| Objective A great deal of accumulated evidence implicate opening of mitochondrial ATP-sensitive potassium (MitoKATP) channel as an important step in the anti-infarct effect of ischemic preconditioning, it is the end-effector of preconditioning's cardioprotection. Recent studies, however, reveal that MitoKATP opening prior to ischemia can actually serve as a trigger of preconditioning's signal transduction. The purpose of this study was to investigate the mechanisms of cardioprotection about ROS producing and PKC epsiolon translocation and functional proteomics in myocytes preconditioning by MitoKATP channels opener.Methods cultured adult rat ventricular myocytes and H9C2 myocytes were used as experimental ischemia/reperfusion model and hypoxia/reoxygen model. The study was carried out in Three parts: In the first part, cultured adult rat ventricular cardiomyocytes were randomly divided into five groups, including control group, ischemia/reperfusion group, 200(mol/L diazoxide (DZ) preconditioning group, 200(mol/L DZ and 400(mol/L N-(2-mercaptopropionyl) glycine (MPG) , 200(mol/L DZ and 2(mol/L chelerythrine chloride (CH) preconditioning group.After preconditioning, cardiomyocytes were treated with simulated ischemia for 40min and reperfusion for 30 min. The cell viability, creatine kinase released from cell, activity of Na+-K+ATPase and cytochrome C released from mitochondria were respectively measured by MTT methold, CK test kit, Na+-K+ATPase test kit and western blot. The PKC epsilon translocation in cells inducing by preconditioning was assayed by immunofluorescence and western blot. In the second part, cultured H9C2 myocytes were randomly divided into five groups, including control group, hypoxia/reoxygen group, 200(mol/L DZ preconditioning group, 200(mol/L DZ and 400(mol/L MPG, 200(mol/L DZ and 2(mol/L CH preconditioning group. After preconditioning, cardiomyocytes were treated with hypoxia for 100min and reoxygen for 30 min. The cell viability and mitochondrial membrane potential were assayed by labeling cells with both propidium iodide (PI) and Hoechst33258 or JC-1.In the third part, cultured H9C2 myocytes were randomly divided into two groups, the control group and 200(mol/L DZ preconditioning group. The phosphoproteins were enriched from H9C2 myocytes in either control group or 200(mol/L DZ preconditioning group, and isolated by two-dimensional electrophoresis. Finally, the differentially expressed phosphoproteins were identified by MALDI-TOF-MS.Results1. The cell viability in all groups treated with simulated ischemia/reperfusion was decreased significantly (p<0.01); Preconditioning by DZ markedly increased the number of live cells (p<0.01), but this effect of DZ preconditioning could be suppressed by MPG or CH (p<0.01). 2. Creatine kinase released from cardiomyocytes increased significantly in all groups treated with simulated ischemia/reperfusion, but the level of CK was much lower in DZ preconditioning group than in the others (p<0.01), and was much higher in DZ and MPG preconditioning group than in DZ and CH preconditioning group (p<0.01).3. The activity of Na+-K+ATPase in cardiomyocytes was inhibited significantly in all groups treated with simulated ischemia/reperfusion (p<0.01), but it was kept much higher in DZ preconditioning group than in the others (p<0.01).4. Lots of cytochrome C released from mitochondria after undergoing simulated ischemia/reperfusion (p<0.01), DZ preconditioning induced cardiomyocytes to release less amount of cytochrome C to cytosol than DZ and MPG preconditioning (p<0.01).5. Under a fluorescence microscope,the fluorescence of PKC epsilon was located on myofibrillar-like structures, but there were a few green fluorescence spot in cells in control group. The myofibrillar-like fluorescence is very faint in DZ+MPG preconditioning group and disappeared in DZ+CH preconditioning group. The result of western blot showed that PKC epsilon translocated to particulate fraction more significantly in DZ preconditioning group (p<0.01) than that in both DZ+MPG precon... |
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