The role of protein kinase B and mitochondrial permeability transition pore in ischaemic preconditioning of myocardium

Background: Protein kinase B (PKB) plays a critical role in cell survival, growth and proliferation. The investigation of its involvement has been limited by the lack of specific pharmacological agents. Similarly mitochondrial permeability transition pore (MPTP) plays a critical role in necrotic and apoptotic cell death. Opposed role of MPTP (transient versus sustained opening) in cardioprotection has been proposed. Therefore the primary aims of this thesis were to (i) to investigate the influence of PKB in the tolerance to ischaemia/reoxygenation (I/R), (ii) to define the relationship of PKB with the mitoKATP channel and with p38 MAPK in the signal transduction mechanism of cardioprotection by IP, (iii) to determine how two structurally different MPTP inhibitors (Cyclosporine A and Bongkrekic Acid) administered for varying time regimes influenced ischemia/reperfusion (I/R)-induced injury and (iv) to explore how pharmacologic MPTP opening at different time points during I/R modulated myocardial injury. Methods & Results: Myocardial slices from rat left ventricle and from the right atrial appendage of patients undergoing elective cardiac surgery were subjected to 90 min ischaemia/120 min reoxygenation at 37°C. Tissue injury was assessed by creatine kinase (CK) released and determination of cell necrosis and apoptosis. Myocardial caspase 3 was also assessed. Muscles were randomized to receive various treatments. The results showed that blockade of PKB activity caused significant reduction of CK release and cell death, a benefit that was as potent as ischaemic preconditioning and could be reproduced by blockade of phosphatidylinositol 3-kinase (PI-3K) with wortmannin and LY 294002. The protection was time dependent with maximal benefit seen when PKB and PI-3K were inhibited before ischaemia or during both ischaemia and reoxygenation. PKB inhibition induced a similar degree of protection in the human and rat myocardium and, importantly, it reversed the unresponsiveness to protection of the diabetic myocardium. The status of MPTP can dramatically influence ischemic/reoxygenation-induced injury and protection of the rat left ventricular myocardium. Importantly, the status of the MPTP during first 10 min of reoxygenation is of critical importance with both opening and closing of the pore being as protective as ischemic preconditioning. Conclusion: Inhibition of PKB plays a critical role in protection of the mammalian myocardium and may represent a clinical target for the reduction of ischaemic injury. Both formation and inhibition of the MPTP can be exploited for therapeutic purposes and that there is a defined therapeutic window, with the first few minutes of reoxygenation being a crucial period to achieve cardioprotection.EThOS - Electronic Theses Online ServiceGBUnited Kingdo