HL-1细胞系IK_（ACh）功能特征及对电刺激的反应
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摘要
HL-1细胞是心房肌细胞系,它来源于AT-1小鼠心房肿瘤组织。HL-1细胞可进行传代培养,培养过程中可观察到HL-1细胞具有自主收缩性,并保持与心房肌细胞相似的生物化学特性和电生理学特征。HL-1细胞具有胚胎期心房肌细胞的超微结构。应用RT-PCR技术研究HL-1细胞的基因表达,其基因表达特征与成年小鼠心房肌细胞基因表达很相近,包括α-肌球蛋白重链、α-肌动蛋白、间隙连接蛋白43以及心房钠脲肽因子。采用膜片钳技术,在HL-1细胞上可记录到延迟内向钾电流Ikr样的电流,在HL-1细胞上还可记录到与Ca2+通道电流相似的电流,但还没有得到确切验证。HL-1细胞系由于其显而易见的优点(可长期传代培养,易获得等),有很多潜在的应用价值。目前的证据表明HL-1细胞在很多方面都与成年小鼠心房肌细胞的生理生化特征和电生理特征很相近,但同时也需要更多的实验证据来丰富对HL-1细胞系的认识,以便更好地利用它。IKACh钾通道在心房肌细胞发挥重要作用,但对于HL-1细胞中是否存在功能性IKACh尚不得知。因此,本部分将针对HL-1细胞的内向整流钾电流IKACh进行研究。
在心房肌细胞存在有乙酰胆碱激活的内向整流钾电流IKACh,它在心房肌细胞动作电位复极化过程中发挥重要作用。现已知道,GIRK1(Kir3.1)和GIRK4(Kir3.4)是构成IKACh的分子基础。在哺乳动物体内,GIRK1和GIRK4主要分布于心房肌细胞和窦房结细胞,对调节心脏兴奋性起重要作用。心脏中分布着交感神经与迷走神经纤维,迷走神经兴奋释放神经递质乙酰胆碱(Acetylcholine, ACh),与心肌细胞膜上的M2受体结合,激活相偶联的对PTX敏感的Gi蛋白,从而诱发内向整流钾电流IKACh。另外,其他与PTX敏感的G(Gi/Go)蛋白偶联的的受体如A1嘌呤受体以及EDG家族的神经鞘脂类受体也可以激活IKACh。IKACh在心房肌细胞动作电位复极化的过程中发挥重要作用。迷走神经张力过高可过度激活IKACh通道,使心房细胞的有效不应期(ERP)和动作电位时程(APD)缩短而离散度增加,导致房内折返的发生,成为可能的心房颤动的诱发和维持机制。心房颤动与IKACh有着密切关系。有两种观点,一种认为IKACh上调是诱发心房颤动的原因,另一种观点认为心房颤动会引起IKACh上调。因此IKACh是心房肌细胞非常重要的钾离子电流,对其功能调节及机制的深入研究对了解包括心房颤动等在内的心脏疾病有重要意义,而HL-1细胞系在这方面有重要的潜在价值。
本研究将首先验证HL-1细胞是否存在IKACh,对此将使用乙酰胆碱及腺苷诱发IKACh,用特异性阻断剂tertiapin-Q对诱发的电流进行鉴别,在此基础上对IKACh的其它特征,如去敏化(desensitization)等进行研究。我们还以电压为6 V频率为3 Hz刺激HL-1细胞12小时,比较刺激前后IKACh的变化。上述研究结果将为应用HL-1细胞系研究心肌细胞电生理特征打下基础。
目的:研究HL-1细胞是否存在IKACh及其特征;研究电刺激对HL-1细胞IKACh功能的影响。
方法:培养HL-1细胞。复苏细胞前一天先在培养瓶中加入明胶和纤维结合蛋白37℃孵育,第二天复苏细胞转入培养瓶中培养,4小时后更换培养基。细胞铺片前先加入明胶和纤维结合蛋白37℃孵育。用0.05% Trypsin-EDTA把培养瓶中的细胞消化下来,将细胞悬液均匀的滴在玻璃片上,37℃温箱中培养。
利用乙酰胆碱和腺苷激活心肌细胞IKACh。乙酰胆碱(Acetylcholine, ACh),与心肌细胞膜上的M2受体结合,激活相偶联的对PTX敏感的Gi蛋白,从而诱发内向整流钾电流IKACh。但是由于ACh对M受体的特异性不强,除M2外还可激活Gq偶联蛋白M1受体等,后者会抑制GIRK通道,因此我们还选用腺苷( adenosine)作为IKACh激活剂。腺苷(adenosine)通过与其A1受体结合,激活相偶联Gi蛋白进而激活GIRK通道。利用腺苷对IKACh电流的激活,以及tertiapin-Q对是IKACh的特异性阻断作用,对HL-1细胞的IKACh进行研究。
利用IonOptix C-Pace 100 pacer以电压为6 V、频率为3 Hz电刺激HL-1细胞,比较刺激的细胞和未刺激的细胞IKACh经上述刺激前后功能的变化。
结果:
(1)基础内向整流钾电流存在的证据。实验所用电压钳制程序为持续钳制电压为-80 mV,然后从-120 mV渐变(ramp)到+20 mV。在HL-1细胞,膜电位在较负时可见明显的内向电流,而在较正时可见外向电流,此外向电流应为外向钾电流,不是本研究的内容。我们首先验证记录到的内向电流中包含内向整流钾电流。首先,所记录到的内向电压电流曲线有明显的内向整流电流特征,用500μM Ba2+阻断该内向电流,记录到的反转电位在-80 mV附近,与Nernst公式计算得到的-82.3 mV的K+平衡电位相近,说明以上记录到的基础内向电流中包括内向整流钾电流。
(2)IKACh电流存在的证据。起初实验时我们使用了10μM乙酰胆碱作为IKACh激动剂,在HL-1细胞上记录到乙酰胆碱激活的内向整流钾电流,此电流可以被500μM Ba2+阻断。我们又观察了IKACh的另一激动剂腺苷的作用,腺苷可以通过与PTX敏感的Gi蛋白偶联的A1受体激活IKACh。与乙酰胆碱相似,腺苷也可激活内向电流且此电流可被500μM Ba2+阻断,该内向电流也可被IKACh特异性阻断剂10 nM tertiapin-Q阻断,而且该电流的翻转电位在-80 mV至-90 mV,与通过Nernst公式计算得到的-82.3 mV的K+平衡电位相近,上述结果说明在HL-1细胞存在能被乙酰胆碱和腺苷激活的IKACh电流。
(3) HL-1细胞系IKACh的去敏特征。心房肌细胞的IKACh的特征之一是该电流的去敏现象。即在激动剂(乙酰胆碱)持续存在的情况下,IKACh电流逐渐衰减。我们观察了HL-1细胞系上的IKACh电流是否也有去敏现象。给予10μM乙酰胆碱后,激活的IKACh电流在乙酰胆碱持续存在的情况下逐渐衰减,洗掉药物待IKACh电流完全消失5 min后,再给予10μM乙酰胆碱,所激活电流的幅度比第一次给予乙酰胆碱减小且在乙酰胆碱持续存在的情况下继续减小,上述结果说明HL-1细胞系的IKACh像心房肌细胞一样有去敏现象。同样的时间间隔给予腺苷,观察到腺苷激活电流没有出现去敏现象。
(4) HL-1细胞系的电压依赖性钠电流和钙电流。我们在实验中发现在膜电位为-60 mV和-30 mV附近出现两个不同的明显的内向电流,-60 mV处的内向电流可能是Na+通道开放引起的Na+电流,因为当在细胞外液中加入终浓度为10 nM TTX(Na+通道阻断剂),此电流消失;在-30 mV附近出现的内向电流可能是Ca2+通道开放引起的Ca2+电流,因为在细胞外液中加入终浓度为10μM nimodipine(Ca2+通道阻断剂)后,该电流被阻断。
(5)电刺激对HL-1细胞系IKACh功能的影响。实验采用C-Pace 100 Pacer电刺激器,以电压为6 V频率为3 Hz的方波刺激HL-1细胞12小时,同时与同样培养条件下的HL-1细胞进行比较。采用膜片钳技术分别记录用乙酰胆碱和腺苷诱发的IKACh,然后给予Ba2+和tertiapin-Q阻断电流,比较刺激前后电流的变化。结果表明未刺激和给予刺激的HL-1细胞由乙酰胆碱诱发的IKACh的电流密度分别为3.31±0.08和2.41±0.15(pA/pF,p<0.05);腺苷诱发的IKACh的电流密度分别为3.37±0.29和2.33±0.05(pA/pF,p<0.05)。这些结果表明在HL-1细胞电刺激可以降低IKACh的功能。
结论:HL-1细胞存在乙酰胆碱激活的内向整流钾电流IKACh,它可被Ba2+ 500μM和tertiapin-Q 10 nM阻断,tertiapin-Q是IKACh特异性阻断剂,因此可用来鉴定HL-1细胞是否存在乙酰胆碱激活的内向整流钾电流IKACh。同心房肌的IKACh一样,由乙酰胆碱激活的HL-1细胞的IKACh也存在去敏现象,即在乙酰胆碱持续存在的情况下IKACh逐渐衰减。电刺激可以使HL-1细胞的IKACh电流密度减少。
HL-1 cells are a cardiac muscle cell line derived from AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged while preserving the ability to contract and retaining their biochemical and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found in the cultured HL-1 cells. Reverse- transcriptase–PCR analyses confirmed that the pattern of their gene expression is similar to that of adult atrial myocytes, including expression of a-cardiac myosin heavy chain, a-cardiac actin, and connexin43. A IKr-like current was recorded in HL-1 cells; L-type calcium current was also recorded in HL-1 cells but has yet to be defined. HL-1 cells have many potential applications because of its obvious advantages e.g. their easy availability and passageability. There are evidences showing the similarity of HL-1 cells to the adult mice atrial myocytes in their physiological, biochemical and electrophysiological characteristics. But we need to know more about HL-1 cell line in order to use it well. IKACh plays an important role in atrial myocytes, but whether there is IKACh in HL-1 cells remains unknown. The present work aims to investigate the inward rectifier potassium current IKACh in HL-1 cells.
Inward rectifier potassium current IKACh activated by acetylcholine is important in action potential repolarization in atrial myocytes. Muscarinic K+ channels (KACh) in the heart are heterotetrameric and composed of two subunits, GIRK1 (Kir3.1) and GIRK4 (Kir3.4). In mammals, GIRK1 and GIRK4 are distributed in the atrial myocytes and sinoatrial node cells and play an important role in regulating the excitability of the heart. The KACh channels are opened by M2 receptor activation in response to parasympathetic stimulation. This is believed to be the main pathway to activate GIRK channels via PTX-sensitive G proteins. In addition, other PTX-sensitive G (Gi / Go) protein-coupled receptors such as the A1 purinergic receptors, the EDG receptor family and spingomyline receptors can also activate IKACh. IKACh plays an important role in the action potential repolarization in atrial myocytes. Excessive activation of IKACh caused by hypertonicity of vagus nerves may shorten ERP and APD, resulting in the occurrence of reentry. This is a possible underlying mechanism for atrial fibrillation. Currently there are two viewpoints: one is that upregulation of IKACh causes atrial fibrillation, the other is that atrial fibrillation can cause IKACh upregulation. So IKACh is a very important potassium ion current in atrial myocytes. Research of the mechanism and regulation of IKACh is of great significance to the investigation of atrial fibrillation. HL-1 cells have potential usefulness for the research of atrial fibrillation.
This study first verified the existence of IKACh in HL-1 cells. We used acetylcholine and adenosine to induce IKACh and used tertiapin-Q to identify this current.We also studied other characteristics of IKACh in HL-1 cells such as its desensitization. Furthermore, we stimulated the HL-1 cells using a voltage of 6 V at 3 Hz for 12 hours, and compared IKACh between paced cells and non-paced cells. The results are the groundwork for future research.
Objective: To study the existence of IKACh in HL-1 cells and its characteristics; to study the effects of electrical stimulation on the function of IKACh in HL-1 cells.
Methods: HL-1 cells are cultured in Claycomb Medium. Before culturing cells, tissue culture flasks were coated with gelatin/fibronectin (2 ml/T25 or 6 ml/T75 flask). The flasks were capped and incubated at 37℃overnight. The next morning, the gelatin/fibronectin was removed from the culture flask, and the cells were thawed and transferred into the flask quickly. The medium was replaced by 2 ml of fresh supplemented Claycomb Medium 4 hours later. Before culturing cells, cover glasses were coated with gelatin/fibronectin. The cells were transfered from the flask to the cover glasses, and incubated at 37℃.
We used acetylcholine and adenosine to activate IKACh. Acetylcholine (ACh) binds to M2 receptors in the cell membrane, which in turn activates PTX-sensitive Gi proteins and thus induces inward rectifier potassium current IKACh. However, because ACh not only activates M2 receptors but also activates Gq-coupled M1 receptors that inhibit GIRK channels, we also used adenosine to activate IKACh. Adenosine (Ado) binds to A1 receptor, and activates Gi protein resulting in the activation of the GIRK channels.We used tertiapin-Q to inhibit IKACh in HL-1 cells research.
We stimulated HL-1 cells using a voltage of 6 V at 3 Hz with a IonOptix C-Pace 100 pacer, and compared IKACh in paced and non-paced cells.
Results:
(1) The evidence for the existence of the inward rectifier potassium current. The protocol for current recordings: the HL-1 cells were held at -80 mV, and then a ramp voltage from -120 to +20 mV was applied. In the HL-1 cells, an obvious inward current was seen at negative membrane potentials while an outward current was observed at possitive membrane potentials, the latter not being the subject of this study. First,the current was characteristic of inward rectifier potassium channels. Its inhibition by 500μM Ba2+ suggests that this current was mediated by inwardly rectifying potassium channels. Ba2+-sensitive component of the current, which was subtraction-constructed, reversed at -80mV, a value close to the equilibrium potential for potassium channels (Ek). So there existed an inward rectifier potassium current in the basal current.
(2) Existence of IKACh current in HL-1 cells. First we used 10μM ACh to induce IKACh. There was an inward rectifier potassium current. It was inhibited by 500μM Ba2+. Then we used 10μM Ado to activate IKACh. Ado activated A1 receptor that coupled to Gi protein and IKACh was induced. Similar to the ACh-induced current, the current induced by Ado was inhibited by 500μM Ba2+ . It was also inhibited by 10 nM tertiapin-Q. Its reversal potential was around -80 mV, a value close to Ek. All the evidence indicates IKACh existed in HL-1 cells.
(3) The desensitisation of IKACh in HL-1 cells. In the presence of the agonist ACh (10μM), the amplitude of IKACh gradually declined. Besides, the IKACh manifested a smaller amplitude following the application of 10μM ACh for a second time. It was similar to the desensitisation of ACh in atrial myocytes.We also used 10μM Ado to activate IKACh, but no desensitisation occurred.
(4)Voltage-dependent sodium current and calcium current in HL-1 cells. We found two different inward currents at -60 mV and -30 mV respectively. Application of TTX 10 nM blocked the inward current activated at -60 mV, suggesting involvement of a voltage-dependent Na+ current. Application of both TTX (10 nM) and nimodipine (10μM), a Ca2+ current antagonist, blcoked both inward currents, suggesting involvement of a voltage-dependent Ca2+ current at -30 mV.
(5)We stimulated HL-1 cells for 12 hours using a voltage of 6 V at 3 Hz with a C-Pace 100 Pacer. We stimulated HL-1 cells and meanwhile we cultured non-paced cells in the same condition for comparison. IKACh was induced by 10μM ACh and 10μM Ado respectively. IKACh was inhibited either by 500μM Ba2+ or by 10nM tertiapin-Q. The results showed that ACh-induced IKACh current density was 3.31±0.08 and 2.41±0.15 (pA / pF, p <0.05); adenosine-induced IKACh current density was 3.37±0.29 and 2.33±0.05 (pA/pF, p<0.05). These results indicate that electrical stimulation reduced the IKACh function in HL-1 cells.
Conclusion: acetylcholine-activated inward rectifier potassium current IKACh blocked by 500μM Ba2+ or by 10 nM tertiapin-Q existences in HL-1 cell. Tertiapin-Q is a specific inhibitor of acetylcholine-activated inward rectifier potassium current IKACh.It can be used to identify IKACh in HL-1 cell. IKACh activated by 10μM ACh desensitized in HL-1 cells as same as atrial myocyte. We stimulated HL-1 cells and meanwhile we cultured non-paced cells in the same condition for comparison. The result indicates that electrical stimulation reduced the IKACh function in HL-1 cells.
在心房肌细胞存在有乙酰胆碱激活的内向整流钾电流IKACh,它在心房肌细胞动作电位复极化过程中发挥重要作用。现已知道,GIRK1(Kir3.1)和GIRK4(Kir3.4)是构成IKACh的分子基础。在哺乳动物体内,GIRK1和GIRK4主要分布于心房肌细胞和窦房结细胞,对调节心脏兴奋性起重要作用。心脏中分布着交感神经与迷走神经纤维,迷走神经兴奋释放神经递质乙酰胆碱(Acetylcholine, ACh),与心肌细胞膜上的M2受体结合,激活相偶联的对PTX敏感的Gi蛋白,从而诱发内向整流钾电流IKACh。另外,其他与PTX敏感的G(Gi/Go)蛋白偶联的的受体如A1嘌呤受体以及EDG家族的神经鞘脂类受体也可以激活IKACh。IKACh在心房肌细胞动作电位复极化的过程中发挥重要作用。迷走神经张力过高可过度激活IKACh通道,使心房细胞的有效不应期(ERP)和动作电位时程(APD)缩短而离散度增加,导致房内折返的发生,成为可能的心房颤动的诱发和维持机制。心房颤动与IKACh有着密切关系。有两种观点,一种认为IKACh上调是诱发心房颤动的原因,另一种观点认为心房颤动会引起IKACh上调。因此IKACh是心房肌细胞非常重要的钾离子电流,对其功能调节及机制的深入研究对了解包括心房颤动等在内的心脏疾病有重要意义,而HL-1细胞系在这方面有重要的潜在价值。
本研究将首先验证HL-1细胞是否存在IKACh,对此将使用乙酰胆碱及腺苷诱发IKACh,用特异性阻断剂tertiapin-Q对诱发的电流进行鉴别,在此基础上对IKACh的其它特征,如去敏化(desensitization)等进行研究。我们还以电压为6 V频率为3 Hz刺激HL-1细胞12小时,比较刺激前后IKACh的变化。上述研究结果将为应用HL-1细胞系研究心肌细胞电生理特征打下基础。
目的:研究HL-1细胞是否存在IKACh及其特征;研究电刺激对HL-1细胞IKACh功能的影响。
方法:培养HL-1细胞。复苏细胞前一天先在培养瓶中加入明胶和纤维结合蛋白37℃孵育,第二天复苏细胞转入培养瓶中培养,4小时后更换培养基。细胞铺片前先加入明胶和纤维结合蛋白37℃孵育。用0.05% Trypsin-EDTA把培养瓶中的细胞消化下来,将细胞悬液均匀的滴在玻璃片上,37℃温箱中培养。
利用乙酰胆碱和腺苷激活心肌细胞IKACh。乙酰胆碱(Acetylcholine, ACh),与心肌细胞膜上的M2受体结合,激活相偶联的对PTX敏感的Gi蛋白,从而诱发内向整流钾电流IKACh。但是由于ACh对M受体的特异性不强,除M2外还可激活Gq偶联蛋白M1受体等,后者会抑制GIRK通道,因此我们还选用腺苷( adenosine)作为IKACh激活剂。腺苷(adenosine)通过与其A1受体结合,激活相偶联Gi蛋白进而激活GIRK通道。利用腺苷对IKACh电流的激活,以及tertiapin-Q对是IKACh的特异性阻断作用,对HL-1细胞的IKACh进行研究。
利用IonOptix C-Pace 100 pacer以电压为6 V、频率为3 Hz电刺激HL-1细胞,比较刺激的细胞和未刺激的细胞IKACh经上述刺激前后功能的变化。
结果:
(1)基础内向整流钾电流存在的证据。实验所用电压钳制程序为持续钳制电压为-80 mV,然后从-120 mV渐变(ramp)到+20 mV。在HL-1细胞,膜电位在较负时可见明显的内向电流,而在较正时可见外向电流,此外向电流应为外向钾电流,不是本研究的内容。我们首先验证记录到的内向电流中包含内向整流钾电流。首先,所记录到的内向电压电流曲线有明显的内向整流电流特征,用500μM Ba2+阻断该内向电流,记录到的反转电位在-80 mV附近,与Nernst公式计算得到的-82.3 mV的K+平衡电位相近,说明以上记录到的基础内向电流中包括内向整流钾电流。
(2)IKACh电流存在的证据。起初实验时我们使用了10μM乙酰胆碱作为IKACh激动剂,在HL-1细胞上记录到乙酰胆碱激活的内向整流钾电流,此电流可以被500μM Ba2+阻断。我们又观察了IKACh的另一激动剂腺苷的作用,腺苷可以通过与PTX敏感的Gi蛋白偶联的A1受体激活IKACh。与乙酰胆碱相似,腺苷也可激活内向电流且此电流可被500μM Ba2+阻断,该内向电流也可被IKACh特异性阻断剂10 nM tertiapin-Q阻断,而且该电流的翻转电位在-80 mV至-90 mV,与通过Nernst公式计算得到的-82.3 mV的K+平衡电位相近,上述结果说明在HL-1细胞存在能被乙酰胆碱和腺苷激活的IKACh电流。
(3) HL-1细胞系IKACh的去敏特征。心房肌细胞的IKACh的特征之一是该电流的去敏现象。即在激动剂(乙酰胆碱)持续存在的情况下,IKACh电流逐渐衰减。我们观察了HL-1细胞系上的IKACh电流是否也有去敏现象。给予10μM乙酰胆碱后,激活的IKACh电流在乙酰胆碱持续存在的情况下逐渐衰减,洗掉药物待IKACh电流完全消失5 min后,再给予10μM乙酰胆碱,所激活电流的幅度比第一次给予乙酰胆碱减小且在乙酰胆碱持续存在的情况下继续减小,上述结果说明HL-1细胞系的IKACh像心房肌细胞一样有去敏现象。同样的时间间隔给予腺苷,观察到腺苷激活电流没有出现去敏现象。
(4) HL-1细胞系的电压依赖性钠电流和钙电流。我们在实验中发现在膜电位为-60 mV和-30 mV附近出现两个不同的明显的内向电流,-60 mV处的内向电流可能是Na+通道开放引起的Na+电流,因为当在细胞外液中加入终浓度为10 nM TTX(Na+通道阻断剂),此电流消失;在-30 mV附近出现的内向电流可能是Ca2+通道开放引起的Ca2+电流,因为在细胞外液中加入终浓度为10μM nimodipine(Ca2+通道阻断剂)后,该电流被阻断。
(5)电刺激对HL-1细胞系IKACh功能的影响。实验采用C-Pace 100 Pacer电刺激器,以电压为6 V频率为3 Hz的方波刺激HL-1细胞12小时,同时与同样培养条件下的HL-1细胞进行比较。采用膜片钳技术分别记录用乙酰胆碱和腺苷诱发的IKACh,然后给予Ba2+和tertiapin-Q阻断电流,比较刺激前后电流的变化。结果表明未刺激和给予刺激的HL-1细胞由乙酰胆碱诱发的IKACh的电流密度分别为3.31±0.08和2.41±0.15(pA/pF,p<0.05);腺苷诱发的IKACh的电流密度分别为3.37±0.29和2.33±0.05(pA/pF,p<0.05)。这些结果表明在HL-1细胞电刺激可以降低IKACh的功能。
结论:HL-1细胞存在乙酰胆碱激活的内向整流钾电流IKACh,它可被Ba2+ 500μM和tertiapin-Q 10 nM阻断,tertiapin-Q是IKACh特异性阻断剂,因此可用来鉴定HL-1细胞是否存在乙酰胆碱激活的内向整流钾电流IKACh。同心房肌的IKACh一样,由乙酰胆碱激活的HL-1细胞的IKACh也存在去敏现象,即在乙酰胆碱持续存在的情况下IKACh逐渐衰减。电刺激可以使HL-1细胞的IKACh电流密度减少。
HL-1 cells are a cardiac muscle cell line derived from AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged while preserving the ability to contract and retaining their biochemical and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found in the cultured HL-1 cells. Reverse- transcriptase–PCR analyses confirmed that the pattern of their gene expression is similar to that of adult atrial myocytes, including expression of a-cardiac myosin heavy chain, a-cardiac actin, and connexin43. A IKr-like current was recorded in HL-1 cells; L-type calcium current was also recorded in HL-1 cells but has yet to be defined. HL-1 cells have many potential applications because of its obvious advantages e.g. their easy availability and passageability. There are evidences showing the similarity of HL-1 cells to the adult mice atrial myocytes in their physiological, biochemical and electrophysiological characteristics. But we need to know more about HL-1 cell line in order to use it well. IKACh plays an important role in atrial myocytes, but whether there is IKACh in HL-1 cells remains unknown. The present work aims to investigate the inward rectifier potassium current IKACh in HL-1 cells.
Inward rectifier potassium current IKACh activated by acetylcholine is important in action potential repolarization in atrial myocytes. Muscarinic K+ channels (KACh) in the heart are heterotetrameric and composed of two subunits, GIRK1 (Kir3.1) and GIRK4 (Kir3.4). In mammals, GIRK1 and GIRK4 are distributed in the atrial myocytes and sinoatrial node cells and play an important role in regulating the excitability of the heart. The KACh channels are opened by M2 receptor activation in response to parasympathetic stimulation. This is believed to be the main pathway to activate GIRK channels via PTX-sensitive G proteins. In addition, other PTX-sensitive G (Gi / Go) protein-coupled receptors such as the A1 purinergic receptors, the EDG receptor family and spingomyline receptors can also activate IKACh. IKACh plays an important role in the action potential repolarization in atrial myocytes. Excessive activation of IKACh caused by hypertonicity of vagus nerves may shorten ERP and APD, resulting in the occurrence of reentry. This is a possible underlying mechanism for atrial fibrillation. Currently there are two viewpoints: one is that upregulation of IKACh causes atrial fibrillation, the other is that atrial fibrillation can cause IKACh upregulation. So IKACh is a very important potassium ion current in atrial myocytes. Research of the mechanism and regulation of IKACh is of great significance to the investigation of atrial fibrillation. HL-1 cells have potential usefulness for the research of atrial fibrillation.
This study first verified the existence of IKACh in HL-1 cells. We used acetylcholine and adenosine to induce IKACh and used tertiapin-Q to identify this current.We also studied other characteristics of IKACh in HL-1 cells such as its desensitization. Furthermore, we stimulated the HL-1 cells using a voltage of 6 V at 3 Hz for 12 hours, and compared IKACh between paced cells and non-paced cells. The results are the groundwork for future research.
Objective: To study the existence of IKACh in HL-1 cells and its characteristics; to study the effects of electrical stimulation on the function of IKACh in HL-1 cells.
Methods: HL-1 cells are cultured in Claycomb Medium. Before culturing cells, tissue culture flasks were coated with gelatin/fibronectin (2 ml/T25 or 6 ml/T75 flask). The flasks were capped and incubated at 37℃overnight. The next morning, the gelatin/fibronectin was removed from the culture flask, and the cells were thawed and transferred into the flask quickly. The medium was replaced by 2 ml of fresh supplemented Claycomb Medium 4 hours later. Before culturing cells, cover glasses were coated with gelatin/fibronectin. The cells were transfered from the flask to the cover glasses, and incubated at 37℃.
We used acetylcholine and adenosine to activate IKACh. Acetylcholine (ACh) binds to M2 receptors in the cell membrane, which in turn activates PTX-sensitive Gi proteins and thus induces inward rectifier potassium current IKACh. However, because ACh not only activates M2 receptors but also activates Gq-coupled M1 receptors that inhibit GIRK channels, we also used adenosine to activate IKACh. Adenosine (Ado) binds to A1 receptor, and activates Gi protein resulting in the activation of the GIRK channels.We used tertiapin-Q to inhibit IKACh in HL-1 cells research.
We stimulated HL-1 cells using a voltage of 6 V at 3 Hz with a IonOptix C-Pace 100 pacer, and compared IKACh in paced and non-paced cells.
Results:
(1) The evidence for the existence of the inward rectifier potassium current. The protocol for current recordings: the HL-1 cells were held at -80 mV, and then a ramp voltage from -120 to +20 mV was applied. In the HL-1 cells, an obvious inward current was seen at negative membrane potentials while an outward current was observed at possitive membrane potentials, the latter not being the subject of this study. First,the current was characteristic of inward rectifier potassium channels. Its inhibition by 500μM Ba2+ suggests that this current was mediated by inwardly rectifying potassium channels. Ba2+-sensitive component of the current, which was subtraction-constructed, reversed at -80mV, a value close to the equilibrium potential for potassium channels (Ek). So there existed an inward rectifier potassium current in the basal current.
(2) Existence of IKACh current in HL-1 cells. First we used 10μM ACh to induce IKACh. There was an inward rectifier potassium current. It was inhibited by 500μM Ba2+. Then we used 10μM Ado to activate IKACh. Ado activated A1 receptor that coupled to Gi protein and IKACh was induced. Similar to the ACh-induced current, the current induced by Ado was inhibited by 500μM Ba2+ . It was also inhibited by 10 nM tertiapin-Q. Its reversal potential was around -80 mV, a value close to Ek. All the evidence indicates IKACh existed in HL-1 cells.
(3) The desensitisation of IKACh in HL-1 cells. In the presence of the agonist ACh (10μM), the amplitude of IKACh gradually declined. Besides, the IKACh manifested a smaller amplitude following the application of 10μM ACh for a second time. It was similar to the desensitisation of ACh in atrial myocytes.We also used 10μM Ado to activate IKACh, but no desensitisation occurred.
(4)Voltage-dependent sodium current and calcium current in HL-1 cells. We found two different inward currents at -60 mV and -30 mV respectively. Application of TTX 10 nM blocked the inward current activated at -60 mV, suggesting involvement of a voltage-dependent Na+ current. Application of both TTX (10 nM) and nimodipine (10μM), a Ca2+ current antagonist, blcoked both inward currents, suggesting involvement of a voltage-dependent Ca2+ current at -30 mV.
(5)We stimulated HL-1 cells for 12 hours using a voltage of 6 V at 3 Hz with a C-Pace 100 Pacer. We stimulated HL-1 cells and meanwhile we cultured non-paced cells in the same condition for comparison. IKACh was induced by 10μM ACh and 10μM Ado respectively. IKACh was inhibited either by 500μM Ba2+ or by 10nM tertiapin-Q. The results showed that ACh-induced IKACh current density was 3.31±0.08 and 2.41±0.15 (pA / pF, p <0.05); adenosine-induced IKACh current density was 3.37±0.29 and 2.33±0.05 (pA/pF, p<0.05). These results indicate that electrical stimulation reduced the IKACh function in HL-1 cells.
Conclusion: acetylcholine-activated inward rectifier potassium current IKACh blocked by 500μM Ba2+ or by 10 nM tertiapin-Q existences in HL-1 cell. Tertiapin-Q is a specific inhibitor of acetylcholine-activated inward rectifier potassium current IKACh.It can be used to identify IKACh in HL-1 cell. IKACh activated by 10μM ACh desensitized in HL-1 cells as same as atrial myocyte. We stimulated HL-1 cells and meanwhile we cultured non-paced cells in the same condition for comparison. The result indicates that electrical stimulation reduced the IKACh function in HL-1 cells.
引文
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2 Claycomb WC Control of Cardiac Muscle Cell Division.TCM,1992,2:231-236
3 Claycomb WC ,Moses RL Growth Factors and TPA Stimulate DNA Synthesis and Alter Morphology of Cultured Terminally Differentiated Adult Rat Cardiac Muscle Cells.Decelopment Biology,1988,127:257-265
4 Koh GY,Soonpaa MH,Klug MG et al. Long-term Survival of AT-1 Cardiomocyte Graftsin Syngeneic Myocardium. Am.J. Physiology, 1993,264:1727-1733
5 Delcarpio JB,Lanson NA,Claycomb WC et al. Morpholofical Characterization of Cardiomyocytes Isolated From a TransplantableCardiac Tumor Derived from Transgenic Mouse Aria(AT-1 Cells).Circulation Research,1991,69:5
6 Claycomb WC HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Cell Biology,1998,95:2979-2984
7 Corey S, Krapivinsky G, Krapivinsky L, et al. Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh. J Biol Chem, 1998, 273:5271-5278
8 Dobrev D, Ravens U Remodeling of cardiomyocyte ion channels in human atrial fibrillation. Basic Res Cardiol,2003,98:137-148
9 Dobrev D,Friedrich A,Voigt N,et al. The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation. Circulation,2005,112:3697-3706
10 Krapivinsky G,Gordon EA,Wickman K,et al. The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins, Nature, 1995, 374:135-141
11 Kovoor P, Wickman K,Maguire CT, et al. Evaluation of the role of I(KACh) in atrial fibrillation using a mouse knockout model, J. Am.Coll. Cardiol,2001,37:2136-2143
12 Cha TJ, Ehrlich JR, S. Nattel et al. Kir3-based inward rectifier potassium current:potential role in atrial tachycardia remodeling effects on atrial repolarization and arrhythmias.Circulation, 2006,113:1730-1737
13 Sarmast F,Kolli A,Zaitsev A,et al. Cholinergic atrial fibrillation: I(K,ACh) gradients determine unequal left/right atrial frequencies and rotor dynamics,Cardiovasc.Res, 2003,59:863-873
14 Kovoor Pramesh,Wickman Kevin,Colin T,et al. Evaluation of the Role of IKACh in Atrial Fibrillation Using a Mouse Knockout Model .Journal of the American College of Cardiology,2001,37:2136-2143
15 Gaborit N, Steenman M, Lamirault G, et al. Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation. Circulation,2005,112:471-481
16 Dobrev D, Friedrich A, Voigt N, et al. The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation. Circulation,2005,112:3697-3706
17 Brundel BJ, Van Gelder IC, Henning RH, et al. Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels for K+ channels. J Am Coll Cardiol,2001,37:926-932
18 Dobrev D, Graf E, Wettwer E, et al. Molecular basis of downregulation of G-protein-coupled inward rectifying K(+) current I(K,ACh) in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced I(K,ACh) and muscarinic receptor-mediated shortening of action potentials. Circulation,2001,104:2551-2557
19 Hill JJ, EG Peralta. Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled m1 muscarinic acetylcholine receptor. J Biol Chem, 2001, 276: 5505-5510
20 Nattel S. New ideas about atrial fibrillation 50 years on. Nature. 2002,415:219 -226
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2 Dobrev D,Ravens U Remodeling of cardiomyocyte ion channels in human atrial fibrillation. Basic Res Cardiol,2003,98:137-148
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4 Bosch RF, Zeng X, Grammer JB.Ionic mechanisms of electrical remodeling in human atrial fibrillation.Cardiovasc Res,1999,44:121-131
5 Van Wagoner DR, Pond AL, Lamorgese M.Atrial L-type Ca2+-currents and human atrial fibrillation.Circ Res,1999,85:428-436
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2 Claycomb WC Control of Cardiac Muscle Cell Division.TCM,1992,2:231-236
3 Claycomb WC ,Moses RL Growth Factors and TPA Stimulate DNA Synthesis and Alter Morphology of Cultured Terminally Differentiated Adult Rat Cardiac Muscle Cells.Decelopment Biology,1988,127:257-265
4 Koh GY,Soonpaa MH,Klug MG et al. Long-term Survival of AT-1 Cardiomocyte Graftsin Syngeneic Myocardium. Am.J. Physiology, 1993,264:1727-1733
5 Delcarpio JB,Lanson NA,Claycomb WC et al. Morpholofical Characterization of Cardiomyocytes Isolated From a TransplantableCardiac Tumor Derived from Transgenic Mouse Aria(AT-1 Cells).Circulation Research,1991,69:5
6 Claycomb WC HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Cell Biology,1998,95:2979-2984
7 Corey S, Krapivinsky G, Krapivinsky L, et al. Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh. J Biol Chem, 1998, 273:5271-5278
8 Dobrev D, Ravens U Remodeling of cardiomyocyte ion channels in human atrial fibrillation. Basic Res Cardiol,2003,98:137-148
9 Dobrev D,Friedrich A,Voigt N,et al. The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation. Circulation,2005,112:3697-3706
10 Krapivinsky G,Gordon EA,Wickman K,et al. The G-protein-gated atrial K+ channel IKACh is a heteromultimer of two inwardly rectifying K(+)-channel proteins, Nature, 1995, 374:135-141
11 Kovoor P, Wickman K,Maguire CT, et al. Evaluation of the role of I(KACh) in atrial fibrillation using a mouse knockout model, J. Am.Coll. Cardiol,2001,37:2136-2143
12 Cha TJ, Ehrlich JR, S. Nattel et al. Kir3-based inward rectifier potassium current:potential role in atrial tachycardia remodeling effects on atrial repolarization and arrhythmias.Circulation, 2006,113:1730-1737
13 Sarmast F,Kolli A,Zaitsev A,et al. Cholinergic atrial fibrillation: I(K,ACh) gradients determine unequal left/right atrial frequencies and rotor dynamics,Cardiovasc.Res, 2003,59:863-873
14 Kovoor Pramesh,Wickman Kevin,Colin T,et al. Evaluation of the Role of IKACh in Atrial Fibrillation Using a Mouse Knockout Model .Journal of the American College of Cardiology,2001,37:2136-2143
15 Gaborit N, Steenman M, Lamirault G, et al. Human atrial ion channel and transporter subunit gene-expression remodeling associated with valvular heart disease and atrial fibrillation. Circulation,2005,112:471-481
16 Dobrev D, Friedrich A, Voigt N, et al. The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation. Circulation,2005,112:3697-3706
17 Brundel BJ, Van Gelder IC, Henning RH, et al. Alterations in potassium channel gene expression in atria of patients with persistent and paroxysmal atrial fibrillation: differential regulation of protein and mRNA levels for K+ channels. J Am Coll Cardiol,2001,37:926-932
18 Dobrev D, Graf E, Wettwer E, et al. Molecular basis of downregulation of G-protein-coupled inward rectifying K(+) current I(K,ACh) in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced I(K,ACh) and muscarinic receptor-mediated shortening of action potentials. Circulation,2001,104:2551-2557
19 Hill JJ, EG Peralta. Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled m1 muscarinic acetylcholine receptor. J Biol Chem, 2001, 276: 5505-5510
20 Nattel S. New ideas about atrial fibrillation 50 years on. Nature. 2002,415:219 -226
1 Nattel S. New ideas about atrial fibrillation 50 years on. Nature, 2002,415:219-226
2 Dobrev D,Ravens U Remodeling of cardiomyocyte ion channels in human atrial fibrillation. Basic Res Cardiol,2003,98:137-148
3 Van Wagoner DR, Pond AL, McCarthy PM,et al. Outward K-current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res,1997,80:772-781
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