Name: voltage-dependent potassium current recording on h9c2 cardiomyocytes via the whole-cell patch-clamp technique
Uploaded: 2022-11-11T15:00:00
Description: Watch this Scientific Journal Video about Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique at JoVE.com

We appreciate the financial support from the National Natural Science Foundation of China (82130113) and the Key R&#38;D and Transformation Program of the Science &#38; Technology Department of Qinghai Province (2020-SF-C33).

The commercially obtained H9c2 rat cardiomyocytes (see the Table of Materials) were incubated in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 &#176;C in a 5% CO2&#160;humidified atmosphere. The whole-cell patch-clamp technique was then employed to detect the changes in IKv in normal H9c2 cells and 4-AP- or AC-treated cells (Figure 1 and Figure 2).
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<ol>
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The patch-clamp electrophysiological technique is mainly used to record and reflect the electrical activity and functional characteristics of ion channels on the cell membrane25. At present, the main recording methods of the patch-clamp technique include single-channel recording and whole-cell recording26. For the whole-cell mode, the glass microelectrode and negative pressure are used to form a high-resistance seal between a small area of the cell membrane and a pipette ti...

Ion channels are special integrated proteins embedded in the lipid bilayer of the cell membrane. In the presence of activators, the centers of such special integrated proteins form highly selective hydrophilic pores, allowing ions of an appropriate size and charge to pass through in a passive transport manner1. Ion channels are the basis of cell excitability and bioelectricity and play a key role in a variety of cellular activities2. The heart supplies blood to other organs through regular contractions resulting from an excitation-contraction-coupled process initiated by action potentials3. Previous studies have confirmed that the generation of action potentials in cardiomyocytes is caused by the change in intracellular ion concentration, and the activation and inactivation of Na+, Ca2+, and K+ ion channels in human cardiomyocytes lead to the formation of action potentials in a certain sequence4,5,6. Disturbed voltage-gated potassium (Kv) channel currents (IKv) could change the normal heart rhythm, leading to arrhythmias, which are one of the leading causes of death. Therefore, recording the IKv is critical for understanding the mechanisms of drugs for treating life-threatening arrhythmias7.
The Kv channel is an important component of the potassium channel. The coordination function of the Kv channel plays an important role in the electrical activity and myocardial contractility of the mammalian heart8,9,10. In cardiomyocytes, the amplitude and duration of action potentials depend on the co-conduction of outward K+ currents by multiple Kv channel subtypes11. The regulation of the Kv channel function is very important for the normal repolarization of the cardiac action potential. Even the slightest change in Kv conductance greatly impacts cardiac repolarization and increases the possibility of arrhythmia12,13.
Representing a fundamental method in cellular electrophysiological research, a high-resistance seal between a small area of the cell membrane and a pipette tip for whole-cell patch-clamp recording can be established by applying a negative pressure. The continuous negative pressure makes the cell membrane come into contact with the pipette tip and stick onto the inner wall of the pipette. The resulting complete electrical circuit allows one to record any single ion channel current across the surface of the cell membrane14. This technique has a very high sensitivity for the cell membrane ion channel current and can be used to detect currents in all ion channels, and the applications are extremely broad15. Moreover, compared with fluorescent labeling and radioactive labeling, patch-clamp has higher authority and accuracy16. At present, the whole-cell patch-clamp technique has been used to detect the traditional Chinese medicine components acting on Kv channel currents17,18,19. For example, Wang et al. used the whole-cell patch-clamp technique and confirmed that the effective component of the lotus seed might achieve the inhibition of the Kv4.3 channel by blocking the activated state channels19. Aconitine (AC) is one of the effective and active ingredients of Aconitum species, such as Aconitum carmichaeli Debx and Aconitum pendulum Busch. Numerous studies have shown that overdoses of AC can cause arrhythmias and even cardiac arrest20. The interaction between AC and voltage-gated ion channels leads to the disruption of intracellular ion homeostasis, which is the key mechanism of cardiotoxicity21. Therefore, in this study, the whole-cell patch-clamp technique is used to determine the effects of AC on the IKv of cardiomyocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-Aminopyridine</td>
			<td>Sigma</td>
			<td>MKCJ2184</td>
			<td></td>
		</tr>
		<tr>
			<td>Aconitine</td>
			<td>Chengdu Lemetian Medical Technology Co., Ltd</td>
			<td>DSTDW000602</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Axon Instrument</td>
			<td>MultiClamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Sartorius</td>
			<td>124S-CW</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP Na2</td>
			<td>Solarbio</td>
			<td>416O022</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament (O.D.: 1.5 mm, I.D.: 1.10 mm, 10 cm length)&#160;</td>
			<td>Sutter Instrument</td>
			<td>163225-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (100 mm)</td>
			<td>Zhejiang Sorfa Life Science Research Co., Ltd</td>
			<td>1192022</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (35 mm)</td>
			<td>Zhejiang Sorfa Life Science Research Co., Ltd</td>
			<td>3012022</td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex software</td>
			<td>Molecular Devices, LLC.</td>
			<td>Version 10. 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit software</td>
			<td>Molecular Devices, LLC.</td>
			<td>Version 10. 6. 0. 13</td>
			<td>data acqusition software</td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Rhawn</td>
			<td>RH289133</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Hamamatsu</td>
			<td>C11440</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Axon Instrument</td>
			<td>Axon digidata 1550B</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>PYG0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (1x)</td>
			<td>Gibco</td>
			<td>8121587</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Biofroxx</td>
			<td>EZ6789D115</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>2166090RP</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/brown micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>Model P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 cells</td>
			<td>Hunan Fenghui Biotechnology Co., Ltd</td>
			<td>CL0111</td>
			<td></td>
		</tr>
		<tr>
			<td>HCImageLive</td>
			<td>Hamamatsu</td>
			<td>4.5.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sichuan Xilong Scientific Co., Ltd</td>
			<td>2106081</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Xiya Chemical Technology (Shandong) Co., Ltd</td>
			<td>20210221</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Chengdu Colon Chemical Co., Ltd</td>
			<td>2020082501</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Chengdu Colon Chemical Co., Ltd</td>
			<td>2020112601</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Tianjin Guangfu Fine Chemical Research Institute</td>
			<td>20160408</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Chengdu Colon Chemical Co., Ltd</td>
			<td>2021020101</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MP-285A</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass (20 &#215; 20 mm)</td>
			<td>Jiangsu Citotest Experimental Equipment Co. Ltd</td>
			<td>80340-0630</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q</td>
			<td>Chengdu Bioscience Technology Co., Ltd</td>
			<td>Milli-Q IQ 7005</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiClamp 700B commander</td>
			<td>Axon Instrument</td>
			<td>MultiClamp commander 2.0</td>
			<td>signal-amplifier software&#160;</td>
		</tr>
		<tr>
			<td>OriginPro 8 software</td>
			<td>OriginLab Corporation</td>
			<td>v8.0724(B724)</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100x)</td>
			<td>Boster Biological Technology Co., Ltd</td>
			<td>17C18B16</td>
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		</tr>
		<tr>
			<td>PH&#160;meter&#160;</td>
			<td>Mettler Toledo</td>
			<td>S201K</td>
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		</tr>
		<tr>
			<td>Phosphate buffered saline (1x)</td>
			<td>Gibco</td>
			<td>8120485</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1x)</td>
			<td>HyClone</td>
			<td>J210045</td>
			<td></td>
		</tr>
	</tbody>
</table>

voltage-dependent potassium current recording on h9c2 cardiomyocytes via the whole-cell patch-clamp technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 &#181;M, 50 &#181;M, 100 &#181;M, and 200 &#181;M) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 &#181;M, 30% inhibitory rate for 50 &#181;M, 46% inhibitory rate for 100 &#181;M and 54% inhibitory rate for 200 &#181;M). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

This protocol allowed the recording of the IKv according to the parameters set in the whole-cell patch-clamp technique. The IKv was triggered by 150 ms of depolarizing pulse stimulus from &#8722;40 to +60 mV at a holding potential of &#8722;60 mV (Figure 3A). The IKv of the H9c2 rat cardiomyocytes first appeared around &#8722;20 mV, and then the amplitude increased with further depolarization. The mean relationship between the IKv and membrane pote...

Watch this Scientific Journal Video about Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique at JoVE.com

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique (Video) | JoVE

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ...

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