The Effects Of Ischemic Preconditioning On The Expression Of Hypoxia Inducible Factor-1α Gene In Ischemic Myocardium

With people's life style and diet habit changing, the incidence of coronary artery disease (CAD) has increased tremendously recently, which is a main threat to human's health. As the most severe subgroup of CAD, acute myocardial infarction (AMI) is featured as sudden happening and serious prognosis in 2020. It is estimated that AMI will increase from the fifth place to the first in all kinds of death causes in the world. Ischemic preconditioning (IP) work through increasing the endogenous ability against ischemic (reperfusion) injury to reduce infarct size and other presentations of postischemic injury. Studies find IP could protect ischemic injure (or reperfusion) of remote organic too. It is considered that IP is the most potent endogenous protective mechanism observed among known strategies. Through the extensive research on IP, people have found its protective effects not only on the organ itself, but also on others in human and mammal. IP is a complex pathophysiologic process that involves a network of intricate regulatory mechanisms at the levels of cell signaling and gene expression. Hypoxia inducible factor-1α(HIF-1α) is a key physiologic regulator under hypoxic and ischemic conditions. It is reported that HIF-1αinduces gene expressions of many protective proteins that are involved in the protective function of IP. The effects of ischemic preconditioning on the expression of hypoxia inducible factor-1αgene in ischemic myocardium are unknown at present. Therefore the study of cardioprotection on IP and HIF-1αmRNA regulation in ischemic myocardium is important meaning to investigate the role and mechanism of HIF-1α.Objective: To investigate the role of HIF-1αin cardioprotection of ischemic preconditioning on early ischemic myocardium by observing changes of myocardial infarct size and the expression of HIF-1αmRNA in ischemic myocardium.Methods: Healthy male Sprague-Dawley rats (270～350) g were randomly divided into four groups:①Normal control (NC) group.②Acute myocardial ischemia (AMI) group: AMI model was established by ligating left anterior descending coronary artery (LAD) of rats.③Renal ischemic precondition group (RIP): after the rats were underwent three cycles of 5 minutes occlusion and 5 minutes reperfusion of renal arteries, LAD was occluded as previously described.④Limb ischemic precondition group (LIP): after the limb arteries was occluded 5 minutes and released 5 minutes four cycles, LAD was occluded as previously described. Rats were executed and took myocardium tissue at 1 hour, 2 hours, 4 hours, 6 hours and 12 hours after ligating LAD except NC group.The changes of epicardial electrography were recorded during the course of the surgery and it showed that LAD was ligated successfully, the ischemic myocardial tissues were harvested at the above-mentioned time points. Myocardial infarct size of 12 hours experimental rats was examined with Evans and triphenyl tetrazolium chloride (TTC). The pathological changes in myocardium were observed. Using reverse transcription polymerase chain reaction (RT- PCR), the changes of HIF-1αmRNA level in ischemic myocardium were measured. All data are presented as mean±S.D. The statistical significance of the change in different models were analysed by SPSS 13.0 statistics software. A value of P<0.05 was accepted as statistically significant.Results:1. Animal model: The color of the myocardial tissues turned to dark. The epicardial electrographic ST segment was elevated significantly during ischemia. The morphology of the myocardial tissues under the suture appeared denaturalization and putrescence. The myocardial tissues were distinguished to three parts that the blue area was normal zone, the red area was ischemic zone and the pale area was infracted zone. Those showed that the AMI model had been established successfully. The color of kidney and limb turned to dark after the renal and limb arteries were occluded. It turned to red after the renal and limb arteries were reperfused. Those showed IP model had been established successfully.2. Infarct size: The myocardial infarct size in RIP and LIP groups were (46.18±6.15)% and (46.92±6.69)% respectively. Both of them were significantly reduced than AMI group [(66.44±19.24)%, (P<0.05)]. It was insignificant difference between RIP and LIP.3. Pathological changes: It was showed from the morphology by the light microscope that cardiac cells arranged orderly, cellular boundary remained complete and cardiac line was clear in NC group. Myocardial cell swelled and little of neutrophilic leukocytes were infiltrated in myocardium at 1 hour after ischemia. Myocardial destruction with focal infiltration of lymphocytes and polymorphonuclear leukocytes was observed in myocardium at 6 hours after ischemia. Infiltration of inflammatory cells was more diffuse and the infarct size extended at 12 hours after ischemia. The degree of the myocardial injury in RPC and LIP groups was lighter than that in AMI group.4. Expression of HIF-1αmRNA:①Extraction of total RNA: There was 28s and 18s distinct straps in agarose gel electrophoresis (AGE), and the ratio of 28s and 18s was about 2:1, and there was insignificant degradation.②The gene expression of HIF-1αwas detected in myocardium of NC (0.65±0.10) and was increased obviously in myocardium at 1 hour (0.75±0.06, vs NC P<0.05) and peaked at 2 hours (0.89±0.03, vs NC P<0.01), remained a high expression at 4 hours (0.74±0.04, vs NC P<0.05) and declined at 6 hours (0.64±0.04) after surgery. The gene expression of HIF-1αwere decreased began to decrease obviously in ischemic myocardium at 1 hour (0.47±0.04, vs AMI 1 hour P<0.05), 2 hours (0.39±0.09, vs AMI 2 hours P<0.01) and decreased the lowest level at 4 hours (0.28±0.02, vs AMI 4 hours P<0.05) and restored to normal level at 6 hours (0.55±0.12, vs AMI 6 hours P<0.05) and 12 hours (0.58±0.03) in RIP. The gene expression of HIF-1αwere decreased began to decrease obviously in ischemic myocardium at 1 hour (0.42±0.05, vs AMI 1 hour P<0.05), 2 hours (0.35±0.05, vs AMI 2 hours P<0.01) and decreased the lowest level at 4 hours (0.28±0.05, vs AMI 4 hours P<0.05) and restored to normal level at 6 hours (0.60±0.02) and 12 hours (0.63±0.03) in LIP. It was insignificant difference between RIP and LIP.Conclusions:1. The upregulation of HIF- 1αgene expression were prior to morphology of myocardial necrosis. It showed that HIF-1αcan be used as a sensitive marker for early acute myocardial ischemia.2. Remote ischemic protectioning could alleviate the myocardium injure after the decrease of HIF-1αgene expression might be one of the molecular mechanism of cardioprotection.