Phenylephrine preconditioning of isolated ventricular myocytes involves modulation of KATP channels through activation of survival kinases
thesisposted on 28.10.2010, 15:53 by Helen Elizabeth Turrell
Pretreatment with the α1-adrenoceptor agonist phenylephrine has been shown to protect cardiac tissue from a subsequent period of ischaemia. This phenomenon, known as pharmacological preconditioning, decreases infarct size and increases the functional recovery of the intact heart. The role of protein kinases and ATP-sensitive potassium channels (KATP) in phenylephrine preconditioning was investigated in isolated ventricular myocytes using whole cell patch clamp, western blotting, fluorescence imaging and measurement of contractile activity. The role of PKC isoforms was examined using isoform-specific PKC activator and inhibitor peptides. Preconditioning with phenylephrine increased the contractile recovery of isolated ventricular myocytes following simulated ischaemia and reperfusion, and this was also correlated with an improvement in calcium homeostasis during reperfusion. The protective effect of phenylephrine preconditioning on contractile recovery was abolished by inhibition of α1-adrenoceptors, PKC or KATP. We observed a sustained activation of PKCε and δ in response to phenylephrine preconditioning, and identified a protective role for PKCε. PKCδ was required for phenylephrine preconditioning but also increased reperfusion injury. CaMKK, AMPK and p38 MAPK were all required for phenylephrine preconditioning, and activation of AMPK by phenylephrine preconditioning required PKCδ. However, both JNK and ERK appear not to be involved in the protective effect. Inhibition of sarcolemmal KATP channels (sarcKATP) or mitochondrial KATP channels (mitoKATP) prevented preconditioning. Peak sarcKATP current activated by metabolic inhibition was increased following phenylephrine preconditioning and inhibition of PKCδ, AMPK or p38 MAPK was sufficient to prevent the increase in current. Increased sarcKATP current can enhance hyperpolarisation of the resting membrane potential, and may be responsible for the observed decrease in calcium loading and improved contractile recovery of phenylephrine preconditioned ventricular myocytes following ischaemia and reperfusion.