NDRG2 Expression Of Rat Heart In Transient And Long Term Ischemia–reperfusion Injury

PartⅠ: NDRG2 expression of rat heart in long term ischemia–reperfusion injuryBackground and objectivesNDRG2 is a new member of NDRG family and is considered as a stress responsive gene to stresses like hypoxia. Its function is largely unknown but is considered to be involved with the process of apoptosis. Clinically, myocardial I/R is a common stimulus for cardiomyoctye and may induce cardiomyocyte apoptosis. Such responsive process is related with the changes of many proteins and kinases expression and the regulation of many signaling pathways. NDRG2 may be involved in the reactive changes induced by MI/R, but the speficity of the change and the possible mechanism involved were never studied before.In the present study, we examined the change of NDRG2 expression post I/R in rat heart and try to find the regulatory mechanism of NDRG2 expression. In addition, we detected myocardial apoptosis post I/R injury in order to explore the possible relationship between NDRG2 and apoptosis in myocardium. Methods1. Thirty rats weighing 260 to 290g were randomly separated into six groups. One group of sham-operated rats was used as control. The remaining five groups undergone 30 min of ischemia followed by reperfusion for 3, 6, 12, 24 or 48 hours.2. We used immunofluorescence and immunohistochemistry to confirm NDRG2 expression, distribution, and localization in normal heart tissue.3. NDRG2 expression was detected over several time periods mentioned above in myocardium post I/R injury by semi-quantitative RT-PCR, western blot and immuno- histochemistry. We also examined c-Myc expression over the same time periods in myocardium post I/R injury by western blot. In order to detect the state of apoptosis in rat heart post I/R stress, injured myocardium from the various time periods was lysed, and cleaved-caspase-3 were examined using western blot analysis. In addition, TUNEL staining was performed in LV tissue sections of injured areas from rats subjected to I/R stress.4. After this simulated ischemia, the primary cardiomyocytes were subjected to reperfusion for a serial of time period (2, 4, 8, 16, 24, 36, or 54 h).The expression kinetics of c-Myc, NDRG2, and activated-caspase-3 over those time periods was detected using western blot analysisResults1. Immunostaining demonstrated high levels of NDRG2 protein expression in normal rat cardiomyocytes and NDRG2 protein is localized primarily in the cytoplasm, with lower levels in the membrane and nucleus.2. Our results showed that the NDRG2 mRNA level decreased with time during reperfusion following 30 min of ischemia; the lowest level was found at 12 hours, and the level then returned towards normal following 24 hours of reperfusion. Following reperfusion, the expression of NDRG2 protein gradually decreased; the lowest level was found at 24 hours i.e. 12 hours after NDRG2 mRNA had reached its lowest level. Immunohistochemical staining was performed and further confirmed these observations obtained by immunoblot analysis.3. We also found the expression of c-Myc was increased with the induction of I/R and its expression was significantly inversely correlated with NDRG2 expression. This reciprocal relationship between c-Myc and NDRG2 suggests that c-Myc has a regulatory effect on the NDRG2 gene.4. Our data also showed cleaved-caspase3 ,an apoptosis related protein level was highly elevated in the early phase of reperfusion (≤12h ),but was decreased in the later phase of reperfusion (24-48h).5. Following reperfusion, myocardial apoptosis suggested by immunoblot analysis of cleaved-caspase3 was also indicated in TUNEL staining.6. All those results in vivo were confirmed in ex vivo cultured cardiomyocytes subjected to simulated I/R.ConclusionWe demonstrate that NDRG2 is highly expressed in cardiomyocytes and this expression is reduced gradually over time following I/R stress. We also demonstrate a negatively correlated relationship between the expression of NDRG2 and that of c-Myc. Based on cardiomyocytes apoptotic rate alteration observed here, we speculate that c-Myc might modulate the expression of NDRG2 protein in cardiomyocyte subjected to I/R, and this modulation may contributes to the regulation of cardiomyocyte apoptosis post I/R. PartⅡ: NDRG2 expression of rat heart in transient ischemia–reperfusion injury: Insulin induces phosphorylation of NDRG2 through activation of AktBackground and objectivesIn the first phase of our study, we have demonstrated the involvement of NDRG2 in myocardial I/R injury。In previous study, Insulin has been found to be able to induce NDRG2 phorsphorylation by activating Akt and may participate in the pathgenesis of diabetes. And it is well known that Akt plays critical role in insulin induced cardioprotection while its own function is not fully understood. Based on the aforementioned knowledge, we speculate that insulin may induced NDRG2 phorsphorylation by the activation of Akt.The objective of this study was first to characterize the relationship between phosphorylation of NDRG2 and Akt induced by myocardial I/R and to delineate the effect of insulin treatment on NDRG2 phosphorylation and the involvement of Akt in this effect.Methods1. Adult male Sprague-Dawley rats weighing 260 to 290g were randomly seperated into five groups in the first phase of our experiment. One group of sham-operated was used as a control. Four groups undergone a 30min period of ischemia followed by reperfusion for 0, 1, 3, and 6 h. Western blot was used to explore the temporal change of the expression of studied proteins and their phosphorated form in myocardium subjected to ischemia/reperfuison.2. In the second phase of the experiment , three more groups of animals were used and subjected to 30 min of ischemia followed by 3h of reperfusion and received one of the following treatment : 1) vehicle (saline); 2) glucose-insulin potassium (GIK); or 3) glucose-potassium (GK). Blood glucose by a glucose meter and plasma insulin concentrations by a commercial radioimmuno-assay kit were measured at the end of 3 h of reperfusion. Western blot was used to examine the changes of Akt and NDRG2 total protein amount and their phosphorylate level in cardiac tissue of each group。3. Primary myocytes were isolated from neonatal (1-3d) SD rats and cultured for 2-3 days. Then, myocytes were exposed to simulated ischemia/reperfusion (SI/R,2h/4h)and randomly subjected to one of the following treatment: vehicle(salt), insulin, insulin plus the PI3K inhibitor wortmannin, insulin plus the Akt inhibitor HIMO, insulin plus the selective eNOS inhibitor Cav-p. At the end of reperfusion, we collected the samples of cardiomyocytes of each group to examine the changes of phosphorylated Akt and NDRG2 using Western blot。Results1. Total Akt and NDRG2 level was also not changed significantly, nevertheless, the phosphorylated Akt and NDRG2 increased significantly following myocardial ischemia (p<0.05 vs control) and reduced gradually after reperfusion, reaching the lowest level at the end of 3 h of reperfusion (p<0.05 vs I 30 min; p>0.05 vs control).2. Infusion of GK during reperfusion significantly increased blood glucose concentration (p<0.01 vs vehicle), but there was no difference between GIK and vehicle groups in I/R rats after 3 h of reperfusion. Compared with the vehicle group, GIK treatment (p<0.01) increased plasma insulin concentration at the end of 3 h of reperfusion, but GK treatment lead to much less increase with no significant derivation ( p>0.05) 3. Then, It was showed that treatment with insulin resulted in a 2.4-fold increase in Akt phosphorylation as well as a 2.2-fold increase in NDRG2 phosphorylation post 30 min of ischemia and 3 h of reperfusion (P<0.05 vs I/R+V) in rat heart, however, there was no difference in total Akt and NDRG2 among the groups studied.4. Insulin stimulated the phosphorylation of NDRG2 in cardiomyocytes. This effect was inhibited by co-treatment with the PI3K inhibitor wortmannin or p-Akt specific inhibitor HIMO. While cavtratin, a selective eNOS inhibitor, did not affect NDRG2 phosphorylation .ConclusionThis series of experiments show that transient I/R injury can lead to the change of Akt and NDRG2 phosphorylation level in myocardium. Futhermore, insulin administration is able to phosphorylated NDRG2 via Akt in the ischemic/ reperfused heart or in primary cardiomyocytes exposed to SI/R stress.