丹参酮ⅡA磺酸钠对稳定表达的人心肌细胞I_（Ks）电流的作用
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摘要
实验背景
缓慢激活延迟整流钾电流(I_(Ks))是参与人心肌细胞动作电位复极,特别是平台期形成与维持的重要外向离子流。I_(Ks)通道由KCNQ1基因编码的α亚单位及KCNE1基因编码的β亚单位组成。hKCNQ1/hKCNE1基因突变或慢性心肌肥厚、慢性心力衰竭等病理过程均可导致I_(Ks)功能异常,引起遗传性或获得性长QT综合征,LQTS易引起尖端扭转型室性心动过速等心律失常,从而危及病人生命。深入研究I_(Ks)通道的药理特性将可能为此类心律失常的防治提供新线索。
丹参酮ⅡA是丹参的脂溶性成分之一,主要分布于丹参根皮部,本质部有少量分布。其磺化产物丹参酮ⅡA磺酸钠易溶于水,在临床上被广泛用于冠心病、心肌梗死等疾病的辅助治疗。但是丹参酮ⅡA磺酸钠对于人心肌细胞I_(Ks)电流的作用目前尚不清楚。
目的与方法
第一部分:将分别编码I_(Ks)通道α亚单位及β亚单位的KCNQ1、KCNE1基因共转染HEK293细胞,潮霉素B筛选,电生理学及药理学方法鉴定,观察hKCNQ1/hKCNE1通道的电生理学特性和药理学特性与野生型人心肌细胞I_(Ks)通道是否相似。
第二部分:主要运用膜片钳技术,探讨丹参酮ⅡA磺酸钠对于稳定表达的人心肌细胞I_(Ks)电流的作用、对I_(Ks)电流激活曲线的影响以及对其激活和去激活时间的影响。
实验结果
第一部分:
1.稳定表达hKCNQ1/hKCNE1基因的细胞上可记录到缓慢激活、无明显失活、具有明显尾电流的外向电流,该电流激活电压大于?40mV,具有明显的电压依赖性;在未转染hKCNQ1/hKCNE1基因的HEK293细胞上只记录到很小的背景电流。
2.细胞外钾离子浓度越大,反转电位负值越大,表明hKCNQ1/hKCNE1电流主要是通道对钾离子通透引起。
3.选择性I_(Ks)通道阻断剂Chromanol293B对hKCNQ1/hKCNE1电流具有明显的、可逆的抑制作用,其IC50(+40mV)为9.1μmol/L。
4.无钾细胞外液能显著增加hKCNQ1/hKCNE1电流,在去极化刺激为+40mV时增加幅度为(28.6±2.0)%(P<0.01,n=8)。
第二部分:
1.丹参酮ⅡA磺酸钠可以增加稳定表达的人心肌细胞I_(Ks)电流的幅度,其作用呈浓度依赖并且可逆,EC50(+40mV)为64.5μmol/L。
2.30μmol/L的丹参酮ⅡA磺酸钠使I_(Ks)电流激活曲线左移而对斜率无影响,30μmol/L的丹参酮ⅡA磺酸钠和对照组的V0.5分别为(16.1±1.2)mV、(24.1±1.9)mV(P<0.05,n=7);S分别为19.1±2.3、17.6±2.4(P>0.05,n=7)。
3.丹参酮ⅡA磺酸钠减小I_(Ks)电流的激活时间常数(-20mV至+40mV,n=7,P<0.05),增大I_(Ks)电流的去激活时间常数(-90mV至-40mV,n=7,P<0.05)。
结论
第一部分:已经成功构建稳定表达人心肌细胞I_(Ks)电流的细胞模型,其电生理学特性和药理学特性与野生型人心肌细胞I_(Ks)电流相似,可以作为研究人心肌细胞I_(Ks)电流功能的细胞模型。
第二部分:丹参酮ⅡA磺酸钠能够可逆地增大稳定表达的人心肌细胞I_(Ks)电流,其作用呈浓度依赖,使I_(Ks)电流激活曲线左移而对斜率无影响,丹参酮ⅡA磺酸钠加快I_(Ks)电流激活过程,减慢I_(Ks)电流的去激活过程。
Background
Slowly activating delayed rectifier potassium current(I_(Ks)) is important in therepolarization of human cardiomyocyte action potential,especially in forming andmaintaining plateau of action potential. I_(Ks) channel is combined byαandβsubunits,which are coded by KCNQ1 and KCNE1 respectively. Mutations ofhKCNQ1/hKCNE1 genes，chronic myocardial hypertrophy and chronic heartfailure will lead to dysfunction of I_(Ks) and ingenital or acquired LQTS which areimportant factor in hazardous ventricular arrhythmia such as Torsade depoints.Research into pharmacological properties of I_(Ks) channel may provide a newclue for protection and treatment of such arrhythmias.
TanshinoneⅡA is a lipophilic form of salvia miltiorrhiza. SodiumtanshinoneⅡA sulfonate is soluble in water and is widely used to treat coronaryartery disease and myocardial infarction in clinic.However, Effects of sodium tanshinoneⅡA sulfonate on human cardiomyocyte slowly activating delayedrectifier potassium current are not known.
ObjectiveObjectivesandMethods
PART I: Human cardiac KCNQ1 and KCNE1 genes, which encodeαandβsubunit of I_(Ks) respectively, were cotransfected into HEK 293 cells.Hygromycin Bwas used to screen the transfected cells.Electrophysiological and pharmacologicalmethods were used to identify whether properties of stably expressedhKCNQ1/hKCNE1 channels are similar to native human cardiac I_(Ks) channel.PARTⅡ：To investigate the effects of sodium tanshinoneⅡA sulfonate on I_(Ks)current by patch clamp. To make clear whether tanshinoneⅡA sulfonate can affectactivation curve of I_(Ks) current and activation and deactivation time constants ofI_(Ks).
Results
PART I：
1. Slowly activating current was recorded in HEK 293 cells stablely expressinghKCNQ1/hKCNE1 genes.The current was voltage-dependent obviously and itsactivation voltage was higher than ?40 mV. We could only record smallbackground current in HEK 293 cells without gene transfection.
2．Its reverse potential linearly correlates with the extracellular potassiumconcentration，indicating that hKCNQ1/hKCNE1 current is a kind of potassiumcurrent.
3．The current was significantly and reversibly inhibited by Chromanol 293B，aspecific I_(Ks) blocker, with an IC50 (+40mV)of 9.1μmol/L.
4．Extacellular fluid without potassium ions significantly increased the magnitudeof I_(Ks). At + 40 mV, the increased level was (28.6±2.0)% (P＜0.01, n = 8).
PARTⅡ：
1．Sodium tanshinoneⅡA sulfonate reversibly enhanced I_(Ks) in a concentrationdependent way,EC50（＋40mV）is 64.5μmol/L.
2．Sodium tanshinoneⅡA sulfonate at 30μmol/L produced a significant leftwardshift in the voltage-dependence of I_(Ks) activation without effect on slope factor. V0.5of sodium tanshinoneⅡA sulfonate at 30μmol/L and control groupwere（16.1±1.2）mV and（24.1±1.9）mV (P＜0.05,n= 7), S of these two groupswere 19.1±2.3 and 17.6±2.4 (P＞0.05,n=7).
3．The activation time constants of I_(Ks) were reduced by tanshinoneⅡA sulfonate at-20 to +40mV(n=7,P＜0.05); however, the deactivation time constants wereincreased by sodium tanshinoneⅡA at potentials of -90 to -40 mV (n=7,P＜0.05).ConclusiConclusion
PART I：Cell model stably expressing human cardiac slowly activating delayedrectifier potassium current has been successfully established, which has similarcharacteristics of native human cardiac I_(Ks). The established cell model is a idealcell model of human cardiac I_(Ks).
PARTⅡ：Sodium tanshinoneⅡA sulfonate reversibly enhanced I_(Ks) in aconcentration dependent way. Sodium tanshinoneⅡA sulfonate at 30μmol/Lproduced a significant leftward shift in the voltage-dependence of I_(Ks) activationwithout effect on slope factor.Sodium tanshinoneⅡA sulfonate reduced activationtime constants of I_(Ks) ,but increased deactivation time constants of I_(Ks).
缓慢激活延迟整流钾电流(I_(Ks))是参与人心肌细胞动作电位复极,特别是平台期形成与维持的重要外向离子流。I_(Ks)通道由KCNQ1基因编码的α亚单位及KCNE1基因编码的β亚单位组成。hKCNQ1/hKCNE1基因突变或慢性心肌肥厚、慢性心力衰竭等病理过程均可导致I_(Ks)功能异常,引起遗传性或获得性长QT综合征,LQTS易引起尖端扭转型室性心动过速等心律失常,从而危及病人生命。深入研究I_(Ks)通道的药理特性将可能为此类心律失常的防治提供新线索。
丹参酮ⅡA是丹参的脂溶性成分之一,主要分布于丹参根皮部,本质部有少量分布。其磺化产物丹参酮ⅡA磺酸钠易溶于水,在临床上被广泛用于冠心病、心肌梗死等疾病的辅助治疗。但是丹参酮ⅡA磺酸钠对于人心肌细胞I_(Ks)电流的作用目前尚不清楚。
目的与方法
第一部分:将分别编码I_(Ks)通道α亚单位及β亚单位的KCNQ1、KCNE1基因共转染HEK293细胞,潮霉素B筛选,电生理学及药理学方法鉴定,观察hKCNQ1/hKCNE1通道的电生理学特性和药理学特性与野生型人心肌细胞I_(Ks)通道是否相似。
第二部分:主要运用膜片钳技术,探讨丹参酮ⅡA磺酸钠对于稳定表达的人心肌细胞I_(Ks)电流的作用、对I_(Ks)电流激活曲线的影响以及对其激活和去激活时间的影响。
实验结果
第一部分:
1.稳定表达hKCNQ1/hKCNE1基因的细胞上可记录到缓慢激活、无明显失活、具有明显尾电流的外向电流,该电流激活电压大于?40mV,具有明显的电压依赖性;在未转染hKCNQ1/hKCNE1基因的HEK293细胞上只记录到很小的背景电流。
2.细胞外钾离子浓度越大,反转电位负值越大,表明hKCNQ1/hKCNE1电流主要是通道对钾离子通透引起。
3.选择性I_(Ks)通道阻断剂Chromanol293B对hKCNQ1/hKCNE1电流具有明显的、可逆的抑制作用,其IC50(+40mV)为9.1μmol/L。
4.无钾细胞外液能显著增加hKCNQ1/hKCNE1电流,在去极化刺激为+40mV时增加幅度为(28.6±2.0)%(P<0.01,n=8)。
第二部分:
1.丹参酮ⅡA磺酸钠可以增加稳定表达的人心肌细胞I_(Ks)电流的幅度,其作用呈浓度依赖并且可逆,EC50(+40mV)为64.5μmol/L。
2.30μmol/L的丹参酮ⅡA磺酸钠使I_(Ks)电流激活曲线左移而对斜率无影响,30μmol/L的丹参酮ⅡA磺酸钠和对照组的V0.5分别为(16.1±1.2)mV、(24.1±1.9)mV(P<0.05,n=7);S分别为19.1±2.3、17.6±2.4(P>0.05,n=7)。
3.丹参酮ⅡA磺酸钠减小I_(Ks)电流的激活时间常数(-20mV至+40mV,n=7,P<0.05),增大I_(Ks)电流的去激活时间常数(-90mV至-40mV,n=7,P<0.05)。
结论
第一部分:已经成功构建稳定表达人心肌细胞I_(Ks)电流的细胞模型,其电生理学特性和药理学特性与野生型人心肌细胞I_(Ks)电流相似,可以作为研究人心肌细胞I_(Ks)电流功能的细胞模型。
第二部分:丹参酮ⅡA磺酸钠能够可逆地增大稳定表达的人心肌细胞I_(Ks)电流,其作用呈浓度依赖,使I_(Ks)电流激活曲线左移而对斜率无影响,丹参酮ⅡA磺酸钠加快I_(Ks)电流激活过程,减慢I_(Ks)电流的去激活过程。
Background
Slowly activating delayed rectifier potassium current(I_(Ks)) is important in therepolarization of human cardiomyocyte action potential,especially in forming andmaintaining plateau of action potential. I_(Ks) channel is combined byαandβsubunits,which are coded by KCNQ1 and KCNE1 respectively. Mutations ofhKCNQ1/hKCNE1 genes，chronic myocardial hypertrophy and chronic heartfailure will lead to dysfunction of I_(Ks) and ingenital or acquired LQTS which areimportant factor in hazardous ventricular arrhythmia such as Torsade depoints.Research into pharmacological properties of I_(Ks) channel may provide a newclue for protection and treatment of such arrhythmias.
TanshinoneⅡA is a lipophilic form of salvia miltiorrhiza. SodiumtanshinoneⅡA sulfonate is soluble in water and is widely used to treat coronaryartery disease and myocardial infarction in clinic.However, Effects of sodium tanshinoneⅡA sulfonate on human cardiomyocyte slowly activating delayedrectifier potassium current are not known.
ObjectiveObjectivesandMethods
PART I: Human cardiac KCNQ1 and KCNE1 genes, which encodeαandβsubunit of I_(Ks) respectively, were cotransfected into HEK 293 cells.Hygromycin Bwas used to screen the transfected cells.Electrophysiological and pharmacologicalmethods were used to identify whether properties of stably expressedhKCNQ1/hKCNE1 channels are similar to native human cardiac I_(Ks) channel.PARTⅡ：To investigate the effects of sodium tanshinoneⅡA sulfonate on I_(Ks)current by patch clamp. To make clear whether tanshinoneⅡA sulfonate can affectactivation curve of I_(Ks) current and activation and deactivation time constants ofI_(Ks).
Results
PART I：
1. Slowly activating current was recorded in HEK 293 cells stablely expressinghKCNQ1/hKCNE1 genes.The current was voltage-dependent obviously and itsactivation voltage was higher than ?40 mV. We could only record smallbackground current in HEK 293 cells without gene transfection.
2．Its reverse potential linearly correlates with the extracellular potassiumconcentration，indicating that hKCNQ1/hKCNE1 current is a kind of potassiumcurrent.
3．The current was significantly and reversibly inhibited by Chromanol 293B，aspecific I_(Ks) blocker, with an IC50 (+40mV)of 9.1μmol/L.
4．Extacellular fluid without potassium ions significantly increased the magnitudeof I_(Ks). At + 40 mV, the increased level was (28.6±2.0)% (P＜0.01, n = 8).
PARTⅡ：
1．Sodium tanshinoneⅡA sulfonate reversibly enhanced I_(Ks) in a concentrationdependent way,EC50（＋40mV）is 64.5μmol/L.
2．Sodium tanshinoneⅡA sulfonate at 30μmol/L produced a significant leftwardshift in the voltage-dependence of I_(Ks) activation without effect on slope factor. V0.5of sodium tanshinoneⅡA sulfonate at 30μmol/L and control groupwere（16.1±1.2）mV and（24.1±1.9）mV (P＜0.05,n= 7), S of these two groupswere 19.1±2.3 and 17.6±2.4 (P＞0.05,n=7).
3．The activation time constants of I_(Ks) were reduced by tanshinoneⅡA sulfonate at-20 to +40mV(n=7,P＜0.05); however, the deactivation time constants wereincreased by sodium tanshinoneⅡA at potentials of -90 to -40 mV (n=7,P＜0.05).ConclusiConclusion
PART I：Cell model stably expressing human cardiac slowly activating delayedrectifier potassium current has been successfully established, which has similarcharacteristics of native human cardiac I_(Ks). The established cell model is a idealcell model of human cardiac I_(Ks).
PARTⅡ：Sodium tanshinoneⅡA sulfonate reversibly enhanced I_(Ks) in aconcentration dependent way. Sodium tanshinoneⅡA sulfonate at 30μmol/Lproduced a significant leftward shift in the voltage-dependence of I_(Ks) activationwithout effect on slope factor.Sodium tanshinoneⅡA sulfonate reduced activationtime constants of I_(Ks) ,but increased deactivation time constants of I_(Ks).
引文
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