| 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. |
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