We would like to thank Dr. Claycomb for providing HL-1 cells and detailed maintenance protocol. We would also like to thank Ms. Elena Carrillo and Dr. Seth Robia for their assistance with generating the cell movie and Mr. Pete Caron for his assistance with making some accessories for our dual channel optical mapping system.&#160;
This work was supported by American Heart Association (10GRNT3770030 &#38; 12GRNT12050478 to XA), National Institutes of Health (HL113640 to XA), and Loyola University Research Development Fund (to XA).&#160;

1. Solution Preparation
<ol>
	<li>Norepinephrine (NP) solution
	<ol>
		<li>Dissolve 40 mg of NP into 25 ml of 30 mM ascorbic acid (0.1475 g ascorbic acid in 25 ml of purified Milli-Q (MQ) water. Avoid direct light exposure during this solution preparation because norepinephrine (NP) is light sensitive.</li>
		<li>Filter the NP solution with a 0.22 &#181;m syringe filter. Aliquot the solution into 1 ml sterile microtubes and store at -20 &#176;C. Once frozen, NP is stable for one month.</li>
	</ol>
	</li>
	<li>Supplemen...

<ol>
	<li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95 (1), 21-33 (2004).</li><li>Salama, G., Hwang, S. 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=Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts.">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.]. 12 (Unit 12 17), (2009).</li><li>Laurita, K. R., Singal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+action+potentials+and+calcium+transients+simultaneously+from+the+intact+heart.">Mapping action potentials and calcium transients simultaneously from the intact heart.</a> Am J Physiol Heart Circ Physiol. 280 (5), H2053-H2060 (2001).</li><li>Fast, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+imaging+of+membrane+potential+and+intracellular+calcium.">Simultaneous optical imaging of membrane potential and intracellular calcium.</a> J Electrocardiol. 38 (4 Suppl), 107-112 (2005).</li><li>Sowell, B., Fast, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+mechanism+of+shock-induced+arrhythmias:+role+of+intracellular+calcium.">Ionic mechanism of shock-induced arrhythmias: role of intracellular calcium.</a> Heart Rhythm. 9 (1), 96-104 (2012).</li><li>Fast, V. G., Kleber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+conduction+in+cultured+strands+of+neonatal+rat+heart+cells+measured+with+voltage-sensitive+dyes.">Microscopic conduction in cultured strands of neonatal rat heart cells measured with voltage-sensitive dyes.</a> Circ Res. 73 (5), 914-925 (1993).</li><li>Hou, L., 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=A+major+role+for+HERG+in+determining+frequency+of+reentry+in+neonatal+rat+ventricular+myocyte+monolayer.">A major role for HERG in determining frequency of reentry in neonatal rat ventricular myocyte monolayer.</a> Circ Res. 107 (12), 1503-1511 (2010).</li><li>Beauchamp, 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=Relative+contributions+of+connexins+40+and+43+to+atrial+impulse+propagation+in+synthetic+strands+of+neonatal+and+fetal+murine+cardiomyocytes.">Relative contributions of connexins 40 and 43 to atrial impulse propagation in synthetic strands of neonatal and fetal murine cardiomyocytes.</a> Circ Res. 99 (11), 1216-1224 (2006).</li><li>Xia, 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=Functional+expression+of+L-+and+T-type+Ca2++channels+in+murine+HL-1+cells.">Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells.</a> J Mol Cell Cardiol. 36 (1), 111-119 (2004).</li><li>Claycomb, W. 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=HL-1+cells:+a+cardiac+muscle+cell+line+that+contracts+and+retains+phenotypic+characteristics+of+the+adult+cardiomyocyte.">HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.</a> Proc Natl Acad Sci U S A. 95 (6), 2979-2984 (1998).</li><li>Fahrenbach, J. P., Ai, X., Banach, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+intercellular+coupling+improves+the+function+of+cardiac+pacemakers+derived+from+mouse+embryonic+stem+cells.">Decreased intercellular coupling improves the function of cardiac pacemakers derived from mouse embryonic stem cells.</a> J Mol Cell Cardiol. 45 (5), 642-649 (2008).</li><li>Yan, 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=c-Jun+N-terminal+kinase+activation+contributes+to+reduced+connexin43+and+development+of+atrial+arrhythmias.">c-Jun N-terminal kinase activation contributes to reduced connexin43 and development of atrial arrhythmias.</a> Cardiovasc Res. 97 (3), 589-597 (2013).</li><li>White, S. M., Constantin, P. E., Claycomb, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+physiology+at+the+cellular+level:+use+of+cultured+HL-1+cardiomyocytes+for+studies+of+cardiac+muscle+cell+structure+and+function.">Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function.</a> Am J Physiol Heart Circ Physiol. 286 (3), H823-H829 (2004).</li><li>Yang, Z., Shen, W., Rottman, J. N., Wikswo, J. P., Murray, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+stimulation+causes+electrical+remodeling+in+cultured+atrial+myocytes.">Rapid stimulation causes electrical remodeling in cultured atrial myocytes.</a> J Mol Cell Cardiol. 38 (2), 299-308 (2005).</li><li>Brundel, B. J., Kampinga, H. H., Henning, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain+inhibition+prevents+pacing-induced+cellular+remodeling+in+a+HL-1+myocyte+model+for+atrial+fibrillation.">Calpain inhibition prevents pacing-induced cellular remodeling in a HL-1 myocyte model for atrial fibrillation.</a> Cardiovasc Res. 62 (3), 521-528 (2004).</li><li>Umapathy, K., 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=Electrogram+fractionation+in+murine+HL-1+atrial+monolayer+model.">Electrogram fractionation in murine HL-1 atrial monolayer model.</a> Heart Rhythm. 5 (7), 1029-1035 (2008).</li><li>Fast, V. G., Cheek, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+arrhythmias+induced+by+strong+electrical+shocks+in+myocyte+cultures.">Optical mapping of arrhythmias induced by strong electrical shocks in myocyte cultures.</a> Circ Res. 90 (6), 664-670 (2002).</li><li>Bayly, P. V., 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=Estimation+of+conduction+velocity+vector+fields+from+epicardial+mapping+data.">Estimation of conduction velocity vector fields from epicardial mapping data.</a> IEEE Trans Biomed Eng. 45 (5), 563-571 (1998).</li></ol>

No conflicts of interest declared.

This article describes the key aspects of optical mapping in a cultured HL-1 atrial myocyte monolayer stained with calcium and voltage sensitive fluorescent dyes. It includes culturing an optimal HL-1 cell monolayer, setup of the mapping equipment, mapping a cultured monolayer, and data analysis.
To successfully map cultured cells, the key is to prepare a uniformly distributed cell monolayer. When seeding the cells on to the coverslips, be sure to evenly disperse the cells. Always map fully-gr...

A unique calcium (Ca) and voltage (Vm) dual channel optical mapping technique1-5 is emerging as an efficient tool to simultaneously record Vm and Ca signals in both intact hearts and cultured cell monolayers. This technique makes it possible to obtain powerful information regarding the relationship between calcium transients and action potentials to better understand the underlying electrophysiological mechanisms of cardiac arrhythmias.
Cultured cell monolayers have proven to be a useful cellular model to study cardiac electrophysiology and the underlying mechanism of arrhythmias.4,6-8 HL-1 cells are a well-characterized atrial myocyte culture line. HL-1 cells also maintain a uniquely differentiated genotype and phenotype that includes morphologic, electrophysiologic, and pharmacologic characteristics of adult atrial myocytes. These cells express cardiac genes and proteins, including important cardiac ion channels (i.e., L- and T-type calcium channels)9 and mature isoforms of sarcomeric contractile proteins normally found in adult atrial myocytes as others and we have previously reported.10-13 In addition, HL-1 cells can be cultured to form a 2-dimensional (2-D) myocyte monolayer. Thus, the advantages of using cultured HL-1 monolayers include: 1) relatively lower cost and easier to maintain a myocyte culture line than&#160;isolating and culturing primary&#160;neonatal myocytes; 2) a confluent monolayer of cells reduces the structural complexities that result from the 3-D structure of the heart; 3) a cell monolayer can eliminate the interference of interstitial fibrosis that occurs in the intact heart. This can be used to dissect specific electrophysiological functions of a group of myocytes without interference of fibroblasts and interstitial matrix; 4) assessment of functional consequences from pharmacological or genetic manipulation in cultured cell monolayers can be effectively achieved. Therefore, HL-1 cells have become a widely used cellular model for studying diverse aspects of myocyte physiology as well as pacing-induced abnormal electrical activities.13-16 However, special handling and care is required to culture healthy cell monolayers that respond to external electrical pacing for optical mapping studies. In addition, dual florescent dye staining procedure may easily damage the integrity of cultured confluent cell monolayers. Thus, performing Vm/CA&#160;dual channel optical mapping in cultured HL-1 monolayers has been a great challenge.
The goal of this method is to provide the key steps for successfully performing dual channel optical mapping in cultured HL-1 monolayers. Here we have provided extensive details on an optimized protocol for HL-1 cell monolayer preparation, dual channel optical mapping of a cultured cell monolayer, and mapping data processing.

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rhod2 dry powder</td>
			<td style="width:103px;">&#160;AAT Bioquest</td>
			<td style="width:119px;">21062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pluronic</td>
			<td style="width:103px;">AAT Bioquest</td>
			<td style="width:119px;">20050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DMSO</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rh237</td>
			<td style="width:103px;">Invitrogen</td>
			<td style="width:119px;">&#160;S-1109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4&#160;</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">P0662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">S6014</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">C3881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPEs</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

voltage and calcium dual channel optical mapping of cultured hl-1 atrial myocyte monolayer

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

A cultured confluent monolayer exhibits a regular intrinsic&#160;rhythm as demonstrated in Movie 1. We then performed Vm/Ca dual channel optical mapping in a fully confluent HL-1 monolayer. Figure 1A shows example traces of Vm and Ca signals from a recorded single beat. Representative isochronal maps of uniformly propagated Vm and Ca signals using the dual channel optical mapping system are shown in Figures 1B and 1C. Rep...

Watch this Scientific Journal Video about Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer at JoVE.com

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

voltage-calcium-dual-channel-optical-mapping-cultured-hl-1-atrial

Research

JoVE Journal

Biology

10.1K 조회수.  Loyola University Chicago. This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.