Studies Of The Mechanisms Of AngⅡ Induces Atrial Fibrosis Through Inhibit Bkca Channel

objective: The proliferation of fibroblasts and their differentiation into myofibroblasts is the main pathological basis of myocardial fibrosis.AngII is the most effective factor in RAAS,which can promote the process of myocar dial fibrosis.Recent studies have found that there are a large number of BKca channels in human atrial fibroblasts,which are associated with the proliferation of fibroblasts.Therefore,the aim of this study is to explore the role of BKca channel in Ang II-induced myocardial fibrosis by culturing human atrial fibroblasts in vitro,whole-cell patch-clamp recording and molecular cloning.Methods: In this study,human atrial fibroblasts were cultured in tissue clumpattached method,subcultured to the second generation,and divided into three group: Control group.The fibroblasts did not receive any treatment;Ang II group,the cells were treated with 500 nmol / L Ang II incubation;BKca αsubunit overexpression group,the cells were transfected with BKca α subunit(CMV/ KCNMA1/IRES/ac GFP1)by lipofectamin 3000.After treatment,q RT-PCR and western blotting were used to detect the m RNA and protein expression levels of fibrotic markers(α-SMA,Collagen I,Collagen III)and BKca α and β subunits before and after intervention,Whole-cell patch clamp technique were used to record BKca current before and after Ang II application to observe: 1)The characteristics of BKca channel current in human atrial fibroblast cells;2)The effect of AngII on BKca Channel Current in Fibroblasts;Results: 1)Ang II up-regulates m RNA and protein expression of α-SMA,Collagen I,Collagen III on human atrial fibroblasts.? Compared with the control group,the mRNA expression of α-SMA(0.893±0.058 vs 0.282±0.072,P=0.004)、Collagen I(0.858±0.060 vs 0.306±0.058,P=0.018)、Collagen III(0.946±0.054 vs 0.447±0.041,P=0.030)on fibroblasts was significantly up-regulated by 500 nmol / L Ang II.? Compared with the control group,the protein expression of α-SMA(0.967±0.038 vs 0.732±0.037,P=0.020)、Collagen I(0.857±0.010 vs 0.505±0.010,P=0.020)、Collagen III(0.975±0.030 vs 0.626±0.010,P=0.009)on fibroblasts was significantly up-regulated by 500 nmol / L Ang II.2)Ang II down-regulate m RNA and protein expression of BKca α and β subunits on human atrial fibroblasts.?Compared with the control group,the m RNA expression level of BKca αsubunit(9.930±0.952 vs 0.709±0.223,P = 0.001)and β subunit 5.039±0.268 vs1.390±0.640,P=0.020)were significantly down-regulated by 500 nmol / L Ang II treatment for 24 h.? Compared with the control group,the protein expression level of BKca α subunit(0.781±0.0728vs1.232±0.043,P=0.006)andβ subunit(0.872±0.0530 vs1.333±0.502,P=0.001)were significantly down-regulated by 500 nmol/L Ang II treatment for 72 h.3)Characteristics of BKca Channel Current in Human Atrial Fibroblasts.The channel has inward rectification and voltage dependency,which can be specifically blocked by IBTX.Pretreatment of Ang II for 24 hours can inhibit BKca macro current amplitude of fibroblasts,at +60 mV voltage,BKca current density decreased from(31.054 ±2.900 p A/p F)to(13.508±3.000 p A/p F).4)Effects ofoverexpression of BKca α subunit(KCNMA1)on m RNA and protein expression of α-SMA,Collagen I,Collagen III on fibroblasts.? compared with the control group,the mRNA expression level of α-SMA(0.524±0.097 vs1.219±0.150,P =0.007)、Collagen I(0.482±0.084 vs 1.023±0.134,P =0.020)、Collagen III(0.664±0.010 vs 1.081±0.138,P =0.036)were significantly down-regulated by overexpression KCNMA1 in fibroblasts.? Compared with the control group,the protein expression level of α-SMA(0.391±0.015 vs0.732±0.0368,P=0.001)、Collagen I(0.227±0.010 vs 0.505±0.010,P=0.001)、Collagen III(0.156±0.040 vs 0.626±0.010,P=0.008)were significantly down-regul at ed by overexpre ssi on KCNMA1 in fi br obl ast s.Conclusions: Our study confirmed: 1)Ang II can induce myocardial fibrosis.2)Ang II can inhibit the mRNA and protein expression of BKca and the current density of BKca on human atrial fibroblasts.3)Overexpression of BKca can downregulate the expression of fibrosis markers.4)Our study suggestion that Ang II may promote the process of myocardial fibrosis by inhibiting the activity and expression of the BKca channel.BKca channel may be a potential target for the prevention and treatment of myocardial fibrosis.