Objective To evaluate the relationship between calmodulin protein kinase Ⅱ (CaMK Ⅱ) and levosimendan against arrhythmias induced by myocardial ischemia-reperfusion (I/R) in rats in vitro.Methods Thirty male Wistar rats,weighing 250-300 g,were randomly divided into 3 groups (n =10 each) using a random number table:control group (group C),I/R group and levosimendan group (group L).Their hearts were rapidly excised and perfused in a langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 36.5-37.5 ℃.At 20 min of equilibration,the hearts were perfused with K-H solution for 60 min in group C.The hearts were subjected to 30 min of ischemia followed by 30 min reperfusion with K-H solution in group I/R.The hearts were subjected to 30 min of ischemia followed by 30 min reperfusion with K-H solution containing 300 nmol/L levosimendan in group L.Left ventricle developed pressure (LVDP),left ventricle end-diastolic pressure (LVEDP),+ dP/dt-dP/dtmax and heart rate (HR) were recorded immediately before ischemia and at 15 and 30 min of reperfusion.Arrhythmia was recorded during reperfusion and scored.Specimens were obtained from the apex of heart at 30 min of reperfusion for determination of the intracellular calcium concentration ([Ca2 +] i).Myocardial specimens were obtained from the left ventricle at 30 min of reperfusion to detect CaMK Ⅱ activity.Results Compared with group C,arrhythmia score,[Ca2+]i and CaMK [Ⅱ activity were significantly increased,and LVDP,+ dP/dtmax,-dP/dtmax and HR were decreased,and LVEDP was increased at 15 and 30 min of reperfusion in group I/R.Compared with group I/R,the number of ventricular premature beat,arrhythmia score,[Ca2+] i and CaMK Ⅱ activity were significantly decreased,and LVDP,+ dP/dtmax,-dP/dtmax and HR were increased,and LVEDP was decreased at 15 and 30 min of reperfusion in group L.Conclusion Inhibition of CaMK Ⅱ activity is involved in the mechanism by which levosimendan decreases the development of arrhythmias induced by myocardial I/R in rats.

Objective To evaluate the effects of sevoflurane preconditioning on wnt/glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signaling pathway during myocardial ischemia-reperfusion (I/R) injury in rats in vitro.Methods Ault male Wistar rats,weighing 220-280 g,were heparinized and anesthetized with intraperitoneal 3％ pentobarbital 30 mg/kg.Their hearts were rapidly excised and perfused in a langendorff apparatus with oxygenated (95％ O2-5％ CO2) K-H solution at 37 ℃.After 15 min of equilibration,36 isolated hearts were randomly divided into 3 groups (n=12 each) using a random number table:sham operation group (group S),group I/R and sevoflurane preconditioning group (group SP).After 30 min of equilibration,the hearts were continuously perfused for 150 min in group S.The isolated hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion.In SP group,the hearts were perfused for 15 min with K-H solution containing 2.4％ sevoflurane,followed by 5 min washout before reperfusion.At the end of equilibration and 30 min of reperfusion,HR,left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP) and ± dp/dtmax were recorded.The severity of arrhythmias was assessed during reperfusion.At 60 min of reperfusion,3 hearts in each group were chosen for measurement of expression of wnt3a,phosphor-GSK-3β (p-GSK-3β) and β-catenin (by Western blot).At 120 min of reperfusion,6 hearts in each group were chosen for determination of myocardial infarct size by TTC staining.Results Compared with group S,HR,LVDP,+dp/dtmax and -dp/dtmax were significantly decreased,and LVEDP was increased at 30 min of reperfusion,arrhythmia scores and the percentage of myocardial infarct size were increased,and the expression of wnt3a,p-GSK-3β and β-catenin was down-regulated in I/R group.Compared with group I/R,HR,LVDP,+dp/dtmax and-dp/dtmax were significantly increased,and LVEDP was decreased at 30 min of reperfusion,arrhythmia scores and the percentage of myocardial infarct size were decreased,and the expression of wnt3a,p-GSK-3β and β-catenin was up-regulated in group SP.Conclusion Sevoflurane preconditioning attenuates myocardial I/R injury by activating wnt/GSK-3β/β-catenin signaling pathway in isolated rat hearts.

Objective To evaluate the effects of sevoflurane preconditioning on zonula occludens-1 (ZO-1) during myocardial ischemia-reperfusion (I/R) in rats in vitro.Methods Adult male Wistar rats,weighing 250-300 g,were anesthetized with intraperitoneal pentobarbital 30 mg/kg and heparinized.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95％ O2-5％ CO2 at 37 ℃.Eighteen isolated rat hearts were randomly assigned into 3 groups (n =6 each) using a random number table:control group (group C),group I/R and sevoflurane preconditioning group (group S).At 10 min of equilibration,the hearts were perfused with K-H solution for 110 min in group C,the hearts were perfused with K-H solution for 20 min,and then subjected to 30 min of ischemia followed by 60 min of reperfusion in group I/R,and the hearts were perfused with K-H solution saturated with 3％ sevoflurane for 15 min followed by 5 min washout,and then subjected to 30 min of ischemia followed by 60 min of reperfusion in group S.At the end of equilibration,immediately before ischemia,and at 15 and 60 min of reperfusion (T1,2),HR,left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP),+ dp/dtmax and-dp/dtmax were recorded.The development of arrhythmias was recorded during reperfusion and scored.At 60 min of reperfusion,myocardial specimens were obtained from the apex of heart for determination of the expression of ZO-1 in myocardial tissues (by Western blot) and for observation of distribution of ZO-1 and connexin43 (Cx43) (by immunofluorescence).Results Compared with group C,HR,LVDP,+ dp/dtmax and-dp/dtmax were significantly decreased and LVEDP was increased at 15 and 60 min of reperfusion,scores of arrhythmia was increased,and ZO-1 expression was down-regulated in I/R group.Compared with group I/R,HR,LVDP,+ dp/dtmax and-dp/dtmax were significantly increased and LVEDP was decreased at 15 and 60 min of reperfusion,arrhythmia was decreased,and ZO-1 expression was up-regulated in group S.ZO-1 and Cx43 were co-localized at the intercalated disk.ZO-1 was redistributed in the lateralization of the membrane and co-localized with Cx43 in group I/R.The incidence of ZO-1 lateralization was significantly decreased in group S.Conclusion The mechanism by which sevoflurane preconditioning decreases reperfusion arrhythmia is related to inhibition of down-regulation of expression and redistribution of ZO-1 and inhibition of redistribution of Cx43 in rats.