AbstractActivation of A1 adenosine receptors have previously been researched to protect the myocardium from ischaemia reperfusion injury in various animal models throughout the pre-conditioning phenomenon. The PI3K-AKT and MEK1/2-ERK1/2 cell survival pathways have been shown to play a critical role in the regulation in myocardial ischaemia reperfusion injury. In this study we investigated whether the A1 adenosine receptor agonist 2’-MeCCPA protects the myocardium from ischaemia reperfusion injury when administered at the onset of reperfusion or post-reperfusion and whether the protection involved the PI3K-AKT or MEK1/2-ERK1/2 cell survival pathways and if this protection was definitely mediated through the A1 adenosine receptor. In the Langendorff model of ischaemia reperfusion injury, the isolated perfused rat hearts underwent 20 minutes stabilisation, 35 minutes of ischaemia and 120 minutes of reperfusion. The administration of 2’-MeCCPA (10nM) at the onset of reperfusion significantly decreased infarct size to risk ratio in comparison to non-treated ischaemic reperfused control hearts. This protection was abolished in the presence of the PI3K inhibitor Wortmannin or MEK1/2 inhibitor UO126; as well as in the presence of A1 adenosine antagonist DPCPX. Western blot analysis determined that the administration of 2’-MeCCPA (10nM) upregulated AKT phosphorylation. In the adult rat cardiomyocyte model of hypoxia/reoxygenation, cells underwent 1 hour of hypoxia and 3 hours of reoxygenation. Administration of 2’-MeCCPA (10nM) at the onset of reoxygenation significantly decreased cellular apoptosis and necrosis. The administration of 2’-MeCCPA (10nM) in the presence of Wortmannin, UO126 and DPCPX significantly reversed the anti-apoptotic and anti-necrotic effects.
The methods used within this study include the Langendorff technique to investigate cardioprotection within the ischaemia/reperfusion model of an isolated rat heart. Apoptosis and necrosis levels were investigated as well as cleaved caspase-3 activity within the hypoxia/reoxygenation model of isolated rat cardiomyocytes. Western blots were also used to investigate the AKT phosphorylation levels within this study.
Our data furthermore implied that 2’-MeCCPA protects myocytes that were subjected to hypoxia/reoxygenation injury via decreasing cleaved caspase-3 activity that was also abolished in the presence of PI3K inhibitor Wortmannin as well as in the presence of the MEK1/2 inhibitor UO126 and A1 adenosine antagonist DPCPX.
Interestingly, postponing the administration of 2’-MeCCPA to 15 or 30 minutes after the onset of reperfusion significantly protected the isolated perfused rat heart from ischaemia reperfusion injury in a Wortmannin, UO126 and DPCPX sensitive manner. This protection was associated with an increase in AKT phosphorylation.
Administration of the A1 adenosine receptor agonist 2’-MeCCPA 15 or 30 minutes after the onset of reoxygenation significantly protected isolated adult rat cardiomyocytes that were subject 1 hour of hypoxia and 3 hours of reoxygenation from injury in an anti-apoptotic/necrotic manner. This anti-apoptotic and necrotic effects was abolished upon the administration of PI3K inhibitor Wortmannin, MEK1/2 inhibitor UO126 and A1 adenosine antagonist DPCPX. Delaying the administrations of 2’-MeCCPA to 15 or 30 minutes after reoxygenation was also associated with a decrease in cleaved caspase-3 activity which was abolished in the presence of PI3K inhibitor Wortmannin; however MEK1/2 inhibitor UO126 was only able to abolish protective effects at 15 minutes and no effect at 30 minutes post-reoxygenation.
It can be implied for the first time that the administration of 2’-MeCCPA at the onset of reperfusion protects the ischaemic reperfused rat myocardium from lethal ischaemia reperfusion injury in a PI3K and MEK1/2 sensitive manner. Delaying the administration of 2’-MeCCPA to 15 minutes or 30 minutes after the onset of reperfusion or reoxygenation, it significantly protects the isolated perfused rat heart from ischaemia reperfusion injury and also the adult rat cardiomyocyte from hypoxia-reoxygenation injury in an anti-apoptotic/necrotic manner. Moreover, this protection is associate with recruitment of the PI3K-AKT and MEK1/2-ERK1/2 cell survival pathways.
Furthermore, any protective effects observed with the administrations of 2’-MeCCPA at the onset of reperfusion, 15 minutes or 30 minutes post-reperfusion/reoxygenation was significantly abrogated in the presence of the A1 adenosine antagonist DPCPX which implies that protection was definitely occurring through the A1 adenosine receptor subtype.
The work carried out in this thesis has implied the cardioprotective effects of the activation of A1 adenosine receptors in an ischaemia/reperfusion injury model. This is the first study to imply the delayed activation of A1 adenosine receptors with A1AR agonist 2’-MeCCPA to confer cardioprotection in isolated perfused rat hearts. The findings from this study imply clinically important developments in the field of managing myocardial infarction. Urgent studies are required to investigate the potential role of A1 adenosine receptor agonists in ameliorating myocardial ischaemia reperfusion injury in human.
|Date of Award||Aug 2021|
|Supervisor||Afthab Hussain (Supervisor), Derek Renshaw (Supervisor) & Helen Maddock (Supervisor)|