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Both A2a and A2b adenosine receptors at reperfusion are necessary to reduce infarct size in mouse hearts
1Abstract
Pre- and postconditioning depend on the activation of adenosine receptors (ARs) at the end of the index ischemia. The aim of this study was to determine which receptor subtypes must be activated. In situ mouse hearts underwent 30 min of regional ischemia, followed by 2 h of reperfusion. As expected, either ischemic postconditioning (6 cycles of 10 s of reperfusion and 10 s of coronary occlusion) or infusion of the selective A2b adenosine receptor (A2bAR) agonist BAY60-6583 (BAY60) for 60 min, starting 5 min before reperfusion reduced infarct size in wild-type C57Bl/6N mice. Protection from either was abolished by the selective A2bAR antagonist MRS-1754, confirming a role for A2bAR. Additionally, the coadministration of ischemic postconditioning and a selective A2aAR antagonist led to the loss of protection as well. 5′-Ectonucleotidase (CD73) is thought to be necessary for the production of adenosine during ischemia. As predicted, ischemic postconditioning did not protect CD73 knockout mice. Selective agonists of either A2bAR (BAY60) or A2aAR (CGS-21680), as well as the coadministration of ischemic postconditioning and BAY60, also failed to protect hearts of the CD73 knockout mice. But the nonselective A1/A2AR agonist 5′-(N-ethylcarboxamido)adenosine (NECA) was protective, suggesting that the activation of multiple AR subtypes might be required. The coadministration of CGS-21680 and BAY60 also elicited profound protection, indicating that two AR subtypes, A2a and A2b, must be simultaneously activated for protection to occur.
following the discovery of ischemic preconditioning, adenosine was soon identified as a major trigger of its protection (10). It was found that both the A1 and A3 adenosine receptors (ARs) were capable of triggering entrance into the protective phenotype if they were occupied before ischemia (14). Unfortunately, mimicking ischemic preconditioning with an A1AR- or A3AR-selective agonist was not clinically useful because the receptors had to be occupied before the onset of ischemia. More recently, it was noted that the protection of ischemic preconditioning is actually mediated in the first minutes of reperfusion following the lethal ischemic insult (3). Thus it should still be possible to treat a patient with acute myocardial infarction if the mediator pathway could be activated at the time of therapeutic reperfusion. The protection, at least in rats and rabbits, was related to the activation of the reperfusion injury survival kinases (RISK) (4) and the reoccupation of ARs (15). The protective AR during reperfusion, however, appeared to be the Gs-coupled A2bAR rather than either of the Gi-coupled A1AR or A3AR responsible for triggering protection before the index ischemia (12). Because given that the A2bAR-potent but nonselective agonist 5′-(N-ethylcarboxamido)adenosine (NECA) (20) or the highly A2bAR-selective agonist BAY60-6583 (BAY60) (8) at reperfusion was found to mimic the protection of ischemic preconditioning, it was concluded that only the adenosine A2bAR receptor controlled the RISK pathway. Because an A2aAR-selective agonist was not protective in our rabbit heart model, we therefore concluded that the A2aAR was not required (19).
Recently, Xi et al. (16) reported that both A2aAR and A2bAR must be simultaneously activated to realize full cardiac protection in rats. In the rabbit, full protection is achieved with the A2bAR-selective agonist alone (8). However, because the affinity of the A2aAR is so high, the latter could have unknowingly been occupied by the high levels of endogenous adenosine released by the ischemic myocardium. The A2bAR has a much lower affinity, and the level of adenosine produced by even deeply ischemic cardiac tissue is insufficient to bind A2bAR unless the latter is modified by protein kinase C (8). Hence, an exogenous agonist is required. Therefore, our prior observations in rabbits that initially led us to promote the primacy of A2bAR and the lack of importance of A2aAR for cardioprotection might have to be reinterpreted. We wanted to further test the dual receptor hypothesis, but unfortunately a highly selective A2aAR blocker is not available. We, therefore, tried to confirm the hypothesis of Xi et al. with 5′-ectonucleotidase (CD73) knockout mice. CD73 catalyzes phosphohydrolysis of AMP, a primary source of extracellular adenosine production in ischemic myocardium. CD73−/− animals should therefore be incapable of producing large amounts of adenosine during ischemia-reperfusion (7, 9). We reasoned that in the absence of endogenous adenosine, these animals would have little opportunity to occupy A2aAR, and we would, therefore, have to occupy both A2a and A2bAR with exogenously administered agonists to produce protection if the dual receptor hypothesis were correct.
METHODS
This study was performed in accordance with The Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996). The experimental protocols were approved by the local authorities of the state of Mecklenburg-Vorpommern, Germany.
Open-chest mouse model.
We used a recently described in situ open-chest mouse model (13). Briefly, mice were anesthetized with pentobarbital sodium, intubated through a tracheotomy, and ventilated with room air supplemented with oxygen with a pressure- and volume-controlled ventilator. After a left thoracotomy, a prominent branch of the left coronary artery was surrounded with a suture to form a snare. All hearts underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion.
Experimental protocol.
Seven groups were studied in C57Bl/6N mice (Charles River, Kisslegg, Germany). Control mice had only the index ischemia followed by reperfusion. Postconditioning (PostC) was effected with six cycles of 10 s of reperfusion and 10 s of coronary artery reocclusion at the end of the index coronary occlusion. BAY60 (10 μg/kg) was injected into the left atrium as two separate boluses: 5 min before and 15 min after the onset of reperfusion. MRS-1754 (9.5 μg/kg), an A2b-selective antagonist, was administered as an intravenous bolus 5 min before the onset of reperfusion either alone or combined with PostC or BAY60. A2aAR antagonist 2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-C]pyrimidin-5-amine (SCH-442416) was given intraperitoneally at 0.017 mg/kg 2 h before the onset of reperfusion.
CD73−/− knockout mice were used in seven groups as well. The CD73−/− mice are described elsewhere in detail (2). Control, PostC, and BAY60 were studied as above. NECA (2 μg/kg) was given as an intravenous bolus 5 min before the onset of reperfusion followed by 0.2 μg·kg−1·min−1 for 65 min. 2-[p-(2-Carboxyethyl)phenyl amino]-5′-N-ethyl carboxamido adenosine (CGS-21680), a highly selective A2aAR agonist, was given as a bolus of 30 μg/kg iv 5 min before the onset of reperfusion, followed by a 60-min infusion of 3 μg·kg−1·min−1. BAY60 was infused together with either CGS-21680 or PostC.
After the completion of the experiment, the coronary artery was reoccluded and infarct size and risk zone were evaluated as described recently (13).
Materials.
BAY60 was kindly provided by Thomas Krahn of Bayer Healthcare (Wuppertal, Germany). All other chemicals were from Sigma-Aldrich Chemical. NECA was dissolved in water, and all other drugs in DMSO before being diluted in 0.9% NaCl. The final concentration of DMSO was kept below 0.01%.
Statistics.
Data are presented as means ± SE. Differences in infarct size were compared by one-way ANOVA with Fisher least significant difference test. A value of P < 0.05 was considered significant.
RESULTS AND DISCUSSION
First, we confirmed the infarct size-reducing properties of both PostC and the highly selective A2bAR agonist BAY60 in in situ mouse hearts. Figure 1 summarizes the infarct size data for the wild-type C57Bl/6N mice. As expected, PostC as well as BAY60 reduced infarct size (29.2 ± 0.7% of risk zone in untreated controls to 9.7 ± 2.2% in PostC and 8.3 ± 2.1% in BAY60, both P < 0.001). The protection seen with BAY60 does not rule out a relevant role for the high-affinity A2aAR since the latter may have been occupied by the relatively high levels of endogenous adenosine seen after a prolonged period of ischemia. On the other hand, A2aAR and A2bAR seem to be crucial for protection since the PostC effect was abolished by either the selective A2bAR antagonist MRS-1754 or the A2aAR antagonist SCH-442416. Additionally, the A2bAR antagonist MRS-1754 could block the cardioprotective effect of BAY60, showing its selectivity.
Results of open chest experiments in C57Bl/6N wild-type mice. Postconditioning (PostC) as well as the A2b adenosine receptor (A2bAR) agonist BAY60-6583 (BAY60) given at reperfusion reduced infarct size compared with untreated control hearts. The protective effect of PostC could be abolished by the selective A2bAR antagonist MRS-1754 and the selective A2aAR antagonist SCH-442416. While MRS-1754 blocked the protective effect of BAY60, it had no effect when given alone. Open circles represent infarct size as percentage of risk zone for individual hearts, and closed circles show the group means ± SE. ***P < 0.001 vs. control.
The next experiments were performed in CD73−/− knockout mice. It had been previously shown that mice lacking CD73 could no longer be preconditioned (2). Figure 2 reveals that infarct size in untreated control animals was similar to that in wild-type mice but that PostC could not reduce infarct size in these animals.
Results of open-chest experiments in CD73−/− knockout mice. PostC as well as activation of a single AR subtype with either BAY60 or CGS-21680 and coadministration of PostC and BAY60 failed to protect. In contrast, the nonselective adenosine agonist 5′-(N-ethylcarboxamido)adenosine (NECA) or cotreatment with A2aAR and A2bAR agonists produced profound protection, indicating a requirement for both AR subtypes. **P < 0.01 vs. control.
Interestingly, the activation of either A2aAR or A2bAR alone failed to limit infarct size in the CD73−/− mice. Even a coadministration of PostC and BAY60 was not able to protect. The mixed A1/A2AR agonist NECA is known to mimic preconditioning when given at reperfusion (20). Xi et al. (16) obtained strong protection in rats with NECA, which could be blocked by either an A2aAR or A2bAR antagonist. As seen in Fig. 2, NECA was also protective in mice lacking CD73 (infarct size reduction from 33.0 ± 4.1% in untreated hearts to 7.1 ± 1.0%, P < 0.01). This finding is compatible with the two-receptor hypothesis.
To more critically test for the requirement of both A2AR subtypes, highly selective agonists for both A2bAR (BAY60) and A2aAR (CSG-21680) were given simultaneously. This combination reduced infarct size to 10.0 ± 1.1% (Fig. 2), which was comparable with that seen with NECA or PostC in wild-type mice.
These findings support those previously observed with the nonselective adenosine agonist AMP-579 (18). This mixed agonist was originally touted as an A1/A2a adenosine agonist, and its protection was attributed to A2aAR (6). However, it was not possible to duplicate the protection of AMP-579 with CGS-21680 alone (18). Recently, AMP-579 has been shown to be a potent A2bAR agonist as well (11), making its pharmacological properties very similar to those of NECA. There is one report in which an A2aAR-selective agonist alone could limit infarct size (21), but in that study infarct size was measured after a prolonged reperfusion time during which postinfarct inflammation may have further extended the infarct. And A2aAR are known to have a powerful anti-inflammatory effect (21). Typically in hearts treated with an A2aAR agonist alone, the low-affinity A2bAR would not be occupied, thus preventing protection. On the other hand, selective A2bAR agonists have consistently been very protective (2, 8) since endogenous adenosine would easily be able to occupy the high-affinity A2aAR.
It cannot be excluded that other G protein-coupled receptors are involved in the signaling pathway of postconditioning, but it seems that the threshold of protection can only be reached when AR activation is present. Although we have shown a clear involvement of A2ARs, we also cannot fully exclude the involvement of other AR subtypes in the signaling. Previous studies have reported that A1AR and A3AR subtypes may also have a role in PostC (1, 5, 17).
Taken together, our findings confirm the conclusion of Xi and coworkers (16) that both A2AR subtypes, A2aAR and A2bAR, work in concert to protect postconditioned hearts at reperfusion. It is unknown why neither receptor is sufficient for protection since both are Gs coupled and would be expected to trigger similar signaling pathways. The results, however, clearly reveal that the different signaling pathways are activated by each receptor. Certainly, further research will be required to address this question.
GRANTS
The study was supported in part by National Heart, Lung, and Blood Institute Grant HL-20468 (to T. Krieg and J. M. Downey) and a grant from the German Research Foundation (DFG) (to T. Krieg).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
ACKNOWLEDGMENTS
The CD73 knockout mice were kindly provided by Dr. Thompson (Oklahoma Medical Research Foundation, Oklahoma City, OK) and Dr. Eltzschig (Department of Anesthesiology, University of Colorado, Denver, CO). BAY60 was provided by BayerHealthcare (Wuppertal, Germany).