Han Z L, Wu X, Liu X H, Chen Z, Bai J, Chen X, Xu W
Department of Cardiology, Nanjing Drum Tower Hospital, Medical School of Nanjing University, Nanjing 210008, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2020 Nov 24;48(11):954-961. doi: 10.3760/cma.j.cn112148-20200929-00778.
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. 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 1 μg/ml PDK1 short hairpin RNA (shRNA) interference plasmid), SC79 group (treated with 8 μmol/ml SC79), GSK2334470 group (treated with 10 nmol/L GSK2334470) and PDK1 knockdown+SC79 group (8 μmol/ml SC79 and 1 μg/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. (1) the expression levels of HCN4 mRNA (1.46±0.03 vs. 0.99±0.01, <0.001) and protein (1.14±0.02 vs. 1.00±0.06, =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, =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, <0.001) and protein (2.33±0.11 vs. 1.00±0.05, <0.001) in GSK2334470 group were higher than those in drug control group. (4) To reduce the effect of drug-miss target, the cultured atrial myocytes were transfected with shRNA plasmid of PDK1 and intervened with SC79. The results showed that the expression of HCN4 mRNA in PDK1 knockdown group was higher than that in the drug control group (1.76±0.11 vs. 1.00±0.06, <0.001), and PDK1 knockdown+SC79 group (1.76±0.11 vs. 1.33±0.07, =0.003). In PDK1 knockdown+SC79 group, the mRNA expression level was also higher than that in the drug control group (1.33±0.07 vs. 1.00±0.06, <0.001). The expression level of HCN4 protein in PDK1 knockdown group was higher than that in drug control group (1.15±0.04 vs. 1.00±0.05, =0.003). As for the The expression level of HCN4 protein, there was no significantly statistical difference between the PDK1 knockdown+SC79 group and the drug control group (>0.05), but PDK1 knockdown+SC79 group was lower than PDK1 knockdown group (0.95±0.01 vs. 1.15±0.04, <0.001). In patch clamp experiments, the results showed that the HCN current density was (-13.27±1.28) pA/pF in the drug control group, (-18.76±2.03) pA/pF in the PDK1 knockdown group, (-13.50±2.58) pA/pF in the PDK1 knockdown+SC79 group; the HCN current density of PDK1 knockdown group was higher than that of drug control group (<0.001), but there was no significant difference between PDK1 knockdown+SC79 group and drug control group (>0.05). (5) The results of immunofluorescence showed that the brightness of green fluorescence of PDK1 knockdown group was higher than that of drug control group, indicating that the expression of HCN4 localized on cell membrane was increased. However, the green fluorescence of PDK1 knockdown+SC79 group was lighter than that of PDK1 knockdown group, suggesting that the expression of HCN4 in PDK1-knockdown cell membrane decreased after further activating Akt. PDK1-Akt signaling pathway is involved in the regulation of HCN4 ion channel transcription, expression and function.
探讨3-磷酸依赖性蛋白激酶1-蛋白激酶B（PDK1-Akt）信号通路对心脏超极化激活环核苷酸门控4（HCN4）离子通道转录、表达及功能的影响。采用酶解法从健康雄性野生型C57小鼠和心脏特异性PDK1基因敲除小鼠（PDK1-KO）获取心房肌细胞。将心房肌细胞分为空白对照组和PDK1-KO组。在进一步研究中，将分离的心房肌细胞培养后再分为药物对照组（用二甲基亚砜（DMSO）处理）、PDK1敲低组（用1μg/ml PDK1短发夹RNA（shRNA）干扰质粒处理）、SC79组（用8μmol/ml SC79处理）、GSK2334470组（用10nmol/L GSK2334470处理）以及PDK1敲低+SC79组（8μmol/ml SC79和1μg/ml PDK1 shRNA干扰质粒）。采用实时定量PCR（qRT-PCR）检测PDK1和HCN4的mRNA表达水平，蛋白质免疫印迹法检测PDK1、Akt和HCN4的蛋白质表达水平，全细胞膜片钳技术检测HCN的电流密度，免疫荧光法检测心房细胞上HCN4蛋白的表达。（1）PDK1-KO组HCN4 mRNA表达水平（1.46±0.03 vs. 0.99±0.01，<0.001）和蛋白质表达水平（1.14±0.02 vs. 1.00±0.06，=0.017）均高于空白对照组。PDK1-KO组的HCN电流密度高于空白对照组（（-17.47±2.00）pA/pF vs.（-12.15±2.25）pA/pF，=0.038）。（2）通过比较PDK1敲低组和SC79组与药物对照组，验证了PDK1 shRNA和特异性Akt激动剂SC79的作用。结果显示，PDK1敲低组中PDK1 mRNA和蛋白质表达水平低于药物对照组，SC79组中磷酸化Akt（Thr 308）蛋白表达水平高于药物对照组。（3）GSK2334470组中HCN4 mRNA表达水平（3.61±0.46 vs. 1.00±0.08，<0.001）和蛋白质表达水平（2.33±0.11 vs. 1.00±0.05，<0.001）均高于药物对照组。（4）为减少药物脱靶效应，用PDK1的shRNA质粒转染培养的心房肌细胞并给予SC79干预。结果显示，PDK1敲低组中HCN4 mRNA表达高于药物对照组（1.76±0.11 vs. 1.00±0.06，<0.001），PDK1敲低+SC79组（1.76±0.11 vs. 1.33±0.07，=0.003）。在PDK1敲低+SC79组中，mRNA表达水平也高于药物对照组（1.33±0.07 vs. 1.00±0.06，<0.001）。PDK1敲低组中HCN4蛋白表达水平高于药物对照组（1.15±0.04 vs. 1.00±0.05，=0.003）。至于HCN4蛋白表达水平，PDK1敲低+SC79组与药物对照组之间无显著统计学差异（>0.05），但PDK1敲低+SC79组低于PDK1敲低组（0.95±0.01 vs. 1.15±0.04，<0.001）。在膜片钳实验中，结果显示药物对照组的HCN电流密度为（-13.27±1.28）pA/pF，PDK1敲低组为（-18.76±2.03）pA/pF，PDK1敲低+SC79组为（-13.50±2.58）pA/pF；PDK1敲低组的HCN电流密度高于药物对照组（<0.001），但PDK1敲低+SC79组与药物对照组之间无显著差异（>0.05）。（5）免疫荧光结果显示，PDK1敲低组绿色荧光亮度高于药物对照组，表明细胞膜上HCN4的表达增加。然而，PDK1敲低+SC79组的绿色荧光比PDK1敲低组更浅，提示进一步激活Akt后，PDK1敲低细胞膜上HCN4的表达降低。PDK1-Akt信号通路参与HCN4离子通道转录、表达及功能的调控。
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