AngⅡ对心室肌细胞Ikr电流的调节及其信号传导机制

发布时间：2018-01-16 23:25

本文关键词：AngⅡ对心室肌细胞Ikr电流的调节及其信号传导机制　出处：《河北医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 心肌肥厚、心衰是冠心病、高血压、心瓣膜病等多种心血管病的常见合并症。心肌肥厚、心衰时发生病理性电生理重构使得心脏的电生理不稳定性增加,常常伴发心律失常,心衰病人约一半以上因恶性心律失常的发生而猝死(即心性猝死Sudden cardiac death, SCD)。肾素-血管紧张素系统(renin-angiotensin system, RAS)是由肾素、血管紧张素以及其受体构成的重要体液系统,在调节心血管系统的正常生理功能与高血压、心肌肥大、充血性心力衰竭等诸多心血管的病理过程中具有重要作用。RAS不仅存在于体液系统,而且在包括心脏、血管在内的许多组织中也有RAS,在局部调节生理病理过程。血管紧张素II(Ang II)是其该系统发挥作用的主要体液因子,其受体有1型(AT1)和2型(AT2)两种。近几年大规模的临床试验研究发现,通过血管紧张转化酶抑制剂(angiotensin-converting enzyme inhibitors,ACEI)抑制Ang II的合成或AT1受体阻断剂可能因减少致命性心律失常的发生而明显降低病人死亡率。实验表明,抑制RAS可有效治疗缺血再灌诱发的心律失常。ACE抑制剂和AT1受体阻断剂成为目前临床高血压、心衰病人治疗的重要药物。然而,这些药物潜在抗心律失常的机制尚不清楚。正常心脏电活动取决于各种离子有序的跨膜转运,先天遗传基因改变或后天获得性因素(如心脏病理性变化、药物等)均可引起离子通道功能紊乱,构成了心律失常的物质基础。心肌肥厚、心衰时最突出的表现是心肌细胞复极化过程变慢,而导致动作电位的延长,为细胞产生后除极、尖端扭转性室速等心律失常创造了前提条件。心肌复极钾电流存在明显的种属差别。大动物、人的复极化钾电流主要是Iks和Ikr。目前认为,人心肌中Ikr由HERG(human ether-a-go-go-related gene)基因编码,HERG通道的激活引发动作电位3期复极的开始,对心肌的复极化有至关重要的作用。由于HERG基因突变或药物诱导的Ikr阻断减少心脏HERG电流,分别产生与7号常染色体有关的先天性LQTS和获得性LQTS,这两种LQTS的形成都与延迟心脏复极,延长体表心电图的QT间期,有较高的尖端扭转型室性心律失常和猝死的危险。最近的实验表明一些G蛋白偶联受体如α-和β-肾上腺素受体通过细胞内第二信使如cAMP,PKA和PKC调节HERG通道。然而,迄今为止Ang II对心室细胞复极过程起重要作用的Ikr的影响尚了解甚少。故本研究利用膜片钳技术,在急性分离的豚鼠心室肌细胞和异源表达系统上观察了Ang II对kr/HERG电流的影响,并分析了中介Ang II作用的细胞内信号途经,为病理情况下心律失常发生的分子机制及解释ACEI、AT1受体阻断剂的药效提供实验依据。第一部分表达HERG蛋白稳定细胞株的建立及其鉴定目的:建立稳定表达HERG通道蛋白的HEK293细胞株。方法:用Lipofectin 2000转染试剂将HERG基因转染到HEK293细胞,经G418筛选,用荧光显微镜,Western Blot和全细胞膜片钳技术检测HERG基因在HEK293细胞中的稳定表达情况。结果:G418筛选,培养4周阳性克隆形成。荧光显微镜,Western Blot和全细胞膜片钳技术检测,证实HERG基因得到了稳定表达并记录到了转染成功的HERG电流,通过HERG通道对其特异性阻断剂E-4031的IC50,进一步表明在HEK细胞上正常HERG通道蛋白表达。结论:建立了稳定表达HERG基因的HEK293细胞株,表达的HERG蛋白能形成有功能的通道,产生HERG电流。第二部分Ang II作用于AT1受体抑制心室肌细胞的快速延迟整流钾(Ikr)电流目的:观察Ang II对豚鼠心室肌细胞Ikr的影响。方法:在急性分离的豚鼠心室肌细胞及共表达HERG和人Ang II AT1受体基因的HEK293细胞上,全细胞膜片钳技术记录Ikr/HERG电流,观察Ang II对电流影响。结果:Ang II以浓度依赖的方式抑制Ikr,IC50是8.9 nM。Ang II (100 nM)延长心室肌细胞动作电位APD50和APD90,分别延长20%和16%。AT1受体阻断剂losartan(1μM)完全取消AngII对Ikr电流的抑制作用。在共表达HERG和AT1受体基因的HEK293细胞上,Ang II也抑制HERG电流,减慢通道的激活、去活和再恢复。PKC抑制剂stausporine和Bis-1明显减弱Ang II对Ikr电流的抑制作用。结论:Ang II作用于AT1受体,经过PKC途经抑制心室肌细胞的Ikr电流。这对解释病理情况下,如心肌肥厚心衰时,Ang II的升高导致心律失常的发生提供了一个可能的机制。第三部分Ang II通过PKCε亚型抑制Ikr/HERG电流目的:分析中介Ang II抑制Ikr/HERG电流的PKC亚型及其下游分子机制。方法:在急性分离的豚鼠心室肌细胞及共表达HERG和人Ang II AT1受体基因的HEK293细胞上,观察选择性PKC抑制剂、激动剂及PKC亚型转位抑制肽对Ang II作用的影响,分析中介其作用的PKC亚型;观察HERG通道磷酸化位点突变对Ang II作用的影响,分析PKC下游的分子机制。结果:在HEK293细胞上,应用PKC抑制剂stausporine和Bis-1明显减小了Ang II对HERG电流的抑制作用;长期孵育PMA下调PKC也明显减小了Ang II对HERG电流的抑制作用。细胞内钙螯合剂浓度增加不影响Ang II抑制HERG电流。特异性亚型PKC抑制剂G?-6976和G?-6983不影响Ang II对HERG电流的抑制作用;PKCε亚型的转位抑制剂明显对抗Ang II对HERG电流的抑制作用。在豚鼠心室肌细胞上,PKCε亚型的转位抑制剂明显减小Ang II对Ikr的抑制作用。HERG通道的磷酸化位点突变后,几乎取消了Ang II对HERG电流的抑制作用。结论:(1)Ang II主要通过PKCε的激活抑制Ikr/HERG。(2)Ang II通过PKCε使HERG通道蛋白磷酸化,调节HERG通道的功能。结论 1.本研究建立了表达HERG基因的稳态HEK293细胞株,表达的蛋白能形成有功能的通道,产生HERG电流。 2. Ang II作用于AT1受体,经过PKC途经抑制心室肌细胞的Ikr电流。这对解释病理情况下,如心肌肥厚心衰时,Ang II的升高导致心律失常的发生提供了一个可能的机制。 3. Ang II主要通过PKCε的激活抑制Ikr/HERG。 4. Ang II有可能通过PKCε使HERG通道磷酸化,调节HERG通道的功能。
[Abstract]:Cardiac hypertrophy, heart failure is a common complication of coronary heart disease, hypertension, heart disease and other cardiovascular disease. Pathological myocardial hypertrophy, electrophysiological remodeling increases cardiac electrophysiological instability is often associated with heart failure, arrhythmia, heart failure patients about more than half due to the occurrence of malignant arrhythmia and sudden death (i.e. sudden cardiac death Sudden cardiac death, SCD). The renin-angiotensin system (renin-angiotensin system RAS) is a renin angiotensin system consisting of an important body fluid and its receptor, in normal physiological function and high blood pressure, regulating cardiovascular hypertrophy, the pathological process of congestive heart failure and other cardiovascular plays an important role in not only.RAS in fluid system, but also in many tissues including heart, blood vessels, there are RAS, regulation of physiological and pathological processes in local angiotensin II (Ang. II) is the main body factors play its role in the system, its receptor type 1 (AT1) and type 2 (AT2) two. In recent years, large-scale clinical trials found by angiotensin converting enzyme inhibitors (angiotensin-converting enzyme, inhibitors, ACEI) inhibited Ang synthesis of II or AT1 receptor antagonist may because of the reduction of fatal arrhythmia and reduce patient mortality. Experimental results show that the inhibition of RAS arrhythmia.ACE inhibitors and AT1 receptor induced effective treatment of reperfusion blockers has become important clinical hypertension, drug therapy in patients with heart failure. However, the mechanism of these potential antiarrhythmic drugs is not clear.
Normal cardiac electrical activity depends on the transmembrane transport in various ions orderly, congenital genetic change or acquired factors (such as cardiac pathological changes, drug etc.) can cause ion channel dysfunction, constitute the material basis of arrhythmia. Myocardial hypertrophy, heart failure is the most prominent manifestation of myocardial repolarization process slowly, which led to the prolongation of the action potential, as the cells produced after depolarization, torsades de pointes arrhythmias such as creating preconditions. There is obvious difference between species. Large animal myocardial repolarization potassium current, repolarising potassium current one is mainly Iks and Ikr. currently believe that the human heart by HERG (Ikr human ether-a-go-go-related gene) gene encoding, to activate the HERG channel lead to action potential repolarization of phase 3, have crucial effect on myocardial repolarization. Due to mutations in the HERG gene or drug induced by blocking Ikr Reduce the cardiac HERG current, respectively, and 7 chromosome associated congenital and acquired LQTS LQTS, the two LQTS Chengdu and delayed cardiac repolarization electrocardiogram, prolonged QT interval, have a higher risk of torsade de pointes ventricular arrhythmias and sudden death. Recent experiments showed that G protein coupled receptors such as alpha and beta adrenergic receptors by intracellular second messengers such as cAMP, PKA and PKC regulate HERG channels. However, so far Ang II cells on ventricular repolarization process effect of Ikr is still poorly understood.
This study utilizes the patch clamp technique in isolated guinea pig ventricular myocytes and heterologous expression system on the effect of Ang II on kr/HERG current, and analyzes the role of II Ang mediated intracellular signal pathway for the molecular mechanism of pathogenesis of cases of arrhythmia and explain ACEI, AT1 receptor antagonist effects provide on the basis of experiment.
Establishment and identification of a stable cell line expressing HERG protein in the first part
Objective: to establish a HEK293 cell line that is stable to express HERG channel protein.
Methods: HERG gene was transfected into HEK293 cells by Lipofectin 2000 transfection reagent. After G418 screening, the stable expression of HERG gene in HEK293 cells was detected by fluorescence microscopy, Western Blot and whole cell patch clamp technique.
Results: G418 screening, positive clones were cultured for 4 weeks. The fluorescence microscope, detection of Western Blot and whole cell patch clamp technique, stable expression of HERG was confirmed and recorded the HERG current after transfection, the IC50 antagonist E-4031 on its specificity by HERG channel, further showed normal expression of HERG channel protein in HEK cells.
Conclusion: the HEK293 cell line that expresses the HERG gene is established. The expression of HERG protein can form a functional channel and produce the HERG current.
The second part Ang II acts on the rapid delayed rectifier potassium (Ikr) current of the AT1 receptor inhibiting ventricular myocytes
Objective: To observe the effect of Ang II on the Ikr of ventricular myocytes of guinea pigs.
Methods: in acute isolated guinea pig ventricular myocytes and HEK293 cells co expressing HERG and human Ang II AT1 receptor genes, Ikr/HERG current was recorded by whole cell patch clamp technique, and the effect of Ang II on current was observed.
Results: Ang II in a concentration dependent manner inhibited Ikr, IC50 is 8.9 nM.Ang II (100 nM) prolonged ventricular cell action potentials of APD50 and APD90, respectively, to extend the 20% and 16%.AT1 receptor antagonist losartan (1 M) completely abolished the inhibitory effect of AngII on Ikr current. The co expression of HERG and AT1 receptor gene HEK293 cells, Ang II also inhibited HERG activation current, slow down the channel, to live and to restore the.PKC inhibitor stausporine and Bis-1 significantly reduced the inhibitory effect of Ang II on Ikr current.
Conclusion: Ang II acts on the AT1 receptor and inhibits the Ikr current in ventricular myocytes through PKC. This provides a possible mechanism for explaining the occurrence of arrhythmia in the pathological condition, such as myocardial hypertrophy, heart failure, and the increase of Ang II.
Third part Ang II suppression of Ikr/HERG current through PKC epsilon
Objective: to analyze the PKC subtype and its downstream molecular mechanism of the intermediate Ang II to inhibit the Ikr/HERG current.
Methods: in isolated guinea pig ventricular myocytes and co expression of HERG and Ang II AT1 receptor gene on HEK293 cells, observe the selective inhibitor of PKC agonist, and PKC subtype translocation inhibitory effects of peptides on Ang II, analysis of the role of intermediary PKC subtype; observe the HERG channel phosphorylation site mutation influence of Ang II effect, analysis of molecular mechanism of PKC downstream.
Results: in HEK293 cells, using PKC inhibitors stausporine and Bis-1 significantly reduced the inhibitory effect of Ang II on HERG current; long-term incubation PMA down-regulation of PKC significantly reduced the inhibitory effects of Ang II on HERG current. The intracellular calcium chelator concentration does not affect Ang II inhibition of HERG subtype specific PKC current. -6976 and G inhibitor G?? -6983 does not affect the inhibitory effect of Ang II on HERG current; translocation inhibitor PKC epsilon isoform significantly against Ang II inhibition of HERG currents. In guinea pig ventricular myocytes, translocation inhibitor PKC epsilon isoform significantly reduced the phosphorylation site of.HERG inhibition of Ang II on Ikr channel the mutation, almost abolished the inhibitory effect of Ang II on HERG current.
Conclusion: (1) Ang II mainly inhibits Ikr/HERG. (2) Ang II through PKC epsilon phosphorylation of HERG channel protein through the activation of PKC epsilon, regulating the function of HERG channel.
conclusion
1. this study has established a stable HEK293 cell line expressing the HERG gene. The expressed protein can form a functional channel to produce a HERG current.
2. Ang II acts on the AT1 receptor and inhibits the Ikr current of ventricular myocytes through PKC. This provides a possible mechanism for explaining the occurrence of arrhythmia in the pathological condition, such as myocardial hypertrophy, heart failure, and the increase of Ang II.
3. Ang II mainly inhibits Ikr/HERG. through the activation of PKC epsilon
4. Ang II may be used to phosphorylate the HERG channel through PKC epsilon, regulating the function of the HERG channel.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R33

【共引文献】