–VII during the day 1
protocol and seven times for all groups during ischemiareperfusion. These measurements were taken to ensure proper
physiological respiration during the experiment.
Measurement of hemodynamics. Hemodynamic measurements included heart rate, systolic arterial pressure, and
mean arterial pressure (MAP). The rate-pressure product
was determined as the product of the heart rate and peak
arterial pressure. These parameters were continuously measured throughout the duration of the experimental protocol
with a strip-chart recorder.
Statistics. All measurements of infarct size, risk areas, and
APD are expressed as group means ⫾ SE. Changes in
hemodynamics, APD, and infarct size variables were analyzed by a one-way repeated-measures ANOVA to determine
the effect of time, group, and time by group interaction. If the
global tests showed major interactions, post hoc contrasts
H1325
H1326
KATP CHANNEL IN DELAYED ISCHEMIC PRECONDITIONING
Table 1. Mortality and exclusions
Group
No. of
No.
Survival,
Animals Excluded
%
14
13
1
2
93
85
5-HD ( group III )
13
2
85
Sham ( group IV )
12
3
75
PC ( group V )
16
4
75
PC ⫹ Glib ( group VI )
PC ⫹ 5-HD ( group VII )
Total
14
13
95
2
1
15
86
92
84
Animal died of ventricular tachycardia/fibrillation during infarction protocol.
One rabbit died right after induction of anesthesia; the other animal died of congestive heart failure during reperfusion stage.
Hearts from the two animals did not have any risk zone as ligature came off during
injection of Evans blue dye.
One animal died during the surgical preparation before ischemia; one animal’s heart
was all stained with Evans blue dye; third rabbit was in cardiogenic shock after
30-min sustained ischemia.
Two rabbits died during the day 1 preconditioning protocol of respiratory cause (one
animal was in congestive heart failure after closure of chest, and other rabbit died
because of ventilator failure); one animal died of cardiogenic shock during reperfusion period on 2nd day; fourth rabbit’s heart was all blue, precluding any measurements.
Hearts of both animals were all blue from Evans blue dye.
Heart was all blue because suture came off during injection of Evans blue dye.
Glib, glibenclamide; 5-HD, 5-hydroxydecanoate; sham, sham operated; PC, preconditioned.
infarct size of 39.0 ⫾ 3.4 and 37.8 ⫾ 1.5%, respectively.
These values were not significantly different compared
with control (40.0 ⫾ 3.8%) or sham (41.2 ⫾ 2.6%)
ischemic-reperfused hearts. A similar trend in infarct
size was observed when it was expressed as a percentage of the LV. The mean infarct size values in control
(21.5 ⫾ 2.2%) and sham-operated (21.7 ⫾ 2.6%) hearts
were not significantly different (P ⬎ 0.05). Preconditioning significantly reduced the infarct size to 6.5 ⫾ 1.0%
(P ⬍ 0.01 vs. control and sham). Both glibenclamide
and 5-HD blocked preconditioning-induced protection
without inducing significant effects in the nonprecondi-
tioned rabbits. Moreover, these drugs did not alter the
infarct size in the nonpreconditioned control hearts.
The risk areas ranged from 50 to 56% with no significant differences among all groups (P ⬎ 0.05). These
data suggest that changes in the size of infarct observed among various groups were not related to the
percentage of area of LV occluded by our technique.
APD. The APD50 significantly shortened by 28.7,
26.6, and 23.3% in sham animals during 10, 20, and 30
min after ischemia, respectively (Fig. 3). A similar
degree of APD50 shortening was observed in the control
animals (not shown). By 30 min of reperfusion, the APD
Table 2. Hemodynamic data
Baseline
Control ( group I )
HR
MAP
RPP
Glib ( group II )
HR
MAP
RPP
5-HD ( group III )
HR
MAP
RPP
Sham ( group IV )
HR
MAP
RPP
PC ( group V )
HR
MAP
RPP
PC ⫹ Glib ( group VI )
HR
MAP
RPP
PC ⫹ 5HD ( group VII )
HR
MAP
RPP
Preischemia
30-min Isch
60-min Rep
120-min Rep
180-min Rep
170 ⫾ 7
91 ⫾ 2
17,827 ⫾ 845†
184 ⫾ 8
89 ⫾ 4
18,625 ⫾ 1,049
202 ⫾ 12
67 ⫾ 5
16,345 ⫾ 1,579
189 ⫾ 9
77 ⫾ 4
16,653 ⫾ 1,073
186 ⫾ 9
64 ⫾ 5
13,899 ⫾ 1,311
182 ⫾ 11
62 ⫾ 4
13,591 ⫾ 894
178 ⫾ 10
89 ⫾ 4
18,072 ⫾ 1,425†
199 ⫾ 11
87 ⫾ 6
20,259 ⫾ 2,066
195 ⫾ 8
65 ⫾ 4
15,329 ⫾ 1,127
209 ⫾ 13
67 ⫾ 7
17,136 ⫾ 1,731
216 ⫾ 14
57 ⫾ 5
15,175 ⫾ 1,482
206 ⫾ 16
54 ⫾ 4
14,722 ⫾ 1,480
180 ⫾ 8
80 ⫾ 4*
16,756 ⫾ 1,094†
197 ⫾ 9
76 ⫾ 4
17,498 ⫾ 1,181
180 ⫾ 12
63 ⫾ 4
13,708 ⫾ 1,544
192 ⫾ 9
66 ⫾ 5
15,389 ⫾ 1,469
187 ⫾ 11
64 ⫾ 4
14,518 ⫾ 1,388
184 ⫾ 13
55 ⫾ 2
12,401 ⫾ 1,126
161 ⫾ 8
74 ⫾ 3*‡
14,160 ⫾ 801
176 ⫾ 9
71 ⫾ 3*‡
15,273 ⫾ 939
187 ⫾ 7
63 ⫾ 4
14,963 ⫾ 1,315
187 ⫾ 7
64 ⫾ 4
14,689 ⫾ 1,032
199 ⫾ 12
60 ⫾ 4
15,427 ⫾ 1,318
186 ⫾ 12
58 ⫾ 5
13,722 ⫾ 879
198 ⫾ 9*†
77 ⫾ 4*
18,092 ⫾ 908†
194 ⫾ 6
73 ⫾ 4*
17,325 ⫾ 766
206 ⫾ 5
66 ⫾ 4
16,860 ⫾ 934
203 ⫾ 8
65 ⫾ 4
16,406 ⫾ 936
196 ⫾ 8
64 ⫾ 4
15,820 ⫾ 916
194 ⫾ 7
59 ⫾ 4
14,539 ⫾ 804
199 ⫾ 7*†
75 ⫾ 4*‡
17,598 ⫾ 813†
200 ⫾ 8
72 ⫾ 3*‡
17,256 ⫾ 750
203 ⫾ 8
61 ⫾ 4
15,222 ⫾ 1,001
193 ⫾ 10
64 ⫾ 3*
15,157 ⫾ 982
186 ⫾ 11
63 ⫾ 4
14,529 ⫾ 1,066
188 ⫾ 11
60 ⫾ 4
14,172 ⫾ 1,125
200 ⫾ 8*†
65 ⫾ 2*‡
16,857 ⫾ 500†
192 ⫾ 8
72 ⫾ 2*‡
16,552 ⫾ 772
197 ⫾ 9
58 ⫾ 5
14,968 ⫾ 826
190 ⫾ 9
56 ⫾ 3*
13,741 ⫾ 856§
192 ⫾ 9
54 ⫾ 4
13,463 ⫾ 842§
191 ⫾ 15
51 ⫾ 5
12,663 ⫾ 1,077
Values are means ⫾ SE. Isch, ischemia; Rep, reperfusion; HR, heart rate (in beats/min); MAP, mean arterial pressure (in mmHg); RPP,
rate-pressure product (in mmHg/min). * P ⬍ 0.05 vs. control; † P ⬍ 0.05 vs. sham; ‡ P ⬍ 0.05 vs. Glib; § P ⬍ 0.05 vs. PC.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on October 1, 2016
Control ( group I )
Glib ( group II )
Reason for Exclusion
KATP CHANNEL IN DELAYED ISCHEMIC PRECONDITIONING
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returned to nearly baseline preischemic levels. In the
preconditioned hearts, the APD50 also shortened but
was not significantly different compared with the shamoperated group. A similar trend in the changes in the
APD90 was observed during ischemia-reperfusion (Fig.
3B). Pretreatment with glibenclamide and 5-HD did
not produce significant changes in baseline APD50 or
APD90. However, these drugs significantly suppressed
the ischemia-induced shortening of APD50 and APD90 in
the sham-operated and preconditioned hearts.
DISCUSSION
The major findings are summarized as follows. 1)
Ischemic preconditioning, 24 h before ischemia-reperfusion, resulted in a significant protection of the
heart as indicated by reduced infarct size. 2) A significant shortening of APD was observed during 10–30 min
of sustained ischemia in the control and sham-preconditioned hearts; the preconditioned hearts did not demonstrate further augmentation in APD shortening during
this period. 3) The KATP blockers glibenclamide and
5-HD abrogated the protective effect of preconditioning.
The epicardial APD shortening was equally suppressed
by glibenclamide and 5-HD, suggesting that these
drugs did not discriminate between the sarcolemmal
versus the mitochondrial KATP channel in vivo. These
results suggest that opening of KATP channels plays an
important role in the second window of preconditioning
in the rabbit heart. It is not clear whether this protection results from the opening of the sarcolemmal or the
mitochondrial KATP channel.
The phenomenon of delayed preconditioning in the
myocardium is an area of investigation that has
branched from the study of early or classical preconditioning. The early phase of preconditioning has been
consistently demonstrated in every species studied
(28). On the other hand, delayed preconditioning appears to be a species-specific phenomenon. It has been
consistently observed in rabbits (6, 30, 44) and dogs (27)
but remains controversial in rats (23, 42, 52) and
appears to be absent in pigs (45). Delayed preconditioning has also been observed in cultured myocytes (36,
37). Early ischemic preconditioning appears to engage
a final common pathway involving the activation of
G-coupled receptors such as adenosine, ␣-adrenergic,
bradykinin, and opioids (39). These receptors activate
protein kinase C (PKC), which appears to play a central
role in conferring ischemic tolerance via a variety of
kinases (14). PKC and tyrosine kinase inhibition has
been shown to abolish phenylephrine-induced functional preconditioning in rat and infarct size reduction
in vivo in rabbit hearts (33, 38). Delayed preconditioning against infarction with ischemic and heat shock
preconditioning was abolished by chelerythrine, a specific inhibitor of PKC (2, 26). Conversely, administration of 1,2-dioctanyol-sn-glycerolin, the physiological
activator of PKC, induced delayed preconditioning
against subsequent ischemia-reperfusion (3). Activation of the KATP channel appears to be an endogenous
adaptive mechanism that protects the myocardium
against ischemia-reperfusion damage (11). PKC is a
potential candidate that links the receptors stimulated
by mediators released during ischemia to the activation
of the KATP channel. De Weille et al. (13) reported that
stimulation of PKC with a phorbol ester resulted in the
activation of the KATP channel in rat insulinoma cells.
Hu et al. (21) showed that PKC-activating phorbol ester
induced currents with properties of a KATP-channel
current at a reduced intracellular ATP concentration in
rabbit and human ventricular myocytes. Similarly,
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Fig. 2. Risk area [% of left ventricle (LV)] and infarct size expressed as % of area at risk and % of LV. In control
group, hearts were subjected to 30-min ischemia followed by 3-h reperfusion. In sham group, chest was opened and a
suture was placed around coronary artery. However, artery was not occluded and thus animals were not
preconditioned. In PC group, animals received preconditioning with four 5-min coronary artery occlusions each
followed by 10-min reperfusion. In PC ⫹ Glib group, preconditioned animals received Glib (0.3 mg/kg ip) 30 min
before sustained ischemia and reperfusion. In PC ⫹ 5-HD group, preconditioned animals were treated with 5-HD (5
mg/kg iv) 15 min before sustained ischemia and reperfusion. In Glib group, nonpreconditioned control rabbits were
treated with Glib (0.3 mg/kg) 30 min before sustained ischemia and reperfusion. In 5-HD group, nonpreconditioned
control rabbits were treated with 5-HD (5 mg/kg) 15 min before sustained ischemia and reperfusion. Each bar
represents mean ⫾ SE of 9–13 rabbits. * P ⬍ 0.01 from control, sham, PC ⫹ Glib, PC ⫹ 5-HD, Glib, and 5-HD
groups.
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KATP CHANNEL IN DELAYED ISCHEMIC PRECONDITIONING
Sato et al. (47) demonstrated that phorbol myristate
acetate, the activator of PKC, potentiated the diazoxideinduced mitochondrial KATP channel activity in the
ventricular myocytes. Preconditioning-induced activation of membrane receptors (adenosine/␣-adrenergic)
may potentially activate nitric oxide (NO) synthase via
a PKC-sensitive mechanism (40). It has been shown
that ␣1-adrenergic stimulation causes an upregulation
of cytokine-induced NO production by cardiac myocytes, which is mediated via activation of PKC (22). NO
has also been suggested to modulate KATP channels by
increasing the second messenger cGMP. Using patchclamp techniques, Cameron et al. (9) provided direct
evidence that NO enhances KATP channel activity in
hypertrophied ventricular myocytes. It has recently
been reported that NO may be an important mediator
of the delayed preconditioning induced by ischemia as
well as the pharmacological agent MLA (51, 55). The
cGMP-dependent protein kinases may be capable of
phosphorylating KATP channels and priming the channel to offer cardioprotection (8, 31).
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.2 on October 1, 2016
Fig. 3. Percent change in shortening of action potential duration
(APD). APD was measured with a hand-held placement electrode and
recorded at a chart speed of 100 mm/s. Electrode was placed with a
constant pressure to perceived center of ischemic zone. APD was
determined during preischemia and after every 10 min of LAD
occlusion. A: APD shortening at 50% repolarization (APD50 ). Baseline
APD50 values (in ms, means ⫾ SE) were 126.6 ⫾ 2.9, 124.3 ⫾ 9.5,
118.0 ⫾ 8.2, and 115.4 ⫾ 8.5 for sham (j), PC (l), PC ⫹ Glib (m), and
PC ⫹ 5-HD (p) groups, respectively (P ⬎ 0.05). * P ⬍ 0.05 vs. sham
group; **P ⬍ 0.05 vs. PC and sham groups. B: APD shortening at 90%
repolarization (APD90 ). Baseline APD90 values (in ms, means ⫾ SE)
were 180.4 ⫾ 4.6, 182.3 ⫾ 11.3, 170.0 ⫾ 9.3, and 151.4 ⫾ 12.3 for
sham, PC, PC ⫹ Glib, and PC ⫹ 5-HD groups, respectively (P ⬎ 0.05,
n ⫽ 5–8/group). * P ⬍ 0.05 vs. PC and sham groups.
Activation of the KATP channel is at least partially
responsible for the increase in outward K⫹ currents,
shortening of APD, and increase in extracellular K⫹
concentration during anoxic or globally ischemic conditions (5); it also modulates arrhythmogenesis in a
variety of experimental conditions (10, 25). Miyoshi et
al. (34) showed that ischemia-induced APD shortening
was observed at both the endocardial and epicardial
layers in response to KATP-channel modulators during
regional ischemia, although greater shortening was
observed at the epicardium. 5-HD suppressed the shortening preferentially at the epicardial layer, suggesting
that the drug was blocking the sarcolemmal KATP
channel in vivo. Sakamoto et al. (46) showed that 5-HD
reduced early accumulation of K⫹ during ischemia, an
effect that was similar to that of glibenclamide. In the
present investigation also, epicardial APD50 and APD90
were significantly shortened during ischemia, which
effect was suppressed not only by glibenclamide but
also by 5-HD. These data raise the possibility that
5-HD may not be the selective blocker of mitochondrial
KATP channel in vivo.
In the present investigation, we observed no further
augmentation in APD shortening in the preconditioned
hearts, suggesting two possibilities. First, the additional channels may not have been opened in the
preconditioned hearts during ischemia. Second, the
cardioprotection caused by opening of the KATP channel
is independent of APD shortening. These data are in
accord with the reports suggesting a lack of correlation
between the APD shortening and cardioprotection with
bimakalim and cromakalim, openers of the KATP channel (19, 53). Similarly, pyranyl cyanoguanidine analogs
have been shown to retain the glibenclamide-reversible
cardioprotective effects but lack the APD shortening
effect. Garlid et al. (16) first proposed that mitochondrial KATP channels could be involved in the cardioprotective effect of preconditioning. Using the mitochondrial KATP channel opener diazoxide, they demonstrated
a significant cardioprotective effect of the drug in the
isolated, perfused heart (16). A similar protective effect
of diazoxide has been shown in ventricular myocytes
(29, 47) and in rabbit heart in vivo (1). The cardioprotective effect of diazoxide was blocked by 5-HD, thereby
further confirming the role of the mitochondrial KATP
channel.
We used glibenclamide as one of the inhibitors of the
KATP channel. Recently, glibenclamide has been demonstrated to block the cAMP-activated chloride conductance (ICl,cAMP; 49). The Hill coefficients for the effect of
glibenclamide on ICl,cAMP and KATP channels are roughly
similar, but the effective concentration range is considerably higher (half-maximal inhibition of ICl,cAMP ⬃30
µM compared with 5–10 µM for half-maximal inhibition of KATP channels). We as well as several other
investigators have used 0.3 mg/kg glibenclamide to
block the channel function. Although we have not
directly measured levels of glibenclamide in the plasma,
Bekheit et al. (5) reported 0.4 ⫾ 0.03 µM in dog plasma
after 30 min of intravenous injection with 0.15 mg/kg
glibenclamide. We used a double concentration of gliben-
KATP CHANNEL IN DELAYED ISCHEMIC PRECONDITIONING
This work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-51045 and HL-59469 (R. C. Kukreja).
N. L. Bernardo was supported by a fellowship from the American
Heart Association, Virginia affiliate.
Address for reprint requests and other correspondence: R. C.
Kukreja, Box 282, Div. of Cardiology, Medical Col. of Virginia,
Virginia Commonwealth Univ., Richmond, VA 23298 (E-mail:
Rakesh@email.hsc.vcu.edu).
Received 5 October 1998; accepted in final form 23 December 1998.
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