Evidence that the acute phase of ischemic preconditioning does not require signaling by the A 2B adenosine receptor

Abstract

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A(2B)AR gene knock-out (A(2B)KO)/β-galactosidase reporter gene knock-in mice and the A(2B)AR antagonist ATL-801 to investigate the potential involvement of the A(2B)AR in IPC, focusing on the acute phase of protection. Cardioprotection provided by acute IPC elicited by two 3-min occlusion/3-min reperfusion cycles was readily apparent in an isolated, Langendorff-perfused mouse heart model in studies using hearts from A(2B)KO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A(2B)KO hearts by ~30-40%, and equivalently decreased the release of cardiac troponin I during the reperfusion period (from 5969 ± 925 to 1595 ± 674 ng/g and 4376 ± 739 to 2278 ± 462 ng/g using WT and A(2B)KO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifested in experiments using A(2B)KO mice, as well as in experiments using rats pretreated with ATL-801. We did observe, however, a marked reduction in infarct size in rats following administration of the selective A(2B)AR agonist BAY 60-6583 (~25% reduction at a dose of 1.0mg/kg). While supportive of its concept as a cardioprotective receptor, these experiments indicate that the mechanism of the early phase of IPC is not dependent on signaling by the A(2B)AR. We present the idea that the A(2B)AR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.

Figure 1

Schematic diagram of the experimental protocols. Abbreviations: IPC, ischemic preconditioning; O, occlusion; R, reperfusion.

Figure 2

ATL-801 effectively blocks the hypotensive effect of the A_2BAR agonist BAY 60-6583 in rats. Rapid bolus administration of BAY 60,6583 (1 mg/kg i.v.) produced a transient (~5 min) reduction in mean arterial blood pressure in anesthetized rats. This effect of ATL-801 was blocked completely in rats pretreated with ATL 801 (1 mg/kg). Blockade persisted for at least 2 h. Shown is a representative tracing from three different experiments.

Figure 3

LV functional recovery data at 45 min of reperfusion (A–D) and cTnI release data (E) from the isolated mouse hearts studies of IPC. Functional data are expressed as a percentage of baseline values. Refer to Fig. 1 for a detailed description of the experimental protocol. IR, non-preconditioned control group; IPC, preconditioned group. *P<0.05 versus the respective non-preconditioned control group (IR). n = 8–10 hearts/group.

Figure 4

Myocardial infarct size data from the in vivo mouse (A) and rat (B) studies of IPC. Refer to Fig. 1 for a detailed description of the experimental protocols. The data are presented as a percentage of the AAR. *P < 0.05 vs. the respective control (Ctrl) group.

Figure 5

Pretreating with the A_2BAR agonist BAY 60-6583 reduced myocardial infarct size in rats. Anesthetized rats were subjected to 30 min of coronary occlusion and 2 h of reperfusion. Vehicle (control) or BAY 60-6583 (0.3 or 1.0 mg/kg i.v.) were administered 10 min before occlusion. In the combination group (BAY+ATL), ATL-801 was administered (1.0 mg/kg i.v.) 10 min before administering BAY 60-6583. The data are presented as a percentage of the AAR. *P<0.05 vs the vehicle-treated control group.