| Objective The research is aimed at evaluating different cardioprotective effects caused by ischemia postconditioning (IPC) of different ischemia duration on mouse heart in vivo suffering from ischemia/reperfusion (I/R) injury and the effects of ischemia postconditioning, and further exploring cardioprotective mechanisms induced by reperfusion injury salvage kinase(RISK) signal transduction pathways changes in isolated mouse hearts. The present study focuses in on the following issues: 1) To establish the mouse model of ischemia postconditioning, evaluate different cardioprotective effect caused by postconditioning of different ischemia duration on mouse heart suffering from reperfusion injury, and assess the cardioprotective function and effective factors of IPC. 2) To establish the advanced Langendorff model of mouse heart, investigate the cardioprotective effect caused by IPC on mouse heart from reperfusion injury (I/R) in isolated heart, and explore the cardioprotective mechanisms associated with express of apoptosis gene Bcl-2/Bax. 3) To explore cardioprotective molecular mechanisms in decreasing apoptosis induced by the upstream target enzyme of Bcl-2 and Bax, extracellular signal-regulated kinase 1/2( ERK1/2) of RISK signal transduction pathways.Methods:Part one: The part is about the ischemia reperfusion model of mouse heart in vivo. 1) The ischemia reperfusion model of mouse heart in vivo has been established, in which 120 adult C57BL mice were randomly divided into three different groups according to myocardial ischeamia duration: 30min, 45min and 60min. The mice of each group undergone two different treatments: postconditioning or ischeamia reperfusion, followed 3h reperfusion. Postconditioning groups undergone open-chest surgery to induce left coronary artery occlusion and reperfused for 3 h, after LCA occlusion, several cycles (10 sec reperfusion and 10 sec reocclusion) were applied during the first one minute of reperfusion.Ischemic size and infarction size were measured by Evans blue and TTC staining respectively, assess the level of plasma CK, the level MDA and SOD activity in myocardium,evaluate hemodynamics of different groups. 2) 120 adult C57BL mice were undergone 30min myocardial ischeamia duration in vivo(except for sham group) and randomly were divided into six different treatment and then reperfusion 3h:①I/R control: 30 min ischemia and 3 h reperfusion (I/R);②3IPC, IPC with 3 episodes of 10 s of ischemia and 10 s reperfusion during the first one minute of 3h reperfusion;③6IPC , IPC with six episodes of 10 sec of ischemia and 10 sec reperfusion after 30 min ischemia;④delayed IPC, IPC with 3 episodes of 10 sec of ischemia and 10 sec reperfusion after the first one minute of the 3h reperfusion;⑤IPC+ Preconditioning: 5 min ischemia followed by 10 min of reperfusion before the 30 min of prolonged ischemia, then followed by IPC with 3 episodes;⑥Sham group. Infarct size, the level of plasma CK, the level MDA and SOD activity in myocardium , hemodynamics of hearts were measured.Part two: The second part is about the ischemia reperfusion model of Langendorff perfused mouse heart. The innovative model of mouse heart has been established, 72 adult C57BL mice randomly divided into three different groups: 1) IPC group: IPC with 3 episodes of 10 s of ischemia and 10 s reperfusion after 30min global ischemia and 3 h reperfusion;②I/R group: 30 min global ischemia and 3 h reperfusion;③control group: only received perfusion and no ischemia. The effects of IPC on infarct size,hemodynamics by mini-balloon were measured; the myocardial cell apoptosis was determined with terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) method; reverse transcription polymerase chain reaction(RT-PCR) technique combined with computerized Gel Imaging system was used to detect the expression changes of Bcl-2 and Bax mRNA in mouse myocardium of left ventricle; expressions of apoptosis related proteins, including Bax, Bcl-2 and cytochrome-C (Cyt.C), in cytosolic and membrane fraction were detected by Western Blot.Part three: 96 Langendorff perfused C57BL mouse hearts were divided into 4 groups: 1) I/R control: 30 min global ischemia and 3 h reperfusion; 2) IPC:IPC with 3 episodes (10 sec of ischemia and 10 sec reperfusion) after 30 min ischemia and 3 h reperfusion; 3) IPC+ERK1/2 inhibitor PD98059;4) I/R+ERK1/2 inhibitor PD98059. The effects of IPC on ischemic size, myocardial cell apoptosis index, phosphor-ERK1/2 and phosphor-Akt were measured. Expressions of apoptosis related proteins, including Bcl-2, Bax and Cyt.C, in cytosolic and membrane fraction were detected by Western blot. Results: Four results have been obtained through the study, 1) With 30min occlusion and 45min occlusion infarct sizes of 30minIPC and 45minIPC groups were 19.4±2.0%, 38.3±4.0%,respectively, and were significantly smaller than infarct sizes of 30minI/R and 45minI/R groups (41.4±3.9%,55.4±5.3%, respectively, P<0.05), and decreased by 53.1%, 31.2%,respectively(P<0.05).Meanwhile, the level of plasma CK and the level of MDA in myocardium were decreased, and the activity of SOD was increased, hemodynamics was improved by IPC. But here were no reduction in infarct size in 60minIPC group compared with 60I/R group(P>0.05). 2) After 30min ischemia,IPC with 3 episodes and IPC with 6 episodes equally improved myocardial function, CK release and myocardial infarction size were reduced, the activity of SOD was increased compared with I/R group (all P<0.05) while these parameters were similar between I/R hearts and delayed IPC hearts. The protection of IPC only exist during the first one minute of 3h reperfusion. 3) the Langendorff model of mouse heart was successfully established, infarct size was significantly reduced in postconditioning group (32.8±3. 8)% compared with I/R group hearts [(48.1±4.8)%, P <0.05], and myocardial function was equally improved compared with I/R group (all P<0.05); apoptotic index(AI) was markedly decreased in IPC group (P<0.05); the mRNA expression of Bcl-2 gene was increased (P<0.05); the ratio of Bcl-2 to Bax was incidentally up-regulated (P<0.05); expression of Bcl-2 in membrane fraction was up-regulated in IPC group compared with I/R group; the releasing of mitochondrial Cyt.C into cytosol induced by I/R was significantly reduced by IPC. The histopathological changes in mitochondria and myocardium destroyed by ischemia repefusion were alleviated markedly by ischemic postconditioning. 4) ERK1/2 phosphorylation of myocardial was significantly increased in IPC group. PD98059 inhibited the phosphorylation of ERK1/2 and abolished the most cardioprotective effect induced by IPC, expression of Bcl-2 in membrane fraction was down-regulated, the releasing of mitochondrial Cyt.C into cytosol was increased.Conclusions: The conclusion of the research can be drawn from the following four aspects, 1) Myocardial reperfusion injury is obviously reduced by IPC at onset of reperfusion in mouse model. The cardioprotection afforded by IPC is limited to mild to moderate myocardial injury and the cardioprotection is lost because of severe myocardial injury with longer ischemia duration. 2) The cardioprotection is not magnified in more postconditioning cycles group, only exists in the first one minute of reperfusion, and is not enhanced in the IPC group added with IP. 3) It is available to establish the Langendorff postconditioning model of mouse heart. IPC plays a pivotal role in reducing ischemia/reperfusion injury. IPC at onset of reperfusion obviously can reduce myocardial infarct size and myocardial apoptosis. IPC attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax in mitochondrial, inhibiting mitochondrial Cyt.C release. 4) IPC right before reperfusion attenuates I/R injury of isolated mouse hearts via the upstream target enzyme, ERK1/2 of RISK which mediated the ratio of Bcl-2/Bax in mitochondrial and the release of mitochondrial Cyt.C.
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