心肌梗死后心肌细胞内向整流钾通道重构以及缬沙坦干预机制的研究

发布时间：2018-06-03 03:31

  本文选题：心肌梗死 + Kir2.1　； 参考：《山东大学》2016年博士论文

【摘要】：研究背景尽管当代社会医疗水平不断进步,人们对健康生活方式的认识逐步加深,但心肌梗死(myocardial infarcion, MI)仍是临床上最常见的心血管疾病之一,是我国人民健康面临的重大威胁。我国每年约有54万人死于心脏性猝死,近九成猝死原因是急性MI后发生的恶性心律失常。以往的研究发现室性心律失常及心源性猝死与钾离子通道的重构有关。尤其是内向整流钾通道(the inward rectifier potassium channel, Kir),它是维持正常的动作电位的主要成分,维持静息膜电位,参与动作电位早期和终末复极化。在心肌细胞中最丰富的内向整流钾通道是IK1通道,心脏型为Kir2.1蛋白,被KCNJ2基因编码。MI后心室肌细胞电重构的主要特点之一是IK1电流密度的下降,导致室性心律失常的发生或易感性增加。尽管目前临床上已有多种抗心律失常药物的应用,但这些成熟的抗心律失常药物同时存在致心律失常作用,尤其是在QT间期延长者中应用。而MI后患者QT间期延长是提示恶性室性心律失常甚至猝死发生的重要指标。因此目前已知的抗心律失常药物在MI患者中的应用颇受局限。临床MI患者多数伴有高血压病等危险因素,而肾素—血管紧张素—醛固酮系统(Renin-angiotensin-aldosterone system, RAAS)拮抗剂可以显著降低恶性心律失常的发生率,但具体机制尚未明确。因此,如能明确RAAS拮抗剂发挥抗心律失常作用的靶点,探明其作用机制,将对发现新的抗心律失常药物、改善MI预后具有重要意义。第一部分 大鼠也肌梗死后内向整流钾通道重构以及缬沙坦的干预作用目的明确急性MI后大鼠梗死灶周和非梗死左心室游离壁区心肌细胞中内向整流钾通道(Kir2.1蛋白)的表达水平,以及缬沙坦干预对该通道的效果。并且评价药物干预后大鼠心率、血压、室性心律失常易感性及血流动力学指标。方法通过永久性结扎大鼠左冠状动脉前降支建立急性MI模型,评价造模情况。造模成功后随机分为假手术组(Control组)、假手术组+缬沙坦组(Control+ Valsartan组)、心肌梗死组(MI组)、心肌梗死+缬沙坦组(MI+Valsartan组)。利用小动物遥测及尾动脉测压法记录大鼠术后心率、血压和心电图。7天后提取梗死灶周和非梗死左心室游离壁区心肌组织,利用qPCR和Westen blot法检测四组KCNJ2 mRNA水平和Kir2.1蛋白水平。结果冠脉结扎术中心电监测和术后Masson's染色证明造模成功。MI后7天,MI组大鼠心率、血压均较Control组和Control+Valsartan组升高,而MI+Valsartan组有所下降。MI后延长的QTc和QTcd亦在缬沙坦干预后趋于正常。此外缬沙坦有效改善MI后心脏射血分数等血流动力学指标。同时,MI组中KCNJ2 mRNA和Kir2.1蛋白水平较Control组和Control+Valsartan组降低,而MI+Valsartan组中缬沙坦改善了该离子通道的重构。结论大鼠MI后IK1通道表达水平显著降低,缬沙坦干预可逆转这一现象,同时伴随着心律失常易感性降低。而在正常大鼠心肌中,缬沙坦对IKI通道的调控作用并无显著统计学差异。机制一：缬沙坦通过抑制心肌梗死后激活的NF-κB-miR-16信号通路调控内向整流钾电流第二部分 缬沙坦改善心肌梗死后内向整流钾通道重构的机制研究目的MicroRNAs是调控心律失常的重要因素之一。计算机预测microRNA-16(miR-16)可以靶向调控KCNJ2基因。NF-κB可以调控miR-16的启动子。本实验主要验证缬沙坦是否可以通过抑制MI后激活的NF-κB来下调miR-16的表达,且miR-16是否可以靶向调控KCNJ2基因的表达。方法MI大鼠给予缬沙坦或生理盐水灌胃7天,提取梗死灶周心肌组织RNA和蛋白。体外培养H9c2心肌细胞和急性分离的新生大鼠心室肌细胞,转染miR-16或给予血管紧张素II及缬沙坦干预。Western blot法检测NF-κB p65, inhibitor KBa (IκBα)和Kir2.1蛋白水平,qPCR检测KCNJ2 mRNA和miR-16表达水平。全细胞膜片钳记录IK1电流。荧光素酶检测验证KCNJ2是否为miR-16靶基因。CHIP验证NF-κB对miR-16的DNA结合水平。结果梗死灶周miR-16表达水平升高,伴随KCNJ2/Kir2.1水平下降。体外转染miR-16使之过表达,导致KCNJ2/Kir2.1表达下调,伴随着IK1电流密度减低。相反的,抑制miR-16或导致其结合位点突变增强了KCNJ2/Kir2.1的表达。MI大鼠接受缬沙坦干预后,升高的NF-κB p65和miR-16水平下调,而降低的IκBα和Kir2.1蛋白水平升高。体外试验中,血管紧张素II诱导的miR-16表达升高和KCNJ2/Kir2.1表达下降,同样被缬沙坦抑制。而体外经缬沙坦处理的细胞中过表达miR-16,可消除缬沙坦对KCNJ2/Kir2.1的保护作用。体内和体外实验均证明缺氧环境下NF-κB p65表达上调,而IκBα表达下降,缬沙坦干预抑制了这一现象。而抑制NF-κB后,缬沙坦和NF-κB抑制剂对miR-16和KCNJ2/Kir2.1的调节作用相似。CHIP进一步验证缬沙坦抑制NF-κB对miR-16的DNA结合水平。结论MiR-16靶向调节KCNJ2基因的表达,MI后缬沙坦改善KCNJ2/Kir2.1的重构一定程度上依赖于NF-KB-miR-16信号通路。机制二：缬沙坦通过抑制心肌梗死后激活的酪蛋白激酶2调控内向整流钾电流目的MI后细胞内PKC等蛋白激酶对IKI的调节主要依赖于PIP2,并且其下游的调控机制鲜有报道。研究发现酪蛋白激酶2(CK2)结合并磷酸化编码Kir2.1蛋白的KCNJ2基因转录因子Spl。然而缬沙坦是否可以抑制MI后激活的CK2蛋白活性来影响KCNJ2基因表达以直接改善IK1通道重构还尚不明确。方法MI大鼠给予缬沙坦或生理盐水灌胃7天,提取梗死灶周和非梗死左心室游离壁心肌组织RNA和蛋白。体外培养H9c2心肌细胞和急性分离的新生大鼠心室肌细胞,转染CK2或给予CoCl2、CK2抑制剂TBB及缬沙坦干预。Western blot法检测CK2和Kir2.1蛋白水平,qPCR检测CK2 mRNA和KCNJ2 mRNA表达水平。全细胞膜片钳记录IK1电流。EMSA检测体内及体外Sp1的DNA结合活性。结果梗死灶周和非梗死左心室游离壁区心肌组织CK2蛋白表达升高,Kir2.1蛋白表达下降,伴随着IK1电流密度减低,同时伴随二者mRNA水平的变化。缬沙坦干预后逆转了这一现象。体外培养的H9c2细胞中过表达CK2蛋白抑制了KCNJ2/Kir2.1的表达。相反的,抑制CK2蛋白可以增加KCNJ2/Kir2.1的表达。同样的,在体外诱导缺氧环境中,缬沙坦同样抑制缺氧后升高的CK2蛋白水平。体外经缬沙坦处理的细胞中过表达CK2,可消除缬沙坦对KCNJ2/Kir2.1的保护作用。EMSA检测证明CK2抑制Sp1的DNA结合活性,TBB抑制CK2后,Sp1的DNA结合活性升高。而MI后大鼠心肌组织中Sp1的DNA结合活性下降,缬沙坦干预后其活性上升。结论AT1受体拮抗剂缬沙坦抑制CK2蛋白活性,增加Kir2.1蛋白表达,从而改善MI后IK1通道重构。机制三：缬沙坦通过抑制心肌梗死后激活的I型辅助性T细胞免疫反应调控内向整流钾电流目的MI导致Kir2.1蛋白介导的IK1电流密度减低,伴随着T细胞水平上调。细胞因子IFN-γ主要Thl细胞分泌。小胶质细胞中IFN-γ导致IK1电流密度下降。本实验主要验证MI后Th1细胞是否可以通过其分泌的细胞因子介导IK1通道重构,以及缬沙坦是否可以改善这一现象。方法空白对照大鼠和MI大鼠给予缬沙坦或生理盐水灌胃7天,提取梗死灶周心肌组织RNA和蛋白。体外培养急性分离的梗死灶周大鼠心室肌细胞和新生大鼠心室肌细胞,分别给予缬沙坦干预和与分离的淋巴细胞共培养。流式细胞术分析大鼠心肌组织中Thl细胞数目。Elisa法检测大鼠血浆中IFN-γ、IL-2和TNF-α水平。Western blot法检测Kir2.1蛋白水平,qPCR检测T-bet、GATA-3、IFN-γ和IL-10的mRNA表达水平。全细胞膜片钳记录IK1电流。结果MI后Th1细胞数目及其分泌的细胞因子水平升高,Kir2.1蛋白水平下降。而MI大鼠缬沙坦干预后,Thl细胞数及其细胞因子水平下降,同时Kir2.1蛋白表达及IK1电流密度升高。在体外淋巴细胞与心肌细胞共培养后,缬沙坦干预对Kir2.1/IK1的调控同上。在体外培养的新生大鼠心室肌细胞中,IFN-γ干预可抑制IK1电流密度,而IL-2和TNF-α对该通道无明显作用。结论缬沙坦可以通过抑制MI后激活的Thl免疫反应、减低IFN-γ水平,来改善IK1通道的重构。
[Abstract]:Background although the level of medical treatment is progressing in the contemporary society, people's understanding of the healthy lifestyle is gradually deepened, but myocardial infarcion (MI) is still one of the most common cardiovascular diseases in the clinic. It is a major threat to the health of the people in our country. About 540 thousand people die of sudden cardiac death every year in China and nearly 90% of them are sudden. The cause of death is malignant arrhythmia after acute MI. Previous studies have found that ventricular arrhythmias and sudden cardiac death are related to the reconstruction of potassium channels. Especially the the inward rectifier potassium channel (Kir), which is the main component of the normal action potential, maintains the resting membrane potential and participates in the action. The most abundant inward rectifier potassium channel in cardiac myocytes is the IK1 channel, the heart type is Kir2.1 protein. One of the main characteristics of the electrical remodeling of ventricular myocytes after the KCNJ2 gene encoding.MI is the decrease of the IK1 current density, which leads to the occurrence of ventricular arrhythmia or the increase of susceptibility. There are a variety of antiarrhythmic drugs, but these mature antiarrhythmic drugs also have arrhythmia effects, especially in QT interphase prolongation. The prolongation of QT interval in patients after MI is an important indicator of malignant ventricular arrhythmias or even sudden death. The current known antiarrhythmic drugs are in MI Most of the clinical MI patients are associated with the risk factors such as hypertension, and the renin angiotensin aldosterone system (Renin-angiotensin-aldosterone system, RAAS) antagonists can significantly reduce the incidence of malignant arrhythmia, but the specific mechanism is not clear. Therefore, it is clear that the RAAS antagonist can play the role of anti arrhythmia. The target of arrhythmia effect and the mechanism of its action will be of great significance for the discovery of new antiarrhythmic drugs and the improvement of the prognosis of MI. The expression level of inward rectifying potassium channel (Kir2.1 protein) in the cell and the effect of valsartan intervention on this channel. And evaluate the heart rate, blood pressure, ventricular arrhythmia susceptibility and hemodynamic indexes after drug intervention. Methods an acute MI model was established by permanent ligation of the left coronary artery descending branch in rats, and the model was evaluated. After the success, the model was randomly divided into sham operation group (group Control), sham operation group + valsartan group (Control+ Valsartan group), myocardial infarction group (group MI), myocardial infarction + valsartan group (group MI+Valsartan). Using small animal telemetry and tail artery pressure measurement to record the heart rate, blood pressure and.7 days after.7 extraction of infarct and non infarct left ventricular travel. The levels of KCNJ2 mRNA and Kir2.1 protein in four groups were detected by qPCR and Westen blot. Results the heart rate and blood pressure of group MI rats were higher than that of Control and Control+Valsartan groups at 7 days after coronary artery ligation and postoperative Masson's staining. QTc and QTcd also tended to be normal after valsartan intervention. In addition, valsartan effectively improved the hemodynamic index of cardiac ejection fraction after MI. Meanwhile, the levels of KCNJ2 mRNA and Kir2.1 in the MI group were lower than those in the Control and Control+Valsartan groups, while valsartan in the MI+Valsartan group improved the reconstruction of the ion channel. Valsartan intervention can be significantly reduced and valsartan intervention can reverse this phenomenon, accompanied by a decrease in cardiac arrhythmia susceptibility. In normal rat myocardium, there is no significant difference in the regulatory effect of valsartan on IKI channel. Mechanism 1: Valsartan regulates inward by inhibiting the NF- kappa B-miR-16 signaling pathway activated after myocardial infarction. The mechanism of valsartan second part of valsartan to improve the remodeling of inward rectifier potassium channel after myocardial infarction objective MicroRNAs is one of the important factors in regulating arrhythmia. The computer predicted that microRNA-16 (miR-16) can regulate the KCNJ2 gene.NF- kappa B to regulate the promoter of miR-16. This experiment mainly verifies the possibility of valsartan. To reduce the expression of miR-16 by inhibiting the activation of NF- kappa B after MI, and whether miR-16 can target the expression of KCNJ2 gene. Methods MI rats were given valsartan or physiological saline for 7 days to extract the RNA and protein of Zhou Xinji tissue from the infarct. In vitro culture of H9c2 myocytes and acute isolated neonatal rat ventricular myocytes were transfected to miR-16. The levels of NF- kappa B p65, inhibitor KBa (I kappa B alpha) and Kir2.1 protein were detected by angiotensin II and valsartan, and inhibitor KBa (I kappa B alpha) and Kir2.1 protein levels were detected. The level of miR-16 expression in the infarct area increased and the level of KCNJ2/Kir2.1 decreased. The over expression of miR-16 in vitro resulted in the down expression of KCNJ2/Kir2.1 and the decrease of the IK1 current density. On the contrary, the inhibition of miR-16 or the mutation of its binding site enhanced the prognosis of KCNJ2/Kir2.1 in the.MI rats, and the increase of NF- kappa B. The levels of p65 and miR-16 decreased, while the decreased levels of I kappa B A and Kir2.1 protein were elevated. In vitro, the elevation of miR-16 expression induced by angiotensin II and the decrease of KCNJ2/Kir2.1 expression were also inhibited by valsartan. The over expression of miR-16 in valsartan treated cells in vitro could eliminate the protective effect of valsartan on KCNJ2/Kir2.1 in vivo and in vivo. In vitro, the expression of NF- kappa B p65 was up-regulated in anoxic environment, while the expression of I kappa B alpha decreased, and the intervention of valsartan inhibited this phenomenon. After the inhibition of NF- kappa B, the regulatory effect of valsartan and NF- kappa B inhibitors on miR-16 and KCNJ2/Kir2.1 was similar. The expression of the node KCNJ2 gene, after MI, the improvement of the remodeling of KCNJ2/Kir2.1 depends partly on the NF-KB-miR-16 signaling pathway. Mechanism two: Valsartan's regulation of IKI is mainly dependent on PIP2, and is dependent on PIP2, by inhibiting the activation of the casein kinase 2 after myocardial infarction, and the regulation of IKI is mainly dependent on PIP2. There are few reports on the regulation mechanism of swimming. The study found that casein kinase 2 (CK2) binding and phosphorylation of Kir2.1 protein KCNJ2 gene transcription factor Spl., however, whether valsartan can inhibit the activity of CK2 protein activated after MI to affect the KCNJ2 gene expression to directly improve the IK1 channel remodeling is still unclear. RNA and non infarcted left ventricular free wall myocardial tissue RNA and protein were extracted by irrigated saline for 7 days. H9c2 cardiomyocytes and acute isolated neonatal rat ventricular myocytes were cultured in vitro, CK2 or CoCl2, CK2 inhibitor TBB and valsartan were used to detect CK2 and Kir2.1 protein levels by.Western blot method. RNA expression level. The whole cell patch clamp recording IK1 current.EMSA detected the DNA binding activity of Sp1 in the body and in vitro. Results the expression of CK2 protein in the myocardial tissue of the infarct peripheral and non infarcted left ventricular wall increased, the expression of Kir2.1 protein decreased, accompanied by the decrease of the current density of IK1 and the change of the mRNA level in the two cases. The overexpression of CK2 protein in H9c2 cells in vitro inhibited the expression of KCNJ2/Kir2.1. On the contrary, the inhibition of CK2 protein could increase the expression of KCNJ2/Kir2.1. Similarly, in the hypoxia environment, valsartan also inhibited the level of CK2 protein increased after hypoxia. In vitro, the expression of CK2 in valsartan cells was overexpressed in the cells treated by valsartan. The.EMSA detection of valsartan to KCNJ2/Kir2.1 showed that CK2 inhibited the DNA binding activity of Sp1, and the DNA binding activity of Sp1 increased after TBB inhibition of CK2, and Sp1 DNA binding activity in the myocardium of rats after MI decreased and the activity of valsartan increased after the intervention of valsartan. Protein expression, thus improving the IK1 channel reconstruction after MI. Mechanism three: Valsartan by inhibiting the I type auxiliary T cell immune response after myocardial infarction regulates the inward rectifier potassium current purpose MI leads to the Kir2.1 protein mediated IK1 current density, accompanied by the level of T cells. The microglia is secreted by the microglia and the microglia. IFN- gamma leads to the decrease of IK1 current density in the cells. This experiment is mainly to verify whether Th1 cells can mediate IK1 channel reconstruction through its secretory cytokines after MI, and whether valsartan can improve this phenomenon. Methods blank control rats and MI rats were given valsartan or physiological saline for 7 days to extract RNA from myocardial tissue of infarct In vitro culture of ventricular myocytes and neonatal rat ventricular myocytes from acute isolated infarcted rats, valsartan intervention and co culture with isolated lymphocytes were given respectively. Flow cytometry was used to determine the number of Thl cells in rat myocardial tissue by.Elisa method to detect IFN- gamma, IL-2 and TNF- alpha levels by.Western blot assay. The level of Kir2.1 protein and the level of mRNA expression of T-bet, GATA-3, IFN- gamma and IL-10 were measured by qPCR. IK1 current was recorded by whole cell patch clamp. The results showed that the number of Th1 cells and the level of cytokines secreted and the level of Kir2.1 protein decreased after MI. IK1 current density increased. After co culture of lymphocytes and cardiomyocytes in vitro, valsartan intervention on Kir2.1/IK1 was involved. In the cultured neonatal rat ventricular myocytes, IFN- gamma intervention could inhibit the current density of IK1, while IL-2 and TNF- alpha were not significantly used for this channel. Conclusion valsartan can inhibit the T activated by MI. HL immune response, reduce IFN- gamma level, to improve the IK1 channel remodeling.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R542.22
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本文编号：1971302