The Effects Of PKC-HSP90 Signal Pathway On Mitochondrial Connexin 43 In Myocardial Ischemic Preconditioning In Rats

Objective:Myocardial ischemia/reperfusion injury and ischemic preconditioning model in rats were prepared.The structure and damage of cardiomyocyte mitochondria were observed.The changes of mitochondrial connexin 43 of myocardium were detected by using protein kinase C and heat shock protein90 specific inhibitor , to investigate the possible regulation mechanism of mitochondrial Cx43 in PKC-HSP90 signal pathway of ischemic preconditioning.Methods:A rat model of myocardial ischemia reperfusion injury was established. 90 healthy SD rats were randomly divided into 6 groups: sham operation group(sham group, n=10), ischemia reperfusion group(IR group,n=16), ischemic preconditioning group(IP group, n=16), chelerythrine treatment+ischemic preconditioning group(CH+IP group, n=16), geldenamycin treatment+ischemic preconditioning group(GA+IP group, n=16),and chelerythrine treatment + geldenamycin treatment+ischemic preconditioning group(CH+GA+IP group, n=16). Myocardial ischemia was induced for 30min followed by reperfusion for 60min(R60min)or 120min(R_120min). Cellular morph was analyzed by light microscope, CK-MB and LDH were assayed by automatic biochemistry analyzer.The degree of damage in mitochondria and the ultrastructure of myocardium were observed semiquantitatively by transmission electron microscope.The integrity and purity of the mitochondrial extraction were tested by transmission electron microscope.The expression of total Cx43 protein was detected by Western Blot.Results: 1.The double membrane of mitochondria wich were isolated from rat heats by using differential centrifugation percoll density gradient centrifugation was clear and integrity. 2. As compared with Sham group,the CK-MB and LDH in IR_120min group were significantly increased ( P<0.05); The CK-MB and LDH in IP_120min group were significantly lower than in IR120min group( P<0.05); The CK-MB and LDH in CH+IP_120min group,GA+IP_120min group and CH+GA+IP_120min group were significantly higher as compared with IP_120min group(P<0.05);But as compared with IR120min group there was no statistical difference between them in CK-MB and LDH; There was no significant difference in CK-MB and LDH in CH+IP_120min group,GA+IP_120min group and CH+GA+IP_120min group. 3. It was demonstrated that the ultrastructure of rat myocardium in Sham group was normal with the damageable degree and semiquantitative analyses of myocardium mitochondria. It was main tendency for the injury of rat myocardium mitochondria in IP_120min group rang from 1 to 2 grade with mitochondrial average 1.42±0.19. It was main tendency for the injury of rat myocardium mitochondria in IR120min group,CH+IP_120min group,GA+IP_120min group,CH+GA+IP_120min group rang from 2 to 3 grade with some mastocyte and apoptosis with higher score than that in IP_120min group (respectively, 2.63±0.13, 2.29±0.23 , 2.33±0.18 versus 2.59±0.48, F=36.83,P<0.01). Compared with IR120min group,the damage of cardiomyocyte mitochondria in IP_120min group was significantly lower(P<0.01) with fewer apoptosis. The damage of cardiomyocyte mitochondria in CH+IP_120min group and GA+IP_120min group were significantly lower than that in IR120min( P<0.01),but were significantly higher than that in IP_120min group( P<0.01);There was no significant difference between CH+GA+IP_120min group and IR120min group(P= 0.72). There was also no significantly difference between CH+IP_120min group and GA+IP_120min group(P= 0.07). As compared with CH + IP_120min group and GA + IP_120min group, the damage of cardiomyocyte mitochondria in CH+GA+IP_120min group was significantly higher(P<0.05). 4. Compared with Sham group, mitochondrial TCx43 protein of ischemic area in R60min and R120min in IR group decreased significantly(P<0.05), and with a decrease tendency as reperfusion. Mitochondrial TCx43 protein of ischemic area in R120min was lower significantly than that in R60min (P<0.05); Compared with the IR group,the mitochondrial expression of TCx43 in R60min and R120min in IP group was increased gradually(P<0.05);Compared with IP group,the expression of mitochondrial Cx43 decreased gradually with reperfusion in CH+IP group, GA+IP group and CH+GA+IP group(P<0.05), and with a descendent tendency with time;Compared with the GA+IP group and CH+IP group ,mitochondrial Cx43 in CH+GA+IP group decreased gradually(P<0.05).Conclusion: 1.The mitochondria were sussessfully extracted from rats heart by using differential centrifugation and percoll density gradient centrifugation. 2.The score of impared mitochondria in ischemic reperfusion injury group was significantly higher than that in ischemic preconditioning group. Then, the score of impared mitochondria in protein kinase C and heat shock protein90 specific inhibitor treatment group or protein kinase C combining heat shock protein90 group was significantly higher than that in ischemic preconditioning group. 3. Ischemic preconditioning could increase mitochondrial TCx43 protein and PCx43 protein .Howere,with protein kinase C and heat shock protein90 specific inhibitor treatment respectively or symphysially the expression of mitochondrial TCx43 protein and PCx43 protein were significantly decreased.The present study suggest that isolation of mitochondria from heart in rats by using differential centrifugation and percoll density gradient centrifugation is practically.Thus,mitochondrial Cx43 plays a role in the protection by ischemic preconditioning in rat and the mechanism may involves the PKC-HSP90 signal pathway.