Post-Ischemic Cardioprotection by A2A Adenosine Receptors: Dependent of Phosphatidylinositol 3-Kinase Pathway

Boucher, Matthieu MSc; Pesant, Stéphanie BSc; Falcao, Stéphanie BSc; de Montigny, Chantal TSA; Schampaert, Érick MD; Cardinal, René PhD; Rousseau, Guy PhD

Journal of Cardiovascular Pharmacology:
Original Article

Activation of myocardial A2A adenosine receptors during reperfusion has been shown to be cardioprotective. The intracellular mechanisms underlying this protection remain unknown. To understand the beneficial effects of activated A2A adenosine receptors in such a state, we investigated whether the enzymes phosphatidylinositol 3-kinase (PI3K) and caspase-3 can account for this post-ischemic cardioprotective effect in an anesthetized rabbit model of myocardial infarction (30 minutes ischemia; 5 hours reperfusion). Administration of the A2A agonist CGS21680 (0.2 μg/kg/min) 5 minutes before reperfusion began (Early) reduced infarct size expressed as a percentage of the area at risk (25.7 ± 5.3% versus 46.5 ± 5.3% for the control group; * P < 0.05). Treatment with the A2A agonist 5 minutes after the onset of reperfusion (Late) had no effect on infarct size (38.2 ± 6.2%). In the presence of a selective inhibitor of PI3K (LY294002), the beneficial effects of CGS21680 on infarct size was no longer observed (43.9 ± 7.9%). After 5 hours of reperfusion, higher PI3K activity in the ischemic region was observed in the Early group compared with the other experimental groups. Caspase-3 activity was not observed in these different groups. In another set of experiments, PI3K activity was significantly higher during the first 15 minutes of reperfusion in the Early group as compared with the Control group. Caspase-3 activity increased rapidly during the first 15 minutes of reperfusion in the Control group and remained stable in the Early group. These results indicated that post-ischemic cardioprotection afforded by A2A adenosine receptor activation is PI3K-dependent and modulate rapidly other signaling pathways such as caspase-3.

Cardioprotection can be afforded before or after ischemia by adenosine receptors activation. Activation of A1 or A3 adenosine receptor subtypes before the onset of sustained ischemia reduces infarct size significantly. 1,2 Intracellular signaling pathways involved in these beneficial effects have been identified to involve kinases followed by phosphorylation and activation of membrane end-effectors possibly via KATP channels. 3 Activation of A2A adenosine receptors is effective at doing so during reperfusion. 4 A2A adenosine receptor activation post-ischemia may exert beneficial effects by interfering with the deleterious events associated with neutrophil accumulation, 5,6 oxygen-free radical production, 6,7 or apoptosis. 4 The molecular signaling pathways involved in any post-ischemic cardioprotection afforded by A2A adenosine receptor activation remain unknown.

Experimental evidence suggests that cell death occurring during reperfusion is due to apoptosis. 8,9 Early after the onset of reperfusion, 10 apoptotic stimuli activate the caspase cascade that involves caspase-3, which cleaves substrates important to the maintenance of cellular integrity. 11 Post-ischemic pharmacological interventions that reduce infarct size may thus interfere with apoptosis. 4,12 To determine the intracellular signaling pathways involved in the cardioprotection afforded by A2A adenosine receptor activation, we studied two enzymes that can be modulated by stimulation of G protein-coupled receptors and involved in cell survival.

PI3K is a key signaling enzyme that modifies downstream effectors such as kinases that are involved in cell growth and protein synthesis, thus cell survival. 13 Among the different downstream effectors, PKB/Akt can inhibit caspase-9, a protease crucial in the initiation of apoptosis. PKB/Akt can also phosphorylate Bad, which can no longer, as a consequence, interact with Bcl-2 or Bcl-xl, allowing them to inhibit apoptosis.

The second enzyme, caspase-3, is rapidly activated during the early phase of apoptosis, thereby contributing to cell death. Inhibition of caspase-3 during reperfusion induces a significant reduction of infarct size. This reduction suggests an important role for this enzyme in reperfusion injury. 12,14 Interestingly, it has been reported that activation of PI3K reduces caspase-3 activity. 15–17 According to these findings, PI3K seems to be upstream of caspase-3 and thus may act as an important enzyme in post-ischemic cardioprotection.

The present study was conducted to determine the role of A2A adenosine receptors in modulating PI3K and caspase-3 activities and thus the role of the former in cardioprotection afforded by A2A adenosine receptor activation.

Author Information

From the Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, Département de pharmacologie and Groupe de Recherche sur le Système Nerveux Autonome, Faculté de médecine, Université de Montréal, Montréal, Québec, Canada.

Received for publication June 9, 2003; accepted December 3. 2003.

Supported by the Fondation des Maladies du Coeur du Québec.

Reprints: Guy Rousseau, PhD, Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, 5400, boul. Gouin Ouest, Montréal (Québec), Canada, H4J 1C5 (e-mail:

© 2004 Lippincott Williams & Wilkins, Inc.