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Circulation. 1995;92:1236-1245

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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

Tsuneo Mizumura, MD, PhD; Kasem Nithipatikom, PhD; Garrett J. Gross, PhD

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.


*    Abstract
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 *Abstract
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 down arrowMethods
down arrowResults
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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


*    Introduction
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 *Introduction
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down arrowResults
down arrowDiscussion
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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.


*    Methods
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 *Methods
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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 1Down 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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  		Figure 1. Schematic diagrams of the experimental protocols. All dogs were subjected to 60 minutes of left anterior descending coronary (LAD) artery occlusion (Occ) followed by 3 hours of reperfusion (Rep). In the preconditioning (PC) group, a 5-minute period of LAD occlusion followed by 10 minutes of reperfusion was elicited before a subsequent 60-minute period of ischemia. In BK/pre or control group, bimakalim 1 µg/kg followed by a 0.05 µg · kg-1 · min-1 infusion or an equivalent dose of saline was administered intravenously approximately 15 minutes before occlusion and continued until the time of reperfusion. In the BK/post group, the same dose of bimakalim was administered 10 minutes before reperfusion and continued until the end of the experiment.

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.


*    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 1Down 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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  		Table 1. Hemodynamics in the Different Groups

Myocardial Infarct Size
Fig 2aDown and 2bDown 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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  		Figure 2. Bar graphs illustrating the effects of ischemic preconditioning and bimakalim on the area at risk expressed as a percent of the left ventricle (AAR/LV) and infarct size expressed as a percent of the area at risk (IS/AAR). a, There were no significant differences in AAR/LV between groups. b, Preconditioning resulted in a marked reduction in IS/AAR ({dagger}P<.01 vs control group), and both BK/pre and BK/post groups (see Fig 1Up) showed a significant reduction in IS/AAR (*P<.05 vs control group). CONT indicates control; PC, preconditioning; and BK, bimakalim.

Fig 3Down 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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  		Figure 3. Plots of the relation between transmural collateral blood flow (in milliliters per minute per gram) and infarct size expressed as a percent of the area at risk (IS/AAR). In all groups, there was an inverse relation between transmural blood flow and IS/AAR. The regression lines for the preconditioning (PC) group and the BK/pre group (see Fig 1Up) were shifted significantly downward compared with those for the control group by ANCOVA (P<.01).

Regional Myocardial Blood Flow
Table 2Down 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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  		Table 2. Transmural Myocardial Blood Flow

Coronary Venous Adenosine Concentrations
Fig 4aDown, 4bDown, and 4cDown 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 4aDown. 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 4bDown. 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 4cDown). 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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  		Figure 4. Plots illustrating venous adenosine concentrations from the ischemic-reperfused area at various times during ischemia and after reperfusion. a, Ischemic preconditioning resulted in a significant reduction in adenosine concentrations from the area at risk during reperfusion after the prolonged ischemic period compared with the control group. There were no significant differences during ischemia between the two groups. b, Pretreatment with bimakalim (BK/pre) also resulted in a significant reduction in adenosine concentration at 5, 10, and 15 minutes of the reperfusion period (*P<.05, {dagger}P<.01, vs control group). Again, there were no differences in adenosine concentrations during ischemia between the two groups. c, There were no significant differences in adenosine concentrations throughout the experiment when bimakalim was administered at reperfusion (BK/post). Oc indicates occlusion; Cont, control.

MPO Activity
Fig 5Down 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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  		Figure 5. Graph illustrating transmural myeloperoxidase (MPO) activity in the area at risk. Ischemic preconditioning resulted in a marked reduction in transmural MPO activity in the ischemic-reperfused area. Treatment with bimakalim also produced a marked reduction in transmural MPO activity regardless of the timing of drug administration (*P<.05 vs control group). CONT indicates control; PC, preconditioning; BK/pre, pretreatment with bimakalim; and BK/post, treatment with bimakalim at reperfusion.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
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.


*    References
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*References
 

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