Ang II和Ang-(1-7)对心房成纤维细胞钙平衡和钙处理相关蛋白的影响和相互作用机制研究

Atrial interstitial fibrosis is one of the basic mechanisms of occurrence and maintenance of atrial fibrillation, atrial fibroblasts are regarded as the direct executor of interstitial fibrosis remodeling, and become a new research hotspot. Ang- (1-7) as an endogenous antagonist of Ang II can improve myocardial remodeling, but its mechanism mediated has not been fully elucidated. Inspired from the different effects of Ang II and Ang-(1-7) on calcium ions, the present study for the first time will focus on dynamic regulation of calcium ion balance by profibrotic proliferative of Ca2+/CaMKIIδ and antifibrotic NO/cGMP/PKGβ1, and will explore theirs roles and mechanisms in fibroblasts proliferation and phenotype transformation. From atrial fibroblasts and animal models of two aspects, this study will focus on the effects of Ang II and Ang-(1-7) on Ca2+/CaMKIIδ and NO/cGMP/PKGβ1, and theirs mechanisms of interaction, and eventually clarify the mechanisms of Ang-(1-7) improving atrial fibrosis and remodeling. With the application and development of clinical drug based on Ang-(1-7), the elucidation of the mechanisms is important for opening new ideas of prophylaxis and treatment and finding new therapeutic targets.

心房肌间质纤维化是心房纤颤发生与维持的基本机制之一，成纤维细胞则是间质纤维化重构的直接执行者，并成为新的研究热点。Ang-(1-7)作为内源性Ang II的拮抗剂具有改善心肌重构的作用，然而其介导的信号转导机制尚未完全阐明。本研究从Ang II和Ang-(1-7)对钙离子的不同作用中受到启发，在国内外首次聚焦促纤维化增殖的Ca2+/CaMKIIδ与抗纤维化的NO/cGMP/PKGβ1对钙离子平衡的动态调节及其在成纤维细胞增殖与表型转化中的作用机制，分别从细胞和动物两个层面，着力探讨Ang II和Ang-(1-7)对Ca2+/CaMKIIδ、NO/cGMP/PKGβ1的影响和相互作用机制，最终阐明Ang-(1-7)改善心房纤维化重构的分子机制。随着基于Ang-(1-7)的临床药物应用与开发，这一机制的阐明对于开启心房纤颤防治的新思路和寻找新的治疗靶点具有重要意义。

脏成纤维细胞的表型转化所致心脏纤维化是诱发心律失常、心脏收缩功能损害的重要病理基础。近年来发现NCX、CaMKII是介导成纤维细胞的表型转化重要的信号分子。本研究以心脏成纤维细胞为研究对象，以CaMKIIδ为核心，分别在体内外实验中评估在高RAAS活化状态下，Ang-(1-7)对Ca2+、CaMKIIδ、NCX1、TRPV4等与钙离子及钙处理相关蛋白影响，阐明 Ang-(1-7)对钙离子平衡的动态调节机制，明确Ang-(1-7)改善心脏纤维化重构的分子机制。在Ang II诱导下，体外培养的原代心脏成纤维细胞快速向肌成纤维细胞转化，伴随着细胞内Ca2+、ROS、NCX1、p-CaMKII、ox-CaMKII表达的显著增加，CaMKII活化促进了TGF-β1、α-SMA、Collagen、CX43等细胞表型转化相关蛋白表达；通过Fluo 4-AM探针实时监测成纤维细胞内Ca2+变化，发现在Ang II诱导下NCX1通过参与SOCE机制介导下的Ca2+内流，促使细胞内Ca2+持续升高而保持CaMKII的过度激活；而 NCX1抑制剂则显著抑制Ang II诱导下的Ca2+内流，降低成纤维细胞表型转化，表明Ang II/NCX1/Ca2+/CaMKII是重要的促成纤维细胞增殖和转化的通路。在细胞内Ca2+层面，Ang-(1-7)预处理细胞48h可抑制 NCX1的表达，因而减少SOCE过程中Ca2+的内流，同时Ang-(1-7)/eNOS/NO通路降低细胞内ROS水平；因而低水平Ca2+、ROS抑制了CaMKII的钙激活及氧化激活，降低成纤维细胞增殖及表型转化，表明Ang-(1-7)/eNOS/NO可通过细胞内钙稳态的再平衡及抗氧化机制拮抗Ang II/NCX1/Ca2+/CaMKII促增殖、促表型转化作用。上述机制也在SD大鼠体内水平也得到了进一步验证。在可预期的成果外，我们创新性的联合应用细胞代谢组学、KEGG数据库评估Ang II诱导下的心脏成纤维代谢产物的变化，发现花生四烯酸代谢及γ-谷氨酰循环紊乱与Ang II所致与心脏成纤维细胞异常激活相关。实验研究则证实了Ang II/AA/ TRPV4/Ca2+通路诱导下的Ca2+入胞在促表型转化中的重要作用，Ang-(1-7)通过减少AA、ROS堆积抑制心脏成纤维细胞CaMKIIδ异常钙激活及氧化激活。本研究明确了钙离子及钙处理