血管紧张素Ⅱ调控钙激活钾通道参与心房纤颤的作用与机制

Atrial fibrillation (AF) is the most common arrhythmia in clinical setting, and traditional pharmacological approaches have proved to have important weaknesses. Ca2+-activated K+ (KCa) channels are divided into three classes based on their single channel conductance: small ('SK')-, intermediate ('IK')- and big ('BK')-conductance channels. SK channels selectively distribute in human and mouse atrium and play a role in late repolarization phase of action potential, while IK and BK channels are present in cardiac fibroblasts and participate in the proliferation and differentiation of the cells. Increased local production of angiotensin II (Ang II) in the heart is involved in the promotion of atrial fibrosis and AF. However, there is no evidence whether or not Ang II regulates the KCa channels in atrium. We hypothesize that Ang II would enhance activator protein-1 (AP-1) via Ang II type 1 receptors (AT1R), then regulate the expression and function of different KCa channels in atrium, promote electrical and structural remodeling in atrium, and persistent AF. Using patch clamp, real time PCR, western blot, immunohistochemisty, immunocytochemistry and gene knockdown techniques, we attempt to investigate the alterations of KCa channel expression and function in freshly separated cardiac myocytes and fibroblasts from sinus rhythm and AF patients, rabbits of rapid artial pacing chronic AF model, and cultured cardiac fibroblasts of human and rabbits, and the modulation by Ang II. The results will elucidate the role of KCa channels in AF, the regulatory effect of Ang II on KCa channels and the underlying mechenisms, and provide valuable insights for therapeutic approaches to prevent AF.

心房纤颤（AF）是最常见的心律失常，尚无安全有效的治疗药物。钙激活钾（KCa）通道中的SK选择性分布于人和动物心房肌细胞，BK表达于人心脏成纤维细胞，但他们在AF中的作用与调控不清楚。我们发现大鼠心脏成纤维细胞的IK参与细胞增殖并受血管紧张素Ⅱ（Ang II）调控。因而假设：AngⅡ经AT1R介导AP-1活化，调节心房不同KCa通道的表达与功能，促进心房电和结构重构，导致持续性AF。本项目拟采用新鲜分离的窦性节律和AF患者的心房肌细胞和成纤维细胞、家兔左心房快速起搏慢性AF模型及Ang II刺激培养的人和家兔心房成纤维细胞，运用膜片钳、细胞和分子生物学技术，观察AF患者和家兔心房不同KCa通道的表达和功能变化及Ang II的调控作用，Ang II对培养的心房成纤维细胞功能的影响及与KCa通道的关系。以阐明血管紧张素Ⅱ调控钙激活钾通道参与心房纤颤的作用与机制，为AF防治提供理论依据与线索。

心房纤颤（Atrial fibrillation, AF）是最常见的持续性心律失常，目前尚无安全有效的药物。心房纤维化是永久性AF的根本决定因素。但有关心房成纤维细胞（Atrial Fibroblasts, AFs）分化及增殖的分子机制尚知之甚少。钙激活的钾通道（Ca2+-activated K+ channels, KCa）分为小电导（SK）、中电导（IK）和大电导（BK）三个亚类。我们前期细胞实验提示血管紧张素Ⅱ（Ang II）通过AT1R调控IK（KCa3.1）促进心室肌成纤维细胞增殖。我们假设AngⅡ经AT1R介导相关的信号途径调节AFs上的KCa通道的表达与功能，促进心房结构重构导致持续性AF。.我们采用比格犬左心房手术植入起搏器，持续8周快速起搏Burst刺激诱发AF，建立犬慢性快速起搏AF模型。 模型犬心房肌组织纤维化明显，KCa2.2 （SK）及KCa1.1（BK）表达上调，这些变化可被AT1R阻断剂Losartan缓解。采用SD大鼠皮下埋置AngⅡ恒速释放微量泵（500ng/kg/min，4周）的方法建立AngⅡ灌注诱导大鼠心房纤维化模型。模型大鼠心房组织发生明显纤维化；I型和III型胶原蛋白表达增多，I/III胶原比值升高；KCa3.1、KCa2.2和KCa2.3（SK）表达上调，这些变化可被KCa3.1通道阻断剂TRAM-34或Losartan缓解。建立酶消化组织块法培养人AFs及酶消化法培养成年大鼠AFs。AngⅡ孵育大鼠AFs，可促进其增殖，上调KCa3.1表达，促进胶原合成与分泌，这些作用可被TRAM-34逆转。进一步研究表明AngⅡ通过激活Nox/ERK1/2/NF-κB及Nox/JNK/AP-1信号通路上调成年AFs上KCa3.1通道的表达。此外，我们通过复制压力超负荷大鼠模型证实，在体阻断KCa3.1通道可逆转压力超负荷所致：血压、心体比值及血浆和心肌AngⅡ含量升高；心肌结构改变及胶原沉积；心肌组织KCa3.1表达明显增加；炎细胞浸润。.上述已完成的研究证实AngⅡ调控IK/BK通道促进心房纤维化，为AF的发生提供结构基础；提示AngⅡ调控SK通道促使心房发生电重塑，为AF的发生提供电生理基础。