Original articleEvidence that the acute phase of ischemic preconditioning does not require signaling by the A2B adenosine receptor
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.).
References (29)
- et al.
Preconditioning of isolated rabbit cardiomyocytes: no evident separation of induction, memory and protection
J Mol Cell Cardiol
(1997) - et al.
Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology
Am J Cardiol
(1997) - et al.
Attenuated purine production during subsequent ischemia in preconditioned rabbit myocardium is unrelated to the mechanism of protection
J Mol Cell Cardiol
(1996) - et al.
Protein kinase C protects preconditioned rabbit hearts by increasing sensitivity of adenosine A2b-dependent signaling during early reperfusion
J Mol Cell Cardiol
(2007) - et al.
Adenosine A2A and A2B receptors work in concert to induce a strong protection against reperfusion injury in rat hearts
J Mol Cell Cardiol
(2009) - et al.
Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium
Circulation
(1986) - et al.
Does myocardial stunning contribute to infarct size limitation by ischemic preconditioning?
Circulation
(1991) - et al.
Myocardial protection is lost before contractile function recovers from ischemic preconditioning
Am J Physiol
(1991) - et al.
The natural history of preconditioning: cardioprotection depends on the duration of transient ischemia and time to subsequent ischemia
Cor Art Dis
(1991) The late phase of preconditioning
Circ Res
(2000)