Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / 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 – 40min@36.5ºC), 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.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: The reperfusion injury after myocardial ischemia is relevant in the clinical setting, after cardiopulmonary bypass for cardiac surgery, after PTCA and stenting and after cardiopulmonary resuscitation. The components of the reperfusion injury considered in this study were myocardial stunning and reperfusion arrhythmias. Calcium antagonists have been shown to be beneficial in attenuating the myocardial reperfusion injury in the in vitro and in vivo laboratory setting (Lamping, Gross 1985, Przyklenk and Kloner 1988, Taylor 1990, Ehring 1992, Gross and Piper 1992). However systemic administration of a dose of calcium antagonist, large enough to attenuate the myocardial reperfusion injury in the clinical setting, would inevitably lead to unwanted systemic side effects of the drug. The aim of this study was to investigate the hypothesis that an adequate dose of verapamil administered timeously, directly into the ischemic myocardium, would attenuate the reperfusion injury, either when administered from the onset of ischemia, or from 3 minutes before reperfusion. The anesthetized open chest porcine model of myocardial ischemia (15 min total LAD occlusion) and reperfusion was employed in this study. A low dose of verapamil (0.5 mg/8mt or 0.0625mg/mt), a high dose of verapamil (2mg/8m or O.25mg/ml), or vehicle (saline) (8ml) was infused over 8 minutes, directly into the LAD coronary artery supplying the ischemic segment. The infusion was started either at the onset of ischemia, or from 3 minutes before reperfusion. The time taken for the various parameters to return to pre ischemic values was compared between the different groups. The results showed that the high dose of verapamil (2mg) attenuated the reperfusion injury both when administered from the onset of ischemia, and when administered from 3 minutes before reperfusion, compared to either the low dose of verapamil, or the saline infusions. The high dose of verapamil groups had a faster recovery of both systolic contractile function and diastolic function and a lower incidence of ventricular fibrillation on reperfusion. There were no systemic effects of verapamil infusion in any of the groups. The clinical setting of cardiac surgery expressly lends itself to the clinical application of this finding. There is direct access to the coronary arteries both before ischemia and before reperfusion. A small dose of calcium channel blocking drug, with no systemic effect can be administered into the aortic root at the onset of ischemia, just prior to cardioplegia (when the heart is still warm), and after rewarming a few minutes prior to removal of the aortic cross clamp. / AFRIKAANSE OPSOMMING: Die reperfusie besering na miokardiale isgemie is klinies relevant na kardiopulmonêre omleiding vir hart chirurgie, na kardiologiese PTKA en stut prosedures en na kardiopulmonale ressussitasie. Die komponente van die reperfusie besering wat in hierdie studie oorweeg is, is miokardiale tydelike omkeerbare onderdrukking (stunning) en reperfusie arritmieë. Kalsium antagoniste is gewys om effektief te wees in beperking van die reperfusie besering in beide in vitro en in vivo laboratorium eksperimente (Lamping, Gross 1985, Przyklenk en Kloner 1988, Taylor 1990, Ehring 1992, Gross en Piper 1992). Sistemiese toediening van 'n dosis kalsium kanaal blokker, voldoende om die miokardiale reperfusie besering in die pasiënt te beperk, lei egter tot ongewenste sistemiese newe effekte van die middel. Die doel van die studie was om die hipotese te ondersoek dat 'n voldoende dosis verapamil, wat betyds direk toegedien is aan die isgemiese miokardium, die reperfusie besering sal beperk, ongeag of dit toegedien is vanaf die begin van isgemie, of van 3 minute voor reperfusie. Die vark model van miokardiale isgemie en reperfusie is aangewend in die studie. Die varke was tydens die eksperiment onder narkose, met die borskas oop, en 15 minute totale LAD okklusie is toegepas. 'n Lae dosis verapamil (0.5mg/8ml of 0.0625 mg/mt), of hoë dosis veraparnil (2mg/8mt of 0.25mg/mt), of saline (8mt) is oor 8 minute toegedien direk in die LAD arterie wat die isgemiese segment voorsien. Die infuus is begin direk na die aanvang van isgemie, of 3 minute voor die aanvang van reperfusie. Die tyd geneem vir terugvoer van parameters na pre isgemiese waardes is tussen die groepe vergelyk. Die resultate toon dat die hoë dosis veraparnil die reperfusie besering beperk in vergelyking met die lae dosis veraparnil of saline infusies, ongeag of dit van die begin van isgemie, of van 3 minute voor reperfusie toegedien word. Die groepe wat die hoë dosis veraparnil ontvang het, het vinniger herstel van sistoliese en diastoliese funksie getoon en het'n laer insidensie van reperfusie disritmieë, gewys. Geen sistemiese effekte van veraparnail infuus is waargeneem nie. Die kliniese toepassing van hierdie bevinding is by uitstek geskik vir toepassing tydens kardiopulmonale omleiding by kardiale chirurgie. Daar is direkte toegang tot koronêre arteries voor isgemie en voor reperfusie. 'n Klein dosis kalsium antagonis, met weglaatbare sistemiese effekte, kan toegedien word in die aorta wortel met die aanvang van isgemie, net voor kardioplegie toediening (hart steeds warm), en na verwarming, 'n paar minute voor verwydering van die aorta kruis klem.