血管紧张素Ⅱ通过抑制人心房成纤维细胞BKCa通道诱导心房纤维化

Angiotensin Ⅱ Induces Atrial Fibrosis by Inhibiting BKCa Channel in Human Atrial Fibroblast

目的探讨在血管紧张素Ⅱ(AngⅡ)诱导心房纤维化的过程中,大电导钙激活钾通道(BKCa通道)的作用机制。方法通过组织块贴壁法获取原代人心房成纤维细胞,使用免疫荧光染色进行鉴定。用浓度为500 nmol/L的AngⅡ处理人心房成纤维细胞24 h,实时荧光定量PCR与蛋白质印迹法用于检测处理前后纤维化标志基因α-平滑肌肌动蛋白(α-SMA)、胶原蛋白Ⅰ(collagenⅠ)和胶原蛋白Ⅲ(collagenⅢ),以及BKCa通道的α与β亚基的mRNA和蛋白表达水平,全细胞膜片钳技术检测AngⅡ处理前后的BKCa通道的电流变化。结果(1)人心房成纤维细胞经AngⅡ处理后,α-SMA、collagenⅠ和collagenⅢ的mRNA和蛋白表达水平升高;(2)经过AngⅡ处理后,BKCa通道α及β亚基mRNA和蛋白表达水平降低;(3)人心房成纤维细胞存在功能正常的BKCa通道,具有电压依赖性;(4)人心房成纤维细胞BKCa通道的宏观电流幅度在经AngⅡ处理后降低;(5)在人心房成纤维细胞上过表达BKCa通道α亚基后,纤维化标志物α-SMA、collagenⅠ和collagenⅢ的表达受到了明显抑制。结论AngⅡ可能通过抑制人成纤维细胞BKCa通道的表达和功能来诱导人心房成纤维细胞的纤维化,并最终导致心房纤维化。

Objective To investigate the mechanism of large conductance calcium-activated potassium channel(BKCa)in angiotensin Ⅱ(AngⅡ)-induced atrial fibrosis.Methods Primary human atrial fibroblasts were obtained by tissue block attachment method and identified by immunofluorescence staining.Human atrial fibroblasts were treated with AngⅡ(500 nmol/L)for 24 h.Real-time fluorescent quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of fibrosis marker genes α-SMA,collagen Ⅰ and collagen Ⅲ,as well asαandβsubunits of BKCa channels before and after treatment.And whole cell patch clamp technique was used to detect the current changes of BKCa channels before and after AngⅡ treatment.Results(1)After AngⅡ treatment of human atrial fibroblasts,the mRNA and protein expression levels ofα-SMA,collagen Ⅰ and collagen Ⅲ increased;(2)After AngⅡ treatment,the mRNA and protein expression of BKCa channel α and β subunits decreased;(3)Human atrial fibroblasts exist normal BKCa channel,which are voltage dependent;(4)Macro current amplitude of BKCa channel in human atrial fibroblasts decreased after AngⅡ treatment;(5)After overexpression of BKCa channelαsubunit on human atrial fibroblasts,the mRNA and protein expression levels of fibrosis markerα-SMA,collagen Ⅰ and collagen Ⅲ decreased significantly.Conclusion AngⅡ may induce fibrosis in human atrial fibroblasts by inhibiting the expression and function of BKCa channel,and finally induce atrial fibrosis.