Experimental Study On Mechanisms Of Cardioprotection Of The Early Protection Of Myocardial Ischemic Preconditioning

Myocardial Ischemic preconditioning (IP) induced by single or repetitive short periods of ischemia followed by intermittent reperfusion, renders the heart more resistant to a subsequent longer ischemic period. This cardioprotection limits infarct size, reduces the risk of ischemia-reperfusion arrhythmias and improves recovery of ventricular function. This cardioprotective effect has been observed in different species, including rats, rabbits, dogs, pigs and human beings. IP can be classified into two phases: early protection and delayed protection. The early cardioprotection appears soon and lasts shortly. The maximum effect of the delayed preconditioning appears 24 hours later and sustains for 2-3 days. However, litter is known about the cardioprtoection of the early cardioprotection.The possible mechanisms of action should be furtherly studied.Objective : 1.To establish a rat model of myocardial ischemic preconditioning in vivo.2. To investigate mechanism and efficacy of protein phosphorylation and protein tyrosine kinase on myocardial protection in ischemic preconditioning in rat myocardial in vivo. 3. To discuss effects of nitric oxide and nitricoxide synthase during early phase of myocardial ischemic preconditioning in rats. 4. To study the variation and effects of second messenger on ischemic preconditioning of myocardium in rats in vivo. Methods and Results:Part one : Establishment and evaluation the models of ischemic preconditioning of myocardium in ratsMethods:A rat model of myocardial ischemia-reperfusion injury in vivo was used for these studies. Myocardial ischemia was confirmed by S-T segment of lead II elevation of the ECG as well as observation of regional cyanosis over the myocardial surface. Reperfusion was confirmed visually by the return of a red color in the region .After surgical preparation, Rats were randomly allocated to one of the three experimental groups: SHAM group; Ischemic preconditioning group(IP group); Ischemia /reperfusion group(I/R group). At the end of the experiment, the rat hearts were excised. We measured serum myocardial enzymes and the changes of myocardial ultrastructure.Results: CK, CK-MB, LDH activity were significantly high in I/R group(P<0.01). Compared with I/R group, injury of myocardial ultrastructure was reduced significantly by IP and SHAM group.Part two :Mechanism and efficacy of protein phosphorylation and protein tyrosine kinase on myocardial protection in ischemic preconditioning in rat myocardial in vivo Methods: Rats were randomly allocated to one of the four experimental groups: ischemic preconditioning group,ischemia/reperfusion group, SHAM group and preconditioning procedure group. In this group, The rats were further divided into six subgroups. At the end of the expriment, excised the hearts, homogenized and centrifuged to get cytoplasm. The protein content of cytoplasm were determined by Lowry technique. The activity of PTK was checked by biochemistry method.Results: In IP group, the activity of PTK was much higher than in I/R group(P < 0.001).During the procedure of preconditioning before the prolonged ischemia, the activity of PTK varied with ischemia and reperfusion, presented marked fluctuation. It showed repeated significant increase when hearts exposed to three episodes of 5min ischemia, and almost complete reversed by each reperfusion period.Both had significant difference(P<0.01)between the period of ischemia and the same period of reperfusion. Part three:Effects of myocardial ischemic preconditioning on nitric oxide-nitric oxide synthase in ratsMethods: After the procedures, NOS activity and NO contents were assessed with spectrophotometric analysis. eNOS immunohistochemistry was studyed by SABC.Results: NO, NOS in IP group were significantly higher than those in I/R group. NO content and NOS activity were significantly higher in IP group than those in SHAM group.During the procedure of preconditioning before prolonged ischemia, NO varied with ischemia and reperfusion, presented marked fluctuation. Both showed repeated significant increase when hearts exposed to three episodes of 5 minutes ischemia, and almost complete reversed by each reperfusion period. Both had significant difference between the period of 5 minutes ischemia and the same period of reperfusion during the procedure of preconditioning. Immunohistochemistry staining for eNOS were performed in both IP and I/R group. Positive staining was observed in both groups.The content of eNOS was much higher in IP group than in I/R group. The difference was significant (P<0.001).Part four: The variation and effects of second messenger on the early protection of myocardial ischemic preconditioning in ratsMethods: For cytoplasm, the content of cAMP,cGMP were checked by radioimmunoassay.Results: The content of cAMP,cGMP were much higher in IP group than I/R group. During the procedure of preconditioning before prolonged ischemia,the content of cAMP,cGMP varied with ischemia and reperfusion, presented marked fluctuation. Both showed repeated significant increase when hearts exposed to three episodes of 5min ischemia, and almost complete reversed by each reperfusion period. Both had significant diference ( P< 0 .01) between the period of ischemia and the same period of reperfusion.Conclusions:1. we used rats'left major coronary artery occlusion and reperfusion to make the models of myocardial ischemic preconditioning in vivo. 2. We measured serum myocardial enzymogram, showed that myocardial ischemic preconditioning could reduce enzyme leakage and have protection against ischemic damage. Compared with ischemia-reperfusion group, the ultrastructures of myocardial ischemic preconditioning group were improved to different degree.3. Protein tyrosine kinase may take part in mediating the protection of IPC.The cyclic fluctuation of the activity of protein tyrosine kinase may be one of the triggers of IPC.4. NO could be the triggers and mediators of classic IPC. The main reason for the high level of NO after the ischemic stress may be the change of the activity of NOS.5.CAMP,cGMP might potentially play an important cardioprotective role in preconditioning.In the procedure of preconditioning, the content of cAMP ,cGMP have the characteristic of marked fluctuation, which probably elicit cardiac protection.The procedure of preconditioning could maintain the content of cAMP,cGMP at a relatively high level, which showed that both could be mediators of IPC.