蛋白激酶C亚型特异性调控心肌延迟整流钾电流及其分子机制研究

The delayed rectifier K+ current is responsible for the late repolarization phase of the action potential (AP) and predominantly contributes to AP duration and shape in many species. This current is separated into two components, the rapidly IKr and slowly IKs activating delayed rectifier K+ current. The human ether-a-go-go-related gene hERG encodes the IKr channel and KCNQ1 co-assembles with the KCNE1 to generate IKs. Both IKr and IKs channel are highly regulated by the protein kinase C (PKC), a family of serine/threonine kinases, which has been implicated as mediators of the neurotransmitters, hormones and biophysical stress in cardiovascular diseases. Currently, at least 12 isoforms of PKC have been identified in various tissues and are classified into three groups based on their structure and activation mechanisms. There is accumulating evidence to indicate that PKC isoforms in the heart are differentially regulated by various stimuli under physiological and pathological conditions. The individual PKC isoforms play distinct roles, some of which oppose each other in the modulation of electrical activity. However, it is little known about the outlines that distinct PKC isoform regulates IKr or IKs channel. By using pharmacological tools and genetic manipulation, the present study was designed to perform in native guinea-pig cardiomycytes and heterologous expression system to answer the following questions: what is the modulatory action of the most abundant PKC isoform α, β, and ε in cardiomycytes on IKr or IKs channel; what is molecular mechanism underlying specific regulatory action of PKC isozymes. The results will be of importance for the understanding of the mechanism of ion channel regulation and the development of new therapeutic agents targeting PKC isoform.

延迟整流K+电流是心室动作电位晚期复极的主要电流，包括快激活IKr和慢激活IKs两种成分，Human ether-a-go-go-related (hERG) 基因编码IKr，Iks通道则由KCNQ1和KCNE1共同形成。已知蛋白磷酸激酶C（PKC）中介神经、体液以及病理因素对IKr和Iks通道的调控，且却来越多的证据显示，生理和病理情况下不同刺激因素特异性激活心脏PKC不同亚型，这些PKC亚型又中介不同、甚至相反的心肌电生理效应。然而，目前对心肌不同PKC亚型特异性调控IKr和IKs通道的认识非常有限。因此，本课题拟在豚鼠心肌细胞和异源表达系统上，采用药理和基因干预手段解决以下问题：心肌富含的PKC α、β以及ε亚型对IKr和IKs钾通道具有什么样的调节作用；调节作用的分子机制；实验结果将对理解心肌离子通道功能的生理调控、解释病理情况下心肌离子通道功能改变具有重要意义。

延迟整流K+电流是心室动作电位晚期复极的主要电流，包括快激活IKr和慢激活IKs两种成分，先天性基因突变和疾病所致的IKr和IKs功能改变是心律失常的分子基础。已知蛋白磷酸激酶C（PKC）中介G蛋白偶联受体、病理因素等对IKr和IKs通道的调控，然而目前PKC调控IKr和IKs通道的结果十分矛盾。心脏存在诸多的PKC亚型，目前对心肌不同PKC亚型特异性调控IKr和IKs通道的认识非常有限。运用电生理和分子生物技术，本项目首次系统阐明了心脏富含的cPKC和PKC-epsilon亚型对IKr和IKs通道的调控作用和机制，并分析了包括血管紧张素II AT1和肾上腺素alpha1A受体等G蛋白偶联受体通过特定PKC亚型调控IKr和IKs通道的作用。此外，还研究了醛固酮对心肌IKr和IKs通道的调控作用及醛固酮拮抗剂对心脏的保护作用。主要创新性发现为：（1）PKCalpha和PKCepsilon亚型以不同分子途径抑制IKr，PKCalpha和PKCepsilon亚型分别中介两个G-蛋白偶联受体alpha1A受体和AT1受体对IKr的下调作用；（2）cPKC和PKCepsilon分别通过KCNQ1和/或KCNE1不同的磷酸化位点对IKs电流分别发挥相反的上调、下调作用，Ang II主要通过PKCepsilon对IKs发挥抑制作用；（3）醛固酮激活盐皮质激素受体通过基因转录途径抑制KCNQ1和KCNE1的表达从而下调IKs引起心肌复极化的延迟；（4）低剂量螺内酯（为心衰治疗指南推荐用量的一半）与厄贝沙坦联用具有与常规剂量相似的抗心肌肥厚作用，但低剂量并不引发血钾升高。.上述研究发现为理解心肌PKC亚型特异性调控IKr和IKs通道以及病理情况下通道功能改变的机制提供了新的认识，同时为Ang II-醛固酮系统抑制药物的抗心律失常有益作用提供了机制性解释，并为醛固酮拮抗剂的临床应用剂量提供参考。