cGMP介导的PI3K/Akt/GSK-3β信号途径在房颤心房重构中的作用研究

Our Previous study has demonstrated that atrial cGMP level is increased at initial stage of pacing and then decreased in rapid pacing in induced rabbit atrial fibrillation (AF). However the role of cGMP in AF induced atrial remodeling has not been fully understood. Other disease model have showed that cGMP regulates PI3K/Akt/GSK-3β signaling pathway, and this effect includes inhibition of GSK-3 β through the activity of PKG or by a dual mechanism of protein phosphatase negative regulation of Akt. This project is based on previous study: (1) Build a model of rabbit rapid pacing AF to detect the changes of cGMP, effects of changes on the distribution of calcium and electrophysiology atrial structural changes, open mPTP, intracellular cGMP, analysis of influence caused by the change of the activity of PKG and PI3K/Akt/GSK-3β signal molecular changes of atrial remodeling; (2) Obseve pharmacological and physiological effects of atrial dynamics and ANP PI3K/Akt/GSK-3 signaling induced changes in cGMP concentrations secretion by using the AF model of isolated perfused heart. This will provide the scientific evidence of cGMP signal pathway induced by PI3K/Akt/GSK-3β reliable of atrial remodeling, and new targets and the theoretical basis for further understand the mechanism of atrial remodeling in AF.

我们在前期研究发现房颤模型家兔心房组织cGMP水平先增后减，但是cGMP浓度变化在房颤心房重构中的作用及机制尚不清楚。其他疾病模型研究表明cGMP通过PKG和蛋白磷酸酯酶调控PI3K/Akt/GSK-3β，即通过PKG抑制GSK-3β的活性和通过蛋白磷酸酯酶负性调控Akt的双重机制。本项目拟在现有研究基础上：（1）采用家兔快速起搏房颤模型，检测cGMP变化对心房结构变化、mPTP开放、胞内钙分布及电生理学改变的影响，分析cGMP变化造成的PKG和蛋白磷酸酯酶活性及PI3K/Akt/GSK-3β信号分子的改变对心房重构的影响；（2）采用房颤模型离体灌流心房，检测心房动力学和ANP分泌，探讨房颤中cGMP调控PI3K/Akt/GSK-3β信号通路的分子机制，旨在获得cGMP通过PI3K/Akt/GSK-3β信号途径引起房颤心房重构的可靠证据。本项目有助于揭示cGMP在房颤心房重构中的作用机制。

心房颤动是最常见的心律失常之一，临床危害严重，迄今为止其确切的发病机制仍不清楚。心房重构（AR）是房颤发生、持续及复律后复发的关键因素。PI3K/Akt/GSK-3β信号通路的激活通过调控胞内钙浓度、线粒体膜稳定性以及细胞凋亡发挥心肌保护作用，多种心肌保护因子都通过此信号途径的激活发挥保护作用。cGMP是参与心脏功能调节的一种细胞内第二信使，可通过PKG或磷酸酯酶调控PI3K/Akt/GSK-3β信号途径而保护心肌。本研究（1）成功制备了家兔快速起搏心房房颤模型，证实血中ANP浓度增高、心房肌组织中cGMP先增高后降低，透射电子显微镜观察到心房肌超微结构发生改变，心房肌mPTP异常开放、胞内Ca2+异常分布，VASP（PKG底物）、Akt和GSK-3β磷酸化程度降低，表明cGMP- PKG和Akt/GSK-3β参与快速起搏引起的心房重构。（2）制备了房颤模型离体灌流心房，观察阻断PI3K/Akt/GSK-3β信号转导对cGMP浓度变化引起的心房动力学和ANP分泌的影响，结果发现高频刺激增加ANP分泌，降低了心房搏出量；cGMP抑制ANP分泌，降低心房搏出量； Akt和GSK-3β抑制剂分别预处理后，阻断cGMP诱导的ANP分泌减少，增强cGMP对心房动力的抑制效应，证实了cGMP介导的PI3K/Akt/GSK-3β信号途径参与房颤心房动力学改变和ANP分泌的调节。（3）西地那非（sildenafil）能减轻快速心房起搏诱导的房颤的发生，Western blot结果显示，心房肌组织中VASP、Akt和GSK-3β磷酸化程度与对照组相比有所提高，进一步证实了cGMP通过增强Akt的活性拮抗房颤发生发展过程中的心肌损伤。总之，通过本研究证实cGMP介导的PI3K/Akt/GSK-3β信号通路参与房颤心房重构，初步阐明了cGMP变化在房颤心房重构中的作用及其下游信号靶点调控房颤心房动力变化和ANP分泌异常的分子机制，为了解cGMP在房颤发生、发展和维持中的作用提供了科学的实验依据。