Kv7.1/Iks负调控Kv1.5/Ikur的机制及其降低房颤易感性的研究

Atrial electrical remodeling plays a critical role in initiating and promoting atrial fibrillation(AF). The essence of AF is multiple ion channels'reconstruction leading to a series of electrical abnormalities. It has been testified that Iks and Ikur patassium channel currents are related to AF. However, the relationship between Iks current and Ikur current is unknown yet. We have found that the change of Kv7.1 protein in the early and late stage of AF is related to Kv1.5 and silencing of Kv7.1 gene by relevant siRNA increases the amount of Kv1.5 protein. Meanwhile, our previous research has shown that AngⅡ/TGF-β1/Smad2/3 pathway may upregulate Kv1.5/Ikur by increasing the expression of SAP97 protein and promote the susceptivity of AF. We assume that Kv7.1 may have an adverse effect on it by the competitive inhibition of SAP97. We aim to observe the changes of Kv7.1、Kv1.5、Smad2/3 and Smad7 by establishing a rabbit AF model and culture of rabbit atrial myocytes which are sujected to AngⅡ and TGF-β1 or siRNA transfection. Quantitative PCR, western blot, immunofluorescence and patch clamp techniques are adopted to investigate the alteration of Kv7.1/Iks,Kv1.5/Ikur and SAP97 in order to clarify the mechanisms underneath. It may provide useful scientific evidence to the development of new therapeutic targets for AF.

心房电重塑是房颤发生发展的主要病理基础，其本质是由于各种离子通道重塑导致的一系列心电活动异常。已证明Iks与Ikur该两种钾离子电流各与房颤有关，但两者的相互影响目前未见报道。我们发现：房颤过程Kv7.1/Iks与Kv1.5/Ikur均有先升后降的规律,应用Kv7.1siRNA转染家兔心房肌细胞24小时后，细胞膜上Kv1.5表达上调；AngⅡ/TGFβ1/Smad2/3通路的激活上调SAP97,进而上调Kv1.5/Ikur电流，增加了房颤的易感性。由此假设Kv7.1通过竞争性抑制SAP97蛋白,与Smad7负调控这一过程殊途同归。本研究建立家兔房颤模型及培养心房肌细胞，予AngⅡ、TGFβ1处理或基因转染改变Kv7.1、Smad2/3、Smad7表达，用定量PCR、蛋白印迹、免疫荧光、膜片钳等技术观察Kv7.1/Iks、Kv1.5/Ikur、SAP97的变化并阐明其机制，开创房颤治疗新靶点

已证明Kv7.1钾通道负载的Iks电流与Kv1.5钾通道负载的Ikur电流均与房颤有关，但两者的相互影响目前未见报道。本研究拟探讨房颤时Kv7.1/Iks、Kv1.5/Ikur、SAP97的相互作用及调控机制。研究内容：房颤过程中SAP97如何调节Kv7.1、Kv1.5的表达；AngII/TGF-β1/Smad2/3及Smad7信号通路参与调控SAP97、Kv7.1、Kv1.5的机制；Kv7.1是否可通过竞争性抑制SAP97而下调Kv1.5的表达；氧化应激参与调控心房Kv1.5表达而影响房颤易感性的机制。我们发现：1.家兔房颤模型中（起搏0-8周），Kv1.5、Kv7.1及SAP97的蛋白表达随起搏时间的延长呈现先增加后减少的特点，在第3周时表达量达到顶峰。Smad7的蛋白表达呈现出先减少后增加的特点,在第3周时表达量最低；2.SAP97蛋白上调可增加心房肌细胞中Kv1.5、Kv7.1的蛋白表达。Kv7.1的蛋白上调可与SAP97结合，竞争性抑制Kv1.5的蛋白表达。3.Ang II干预可上调心房肌细胞Kv1.5及Kv7.1的蛋白表达，沉默Smad2或Smad3可下调Kv1.5的表达，沉默Smad7可抑制Kv7.1的表达；4.沉默SAP97的表达可抑制由Ang II介导的Kv1.5表达。与单纯使用Ang II组比较，使用Kv7.1 siRNA预处理心房肌细胞后再加入Ang II干预，可进一步上调Kv1.5的蛋白表达。与单纯使用Ang II组比较，过表达Kv7.1后再加入Ang II干预，可抑制Ang II诱导的Kv1.5蛋白表达；5.心房颤动时，H2S可通过抑制由NOX-4介导的ROS生成而抑制Ang II诱导的心房Kv1.5的蛋白表达。科学意义：过表达Kv7.1蛋白可通过与SAP97蛋白结合，竞争性抑制Kv1.5的蛋白表达而降低房颤易感性。抑制Ang II/TGF-β1/P-Smad2/3信号通路可下调Kv1.5的表达，提示临床上使用ACEI或ARB类药物可能通过抑制心房Kv1.5而抑制房颤发生。此外，我们的研究提示阻断NOX-4-ROS轴介导的氧化应激有望成为新的房颤治疗途径。