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(Circulation. 1995;92:1236-1245.)
© 1995 American Heart Association, Inc.
Articles |
Bimakalim, an ATP-Sensitive Potassium Channel Opener, Mimics the Effects of Ischemic Preconditioning to Reduce Infarct Size, Adenosine Release, and Neutrophil Function in Dogs
From the Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee.
Correspondence to Garrett J. Gross, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background The primary goal of the present study was to determine whether the infarct size–reducing effect of preconditioning is associated with an increase in adenosine release from the ischemic myocardium during a prolonged occlusion period or the subsequent reperfusion period and by a decrease in neutrophil infiltration. A second objective was to determine whether bimakalim, a KATP channel opener, mimics the effects of ischemic preconditioning.
Methods and Results Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 3 hours of reperfusion. In the preconditioning group, 5 minutes of LAD occlusion followed by 10 minutes of reperfusion was elicited before the 60-minute occlusion period. In two other groups, bimakalim 1 µg/kg bolus followed by a 0.05 µg · kg-1 · min-1 infusion or an equivalent volume of saline was administered intravenously 15 minutes before occlusion and continued until the time of reperfusion. In a final group, bimakalim was administered 10 minutes before reperfusion and continued until the end of the experiment. To measure the release of adenosine from the ischemic region, coronary venous blood samples were collected at various times during ischemia and after reperfusion, and the concentration of adenosine was measured. Myocardial infarct size was determined by triphenyl tetrazolium chloride; transmural myocardial blood flow, by radioactive microspheres. Transmural myeloperoxidase (MPO) activity, an index of neutrophil infiltration in the area at risk, was also measured. Preconditioning produced a marked reduction in infarct size (9.8±3.0% versus 28.6±5.2% in the control group, mean±SEM); adenosine release at 5, 10, 15, and 30 minutes of the 3-hour reperfusion period; and transmural MPO activity in the risk area. Similarly, pretreatment with bimakalim resulted in reductions in infarct size, adenosine release, and transmural MPO activity to an extent almost identical to that of preconditioning. When bimakalim was administered 10 minutes before reperfusion, the drug also produced a significant reduction in infarct size and transmural MPO activity; however, no significant reduction in coronary venous adenosine concentrations was observed. There were no significant differences in collateral blood flow between groups.
Conclusions These results indicate that myocardial preconditioning in the canine heart produced by a short period of ischemia or a KATP channel opener is not mediated by an increase in adenosine release, as measured by coronary venous adenosine concentrations, during 60 minutes of occlusion or the initial 30 minutes of reperfusion. A significant reduction in transmural MPO activity in the ischemic area also appears to result from KATP channel activation and may play a role, at least in part, in the reduction in infarct size observed, particularly when a KATP channel opener is administered just before reperfusion.
Key Words: infarction • adenosine • reperfusion • occlusions • ischemia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Single or multiple brief periods of myocardial ischemia and reperfusion that limit infarct size after a subsequent, more sustained ischemic insult have been called ischemic preconditioning.1 This phenomenon has been shown to occur in a variety of animal species, including dogs,1 2 3 rabbits,4 rats,5 and pigs.6 Although myocardial infarction was not the end point, a preconditioning-like effect has also been demonstrated in patients subjected to repeated episodes of coronary occlusion during percutaneous transluminal coronary angioplasty.7 However, despite these observations, the precise mechanisms responsible for ischemic preconditioning are still unknown and are currently under investigation.
Initially, Liu et al8 suggested that preconditioning is mediated by activation of adenosine receptors because blockade of these receptors by 8-sulfophenyl theophylline prevents this phenomenon. Kirsch et al9 demonstrated that adenosine receptors are coupled to KATP channels by a Gi protein in rat ventricular myocytes. Results from our laboratory10 showed that ischemic preconditioning was completely blocked by glibenclamide, a selective KATP channel antagonist, in dogs. A number of studies also suggested that opening of KATP channels by ischemia or hypoxia11 or by selective potassium channel openers12 13 exerts a protective effect on the ischemic-reperfused heart. These results have led to the hypothesis that preconditioning occurs as a result of adenosine acting on its A1 receptor, which subsequently enhances opening of the KATP channel during ischemia.
Alternatively, preliminary data of Kitakaze et al14 led to the hypothesis that opening of KATP channels by ischemic preconditioning or by KATP channel openers increases the formation of adenosine through enhanced 5'-ectonucleotidase activity, and the increase in interstitial adenosine concentrations during preconditioning ischemia or the prolonged occlusion period is responsible for the cardioprotective effect of KATP channel opening. Using changes in local coronary venous adenosine concentrations and release during reperfusion as an index of interstitial adenosine during prolonged ischemia, Kitakaze et al15 also showed that adenosine release from the ischemic region after reperfusion was significantly increased in preconditioned dogs. Therefore, the first objective of the present study was to determine whether the cardioprotective effects of ischemic preconditioning or bimakalim, a KATP channel opener, are mediated by an increase in adenosine release from the ischemic myocardium during prolonged ischemia or after reperfusion in dogs. Coronary venous concentrations of adenosine were measured before and during the prolonged ischemic period and after reperfusion as an index of interstitial adenosine concentrations, recognizing that, in addition to cardiomyocyte adenosine metabolism, the endothelium markedly influences the levels of adenosine collected in venous blood draining the ischemic area and is likely to influence data interpretation.16
Previous results10 17 from our laboratory also showed that two KATP channel openers, aprikalim and bimakalim, and ischemic preconditioning10 result in a reduction in neutrophil infiltration into the ischemic myocardium after a prolonged period of coronary artery occlusion and reperfusion. Thus, a second objective of the present study was to examine the effect of preconditioning and bimakalim on neutrophil infiltration as assessed by myeloperoxidase (MPO) activity in the ischemic-reperfused area.
Finally, it is generally thought that beneficial effects of ischemic preconditioning and KATP channel openers occur primarily during the ischemic period and not during reperfusion. In support of this hypothesis, Grover et al13 and Auchampach and Gross17 showed that cromakalim and bimakalim administered just before reperfusion did not reduce infarct size in dogs subjected to 90 minutes of coronary artery occlusion and 5 hours of reperfusion. Similarly, Auchampach et al18 showed that aprikalim did not improve myocardial stunning when administered at reperfusion in dogs. In contrast, recent work of Iwamoto et al19 showed a beneficial effect of nicorandil on the recovery of postischemic contractile function when the drug was given during reperfusion in rabbit hearts; recent results from our laboratory20 demonstrated that nicorandil produced a significant reduction in infarct size when administered 10 minutes before and throughout reperfusion in dogs subjected to 60 minutes of coronary artery occlusion and 3 hours of reperfusion. Recent evidence also suggested that part of the effect of adenosine to produce preconditioning may occur during the reperfusion period21 ; however, similar studies have not been performed to determine whether KATP channel activation during reperfusion might also contribute to the cardioprotective effects of preconditioning. Therefore, the final objective of the present study was to determine whether activation of the KATP channel with bimakalim, a more selective and potent KATP channel opener than nicorandil, could also reduce myocardial infarct size, adenosine release, and MPO activity when administered 10 minutes before and throughout the reperfusion period.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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General Preparation of Dogs
All experiments conducted in the current study were in accordance with the "Position of the American Heart Association on Research and Animal Use" adopted in 1984 and the guidelines of the Animal Care Committee of the Medical College of Wisconsin. The Medical College of Wisconsin is accredited by the American Association of Laboratory Animal Care.
Adult mongrel dogs of either sex, weighing 19.5 to 29.3 kg, were fasted overnight, anesthetized with a combination of sodium barbital (200 mg/kg) and sodium pentobarbital (15 mg/kg), and ventilated by a respirator with room air supplemented with 100% oxygen. Atelectasis was prevented by maintaining an end-expiratory pressure of 5 to 7 cm H2O with a trap. Arterial blood pH, PCO2, and PO2 were monitored at selected intervals by an automatic blood gas system (AVL 995, AVL Scientific Corp) and maintained within normal physiological limits (pH 7.35 to 7.45; PCO2, 30 to 35 mm Hg; and PO2, 85 to 100 mm Hg) by adjustment of the respiration rate and oxygen flow or by intravenous administration of 1.5% sodium bicarbonate if necessary. Body temperature was maintained at 38±1°C with a heating pad. Aortic blood pressure and left ventricular (LV) pressure were monitored by insertion of a double-pressure transducer-tipped catheter (PC 771, Millar Instruments) into the aorta and left ventricle through the left carotid artery. LV dP/dt was recorded by electronic differentiation of the LV pressure pulse, and heart rate was determined by a tachometer. The right femoral vein and artery were cannulated for drug administration and for blood gas analysis and measurement of the reference blood flow used to determine myocardial tissue blood flow, respectively. A left thoracotomy was performed at the fifth intercostal space, the lung was carefully retracted, the pericardium was incised, and a catheter was inserted into the left jugular vein and gently advanced into the great cardiac vein at its junction with the anterior interventricular vein for the subsequent collection of blood samples for adenosine determination. The position of the catheter tip in the great cardiac vein was confirmed by visual inspection throughout the sampling portion of the protocol. The heart was then suspended in a pericardial cradle. A proximal portion of the left anterior descending coronary artery (LAD) distal to the first diagonal branch was isolated from surrounding tissue, and a calibrated electromagnetic flow probe (Statham SP 7515, Gould-Statham) was placed around the vessel. A flowmeter (Statham 2202) was used to measure LAD blood flow. A mechanical occluder was placed distal to the flow probe so that there were no branches between the flow probe and the occluder. The occluder was used to set the flow probe to zero (20 minutes before coronary occlusion, the LAD was occluded for 10 seconds), to occlude the LAD, and to reperfuse the myocardium. If the basal heart rate was <150 beats per minute (bpm), the heart was paced at that rate with rectangular pulses of 4-millisecond duration and with a voltage twice the threshold through bipolar electrodes clipped to the left atrial appendage. Pacing was not used in the few animals with initial rates >150 bpm. Hemodynamics, heart rate, and LAD blood flow were monitored and recorded by a polygraph (model 7, Grass Instrument) throughout the experiment. The left atrium was cannulated through the appendage for radioactive microsphere injection.
Experimental Design
Fig 1
shows the protocols
used in this study.
Animals were randomly assigned to one of four groups. The experimental
protocol included initial hemodynamic measurements and
arterial blood gas analysis before LAD occlusion.
Preconditioning was elicited by 5 minutes of LAD occlusion with 10
minutes of reperfusion before a 60-minute LAD occlusion period followed
by 3 hours of reperfusion (PC group). In another two groups,
approximately 15 minutes before the 60-minute LAD occlusion period,
bimakalim (BK/pre group) 1 µg/kg bolus followed by a 0.05
µg · kg-1 · min-1 infusion
or an
equivalent volume of saline (control group) was administered
intravenously and continued until the time of reperfusion.
In the last group, the same dose of bimakalim was given 10 minutes
before reperfusion and throughout the remainder of the 3-hour
reperfusion period (BK/post group). In all groups,
hemodynamics, blood gas analyses, and
myocardial blood flow were determined 30 minutes into the 60-minute
occlusion period. After reperfusion, hemodynamics were
measured every hour, and myocardial blood flow was determined at the
end of the experiment. Finally, the hearts were electrically
fibrillated, removed, and prepared for infarct size determination and
regional myocardial blood flow measurement; samples were obtained by
biopsy for analysis of MPO content.
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Infarct Size Determination
At the end of the 3-hour
reperfusion period, the LAD was
cannulated. To determine the anatomic area at risk (AAR) and the
nonischemic area, 5 mL of patent blue dye and 5 mL of saline
were injected at equal pressure into the left atrium and LAD,
respectively. The heart was immediately fibrillated and removed. The
left ventricle was dissected and sliced into serial transverse sections
6 to 7 mm wide. The nonstained ischemic area and the
blue-stained normal area were separated, and both regions were
incubated at 37°C for 15 minutes in 1% 2,3,5-triphenyl
tetrazolium chloride (TTC, Sigma Chemical Co) in 0.1 mol/L phosphate
buffer adjusted to pH 7.4. The TTC stains the noninfarcted
myocardium brick red, indicating the presence of a formazin
precipitate that results from the reduction of TTC by dehydrogenase
enzymes in viable tissue. After storage overnight in 10% formaldehyde,
infarcted and noninfarcted tissues within the AAR were separated and
determined gravimetrically. Infarct size was expressed as a percent of
the AAR.
Regional Myocardial Blood Flow
Regional myocardial blood flow
was measured by the radioactive
microsphere technique as described previously in this
laboratory.22 Microspheres were administered 30
minutes into the prolonged 60-minute occlusion period and at the end of
reperfusion. Carbonized plastic microspheres (15-µm diameter,
New England Nuclear) labeled with 141Ce or 95Nb
were suspended in isotonic saline with 0.01% Tween 80 added to prevent
aggregation. The microspheres were ultrasonicated for 5 minutes
and vortexed for another 5 minutes before injection. One milliliter of
the microsphere suspension (2 to 4x106
spheres) was given through the left atrial catheter and flushed by 5 mL
of saline. A reference blood flow sample was drawn from the right
femoral artery at a constant rate of 9.4 mL/min starting 30 seconds
before microsphere injection and continuing for 3 minutes. The
next day, the tissue slices were sectioned into subepicardium,
midmyocardium, and subendocardium of nonischemic
(three pieces) and ischemic (five pieces) regions. Transmural
pieces were obtained from the center of several transverse sections
used to determine the AAR and were at least 1 cm from the perfusion
boundaries as indicated by patent blue dye. All samples were counted in
a gamma counter (Tracor Analytic 1195) to determine the activity of
each isotope in each sample. The activity of each isotope was also
determined in the reference blood flow samples. Myocardial blood flow
was calculated by use of a preprogrammed computer to obtain the true
activity of each isotope in individual samples, and tissue blood flow
was calculated from the equation
Qm=QrxCm/Cr,
where Qm is myocardial blood flow (in milliliters per
minute per gram of tissue), Qr is the rate of withdrawal of
the reference blood flow (9.4 mL/min), Cr is the activity
of the reference blood flow sample (counts per minute), and
Cm is the activity of the tissue sample (counts per minute
per gram). Transmural blood flow was calculated as the weighted average
of the three layers in each region.
Adenosine Measurements
Sample Collection and
Preparation
Before LAD occlusion, arterial blood was sampled
from the right femoral artery, and coronary venous blood was
withdrawn through an 8F single-lumen (length 100 cm) catheter placed
into the great cardiac vein. Once the catheter was cleared of residual,
stagnant blood, 1 mL of blood was aspirated over a 3- to 5-second
period into a chilled 3-mL syringe containing stop solution that
consisted of a small amount of heparin (about 2 µL), 11 µmol/L
dipyridamole, and 0.6 µmol/L
erythro-9(2-hydroxy-3-nonyl) adenine. Immediately after the sample was
collected, the syringe was inverted back and forth gently and placed in
an ice bucket. Coronary venous blood was sampled after 5, 15,
and 60 minutes of the LAD occlusion period and at 5, 10, 15, and 30
minutes after reperfusion. In addition to these samples, blood was
drawn at 5 minutes of the initial occlusion period and at 5 and 10
minutes after reperfusion in the PC group. The technique used for
measuring plasma adenosine concentrations was a modified method
of Hermann and Feigl.23 After the last sample was drawn,
all samples were centrifuged for 2 minutes at
30 000g at 0°C to separate the plasma and stop solution
mixture from the cellular elements. Subsequently, 1 mL of the
supernatant was removed and transferred to a tube containing 25 µL of
cold 7 mol/L perchloric acid to precipitate plasma proteins. After
centrifugation (30 000g, 0°C for 10
minutes) to separate proteins, 0.6 mL of the supernatant was removed
and transferred to a tube containing 25 µL of 5N NaOH to neutralize
the solution. Then, 250 µL of the solution was transferred to an
autosample vial and mixed with 20 µL of 2.0 mol/L acetate buffer, pH
4.5, and 10 µL of chloroacetaldehyde. The vial was capped and heated
in an oven at 60°C for 4 hours. Adenosine reacts with
chloroacetaldehyde to form a strong fluorescent 1,
N6-ethanoadenosine. The sample was injected
directly into the high-performance liquid
chromatography (HPLC) from the sample vial.
HPLC Analysis
A newly developed HPLC method was used to
determine
adenosine concentrations in plasma.24 Briefly, 5
µL of the samples was injected and chromatographed on a 1090
series II Liquid Chromatograph (Hewlett-Packard Co) with an
autosampler and a column switching valve. A shielded hydrophobic phase
column, HiSep, 250x2.1 mm (Supelco, Inc), and an ODS-2 C18,
250x2.0
mm (Metachem Technologies, Inc), with an isochratic mobile phase of
10% acetonitrile and 90% of 0.1 mol/L sodium acetate and 0.002 mol/L
1-octanesulfonic acid, sodium salt was used for separation. The flow
rate was 0.20 mL/min. The eluent from the HiSep column was bypassed to
waste 6 seconds after injection. After 5 minutes, the eluent was
switched back to the C18 column for further separation and to the
detector. The fluorescence was detected by an FS 970 LC fluorometer
(Kratos Analytical Instruments) with an excitation wavelength of 274 nm
and a 370-nm long-pass filter for emission. The chromatograms were
recorded and the peaks integrated on a 3392 integrator
(Hewlett-Packard). The run time was 20 minutes with 5 minutes after run
time.
MPO Determination
MPO activity, an index of neutrophil
infiltration, was
determined in transmural tissue biopsies weighing 50 to 500 mg obtained
from the center of the AAR. The samples were immediately frozen in
liquid nitrogen and stored at -70°C.
MPO was extracted from the tissue samples by two separation procedures.25 26 Initially, the samples were homogenized in 50 mmol/L EDTA and centrifuged at 40 000g for 15 minutes. The supernatant, which contains the water-soluble heme-containing proteins hemoglobin and myoglobin, was decanted, and the pellet was resuspended in 50 mmol/L potassium phosphate buffer (pH 6) containing 5 mmol/L EDTA and 0.5% hexadecylammonium bromide to solubilize the MPO. Subsequently, the suspension was rehomogenized four times for 10 seconds each time, sonicated three times for 10 seconds each, and centrifuged at 40 000g for 15 minutes. The supernatant was then heated in a water bath at 60°C for 2 hours, a procedure previously shown to increase the recovery of MPO.26 MPO activity was assayed in triplicate using o-dianisidine as the substrate. A 0.1-mL aliquot of the supernatant was mixed with 2.9 mL of 50 mmol/L potassium phosphate buffer (pH 6) containing 0.167 mg/mL o-dianisidine and 0.005% hydrogen peroxide. The change in absorbance was measured spectrophotometrically at 460 µm and recorded on a chart recorder for approximately 3 minutes. Results were expressed as units of MPO activity per gram of wet tissue weight, where 1 unit is defined as that quantity of enzyme that degrades 1 µmol hydrogen peroxide per minute at 25°C.
Exclusion Criteria
Dogs were excluded if (1) heartworms were
found after the dogs
were killed, (2) transmural collateral blood flow was >0.20
mL · min-1 · g-1, (3) heart
rate was >180 bpm at the beginning of the experiment, or (4) more than
three consecutive attempts were needed to convert
ventricular fibrillation with low-energy DC pulses applied
directly to the heart.
Statistical Analysis
All values are expressed as
mean±SEM. Differences between
groups in hemodynamics and adenosine
concentrations were compared by use of a two-way (for time and
treatment) ANOVA with repeated measures and Fisher's least significant
difference test if significant F ratios were obtained. Differences
between groups in tissue blood flows, AAR, infarct size, and MPO
activity were compared by one-way ANOVA, and comparisons between groups
were made with Fisher's least significant difference test. ANCOVA was
used to determine whether the relation between transmural collateral
blood flow and infarct size differed between the control and
drug-treated groups. Differences between groups were considered
significant if the probability value was P<.05.
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Results |
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Mortality and Exclusions
Forty dogs were used initially in this study. We excluded 6 dogs because transmural collateral blood flow was >0.20 mL · min-1 · g-1 (2 each in the control and the BK/post groups, and 1 each in the PC group and the BK/pre groups), 1 dog in the control group because heart rate was >180 bpm, and 1 dog in the BK/pre group because more than three consecutive attempts were needed to convert ventricular fibrillation with DC pulses. Thus, 32 dogs, 8 in each group, were used for data analysis.
Hemodynamic Responses
Table 1 summarizes the
hemodynamic
data. There were no significant differences in heart rate, mean blood
pressure, pressure-rate product, LV dP/dt, or LAD blood flow
between groups throughout the experiment, except for the following
parameters. In the BK/post group, mean blood pressure was
significantly lower at 1, 2, and 3 hours of reperfusion, and there was
a significant increase in LAD blood flow at 2 and 3 hours of
reperfusion compared with the control group.
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Myocardial Infarct Size
Fig 2a and
2b shows the AAR and myocardial infarct
size, respectively. The anatomic AAR expressed as a percent of the left
ventricle was not significantly different between groups: control
group, 30.1±1.1%; PC group, 27.2±0.6%; BK/pre group,
31.5±1.4%;
and BK/post group, 32.3±1.5%. Myocardial infarct size expressed as a
percent of the AAR was 28.6±5.2% in the control group. In contrast,
the PC and BK/pre groups exhibited a marked reduction in infarct size:
PC group, 9.8±3.0% (P<.01 versus the control group);
BK/pre group, 14.3±3.4% (P<.05). Similarly, the BK/post
group also demonstrated a smaller infarct (17.1±3.4%,
P<.05).
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P<.01 vs
control group), and both BK/pre and BK/post groups (see Fig 1
Fig 3 shows the relation between
transmural collateral
blood flow measured at 30 minutes of occlusion and infarct size as a
percent of the AAR. In all groups, an inverse relation existed between
these two variables; however, the regression lines of the PC and
BK/pre groups were shifted downward compared with the control group by
ANCOVA (P<.05). These data indicate that infarcts were
smaller for any given collateral flow in the PC and BK/pre groups.
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Regional Myocardial Blood Flow
Table 2
summarizes the transmural collateral blood
flow data in the nonischemic (left circumflex coronary
artery) and ischemic (LAD) regions. There were no significant
differences in transmural collateral blood flows during occlusion
between groups, which indicates that all groups were subjected to
similar degrees of ischemia. At 3 hours of reperfusion,
regional myocardial blood flow in the ischemic-reperfused
region was significantly higher in the BK/pre and BK/post groups.
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Coronary Venous Adenosine Concentrations
Fig
4a, 4b, and 4c shows the
adenosine
concentrations obtained from the great cardiac vein at various times
before preconditioning, during preconditioning, after preconditioning,
during the prolonged ischemic period, and at 5, 10, 15, and 30
minutes after reperfusion in the PC group, and before and during the
prolonged ischemic period and at 5, 10, 15, and 30 minutes of
reperfusion in the BK/pre and BK/post groups, respectively. A single
5-minute preconditioning episode resulted in no change in
adenosine concentrations during prolonged ischemia and
a significant reduction in adenosine concentration at 5, 10,
15, and 30 minutes of reperfusion (at 5 and 30 minutes,
P<.01; at 10 and 15 minutes, P<.05 versus
control group), as shown in Fig 4a. The BK/pre group also
showed no
change in adenosine concentration during the prolonged
ischemic period and a significant reduction in
adenosine concentration at 5, 10, and 15 minutes of reperfusion
(P<.05 versus control group), as illustrated in Fig
4b. On
the other hand, the "posttreated" group (BK/post group) showed no
significant differences in adenosine concentrations during
ischemia or reperfusion compared with the control group (Fig
4c). Because coronary blood flows during occlusion and the
first 30 minutes of reperfusion were not significantly different in the
four groups (data not shown), these changes in coronary venous
adenosine concentrations are a reflection of adenosine
release during occlusion and reperfusion and suggest that PC and BK/pre
reduced adenosine release during early reperfusion after the
prolonged ischemic period.
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MPO Activity
Fig 5 summarizes the data
illustrating transmural
MPO activity, an index of neutrophil infiltration into the AAR.
Preconditioning and treatment with bimakalim resulted in a significant
reduction in transmural MPO activity compared with control animals
(P<.05 versus control group).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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In the present study, we compared the effects of ischemic preconditioning and the KATP channel opener bimakalim on infarct size, adenosine release from the ischemic-reperfused area, and MPO activity in the risk zone. The results indicate that a single 5-minute period of coronary artery occlusion 10 minutes before a subsequent 60-minute period of ischemia and 3 hours of reperfusion produced a marked reduction in infarct size, adenosine release after prolonged ischemia, and MPO activity. Similarly, bimakalim mimicked the effects of ischemic preconditioning when administered before and throughout the coronary occlusion period. We also observed that bimakalim, when given 10 minutes before and throughout reperfusion, failed to show any reduction in adenosine release from the ischemic area during ischemia or reperfusion; however, the drug still produced a significant reduction in infarct size and MPO activity.
These results suggest that the cardioprotective effects of ischemic preconditioning and bimakalim may have similar mechanisms but are unlikely to be mediated by an increase in adenosine release from the ischemic-reperfused area during reperfusion. Because the KATP channel was previously shown to be an important mediator of ischemic preconditioning and bimakalim mirrored its effect, it is likely that the KATP channel is primarily responsible for the powerful infarct size–limiting effect of preconditioning in this canine model. The additional finding that bimakalim still produced a significant reduction in infarct size while reducing MPO activity in the ischemic-reperfused area when the drug was given at reperfusion suggests that a decrease in neutrophil activation and/or infiltration into the reperfused myocardium may also contribute, at least in part, to the beneficial effects of bimakalim.
Ischemic Preconditioning and Myocardial Infarct
Size
A number of studies were performed to determine the cellular
mechanism of ischemic preconditioning. Previous work of Murry
and colleagues1 indicated that preconditioning in dogs
produced by four 5-minute episodes of coronary artery occlusion
followed by a subsequent 40-minute ischemic period and 72 hours
of reperfusion resulted in a marked reduction in infarct size. These
investigators also observed that preconditioned hearts developed
ultrastructural damage more slowly than
nonpreconditioned hearts and that the rate of ATP
depletion during the initial phase of the sustained 40-minute
ischemic period was reduced as a result of a decrease in ATP
utilization.27 In addition, they found a decrease in the
rate of glycogen breakdown and anaerobic glycolysis and
suggested that a preservation of ATP levels or a reduction in the
cellular content of toxic metabolites may be responsible for
preconditioning. Recently, Liu et al8 showed that the
adenosine A1-receptor agonist R-PIA mimicked the
effects of preconditioning and that several adenosine receptor
antagonists blocked its effect, suggesting that
ischemic preconditioning is mediated by adenosine
A1 receptors. Preliminary results of Kitakaze et
al14 showed that the KATP channel openers
nicorandil and cromakalim mimicked the effect of ischemic
preconditioning in the canine heart and suggested that this beneficial
effect was, at least partially, the result of an increase in
5'-ectonucleotidase activity and subsequent increase in
adenosine release during reperfusion after the prolonged
ischemic period. These authors15 also demonstrated
that adenosine release from the ischemic-reperfused
area at similar times during reperfusion was significantly increased in
preconditioned dogs compared with control animals. More recently, Walsh
et al28 found that pinacidil, a KATP channel
opener, reduced myocardial infarct size in rabbits and that this effect
could be blocked by 8-sulfophenyl theophylline, a nonselective
adenosine receptor antagonist. These authors
suggested that pinacidil may be protecting the ischemic
myocardium by increasing adenosine release.
However, the results of the present study with preconditioning or
bimakalim do not support this hypothesis because we observed that
adenosine release from the ischemic
myocardium during reperfusion was significantly reduced in
the preconditioning and bimakalim-treated groups compared with the
vehicle-treated control animals.
Previous results from our laboratory10 showed that a single 5-minute period of preconditioning before a subsequent 60-minute ischemic period produced a marked reduction in infarct size and that this phenomenon was abolished by glibenclamide, a selective KATP channel antagonist. Kirsch et al9 demonstrated that adenosine A1 receptors are coupled to KATP channels in neonatal rat ventricular myocytes. Recent preliminary results of Snell et al29 showed that interstitial adenosine, which was markedly increased during 30 minutes of ischemia, was not altered by blockade of KATP channels with glibenclamide in rabbits. More recently, Schulz et al30 reported that blockade of KATP channels with glibenclamide abolished the infarct-reducing effect of preconditioning accompanied by an increase in adenosine release into the coronary venous effluent after reperfusion in swine. Finally, Van Wylen31 showed that interstitial levels of adenosine were actually decreased during the initial 20 to 30 minutes of the prolonged occlusion period in preconditioned dog hearts. Therefore, from these previous results and those of the present study, it is unlikely that ischemic preconditioning is mediated through an increase in adenosine release from the ischemic-reperfused myocardium during coronary artery occlusion and/or reperfusion and is more likely to be mediated by direct activation of myocardial KATP channels in this canine model. In fact, the present results suggest that adenosine release is directly correlated with the size of the myocardial infarction, which supports the previous findings of Bardenheuer and Schrader32 in animals and Bardenheuer et al33 in man that suggest that adenosine release from the heart, particularly during reperfusion, is a sensitive marker of the intensity of ischemia during the occlusion period. Therefore, from these results, one would expect that adenosine release during reperfusion would be decreased in hearts with smaller infarcts in the ischemic PC and BK/pre groups as a result of the antiischemic effects of these two interventions; the present results support this hypothesis. In addition, studies by Grover et al34 in isolated rat hearts and McPherson et al35 in guinea pig right ventricular free-wall preparations showed that KATP channel openers reduced ATP breakdown during ischemia, which would most likely result in less adenosine formation and subsequent release during reperfusion, a supposition also supported by the present findings.
The reasons for the differences between the present results and those of Kitakaze et al14 15 are not apparent but may be related to a number of factors, including experimental design, sampling techniques, and methods used for adenosine determinations. In the studies performed by Kitakaze et al,14 15 preconditioning produced by ischemia or KATP channel openers was produced by four 5-minute periods of coronary occlusion or intracoronary drug infusions as opposed to the one period of occlusion or a continuous infusion of bimakalim in this study. Also, different periods of prolonged ischemia (40, 60, and 90 minutes) were used in the three studies and could influence the pattern of adenosine release after reperfusion. Nevertheless, in all three studies, a similar reduction in infarct size was produced by preconditioning with ischemia or by KATP channel openers, and a similar effect on adenosine metabolism and release might be expected. Obviously, more experiments are needed in which identical protocols are used to resolve these issues.
Another important finding in the present study was that the KATP channel opener bimakalim mimicked the effect of ischemic preconditioning when the drug was given before and throughout occlusion at a low, clinically relevant, nonhypotensive dose. The effect of KATP channel openers on infarct size is controversial, although the weight of evidence suggests that these agents produce a reduction in irreversible injury after a prolonged period of coronary artery occlusion and reperfusion. Previous work of Endo et al36 and our laboratory37 showed that nicorandil or bimakalim produced a marked reduction in infarct size in dogs. Because nicorandil and bimakalim reduced the loading conditions on the heart in this previous study, the mechanism by which these agents reduced infarct size, direct activation of myocyte KATP channels, or the reduction in preload and/or afterload was not clear. The present results obtained with a nonhypotensive dose of bimakalim (BK/pre group) suggest that the hemodynamic effects of this agent are not essential for its cardioprotective properties and that these beneficial actions may be the result of direct activation of myocardial KATP channels. In support of this hypothesis, Yao and Gross38 also found that bimakalim reduced infarct size when administered intracoronarily at three nonhypotensive doses only during the first 10 minutes of a 60-minute occlusion period in dogs. Therefore, the results of the present study that demonstrate that pretreatment with a nonhypotensive dose of bimakalim produces a marked reduction in infarct size, adenosine release, and MPO activity to an extent almost identical to that of the preconditioning group are further indirect evidence to support the hypothesis that ischemic preconditioning is mediated primarily mediated through activation of KATP channels in canine hearts. On the other hand, a similar profile of activity between KATP channel openers and ischemic preconditioning does not necessarily mean that the two interventions are reducing infarct size by identical mechanisms. Several studies39 40 also were performed in which ß-blockers and calcium channel blockers were shown to reduce infarct size and MPO activity. However, in these studies, one could not rule out a primary hemodynamic effect as being responsible for the cardioprotection observed with these two classes of drugs. Obviously, further studies are needed in which direct comparisons between KATP channel openers, ß-blockers, calcium channel blockers, and ischemic preconditioning are performed to determine whether similar profiles exist in the presence of these interventions on infarct size, adenosine release, and MPO activity.
Neutrophil Function and Infarct Size
Another potentially
important mechanism by which ischemic
preconditioning or bimakalim may reduce infarct size is through an
inhibitory effect on neutrophil function. ATP-sensitive
potassium channel openers have been reported to attenuate neutrophil
function in ischemic myocardium. The results of in
vitro studies showed that nicorandil produced a concentration-dependent
(10-6 to 10-3 mol/L) inhibition of
superoxide
production by human and canine neutrophils.37
Bimakalim was also shown to inhibit superoxide production in canine
neutrophils.41 More recently, aprikalim,10
bimakalim,17 and nicorandil20 were shown to
reduce neutrophil infiltration into the ischemic-reperfused
myocardium of dogs subjected to 60 or 90 minutes of
coronary artery occlusion and 4 to 5 hours of reperfusion. The
effect on neutrophil infiltration with aprikalim and bimakalim occurred
when the drugs were given throughout the occlusion and reperfusion
periods, or in the case of bimakalim, MPO activity was also reduced
when it was given at reperfusion. Nicorandil reduced MPO activity when
given throughout the occlusion period or only at reperfusion. The
present results with bimakalim are similar to those from a previous
study from our laboratory17 and indicate that MPO activity
in the AAR was significantly lower in both the BK/pre and BK/post
groups. Because bimakalim had a significant effect on reducing infarct
size when administered immediately before and during reperfusion, it is
possible that this reduction in infarct size may have resulted, at
least in part, from a decrease in neutrophil activation and/or
infiltration into the reperfused myocardium.
Adenosine and its analogues were also demonstrated to diminish
the generation of toxic oxygen products by activated neutrophils.
Previous work of Babbitt et al42 showed that
intracoronary adenosine administration at reperfusion
resulted in a significant reduction in vascular injury in a canine
model of ischemia–reperfusion injury. Recent preliminary
results of Akimitsu and coworkers43 indicated that the
protective effects of ischemic preconditioning on leukocyte
adhesion to and emigration across venules in mouse cremaster muscles
were blocked by either adenosine deaminase or glibenclamide,
suggesting that both adenosine receptors and KATP
channels may play important roles in reducing neutrophil adhesion and
emigration induced by ischemic preconditioning. The results of
the present study, which demonstrated that posttreatment with
bimakalim still produced a significant reduction in infarct size and
MPO activity in the AAR whereas adenosine release from the
ischemic-reperfused area during reperfusion was not different
between the BK/post and control groups, suggest that the beneficial
effect of bimakalim could not be explained by an increase in
adenosine release from the AAR.
On the other hand, several findings suggest that neutrophils are not absolutely essential for the infarct size–reducing effect of KATP channel openers. First, in a recent study from our laboratory,17 bimakalim produced a significant reduction in MPO activity after 90 minutes of coronary artery occlusion and 5 hours of reperfusion when it was administered throughout the occlusion and reperfusion period or only at reperfusion; however, bimakalim reduced infarct size in the pretreated dogs only. On the other hand, because the ischemic period was longer in the previous study compared with the present one (90 versus 60 minutes), it is possible that the effect of bimakalim on neutrophils may be protective in the present protocol only when the total ischemic burden was less. That bimakalim reduced infarct size only modestly in our previous study suggests that this may be the case. Second, we recently reported that a nonhypotensive dose of nitroglycerin produced a significant reduction in infarct size without attenuating MPO activity in the ischemic-reperfused area when the drug was administered just before reperfusion in dogs.20 Finally, a number of studies44 45 performed in isolated hearts with buffer perfusion indicated that the protective effect of KATP channel openers or preconditioning can occur in the absence of neutrophils or other blood-borne elements. Therefore, although the reduction in MPO activity observed in the preconditioned and bimakalim-treated dogs is intriguing and may contribute to the infarct-reducing effect of these two interventions in this model, it is unlikely that neutrophils are absolutely necessary for the protective effect of KATP channel openers to be manifest in all models of ischemia-reperfusion injury.
Conclusions
The results of the present study indicate that
the marked
reduction in infarct size after a single 5-minute period of
preconditioning before a subsequent 60-minute period of
ischemia or after infusion of a selective KATP
channel opener before and throughout the prolonged occlusion period is
not mediated by an increase in adenosine release from the
ischemic-reperfused area after the prolonged occlusion period
but by direct activation of myocardial KATP channels in
canine hearts. A reduction in neutrophil infiltration into the
ischemic-reperfused myocardium may play a role in
the cardioprotective actions of KATP openers and in
ischemic preconditioning in this model. Because bimakalim
mimicked the cardioprotective effect of ischemic
preconditioning, this is further evidence that the KATP
channel is likely to be responsible, at least partially, for the
cardioprotective effect of ischemic preconditioning in the
canine heart in vivo.
|
Acknowledgments |
|---|
This study was supported by a grant from E. Merck and by NIH grant HL-08311. Dr Pierre Schelling, E. Merck, Darmstadt, Germany, generously provided the bimakalim. We would like to thank Anna Hsu and Jeannine Moore for their excellent technical assistance. We also thank Carol Knapp for assistance in the preparation of this manuscript.
Received January 19, 1995; accepted February 15, 1995.
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References |
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A. W. Gomoll, R. A. Roth, R. E. Swillo, A. J. Baird, C. S. Sargent, R. W. Behling, H. J. Malone, and G. J. Grover Effect of Timing of Treatment of the Glyburide-Reversible Cardioprotective Activity of BMS-180448 J. Pharmacol. Exp. Ther., April 1, 1997; 281(1): 24 - 33. [Abstract] [Full Text]
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G. J. Gross, D. A. Mei, P. G. Sleph, and G. J. Grover Adenosine A1 Receptor Blockade Does Not Abolish the Cardioprotective Effects of the Adenosine Triphosphate-sensitive Potassium Channel Opener Bimakalim J. Pharmacol. Exp. Ther., February 1, 1997; 280(2): 533 - 540. [Abstract] [Full Text]
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T. Mizumura, J. A. Auchampach, J. Linden, R. F. Bruns, and G. J. Gross PD 81,723, an Allosteric Enhancer of the A1 Adenosine Receptor, Lowers the Threshold for Ischemic Preconditioning in Dogs Circ. Res., September 1, 1996; 79(3): 415 - 423. [Abstract] [Full Text]
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M. Murata, M. Akao, B. O'Rourke, and E. Marban Mitochondrial ATP-Sensitive Potassium Channels Attenuate Matrix Ca2+ Overload During Simulated Ischemia and Reperfusion: Possible Mechanism of Cardioprotection Circ. Res., November 9, 2001; 89(10): 891 - 898. [Abstract] [Full Text] [PDF]
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