钠泵α2亚基介导低浓度哇巴因影响大鼠心室肌细胞收缩力的作用研究

Effect of Sodium Pump α2 Subunit Mediating Low- concentration Ouabain on the Contractility of Rat Myocardial Cells

背景强心苷类(CGs)药物临床治疗心力衰竭因其治疗窗窄使应用受到很大限制。心肌钠泵是CGs药物作用的靶点,然而CGs药物与钠泵相互作用的机制仍有待阐明。目前认为,钠泵的第一个膜外区H1-H2是哇巴因可能的结合位点。目的利用作用在抗心肌钠泵α2亚基H1-H2膜外区结构域的多克隆WJS为工具,封闭该结合位点,有效拮抗哇巴因对钠泵的抑制作用,比较封闭前后哇巴因对心室肌细胞钠泵活性的影响及心室肌细胞收缩力、细胞内钙的变化,探讨钠泵与哇巴因的作用机制。方法 2013年1月-2015年1月急性分离大鼠心室肌细胞,将心室肌细胞分为4组,1μmol/L哇巴因组、WJS+1μmol/L哇巴因组、1 mmol/L哇巴因组、WJS+1μmol/L哇巴因组。1μmol/L哇巴因组给予哇巴因(1μmol/L)进行灌流;WJS+1μmol/L哇巴因组以WJS(稀释浓度1:1000)孵育心室肌细胞0.5 h后,再给予哇巴因(1μmol/L)进行灌流;1 mmol/L哇巴因组给予哇巴因(1mmol/L)进行灌流;WJS+1 mmol/L哇巴因组以WJS(稀释浓度1:1000)孵育心室肌细胞0.5h后,再给予哇巴因(1 mmol/L)进行灌流。采用全细胞膜片钳技术记录钠泵电流(Ip),观察WJS对哇巴因抑制Ip作用的影响;采用激光共聚焦显微镜测定心室肌细胞游离钙浓度([Ca^(2+)]i);采用细胞动缘探测系统测定心室肌细胞收缩力的大小。结果 Western blotting法检测结果显示,纯化后的WJS在电泳时分别出现2条清晰的条带,其分子量均为110 kD左右。细胞免疫荧光法测定结果显示,WJS均结合在心室肌细胞钠泵的膜外区,而且这种结合并不被1μmol/L哇巴因影响,但却被在H1-H2膜外区特异性抗原点具有相同成分的多肽阻断剂RE2取消。WJS+1μmol/L哇巴因组高亲和力钠泵电流(Iph)低于1μmol/L哇巴因组(P<0.05);1 mmol/L哇巴因组、WJS+1 mmol/L哇巴因组Iph高于1μmol/L哇巴因组和WJS+1μmol/L哇巴因组,低亲和力钠泵电流(Ipl)低于1μmol/L哇巴因组和WJS+1μmol/L哇巴因组(P<0.05);WJS+lmmol/L哇巴因组Iph高于1 mmol/L哇巴因组,Ip1低于1 mmol/L哇巴因组(P<0.05)。灌流5、15 min WJS+1μmol/L哇巴因组心室肌细胞[Ca^(2+)]i低于1μmol/L哇巴因组(P<0.05);灌流5、15 min 1 mmol/L哇巴因组、WJS+1 mmol/L哇巴因组心室肌细胞[Ca^(2+)]i高于1μmol/L哇巴因组和WJS+1μmol/L哇巴因组(P<0.05);灌流5、15min WJS+1 mmol/L哇巴因组心室肌细胞[Ca^(2+)]i高于1 mmol/L哇巴因组(P<0.05)。灌流5、15min WJS+1μmol/L哇巴因组心室肌细胞收缩力低于1μmol/L哇巴因组(P<0.05);灌流5 min 1 mmol/L哇巴因组、WJS+1 mmol/L哇巴因组心室肌细胞收缩力高于1μmol/L哇巴因组和WJS+1μmol/L哇巴因组,灌流15min心室肌细胞收缩力低于1μmol/L哇巴因组和WJS+1μmol/L哇巴因组(P<0.05);灌流5、15min WJS+1 mmol/L哇巴因组心室肌细胞收缩力低于1 mmol/L哇巴因组(P<0.05)。结论抗心肌钠泵α2亚基H1-H2膜外区结构域的多克隆抗体WJS取消Iph和低浓度哇巴因所致的心室肌细胞收缩力增强作用,表明钠泵α2亚基介导低浓度哇巴因治疗心力衰竭的作用。

Backgroud The application of CGs has much limitation in the treatment of heart failure due to its therapeutic window. Myocardial sodium pump is the target spot of the drug effect of CGs, while the mechanism of the interaction between CGS and sodium pump needs further elaboration. The first extramembrane fragment H1 - H2 is currently considered as a possible binding site for ouabain （ OUA ） . Objective The polyclonal antibody WJS which has effect on the extramembrane fragment H1 - H2 of anti - myocardial sodium pump α2 subunit was employed, and the binding site was closed, which had effective antagonism on ouabain＇s inhibiting effect on sodium pump. Comparison was made in the influence of ouabain on the activity of sodium pump of myocardial cells, cell contractility and the variation of intracellular calcium, and the effect mechanism of sodium pump and ouabain was investigated. Methods The ventricle muscle cells of rates were acutely isolated from January 2013 to January 2015 and were divided into four groups： 1 μmol/L ouabain group, WJS ＋ 1 μmol/L ouabain group, 1 mmol/L ouabain group and WJS ＋ 1 mmol/L ouabain group. The 1 μmol/L ouabain group was given 1 μmol/L ouabain by perfusion ; the WJS ＋ 1 μ mol/L group was given ouabain after incubating myocardial cell by antibody WJS （ diluted concentration 1 ： 1 000） ; the 1 mmol/L ouabain group was given 1 mmol/L ouabain by perfusion; the WJS ＋ 1 mmol/L ouabain group was given 1 mmol/L ouabain by perfusion after incubating myocardial cells for 0.5 hour by antibody WJS （ diluted concentration 1 ： 1 000 ） . Sodium pump current was recorded using whole - cell patch clamp technique, and the influence of WJS on the inhibiting effect of ouabain on Ip was observed; laser scanning confocal microscope was used to determine the concentration of free calcium （ [ Ca2＋] i） of myocardial cells ; video - based motion edge - detection system was employed to measure the length of myocardial ceils and the magnitude of contractility. Results The results of western blotting showed that two clear stripes showed in the electrophoresis of WJS after purification, with a molecular weight of 110 kD each. The results of cell immunofluorescence method showed that WJS all bonding in the extramembrane fragment of the sodium pump of myocardial cells; the bonding was not influenced by 1 μmol/L ouabain but was undone by polypeptide blockers of RE2 with the same components at the specific antigen cutoff point in the extramembrane fragment H1 - H2. The WJS ＋ 1 μmol/L ouabain group was lower than the 1 μmol/L ouabain group in the high - affinity sodium pump current （fph） （ P 〈 0. 05 ） ; the 1 mmol/L ouabain group and the WJS ＋ 1 mmol/L ouabain group were higher in Iph and lower in low -affinity sodium pump current （Iph） than the 1 μmol/L ouabain group and the WJS ＋ 1 μmol/L ouabain group （P 〈 0. 05 ） ; the WJS ＋ 1 mmol/L ouabain group was higher in Iph and was lower in Ipl than the 1 mmol/L ouabain group （P 〈0. 05 for all） . The WJS ＋ 1 μmol/L ouabain group was lower than the 1 μmol/L ouabain group in 5 min and 15 min [ Ca2＋ ] i in myocardial cells （P 〈0.05 ） ; the 1 mmol/L ouabain group and the WJS ＋ 1 mmol/L ouabain group were higher than the 1 μmol/L ouabain group and the WJS ＋ 1 μmol/L ouabain group in 5 min and 15 min [ Ca2 ＋ ] i in myocardial cells （P 〈0.05） ; the WJS ＋ 1 mmol/L group was higher than the 1 mmol/L ouabain group in 5 rain and 15 min [ Ca2＋] i in myocardial cells （ P 〈 0.05 ） . The WJS ＋ 1 p, mol/L ouabain group was lower than the 1 μmol/L ouabain group in 5 min and 15 min contractility of myocardial ceils （ P 〈 0. 05 ） ; 1 mmol/L ouabain group and WJS ＋ 1 mmol/L ouabain group were higher in 5 min contractility and lower in 15 min contractility of myocardial cells than 1 p, moL/L ouabain group and WJS ＋ 1 μmol/L ouabain group （P 〈 0.05） ; the WJS ＋ 1 mmol/L ouabain group was higher in 5 min contractility of myocardial ceils and was lower in 15 min contractility of myocardial cells than 1 mmol/L ouabain group （ P 〈 0. 05 ） . Conclusion The polyclonal antibody WJS in the H1 - H2 extramembrane fragment of anti - myocardial sodium pump α2 subunit could undo the high - affinity sodium pump currency and the reinforcement of the contractility of myocardial cells induced by low - concentration uabain, which indicates the therapeutic effect of sodium pump a2 aubunit mediating low - concentration ouabain.