PDK1-Akt信号通路干预对心肌细胞HCN4离子通道的影响  

The effects of PDK1-Akt signaling pathway intervention on cardiomyocyte HCN4 ion channels

作　　者：韩钟霖 吴翔 刘雪华 陈诤 白剑[1] 陈鑫[1] 徐伟 Han Zhonglin;Wu Xiang;Liu Xuehua;Chen Zheng;Bai Jian;Chen Xin;Xu Wei(Department of Cardiology,Nanjing Drum Tower Hospital,Medical School of Nanjing University,Nanjing 210008,China)

机构地区：[1]南京大学医学院附属南京鼓楼医院心血管内科,210008

出　　处：《中华心血管病杂志》2020年第11期954-961,共8页Chinese Journal of Cardiology

基　　金：国家自然科学青年基金(81600267)。

摘　　要：目的探讨3-磷酸肌醇依赖性蛋白激酶1-蛋白激酶B(PDK1-Akt)信号通路干预对心肌细胞超极化激活环核苷酸门控离子通道4(HCN4)转录、表达及功能的影响。方法使用酶解法从健康雄性野生型C57小鼠和心脏特异性敲除PDK1小鼠获得心房肌细胞,分别为空白对照组和PDK1-KO组;另外,对急性分离自C57小鼠的心房肌细胞进行培养,将其分为药物对照组[予二甲基亚砜(DMSO)干预)]、PDK1敲低组[予1μg/ml PDK1的短发夹状RNA(shRNA)干扰质粒干预]、SC79组[予Akt激动剂SC79(8μmol/ml)干预]、GSK2334470组[予PDK1抑制剂GSK2334479(10 nmol/ml)干预]和PDK1敲低+SC79组(予8μmol/ml SC79和1μg/ml PDK1的shRNA干扰质粒干预)。为进一步减少药物脱靶的可能,故运用PDK1的shRNA质粒转染培养的心肌细胞,并在此基础上加用SC79干预。采用实时荧光定量PCR(qRT-PCR)检测相应组心房肌细胞PDK1及HCN4的mRNA表达水平,Western blot检测PDK1、Akt及HCN4蛋白表达水平,全细胞膜片钳检测HCN的电流密度,免疫荧光技术检测HCN4蛋白表达情况。结果(1)PDK1-KO组的HCN4的mRNA(1.46±0.03比0.99±0.01,P<0.001)及蛋白(1.14±0.02比1.00±0.06,P=0.017)表达水平高于空白对照组。全细胞膜片钳结果显示,PDK1-KO组的HCN电流密度大于空白对照组[(-17.47±2.00)pA/pF比(-12.15±2.25)pA/pF,P=0.038]。(2)通过比较PDK1敲低组、SC79组和药物对照组细胞,对PDK1 shRNA及Akt特异性激动剂SC79进行功能验证,结果显示PDK1敲低组细胞的PDK1 mRNA及蛋白表达水平低于药物对照组,SC79组细胞的磷酸化-Akt(Thr 308)蛋白表达水平高于药物对照组。(3)GSK2334470组细胞的HCN4 mRNA(3.61±0.46比1.00±0.08,P<0.001)及蛋白(2.33±0.11比1.00±0.05,P<0.001)表达水平高于药物对照组。(4)PDK1敲低组细胞的HCN4 mRNA表达水平高于药物对照组(1.76±0.11比1.00±0.06,P<0.001)及PDK1敲低+SC79组(1.76±0.11比1.33±0.07,P=0.003),PDK1敲低+SC79组的HCN4 mRNA表达水平亦高于药物对照组(1.3Objective To explore the effects of 3-phosphate dependent protein kinase 1-protein kinase B(PDK1-Akt)signaling pathway on the transcription,expression and function of cardiac hyperpolarized activated cyclic nucleotide gated 4(HCN4)ion channels.Methods Atrial myocytes were obtained from healthy male wild-type C57 mice and heart-specific PDK1 knockout mice(PDK1-KO)by enzymolysis.Then the atrial myocytes were divided into blank control group and PDK1-KO group.In further studies,the isolated atrial myocytes were cultured and further divided into drug control group(treated with dimethyl sulfoxide(DMSO))and PDK1 knockdown group(treated with 1μg/ml PDK1 short hairpin RNA(shRNA)interference plasmid),SC79 group(treated with 8μmol/ml SC79),GSK2334470 group(treated with 10 nmol/L GSK2334470)and PDK1 knockdown+SC79 group(8μmol/ml SC79 and 1μg/ml PDK1 shRNA interference plasmid).Real time quantitative PCR(qRT-PCR)was used to detect the mRNA expression levels of PDK1 and HCN4,Western blot was used to detect the protein expression levels of PDK1,Akt and HCN4,the whole cell patch clamp was used to detecte the current density of HCN,and immunofluorescence was used to detecte the expression of HCN4 protein on atrial cells.Results(1)the expression levels of HCN4 mRNA(1.46±0.03 vs.0.99±0.01,P<0.001)and protein(1.14±0.02 vs.1.00±0.06,P=0.017)in PDK1-KO group were higher than those in blank control group.The HCN current density in PDK1-KO group was higher than that in blank control group((-17.47±2.00)pA/pF vs.(-12.15±2.25)pA/pF,P=0.038).(2)The functions of PDK1 shRNA and specific Akt agonist SC79 were verified by comparing the PDK1 knockdown group and SC79 group with the drug control group.The results showed that the expression levels of PDK1 mRNA and protein in PDK1 knockdown group were lower than those in drug control group,and the expression level of phosphorylated Akt(Thr 308)protein in SC79 group was higher than that in drug control group.(3)The expression levels of HCN4 mRNA(3.61±0.46 vs.1.00±0.08,P<0.001)and protein

关 键 词：肌细胞 心脏 HCN4 PDK1 AKT 

分 类 号：R542.2[医药卫生—心血管疾病]