Caspase Inhibition Via A3 Adenosine Receptors: a New Cardioprotective Mechanism Against Myocardial Infarction

Purpose: 2-CL-IB-MECA, (A3 adenosine receptor agonist)(A3AR) mediated cardioprotection is well documented although the associated intracellular signalling pathways remain unclear. Here we demonstrate a role of the pro-survival signalling pathways MEK1/2-ERK1/2 and PI3K/AKT and their effect on modifying Caspase-3 activity in A3AR mediated cardioprotection.

Methods: Isolated perfused rat hearts or primary adult rat cardiac myocytes were subjected to ischaemia/hypoxia and reperfusion/reoxygenation, respectively. 2-CL-IB-MECA (1 nM) was administered at the onset of reperfusion/reoxygenation in the presence and absence of either the PI3K inhibitor Wortmannin (5 nM) or MEK1/2 inhibitor UO126 (10 μM). Heart tissues were harvested for assessment of p-ERK1/2(Thr202/Tyr204) or p-AKT (Ser-473) status or underwent infarct size assessment. Cardiac myocytes underwent flow-cytometric analysis for apoptosis, necrosis, cleaved-caspase 3/p-BAD (Ser-112 and Ser-136) activity post-reoxygenation.

Results: 2-CL-IB-MECA significantly reduced infarct size compared to non-treated controls, where co-administration with either of the kinase inhibitors abolished the infarct sparing effects. Administration of 2-CL-IB-MECA at reperfusion significantly upregulated the status of p-ERK1/2 and p-AKT compared to time matched controls in a UO126 and Wortmannin sensitive manner respectively. 2-CL-IB-MECA when administered throughout reoxygenation significantly reduced apoptosis, necrosis, cleaved-caspase 3 activity and increased p-BAD (Ser-112) and p-BAD (Ser-136) activity in myocytes subjected to hypoxia/reoxygenation injury. The cytoprotective effect was abolished by co-administration with the kinase inhibitors Wortmannin and/or UO126.

Conclusions: We have described the molecular mechanisms associated with A3AR mediated cardioprotection indicating a role for the pro-survival signalling pathways that decrease caspase-3 activity. These observations provide novel insight into the pharmacological effects of A3ARs in ameliorating myocardial ischaemia/reperfusion injury.