Src激酶参与肿瘤坏死因子α诱导的心房肌细胞超快速延迟整流钾电流下调  

Involvement of Src kinase in the down-regulation of ultra-rapid delayed rectifier K^+current induced by tumor necrosis factor-αin cardiomyocytes

作　　者：周慧珊 王钊煜 高小燕 邓春玉[3] 薛玉梅[2] 杨慧[3] 李昕 邝素娟[3] 彭德威 饶芳[3] 吴书林 Zhou Huishan;Wang Zhaoyu;Gao Xiaoyan;Deng Chunyu;Xue Yumei;Yang Hui;Li Xin;Kuang Sujuan;Peng Dewei;Rao Fang;Wu Shulin(School of Medicine,South China University of Technology,Guangdong 510006,China;Department of Cardiology,Guangdong Cardiovascular Institute,Guangdong 510080,China;Research Department of Medical Sciences,Guangdong Provincial People′s Hospital,Guangdong 510080,China)

机构地区：[1]华南理工大学医学院,广州510006 [2]广东省心血管病研究所心内科,广州510080 [3]广东省人民医院医学研究部,广州510080

出　　处：《中华心血管病杂志》2020年第4期323-328,共6页Chinese Journal of Cardiology

基　　金：国家自然科学基金(81670314,81870254);广州市科技计划项目(201804010059)。

摘　　要：目的探讨肿瘤坏死因子α(TNF-α)是否通过调控超快速延迟整流钾电流(Ikur)参与心房肌细胞的电重塑,以及Src激酶是否参与其中。方法将大鼠胚胎心肌细胞株H9c2细胞置于DMEM培养基中,将小鼠心房肌细胞株HL-1细胞置于Claycomb培养基中,于37℃5%CO2培养箱中培养。以正常培养的细胞为对照组。予以不同浓度TNF-α干预细胞24 h,分别为TNF-α25 ng/ml组、TNF-α50 ng/ml组和TNF-α100 ng/ml组。为观察Src抑制剂PP1能否逆转TNF-α作用,设置PP1+TNF-α组:先予10μmol/L的PP1预处理1 h后再加入100 ng/ml TNF-α干预细胞24 h。采用Western blot检测各组细胞中形成Ikur的主要钾通道蛋白Kv1.5、Src的表达水平。采用全细胞膜片钳检测各组细胞的Ikur密度。结果(1)H9c2细胞中,TNF-α100 ng/ml组细胞的Kv1.5蛋白表达水平低于对照组及TNF-α25 ng/ml组(P均<0.05)。各组细胞间Src蛋白表达水平差异无统计学意义(P>0.05),但TNF-α25 ng/ml组、TNF-α50 ng/ml组及TNF-α100 ng/ml组细胞磷酸化Src(p-Src)蛋白表达水平均高于对照组(P均<0.05)。TNF-α50 ng/ml组及TNF-α100 ng/ml组细胞Ikur电流密度均小于对照组(P均<0.05)。PP1+TNF-α组细胞Kv1.5蛋白表达水平高于TNF-α100 ng/ml组(P<0.05),Ikur电流密度大于TNF-α100 ng/ml组(P<0.01)。PP1+TNF-α组细胞Kv1.5蛋白表达水平及Ikur电流密度与对照组比较差异均无统计学意义(P均>0.05)。(2)在心房肌细胞株HL-1细胞中,TNF-α100 ng/ml组细胞Kv1.5蛋白表达水平低于对照组和TNF-α25 ng/ml组(P均<0.01)。TNF-α100 ng/ml组细胞p-Src蛋白表达水平高于对照组(P<0.05)。各组细胞Src蛋白表达水平差异无统计学意义(P>0.05)。PP1+TNF-α组细胞Kv1.5蛋白表达水平高于TNF-α100 ng/ml组(P<0.05)。结论在心房肌细胞中,TNF-α可能通过激活Src激酶降低Kv1.5蛋白表达水平,进而下调Ikur,参与心房颤动的发生及发展过程。Objective To investigate whether inflammatory factor tumor necrosis factor-α(TNF-α)is involved in the electrical remodeling of cardiomyocytes by regulating ultra-rapid delayed rectifier K+current(Ikur)and the role of Src kinase.Methods H9c2 cells,embryonic cardiomyocytes of rat,were cultured in Dulbecco′s modified Eagle′s medium(DMEM)and atrium-derived HL-1 cells were cultured in Claycomb medium.Both H9c2 and HL-1 cells were cultured at 37℃with 5%CO2.Cells cultured in normal conditions without additional treatment served as control group.Experimental groups were treated with different concentration of TNF-α(25 or 50 or 100 ng/ml)for 24 hours.To study whether Src specific inhibitor PP1 could abrogate the effect of TNF-α,cells were pre-treated with 10μmol/L PP1 for 1 hour,followed by TNF-α(100 ng/ml)for 24 hours.Western blot and the whole cell patch clamp technique were used to detect the protein expression of Kv1.5 and Src and Ikur in each group.Results(1)In H9c2 cells,high concentration of TNF-αtreatment(100 ng/ml)significantly reduced the Kv1.5 protein expression compared with control group and TNF-α25 ng/ml group(both P<0.05).Compared with control group,the expression of p-Src protein was higher in 25 ng/ml,50 ng/ml,100 ng/ml TNF-αgroup(all P<0.05),but there was no statistical difference in the expression of Src protein among groups(P>0.05).In addition,the current density of Ikur was decreased in 50 ng/ml,100 ng/ml TNF-αgroup(both P<0.05).Furthermore,the expression of Kv1.5 protein and the current density of Ikur were increased in PP1+TNF-αgroup compared with TNF-α100 ng/ml group(both P<0.05).There was no statistical difference in the expression of Kv1.5 protein and the current density of Ikur between the control group and PP1+TNF-αgroup(both P>0.05).(2)In atrium-derived HL-1 cells,the expression of Kv1.5 protein was reduced in 100 ng/ml TNF-αgroup compared with control group and TNF-α25 ng/ml group(both P<0.01).In addition,the expression of p-Src protein was increased in TNF-α100 ng/ml gr

关 键 词：心房颤动 肿瘤坏死因子Α SRC激酶 超快速延迟整流钾电流 心房肌细胞 

分 类 号：R541[医药卫生—心血管疾病]