Caspase inhibition via A3 adenosine receptors: a new cardioprotective mechanism against myocardial infarction.
OBJECTIVE
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.
Biomolecular Sciences Applied Research Group, Faculty of Health and Life Sciences, Coventry University, Coventry, CV1 5FB, UK.
Afthab
A
Hussain
Ahmed Mayel
AM
Gharanei
Aarondeep Singh
AS
Nagra
Helen L
HL
Maddock
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058909:Adenosine A3 Receptor Agonists / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002070:Butadienes / Q000494:pharmacology; D002316:Cardiotonic Agents / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D004195:Disease Models, Animal; D008297:Male; D009203:Myocardial Infarction / Q000209:etiology / Q000473:pathology / Q000517:prevention & control*; D015428:Myocardial Reperfusion Injury / Q000503:physiopathology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000473:pathology; D009570:Nitriles / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043684:Receptor, Adenosine A3 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000077191:Wortmannin
D058909:Adenosine A3 Receptor Agonists; D000730:Androstadienes; D002070:Butadienes; D002316:Cardiotonic Agents; D009570:Nitriles; D043684:Receptor, Adenosine A3; C113580:U 0126; D053148:Caspase 3; D000241:Adenosine; D000077191:Wortmannin; C090034:2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide