| Backgroud:Hypoxia is the most common pathophysiological changes of cardiovascular disease, chronic hypoxia occurs not only in high altitude areas, but also in cyanotic congenital heart disease(such as tetralogy of Fallot) and chronic ischemia heart disease. Previous epidemiologic evidence has revealed that people living at high altitude are less prone to chronic ischemic heart disease and have lower mortality risks from heart disease. Several animal experimental studies have demonstrated that myocardial chronic intermittent hypoxia is associated with more tolerance to acute hypoxia/ischemia injury. Furthermore, clinical observation has shown long-term survival with rare occurrences of heart failure in children with cyanotic congenital heart defects. In our experience these children can tolerate the ischemia/hypoxia injury caused by cardiac surgery. These phenomenons above suggest that chronic hypoxia might induce a variety of adaptive changes in the myocardium playing a crucial role in cardioprotection. However, the details of the molecular mechanisms for this protective effect still need to be elucidated.Therefore,further study on the molecular mechanisms of cardioprotection induced by chronic hypoxia may conctribute to provide additional myocardial protective options and improve the treatment of perioperative hypoxia/ischemia injury caused by cardiac surgery for the patients suffering from chronic ischemia heart disease.Hypoxia leads to cellular endoplasmic reticulum(ER) stress and activates unfolded protein response(UPR). ATF6(activating transcription factor 6), a key transcriptional activator involved in maintaining cellular homeostasis, is a 670-amino acid endoplasmic reticulum transmembrane protein that is cleaved in response to ER stress. The cleaved transcriptional factor translocates to the nucleus and activates a series of ER stress response genes. Recent studies have revealed that ATF6α modulates ER function to protect cells against chronic stress, which suggests that ATF6 mediates cytoprotective and pro-survival functions. In previous studies we have demonstrated that the cardiac ER stress responses are activated by long-term hypoxia stress and that ATF6α is continuously activated in primary human cardiac myocytes and rat myoblast H9c2 cells,which shows it play a role of self adaptation and protection in cardiomyocytes subjected to chronic hypoxia. However, its potential protective effect and exact mechanisms need further research.Numerous studies have already demonstrated the importance of the serine/threonine specific protein kinase, Akt(also known as protein kinase B), in cardiomyocyte survival and cardioprotection. Recent studies have also revealed that the expression of Akt was induced in cardiac tissue by chronic hypoxia stress and involved in the mechanisms which protect cardiomyocyte against hypoxia/reoxygenation injury. For a long time, the UPR and the Akt signal pathway have been known to share similar regulatory function and influence cell fate under special conditions, but a functional link between these two pathways has been reported just recently. As for the involvement of Akt in the cardioprotective effects of ATF6 against myocardial ischemia/reperfusion(I/R) injury following chronic hypoxia, no reports have been published yet. Therefore, in order to demonstrate the hypothesis further experimental studies need to be performed.Objective:Our present studies have intended to develope animal models of chronic hypoxia, and investigate the expression of ATF6 in myocardial tissue induced by chronic hypoxia.In vivo and in vitro animal experiments,we have tested whether chronic hypoxia increase myocardial tolerance to acute I/R injury or not. We have also investigated, by using a cultured cardiac myocyte model system, the effect of ATF6 on the mechanism through which chronic hypoxia increases tolerance to myocardial I/R. And the effect of Akt in this process has also been investigated in a cultured cardiac myocyte model system by using siRNA technology.Methods: 1. We set up chronic hypoxia a animal model, and investigated the expression of ATF6 in Myocardial tissue.Health male Sprague–Dawley rats were used as chronic hypoxia Model. Rats were randomly assigned to either the normoxia group or the chronic hypoxia group. Rats in the chronic hypoxia group were housed for 4 weeks in a continuous hypobaric hypoxic chamber(equivalent to an altitude of 5000 m, with a barometric pressure of 404 mm Hg, PO2=84.98 mm Hg,temperature of 25℃). Barometric pressure in the chamber was adjusted weekly(equivalent to an altitude of 3000 m over a period of 1 h) for cage maintenance. Age-matched rats in the Normoxia group were housed under normoxic conditions(78%N2、21%O2) for the duration of the experiments. At the end of the fourth week, the body weight,heart weight,blood oxygen index, Hemoglobin and Hematocrit value of the animals were measured to access the degree of hypoxia. The expression of ATF6 and GRP78/Bip in chronic hypoxic myocardial tissue is investigated by using Western Blot and inmunofluoescence techniques. 2. We studied the effect of chronic hypoxia on myocardial telorance to acute I/R injury.To study the role,we firstly set up a model of myocardial I/R in vivo by ligating the left anterior descending coronary artery,cardiac function was measured by transthoracic echocardiography before ischemia and after 24-h reperfusion,we detected blood myocardial enzyme and by Triphenyl tetrazolium chloride staining, we evaluated the degree of myocardial necrosis. That way, we could prove the effect of chronic hypoxia on the mayocardial telorance to acute I/R injury. Then, we set up isolated rat hearts I/R model according to Langendorff perfusion system. We monitored the changes of left ventricular pressure before ischemia and after reperfusion,assayed lactate dehydrogenase(LDH) activity from effluent.We also evaluated the degree of myocardial necrosis by Triphenyl tetrazolium chloride staining. In this way, we were able to study about how chronic hypoxia increased the mayocardial telorance to acute I/R injury.3. We Investigated the effect and the potential mechanism of ATF6 in which chronic Hypoxia increase myocardial tolerance to I/R.H9c2 myocardial cell lines were used to set up chronic hypoxia cell model. First,H9c2 cells were cultured in an incubator of 13% O2 for 12, 24, 48, 72 hours respectively, With Western Blot techniques, at the above 4 different hours, we assayed the effect of hypoxia on the expression of ATF6,Bip and p-Akt. Then we set up the I/R models. H9c2 cells were randomly divided into Normoxia and mild hypoxia groups, the cells were cultured in incubators where the fraction of oxygen(Fi O2) levels were 21% and 13%, respectively. After 48 hours, that is, when the expression of ATF6 reached the maximum, was performed the subsequent experiments to investigate the protective effect of ATF6 on cells subjected to I/R injury. I/R was simulated by placing cells in ischemia buffer for 1h. Subsequently, cells were reperfused for 3h under normal culture conditions. Cell viability was assessed by Cell Counting Kit-8 assay to know about the living condition of the cells and the activity of LDH released into the culture medium was also assessed to know about the degree of cells necrosis. So we could evaluate the influence of chronic hypoxa on the tolerance of myocardial cells to acute I/R injury. With si RNA technology, H9c2 cells were transfected with siRNA-ATF6 prior to culture,then were cultured under normoxia and hypoxia condition for 48 hours and then simulated I/R on the cells. Cell viability and LDH activity could be assessed to illustrate how ATF6 worked to help chronic hypoxia increase cells tolerance to acute I/R injury. We also assessed the differentces of the expression of Bip and Akt/p-Akt in the cells in order to illustrate the potential mechanism of ATF6 working out that effect.Results:1. There were no differences in the initial body weights of the rats before the experiment. After 4-week of exposure to hypoxia, the final body weight, arterial oxygen tension and oxygen saturation of the chronic bypoxia group were significantly lower than those of normoxia group, while the blood hemoglobin levels, hematocrit values, and the heart weight/body weight ratios were significantly higher.2. In the model of I/R in vivo, pre-ischemic cardiac function was lower in the chronic hypoxia group than that in the normoxia group.After 30 min LAD ligation followed by 24-hour coronary reperfusion, the LV systolic function was reduced in both groups,but the decrease in EF and FS in chronic hypoxia group was less than that in the normoxia group. Notably, both the EF and the FS after I/R in the chronic hypoxia group was higher than that in the normoxia group. CK-MB activity and the infarct size following I/R were significantly lower in the chronic hypoxia group than in the normoxia group, the result indicates less myocardial damage in the chronic hypoxia group and chronic hypoxia preserves rat cardiac function after I/R.3. In Langendorff model, the left ventricular developed pressure(LVDP) of preischemia in the chronic hypoxia group was lower than that in Normoxia group. However, at different time-point during the reperfusion period, greater recovery of LVDP was observed in the chronic hypoxia group.Lower LDH activity in the effluent and Smaller extension of the infarct size of all reperfused hearts at the end of reperfusion were observed in the chronic hypoxia group, The result indicates that chronic hypoxia improves the post-ischemic recovery of myocardial performance in isolated rat hearts.4. ATF6α expression in tissue samples taken from the right ventricle(RV) and left ventricle(LV) of rat hearts exposed to normoxic and chronic hypoxic conditions was performed by western blotting. Myocardial cleaved ATF6α(50 k Da) protein expression was significantly higher in the chronic hypoxia group compared with the normoxia group, but there are no statistical differences between the expression in the LV and RV, immunofluorescent staining revealed a significant increase in ATF6α expression both in the LV and RV induced by chronic hypoxia. BiP expression was also found to be increased in the chronic hypoxia group in both the LV and RV in comparison with the normoxia group. Cleaved ATF6α protein expression was significantly upregulated in cultured H9c2 cells after 24 h of exposure to mild hypoxia, and its expression increase with the extension of exposure to hypoxia. Similar results were also seen with Bi P expression.5. ATF6 protein expression was induced in H9c2 cells after exposure to mild hypoxia, ATF6 expression was blocked by transfected with siRNA-ATF6. H9c2 cells exposed to hypoxia for 48 h(the time determined for high ATF6 expression) were subjected to ischemia for 1 h followed by reperfusion for 3 h, mild hypoxia increased H9c2 cell viability and reduced LDH activity, while the viability of H9c2 cells and LDH activity were opposite when H9c2 cells were transfected with siRNA-ATF6,the result indicates that involvement of ATF6 in the cardioprotection is against myocardial I/R injury through mild hypoxia.6. To elucidate the molecular mechanism by which ATF6 is involved in cardioprotection against I/R injury induced by chronic hypoxic, the Akt signaling pathway was examined. The p-Akt expression was induced in H9c2 cells cultured under chronic mild hypoxia conditions,and the increased expression of p-Akt were consistent with the changes observed in ATF6 and Bip protein levels. Transfection of H9c2 cells with ATF6-si RNA suppressed hypoxia-induced p-Akt expression.The result indicate that ATF6 exerts cardiac protection against I/R injury induced by mild hypoxia via upregulation of p-Akt.3. Conclusion:1. We have successfully set up chronic hypoxic rats model during our present study.We evaluated the hypoxic degree by analyzing the changes of physical parameters,such as body weight,heart weight, blood oxygen index, Hemoglobin and Hematocrit value. The onstructed model is in conformity with pathophysiological changes of cyanotic congenital heart disease, it may be used for experimental study for cyanotic congenital heart disease.2. The expression of ATF6 and Bip in cardiac tissue was upregulated by chronic hypoxia. For H9c2 cells cultured in hypoxia condition, the longer the cells are cultured under hypoxia condition, the more its expression will be.3. In I/R model in vivo, chronic hypoxia preserves rat cardiac function after acute I/R,and in Langendorff model, chronic hypoxia improves the post-ischemic recovery of myocardial performance, The result indicates that chronic hypoxia contributes to increase the myocardial tolerance to acute I/R injury.4. Chronic mild hypoxia increase the H9c2 cells tolerance to acute I/R injury, and ATF6 plays the role of the protection for the cells.5. p-Akt expression was upregulated in H9c2 cells cultured under chronic hypoxia conditions,Transfection of H9c2 cells with ATF6-si RNA suppressed hypoxia-induced p-Akt expression. ATF6 exerts cardiac tolerance to acute I/R injury induced by chronic hypoxia partly via upregulating p-Akt. |
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