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).
<p class="jove_titl...

<ol>
	<li>Luan, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+transport+and+ion+channels+in+biofilms.">Passive transport and ion channels in biofilms.</a> Acta Scientiarum Naturalium Universitatis Intramongoljcae. 2, 215-235 (1984).</li><li>Lei, M., Sun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+mechanism+of+arrhythmia+induced+by+sodium+channel+disease.">Advances in the mechanism of arrhythmia induced by sodium channel disease.</a> Journal of Clinical Cardiology. 21 (4), 246-248 (2005).</li><li>Varr&#243;, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+transmembrane+ion+channels+and+action+potentials:+Cellular+physiology+and+arrhythmogenic+behavior.">Cardiac transmembrane ion channels and action potentials: Cellular physiology and arrhythmogenic behavior.</a> Physiological Reviews. 101 (3), 1083-1176 (2021).</li><li>Campuzano, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+autopsy+and+sudden+cardiac+death.">Negative autopsy and sudden cardiac death.</a> International Journal of Legal Medicine. 128 (4), 599-606 (2014).</li><li>Amin, A. S., Asghari-Roodsari, A., Tan, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+sodium+channelopathies.">Cardiac sodium channelopathies.</a> Pflu&#776;gers Archiv: European Journal of Physiology. 460 (2), 223-237 (2010).</li><li>Benitah, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+gated+Ca2++currents+in+the+human+pathophysiologic+heart:+A+review.">Voltage gated Ca2+ currents in the human pathophysiologic heart: A review.</a> Basic Research in Cardiology. 97 (1), 111-118 (2002).</li><li>Banyasz, T., Horvath, B., Jian, Z., Izu, L. T., Chen-Izu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+dissection+of+multiple+ionic+currents+in+single+cardiac+myocytes+under+action+potential-clamp.">Sequential dissection of multiple ionic currents in single cardiac myocytes under action potential-clamp.</a> Journal of Molecular and Cellular Cardiology. 50 (3), 578-581 (2011).</li><li>Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+functional+myocardial+potassium+channel+diversity.">Molecular basis of functional myocardial potassium channel diversity.</a> Cardiac Electrophysiology Clinics. 8 (2), 257-273 (2016).</li><li>Grant, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+ion+channels.">Cardiac ion channels.</a> Circulation: Arrhythmia and Electrophysiology. 2 (2), 185-194 (2009).</li><li>Olson, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv1.5+channelopathy+due+to+KCNA5+loss-of-function+mutation+causes+human+atrial+fibrillation.">Kv1.5 channelopathy due to KCNA5 loss-of-function mutation causes human atrial fibrillation.</a> Human Molecular Genetics. 15 (14), 2185-2191 (2006).</li><li>Christophersen, I. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+KCNA5:+impact+on+the+atrial-specific+potassium+current+IKur+in+patients+with+lone+atrial+fibrillation.">Genetic variation in KCNA5: impact on the atrial-specific potassium current IKur in patients with lone atrial fibrillation.</a> European Heart Journal. 34 (20), 1517-1525 (2013).</li><li>Barry, D. M., Xu, H., Schuessler, R. B., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+knockout+of+the+transient+outward+current,+long-QT+syndrome,+and+cardiac+remodeling+in+mice+expressing+a+dominant-negative+Kv4+alpha+subunit.">Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit.</a> Circulation Research. 83 (5), 560-567 (1998).</li><li>Abbott, G. W., Xu, X., Roepke, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+ancillary+subunits+on+ventricular+repolarization.">Impact of ancillary subunits on ventricular repolarization.</a> Journal of Electrocardiology. 40, 42-46 (2007).</li><li>Jia, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+studies+on+the+application+of+patch-clamp+technique+in+cellular+electrophysiology.">Recent studies on the application of patch-clamp technique in cellular electrophysiology.</a> Journal of Chemical Engineering of Chinese Universities. 32 (4), 767-778 (2018).</li><li>Leuthardt, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+electrocorticographic+speech+network+to+control+a+brain-computer+interface+in+humans.">Using the electrocorticographic speech network to control a brain-computer interface in humans.</a> Journal of Neural Engineering. 8 (3), 1-3 (2011).</li><li>Tian, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+applying+progress+of+patch-clamp+technique.">The applying progress of patch-clamp technique.</a> Journal of Jilin Medical University. 4, 227-229 (2008).</li><li>Wang, Z. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+shensong+yangxin+capsule+on+c-type+Kv1.4+potassium+channel.">Effects of shensong yangxin capsule on c-type Kv1.4 potassium channel.</a> Chinese Heart Journal. 21 (6), 782-785 (2009).</li><li>Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+resveratrol+on+Kv2.1+potassium+channels+in+cardiac+myocytes.">The effect of resveratrol on Kv2.1 potassium channels in cardiac myocytes.</a> Chinese Journal of Cardiac Pacing and Electrophysiology. 34 (5), 484-487 (2020).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neferine+on+Kv4.3+channels+expressed+in+HEK293+cells+and+ex+vivo+electrophysiology+of+rabbit+hearts.">Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts.</a> Acta Pharmacologica Sinica. 36 (12), 1451-1461 (2005).</li><li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aconitine:+A+review+of+its+pharmacokinetics,+pharmacology,+toxicology+and+detoxification.">Aconitine: A review of its pharmacokinetics, pharmacology, toxicology and detoxification.</a> Journal of Ethnopharmacology. 293, 115270 (2022).</li><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+efficacy+and+toxicity+of+aconitine:+A+new+frontier+for+the+ancient+poison.">Cardiac efficacy and toxicity of aconitine: A new frontier for the ancient poison.</a> Medicinal Research Reviews. 41 (3), 1798-1811 (2021).</li><li>An, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+tegaserod,+a+gastrokinetic+agent,+on+voltage-gated+K++channels+in+rabbit+coronary+arterial+smooth+muscle+cells.">The effects of tegaserod, a gastrokinetic agent, on voltage-gated K+ channels in rabbit coronary arterial smooth muscle cells.</a> Clinical and Experimental Pharmacology &amp; Physiology. 48 (5), 748-756 (2021).</li><li>Sun, Q., Liu, F., Zhao, J., Wang, P., Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+Kv2.1+by+BACE1+decreases+potassium+current+and+reduces+neuronal+apoptosis.">Cleavage of Kv2.1 by BACE1 decreases potassium current and reduces neuronal apoptosis.</a> Neurochemistry International. 155, 105310 (2022).</li><li>Manz, K. M., Siemann, J. K., McMahon, D. G., Grueter, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+and+multi-electrode+array+electrophysiological+analysis+in+acute+mouse+brain+slices.">Patch-clamp and multi-electrode array electrophysiological analysis in acute mouse brain slices.</a> STAR Protocols. 2 (2), 100442 (2021).</li><li>Kanda, H., Tonomura, S., Dai, Y., Gu, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+pressure-clamped+patch-clamp+recording+at+the+node+of+Ranvier+of+rat+myelinated+nerves.">Protocol for pressure-clamped patch-clamp recording at the node of Ranvier of rat myelinated nerves.</a> STAR Protocols. 2 (1), 100266 (2021).</li><li>Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+purity+human-induced+pluripotent+stem+cell-derived+cardiomyocytes:+electrophysiological+properties+of+action+potentials+and+ionic+currents.">High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents.</a> American Journal of Physiology-Heart and Circulatory Physiology. 301 (5), 2006-2017 (2011).</li><li>Yoshimura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+vivo+patch-clamp+technique+to+pharmacological+analysis+of+synaptic+transmission+in+the+CNS.">Application of in vivo patch-clamp technique to pharmacological analysis of synaptic transmission in the CNS.</a> Nihon Yakurigaku Zasshi. Folia Pharmacologica Japonica. 124 (2), 111-118 (2004).</li><li>Aziz, Q., Nobles, M., Tinker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+and+perforated+patch-clamp+recordings+from+acutely-isolated+murine+sinoatrial+node+cells.">Whole-cell and perforated patch-clamp recordings from acutely-isolated murine sinoatrial node cells.</a> Bio-protocol. 10 (1), 3478 (2020).</li><li>Witchel, H. J., Milnes, J. T., Mitcheson, J. S., Hancox, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+problems+with+in+vitro+screening+of+drugs+for+QT+interval+prolongation+using+HERG+K++channels+expressed+in+mammalian+cell+lines+and+Xenopus+oocytes.">Troubleshooting problems with in vitro screening of drugs for QT interval prolongation using HERG K+ channels expressed in mammalian cell lines and Xenopus oocytes.</a> Journal of Pharmacological and Toxicological Methods. 48 (2), 65-80 (2002).</li><li>Rodriguez-Menchaca, A. A., Ferrer, T., Navarro-Polanco, R. A., Sanchez-Chapula, J. A., Moreno-Galindo, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+whole-cell+patch-clamp+configuration+on+the+pharmacological+assessment+of+the+hERG+channel:+Trazodone+as+a+case+example.">Impact of the whole-cell patch-clamp configuration on the pharmacological assessment of the hERG channel: Trazodone as a case example.</a> Journal of Pharmacological and Toxicological Methods. 69 (3), 237-244 (2014).</li><li>Yang, S., Liu, Z. W., Zhang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+in+vivo+patch+clamp+technique.">The development of in vivo patch clamp technique.</a> Chinese Remedies &amp; Clinics. 5, 399-401 (2003).</li><li>Lin, Y. F., Ouyang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+and+application+of+patch+clamp+technique.">Research progress and application of patch clamp technique.</a> Strait Pharmaceutical Journal. 9, 8-11 (2008).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+insight+into+current+advances+on+pharmacology,+pharmacokinetics,+toxicity+and+detoxification+of+aconitine.">An insight into current advances on pharmacology, pharmacokinetics, toxicity and detoxification of aconitine.</a> Biomedicine &amp; Pharmacotherapy. 151, 113115 (2022).</li><li>Chan, T., Chan, J., Tomlinson, B., Critchley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinese+herbal+medicines+revisited:+A+Hong+Kong+perspective.">Chinese herbal medicines revisited: A Hong Kong perspective.</a> Lancet. 342 (8886-8887), 1532-1534 (1993).</li><li>Jiang, H., Zhang, Y. T., Zhang, Y., Wang, X. B., Meng, X. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+updated+meta-analysis+based+on+the+preclinical+evidence+of+mechanism+of+aconitine-induced+cardiotoxicity.">An updated meta-analysis based on the preclinical evidence of mechanism of aconitine-induced cardiotoxicity.</a> Frontiers in Pharmacology. 13, 900842 (2022).</li><li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+toxicity+of+aconite+alkaloids.">Myocardial toxicity of aconite alkaloids.</a> Shenyang Pharmaceutical University. , (2007).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aconitine+blocks+HERG+and+Kv1.5+potassium+channels.">Aconitine blocks HERG and Kv1.5 potassium channels.</a> Journal of Ethnopharmacology. 131 (1), 187-195 (2010).</li><li>Campbell, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+kinetics+and+selectivity+of+sodium+channels+in+frog+skeletal+muscle+fibers+treated+with+aconitine.">Modified kinetics and selectivity of sodium channels in frog skeletal muscle fibers treated with aconitine.</a> The Journal of General Physiology. 80 (5), 713-731 (1982).</li><li>Huang, X. Y., Ying, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+specific+protein+1+on+Kv2.1+potassium+channel+in+cardiac+myocytes.">The effect of specific protein 1 on Kv2.1 potassium channel in cardiac myocytes.</a> Journal of Electrocardiology and Circulation. 39 (4), 338-341 (2020).</li><li>Cao, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+patch+clamp+technique.">Development and application of patch clamp technique.</a> Journal of Yuncheng University. 27 (2), 53-55 (2009).</li></ol>

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>
			<td></td>
		</tr>
		<tr>
			<td>PH&#160;meter&#160;</td>
			<td>Mettler Toledo</td>
			<td>S201K</td>
			<td></td>
		</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

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 ...

This protocol successfully records the effects of the aconitine on the IKv of H9C2 cells. Promoting new phases for investigating the mechanism of cardiotoxicity of drugs from the perspective of ion channels. The whole-cell patch-clamp technique is the gold standard of ion channel research with high sensitivity, high precision, and high specificity.The whole-cell patch-clamp technique can be employed to explore the pharmacological or toxic mechanisms of juxta gating specific ion channels for multisystem diseases, such as cardiovascular system and nervous system. To begin, digest the cells with 0.25%trypsin for 30 seconds, once the H9C2 culture dish becomes 80%confluent. Culture 2 times 10 to the 5th cells per milliliter with normal medium or drug containing medium in a 35 millimeter dish carpeted with glass plates for 24 hours at 37 degrees celsius, with a 5%carbon dioxide atmosphere and 70 to 80%relative humidity.Turn on the micropipette puller and preheat for 30 minutes. Then put a borosilicate glass capillary with a filament on the micropipette puller. Select the program 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. Write the program for pulling the electrode according to the value determined by the ramp test and click on pull to start fabricating the pipettes. Turn on the digital analog converter, the signal amplifier, the micro manipulator, the microscope, and the camera.Open the imaging application, the signal amplifier software and the data acquisition software in sequence. Click on acquire and select edit protocol in the data acquisition software. Edit the program in the waveform interface.Enter values for first level, delta level, first duration, and delta duration for epoch A, epoch B, and epoch C.Then click on the mode rate interface and set the trial hierarchy data. Next, click on edit protocol to select the stimulus button and set the leak subtraction program in the PM leak subtraction dialogue box. To establish the data storage path, click on file and select set data file names in the data acquisition software.Then open the established lKv protocol by selecting acquire 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. Add the extracellular solution to the cell bath on the whole-cell patch-clamp apparatus and place the cover slip upward with the H9C2 cells in the bath.Fill 30%of the pipette with the extracellular solution and install it on the recording electrode holder integrated into the patch-clamp apparatus. Tighten the pipette with the recording electrode holder. Then drop the pipette into the bath with the micro manipulator.Click on the pipette offset interface of the signal amplifier software to maintain the lKv current baseline at zero picoamps. Deliver an appropriate positive pressure manually with a 1.0 milliliter 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.Once the membrane tests square wave drops by one third to a half 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 gigaseal. Use the capacitance compensation fast and capacitance compensation slow signal amplifier software during cell ceiling to compensate for the fast and slow capacitance.Apply brief pulses of negative pressure to rupture a patch of the cell membrane. 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.Open the saved lKv data with the data analysis software. Save the current voltage relationships. Click on analyze to select statistics.Select the range as Cursors 1 2 for data analysis, click on the peak amplitude and mean buttons and then click on okay to view the data in the results page. Finally, copy the column of lKv mean data into the function drawing software for further analysis. Save the representative lKv 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 in signals. Finally, click on okay to view the data in the results page and copy the total data into the function drawing software to draw the current traces.Save the lKv protocol. Click on edit to select create stimulus waveform signal and select okay. Then repeat the step for drawing current traces without selecting the signal.The lKv was triggered by 150 milliseconds of depolarizing pulse stimulus from minus 40 to plus 60 millivolts at holding potential of minus 60 millivolts. The lKv of the H9C2 rat cardiomyocytes, first appeared around minus 20 millivolts, and then the amplitude increased with further depolarization. In comparison with the control group, the lKv amplitude was observably reduced after the five minutes of treatment with 1.5 millimolar 4-AP.Additionally, the lKv decreased significantly at membrane potentials from 10 to 60 millivolts in a dose-dependent manner, after the 24 hours AC treatment. The individual cell after recording ion channel, can be used for further single-cell omics assess, such as single-cell genomics, transcriptomics, proteomics, and the metabolomics.

voltage-dependent-potassium-current-recording-on-h9c2-cardiomyocytes

Research

JoVE Journal

Medicine

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation (HD-tDCS)

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller "high-definition" electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients. Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes.
Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects. The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets.

Technique and Considerations in the Use of 4x1 Ring High-definition Transcranial Direct Current Stimulation HD-tDCS

High-definition transcranial direct current stimulation (HD-tDCS), with its 4x1-ring montage, is a noninvasive brain stimulation technique that combines both the neuromodulatory effects of conventional tDCS with increased focality. This article provides a systematic demonstration of the use of 4x1 HD-tDCS, and the considerations needed for safe and effective stimulation.

High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of brain stimulation presents several potential advantages compared to other techniques, as it is noninvasive, cost-effective, broadly deployable, and well-tolerated provided proper equipment and protocols are administered. Even though tDCS is apparently simple to perform, correct administration of the tDCS session, especially the electrode positioning and preparation, is vital for ensuring reproducibility and tolerability. The electrode positioning and preparation steps are traditionally also the most time consuming and error-prone. To address these challenges, modern tDCS techniques, using fixed-position headgear and pre-assembled sponge electrodes, reduce complexity and setup time while also ensuring that the electrodes are consistently placed as intended. These modern tDCS methods present advantages for research, clinic, and remote-supervised (at home) settings. This article provides a comprehensive step-by-step guide for administering a tDCS session using fixed-position headgear and pre-assembled sponge electrodes. This guide demonstrates tDCS using commonly applied montages intended for motor cortex and dorsolateral prefrontal cortex (DLPFC) stimulation. As described, selection of the head size and montage-specific headgear automates electrode positioning. Fully assembled pre-saturated snap-electrodes are simply affixed to the set position snap-connectors on the headgear. The modern tDCS method is shown to reduce setup time and reduce errors for both novice and expert operators. The methods outlined in this article can be adapted to different applications of tDCS as well as other forms of transcranial electrical stimulation (tES) such as transcranial alternating current stimulation (tACS) and transcranial random noise stimulation (tRNS). However, since tES is application specific, as appropriate, any methods recipe is customized to accommodate subject, indication, environment, and outcome specific features.

When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS).

Transcranial direct current stimulation (tDCS) is a noninvasive method of neuromodulation using low-intensity direct electrical currents. This method of ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...