Original article
Evidence that the acute phase of ischemic preconditioning does not require signaling by the A2B adenosine receptor

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Abstract

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A2BAR gene knock-out (A2BKO)/β-galactosidase reporter gene knock-in mice and the A2BAR antagonist ATL-801 to investigate the potential involvement of the A2BAR 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 A2BKO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A2BKO 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 A2BKO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifested in experiments using A2BKO 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 A2BAR agonist BAY 60-6583 (~ 25% reduction at a dose of 1.0 mg/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 A2BAR. We present the idea that the A2BAR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.

Research Highlights

►Protection by early preconditioning can be elicited in A2B receptor knock-out mice. ►Preconditioning in rats is also present during A2B receptor blockade with ATL-801. ►Giving the A2B receptor agonist BAY 60-6583, however, reduces infarct size in rats.

Introduction

Ischemic preconditioning is a phenomenon whereby exposure to brief periods of ischemia renders the myocardium resistant to subsequent ischemic insults, manifest as a reduction in myocardial infarct size [1]. IPC appears to consist of two phases, an acute phase (early IPC) that develops immediately but wanes within 1–2 h, and a delayed phase (late IPC) that appears 12–24 h later but lasts for several days [2], [3], [4]. The time-course and duration of the delayed phase of IPC is consistent with a mechanism involving the synthesis of cardioprotective proteins [5], whereas the early phase is explained by metabolic slowing that preserves stores of high energy phosphates thereby promoting cell survival [6]. The early phase of IPC can be elicited in isolated heart and cardiomyocyte models of ischemic injury, inferring that the mechanism of protection is intrinsic to the cardiac muscle [7], [8].

Current evidence suggests that adenosine and other factors (i.e., opioid peptides and bradykinin) released during preconditioning ischemia serve to initiate the development of the cardioprotected phenotype associated with IPC [9]. Although there is support for involvement of the A3AR, most evidence implicates the A1 in IPC [10], [11], [12], [13], which is the predominant AR subtype expressed in cardiac myocytes well-known to regulate heart rate and to suppress responses to β-adrenergic stimulation [14], [15]. Previous studies have identified the importance of the A1AR in IPC using pharmacological strategies and gene knock-out mice [9], [10], [11], [12], [13], [16].

It has recently been reported by Eckle and colleagues [16], [17], however, that cardioprotection by what appears to be the early phase of IPC is completely lost in a commercially available line of A2BKO mice, suggesting that the A2BAR also plays an important role in the mechanism of IPC. These studies also reported that IPC protection is absent in gene-ablated mice lacking the extracellular adenosine-generating enzyme ecto-5′-nucleotidase (CD73). Based on these observations, the authors developed the intriguing hypothesis that, as a result of the presence of hypoxia-inducible-1 (HIF-1) elements within their respective gene promoters, the level of expression of the A2BAR and CD73 is increased following IPC. Through a combined increase in adenosine production by CD73 and expression of functional A2BARs in the myocardium, the theory proposes that signaling via the A2BAR is increased during reperfusion providing physiological cardioprotection. While this hypothesis is relevant to the late phase of IPC, it does not account for the finding that the early phase of IPC is not prevented by protein synthesis inhibitors [18].

In this study, we sought to confirm and extend our understanding of the contribution of the A2BAR in IPC, focusing exclusively on the acute phase of protection. Our studies utilized A2BAR gene knock-out/β-galactosidase reporter gene knock-in mice (A2BKO; [19]) and the selective A2BAR antagonist ATL-801 [20].

Section snippets

Animals

All experiments were performed with male mice weighing ~ 25–30 g (12–16 weeks of age) and male rats weighing ~ 250–350 g (8–12 weeks). C57BL/6J wild-type (WT) mice were purchased from Jackson Laboratories, (Bar Harbor ME) and Sprague–Dawley rats were purchased from Harlan Laboratories (Madison, WI). A2BAR gene KO/β-galactosidase reporter gene “knock-in” mice made congenic on the C57BL/6J genetic background were a kind gift from Dr. Katya Ravid (Boston University; [19]. All animals in the study

Isolated mouse heart studies

Baseline functional data from the isolated mouse heart studies are reported in Table 1. During pacing at 420 bpm, there were no differences in any of the parameters measured at baseline including LV developed pressure (DP), maximal LV ±dP/dt, and coronary flow. Following 20 min of global ischemia and 45 min of reperfusion, recovery of contractile function averaged ~ 50% of baseline values in the control, non-preconditioned group (Fig. 3). Although not statistically significant, tolerance to

Discussion

The possibility has arisen in recent years that the A2BAR subtype may play an important role in the mechanism of the early phase of IPC [9], [16], [17], [24]. To further test this theory, we examined the potential involvement of the A2BAR in acute IPC using a comprehensive experimental approach. We first examined whether IPC can be demonstrated in A2BKO mice. For our studies we used a well-characterized line of A2BKO/β-galactosidase reporter gene “knock-in” mice developed by Yang and colleagues

Disclosure

Dr. Figler is an employee and shareholder of PGxHealth.

Acknowledgments

We thank Ms. Anna Hsu for technical assistance and Dr. Thomas Krahn (Bayer HealthCare) for supplying BAY 60-6583. We also thank Drs. Holger Eltzschig (University of Colorado) and Katya Ravid (Boston University) for critical evaluation of this work. This study was supported by grants from the National Institutes of Health (R01 HL077707 to J.A.A., R37 HL074314 to G.J.G., and R01 HL 008311 to G.J.G.) and by a pre-doctoral fellowship from the American Heart Association (0810035Z to J.E.M.).

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