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CARDIOVASCULAR
Departments of Pharmacology and Toxicology (T.C.W., Z.-D.G., G.J.G., W.-M.K., M.W.B., J.A.A.) and Anesthesiology (A.T., Y.M., M.W.B., W.-M.K.), Medical College of Wisconsin, Milwaukee, Wisconsin; and Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, Connecticut (W.R.T.)
Received June 19, 2007; accepted September 27, 2007.
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Abstract |
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, 2003; Tracey et al., 1997, 1998, 2003; Jordan et al., 1999; Thourani et al., 1999; Ge et al., 2004, 2006). A3AR agonists are attractive as cardioprotective agents because they do not alter systemic hemodynamic parameters in nonrodent species and are effective if administered before the ischemic event or only during reperfusion (Auchampach et al., 1997b, 2003; Tracey et al., 1997, 1998, 2003; Jordan et al., 1999; Thourani et al., 1999; Ge et al., 2004, 2006). The most widely available A3AR agonists that have been tested in experimental animal models of ischemia/reperfusion injury include the N6-benzyladenosine-5'-N-methylcarboxamide derivative IB-MECA and its 2-chloro derivative Cl-IB-MECA (Fig. 1). Although IB-MECA and Cl-IB-MECA are potent A3AR agonists, they exhibit moderate selectivity. In radioligand binding assays, the selectivity of IB-MECA and Cl-IB-MECA has been reported to range from 6 to 2500-fold versus the A1AR and 4 to 1400-fold versus the A2AAR, depending on the species and the assay conditions (Gallo-Rodriguez et al., 1994; Kim et al., 1994; Hill et al., 1997; Takano et al., 2001; Murphree et al., 2002).
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In the present investigation, we characterized the cardioprotective profile of the A3AR agonist CP-532,903 in an isolated mouse heart model of global ischemia and reperfusion and an in vivo mouse model of infarction. The goal of this work was to examine the cardioprotective effectiveness of CP-532,903 and to confirm whether it mediates cardioprotection via the A3AR. A second goal of this investigation was to determine whether A3AR activation provides ischemic protection by facilitating opening of the sarcolemmal isoform of the ATP-sensitive potassium (KATP) channel. This second question was addressed using Kir6.2 gene knockout (KO) mice lacking the pore-forming subunit of the sarcolemmal KATP channel (Suzuki et al., 2002).
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Materials and Methods |
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,b; Kreckler et al., 2006). cAMP and histamine radioimmunoassay kits were obtained from GE Healthcare (Piscataway, NJ) and Immunotech (Marseille, France).
Animals
All experiments were performed with 10 to 14-week-old male mice weighing 
24 to 32 g. Wild-type C57BL/6 mice were purchased from Taconic Farms Inc. (Germantown, NY). A3KO mice were provided by Dr. Marlene Jacobson (Merck Research Laboratories, West Point, PA). Kir6.2 KO mice were provided by Dr. Susumu Seino (Kobe University, Kobe, Japan) and were generated by Dr. Aaron Fisher (University of Pennsylvania, Philadelphia, PA), as described previously (Miki et al., 1998). All animals used in the study received humane care in accordance with the guidelines established by the Medical College of Wisconsin, which conform to the Institute of Laboratory Animal Resources (1996).
Radioligand Binding Assays
Competition radioligand binding assays were conducted with membranes prepared from HEK 293 cells expressing recombinant mouse A1 or A3ARs using the agonist radioligand [125I]I-AB-MECA (Auchampach et al., 1997a,b; Kreckler et al., 2006). Incubations were conducted in 100 µl of buffer (10 mM Na-HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl2, 1 U/ml adenosine deaminase) with 0.3 nM [125I]I-AB-MECA and competitors at room temperature for 3 h, after which bound and free radioligand were separated by filtration over GF/C grade glass fiber filters (Brandel, Gaithersburg, MD). Nonspecific binding was defined by the presence of 100 µM adenosine-5'-N-ethylcarboxamide in the assays. Ki values for high-affinity agonist binding were calculated as described previously (Auchampach et al., 1997a,b; Kreckler et al., 2006).
cAMP Assays
HEK 293 cells expressing recombinant mouse ARs were detached using phosphate-buffered saline containing 5 mM EDTA and resuspended at 50,000 cells/tube in Dulbecco's modified Eagle's medium with 25 mM HEPES, pH 7.4, 1 U/ml adenosine deaminase, and 20 µM Ro 20,1724 to inhibit phosphodiesterases. Cells were incubated with agonists for 15 min at 37°C with shaking. Reactions were terminated by addition of 0.15 N HCl. [cAMP] in the acid extract was determined by radioimmunoassay. MRS 1754 (1 µM) was included in assays with HEK 293 cells expressing A2A or A3ARs to block endogenous A2BARs expressed in HEK 293 cells.
Langendorff-Perfused Mouse Heart Model
Experimental Preparation. Male mice (10–12 weeks of age, 23.7 ± 0.4 g body weight) were anesthetized with sodium pentobarbital (100 mg/kg i.p.). As soon as deep anesthesia was achieved, the hearts were removed and arrested in ice-cold perfusion solution. The hearts were cannulated via the aorta and perfused retrogradely by the Langendorff method at a continuous pressure of 80 mm Hg using Krebs-Henseleit buffer containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 0.5 mM EDTA, 25 mM NaHCO3, and 11 mM glucose. The buffer was equilibrated with 95% O2/5% CO2 at 37°C to maintain the pH at 7.4 and filtered through an in-line Sterivex filter unit (0.22 µm; Millipore, Bedford, MA) to remove particulate matter. For each heart, the left ventricle was drained by inserting a short polyethylene tube through the apex, and a fluid-filled balloon connected to a pressure transducer was inserted into the left ventricle via the mitral valve. The balloon was connected to a pressure transducer (ADInstruments, Colorado Springs, CO) for continuous measurement of left ventricular pressure. The hearts were immersed in perfusion buffer maintained at 37°C, and the balloons were inflated to achieve end-diastolic pressures of 5 to 10 mm Hg. Coronary flows were monitored by an in-line flow probe connected to a flowmeter (model T206; Transonics Systems Inc., Ithaca, NY). The left ventricular pressure signals were acquired continuously using a PowerLab data acquisition system (ADInstruments) and processed (Chart software) to yield heart rates and left ventricular dP/dts.
Global Ischemia/Reperfusion Protocol. 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 with 2-ms square waves at a, voltage of 20% above threshold). Baseline functional measurements were acquired immediately before subjecting the hearts to 20 min of normothermic no-flow ischemia and 45 min of reperfusion achieved by closing and opening an in-line stopcock. To examine the effect of CP-532,903 on functional recovery, hearts were perfused with buffer containing the indicated concentrations of agonists for 10 min before ischemia. In studies of ischemic preconditioning (IPC), the hearts were subjected to three cycles of 3-min occlusion/2-min reperfusion before the 20-min occlusion.
In Vivo Mouse Model of Infarction
Experimental Preparation. Male mice were anesthetized with sodium pentobarbital (100 mg/kg i.p.) and prepared for surgery, as described previously (Black et al., 2002; Ge et al., 2006). In brief, the mice were intubated using PE-60 tubing, ventilated (model 845; Hugo Sachs Elektronic, Hugsteten, Germany) at a rate of 100 to 110 beats/min and a tidal volume of 200 to 250 µl using room air supplemented with oxygen, and fitted with electrodes to obtain the electrocardiogram (ECG) using the limb lead II configuration. The heart was exposed via a lateral incision at the level of the fourth intercostal space and a ligature (8-0 nylon suture) was placed around the left coronary artery 1 to 3 mm from the tip of the left atria. The ligature was used to induce coronary occlusion and reperfusion by gently tightening the snare around a piece of wetted gauze. Approximately 15 min after completing the ischemia/reperfusion protocol, the chest cavity was closed using 6-0 polypropylene suture, and the mice were allowed to recover.
Ischemia/Reperfusion Protocol. After a 30-min stabilization period after surgery, all mice were subjected to 30 min of LAD occlusion and 24 h of reperfusion. Successful performance of occlusion and reperfusion was verified by visual inspection (i.e., change in color of the ischemic myocardium) and by changes in the ECG. CP-532,903 or equivalent vehicle was administered as an i.v. bolus 10 min before the coronary artery occlusion. 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, infarct size was measured by dual staining with phthalo blue dye and triphenyltetrazolium chloride (Black et al., 2002; Ge et al., 2006). To delineate the ischemic area at risk, the LAD was reoccluded in situ while a 5% solution of phthalo blue dye was injected into the aortic root. To delineate infarcted tissue, the left ventricle was sliced into five to six transverse sections and stained for exactly 10 min in a solution of 1% triphenyltetrazolium chloride at 37°C. Triphenyltetrazolium chloride stains viable tissue red, leaving infarcted tissue unstained. The slices were weighed and photographed from both sides using 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 studies with separate groups of mice were conducted to examine the effect of CP-532,903 on systemic blood pressure and plasma histamine levels. Blood pressure was measured in pentobarbital-anesthetized mice via a catheter inserted into the left femoral artery, and plasma histamine levels were measured by enzyme-linked immunosorbent assay (Immuno-Biological Laboratories, Hamburg, Germany) from blood samples obtained by cardiac puncture (Ge et al., 2006).
Electrophysiology
Mouse Cardiomyocyte Isolation. Myocytes from adult mouse hearts were isolated according to methods established by the Alliance for Cellular Signaling (http://www.signaling-gateway.org; protocol no. PP00000015). In brief, hearts were excised from pentobarbital-anesthetized mice, cannulated via the aorta onto a blunted needle, and perfused for 10 min with warmed (37°C) perfusion buffer (113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4-7H2O, 0.032 mM phenol red, 12 mM NaHCO3, 10 mM KHCO3, 10 mM HEPES, pH 7.4, 30 mM taurine, 10 mM 2,3-butanedione monoxime, 5.5 mM glucose) containing 0.25 mg/ml Liberase blendzyme I, 0.14 mg/ml trypsin, and 12.5 µM CaCl2. After perfusion, the ventricles were dissected free from the atria and repeatedly passed through a plastic transfer pipette to disaggregate the cells into a single-cell suspension. Subsequently, myocytes were enriched by sedimentation in perfusion buffer containing 5% bovine calf serum while slowly exposing the cells to increasing concentrations of CaCl2 to achieve a final concentration of 1.2 mM. The final cell pellet containing calcium-tolerant myocytes was resuspended in minimal essential medium culture media containing Hanks' salts, 2 mM L-glutamine, 5% bovine calf serum, 10 mM 2,3-butanedione monoxime, and 100 U/ml penicillin.
Recording Solutions. Myocytes were placed in an external bath solution containing 5 mM KCl, 132 mM N-methyl-D-glucamine, 1 mM CaCl2, 2 mM MgCl2, 5 mM 4-aminopyridine, 5 µM E-4031, 200 nM nisoldipine, and 10 mM HEPES with pH adjusted to 7.4 with HCl. Pinacidil (KATP channel opener), glibenclamide (KATP channel antagonist), 1,3-dipropyl-8-cyclopentylxanthine (CPX; A1AR antagonist), ZM 241385 (A2AAR antagonist), and CP-532,903 were all prepared as stock solutions in dimethyl sulfoxide and diluted to the desired concentrations before use. The final concentration of dimethyl sulfoxide was <0.05%. The pipette solution contained 60 mM K-glutamate, 50 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.01 K2-ATP, 11 mM EGTA, and 10 mM HEPES with pH adjusted to 7.4 with KOH. For the experiments investigating coupling between the A3AR and the KATP channel, 0.2 mM K2-ATP, and 0.5 mM Na-GTP were included in the pipette solution.
Recording Procedure and Data Analysis. ATP-sensitive potassium current, IKATP, was recorded using the whole-cell configuration of the patch-clamp technique. Pipettes were pulled from borosilicate glass capillary tubes (Garner Glass, Claremont, CA) using a horizontal two-stage puller (Sachs-Flaming P-97; Sutter Instruments, Novato, CA) and heat polished (microforge MF-830; Narishige, Tokyo, Japan). In standard solutions, the pipette resistance ranged from 3 to 5 M
. Current was measured using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City, CA) interfaced to a computer via a digitizer (Digidata 1322 A; Axon Instruments). Data acquisition and analysis were conducted using the pClamp software package version 9.0 (Axon Instruments). Additional analyses were performed on Origin version 7 (OriginLab, Northampton, MA).
Whole-cell IKATP was monitored during 100-ms test pulses from –120 to + 60 mV from a holding potential of –40 mV. Current-voltage relationships were obtained from current amplitude measured at the end of the test pulse and plotted against membrane potential.
Mitochondrial Isolation and Functional Assays
Cardiac mitochondria were isolated from adult mouse hearts using the MITOISO1 kit (Sigma-Aldrich). Respiration of isolated mitochondria (1 mg/ml) was monitored at 30°C with an oxygen electrode (Hansatech Instruments Ltd., Norfolk, UK) in respiration buffer containing 130 mM KCl, 5 mM K2HPO4, 20 mM MOPS, pH 7.2, 2.5 mM EGTA, 0.001 mM Na4P2O7, and 0.1% bovine serum albumin. State 2 respiration was stimulated with a combination of pyruvate and malate (5 mM each) as substrates. ADP-stimulated state 3 respiration was measured in the presence of 250 µM ADP, and state 4 respiration was measured after added ADP was consumed. The respiratory control ratio was calculated as a ratio of the state 3 rate divided by the state 4 rate.
ATP synthesis rates by isolated mitochondria was assessed using a chemiluminescence-based method utilizing firefly luciferase and luciferin (ATP Determination Kit; Invitrogen), as described previously (Ljubkovic et al., 2007). The reaction solution contained respiration buffer, 0.2 µM diadenosine pentaphosphate, 5 mM pyruvate, 5 mM malate, 2 mg/l mitochondria, 200 µM luciferin, and 1.25 mg/l luciferase. The reaction was initiated by the addition of 30 µM ADP (made ATP-free by hexokinase treatment). Blanks were obtained through measurements in the absence of substrates. Chemiluminescence was monitored in a Modulus Luminometer (Turner Biosystems, Sunnyvale, CA) at room temperature for 120 s. The rate of mitochondrial ATP production was calculated from standard curves generated with defined ATP concentrations.
Data Analysis
All data are reported as means ± S.E.M. In vivo blood pressure measurements, plasma histamine concentration, and left ventricular recovery of developed pressure were analyzed by two-way repeated measures analysis of variance (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 analysis was performed using unpaired or paired analyses, as appropriate. Infarct size, risk region size, and left ventricular functional recoveries at 45 min of reperfusion in the isolated heart studies were compared using one-way analysis of variance followed by Student's t test with the Bonferroni correction or an unpaired Student's t test, as appropriate.
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Results |
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100-fold higher affinity to the mouse A3AR compared to the mouse A1AR (Fig. 2). In HEK 293 cells overexpressing the mouse A3AR, CP-532,903 inhibited forskolin-stimulated cAMP production with greater than 200-fold higher potency compared with assays conducted with HEK 293 cells overexpressing the mouse A1AR (Fig. 3). CP-532,903 did not stimulate cAMP production in cells transfected with mouse A2A or A2BARs at concentrations as high as 10 µM, demonstrating that it has little agonist activity for mouse A2ARs (Fig. 3).
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Since it has been suggested that some A3AR agonists with limited selectivity (i.e., IB-MECA) may achieve cardioprotection by activating the A2AAR rather than the A3AR (Yang et al., 2003), in an additional series of experiments, we examined whether the A2AAR system is functional in A3KO mice by measuring coronary flow responses to the A2AAR agonist CGS 21680. As shown in Fig. 4, administration of CGS 21680 (100 nM) increased coronary flow in hearts from A3KO mice to a similar extent compared with wild-type hearts.
Ischemia/Reperfusion Studies. Administration of CP-532,903 concentration dependently improved functional recovery of hearts subjected to 20 min of global ischemia and 45 min of reperfusion (Fig. 5). At a concentration of 100 nM, developed pressure, +dP/dt, and –dP/dt were improved from 47.5 ± 1.1 to 58.2 ± 1.2%, from 49.4 ± 1.3 to 61.3 ± 1.5%, and from 39.7 ± 1.3 to 50.5 ± 1.7% of baseline at 45 min of reperfusion, respectively. Treatment with 30 nM CP-532,903 provided no benefit in studies using hearts obtained from A3AR KO mice (Fig. 6). Noticeably, recovery of function during the first 10 min of reperfusion was impaired in A3KO compared with wild-type mice, suggesting that endogenous adenosine acting through native A3ARs may provide some degree of protection during ischemia/reperfusion injury.
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). For purposes of comparison, we also examined whether protection provided by IPC is functional in Kir6.2 KO hearts. As shown in Fig. 8, recovery of left ventricular developed pressure, +dP/dt, and –dP/dt was not improved by treatment with 30 nM CP-532,903 in Kir 6.2 KO hearts subjected to 20 min of global ischemia and 45 min of reperfusion. Postischemic functional recovery was also not improved by IPC induced by three 3-min occlusion/2-min reperfusion cycles, whereas the same IPC protocol improved all measures of contractile function of wild-type hearts from 45 to nearly 60% of preischemic levels (Fig. 9). Collectively, these results demonstrate that protection provided by activation of the A3AR as well as by IPC in isolated hearts is mediated by a mechanism involving the sarcolemmal KATP channel.
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In Vivo Infarction Studies
Ischemia/Reperfusion Studies. In in vivo studies, infarct size was 59.2 ± 2.1% of the risk region in vehicle-treated mice subjected to 30 min of LAD occlusion and 24 h of reperfusion. Administration of 30 or 100 µg/kg CP-532,903 significantly reduced infarct size to 42.5 ± 2.3 and 39.0 ± 2.9%, respectively (Fig. 10). At a dose of 100 µg/kg, administration of CP-532,903 did not reduce infarct size in studies using A3AR KO mice (Fig. 10). The size of the risk region, which ranged from 37.4 ± 2.9 to 40.6 ± 2.4% of the left ventricle, was not significantly different among the five experimental groups.
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Hemodynamic and Plasma Histamine. In parallel studies, we examined the effect of 100 µg/kg CP-532,903 on systemic hemodynamic parameters and plasma histamine levels. Although A3AR agonists do not alter hemodynamic variables in nonrodent species including humans, rabbits, and dogs (Auchampach et al., 1997b, 2003; van Troostenburg et al., 2004), activation of the A3AR evokes the release of mediators from mast cells in rodents, which indirectly causes hypotension (Hannon et al., 1995; Van Schaik et al., 1996; Ge et al., 2006). As shown in Fig. 10, administration of 100 µg/kg CP-532,903 produced a 30% decrease in mean arterial blood pressure that persisted for at least 45 min without changing heart rate (data not shown). Concomitantly, administration of CP-532,903 significantly increased plasma histamine levels, from 198 ± 22 nM at baseline to 3104 ± 559 nM 15 min after administration of the drug (Fig. 10). In A3KO mice, administration of CP-532,903 had no effect on blood pressure or plasma histamine levels.
Electrophysiology Studies
Our results obtained with Kir6.2 KO mice suggest that the A3AR is functionally coupled to the sarcolemmal KATP channel. To test this hypothesis, whole-cell recordings were obtained from myocytes isolated from wild-type mice in the presence of CPX (500 nM) and ZM 241385 (500 nM), antagonists of mouse A1 and A2AARs, respectively (Kreckler et al., 2006). Basal whole-cell current was initially monitored for 20 min to allow for the diffusional exchange of ATP between the pipette solution and the intracellular milieu. Myocytes that exhibited spontaneous activation of outward current during this time period were discarded. Extracellular application of CP-532,903 elicited an outward current that was blocked by glibenclamide (Fig. 11). This current was identified as IKATP. To confirm that the IKATP elicited by CP-532,903 was indeed due to activation of the A3AR, the experiments were repeated with myocytes isolated from A3KO mice. As shown in Fig. 11, the ability of CP-532,903 to elicit opening of the KATP channel was markedly attenuated. These results demonstrate a functional coupling between the A3AR and the KATP channel.
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Mitochondrial Function Studies
Since it has been hypothesized that mitochondria may express a related isoform of the KATP channel that has yet to be molecularly identified (Hanley and Daut, 2005), we examined respiration and ATP synthesis capacity of mitochondria isolated from Kir6.2 KO mice to determine whether deletion of KCNJ11 affects normal function of cardiac mitochondria. Compared with wild-type mice, we observed no differences in state 2, 3, or 4 respiration, the respiratory control ratio, or ATP synthesis rates of cardiac mitochondria isolated from Kir6.2 KO mice (Fig. 12).
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Discussion |
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40% reduction in infarct size and significant improvement in all parameters of postischemic functional recovery of isolated mouse hearts. In the isolated mouse heart studies, CP-532,903 was effective when used at low concentrations that did not influence A1AR-mediated bradycardia or A2A/A2BAR-mediated coronary dilation. The protection against injury provided by CP-532,903 was not apparent in isolated heart studies using hearts from A3KO mice or from Kir6.2 KO mice lacking expression of the poreforming subunit of the sarcolemmal KATP channel. These observations demonstrate that CP-532,903 provides a direct cardioprotective effect in the ischemic myocardium by a mechanism involving activation of the A3AR and sarcolemmal KATP channels.
CP-532,903 is a member of a new series of N6-benzylsubstituted adenosine 5'-N-methylcarboxamide AR agonists developed by Hill and colleagues (DeNinno et al., 2003, 2006; Tracey et al., 2003) that resulted from a search for more selective human A3AR agonists. The unique structural feature of CP-532,903 as well as other members within this series is an amino substitution at the 3' position of the ribose ring rather than a hydroxyl group (Fig. 1). Unlike other members in this series, including CP-608,039, CP-532,903 has been reported to maintain relatively high A3AR selectivity in some nonhuman species including the rabbit (90-fold), leading to the selection of this compound as a potentially useful agent for study of the A3AR in preclinical animal models (DeNinno et al., 2003, 2006; Tracey et al., 2003). In the present investigation, we confirmed that CP-532,903 also displays high selectivity for the mouse A3AR. In radioligand binding assays, CP-532,903 bound with high affinity (Ki = 9.6 nM) to the mouse A3AR and with 100-fold selectivity compared with its closest pharmacological relative the A1AR. The A3 versus A1AR selectivity of CP-532,903 in the mouse is comparable with that of the first generation A3AR agonists IB-MECA (68-fold) and Cl-IB-MECA (210-fold; Ge et al., 2006). One important finding of our studies is that we observed that CP-532,903 exhibited very weak agonist activity for mouse A2A and A2BARs producing no stimulation up to a concentration of 10 µM (Fig. 3). This is a significant observation since it has been suggested that IB-MECA and Cl-IB-MECA may stimulate A2AARs with relatively high potency in some species (Murphree et al., 2002). Indeed, we have previously shown that IB-MECA stimulated cAMP production in HEK 293 cells transfected with the mouse A2AAR with an EC50 value of 700 nM (Ge et al., 2006). Thus, CP-532,903 exhibits superior A3 versus A2A/A2BAR selectivity compared with currently available first generation A3AR agonists.
Tracey et al. (2003) have previously reported that CP-532,903 effectively reduced infarct size in an isolated rabbit heart model of regional infarction. In these studies, pretreatment with CP-532,903 was shown to produce a maximal reduction in infarct size of 77% at a concentration of 150 nM and an EC50 value of 1 nM (Tracey et al., 2003). Pretreatment with CP-532,903 was also shown to reduce infarct size in an in vivo rabbit model of infarction at doses (0.25 and 1 mg/kg) that were devoid of hemodynamic effects (Tracey et al., 2003). Although CP-532,903 was shown to be an effective cardioprotective agent in this study, it remained uncertain whether it reduced ischemic injury via activation of the A3AR or via interaction with the other AR subtypes. This issue could not be addressed directly since selective A3AR agonists proven to be useful in the rabbit are not currently available. The results of the present investigation clearly demonstrate that the protection provided by CP-532,903 in both the isolated mouse heart model as well as the in vivo mouse model of infarction was completely lost in studies using A3AR KO mice, supporting the theory that CP-532,903 alleviates ischemic injury via the A3AR.
The inward rectifying K+ channel Kir6.2 is the pore-forming subunit of the KATP channel (Inagaki et al., 1995; Seino, 1999; Alekseev et al., 2005; Kane et al., 2005). In cardiac myocytes, Kir6.2 associates with the glibenclamide-sensitive sulfonylurea SUR2A subunit to form functional KATP channels in the sarcolemmal membrane (Inagaki et al., 1996; Alekseev et al., 2005; Kane et al., 2005). KATP channels, which are tightly coupled to the metabolic state of the cell, open in response to various stresses functionally shortening the duration of the action potential lessening time for calcium influx (Alekseev et al., 2005; Kane et al., 2005). Thus, the KATP channel serves to adjust cellular excitability to match metabolic demand. With the aid of Kir6.2 KO mice, the sarcolemmal KATP channel has been shown to importantly participate in normal stress responses, including IPC (Suzuki et al., 2002; Hanley and Daut, 2005; Kane et al., 2005). Although it remains controversial, a related isoform of the KATP channel has also been proposed to be expressed in mitochondria (Hanley and Daut, 2005). Similar to the sarcolemmal KATP channel, the mitochondrial KATP channel is thought to open in response to metabolic stress, thereby preserving mitochondrial integrity and reducing apoptosis secondary to changes caused by increased potassium influx. We and others (Tracey et al., 1998; Thourani et al., 1999; Auchampach et al., 2003) have previously observed that protection provided by A3AR agonists is blocked by coadministration of glibenclamide, which equally inhibits both isoforms of the KATP channel. However, the relative importance of the two KATP channel isoforms in A3AR-mediated cardioprotection remained unknown. In the present investigation, we have shown that, like IPC, administration of CP-532,903 does not provide cardioprotection in the isolated heart model using Kir6.2 KO mice confirmed to lack functional sarcolemmal IKATP. We have also provided evidence suggesting that the A3AR is expressed in cardiomyocytes and that it couples to opening of the sarcolemmal KATP channel using electrophysiological techniques. Suzuki et al. (2002) have previously shown that the mitochondrial KATP channel is functional in Kir6.2 KO mice, based on diazoxide-induced changes in flavoprotein oxidation. We have also shown in the present investigation that mitochondrial respiration and ATP-synthesizing capacity of mitochondria from Kir6.2 KO mice are normal. Thus, our data suggest that CP-532,903 provided a direct cardioprotective effect in the isolated heart model via activation of the sarcolemmal KATP channel, rather than via the putative mitochondrial KATP channel.
The data from the isolated heart experiments indicate that CP-532,903 produced a direct cardioprotective effect involving the sarcolemmal KATP channel. However, it is possible that CP-532,903 reduced infarct size in vivo by additional or alternative mechanisms. Administration of CP-532,903 increased plasma histamine levels and produced a 30% decrease in mean arterial blood pressure, responses that were not observed in A3AR KO mice. Thus, improvement in the oxygen supply-demand balance or depletion of mast cell contents may have contributed to the reduction in infarct size in the in vivo model, although we think these potential mechanisms are unlikely based on previous work performed by our laboratory (Ge et al., 2006). Since it has been shown that A3AR agonists including CP-532,903 reduce infarct size when administered at the time of reperfusion (Auchampach et al., 2003; Tracey et al., 2003), it is also possible that CP-532,903 protected against infarction in the present investigation by reducing reperfusion-mediated injury. Even though CP-532,903 was administered 10 min before the ischemic period in our studies, it was likely present during the initial phase of reperfusion since hemodynamic responses to CP-532,903 persisted for over 45 min. Numerous studies in various models of inflammation have suggested that A3AR activation suppresses inflammatory responses and may regulate neutrophil-mediated injury (Hasko and Cronstein, 2004). Thus, CP-532,903 may have acted, in part, by suppressing injury caused by reperfusion-induced inflammation. In support of this theory, we have shown in preliminary experiments that Cl-IB-MECA does not reduce infarct size when administered at the time of reperfusion in bone marrow chimeric mice lacking the expression of A3ARs in bone marrow-derived cells (Ge et al., 2004). Although the results of the present investigation clearly implicate the involvement of the A3AR, additional studies are required to investigate the multiple different mechanisms by which CP-532,903 effectively limits infarct size under in vivo conditions and to determine the relative contribution of the sarcolemmal KATP channel.
In summary, CP-532,903 has been shown to be a highly selective agonist for the mouse A3AR with improved selectivity versus the A2AR subtypes. Thus, CP-532,903 should be useful for discerning the biological role of the A3AR in mice. The results further confirm that CP-532,903 exerts a direct protective effect on the ischemic myocardium by activation of the A3AR by a mechanism that involves opening the sarcolemmal KATP channel. The importance of this direct cardioprotective action under in vivo circumstances awaits further investigation. Finally, the results of the present investigation support the contention that A3AR agonists may be useful agents for treating acute ischemia/reperfusion injury.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: AR, AR, adenosine receptor; IB-MECA, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide; CP-532,903, N6-(2,5-dichlorobenzyl)-3'-aminoadenosine-5'-N-methylcarboxamide; KATP, ATP-sensitive potassium; KO, knockout; HEK, human embryonic kidney; [125I]AB-MECA, N6-(4-amino-3-[125I]iodobenzyl)adenosine-5'-N-methylcarboxamide; Ro-20,1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; ±dP/dt, maximal rate of contraction/relaxation; IPC, ischemic preconditioning; ECG, electrocardiogram; LAD, left anterior descending; CPX, 1,3-dipropyl-8-cyclopentylxanthine; ZM 241385, 4-[2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl]phenol; CCPA, 2-chloro-N6-cyclopentyladenosine; CP-608,039, N6-[2-(3-methylisoxazol-5-ylmethoxy)-5-chloro]benzyl-3'-aminoadenosine-5'-N-methylcarboxamide; DP, developed pressure; MRS 1754, 1,3-dipropyl-8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]xanthine.
Address correspondence to: Dr. John A. Auchampach, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail: jauchamp{at}mcw.edu
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) and A3AR KO (
) myocytes. Current is depicted as current density (pA/pF) to normalize for differences in cell size.