Hypoxia-inducible Factor-1α Mediates The Protective Effect Of Ischemic Postconditioning On Reperfused Myocardium In Hypercholesterolemic Rats

BackgroundIschemic heart disease is expected to become the leading worldwide cause of death, and acute myocardial infarction is a major cause of such mortality. Hypercholesterolemia and the corresponding atherosclerosis are the pathological foundation of coronary atherosclerotic heart disease (CAHD) and heart muscle ischemia, and are critical risk factors of ischemic heart disease. We and other researchers have found the myocardium has increased susceptibility to ischemia/reperfusion injury in hypercholesterolemia. For this reason, it is very impotant to know how to reduce the ischemia/reperfusion injury in hypercholesterolemia.In 2003, Zhao et al. reported ischemic postconditioning (PostC) as a novel strategy to harness nature's protection against myocardial I/R injury. PostC is induced by a short series of repetitive cycles of brief reperfusion and re-occlusion of the coronary artery applied immediately at the onset of reperfusion. Since its introduction, PostC has been consistently reproduced in several experimental preparations and different animal species.Therefore, when myocardium underwent ischemia, it would induce serious myocardial injury and lead to contraction dysfunction due to the oxygen disequilibrium between supplement and requirement. A critical component of myocardial ischemia is hypoxia, while hypoxia inducible factor-1 (HIF-1) is the principal transcription factor involved in the regulation of adaptive responses to hypoxia. HIF-1 is a dimeric transcriptional complex composed of HIF-1αand HIF-1βsubunits that has been recognized primarily for its role in the maintenance of oxygen homoeostasis. Although HIF-1βsubunit is constitutively expressed and maintained at constant level regardless of oxygen availability, the expression and function of HIF-1 a are precisely regulated by cellular oxygen concentrations, and hence HIF-1αis the key determinant of HIF-1 activity.Although the effectiveness of the PostC has been verified under normal conditions in every species, there are several concerns regarding its effectiveness under pathological circumstances. Rafal et al have shown that PreC improves the myocardial contractibility and decreases cardiocytes apoptosis in hypercholesterolemic animals, although there are some opposing reports. But very little is known about the effect of PostC on the myocardial injury under hyperlipidemic condition. Martin et al have recently shown that PostC reduces infarct size in hypercholesterolemic animals through activation of A1 and K-ATP channels. But it is very interesting that Iliodromitis et al have found that the infarct size-limiting effect of PostC is lost in rabbits with experimental hyperlipidemia and atherosclerosis.Overall, whether PostC can reduces the myocardial injury by I/R in hyperlipidemic animals, and what is the relationship between HIF-la expression and cardiac dysfunction in hyperlipidemic rats,we are not know.Objective1. To investigate the effects of postconditioning on the myocardial ischemia/reperfusion injury in the hyperlipidemic rats;and to investigate the effects of postconditioning on the expression of HIF-la;2. After upregulation of HIF-la in myocardium with pharmacological agent (DMOG, a prolyl hydroxylase inhibitor), to investigate the effects of it on cardioprotection against ischemia/reperfusion-injury by postconditioning in the hyperlipidemic rats;3. After silence HIF-la gene by siRNA method in the cultured hyperlipidemic cardiocyte in vitro,to investigate the effects of postconditioning on the insult of cardiocytes.Material and Methods1. In vivo experiment(1) Male Wistar rats (4-5 weeks) weighing 110±10g randomly assigned to one of five groups, provided by Shanxi Medical University Laboratory Animal Center:1) Sham group(n=10):rats were given a normal diet for 1 Oweeks and underwent the same surgical procedures except that the suture passed under the LAD was not tightened for 210 min; 2)HC+Sham group(n=10):rats were given a HC diet for 10weeks and underwent the same surgical procedures except that the suture passed under the LAD was not tightened for 210 min; 3) HC+I/R group (n=10):rats were given a HC diet for 10weeks and subjected to 30 min of LAD occlusion followed by 180 min of reperfusion; 4) HC+PostC group (n=10):rats were given a HC diet for 10weeks and at the onset of reperfusion,3 cycles of 10 s reperfusion and 10 s LAD re-occlusion preceded 180 min of reperfusion; 5) HC+DMOG+PostC (n=10):rats were given a HC diet for 10weeks,24 hours before I/R treatment, rats were randomized to receive 40mg/kg DMOG and then these rats underwent the same treatment as PostC group.(2) Determination of area at risk (AAR) and infarct size using TTC staining, the AAR was expressed as a percentage of the left ventricular area (AAR/LV), and the An as a percentage of the AAR (An/AAR, infarct size);(3) Determination of plasma creatine kinase (CK) activity to reflect the degree of myocardial injury;(4) Myocardial apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining and Caspase-3 activity assay;(5) The protein expression level of HIF-1αwas determined by Western-blot;(6) The gene expression level of HIF-la was determined by real time-PCR.2. In vitro experiment(1) The groups of H9c2(2-1) cell:1) Sham:Cells were given a normal diet for 48h and cultured in atmosphere of 5%CO2,95%air for 5h; 2) HC+Sham:Cells were given a HC diet for 48h and cultured in atmosphere of 5%CO2,95%air for 5h; 3) HC+H/R:Cells were given a HC diet for 48h and hypoxia with 95%N2 and 5%CO2 for 3 h, then followed by 2h reoxygenation with 5%CO2 and 95%air; 4) HC+PC:Cells were given a HC diet for 48h and hypoxia for 3h, then received 3 cycles of 5min reoxygenation and 5min hypoxia preceded the 1.5 h reoxygenation; 5) HC+DMOG+PC:Cells were given a HC diet for 48h and before hypoxia, 0.5mM DMOG was added into the medium respectively, then followed by similar postconditioning treatment; 6) HC+RNAi+PC:Cells were given a HC diet for 48h and Silence HIF-1αgene using RNAi method followed before hypoxia,0.5mM DMOG was added into the medium respectively, then followed by similar postconditioning treatment.(2) Detected H9c2 (2-1) cell proliferation by Cell counting Kit-8, Correlation analysis of LDH leakage, caspase-3 activity(3) The protein expression level of HIF-1αwas determined by Western-blot;(4) The gene expression level of HIF-la was determined by real time-PCR.Results:1. The levels of plasma lipidThere were no significant differences in plasma lipid concentrations among the groups at the beginning of the 10-week feeding period. After the 8-week feeding period, plasma total cholesterol (TC), total triglyceride (TG) and low density lipoprotein (LDL) levels were markedly increased in high fat diet-fed rats than in normally fed rats.Hyperlipidemia enhanced the susceptibility of myocardium to ischemia/reperfusion injury.1.1 Hyperlipidemia further increased the myocardial infarct size induced by I/R.The AAR was comparable in all groups, averaging between 29%and 40%, indicating that a comparable degree of ischemia was induced in these groups. Infarct size in the Control group was greatly increased than that in the Sham group both in normal diet group and in high fat diet group (33.38±1.4%vs.3.05±0.99%in normolipidemia and 39.54±1.16%vs.4.98±0.83%in hyperlipidemia. Hyperlipidemia itself increased the extension of myocardial infarct size (4.98±0.83%vs.3.05±0.99%), but there was no significant difference. However, hyperlipidemia amplified the increase in infarct size induced by I/R than control normolipidemia (P＜0.05)1.2 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＜0.05). 1.3 Hyperlipidemia reinforced the increase of plasma creatine kinase (CK) activity induced by I/R.I/R increased plasma CK activity, which was a surrogate measure of myocardial morphological injury,in hyperlipidemic group.CK activity in the PostC group was significantly lower than that in the sham group (0.68±0.15 U/ml vs.0.±0.05U/ml, P＜0.05), which was consistent with those observed for infarct size.This suggested that cardiomyocyte membrane may be injured by ischemia/reperfusion leading to the leakage of CK,while PostC appeared to protect the membrane from I/R-induced injury.1.4Postconditioning attenuated myocardial apoptosis induced by I/R.Apoptosis plays a key role in the formation of infarct area. Therefore, in the present study, myocardial apoptosis was first determined by highly sensitive terminal dUTP nick end-labeling (TUNEL) assay. Cardiomyocytes exhibited significant TUNEL positive staining in the Control group. The myocardial apoptosis index in the I/Rgroup was significantly higher than that in the sham group (15.21±2.47%vs.1.96±0.23%, P＜0.05). Postconditioning greatly reduced the apoptosis index relative to the I/Rgroup (9.26±0.94%vs.15.21±2.47%, P<0.05).Caspase-3 has been identified as being a key protein in the final pathway of cell apoptosis. To further verify the I/R-induced myocardial cell apoptosis, we also performed Caspase-3 activity assay. The activity of Caspase-3 significantly increased in the I/R group compared to the sham group (4.02±0.59 vs.1.19±0.20, P＜0.05). Postconditioning reduced remarkably the Caspase-3 activity in comparison to that in the I/R group (2.21±0.24 vs.4.02±0.59, P＜0.05). Coupled with the above-mentioned TUNEL assay, these results indicate that postconditioning is able to attenuate the I/R-induced apoptosis.These results suggested that postconditioning attenuated the myocardial injury induced by I/R in HC rats.2. Effects of postconditioning on HIF-la expression2.1 HIF-la protein levels were further increased by postconditioning.The HIF-la protein level in Sham group was extremely low because of its rapid degradation under normoxic condition. When the heart was subjected to ischemia/reperfusion insult, HIF-la protein level was greatly increased to (2.29.±0.32) as compared to that of Sham group(.1.15±0.31, P＜0.05). Whereas after postconditioning treatment, HIF-la protein level was further significantly increased to 4.07±0.33. This indicates that after a prolonged "index" ischemia, the postconditioning procedure is still able to trigger the expression of HIF-1αprotein. The elevation of HIF-1αprotein levels by postconditioning was comparable to that induced by preconditioning.Consistent with these results, immunohistochemical staining showed enhanced HIF-lαexpression in area at risk myocardium in the Control animals. However, HIF-1αexpression was markedly increased in the PostC group.2.2 No changes in HIF-1 a mRNA level were observed in all groups.To determine whether the change in HIF-la protein expression in both Control and PostC groups is regulated at a transcriptional level, steady-state mRNA level of HIF-1αwas analyzed by a real time-PCR assay. There were no significant changes in HIF-lαmRNA level among all groups.These results suggested that the changes of HIF-la expression by I/R and postconditioning treatment were regulated at its post-translational level but not at gene level. Conclusions1,Postconditioning reduced the myocardial ischemia/reperfusiori injury in hyperlipidemic rats.2,Postconditioning reduced the myocardial ischemia/reperfusion injury in hyperlipidemic rats,which may be related to the upregulation of HIF-1α.