去甲肾上腺素和血管紧张素Ⅱ对心肌慢激活延迟整流钾电流的调节作用及机制
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摘要
心脏疾病如心肌肥厚、心力衰竭发生时，可引发严重心律失常如心房纤颤和心室颤动，也可诱发扭转型室性心动过速（Tdp），其中一个显著特点是动作电位时程（APD）延长，这可能与心肌细胞离子通道发生病理性重构有关。去甲肾上腺素（norepinephrine，NE）和血管紧张素II（angiotensin II，AngII）作为重要的体内心脏电生理和功能调节物质，在正常心肌电生理及病理状态下心肌电重构中都发挥重要作用。生理情况下，去甲肾上腺素介导交感神经兴奋时对心脏的兴奋作用，通过激动心肌细胞β1-肾上腺素能受体——腺苷酸环化酶（AC）——环磷酸腺苷（cAMP）途径，通过第二信使PKA使L型钙离子通道开放，钙离子内流增加，通过兴奋——收缩偶联增强心肌细胞收缩力。病理状态下交感神经过度兴奋，心脏中去甲肾上腺素水平显著增加，通过使多种离子通道发生重构，使其功能异常，最终导致心脏电活动和收缩功能异常。已见报道的包括房颤和心衰发生时，交感神经过度兴奋导致NE异常增多引起心肌细胞内cAMP骤增可以增大If，加快舒张期除极，细胞的自律性增高； NE激动α-肾上腺素能受体，使Ito密度减小，心肌动作电位时程延长；心肌细胞NE释放增多，可显著激活PKC，PKC会减小IK1，可能引起动作电位时程延长，自发除极发生概率增加，产生延迟后除极，以上离子通道发生重构都可能诱发心律失常。正常生理情况，交感神经兴奋释放NE，通过β1-肾上腺素能受体——PKA途径可以增大慢激活延迟整流钾电流（IKs），但在NE长期过度刺激心肌致使心衰时IKs反而减小，心脏复极储备功能大大减弱，在心电图上表现为QT间期延长，动作电位时程明显延长。总之，NE对多种离子通道都有调节作用，在生理和病理情况下对心脏的功能都有重要的影响。
AngII是肾素-血管紧张素系统（renin-angiotensin system，RAS）的重要生物活性肽。它可以维持心血管系统动态平衡，当血压突然降低时，可以迅速增高血压。此外它与心肌梗死、心力衰竭和心肌肥厚等心脏疾病紧密联系，是这些病理生理改变的中心环节。它可以使心肌组织纤维积聚，损害心肌电传导。已有研究表明，AngII通过AT1受体延长L型钙通道的激活和失活时间；在大鼠急性分离的心肌细胞，AngII可以减小Ito电流密度，这与过表达人类基因AT1受体的转基因小鼠上所得结果相同，这些都会使心肌复极化变缓，动作电位时程延长，最终导致心律失常。可见AngII对心肌细胞多种离子通道都有调节作用，在心肌纤维化及心律失常方面具有重要作用。
慢激活延迟整流钾电流（IKs）是心肌细胞复极化过程中重要的钾电流，由KCNQ1（KvLQT1）编码的构成孔区的α亚单位和KCNE1（Mink）编码的β亚单位构成。它是构成心脏复极储备功能的重要钾离子通道。先天性缺陷、药物作用和后天获得性IKs缺失都会导致QT间期延长，复极过程减慢，动作电位时程延长。
在NE和AngII所致的心肌肥厚和心衰的动物模型中观察到IKs发生显著变化，但NE和AngII对IKs的调节和信号通路的比较和差异尚不清楚。本实验利用全细胞膜片钳技术，在急性分离的豚鼠心室肌细胞和心房肌细胞分别观察NE和AngII对IKs的作用，并分析、比较两者作用的细胞内信号通路的异同。第一部分NE对豚鼠心肌细胞的慢激活延迟整流钾电流（IKs）的作用
目的：观察NE对豚鼠心室肌细胞和心房肌细胞IKs的作用。
方法：在急性分离的豚鼠心室肌细胞和心房肌细胞，采用吸破式全细胞膜片钳技术记录IKs电流，观察NE对IKs的作用。
记录IKs的细胞外液包括（in mM）：NaCl132，KCl4，MgCl21.2，CaCl21.8，glucose5，Hepes10（pH7.4with NaOH）。外液中加入Nimodipine（1M）用于阻断L-type Ca2+current。IKr用5M E-4031阻断。电极内液包括（in mM）：potassium aspartate125，KCl20，MgCl21，Mg-ATP5EGTA10，Hepes5（pH7.2with KOH）。将细胞钳制在-30mV,从-30mV以阶跃电压10mV的方式去极化至+70mV,维持2s,然后电压复极至-30mV，诱发IKs外向尾电流。以上实验在室温（20℃～25℃）条件下进行。
结果：外液中加入NE（10M）后，豚鼠心室肌细胞去极化外向电
流和复极时的尾电流显著增大，当去极化电压为+70mV时，尾电流由给药前的1.8±0.1pA/pF增大到2.6±0.2pA/pF（n=6）。增大作用在2-3min达到稳态，且电流可被IKs特异性抑制剂Chromanol293B（293B，30M）完全抑制。NE浓度依赖性增大IKs，以IKs尾电流增大百分比为纵坐标做出NE增大IKs的量效曲线，经Hill方程拟合得到EC50=0.66±0.09M。由尾电流制作使用NE前后IKs电流-电压关系曲线并得到给药前后V1/2分别为36.9±1.8mV和35.7±2.4mV （n=6，P>0.05），NE不影响豚鼠心室肌细胞IKs通道电压依赖性激活。
NE（10M）对豚鼠心房肌细胞IKs同样有增大作用。NE增大IKs尾电流，当电压去极至+50mV时，电流密度由0.65±0.10pA/pF增大到0.95±0.10pA/pF（n=5），且电流可被IKs特异性抑制剂Chromanol293B（293B，30M）完全抑制。由尾电流制作使用NE前后IKs电流-电压关系曲线并得到给药前后V1/2分别为34.45±4.24mV和33.05±5.86mV（n=5，P>0.05），NE不影响豚鼠心房肌细胞IKs通道电压依赖性激活。
结论：NE增大豚鼠心室肌细胞和心房肌细胞的IKs电流，这种增大作用不影响通道的电压门控特征。第二部分Ang II对豚鼠心肌细胞的慢激活延迟整流钾电流（IKs）的作用
目的：观察AngII对豚鼠心室肌细胞和心房肌细胞IKs的作用。
方法：在急性分离的豚鼠心室肌细胞和心房肌细胞，采用吸破式全细胞膜片钳技术记录IKs电流，观察AngII对IKs的作用。
结果：外液中加入AngII（100nM）后，去极化外向电流和复极时的尾电流显著减小，当去极化电压为+70mV时，尾电流由给药前的1.9±0.1pA/pF减小到1.4±0.01pA/pF （n=6）。抑制作用2-3min出现，5-10min左右达到稳态，冲洗后AngII对电流的抑制作用不能完全恢复。AngII浓度依赖性抑制IKs，以IKs尾电流抑制百分比为纵坐标做出AngII减小IKs的量效曲线，经Hill方程拟合得到IC50=30.42±5.97nM。由尾电流制作使用AngII前后IKs电流-电压关系曲线并得到给药前后V1/2分别为33.3±0.8mV和31.6±1.9mV（n=6，P>0.05），AngII不影响心室肌IKs通道电压依赖性激活。
AngII（100nM）同样抑制豚鼠心房肌细胞IKs电流。AngII减小IKs尾电流，当电压去极至+70mV时，电流密度由1.76±0.12pA/pF减小到1.29±0.08pA/pF（n=5），冲洗后AngII对电流的抑制作用不能完全恢复。由尾电流制作使用AngII前后IKs电流-电压关系曲线并得到给药前后V1/2分别为37.94±1.19mV和38.90±2.47mV（n=6，P>0.05），AngII不影响IKs通道电压依赖性激活。
结论：AngII抑制豚鼠心室肌细胞和心房肌细胞的IKs电流，这种抑制作用不影响通道的电压门控特征。第三部分NE及Ang II调节豚鼠心肌细胞IKs功能的机制
目的：分别观察NE和AngII对豚鼠心室肌细胞IKs调节作用的细胞信号机制。
方法：在急性分离的豚鼠心室肌细胞上，采用吸破式全细胞膜片钳技术记录IKs电流，观察PKA、PKC、PLC抑制剂对NE和AngII对IKs作用的影响。
结果：α1肾上腺素能受体阻断剂prazosin（1M）和doxazosin（1M）可以部分阻断NE增大IKs的作用，IKs增大作用幅度由42.3±1.9%分别降低至21.9±0.6%和26.1±0.1%。PLC抑制剂U73122（1M）和edelfosine（1M）明显减弱NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%分别降低至21.0±0.4%和12.7±1.2%。PKC抑制剂Bis-1（100nM）也明显减弱NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%降低至22.6±0.6%。
低浓度α1肾上腺素能受体特异性激动剂phenylephrine（PE）（10M）对豚鼠心室肌细胞IKs没有明显的增大作用。
高浓度α1肾上腺素能受体特异性激动剂phenylephrine（PE）（60M）增大IKs百分比为24.4±0.8%。在β肾上腺素能受体阻断剂propranolol（1M）存在情况下，PE不再增大IKs，反而抑制IKs。
β肾上腺素能受体阻断剂propranolol（1M）几乎完全阻断NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%降低至5.78±0.2%。PKA阻断剂H89（30M）也几乎完全阻断NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%降低至5.4±1.1%。
AT1受体阻断剂Losartan（1μM）几乎完全消除AngII对IKs电流的抑制作用。PLC抑制剂U73122（1M）和edelfosine（1M）明显减弱AngII抑制IKs的作用，IKs减小作用幅度由29.8±0.9%分别降低至15.2±2.7%和12.9±0.3%。PKC抑制剂Bis-1（100nM）也明显减弱AngII抑制IKs的作用，AngII抑制IKs的作用由29.8±0.9%降低至8.9±1.0%。
结论：NE激动心肌α1和β肾上腺素能受体增加IKs电流，分别通过α1受体-PLC-PKC和β受体-PKA途径完成，而且β受体-PKA途径交联影响α1受体-PKC途径，共同完成NE对IKs的调节。AngII激动AT1受体对IKs产生抑制作用，这种抑制作用是通过AT1-PLC-PKC途径介导的。
Cardiomyopathies such as cardiac hypertrophy and heart failure are oftenaccompanied with arrhythmia such as atrial fibrillation and ventricularfibrillation and sometimes torsade de pointes (Tdp). An often seencharacteristic of cardiac electrophysiological changes accompanying thesearrhythmias is the prolongation of action potential duration (APD). Theseabnormal electrophysiological activities during the cardiomyopathies are theresults of pathological remodeling of cardiac ion channels. Norepinephrine(NE) and angiotensinⅡ (Ang II), two endogenous important modulators ofcardiac electrophysiology and function, play an major role both in normalphysiological condition as well as in pathological remodeling. Norepinephrinemediates the effect of activation of sympathetic nerves which activatesβ1-adrenoceptor, which in turn activates adenylate cyclase (AC)-cAMP-PKApathway, and increases the activity of L-type calcium channel and contractilityof cardiac myocytes through excitation-contraction coupling. Under thepathological condition, NE level is increased due to the overexcitation ofsympathetic nerve which will promote the remodeling of ion channel, aprocess with characteristic of changes of expression or malfunction of manycardiac ion channels. It has been reported that overly induced NE duringoverexcitation of sympathetic nerve would increase the automaticity throughcAMP-mediated increase of Ifwhich accelerates the diastolic depolarization;Itodensity is reduced by NE through activation of α1-adrenoceptor which leadto prolongation of action potential duration; NE-mediated activation of PKCin pathological condition reduces IK1which results in prolongation of actionpotential duration, and increases the probability of spontaneous depolarizationand delayed after depolarization. These remodeling of cardiacelectrophysiology all can result in arrhythmia. In physiological condition, NE can increase the slowly activating delayed-rectifier K+current (IKs) throughβ1-adrenoceptor-PKA path way. In contrast, NE reduces IKsin heart failure dueto the long exposure to NE stimulation. These effects of NE will reduce thecardiac repolarization reserve accompanied with prolongation of QT intervaland action potential duration. As such, modulation of ion channel functions byNE plays an important role in cardiac function under both physiological andpathological conditions.
Ang II is the main biologically active peptide in the renin-angiotensinsystem (RAS), which is important in maintaining the cardiovascularhomeostasis. In addition, Ang II is involved in diseased conditions such asmyocardial infarction, heart failure, cardiac hypertrophy, where Ang II ispossibly one of the central mechanisms. Furthermore, Ang II-inducedinterstitial fibrous tissue accumulation is noted to impair electrical conduction.Ang II also affects the function of cardiac ion channels. Ang II, throughactivation of AT1receptor, prolongs the time course of activation andinactivation of L-type calcium channel. It has been shown that Ang IIdown-regulates Itodensity in isolated rat ventricular myocytes. The reduced Itodensity has also been reported on mice with cardiac-specific overexpression ofhuman Ang II type1(AT1) receptors. It has been demonstrated that specificoverexpression of Ang II genes in mice hearts tissues can prolong actionpotential duration and induce ventricular arrhythmia.
Slowly activating delayed-rectifier potassium current (IKs) is one of majorrepolarzing currents in the hearts. The IKschannel is formed by thepore-forming α-subunits encoded by KCNQ1(Kv7.1) and β-subunits encodedby KCNE1. It is one of the most important potassium currents in cardiacrepolarization reserve. Congenital defects, pharmacological inhibition, andacquired loss of IKscan all result in prolongation of QT-interval and actionpotential duration.
It has been demonstrated IKschanged significantly in the animal modelsof hypertrophy and heart failure induced NE and Ang II. However the signalpathways for modulation of IKsby NE and Ang II are far from being well understood. This study is planned to investigate the effect of NE and Ang II onIKsin the atrial and vetricular myocytes of guinea pig and to compare theirsignaling mechanisms of actions.Part1The effect of norepinephrine (NE) on IKsin guinea pig cardiac
myocytes
Objective: To assess the effect of NE on IKsin guinea pig ventricular andatrial myocytes.
Methods: Single ventricular and atrial myocytes were enzymaticallydissociated from the hearts of adult guinea pigs. The IKswas recorded by usingthe whole-cell patch-clamp technique. The external solution contained (in mM)NaCl132，KCl4，MgCl21.2，CaCl21.8，glucose5，Hepes10(pH adjustedto7.4with NaOH). L-type Ca2+current was blocked by1μM Nimodipine(Nim). IKrwas blocked by5μM E4031. The pipette solution contained (inmM) potassium aspartate125，KCl20，MgCl21，Mg-ATP5，EGTA10，Hepes5(pH adjusted to7.2with KOH). Cardiomyocytes were depolarizedfrom a holding potential of-30mV to various prepulse potentials of-30to+70mV for2s, and repolarized to-30mV to evoke outward tail currents. Allexperiments were performed at room temperature (22-23℃).
Results: NE markedly potentiated the IKscurrents in guinea pigventricular myocytes. The tail current densities at+70mV were1.8±0.1pA/pF and2.6±0.2pA/pF (n=6) in the absence and presence of NE (10M).The potentiation of IKstail current reached saturation about2-3min afteraddition of NE (10M) and was abolished by a IKsspecific inhibitor,293B(30M). NE increased the amplitude of IKsin a concentration-dependentmanner with an EC50of0.66±0.09M. The V1/2of IKsin the absence andpresence of NE were36.9±1.8mV and35.7±2.4mV (n=6, P>0.05),respectively. The voltage dependence of IKsactivation was found to be notaffected by NE.
NE also potentiated IKsin guinea pig atrial myocytes. The tail currentdensities at+50mV increased from0.65±0.10pA/pF to0.95±0.10pA/pF (n=5)and were abolished by a IKsspecific inhibitor,293B (30M). The V1/2of IKsin the absence and presence of NE were34.45±4.24mV and33.05±5.86mV (n=5,P>0.05), respectively. The voltage dependence of IKsactivation was found to benot affected by NE.
Conclusion: NE activates IKscurrents in both ventricular and atrialmyocytes, which were not achieved through affecting the voltage-dependentactivation.Part2The effect of angiotensin II (AngII) on IKsin guinea pig cardiac
myocytes
Objective: To assess the effect of AngII on IKsin guinea pig ventricularand atrial myocytes.
Methods: Single ventricular and atrial myocytes were enzymaticallydissociated from the hearts of adult guinea pigs. The IKswas recorded by usingthe whole-cell patch-clamp technique. Cardiomyocytes were depolarized froma holding potential of-30mV to various prepulse potentials of-30mV to+70mV for2s, and repolarized to-30mV to evoke outward tail currents.
Results: AngII markedly inhibited the IKscurrents in guinea pigventricular myocytes. The tail current densities at+70mV were1.9±0.1pA/pF and1.4±0.01pA/pF (n=6) in the absence and presence of AngII (100nM). The inhibition of IKstail current reached saturation about5-10min afteraddition of AngII (100nM). AngII decreased the amplitude of IKsin aconcentration-dependent manner with an IC50of30.42±5.97nM. The V1/2of IKsin the absence and presence of AngII were33.3±0.8mV and31.6±1.9mV(n=6, P>0.05), respectively. The voltage dependence of IKsactivation wasfound to be not affected by AngII.
AngII also inhibited IKsin guinea pig atrial myocytes. The tail currentdensities at+70mV were decreased from1.76±0.12pA/pF to1.29±0.08pA/pF(n=5) by AngII. The V1/2of IKsin the absence and presence of AngII were37.94±1.19mV and38.90±2.47mV (n=5, P>0.05), respectively. The voltagedependence of IKsactivation was found to be not affected by AngII.
Conclusion: AngII inhibits IKscurrents in both ventricular and atrialmyocytes, which were not achieved through affecting the voltage-dependent activation.Part3Signaling mechanism for norepinephrine-and
angiotensinII-induced modulation of guinea pig cardiac IKs
Objective: To examine the signal transduction pathways mediating themodulation of IKsby NE and AngII.
Methods: The IKswas recorded in guinea pig ventricular myocytes byusing whole-cell patch-clamp technique to examine the effects of PKAinhibitor, PKC inhibitor and PLC inhibitor on IKsinduced by NE or AngII.
Results: Prazosin (1M) and doxazosin (1M) as α1-adrenoceptorblockers only partially reduced NE-induced increase of IKs. The increase wasreduced from42.3±1.9%to21.9±0.6%or26.1±0.1%by prazosine ordoxazosin, respectively. PLC inhibitors U73122(1M) or edelfosine (1M)reduced NE-induced potentiation of IKsfrom40.9±2.3%to21.0±0.4%or12.7±1.2%by U73122or edelfosine, respectively. PKC inhibitorbisindolylmaleimide (Bis-1)(100nM) reduced NE-induced potentiation of IKsfrom40.9±2.3%to22.6±0.6%.
Phenylephrine (PE), a selective agonist of α1-adrenoceptor did not affectIKsat a concentration of10M. However, IKswas increased by24.4±0.8%by PE at a higher concentration of60M. In the presence of propranolol (1
M), a specific inhibitor of β-adrenoceptor, PE induced an inhibition ratherthan potentiation of IKs. Propranolol (1M) almost totally abolishedNE-induced increase of IKs(potentiation was reduced from40.9±2.3%to5.78±0.2%). NE-induced potentiation of IKswas reduced from40.9±2.3%(n=6) to5.4±1.1%by H89(30M), a PKA blocker.
Specific AT1receptor blocker, losartan (1μM), antagonized the inhibitoryaction of Ang II on IKs. PLC inhibitors U73122(1M) or edelfosine (1M)reduced Ang II-induced inhibition of IKsfrom29.8±0.9%to15.2±2.7%or12.9±0.3%by U73122or edelfosine, respectively. PKC inhibitorbisindolylmaleimide (Bis-1)(100nM) reduced Ang II-induced inhibition ofIKsfrom29.8±0.9%to8.9±1.0%.
Conclusion: NE activates IKsvia α1-PLC-PKC and β-PKA pathways. And a crosstalk between α1-and β-adrenoceptor pathways occurred for theeffect of NE which contributes to the NE-induced enhancement of IKs. Ang IIinhibits IKsvia AT1-PLC-PKC pathway.
AngII是肾素-血管紧张素系统（renin-angiotensin system，RAS）的重要生物活性肽。它可以维持心血管系统动态平衡，当血压突然降低时，可以迅速增高血压。此外它与心肌梗死、心力衰竭和心肌肥厚等心脏疾病紧密联系，是这些病理生理改变的中心环节。它可以使心肌组织纤维积聚，损害心肌电传导。已有研究表明，AngII通过AT1受体延长L型钙通道的激活和失活时间；在大鼠急性分离的心肌细胞，AngII可以减小Ito电流密度，这与过表达人类基因AT1受体的转基因小鼠上所得结果相同，这些都会使心肌复极化变缓，动作电位时程延长，最终导致心律失常。可见AngII对心肌细胞多种离子通道都有调节作用，在心肌纤维化及心律失常方面具有重要作用。
慢激活延迟整流钾电流（IKs）是心肌细胞复极化过程中重要的钾电流，由KCNQ1（KvLQT1）编码的构成孔区的α亚单位和KCNE1（Mink）编码的β亚单位构成。它是构成心脏复极储备功能的重要钾离子通道。先天性缺陷、药物作用和后天获得性IKs缺失都会导致QT间期延长，复极过程减慢，动作电位时程延长。
在NE和AngII所致的心肌肥厚和心衰的动物模型中观察到IKs发生显著变化，但NE和AngII对IKs的调节和信号通路的比较和差异尚不清楚。本实验利用全细胞膜片钳技术，在急性分离的豚鼠心室肌细胞和心房肌细胞分别观察NE和AngII对IKs的作用，并分析、比较两者作用的细胞内信号通路的异同。第一部分NE对豚鼠心肌细胞的慢激活延迟整流钾电流（IKs）的作用
目的：观察NE对豚鼠心室肌细胞和心房肌细胞IKs的作用。
方法：在急性分离的豚鼠心室肌细胞和心房肌细胞，采用吸破式全细胞膜片钳技术记录IKs电流，观察NE对IKs的作用。
记录IKs的细胞外液包括（in mM）：NaCl132，KCl4，MgCl21.2，CaCl21.8，glucose5，Hepes10（pH7.4with NaOH）。外液中加入Nimodipine（1M）用于阻断L-type Ca2+current。IKr用5M E-4031阻断。电极内液包括（in mM）：potassium aspartate125，KCl20，MgCl21，Mg-ATP5EGTA10，Hepes5（pH7.2with KOH）。将细胞钳制在-30mV,从-30mV以阶跃电压10mV的方式去极化至+70mV,维持2s,然后电压复极至-30mV，诱发IKs外向尾电流。以上实验在室温（20℃～25℃）条件下进行。
结果：外液中加入NE（10M）后，豚鼠心室肌细胞去极化外向电
流和复极时的尾电流显著增大，当去极化电压为+70mV时，尾电流由给药前的1.8±0.1pA/pF增大到2.6±0.2pA/pF（n=6）。增大作用在2-3min达到稳态，且电流可被IKs特异性抑制剂Chromanol293B（293B，30M）完全抑制。NE浓度依赖性增大IKs，以IKs尾电流增大百分比为纵坐标做出NE增大IKs的量效曲线，经Hill方程拟合得到EC50=0.66±0.09M。由尾电流制作使用NE前后IKs电流-电压关系曲线并得到给药前后V1/2分别为36.9±1.8mV和35.7±2.4mV （n=6，P>0.05），NE不影响豚鼠心室肌细胞IKs通道电压依赖性激活。
NE（10M）对豚鼠心房肌细胞IKs同样有增大作用。NE增大IKs尾电流，当电压去极至+50mV时，电流密度由0.65±0.10pA/pF增大到0.95±0.10pA/pF（n=5），且电流可被IKs特异性抑制剂Chromanol293B（293B，30M）完全抑制。由尾电流制作使用NE前后IKs电流-电压关系曲线并得到给药前后V1/2分别为34.45±4.24mV和33.05±5.86mV（n=5，P>0.05），NE不影响豚鼠心房肌细胞IKs通道电压依赖性激活。
结论：NE增大豚鼠心室肌细胞和心房肌细胞的IKs电流，这种增大作用不影响通道的电压门控特征。第二部分Ang II对豚鼠心肌细胞的慢激活延迟整流钾电流（IKs）的作用
目的：观察AngII对豚鼠心室肌细胞和心房肌细胞IKs的作用。
方法：在急性分离的豚鼠心室肌细胞和心房肌细胞，采用吸破式全细胞膜片钳技术记录IKs电流，观察AngII对IKs的作用。
结果：外液中加入AngII（100nM）后，去极化外向电流和复极时的尾电流显著减小，当去极化电压为+70mV时，尾电流由给药前的1.9±0.1pA/pF减小到1.4±0.01pA/pF （n=6）。抑制作用2-3min出现，5-10min左右达到稳态，冲洗后AngII对电流的抑制作用不能完全恢复。AngII浓度依赖性抑制IKs，以IKs尾电流抑制百分比为纵坐标做出AngII减小IKs的量效曲线，经Hill方程拟合得到IC50=30.42±5.97nM。由尾电流制作使用AngII前后IKs电流-电压关系曲线并得到给药前后V1/2分别为33.3±0.8mV和31.6±1.9mV（n=6，P>0.05），AngII不影响心室肌IKs通道电压依赖性激活。
AngII（100nM）同样抑制豚鼠心房肌细胞IKs电流。AngII减小IKs尾电流，当电压去极至+70mV时，电流密度由1.76±0.12pA/pF减小到1.29±0.08pA/pF（n=5），冲洗后AngII对电流的抑制作用不能完全恢复。由尾电流制作使用AngII前后IKs电流-电压关系曲线并得到给药前后V1/2分别为37.94±1.19mV和38.90±2.47mV（n=6，P>0.05），AngII不影响IKs通道电压依赖性激活。
结论：AngII抑制豚鼠心室肌细胞和心房肌细胞的IKs电流，这种抑制作用不影响通道的电压门控特征。第三部分NE及Ang II调节豚鼠心肌细胞IKs功能的机制
目的：分别观察NE和AngII对豚鼠心室肌细胞IKs调节作用的细胞信号机制。
方法：在急性分离的豚鼠心室肌细胞上，采用吸破式全细胞膜片钳技术记录IKs电流，观察PKA、PKC、PLC抑制剂对NE和AngII对IKs作用的影响。
结果：α1肾上腺素能受体阻断剂prazosin（1M）和doxazosin（1M）可以部分阻断NE增大IKs的作用，IKs增大作用幅度由42.3±1.9%分别降低至21.9±0.6%和26.1±0.1%。PLC抑制剂U73122（1M）和edelfosine（1M）明显减弱NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%分别降低至21.0±0.4%和12.7±1.2%。PKC抑制剂Bis-1（100nM）也明显减弱NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%降低至22.6±0.6%。
低浓度α1肾上腺素能受体特异性激动剂phenylephrine（PE）（10M）对豚鼠心室肌细胞IKs没有明显的增大作用。
高浓度α1肾上腺素能受体特异性激动剂phenylephrine（PE）（60M）增大IKs百分比为24.4±0.8%。在β肾上腺素能受体阻断剂propranolol（1M）存在情况下，PE不再增大IKs，反而抑制IKs。
β肾上腺素能受体阻断剂propranolol（1M）几乎完全阻断NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%降低至5.78±0.2%。PKA阻断剂H89（30M）也几乎完全阻断NE增大IKs的作用，IKs增大作用幅度由40.9±2.3%降低至5.4±1.1%。
AT1受体阻断剂Losartan（1μM）几乎完全消除AngII对IKs电流的抑制作用。PLC抑制剂U73122（1M）和edelfosine（1M）明显减弱AngII抑制IKs的作用，IKs减小作用幅度由29.8±0.9%分别降低至15.2±2.7%和12.9±0.3%。PKC抑制剂Bis-1（100nM）也明显减弱AngII抑制IKs的作用，AngII抑制IKs的作用由29.8±0.9%降低至8.9±1.0%。
结论：NE激动心肌α1和β肾上腺素能受体增加IKs电流，分别通过α1受体-PLC-PKC和β受体-PKA途径完成，而且β受体-PKA途径交联影响α1受体-PKC途径，共同完成NE对IKs的调节。AngII激动AT1受体对IKs产生抑制作用，这种抑制作用是通过AT1-PLC-PKC途径介导的。
Cardiomyopathies such as cardiac hypertrophy and heart failure are oftenaccompanied with arrhythmia such as atrial fibrillation and ventricularfibrillation and sometimes torsade de pointes (Tdp). An often seencharacteristic of cardiac electrophysiological changes accompanying thesearrhythmias is the prolongation of action potential duration (APD). Theseabnormal electrophysiological activities during the cardiomyopathies are theresults of pathological remodeling of cardiac ion channels. Norepinephrine(NE) and angiotensinⅡ (Ang II), two endogenous important modulators ofcardiac electrophysiology and function, play an major role both in normalphysiological condition as well as in pathological remodeling. Norepinephrinemediates the effect of activation of sympathetic nerves which activatesβ1-adrenoceptor, which in turn activates adenylate cyclase (AC)-cAMP-PKApathway, and increases the activity of L-type calcium channel and contractilityof cardiac myocytes through excitation-contraction coupling. Under thepathological condition, NE level is increased due to the overexcitation ofsympathetic nerve which will promote the remodeling of ion channel, aprocess with characteristic of changes of expression or malfunction of manycardiac ion channels. It has been reported that overly induced NE duringoverexcitation of sympathetic nerve would increase the automaticity throughcAMP-mediated increase of Ifwhich accelerates the diastolic depolarization;Itodensity is reduced by NE through activation of α1-adrenoceptor which leadto prolongation of action potential duration; NE-mediated activation of PKCin pathological condition reduces IK1which results in prolongation of actionpotential duration, and increases the probability of spontaneous depolarizationand delayed after depolarization. These remodeling of cardiacelectrophysiology all can result in arrhythmia. In physiological condition, NE can increase the slowly activating delayed-rectifier K+current (IKs) throughβ1-adrenoceptor-PKA path way. In contrast, NE reduces IKsin heart failure dueto the long exposure to NE stimulation. These effects of NE will reduce thecardiac repolarization reserve accompanied with prolongation of QT intervaland action potential duration. As such, modulation of ion channel functions byNE plays an important role in cardiac function under both physiological andpathological conditions.
Ang II is the main biologically active peptide in the renin-angiotensinsystem (RAS), which is important in maintaining the cardiovascularhomeostasis. In addition, Ang II is involved in diseased conditions such asmyocardial infarction, heart failure, cardiac hypertrophy, where Ang II ispossibly one of the central mechanisms. Furthermore, Ang II-inducedinterstitial fibrous tissue accumulation is noted to impair electrical conduction.Ang II also affects the function of cardiac ion channels. Ang II, throughactivation of AT1receptor, prolongs the time course of activation andinactivation of L-type calcium channel. It has been shown that Ang IIdown-regulates Itodensity in isolated rat ventricular myocytes. The reduced Itodensity has also been reported on mice with cardiac-specific overexpression ofhuman Ang II type1(AT1) receptors. It has been demonstrated that specificoverexpression of Ang II genes in mice hearts tissues can prolong actionpotential duration and induce ventricular arrhythmia.
Slowly activating delayed-rectifier potassium current (IKs) is one of majorrepolarzing currents in the hearts. The IKschannel is formed by thepore-forming α-subunits encoded by KCNQ1(Kv7.1) and β-subunits encodedby KCNE1. It is one of the most important potassium currents in cardiacrepolarization reserve. Congenital defects, pharmacological inhibition, andacquired loss of IKscan all result in prolongation of QT-interval and actionpotential duration.
It has been demonstrated IKschanged significantly in the animal modelsof hypertrophy and heart failure induced NE and Ang II. However the signalpathways for modulation of IKsby NE and Ang II are far from being well understood. This study is planned to investigate the effect of NE and Ang II onIKsin the atrial and vetricular myocytes of guinea pig and to compare theirsignaling mechanisms of actions.Part1The effect of norepinephrine (NE) on IKsin guinea pig cardiac
myocytes
Objective: To assess the effect of NE on IKsin guinea pig ventricular andatrial myocytes.
Methods: Single ventricular and atrial myocytes were enzymaticallydissociated from the hearts of adult guinea pigs. The IKswas recorded by usingthe whole-cell patch-clamp technique. The external solution contained (in mM)NaCl132，KCl4，MgCl21.2，CaCl21.8，glucose5，Hepes10(pH adjustedto7.4with NaOH). L-type Ca2+current was blocked by1μM Nimodipine(Nim). IKrwas blocked by5μM E4031. The pipette solution contained (inmM) potassium aspartate125，KCl20，MgCl21，Mg-ATP5，EGTA10，Hepes5(pH adjusted to7.2with KOH). Cardiomyocytes were depolarizedfrom a holding potential of-30mV to various prepulse potentials of-30to+70mV for2s, and repolarized to-30mV to evoke outward tail currents. Allexperiments were performed at room temperature (22-23℃).
Results: NE markedly potentiated the IKscurrents in guinea pigventricular myocytes. The tail current densities at+70mV were1.8±0.1pA/pF and2.6±0.2pA/pF (n=6) in the absence and presence of NE (10M).The potentiation of IKstail current reached saturation about2-3min afteraddition of NE (10M) and was abolished by a IKsspecific inhibitor,293B(30M). NE increased the amplitude of IKsin a concentration-dependentmanner with an EC50of0.66±0.09M. The V1/2of IKsin the absence andpresence of NE were36.9±1.8mV and35.7±2.4mV (n=6, P>0.05),respectively. The voltage dependence of IKsactivation was found to be notaffected by NE.
NE also potentiated IKsin guinea pig atrial myocytes. The tail currentdensities at+50mV increased from0.65±0.10pA/pF to0.95±0.10pA/pF (n=5)and were abolished by a IKsspecific inhibitor,293B (30M). The V1/2of IKsin the absence and presence of NE were34.45±4.24mV and33.05±5.86mV (n=5,P>0.05), respectively. The voltage dependence of IKsactivation was found to benot affected by NE.
Conclusion: NE activates IKscurrents in both ventricular and atrialmyocytes, which were not achieved through affecting the voltage-dependentactivation.Part2The effect of angiotensin II (AngII) on IKsin guinea pig cardiac
myocytes
Objective: To assess the effect of AngII on IKsin guinea pig ventricularand atrial myocytes.
Methods: Single ventricular and atrial myocytes were enzymaticallydissociated from the hearts of adult guinea pigs. The IKswas recorded by usingthe whole-cell patch-clamp technique. Cardiomyocytes were depolarized froma holding potential of-30mV to various prepulse potentials of-30mV to+70mV for2s, and repolarized to-30mV to evoke outward tail currents.
Results: AngII markedly inhibited the IKscurrents in guinea pigventricular myocytes. The tail current densities at+70mV were1.9±0.1pA/pF and1.4±0.01pA/pF (n=6) in the absence and presence of AngII (100nM). The inhibition of IKstail current reached saturation about5-10min afteraddition of AngII (100nM). AngII decreased the amplitude of IKsin aconcentration-dependent manner with an IC50of30.42±5.97nM. The V1/2of IKsin the absence and presence of AngII were33.3±0.8mV and31.6±1.9mV(n=6, P>0.05), respectively. The voltage dependence of IKsactivation wasfound to be not affected by AngII.
AngII also inhibited IKsin guinea pig atrial myocytes. The tail currentdensities at+70mV were decreased from1.76±0.12pA/pF to1.29±0.08pA/pF(n=5) by AngII. The V1/2of IKsin the absence and presence of AngII were37.94±1.19mV and38.90±2.47mV (n=5, P>0.05), respectively. The voltagedependence of IKsactivation was found to be not affected by AngII.
Conclusion: AngII inhibits IKscurrents in both ventricular and atrialmyocytes, which were not achieved through affecting the voltage-dependent activation.Part3Signaling mechanism for norepinephrine-and
angiotensinII-induced modulation of guinea pig cardiac IKs
Objective: To examine the signal transduction pathways mediating themodulation of IKsby NE and AngII.
Methods: The IKswas recorded in guinea pig ventricular myocytes byusing whole-cell patch-clamp technique to examine the effects of PKAinhibitor, PKC inhibitor and PLC inhibitor on IKsinduced by NE or AngII.
Results: Prazosin (1M) and doxazosin (1M) as α1-adrenoceptorblockers only partially reduced NE-induced increase of IKs. The increase wasreduced from42.3±1.9%to21.9±0.6%or26.1±0.1%by prazosine ordoxazosin, respectively. PLC inhibitors U73122(1M) or edelfosine (1M)reduced NE-induced potentiation of IKsfrom40.9±2.3%to21.0±0.4%or12.7±1.2%by U73122or edelfosine, respectively. PKC inhibitorbisindolylmaleimide (Bis-1)(100nM) reduced NE-induced potentiation of IKsfrom40.9±2.3%to22.6±0.6%.
Phenylephrine (PE), a selective agonist of α1-adrenoceptor did not affectIKsat a concentration of10M. However, IKswas increased by24.4±0.8%by PE at a higher concentration of60M. In the presence of propranolol (1
M), a specific inhibitor of β-adrenoceptor, PE induced an inhibition ratherthan potentiation of IKs. Propranolol (1M) almost totally abolishedNE-induced increase of IKs(potentiation was reduced from40.9±2.3%to5.78±0.2%). NE-induced potentiation of IKswas reduced from40.9±2.3%(n=6) to5.4±1.1%by H89(30M), a PKA blocker.
Specific AT1receptor blocker, losartan (1μM), antagonized the inhibitoryaction of Ang II on IKs. PLC inhibitors U73122(1M) or edelfosine (1M)reduced Ang II-induced inhibition of IKsfrom29.8±0.9%to15.2±2.7%or12.9±0.3%by U73122or edelfosine, respectively. PKC inhibitorbisindolylmaleimide (Bis-1)(100nM) reduced Ang II-induced inhibition ofIKsfrom29.8±0.9%to8.9±1.0%.
Conclusion: NE activates IKsvia α1-PLC-PKC and β-PKA pathways. And a crosstalk between α1-and β-adrenoceptor pathways occurred for theeffect of NE which contributes to the NE-induced enhancement of IKs. Ang IIinhibits IKsvia AT1-PLC-PKC pathway.
引文
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