The Role Of Hypoxia-inducible Factor-1α In The Cardioprotection Of Ischemic Preconditioning And Ischemic Postconditioning Against Ischemia-reperfusion Injury

Introduction:Insufficient oxygen supply to the heart may cause cardiomyocyte apoptosis as well as necrosis and then lead to cardiac dysfunction.Hypoxia plays a crucial role in the pathophysiological consequences induced by myocardial ischemia.Although early restoration of blood flow to ischemic myocardium is supposed to be the essential measure to prevent cell death and cardiac dysfunction,the reperfusion per se may further aggravate myocardial injury resulting in the so-called ischemia/reperfusion injury.Currently,ischemic preconditioning and the subsequently developed ischemic postconditioning have been considered to be the most potent cardioprotective strategy against ischemia-reperfusion injury.However,the mechanisms responsible for these protections are very complex and have not been yet precisely clarified.Hypoxia-inducible factor-1α(HIF-1α)is a transcriptional activator that regulates gene expression of various stress proteins involving in physiological and pathological adaptive responses to hypoxia.It is reported that HIF-1αmay be the first response in molecular level to ischemia and modulates the expression of several target genes that protect tissues against ischemia-reperfusion injury.Accordingly,HIF-1αmay play a pivotal initiative role in the endogenous protective mechanisms.It has been reported that the expression of HIF-1αincreased significantly during ischemic preconditioning.However,no reports are yet available in the literature as to the status of expression of HIF-1αduring ischemic postconditioning and the relationship between HIF-1αand the cardioprotection of ischemic preconditioning and ischemic postconditioning.Objective:1.To examine whether the expression of HIF-1αis increased during both ischemic preconditioning and ischemic postconditioning.2.To explore the correlation of HIF-1αand the cardioprotection of ischemic preconditioning and ischemic postconditioning.Methods:Forty healthy Wistar rats weighing 220-280 g were randomly divided into the following 4 groups:(1)sham group:the left anterior descending artery(LAD)was only exposed without ligation and the total time course lasts for 225 min;(2)ischemia/reperfusion(I/R)group:ischemia 45min followed by reperfusion 3 h;(3)ischemic preconditioning(IP)group:three cycles of 5 rain occlusion-5 min reperfusion was performed prior to ischemia 45min/reperfusion 3 h;(4)ischemic postconditioning(PC)group:after ischemia 45 min,three cycles of 10 s reperfusion -10 s occlusion was given and then followed by reperfusion 3 h;Myocardium was stained with Even's blue and TTC and then the area at risk was determined.The area of necrosis was observed with image analysator.Serum was collected for detection of the activity of creatine kinase(CK)and the contents of malonaldehyde(MDA).The expression of HIF-1αwas examined by Western blot and Immunohistochemistry.TUNEL and caspase-3 activity assay were used to detect cell apoptosis.Results:1.Establishment of myocardial ischemia- reperfusion injury model.1.1 Measurement of area at risk and infarct sizeThe ratio of area at risk to left ventricle area(AAR/LV)was not significantly different between ischemia/reperfusion group and sham group indicating that these groups are comparable.I/R significantly increased the myocardial infarct size with the ratio of AN/AAR in I/R group being significantly higher than sham group(P＜0.01).1.2 Measurement of CK activity and the contents of MDAThe CK activity and the content of MDA were significantly increased in I/R group as compared with sham group(10.17±2.36 U/ml vs.0.037±0.01U/ml,P＜0.01;0.27±0.03 nmol/ml vs.0.04±0.01 nmol/ml,P＜0.01). 1.3 Detection of cell apoptosisTUNEL detection:Apoptotic index was obviously increased to(17.68±2.44)%in I/R group as compared with sham group(1.45±0.41)%;Caspase-3 activity assay:Caspase-3 activity of I/R group was greatly higher than that of sham group(P＜0.01).2.The cardioprotective role of ischemic preconditioning and ischemic postconditioning2.1 Measurement of area at risk and infarct sizeThe ratio of area at risk to left ventriclar area(AAR/LV):No significant differences were observed among these groups,which indicates that the ischemic area induced by coronary ligation is roughly identical among each experimental group and that these groups are comparable.The ratio of myocardial infarction area to area at risk(the ratio of AN/AAR):The ratio of AN/AAR in I/R group was(44.17±3.24)%.In both IP group(23.76%±7.78%)and PC group (19.79%±2.42%),the infarct size was significantly reduced(P＜0.01)as compared with I/R group.However,no statistical difference was observed between IP group and PC group.2.2 Measurement of creatine kinase(CK)activity and malonaldehyde(MDA)contentThe CK activity and MDA content in IP group(7.84±0.87 U/ml;0.16±0.02 nmol/ml)and PC group(7.67±1.15 U/ml:0.16±0.02 nmol/ml)were significantly attenuated as compared with I/R group(10.17±2.36 U/ml;0.27±0.03 nmol/ml).2.3 Detection of cell apoptosisTUNEL detection:The apoptotic indices in both IP group(13.79%±1.29%)and PC group (11.34%±1.54%)were significantly decreased than that in I/R group(17.68%±2.44%) (P＜0.01).Caspase-3 activity assay:Caspase-3 activity in both IP group(1.53±0.25)and PC group (1.23±0.26)were also significantly decreased compared with I/R group(2.22±0.39)(P＜0.01).3.Expression of HIF-1αproteinI/R induced remarkably the expression of HIF-1αprotein.Both IP and PC could further significantly increase the expression of HIF-1αprotein by Western blot assay(2.13±0.39 and 2.07±0.33 vs.1.70±0.34,P＜0.05)and by Immunohistochemistry(9394.61±757.31 and 9847.20±940.66 vs.8331.64±527.40,P＜0.01).4.Correlative analysisThe results of correlative analysis showed that there were significant negative correlations between infarction size,cell apoptosis,release of MDA,CK and the expression level of HIF- 1α(P＜0.01).Conclusion(1).Both IP and PC could significantly relieve the I/R-induced myocardial injuries as evidenced by diminished infarct size,decreased release of MDA,CK and attenuated cell apoptosis.(2).The expression of HIF-1αin rat cardiomyocyte was significantly increased after myocardial ischemia/reperfusion,indicating that it is an important molecular response of cardiomyocyte to myocardial ischemia.(3).I/R induced remarkably the expression of HIF-1α,while IP and PC could even further increase the expression level of HIF-1α.(4).Correlative analysis showed that there were significant negative correlations between infarct size,release of MDA and CK,cell apoptosis and expression level of HIF-1α, suggesting that HIF-1αis intimately involved in the cardioprotection of both ischemic preconditioning and postconditioning.(5).The initial mechanism of cardioprotection of ischemic preconditioning and ischemic postconditioning might be similar.