In acute myocardial infarction (AMI), coronary reperfusion by percutaneous coronary intervention (PCI) or thrombolysis is a preferable therapy for salvaging the myocardium.1 Reperfusion therapy, however, is a double-edged sword; reperfusion itself causes reperfusion injury, such as myocardial cell damage, arrhythmias, myocardial stunning, and no-reflow.2,3 In particular, no-reflow just after PCI is one of the poor prognostic phenomena.4 Hence, adenosine,5 adenosine triphosphate,6 or verapamil,7 which can increase coronary blood flow during the acute phase of a myocardial infarction, are useful for the therapy of AMI. The effects of such drugs are achieved by direct administration to the coronary artery to avoid decreases in systemic blood pressure and heart rate, which are provoked when the drugs are systemically administered. Furthermore, microvascular and myocardial functions are improved by the intracoronary8 or intravenous injection of coronary vasodilators such as nicorandil.9 Thus, maintenance of coronary blood flow and myocardial microcirculation with pharmacological agents during reperfusion may play an important role in the achievement of optimal myocardial salvage in AMI.
Previous studies from our laboratory showed the pharmacological profiles of the 2-alkynyladenosine derivative 2-octynyladenosine (YT-146) in the cardiovascular system.10 This coronary vasodilator is characterized as a selective adenosine A2 receptor agonist on the basis of data obtained from a receptor-binding study.11 There are two possible mechanisms to explain its action following stimulation of the receptor: one mediated an increase in the cAMP level of aortic endothelial cells12 and the other causes opening of K+ channels.13 Indeed, intracoronary injection of YT-146 at the dose of 0.001 to 0.3 μg/kg produced a dose-dependent increase in the coronary blood flow, which reached the peak level within 30 s after the injection and decayed thereafter.10 Hence, we can expect cardioprotective effects of YT-146 through its specific coronary vasodilating action.
In the present study, we examined the effect of YT-146 administered just before reperfusion on ischemia/reperfusion-induced myocardial infarction of open-chest dogs. To evaluate the effect of YT-146, we examined parameters of myocardial infarct size, time course of profiles of hemodynamics, regional tissue blood flow at the territory of the occluded left anterior descending coronary artery (LAD), and plasma creatine kinase (CK) activity as a conventional marker of cardiac cell necrosis in the diagnosis of AMI. Particularly, LAD-perfused tissue blood flow was measured for the assessment of the effects of YT-146 via the adenosine A2 receptor because it is generally accepted that coronary artery dilatation by adenosine is mediated through A2 receptors.14 Furthermore, we compared the results of YT-146 with those of the ischemic preconditioning that is widely recognized to exert remarkable cardioprotective effects against myocardial ischemia/reperfusion injury.15
MATERIALS AND METHODS
All animal experiments were reviewed and approved by the Experimental Animal Committee of the Drug Research Department, Toa Eiyo Research Institute (Fukushima, Japan). Beagle dogs (Ridgilan Research Farms, Madison, WI) of either sex weighing 7.8 to 12.0 kg were housed individually in cages under regulated temperature (23°±3°C), humidity (50%±20%) and a 12-h light/dark cycle before the study. Each dog received a standard laboratory diet (DM-2, Funabashi Farms, Chiba, Japan) once daily and had free access to water.
Drugs and Reagents
YT-146 was synthesized by YAMASA Corporation (Chiba, Japan). The agent was dissolved in dimethyl sulfoxide, the final concentration of which was adjusted to 1% with 0.9% saline. 2,3,5-Triphenyl tetrazolium chloride (TTC) and Evans blue were purchased from Sigma (St Louis, MO), and CK assay reagent (L-Type Wako-CK) was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Other reagents were of the highest quality available (Wako Pure Chemical Industries Ltd.).
The dogs were anesthetized with pentobarbital sodium (30 mg/kg, IV), intubated with a cuffed endotracheal tube, and artificially ventilated with room air by a respirator (SN-480-3, Shinano, Tokyo) at a frequency of 18 strokes/min. The tidal volume was adjusted to maintain the pO2 of arterial blood at approximately 100 mm Hg, as determined by an automatic blood gas analyzer (AVL995, Sysmex, Tokyo, Japan). The body temperature was maintained at 38°C with a thermostat-equipped warming pad. The left femoral artery and vein were cannulated for collection of blood samples and administration of agents, respectively. The arterial blood pressure was measured through a polyethylene catheter inserted into the right femoral artery with a pressure transducer (DX-360, OHMEDA, Tokyo) that was connected to an amplifier (AP-601G, Nihon Kohden, Tokyo). The heart rate was measured by using a cardiotachometer (AT-601G, Nihon Kohden, Tokyo) triggered by the pulse wave of the arterial blood pressure. All hemodynamic parameters were continuously recorded on a multichannel recorder (Linearcorder F WR3701, GRAPHTEC, Tokyo). Throughout the experiment, hydration was maintained by transfusion with Solita-T3 (Takeda Chemical Industries, Osaka) given intravenously.
A left thoracotomy at the fifth intercostal space was performed, and the heart was suspended in a pericardial cradle. Then the proximal portion of the LAD distal to the first branch was isolated from the surrounding tissue. Thereafter, a nylon ligature was placed around the vessel and both ends of the nylon ligature were passed through a small polyethylene tube to make a coronary snare. The myocardial ischemia was achieved by tightening the coronary snare, and reperfusion was performed by releasing the snare after 90 min of ischemia.
Measurement of Regional Blood Flow
Regional myocardial blood flow in the LAD-perfused territory was measured by the hydrogen clearance technique as described previously.16 A platinum wire (UHE-201C, Unique Medical, Tokyo) was embedded into the center of the LAD-perfused endocardial myocardium to a depth of 6 to 8 mm from the surface. Hydrogen for a final concentration of 5% was added to the room air ventilation mixture for 1 min and then discontinued. Regional hydrogen washout from the tissue was measured polarographically (MHG-DI, Unique Medical). Blood flow was calculated as time required for hydrogen levels to fall from 90% maximum to 40% according to the formula below:
Regional myocardial blood flow (mL/min/g)=−ln (myocardial hydrogen levels 40%/90%)/Time (90% to 40%)
Measurements of Plasma CK Activity
The arterial blood samples were collected in heparinized syringes and immediately centrifuged at 1000g for 10 min at 4°C. The separated plasma was stored at –20°C until the assay could be performed. Plasma CK was measured by using a commercially available assay reagent and a clinical chemistry analyzer (CL-8000, Shimazu, Kyoto, Japan).
Determination of Infarct Size
After the reperfusion period, heparin (1000 U/kg) was administered IV, and the dog was then euthanized with an excess dose of pentobarbital given IV. The heart was quickly removed, and the site of occlusion on the LAD and the aorta above the coronary ostium were cannulated and perfused with 0.9% saline and 2% Evans blue in 0.9% saline, respectively, at the pressure of 100 mm Hg. The area at no risk was stained with Evans blue. Thereafter, the left ventricle was cut into 7 transverse slices, which were incubated with 1% TTC in 0.9% saline at 37°C for 20 min to discriminate the infarct area. After the slices had been fixed in 10% formalin, cumulative sizes of the left ventricle, area at risk (negative staining with Evans blue), and infarction (negative staining with TTC) of each slice, were determined by a computed planimetric method (QUANTIMET 500+, Leica Cambridge, Cambridge, England).
The experimental protocol is shown in Figure 1. After stabilization of the hemodynamic parameters, the LAD was occluded for 90 min followed by 300 min of reperfusion. Dimethyl sulfoxide (1% as vehicle control, n=5), a low dose of YT-146 (3 μg/kg, n=5), or a high dose of it (10 μg/kg, n=5) was administered into the dog 1 min before the start of reperfusion. Dogs in the ischemic preconditioning group (n=5) were subjected to 4 episodes of 5-min occlusion followed by 5 min of reperfusion. Regional myocardial blood flow was estimated at 10 different time intervals (before ischemia, after 60 min of ischemia and after 5, 30, 60, 90, 120, 180, 240, and 300 min of reperfusion). Furthermore, 1.5 mL of arterial blood was collected for measuring the plasma CK activity and blood pO2 at the different time intervals given above. In a preliminary study, we tested the effects of YT-146 at different doses (3, 10, and 30 μg/kg) and found that YT-146 at the highest dose elicited a decrease in acute hyperemic coronary flow associated with no reduction in the infarct size. Thus, we employed 3 and 10 μg/kg YT-146 in the present study.
Experiments were terminated or excluded from the final data analysis, if any of the following events occurred: (1) ventricular fibrillation appeared during ischemia, (2) >4 attempts were required to correct ventricular fibrillation with 10 to 20 J DC pulse with a cardiac monitor defibrillator (Life pack 9B, NEC Medical System, Tokyo) applied directly to the heart, and (3) regional myocardial blood flow was <0.5 mL/min/g before ischemia or >0.1 mL/min/g during ischemia.
Each value was represented as the mean±SEM. Statistical significance was estimated by using SPSS (SPSS Japan, Tokyo). Comparison of area at risk or infarct size between the groups was made by one-way analysis of variance followed by Dunnett t test. Comparison of the degree of coronary hyperemic flow at 5 min after the onset of reperfusion among the groups was made by 1-way analysis of variance followed by Dunnett t test. Comparisons between the agent-treated groups and control for the values of the regional blood flow and plasma CK activity after reperfusion and of hemodynamic parameters throughout the experiment between controls and the agent-treated groups were made by 2-way analysis of variance for repeated measures. Intergroup differences in hemodynamics were estimated by using Student paired t test and a modified Bonferroni correction was used to determine significance. Differences reaching P < 0.05 were considered to be statistically significant.
Number of Animals Employed
Twenty-seven dogs were used in the present study. Four dogs were excluded because a considerably large degree of regional myocardial blood flow in the ischemic area (>0.1 mL/min/g) was observed. Two dogs were excluded because of ventricular fibrillation during ischemia. After reperfusion, reperfusion-induced ventricular fibrillation appeared in 3 dogs (2 dogs in the vehicle-treated group and 1 dog in the preconditioning group). One of them had to undergo more than 4 defibrillations (in the vehicle-treated group). In YT-146-treated dogs, lethal arrhythmias did not occur during reperfusion. Accordingly, 20 dogs, 5 each in vehicle, preconditioning, and the 2 treatment groups (ie, with 3 μg/kg [low dose] YT-146 [YT-146 L], and 10 μg/kg [high dose] YT-146 [YT-146 H]), were used for data analysis.
Figure 2 shows changes in the mean blood pressure and heart rate in each group. The initial mean blood pressure and heart rate were maintained at approximately 100 mm Hg and 160 bpm, respectively. YT-146 at doses of 3 and 10 μg/kg per se transiently decreased the diastolic blood pressure immediately after the bolus injection, but did not significantly alter the mean blood pressure or heart rate during the reperfusion period.
Regional Myocardial Blood Flow During Ischemia and Reperfusion
Changes in regional blood flow during ischemia and reperfusion were expressed as percentages of the value before ischemia (Fig. 3a) because the values for regional myocardial blood flow before ischemia in each group ranged from 0.8 to 1.3 mL/min/g. The values for regional collateral blood flow during ischemia in each group were 0 to 0.04 mL/min/g, indicating that the hearts received a similar degree of ischemic insult. After 5 min of reperfusion, coronary hyperemic flow was detected in the vehicle group, and the regional myocardial blood flow increased to 124%±18% of the value before ischemia. YT-146 at doses of 3 and 10 μg/kg increased the coronary hyperemic flow dose dependently to 140%±20% and 180%±25% of each value before ischemia, whereas the ischemic preconditioning did not alter the flow (Fig. 3b).
Plasma CK Activity
Figure 4 shows the time course of changes in the plasma CK activity in each group. The initial plasma CK activities before ischemia ranged from 175 to 262 IU/mL. The CK levels were markedly increased after ischemia/reperfusion and reached the peak value of 17110±501 IU/mL after 180 min of reperfusion in the vehicle group. The ischemic preconditioning and treatment with YT-146 at doses of 3 and 10 μg/kg significantly reduced the CK activity at 180 min of reperfusion to 5905±2364, 9385±2338, and 5981±1612 IU/mL, respectively.
Myocardial Infarct Size
Figure 5 shows the myocardial area at risk/left ventricle and infarct size/area at risk. The area at risk in each group was 36.1% to 39.5%, and was not significantly different among each other. The infarct size of the vehicle group was 56.2%±2.7%, and that of the ischemic preconditioning group was significantly reduced to 19.2%±6.3%. YT-146 at doses of 3 and 10 μg/kg significantly reduced the infarct size to 29.5%±8.7% and 20.2%±7.0%, respectively.
In the present study, we demonstrated that YT-146 at doses of 3 and 10 μg/kg administered intravenously just before reperfusion reduced the myocardial infarct size and attenuated CK leakage from the reperfused heart. The effects were associated with a dose-dependent increase in acute hyperemic coronary flow immediately after reperfusion without significant alterations in the systemic blood pressure or heart rate. The intravenous injection of YT-146 produced a dose-dependent increase in LAD blood flow in the anesthetized and open-chest dog model, and the flow was increased to 3 times the baseline value or more at the dose of 10 μg/kg (data not shown). However, the femoral artery blood flow of the same dog hardly increased, suggesting that the vasodilating action of YT-146 may be coronary selective. The reduction in myocardial infarct size was also detected to a degree similar to that of the ischemic preconditioned heart under the same ischemia/reperfusion protocol. Thus, YT-146, like ischemic preconditioning, appears to be capable of exerting a cardioprotective effect on the ischemic/reperfused heart in vivo, although the mechanisms between the 2 interventions would differ from each other.
Acute hyperemic coronary flow is considered to be a reaction that satisfies oxygen demand of the myocardium and its augmentation is related to an increase in myocardial metabolic requirements during ischemia.17 Previous studies concerning the effects of adenosine or its derivatives indicated that an increase in myocardial flow including acute hyperemic coronary flow is correlated with the reduction in myocardial necrosis18–21 and with the recovery of myocardial contractility22 in the canine myocardial ischemia-reperfusion model. Because a dose-dependent increase in the regional myocardial blood flow immediately after reperfusion of the YT-146-treated animals with concomitant reduction in the infarct size was observed, an increase in the acute hyperemic coronary flow by YT-146 is likely to contribute to the limitation of myocardial infarct size.
The regional myocardial blood flow of low- and high-dose YT-146-treated animals after 30 min of reperfusion was different: a relatively retained increase in regional blood flow was seen in the animals with the low dose of YT-146, whereas a lower level of blood flow was detected in the high dose YT-146-treated animals after 30 min of reperfusion. Some studies have suggested that coronary reflow upon reperfusion is inversely correlated with myocardial infarct size23 and that good reflow after PCI specifies the prognosis of patients after AMI.4 As described above, a good correlation between an increase in acute hyperemic coronary flow immediately after reperfusion and limitation of infarct size was seen under the present experimental conditions. Recently, Boucher et al reported that an adenosine A2 antagonist, administrated 5 min after the onset of reperfusion, was unable to demonstrate postischemic cardioprotection on the rabbit ischemia-reperfusion model.24 Furthermore, Kin et al. reported that postconditioning, which was instituted immediately after the onset of reperfusion, reduced infarct size via adenosine A2 receptor activation.25 Our results also suggest that augmented coronary blood flow for several minutes after the onset of reperfusion is important for the cardioprotective effect of the adenosine A2 receptor agonist. However, no acute hyperemic coronary flow and a decreased regional blood flow during the following period of reperfusion were seen in the animal with ischemic preconditioning. Thus, it does not appear that the mechanism for cardioprotection of adenosine or its derivatives is similar to that for ischemic preconditioning.
Adenosine is recognized to exert its pharmacological effect via 3 adenosine receptor subtypes: A1, A2, and A3. Several studies have been conducted to determine whether the cardioprotective effect of adenosine against ischemia/reperfusion injury may be mediated by the adenosine A1 and/or A2 receptor. Furthermore, adenosine A1 receptor agonists including cyclopentyladenosine26 and GR7923627 are capable of reducing the infarct size in the ischemic animals. The mechanism by which the cardioprotective effect of these agents is achieved has been attributed to A1-stimulated alterations in myocardial glucose metabolism through inhibition of glycolysis,28 an increase in glucose influx,29 and/or reduction in norepinephrine release from sympathetic nerve endings.30 In contrast, some other studies failed to demonstrate cardioprotective effects of adenosine A1 receptor agonists (eg, N6-(phenyl-2R-isopropyl)-adenosine31 and KW-390232) in the rabbit. This difference may be attributable to different actions of adenosine A1 receptor agonists in response to the experimental conditions. This possibility is in line with the findings that occurrence of bradycardia resulting from activation of the adenosine A1 receptor agonist R-PIA may compromise cardioprotective effects33 and that limitation of infarct size by an adenosine A1 agonist GR79236 was markedly affected by the body temperature of the ischemic animal.34
Adenosine A2 receptor activation is also recognized to be cardioprotective against ischemia/reperfusion injury through coronary dilation and inhibition of polymorphonuclear neutrophil activation, O2− generation, coronary endothelial adherence, and/or platelet activation during reperfusion.35,36 Norton et al and Schlack et al reported that the selective adenosine A2 receptor agonist CGS 21680 reduced infarct size when the treatment was started before reperfusion in rabbits and dogs,20,26 but that the adenosine A1 agonist GR79236 did not reduce the infarct size under similar experimental conditions.37 In addition to these findings, the results of a receptor-binding assay in a previous study11 demonstrated that YT-146 bound to adenosine A2 receptor more selectively than to the adenosine A1 receptor (Ki for adenosine A1 receptor=211.04 nmol/L, and Ki for adenosine A2 receptor=12.12 nmol/L). Furthermore, the present study clearly showed that YT-146 reduced the infarct size and concomitantly augmented acute hyperemic coronary reflow, a typical phenomenon of A2 receptor agonists,14 immediately after reperfusion. YT-146 was also reported to inhibit platelet aggregation (IC50=2.2×10−7 mmol/L)10 and this effect is thought to contribute to an increase in coronary flow. Thus, our findings may support the hypothesis that YT-146 exerts its cardioprotective action through activation of adenosine A2 receptors.
Recently Konno et al reported that YT-146 had a 506-fold higher affinity for the adenosine A2a receptor than for the adenosine A2b receptor.38 It has been reported that ATL-146, a selective adenosine A2a agonist, increased coronary flow without any changes in the systemic mean arterial pressure upon bolus intravenous injection.39 In this study, intravenously administered YT-146 also showed an increase in myocardial blood flow and cardioprotective effect; these actions may well be mediated by adenosine A2a receptors.
Another possible mechanism for the cardioprotective effect of YT-146 concerns its affinity for the adenosine A3 receptor. Because there is no vasodilating evidence at adenosine A3 receptor agonists, the activation of the adenosine A3 receptor seems to be irrelevant to the increase in hyperemic coronary flow in the present study. In contrast, adenosine A3 receptor stimulation has been reported to elicit cardioprotection via a cAMP response element-binding protein-dependent Bcl-2 pathway in addition to an Akt-Bcl pathway40 and/or an adenosine triphosphate-sensitive K+ channel-dependent mechanism.41 We do not have any data suggesting the possibility of YT-146 acting as an adenosine A3 receptor agonist at present. In this context, further studies are required for elucidating the contribution of adenosine A3 receptor activation to the cardioprotective effect of YT-146.
In conclusion, the present study demonstrated that the novel adenosine A2 receptor agonist YT-146, when administrated by the intravenous injection just before reperfusion, increased acute hyperemic coronary flow immediately after reperfusion and reduced infarct size. The degree of reduction in infarct size was similar to that of ischemic preconditioning. The effect was elicited by doses that did not affect hemodynamics. In the case of thrombolytic therapy or PCI in patients with AMI, medicines that can prevent the no-reflow phenomenon and that can attenuate myocardial necrosis are apparently beneficial. A hemodynamically silent dose of YT-146 would seem to be preferable for the treatment of ischemia/reperfusion-induced myocardial infarction. Although several other mechanisms underlying cardioprotective effects of YT-146, including reduction in the rise in left ventricular end diastolic pressure, suppression of neutrophile adhesion to endothelium, attenuation of free radical formation, and antiplatelet activity are considered. Further studies must be awaited to explore the exact mechanism for the cardioprotection of YT-146.
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