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JoVE Journal Medicine

Medicine

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

Published: November 11, 2022 doi: 10.3791/64805
Automatic Translation
*1, *1, 1, 2, 3,4, 5, 1,3,4, 4
1State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 2Chengdu Techman Instrument Co., Ltd., 3Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, 4Research Institute of Integrated TCM & Western Medicine, Chengdu University of Traditional Chinese Medicine, 5School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine
* These authors contributed equally

Summary

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.

Abstract

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 µM, 50 µM, 100 µM, and 200 µM) 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 µM, 30% inhibitory rate for 50 µM, 46% inhibitory rate for 100 µM and 54% inhibitory rate for 200 µM). 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.

Introduction

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.

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Protocol

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 °C in a 5% CO2 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).

1. Solution preparation

  1. Prepare the DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin (see Table of Materials).
  2. Prepare 1.5 M 4-AP solution by adding 14.1165 mg of 4-AP to 100 µL of DMSO solution (see Table of Materials). Dilute 1.5 M 4-AP to 1.5 mM by adding the extracellular solution (step 1.5).
  3. Prepare 400 mM AC by adding 5 mg of AC to 19.36 µL of DMSO solution (see Table of Materials).
    NOTE: All the above solutions were stored in a 4 °C refrigerator, and the final concentration of the DMSO must not exceed 0.1% (v/v).
  4. Prepare 50 mL of intracellular solution by adding 0.4100 g of KCl (110.0 mM), 0.0120 g of MgCl2 (1.2 mM), 0.1270 g of Na2 ATP (5.0 mM), 0.1190 g of HEPES (10.0 mM), and 0.1900 g of EGTA (10.0 mM) into 50 mL of double-distilled water (see Table 1 and Table of Materials).
    NOTE: The pH value of the intracellular solution was adjusted to 7.2 using 1 M KOH and was aliquoted into small volumes (1.5-2 mL) and stored at −20 °C.
  5. Prepare 50 mL of extracellular solution by adding 0.0185 g of KCl (5.0 mM), 0.3945 g of NaCl (135.0 mM), 0.0203 g of MgCl2·6H2O (2.0 mM), 0.0595 g of HEPES (5.0 mM), and 0.0990 g of D-glucose (10.0 mM) into 50 mL of double-distilled water (see Table 1 and Table of Materials).
    NOTE: The extracellular solution must be prepared for immediate use, and its pH value needs to be adjusted to 7.4 by using 1 M NaOH.

2. Cell culture

  1. Once the H9c2 culture dish becomes 80% confluent, digest the cells with 0.25% trypsin for 30 s.
  2. Culture 2 x 105 cells/mL with normal medium or drug-containing medium (25 µM, 50 µM, 100 µM, and 200 µM AC) in a 35 mm dish carpeted with glass plates for 24 h at 37 °C, with a 5% CO2 atmosphere and 70% to 80% relative humidity (see Table of Materials).

3. Fabrication of micropipettes

  1. Turn on the micropipette puller (see Table of Materials), and preheat for 30 min.
  2. Put a borosilicate glass capillary with a filament (OD: 1.5 mm, ID: 1.10 mm, 10 cm length) on the micropipette puller. Select the program set in step 3.3, and click on Enter on the control panel. Click on the Ramp program in the upper-right corner to determine the "Heat" value of the glass capillary.
  3. Write the program for pulling the electrode according to the value determined by the "Ramp" test, and use the following steps: "Heat" value = "Ramp" value, Pull = 0, Vel = pulling rod moving speed, Time = 200-250. Click on Pull to start fabricating the pipettes.
    NOTE: Observe the color of the desiccants in the sealed container of the micropipette puller before using it. If the color changes from blue to pink, the desiccants must be replaced. To prevent the contamination of the electrode tip, try not to touch the middle part of the glass capillary during the pipette preparation. Subsequently, carefully remove the produced pipettes, and place them in a closed and sealed container.

4. Instrument setup

  1. Turn on the corresponding instruments in the following order: the digital-analog converter, the signal amplifier, the micromanipulator, the microscope, and the camera (see Table of Materials).
  2. Open the imaging application, the signal-amplifier software, and the data acquisition software in sequence (see Table of Materials).
    NOTE: The instrument must be turned on sequentially; otherwise, the "Demo Digitizer" state will appear after opening the software.

5. IKv parameter setting

  1. Edit the protocol for recording the IKv following the steps below.
    1. Click on Edit, and select Edit Protocol in the data acquisition software (see Table of Materials).
    2. Edit the program in the "Waveform" interface: Epoch A (First level = −60 mV, Delta level = 0 mV, First duration = 20 ms, and Delta duration = 0 ms); Epoch B (First level = −40 mV, Delta level = 10 mV, First duration = 150 ms, and Delta duration = 0 ms), and Epoch C (First level = −60 mV, Delta level = 0 mV, First duration = 30 ms, and Delta duration = 0 ms).
    3. Then, click on the Mode/Rate interface, and set the "Trial Hierarchy" data: Trial delay = 0 s, Runs = 1, Sweeps = 11, and Sweep duration = 0.22 s22,23.
      NOTE: Acquire the IKv channel currents by applying 150 ms depolarizing steps from −40 mV to +60 mV with an increment of 10 mV at a holding potential of −60 mV under control conditions. The total time of the protocol needs to be greater than the time set in the "Waveform" program.
  2. Edit the P/N Leak Subtraction program: click on Edit Protocol to select the Stimulus button, and set the leak subtraction program in the P/N Leak Subtraction dialog box: number of Subsweeps = 2-8 (4 in this study), Settling time = 100-1,000 ms (200 in this study), "Polarity" = "Opposite to waveform", holding level = −80 mV.
    NOTE: The P/N Leak subtraction must have a holding level lower than the "First holding" (−60 mV).

6. Whole-cell patch-clamp recording of the I Kv in voltage-clamp mode

  1. Establish the data storage path: click on File, and select Set Data File Names in the data acquisition software. Open the established IKv protocol (step 5.1) by selecting Edit and clicking on Open Protocol in the data acquisition software. Finally, click on the Tools option, and select Membrane Test to start running the protocol.
  2. Add the extracellular solution (step 1.5) to the cell bath on the whole-cell patch-clamp apparatus (see Table of Materials), and place the coverslip upward with the H9c2 cells (cultured in step 2.1) in the bath.
  3. Fill 30% of the pipette (fabricated in step 3) with the extracellular solution, and install it on the recording electrode holder integrated into the patch-clamp apparatus. Tighten the pipette with the o-ring rubber gasket and plastic washer. Then, drop the pipette into the bath with the micromanipulator. Click on the Pipette offset interface of the signal-amplifier software (see Table of Materials) to maintain the IKv current baseline at 0 pA.
    NOTE: The intracellular solution (step 1.4) must be in contact with the AgCl/Ag portion of the silver wire, and the silver wire must not be close to the inner wall of the pipette. The resistance of the pipette needs to be 2-6 MΩ. To avoid the occlusion of the pipette tip, a continuous positive pressure must be delivered to the pipette using a 1.0 mL syringe connected to the recording electrode holder via plastic tubing24.
  4. Deliver an appropriate positive pressure manually with a 1.0 mL syringe connected to the recording electrode holder through a plastic tube. Move the pipette slightly by manipulating the micromanipulator in three dimensions to contact the cell.
  5. Once the membrane test square wave drops by 1/3 to 1/2 after the pipette touches the cell membrane, remove the positive pressure, and manually deliver an appropriate negative pressure. Then, click on the Patch interface of the data acquisition software to form the GΩ seal. Use the "Cp Fast" and "Cp Slow" signal-amplifier software during cell sealing to compensate for the fast and slow capacitance.
    NOTE: When the membrane test is ≥1 GΩ, a cell-attached configuration is formed between the pipette tip and the cell.
  6. Apply brief pulses of negative pressure to rupture a patch of the cell membrane.
    NOTE: A precipitous reduction in membrane resistance characterizes a successful whole-cell recording pattern. If the access resistance (Ra) ≥30 MΩ, the cells must be reselected for steps 6.3-6.6. In order to ensure the accuracy of the recorded IKv data, the negative pressure must be removed after the cell membrane is broken.
  7. Execute whole-cell membrane capacitance compensation by clicking on the Whole-cell button of the signal-amplifier software. Finally, save and record the data by clicking on the Data recording button.
  8. Open the saved IKv data with the data analysis software. Save the current-voltage (I−V) relationships: click on Analyze to select Statistics to do the following: select the Range as Cousors 1,2 for data analysis; click on the Peak Amplitude and Mean buttons, and then click on OK to view the data in the Results page. Finally, copy the column of IKv mean data into the function drawing software (see Table of Materials) for further analysis.
  9. Save the representative IKv current traces: click on Edit to select Transfer Traces; then, select the Full trace in the Region to transfer; next, click on Select in Trace Selection, and select IN 0 (pA) in Signals; finally, click on OK to view the data in the "Results" page, and copy the total data into the function drawing software to draw the current traces.
  10. Save the IKv protocol: click on Edit to select Create Stimulus Waveform Signal, and select OK. Then, redo step 6.9 except for selecting A0 # 0 (mA) in "Signals".

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Representative Results

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 −40 to +60 mV at a holding potential of −60 mV (Figure 3A). The IKv of the H9c2 rat cardiomyocytes first appeared around −20 mV, and then the amplitude increased with further depolarization. The mean relationship between the IKv and membrane potential was calculated from the measured current amplitudes. The results showed that in comparison with the control group, the IKv amplitude was observably reduced after the 5 min treatment with 1.5 mM 4-AP (Figure 3B). Additionally, the IKv decreased significantly at membrane potentials from 10 mV to 60 mV in a dose-dependent manner after the 24 h AC treatment (Figure 3C-F).

Figure 1
Figure 1: Equipment and instruments required to record the IKv. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Flow chart of the electrophysiological recording of IKv in H9c2 cells using a whole-cell patch-clamp technique. (A) Cell culture. (B) Preparation of the intracellular and extracellular solutions. (C) Schematic illustration of whole-cell recording. C1: Move the pipette close to the cell; C2: Form a high resistance seal between the pipette and the cell; C3: Rupture the cell membrane. (D) Record the IKv. D1: Schematic diagram of K+ current formation; D2: Representative current traces for the IKv recorded in whole-cell voltage-clamp mode. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Representative IKv in H9c2 cells. (A) Representative current traces for the IKv measured in H9c2 cells without any treatment (control group), with AC-containing media for 24 h (25 µM, 50 µM, 100 µM and 200 µM), and with 1.5 mM 4-AP for 5 min. The IKv was triggered by 150 ms of depolarizing pulse from −40 to +60 mV at a holding potential of −60 mV. (B) For 1.5 mM 4-AP stimulation, the IKv descended at membrane potentials from 10 mV to 60 mV. (C-F) AC treatment decreased the IKv in a concentration-dependent manner at membrane potentials from 10 mV to 60 mV. *p < 0.05 versus the control group (n = 6). Please click here to view a larger version of this figure.

Extracellular solution
Chemicals g/50 mL Composition (mM)
NaCl 0.3945 135.0
KCl 0.1865 5.0
HEPES 0.5958 5.0
MgCl2·6H2O 0.2033 2.0
D-glucose 0.9900 10.0
Intracellular solution 
Chemicals g/50 mL Composition (mM)
KCl 0.4100 110.0
MgCl2 0.0120 1.2
Na2-ATP 0.1270 5.0
HEPES 0.1190 10.0
EGTA 0.1900 10.0

Table 1: Intracellular and extracellular solutions for recording the IKv in voltage-clamp mode.

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Discussion

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 tip27. Once the sustained negative pressure causes the tip of the pipette to rupture the cell membrane and the membrane attaches to the inner wall of the pipette, the complete electrical circuit formed between the pipette and the cell allows for recording the current density of individual ion channels on the cell membrane surface26,27. In recent years, the whole-cell patch-clamp technique has been widely used for drug research targeting ion channel-related diseases. Although it has high requirements for operators, this technique still remains the "gold standard" for ion channel research28. In addition, the perforated patch-clamp technique can also record the current changes in target ion channels in relatively stable intracellular environments over prolonged durations by using antibiotics to form permeability pores in the cell membrane29,30. One can record and trace the dynamic changes in the voltage or current of ion channels using the whole-cell patch-clamp technique in the current-clamp or voltage-clamp mode, making this undoubtedly a powerful platform to evaluate the pharmacological activity or toxicity mechanisms of drugs31,32.

AC is one of the main toxic components of Aconitum species, belongs to the group of diester-diterpenoid alkaloids, and is highly toxic20,33. Evidence has indicated that AC can cause cardiovascular toxicity34,35. As a non-selective K+ channel blocker, it has been reported that AC can block the transient outward K+ current, ultra-rapid delayed rectifier K+ current, and fast delayed rectifier outward K+ current, inducing arrhythmias21,36,37. To date, there is no strong evidence that voltage-dependent potassium currents are involved in the cardiotoxicity of AC. Therefore, in this study, the inhibitory effect of AC on the IKv in rat H9c2 cardiomyocytes was examined using the whole-cell patch-clamp technique. The activation of Na+ channels is a widely recognized mechanism by which AC exerts pharmacological or toxicological effects38. Interestingly, there is evidence that AC can act directly on the IKv21,36,37. However, the data presented in this paper do not provide sufficient evidence that AC can directly inhibit the IKv. The IKv inhibitory effect of AC may be due to the direct activation of Na+ channels, which requires further investigation.

Since its inception and development, the whole-cell patch-clamp technique used in this study has become a conventional method to explore the cardiotoxicity of drugs in terms of ion channels. This experiment confirmed that AC effectively inhibited the IKv of H9c2 cells in a concentration-dependent manner in voltage-clamp mode. However, each type of ion channel, including K+ channels, contains several subtypes, and only the total voltage-dependent K+ channel current was recorded in this study. Subsequent studies can explore the pharmacological and toxicological mechanisms of AC by means of model cell lines with a high expression of specific ion channel subtypes37. Alternatively, one can incorporate specific ion channels labeled with fluorescent proteins to investigate the myocardial toxicity of AC visually39. The critical steps in this protocol are steps 6.4-6.6; completing these three steps directly determines whether the subsequent recording of IKv is successful. Compared with other technologies, the whole-cell patch-clamp technique is the gold standard and accepted method for recording the current in single ion channels in the cell membrane or organelle membrane, with the characteristics of high technical requirements and low-throughput recording40. In summary, this technique is not only a basic method for cell electrophysiology research but is also widely used in neuroscience, cardiovascular science, and other fields.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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

Materials

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

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Voltage-dependent Potassium Current H9c2 Cardiomyocytes Whole-cell Patch-clamp Technique Aconitine Effects Ion Channels Cardiotoxicity Mechanism Pharmacological Mechanisms Toxic Mechanisms Specific Ion Channels Multisystem Diseases Cardiovascular System Nervous System Trypsin Digestion Cell Culture Glass Plates Micropipette Puller Borosilicate Glass Capillary Filament Ramp Program Electrode Fabrication
Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes <em>via</em> the Whole-Cell Patch-Clamp Technique
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Jiang, H., Zhang, Y., Hou, Y., Li,… More

Jiang, H., Zhang, Y., Hou, Y., Li, L., Zhang, S., Zhang, Y., Meng, X., Wang, X. Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique. J. Vis. Exp. (189), e64805, doi:10.3791/64805 (2022).

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