Methods: Thirty-six male Wistar rats were selected for the study. Rats’ hearts were rapidly excised after application of ketamine. Aorta was cannulated and infusion of 37 °C Krebs-Henseleit Buffer (KHB) was started to provide heart stabilization for 10 minutes. Then, the isolated hearts were randomly assigned to one of six groups: group 1 (normothermia 37 °C-control), group 2 (hypothermia 28 °C-Control), group 3 (normothermia-ischemic preconditioning), group 4 (hypothermia-ischemic preconditioning), group 5 (normothermialevosimendan), group 6 (hipothermi-levosimendan). Levosimendan was added to KHB solution (24 μg/kg for 10 minutes loading, and 0.1 μg/kg/ minute for maintenance). Data were analyzed by using Kruskal Wallis and Mann-Whitney U test, and p values of <0.05 and <0.008 were accepted significant after using Bonferroni adjustment.

Results: The tissue malondialdehyde (MDA) levels in group 4 were significantly increased compared to group 6 (median range 1.85 vs. 0.70, respectively; p=0.004). Sodium, potassium adenosine triphosphatase (Na⁺-K⁺ A TPase) e nzyme a ctivity w as s ignificantly p rotected i n group 6 compared to group 4 (303.6 vs. 209.1, respectively; p=0.004). Group 4 revealed extensive TUNEL-positive cardiomyocytes compared to group 6. Percentage of apoptotic cell staining was 37.5% and 10%, respectively (p=0.004). Electron microscopic evaluation of group 6 showed normal morphological composition characterized by regular arrangement of myofibrils and protected mitochondrial structure and sarcomer in comparison with other groups.

Conclusion: Results of this study showed that separate use of levosimendan and hypothermia provided protective effects on ischemic myocardium. However, pretreatment with levosimendan provided better protection under the moderate hypothermic condition.