Abstract
We used pharmacological agents and genetic methods to determine whether the potent A3 adenosine receptor (AR) agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A3AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 μg/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A3AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A2AAR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A3AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A3AR gene “knock-out” (A3KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A3KO mice in vivo and did not protect isolated perfused hearts obtained from A3KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A3AR.
Several different A3 adenosine receptor (AR) agonists, including the N6-benzyl adenosine-5′-N-methylcarboxamide derivatives IB-MECA, Cl-IB-MECA, and CB-MECA, have been shown to be effective at protecting against myocardial ischemia/reperfusion injury in animal models of infarction and myocardial stunning (Auchampach et al., 1997b, 2003; Tracey et al., 1997, 1998, 2003; Jordan et al., 1999; Thourani et al., 1999a,b; Kodani et al., 2001; Takano et al., 2001). However, it remains uncertain whether these agents are effective by activating the A3AR or by nonspecific interactions with other AR subtypes. This issue has been difficult to address, because useful A3AR antagonists have only recently been developed.
The goal of this investigation was to test the cardioprotective effectiveness of Cl-IB-MECA in an in vivo mouse model of infarction and in an isolated mouse heart model of global ischemia and reperfusion. A second goal of this investigation was to establish definitively whether Cl-IB-MECA exerts cardioprotection by activating A3ARs. Our experimental approach involved the use of the rodent A3AR antagonist MRS 1523 (Li et al., 1998), the potent A2AAR antagonist ZM 241385, and mice with genetic deletion of the A3AR gene (A3KO mice; Salvatore et al., 2000). We examined whether cardioprotection provided by Cl-IB-MECA is sensitive to blockade by ZM 241385, because the possibility has been raised that N6-benzyladenosine-5′-N-methylcarboxamide A3AR agonists may be beneficial in animal models of tissue injury via interactions with the A2AAR rather than the A3AR (Murphree et al., 2002; Lappas et al., 2005). Finally, because A3AR agonists induce mast cell degranulation in rodents (Linden, 1994; Hannon et al., 1995; Van Schaik et al., 1996), we also examined the cardioprotective profile of Cl-IB-MECA in mice that had been depleted of mast cell contents by chronic treatment with compound 48/80.
Materials and Methods
Materials
Cell culture reagents G418, pcDNA3.1, and Lipofectamine were purchased from Invitrogen (Carlsbad, CA). ZM 241385 was from Tocris Cookson, Inc. (Ellisville, MO), adenosine deaminase was from Roche Applied Science (Indianapolis, IN), and all remaining drugs and reagents were purchased from Sigma-Aldrich (St. Louis, MO). N6-(4-Amino-3-[125I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([125I]I-AB-MECA) was synthesized and purified by HPLC, as described previously (Olah et al., 1994; Auchampach et al., 1997a). cAMP and histamine radioimmunoassay kits were obtained from GE Healthcare (Piscataway, NJ) and Immunotech (Marseille, France), respectively.
Animals
All experiments were performed with 10- to 14-week-old male mice (weighing ∼25–30 g). Wild-type (WT) FVB/N and C57BL/6 mice were purchased from Taconic Farms Inc. (Germantown, NY). A3KO mice were generated by embryonic stem cell targeting and genotyped by Southern blotting, as described previously (Salvatore et al., 2000). A3KO mice used had been transferred to the C57BL/6 genetic background by backcrossing greater than 12 generations. Transgenic mice cardiac-specifically overexpressing the A3AR (A3tg.1) were generated on the FVB/N genetic background, as described previously (Black et al., 2002). All animals in the study received humane care in accordance with the guidelines established by the Medical College of Wisconsin, which conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication 85-23, revised 1996).
Radioligand Binding Assays and cAMP Accumulation Assays
Competition radioligand binding assays using [125I]I-AB-MECA were conducted with membranes prepared from HEK 293 cells expressing recombinant mouse A1 or A3ARs, and cAMP accumulation assays were performed with HEK 293 cells transfected with mouse A2A or A2BARs, as described previously (Auchampach et al., 1997a; Takano et al., 2001; Kreckler et al., 2006).
In Vivo Mouse Model of Infarction
Surgical Preparation. Mice were anesthetized with sodium pentobarbital (100 mg/kg i.p.) and placed on a warm heating pad to maintain body temperature at 37 ± 0.3°C. A polyethylene (PE)-60 tube was inserted into the trachea and connected to a mouse ventilator (model 845; Hugo Sachs Elektronic, Hugstetten, Germany). The mice were respirated (tidal volume = 225 μl; rate ∼100 strokes/min) with room air supplemented with 100% oxygen to maintain blood gases within normal physiological limits. The electrocardiogram (ECG) was obtained with needle electrodes using the limb lead II configuration and recorded continuously using a Powerlab data acquisition system (ADInstruments, Colorado Springs, CO).
A left thoracotomy was performed at the fourth intercostal space to expose the heart, the pericardium was removed, and an 8.0 nylon suture was passed below the left anterior descending (LAD) coronary artery 1 to 3 mm from the tip of the left atrium with the aid of a dissecting microscope. Myocardial ischemia was induced by tying the suture over a piece of wetted gauze, and reperfusion was initiated by loosening the suture. Successful performance of coronary occlusion and reperfusion was verified by visual inspection (i.e., by the development of a pale color in the distal myocardium upon occlusion and the return of a bright red color due to hyperemia after release) and by observing changes in the ECG (i.e., widening of the QRS complex and ST segment changes). The chest wall was then closed with a 7-0 polypropylene suture with one layer through the chest wall and muscle and a second layer through the skin and subcutaneous tissue. Subsequently, the mouse was removed from the ventilator and kept in a warm chamber supplied with 100% oxygen. The endotracheal tube was removed after approximately 60 min when the mice began to recover their righting reflex and respiratory rate was ∼140 breaths/min.
Experimental Protocol. After a 30-min stabilization period, all mice were subjected to 30 min of coronary occlusion and 24 h of reperfusion. Cl-IB-MECA or equivalent vehicle (0.2 ml of 50% dimethyl sulfoxide in normal saline) was administered as i.v. boluses (tail vein injection) at the doses indicated beginning 10 min before the coronary occlusion. In studies using AR antagonists, the drugs were administered by i.v. injection 15 min before the administration of Cl-IB-MECA. Heart rate was monitored at baseline and during the 30-min occlusion period from the ECG recording.
Measurement of Ischemic Area and Infarct Size. After 24 h of reperfusion, the mice were administered heparin (1 U/g i.p.) and anesthetized with pentobarbital. After anesthesia, a thoracotomy was performed and the ascending aorta was cannulated with PE-10 tubing. To delineate the ischemic area at risk, the LAD was reoccluded and a 5% solution of phthalo blue dye in normal saline was injected into the aortic root. The heart was excised, washed with phosphate-buffered saline, embedded in agarose, and frozen at –20°C. To delineate infarcted tissue, the left ventricle was sliced into five to six transverse pieces using a microtome, incubated in a 1% solution of triphenyltetrazolium chloride for 10 min at 37°C to stain viable tissue red, and fixed in 10% formaldehyde overnight. The slices were weighed and photographed from both sides using a dissecting microscope mounted with a SPOT Insight digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). The left ventricular, ischemic, and infarcted areas were measured by digital planimetry. Infarct size is presented as a percentage of the ischemic risk region.
Measurement of Systemic Blood Pressure and Plasma Histamine Levels. Parallel experiments were performed on two separate groups of mice to determine the effect of Cl-IB-MECA on systemic blood pressure and plasma histamine levels. For blood pressure measurements, the mice were anesthetized with pentobarbital and respirated as described above. A saline-filled cannula (stretched PE-10 tubing) was inserted into the left femoral artery and connected to a pressure transducer (ADInstruments model MLT 0699). Blood pressure was recorded continuously by a PowerLab data acquisition system (ADInstruments).
For measurement of plasma histamine levels, the mice were anesthetized, connected to the respirator, and subjected to a thoracotomy to expose the heart. Blood samples (0.15 ml) were collected by cardiac puncture into EDTA-coated syringes containing the histamine N-methyltransferase inhibitor SKF-91488 (final concentration = 20 μM) at baseline and at 15, 30, and 45 min after the administration of Cl-IB-MECA. The blood samples were centrifuged (1000g for 10 min at 4°C), and the plasma was analyzed for histamine content by radioimmunoassay.
Langendorff-Perfused Mouse Heart Model of Global Ischemia and Reperfusion Experimental Setup.
Mice were anesthetized with pentobarbital. As soon as deep anesthesia was achieved, the hearts were excised quickly and arrested in ice-cold perfusion buffer. The hearts were cannulated (20 gauge stainless steel) via the aorta and perfused by the Langendorff method using Krebs-Henseleit buffer containing 118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.2 mM KH2PO4, 0.5 mM EDTA, and 11 mM glucose. The buffer was equilibrated with 95% O2/5% CO2 at 37°C to maintain the pH at 7.4. The left ventricle was ventilated with a PE drain, and a fluid-filled balloon was inserted into the ventricle via the mitral valve. The balloon was connected to a pressure transducer for continuous measurement of left ventricular pressure. The hearts were immersed in perfusate maintained at 37°C, and the balloon was inflated to a diastolic pressure of ∼5 to 10 mm Hg. Coronary flow was monitored by an in-line flow probe connected to a flowmeter (Model T206; Transonics Systems Inc., Ithaca, NY). The left ventricular pressure signal was acquired by a PowerLab data acquisition system (ADIn-struments) and processed to yield heart rate and left ventricular dP/dt.
Protocol. Isolated hearts were perfused for 20 min to allow for stabilization and then perfused for an additional 15 min while pacing at 420 beats/min (ventricular pacing, 2-ms square waves, 20% above threshold). Baseline measurements of function were acquired immediately before subjecting the hearts to 20 min of no-flow global ischemia and 45 min of reperfusion. To examine the effect of Cl-IB-MECA on functional recovery, hearts were perfused with buffer containing 100 nM Cl-IB-MECA for 10 min before ischemia and throughout the reperfusion period.
Statistical Analysis
Ki and EC50 values are reported as geometrical means with corresponding 95% confidence intervals. All other data are presented as arithmetic means ± S.E.M. Hemodynamic variables and plasma histamine concentrations were analyzed by two-way repeated measures ANOVA (time and treatment) to determine whether there was a main effect of time, a main effect of treatment, or a time-treatment interaction. If global tests showed a main effect or interaction, post hoc tests were performed using unpaired or paired analyses, as appropriate. Infarct size, ischemic area at risk size, and left ventricular functional recoveries in the isolated heart studies were compared by one-way ANOVA followed by a Student's t test with the Bonferroni's correction or an unpaired t test, as appropriate.
Results
Selectivity of Cl-IB-MECA for Recombinant Mouse ARs. Preliminary assays were conducted with transfected HEK 293 cells to determine the affinity and selectivity of Cl-IB-MECA and its parent compound IB-MECA for recombinant mouse ARs. Competition radioligand binding assays were performed to compare the binding affinity of the ligands for A1 and A3ARs. Because high-affinity agonist binding to A2AARs can not be accurately measured in heterologous expression systems due to poor receptor coupling (Murphree et al., 2002) and because agonist radioligands are not available for the A2BAR, we determined the potency of Cl-IB-MECA and IB-MECA to stimulate cAMP production in HEK 293 cells expressing mouse A2A or A2BARs. On the basis of radioligand binding analysis, Cl-IB-MECA was determined to be 219-fold selective at binding to the high-affinity form of the mouse A3AR versus the high-affinity form of the mouse A1AR (Fig. 1). The dissociation constants were calculated to be 35 nM (95% confidence intervals = 33 and 36 nM) for the mouse A1AR and 0.18 nM (0.16 and 0.19 nM) for the mouse A3AR. In comparison, IB-MECA was 68-fold more potent at binding to the mouse A3AR versus the mouse A1AR [5.9 nM (4.4 and 7.9 nM) versus 0.087 nM (0.078 and 0.098 nM), respectively]. In cAMP assays (Fig. 1), Cl-IB-MECA and IB-MECA displayed low potency at stimulating cAMP production in HEK 293 cells expressing either A2A or A2BARs. In fact, accurate EC50 values could not be calculated for these ligands due to failure to reach maximal responses at their solubility limit of 10 μM. It is reasonable to estimate that Cl-IB-MECA was at least 1000-fold less potent at stimulating cAMP production in A2AAR-expressing cells compared with the potent A2AAR agonist CGS 21680. In A2BAR-expressing cells, Cl-IB-MECA was considerably less potent at stimulating cAMP (NECA) production compared with adenosine-5′-N-ethylcarboxamide, the most potent A2BAR agonist currently known.
Dose-Dependent Reduction in Infarct Size by Cl-IB-MECA. We initially conducted a dose-response study (10–100 μg/kg) with Cl-IB-MECA examining its ability to reduce infarct size in WT FVB/N mice in response to 30 min of LAD occlusion and 24 h of reperfusion. In separate groups of mice, we assessed the effect of Cl-IB-MECA on systemic blood pressure and plasma histamine concentrations. The data are presented in Fig. 2. In WT FVB/N mice, administration of Cl-IB-MECA produced a dose-dependent reduction in infarct size that was significantly different from vehicle at 30 μg/kg. At a dose of 100 μg/kg, the magnitude of the reduction in infarct size provided by Cl-IB-MECA was 36% compared with vehicle-treated mice (30 ± 3% of the ischemic area at risk versus 47 ± 2%, respectively). Treatment with Cl-IB-MECA had no effect on heart rate during the 30-min ischemic period (Table 1).
In parallel experiments, we observed that Cl-IB-MECA also reduced blood pressure and significantly increased plasma histamine levels (Fig. 2). The decrease in blood pressure was transient peaking at 10 min after Cl-IB-MECA administration (15% reduction by 100 μg/kg Cl-IB-MECA) and returned to normal within 30 min. The increase in plasma histamine concentration was most prominent at a dose of 100 μg/kg (from 151 ± 16 to 1763 ± 298 nM 15 min after Cl-IB-MECA administration), although a small increase was also detected at a dose of 30 μg/kg (from 150 ± 11 to 358 ± 132 nM).
Cl-IB-MECA Reduced Infarct Size in A3AR Transgenic Mice. We have previously created transgenic mice that cardiac-specifically overexpress the A3AR using the α-myosin heavy chain gene promoter and have shown that these mice demonstrate increased tolerance to ischemia/reperfusion injury (Black et al., 2002; Cross et al., 2002), supporting the theory that the A3AR is capable of coupling to protective signaling pathways in cardiomyocytes. In the present study, we examined whether administration of a cardioprotective dose of Cl-IB-MECA provides additional protection in A3AR transgenic mice. We used A3tg.1 mice containing a single copy of the α-myosin heavy chain-A3AR transgene expressing 12.5 ± 3.2 fmol/mg of the high-affinity G protein-coupled form of the A3AR in the heart (Black et al., 2002). As shown in Fig. 3, administration of 100 μg/kg Cl-IB-MECA decreased infarct size in A3tg.1 mice from 35 ± 4to 25 ± 3%. Administration of Cl-IB-MECA caused a 10% reduction in heart rate after 30 min of ischemia (Table 1) that was probably due to activation of exogenously expressed A3ARs in pacemakers cells (Black et al., 2002). Although Cl-IB-MECA produced additional cardioprotection in A3tg.1 mice, there was no difference in infarct size between A3tg.1 and WT mice treated with Cl-IB-MECA reported in Fig. 1.
Cl-IB-MECA Reduces Infarct Size in Vivo via a Specific Interaction with the A3AR. To provide evidence whether or not Cl-IB-MECA effectively reduces infarct size via an A3AR-mediated mechanism, we initially assessed the effect of MRS 1523 (Fig. 4). MRS 1523 is a dihydropyridine compound developed by Li et al. (1998) as a rat A3AR antagonist (Ki = 113 ± 12 nM for rat A3ARs; 140-fold selectivity). In a previous study (Kreckler et al., 2006), we determined that MRS 1523 binds to mouse A3ARs with high affinity (Ki = 731 nM) and with moderate selectivity versus the A1AR (11-fold) but excellent selectivity versus A2A and A2BARs (at least 1000-fold). As shown in Fig. 4, pretreatment with 2 mg/kg MRS 1523 blocked the reduction in infarct size produced by 100 μg/kg Cl-IB-MECA. This dose of MRS 1523 also inhibited the ability of Cl-IB-MECA to increase plasma histamine concentrations and reduce blood pressure (Fig. 4).
We subsequently examined the effect of the A2AAR antagonist ZM 241385 on cardioprotection provided by Cl-IB-MECA. Mice were pretreated with 2 mg/kg ZM 241385 15 min before the administration of 100 μg/kg Cl-IB-MECA. This dose of ZM 241385 was based on the study of Ohta and Sitkovsky (2001). As shown in Fig. 5, we were surprised to observe that 2 mg/kg ZM 241385 blocked the beneficial effect of Cl-IB-MECA on infarct size. However, we observed that this dose of ZM 241385 also antagonized the ability of Cl-IB-MECA to increase plasma histamine levels and to reduce blood pressure (Fig. 5.), suggesting that ZM 241385 at a dose of 2 mg/kg is sufficient to block A3ARs. We subsequently tested ZM 241385 at a lower dose of 0.5 mg/kg. This dose only partially antagonized the hemodynamic and histamine-releasing actions of Cl-IB-MECA (Fig. 5) but blocked completely the hypotensive response induced by i.v. administration of 30 μg/kg A2AAR agonist CGS 21680 (Supplemental Fig. 1). As shown in Fig. 5, treatment with 0.5 mg/kg ZM 241385 failed to block the infarct size-reducing effect of Cl-IB-MECA.
Finally, we examined whether Cl-IB-MECA reduces infarct size in A3KO mice. We used A3KO mice made congenic on the C57BL/6 genetic background by back-crossing greater than 12 generations to the pure strain. Comparisons were made with WT C57BL/6 mice. Similar to FVB/N mice, pretreating WT C57BL/6 mice with 100 μg/kg Cl-IB-MECA reduced infarct size 54% (vehicle = 52 ± 4% of the risk region; Cl-IB-MECA = 24 ± 2%), decreased systemic blood pressure 15% (with no effect on heart rate), and increased plasma histamine levels (Fig. 6; Table 1). The onset of the hypotensive effect of Cl-IB-MECA in C57BL/6 mice was immediate (within 5 min) but more sustained (>45 min) compared with that produced in FVB/N mice. Infarct size induced by 30 min of occlusion and 24 h of reperfusion was similar in magnitude in A3KO mice (56 ± 4% in A3KO mice) compared with WT mice (Fig. 6). However, unlike WT mice, administration of Cl-IB-MECA (100 μg/kg) to A3KO mice subjected to ischemia and reperfusion did not reduce infarct size (infarct size as a percentage of the risk region was 56 ± 2%; Fig. 6); similarly, it did not evoke systemic release of histamine (Fig. 6). These results concur with the data obtained with AR antagonists and indicate that Cl-IB-MECA reduces infarct size by an A3AR-mediated mechanism. It is interesting that we observed that Cl-IB-MECA continued to reduce blood pressure in A3KO mice, although the kinetics of the response was different. Rather than a rapid but sustained hypotensive effect, Cl-IB-MECA produced a delayed reduction in blood pressure that became apparent 15 min after drug administration (Fig. 6). Cl-IB-MECA produced a similar hypotensive profile in WT C57BL/6 mice pretreated with 2 mg/kg A3AR antagonist MRS 1523, as well as in A3KO mice pretreated with a high dose of ZM 241385 (2 mg/kg) that presumably blocks all four AR subtypes (Fig. 6).
Cl-IB-MECA Protects Isolated Mouse Hearts from Global Ischemia/Reperfusion Injury via the A3AR. It has previously been reported that Cl-IB-MECA improves reperfusion contractile function of isolated mouse hearts subjected to global ischemia/reperfusion injury (Harrison et al., 2002; Peart et al., 2002). In this study, we examined whether the protective effect of Cl-IB-MECA in isolated hearts involves a specific interaction with the A3AR. Langendorff-perfused hearts from WT or A3KO mice were subjected to 20 min of normothermic no-flow global ischemia and 45 min of reperfusion; the hearts were treated throughout the experiment with either vehicle or 100 nM Cl-IB-MECA. Postischemic recovery of left ventricular function was assessed via a fluid-filled balloon placed in the left ventricle. As shown in Fig. 7, indices of left ventricular contractile function, including developed pressure, +dP/dt, and –dP/dt returned to ∼50% of baseline values at 45 min of reperfusion in both vehicle-treated WT and A3KO hearts, indicating that 20 min of global ischemia followed by reperfusion results in significant contractile dysfunction in our isolated mouse heart model system and that ischemic tolerance is similar between WT and A3KO mice. Treatment with 100 nM Cl-IB-MECA significantly improved reperfusion contractile function from ∼50 to ∼75% of baseline values in WT mouse hearts; this beneficial effect of Cl-IB-MECA was not apparent in hearts from A3KO mice (Fig. 7). In both WT and A3KO hearts, treatment with Cl-IB-MECA increased reperfusion coronary flow to preischemic levels. Collectively, these results suggest that cardioprotection provided by Cl-IB-MECA in isolated mouse hearts is the result of A3AR activation.
Cardioprotection Provided by Cl-IB-MECA Is Not Mediated by the Release of Stored Mediators from Mast Cells. Additional experiments were conducted to test the possibility that Cl-IB-MECA induces cardioprotection, because of its ability to stimulate mast cells, to degranulate. Theoretically, Cl-IB-MECA could provide cardioprotection by depleting mast cells of noxious mediators that contribute to injury during ischemia or induce adaptive changes that provide protection similar to that of myocardial preconditioning (Linden, 1994). To address this possibility, we conducted a series of experiments testing the cardioprotective effectiveness of Cl-IB-MECA in mice that had been treated chronically with compound 48/80 to deplete mast cells of stored mediators. Compound 48/80 was administered to WT mice intraperitoneally twice daily for 5 days beginning with a dose of 1 mg/kg on the first day, followed by an increment of 1 mg/kg per day, to a maximum of 5 mg/kg on the final day (Riley and West, 1955). On the following day (day 6), the effectiveness of Cl-IB-MECA was examined in both the in vivo mouse model of infarction and the isolated mouse heart model of global ischemia and reperfusion. As shown in Figs. 8 and 9, depletion of mast cell contents with compound 48/80 did not attenuate protection provided by Cl-IB-MECA (100 μg/kg) in intact mice or in isolated mouse hearts. In the in vivo model, treatment with Cl-IB-MECA produced a 30% reduction in infarct size in compound 48/80-treated mice (Fig. 8). In the isolated mouse heart model, Cl-IB-MECA increased postischemic recovery of left ventricular developed pressure from 54 to 71% of preischemic baseline values after treatment with compound 48/80 (Fig. 9). Effective depletion of mast cell mediators was confirmed by the observation that Cl-IB-MECA did not increase plasma histamine concentrations or reduce blood pressure in compound 48/80-treated mice (Fig. 8).
Discussion
We observed that pretreatment with the A3AR-selective agonist Cl-IB-MECA produced a dose-dependent reduction in infarct size in mice subjected to 30 min of coronary artery occlusion and 24 h of reperfusion. This protective effect of Cl-IB-MECA was apparent at a dose of 30 μg/kg and produced a ∼35% reduction in infarct size at a dose of 100 μg/kg. We subsequently observed that cardioprotection provided by Cl-IB-MECA was blocked completely in mice pretreated with the rodent A3AR antagonist MRS 1523 (Li et al., 1998), suggesting involvement of the A3AR. To further explore the cardioprotective profile of Cl-IB-MECA, we examined its effectiveness in an isolated mouse heart model of 20 min of normothermic global ischemia and 45 min of reperfusion. In hearts from WT mice, treatment with 100 nM Cl-IB-MECA significantly improved several indices of left ventricular postischemic contractile function including developed pressure; this protective effect of Cl-IB-MECA was lost completely using hearts obtained from A3KO mice. Collectively, these data are the first to provide definitive evidence that an A3AR agonist provides protection against myocardial ischemia/reperfusion injury by activating the A3AR. These data support the concept that the A3AR plays a protective role in the ischemic myocardium.
Several previous studies have demonstrated that A3AR agonists are effective at protecting against myocardial ischemia/reperfusion injury (Auchampach et al., 1997b, 2003; Tracey et al., 1997, 1998, 2003; Jordan et al., 1999; Thourani et al., 1999a,b; Kodani et al., 2001; Takano et al., 2001). For example, we have reported that pretreatment with IB-MECA effectively reduced infarct size, attenuated myocardial stunning, and induced the late phase of ischemic preconditioning in a clinically relevant conscious rabbit model or a barbital-anesthetized dog model of regional ischemia/reperfusion injury (Auchampach et al., 1997b, 2003; Kodani et al., 2001; Takano et al., 2001). In the dog model, IB-MECA was shown to reduce infarct size even when administered immediately before release of the occlusion, demonstrating that it was also effective at reducing reperfusion-mediated injury (Auchampach et al., 2003). Similar results have been obtained by others using more selective A3AR agonists in in vivo models as well as in vitro models of ischemia/reperfusion injury, including isolated rodent hearts (Tracey et al., 1997, 1998, 2003; Thourani et al., 1999a,b). However, even though the A1AR was excluded using highly selective antagonists (Tracey et al., 1997, 1998, 2003; Kodani et al., 2001), involvement of the A3AR could not be proven definitively in these studies due to the lack of selective antagonists. Using the most recently developed rodent A3AR antagonist MRS 1523 and A3KO mice, we have now confirmed that protection against ischemia/reperfusion injury provided by Cl-IB-MECA in the mouse is mediated predominantly via A3AR.
We conducted additional experiments using ZM 241385 to address the possibility that Cl-IB-MECA may be producing cardiac protection by a mechanism involving the A2AAR. We chose to address this issue, because administration of low doses of A2AAR agonists has been reported to reduce myocardial reperfusion injury by suppressing inflammatory responses (Glover et al., 2004; Yang et al., 2005) and because it has been suggested that N6-benzyladenosine-5′-N-methylcarboxamide A3AR agonists may have higher affinity for the A2AAR than originally appreciated (Murphree et al., 2002). Moreover, Yang et al. (2004) have presented the hypothesis, in abstract form, that A3AR agonists may act by increasing adenosine production, which subsequently provides tissue protection via the A2AAR by an anti-inflammatory mechanism. ZM 241385 is a highly potent A2AAR antagonist with low affinity for the mouse A3AR (Palmer et al., 1995; Kreckler et al., 2006). Surprisingly, we found that pretreating mice with 2 mg/kg ZM 241385 (Ohta and Sitkovsky, 2001) was also capable of blocking the infarct size-reducing effect of Cl-IB-MECA in our in vivo mouse model. However, at this dose, ZM 241385 also inhibited the effect of Cl-IB-MECA to increase plasma histamine levels, a response in rodents known to be due to A3AR-mediated degranulation of mast cells (Linden, 1994; Van Schaik et al., 1996), confirming that a lower dose of ZM 241385 was required to prevent blockade of A3ARs. Using a dose of 0.5 mg/kg ZM 241385 that only partially inhibited Cl-IB-MECA-induced histamine release (but effectively blocked the hypotensive actions of the A2AAR agonist CGS 21680; Supplemental Fig. 1), we found that Cl-IB-MECA continued to produce a significant reduction in infarct size. These results exclude the involvement of the A2AAR in mediating the cardioprotective effects of Cl-IB-MECA and provide further evidence in support of an A3AR-mediated mechanism. Perhaps more importantly, these results demonstrate that ZM 241385 exhibits relatively high in vivo potency in rodents such that doses lower than 2 mg/kg should be used to selectively block A2AARs.
We have previously reported in conscious rabbits and dogs that IB-MECA effectively reduced ischemia/reperfusion injury at doses that produced no changes in systemic hemodynamic parameters (Auchampach et al., 1997b, 2003; Kodani et al., 2001; Takano et al., 2001). This was an important observation, because the clinical use of adenosine as well as subtype-selective agonists for A1 and A2AARs for treating ischemia/reperfusion injury carry the risk of hemodynamic side-effects, including hypotension, bradycardia, and atrioventricular blockade, due to activation of A1ARs expressed in conducting tissue and A2AARs in vascular smooth muscle cells. However, in rodent species (but not in other species), it is well known that activation of A3ARs reduces blood pressure due to the actions of vasoactive mediators released from mast cells (Linden, 1994; Hannon et al., 1995; Fozard et al., 1996). Indeed, in the present investigation, we observed that administration of 100 μg/kg Cl-IB-MECA produced an immediate (within 1–2 min) 10 to 15% reduction in blood pressure, which correlated with increased plasma histamine concentrations, but was absent in compound 48/80-treated mice. Interestingly, however, we observed that the kinetic profile of Cl-IB-MECA to reduce blood pressure differed between the two strains of mice that we used in our studies, in that the response was transient in FVB/N mice (∼15–20 min) but more sustained (at least 45 min) in C57BL/6 mice. Because Cl-IB-MECA continued to produce a delayed reduction in blood pressure in WT or A3KO mice pretreated with MRS 1523 or a nonselective dose of ZM 241385 (2 mg/kg), we conclude that Cl-IB-MECA reduced blood pressure in C57BL/6 mice by a mechanism that may not be receptor-dependent.
We did not address the specific mechanism by which Cl-IB-MECA exerted cardioprotection, although we did determine that it is independent of mast cell degranulation. We have previously shown that the infarct size-reducing effects of IB-MECA are blocked by glibenclamide (Auchampach et al., 1997b, 2003), implicating involvement of ATP-sensitive potassium channels. We have also determined in preliminary studies using our in vivo mouse model of infarction and A3KO bone marrow chimeric mice that Cl-IB-MECA seems to reduce injury when administered at the time of reperfusion by an effect on bone marrow-derived cells, suggesting an anti-inflammatory mechanism (Ge et al., 2004). Therefore, we predict that A3AR agonists function by different mechanisms to protect against ischemia/reperfusion injury depending on whether they are administered before ischemia or during reperfusion. Although it is unlikely to be a significant factor, since we observed protection in the isolated heart model and in compound 48/80-treated mice, it is important to note that the hypotensive action produced by Cl-IB-MECA in the present investigation may have contributed to the reduction in infarct size in the in vivo studies.
One aspect of our ischemia/reperfusion studies using A3KO mice requires particular attention. In an earlier study, we reported that infarct size was smaller in A3KO mice subjected to 30 min of coronary artery occlusion and 24 h of reperfusion, suggesting that deletion of the A3AR gene induces an unexpected cardioprotective phenotype (Guo et al., 2001). However, this earlier study was conducted with A3KO mice on a mixed background of three different genetic strains (C57BL/6, sv129, and D2). Furthermore, controls used in this previous study were not littermates but were from a separate line of mice interbred among the three different genetic strains. This line of control mice was considered to be the best available at that time. Based on the results of the present investigation using congenic (C57BL/6) A3KO mice, we conclude that genetic deletion of the A3AR gene itself has no effect on ischemic tolerance and that the results of our earlier study are probably explained by differences in the genetic background of the mice rather than specific deletion of the A3AR gene (Guo et al., 2001). Harrison et al. (2002) reached a similar conclusion regarding A3KO mice.
In summary, we have demonstrated that pretreatment with the A3AR agonist Cl-IB-MECA protects against ischemia/reperfusion injury in mice and that this occurs through an A3AR-mediated mechanism that is independent of mast cell degranulation. These results further support the contention that therapeutic strategies targeting the A3AR could be a novel and useful approach for protection of the ischemic myocardium.
Acknowledgments
We acknowledge the Cardiovascular Center HPLC Core Facility at the Medical College of Wisconsin for purification of radioligands.
Footnotes
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This work was supported by National Institutes of Health Grants R01 HL60051, R01 HL07707, and T32 HL73643 and by American Heart Association Research Fellowships 0320019Z and 0315274Z.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.111351.
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ABBREVIATIONS: AR, adenosine receptor; CB-MECA, -N6-(3-chlorobenzyl)adenosine-5′-N-methylcarboxamide; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide; ECG, electrocardiogram; IB-MECA, N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide; [125I]I-AB-MECA; N6-(4-amino-3-[125I]iodobenzyl)adenosine-5′-N-methylcarboxamide; LAD, left anterior descending; LV, left ventricle; KO, knockout; PE, polyethylene; WT, wild-type; HEK, human embryonic kidney; MRS 1523, 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate; ZM 241385, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol; compound 48/80, condensation product of p-methoxyphenethyl methylamine with formaldehyde; SKF-91488, 4-(N,N-dimethylamino)butylisothiourea; dP/dt, change in pressure over time; CGS 21680, 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received July 21, 2006.
- Accepted September 18, 2006.
- The American Society for Pharmacology and Experimental Therapeutics