血管紧张素Ⅱ对hERG钾通道的慢性调节机制研究

发布时间：2018-09-07 22:16

【摘要】：心肌肥厚、心力衰竭是冠心病、心瓣膜病、高血压等多种心血管病的常见合并症。心肌肥厚、心力衰竭时发生的病理性电重构使心脏的电不稳定性增加，常伴发各种心律失常。心衰病人在泵功能稳定的情况下半数以上因发生恶性心律失常而猝死（即心源性猝死Sudden Cardiac Death,SCD）。因此，阐明病理情况下心律失常产生机制，寻找药物作用靶点具有重要意义。 已知心肌肥厚、心力衰竭病理发展过程中全身和局部的肾素-血管紧张素系统（renin-angiotensin system, RAS）活性增加是病理性组织重构重要的原因，该系统的关键成分血管紧张素II （AngII）主要通过激活AT1受体从而激活多种细胞内信号通路，对心血管功能发挥广泛的调节作用。实验证明，过表达人AngII基因的大鼠常因严重室性心动过速而猝死；在急性分离的犬和大鼠的心室肌细胞、以及转染编码Ito通道基因Kv4.3哺乳细胞上，AngII均可使Ito电流密度减小。上述研究结果提示，AngII通过刺激AT1受体直接引起离子通道的改变导致病理性电重构是心律失常发生的重要原因。 哺乳类动物心室肌细胞延迟整流钾电流在动作电位复极化过程中发挥关键作用，其中快激活成份（IKr）通道孔区亚单位由human‘ether-a-go-go’ related gene基因(简称hERG)编码，此种hERG钾通道由于特殊的动力学特征对动作电位形状、时程具有重要影响。疾病状况下该通道功能下调引发动作电位时程延长和/或形状改变是室性心律失常发生的重要原因。hERG钾通道由于本身的缺陷或者药物的影响而发生变异或功能异常，产生先天性LQTS和获得性LQTS，均有较高尖端扭转型室性心律失常和猝死的危险。 在前期的研究我们观察到AngII对豚鼠心室肌细胞IKr和表达的IhERG呈现急性抑制作用，提示AngII可能通过直接下调IKr参与病理条件下电生理重构。已知AngII可激活多条细胞内信号通路引发急性和慢性生物效应，慢性效应发生在数小时甚至更晚时间，常涉及蛋白基因转录及转录后的改变。我们已证明AngII长时间作用（12h）可使表达的hERG钾通道成熟膜蛋白明显减少。那么，AngII下调通道膜蛋白含量潜在的机制尚不清楚。 新近实验证明，慢性刺激一些G蛋白偶联受体对hERG钾通道发挥明显的转录后调节作用。因此，本研究主要利用分子生物学的方法，在前期研究AngII作用的基础上又在异源表达系统上进一步研究AT1受体刺激对hERG钾通道的转录后调节作用（包括如何影响hERG钾通道蛋白正向转运及膜蛋白入胞降解等转录后调节过程而改变细胞膜蛋白含量）并对AngII下调hERG钾通道膜蛋白量的信号传导机制进行分析，这对理解离子通道病理重构机制从而解释心律失常发生机制具重要理论意义。第一部分血管紧张素II对hERG钾通道转录后调节作用 目的：研究AngII对hERG钾通道转录后的调节作用。 方法：（1）Lipofectamine2000瞬时转染：将AT1受体质粒转染到稳态转染的hERG-HEK293细胞上，或将AT1受体质粒与hERG质粒以1:1的比例瞬时转染到HEK293细胞上。 （2）细胞免疫荧光：将细胞用4%多聚甲醛固定后，，用0.2%TritonX-100透化处理（不做透化处理省略这一步），之后用PBS配制含5%BSA的封闭液进行封闭，加入特异性一抗和FITC标记的二抗，用共聚焦显微镜观察显色情况。 （3）Western blot：将提取的细胞总蛋白进行SDS-PAGE电泳，电转移到NC膜上，用TBST配制含5%脱脂牛奶进行封闭，加入特异性一抗和红外荧光标记的二抗，用双色红外激光成像系统仪器扫描。 （4）In-cell western：将细胞用4%多聚甲醛固定后用TBST配制含5%脱脂牛奶封闭，加入特异性一抗和红外荧光标记的二抗，用双色红外激光成像系统仪器扫描。 结果：（1）Western blot检测到目的条带约在65kDa出现，与AT1受体分子量大小基本一致。提示AT1受体在hERG-HEK293细胞中成功表达。 （2）AngII长时间孵育对hERG钾通道蛋白含量影响:AngI（I100nM）孵育24小时后In-cell western检测显示膜上成熟蛋白减少为（69%±4%，P0.01）；细胞免疫荧光染色共聚焦显微镜下观察通道蛋白在细胞膜和细胞内分布情况，发现AngII使未透化处理过的细胞膜上的荧光明显减少，透化处理过的细胞膜荧光减少，胞浆荧光变化不明显。 （3）AngII对hERG钾通道蛋白正向转运的影响: AngII孵育24h后western blot检测到成熟蛋白的表达显著减少为（68%±4%，P0.01），胞浆未成熟蛋白表达没有明显变化（P0.05）。而已知能够引起hERG钾通道转运障碍的Fluoxetine（3μM）或在瞬时转染的hERG钾通道突变体A422T和H562P，均可检测到成熟蛋白表达明显减少，同时伴未成熟蛋白表达的明显增加。Fluoxetine使成熟蛋白减少为（64%±2%，P0.01），未成熟蛋白增加为（114%±5%，P0.05）。提示AngII不影响通道蛋白的正向转运。 （4） AngII对hERG钾通道膜蛋白降解的影响：已知Brefeldin A1(BFA)能够阻断通道正向转运，加入BFA（10μM）使用western blot观察不同时间成熟蛋白的降解情况，对照组在2h、4h、8h、12h、24h下降为82%±8%、70%±1%、63%±9%、55%±8%、53%±8%（P0.05），AngII组在2h、4h、8h、12h、24h下降为77%±7%、64%±9%、55%±11%、44%±12%、40%±12%（P0.05），提示AngII加速通道膜蛋白降解。Lactacystin（5μM，蛋白酶体抑制剂）明显能够抑制AngII介导的成熟蛋白减少（67±4%增加为89%±3%，P0.05），而Bafilomycin（1μM，溶酶体抑制剂）对AngII的作用无明显影响（P0.05），提示AngII加速蛋白降解主要通过蛋白酶体途径。 结论：在异源表达系统上，AngII长时间孵育可显著减少hERG钾通道膜上成熟蛋白含量，其原因可能是由于膜蛋白以泛素-蛋白酶体途径降解加速引起。第二部分血管紧张素II下调hERG钾通道膜蛋白含量的细胞内信号传导机制 目的：分析AngII下调hERG钾通道膜蛋白含量的细胞内信号传导机制。 方法：（1）Lipofectamine2000瞬时转染：将AT1受体质粒转染到稳态转染的hERG-HEK293细胞上。 （2）Western blot：方法同第一部分。 结果：Western blot结果显示:（1）PKC抑制剂Bis I（100nM）明显能够抑制AngII介导的成熟蛋白减少(64%±4%增加到90%±4%,P0.01)。 （2）PKC激动剂PMA（100nM）或OAG（100nM），也明显能够抑制AngII介导的成熟蛋白减少（59%±5%增加到101%±5%，P0.01；58%±4%增加到100%±5%, P0.05）。 （3）PKA抑制剂H-89（1μM）不能抑制AngII介导的成熟蛋白减少（P0.05）。 结论：在异源表达系统上，AngII长时间孵育减少hERG钾通道膜上成熟蛋白的表达主要由PKC信号通路介导。
[Abstract]:Cardiac hypertrophy and heart failure are common complications of coronary heart disease, valvular heart disease, hypertension and other cardiovascular diseases. Cardiac hypertrophy and pathological electrical remodeling during heart failure increase electrical instability of the heart and often accompany various arrhythmias. More than half of patients with heart failure develop malignant arrhythmias when the pump function is stable. Sudden cardiac death (SCD). Therefore, it is of great significance to elucidate the mechanism of arrhythmia in pathological conditions and find the target of drug action.
It is known that the increased activity of renin-angiotensin system (RAS) is an important cause of pathological tissue remodeling in cardiac hypertrophy and heart failure. AngII has been shown to reduce Ito current density in acute isolated canine and rat ventricular myocytes and in transfected mammalian cells encoding the Ito channel gene Kv4.3. Pathological electrical remodeling is an important cause of arrhythmia by stimulating AT1 receptor.
Delayed rectifier potassium currents play a key role in action potential repolarization in mammalian ventricular myocytes. The pore region subunit of the fast-activating component (IKr) channel is encoded by the human `ether-a-go-go'related gene (hERG) gene. This kind of hERG potassium channel has special dynamic characteristics for action potential shape and duration. Important effects. The prolongation of action potential duration and/or shape changes caused by the downregulation of the channel function are important causes of ventricular arrhythmias in disease conditions. The risk of arrhythmia and sudden death.
In previous studies, we observed the acute inhibitory effect of AngII on IKr and IhERG expression in guinea pig ventricular myocytes, suggesting that AngII may be involved in electrophysiological remodeling through direct down-regulation of IKr under pathological conditions. AngII is known to activate multiple intracellular signaling pathways leading to acute and chronic biological effects, with chronic effects occurring for hours or even hours. Later, it is often involved in the transcriptional and post-transcriptional changes of protein genes. We have shown that long-term action of AngII (12 hours) can significantly reduce the expression of mature membrane proteins of hERG potassium channels. The underlying mechanism of AngII down-regulation of channel membrane proteins remains unclear.
Recent studies have shown that chronic stimulation of some G-protein-coupled receptors plays a significant posttranscriptional role in the regulation of hERG potassium channels. The signal transduction mechanism of AngII down-regulating the membrane protein content of hERG potassium channel was analyzed, which is important for understanding the mechanism of ion channel pathological remodeling and explaining the mechanism of arrhythmia. Significance. Part one: angiotensin II regulates post transcriptional regulation of hERG potassium channel.
Objective: To study the regulatory effect of AngII on the transcription of hERG potassium channel.
Methods: (1) Lipofectamine 2000 transient transfection: AT1 receptor plasmid was transfected into stable transfected hERG-HEK293 cells, or AT1 receptor plasmid and hERG plasmid were transfected into HEK293 cells in 1:1 ratio.
(2) Cell immunofluorescence: Fixed with 4% paraformaldehyde, the cells were treated with 0.2% Triton X-100 dialysis (without dialysis omitting this step), then blocked with 5% BSA blocking solution prepared with PBS. Specific primary antibody and FITC-labeled secondary antibody were added to the blocking solution, and the pornography was observed by confocal microscopy.
(3) Western blot: SDS-PAGE electrophoresis was carried out on the extracted total cell proteins, which were transferred to NC membrane, sealed with 5% skimmed milk prepared by TBST, and scanned with dual-color infrared laser imaging system.
(4) In-cell western: Fixed cells with 4% paraformaldehyde and sealed them with 5% skimmed milk by TBST, added specific antibodies and infrared fluorescent labeled antibodies, and scanned them with dual-color infrared laser imaging system.
Results: (1) Western blot showed that the target band appeared at about 65 kDa, which was consistent with the molecular weight of AT1 receptor, suggesting that AT1 receptor was successfully expressed in hERG-HEK293 cells.
(2) The effect of long incubation with AngII on the content of potassium channel protein in hERG: In-cell Western assay showed that the mature protein on the membrane decreased to (69%+4%, P 0.01) 24 hours after incubation with AngI (I100nM); the distribution of channel protein in the cell membrane and in the cell was observed by confocal microscopy with immunofluorescence staining, and it was found that AngII made the unpermeable treatment fine. The fluorescence on the cell membrane decreased significantly, the fluorescence of the permeable treated cell membrane decreased, and the cytoplasmic fluorescence did not change significantly.
(3) The effect of AngII on the forward transport of hERG potassium channel protein: Western blot showed that the expression of mature protein was significantly reduced to (68% + 4%, P 0.01) after incubation for 24 hours, and the expression of immature protein was not changed significantly (P 0.05). However, fluoxetine (3 mu M) or transient transfection of hERG potassium channel were known to cause the hERG potassium channel transport disorder. Fluoxetine reduced the expression of mature protein to (64%+2%, P 0.01) and increased the immature protein to (114%+5%, P 0.05). AngII did not affect the positive transport of channel protein.
(4) Effects of AngII on the degradation of hERG potassium channel membrane proteins: Brefeldin A1 (BFA) was known to block the forward transport of the channel. The degradation of mature proteins at different time points was observed by Western blot after adding BFA (10 mu M). In the control group, the degradation rate was 82% + 8%, 70% + 1%, 63% + 9%, 55% + 8%, 53% + 8% (P 0.05) at 2h, 4h, 8h, 12h, 24h, 12h and 24h, respectively. Lactacystin (5 mu M, proteasome inhibitor) significantly inhibited the decrease of mature protein mediated by AngII (67.4% increased to 89% + 3%, P 0.05), while Bafilomycin (1 mu M, lysosomal inhibitor) had no significant effect on AngII (P 0.05). It suggested that AngII accelerated protein degradation mainly through proteasome pathway.
CONCLUSIONS: AngII prolonged incubation can significantly reduce the content of mature proteins on hERG potassium channel membranes in the heterologous expression system, possibly due to the accelerated degradation of membrane proteins via ubiquitin-proteasome pathway. Part II Intracellular signal transduction mechanism of angiotensin II down-regulating the content of hERG potassium channel membrane proteins.
Objective: to analyze the intracellular signal transduction mechanism of AngII down regulating the protein content of hERG potassium channel membrane.
Methods: (1) Lipofectamine 2000 transient transfection: AT1 receptor plasmid was transfected into stable transfected hERG-HEK293 cells.
(2) Western blot: method is the same as the first part.
Results: Western blot showed that: (1) PKC inhibitor Bis I (100nM) could significantly inhibit AngII-mediated decrease of mature protein (64%+4% increased to 90%+4%, P 0.01).
(2) PKC agonists PMA (100nM) or OAG (100nM) also significantly inhibited AngII-mediated decrease of mature protein (59% + 5% to 101% + 5%, P 0.01, 58% + 4% to 100% + 5%, P 0.05).
(3) PKA inhibitor H-89 (1 M) can not inhibit AngII mediated maturation protein decrease (P0.05).
CONCLUSION: Long-term incubation of AngII reduces the expression of mature proteins on hERG potassium channel membranes, which is mediated by PKC signaling pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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相关博士学位论文 前1条

1 张国红;EGF对延迟整流性钾离子通道功能调节的研究[D];河北医科大学;2007年

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