Possible mechanisms for levosimendaninduced cardioprotection
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Background and purpose. To limit ischaemic injury, rapid restoration of coronary blood flow is required, which will in turn reduce infarct size. However, reperfusion itself causes myocyte death – a phenomenon termed lethal reperfusion-induced injury, which limits protection of the ischaemic myocardium. Thus the reperfusion of irreversibly damaged myocytes may accelerate the process of cell necrosis. Additive protection of the ischaemic myocardium in the form of adjunct therapy remains a topic of intensive research. Levosimendan, a calcium sensitizing agent with positive inotropic effects has in several studies been found to alleviate the damaging effects of reperfusion injury. Levosimendan has been shown to be a KATP channel opener. These channels have been implicated to play an important role in ischaemic preconditioning (IPC). With this knowledge, the aim of this study was to determine whether levosimendan and IPC have certain cardioprotective mechanisms in common and whether protection with pharmacological preconditioning could be elicited with levosimendan. In this study, we investigated whether: 1) the isolated guinea pig heart could be protected by ischaemic preconditioning (IPC) and postconditioning (IPostC), 2) the heart could be pharmacologically pre- and postconditioned, using levosimendan (LPC & LPostC), 3) a combination of IPC & LPC had an additive protective effect on the heart, 4) the KATP (both mitochondrial and sarcolemmal) channels are involved in this protection and 5) the pro-survival kinases of the RISK (reperfusion injury salvage kinase) pathway are involved. Experimental approach. Isolated perfused guinea pig hearts were subjected to three different IPC protocols (1x5, 2x5 and 3x5 minutes of ischaemia) or levosimendan (0.1μM) preconditioning, before coronary artery occlusion (CAO – firstname.lastname@example.orgºC), followed by 30 minutes of reperfusion. Hearts were also subjected to a combination of IPC & LPC, to establish whether they had additive protective effects. In addition, hearts were pre-treated with levosimendan directly before induction of sustained ischaemia (without washout of the drug – levosimendan pre-treatment (LPT)) for 10min. With the postconditioning protocol, iii the hearts were subjected to 3x30second cycles of ischaemia/reperfusion or levosimendan/vehicle. In a separate series of experiments, hearts were treated with KATP channel blockers (for both sarcolemmal & mitochondrial), before LPC, LPT and LPostC. The endpoints that were measured were: cardiac reperfusion function, myocardial infarct size and RISK pathway expression and phosphorylation (PKB/Akt and extracellular signal-regulated kinase – ERK42/44). Results. IPC, IPostC, LPC & LPostC decreased myocardial infarct size significantly compared with their controls (21.9±2.2%, 21.4±2.2%, 20.6±3.1% and 20.6±1.8% respectively vs. 46.4±1.8% for controls, p<0.05). The combination of IPC & LPC had no additive protective effect. Pre-treating the hearts with levosimendan (without washout), before index ischaemia, proved to be the most effective method of cardioprotection (infarct size: 5.8±0.9% vs. 46.4±1.8% for controls, p<0.001). With LPT a significant increase (p < 0.05 vs. control) in phosphorylation of ER42/44 was also observed. An increase in the activity of one of the RISK pathway kinases, ERK42/44 seems to be one of the reasons for LPT’s efficacy. Treating the hearts with KATP channel blockers before subjecting them to LPC, LPT & LPostC abolished the protective effects induced by levosimendan, suggesting a role for the sarcolemmal and mitochondrial KATP channels in levosimendan-induced cardioprotection. Conclusions and implications. 1) Isolated guinea pig hearts could be pre- and postconditioned within the setting of ischaemia, 2) Hearts could be pharmacologically pre- and postconditioned with levosimendan, 3) levosimendan pre-treatment is the most effective way to reduce infarct size, possibly acting by increasing the phosphorylation of ERK42/44, 4) Myocardial protection was not increased by combining IPC & LPC (suggesting similar mechanisms of protection), 5) LPC, LPT and LPostC were abolished by both sarcolemmal and mitochondrial KATP channel blockers. .LPC and especially LPT, could be useful before elective cardiac surgery while LPostC may be considered after acute coronary artery events.