Name: murine short axis ventricular heart slices for electrophysiological studies
Uploaded: 2017-06-04T14:00:00
Description: Watch this Scientific Journal Video about Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies at JoVE.com

We acknowledge the support provided by the workshops and the animal facility of the Institute of Neurophysiology. This work was supported by Walter und Marga Boll- Stiftung, K&#246;ln Fortune and Deutsche Stiftung f&#252;r Herzforschung.

Animal handling has to be conform to guidelines of the local animal welfare committee and to the Directive 2010/63/EU of the European Parliament.
1. Prepare Solutions
<ol>
	<li>Prepare Tyrode&#39;s solution without Ca2+ (composition in mM): NaCl 136, KCl 5.4, NaH2PO4 0.33, MgCl2 1, glucose 10, HEPES 5, 2,3-butanedione monoxime (BDM) 30. Adjust pH to 7.4 with NaOH at 4 &#176;C.</li>
	<li>Prepare Tyrode&#39;s solution with Ca2+ (compositio...

<ol>
	<li>Yamamoto, C., McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activities+in+thin+sections+from+the+mammalian+brain+maintained+in+chemically-defined+media+in+vitro.">Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro.</a> J Neurochem. 13, 1333-1343 (1966).</li><li>Colbert, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cortical+brain+slices+for+electrophysiological+recording.">Preparation of cortical brain slices for electrophysiological recording.</a> Methods Mol Biol. 337, 117-125 (2006).</li><li>Ad Graaf, I., Groothuis, G. M., Olinga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+tissue+slices+as+a+tool+to+predict+metabolism+of+novel+drugs.">Precision-cut tissue slices as a tool to predict metabolism of novel drugs.</a> Expert Opin Drug Metab Toxicol. 3, 879-898 (2007).</li><li>Kim, Y. H., 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=Cardiopulmonary+toxicity+of+peat+wildfire+particulate+matter+and+the+predictive+utility+of+precision+cut+lung+slices.">Cardiopulmonary toxicity of peat wildfire particulate matter and the predictive utility of precision cut lung slices.</a> Part Fibre Toxicol. 11, 29 (2014).</li><li>Nembo, E. N., 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=In+vitro+chronotropic+effects+of+Erythrina+senegalensis+DC+(Fabaceae)+aqueous+extract+on+mouse+heart+slice+and+pluripotent+stem+cell-derived+cardiomyocytes.">In vitro chronotropic effects of Erythrina senegalensis DC (Fabaceae) aqueous extract on mouse heart slice and pluripotent stem cell-derived cardiomyocytes.</a> J Ethnopharmacol. 165, 163-172 (2015).</li><li>Wang, 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=Cardiac+tissue+slices:+preparation,+handling,+and+successful+optical+mapping.">Cardiac tissue slices: preparation, handling, and successful optical mapping.</a> Am J Physiol Heart Circ Physiol. 308, H1112-H1125 (2015).</li><li>Bussek, 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=Tissue+slices+from+adult+mammalian+hearts+as+a+model+for+pharmacological+drug+testing.">Tissue slices from adult mammalian hearts as a model for pharmacological drug testing.</a> Cell Physiol Biochem. 24, 527-536 (2009).</li><li>Burnashev, N. A., Edwards, F. A., Verkhratsky, A. N. <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+recordings+on+rat+cardiac+muscle+slices.">Patch-clamp recordings on rat cardiac muscle slices.</a> Pflugers Arch. 417, 123-125 (1990).</li><li>Pillekamp, F., 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=Establishment+and+characterization+of+a+mouse+embryonic+heart+slice+preparation.">Establishment and characterization of a mouse embryonic heart slice preparation.</a> Cell Physiol Biochem. 16, 127-132 (2005).</li><li>Pillekamp, F., 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=Neonatal+murine+heart+slices.+A+robust+model+to+study+ventricular+isometric+contractions.">Neonatal murine heart slices. A robust model to study ventricular isometric contractions.</a> Cell Physiol Biochem. 20, 837-846 (2007).</li><li>Halbach, 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=Ventricular+slices+of+adult+mouse+hearts--a+new+multicellular+in+vitro+model+for+electrophysiological+studies.">Ventricular slices of adult mouse hearts--a new multicellular in vitro model for electrophysiological studies.</a> Cell Physiol Biochem. 18, 1-8 (2006).</li><li>Halbach, 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=Electrophysiological+maturation+and+integration+of+murine+fetal+cardiomyocytes+after+transplantation.">Electrophysiological maturation and integration of murine fetal cardiomyocytes after transplantation.</a> Circ. Res. 101, 484-492 (2007).</li><li>Halbach, 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=Time-course+of+the+electrophysiological+maturation+and+integration+of+transplanted+cardiomyocytes.">Time-course of the electrophysiological maturation and integration of transplanted cardiomyocytes.</a> J. Mol. Cell Cardiol. 53, 401-408 (2012).</li><li>Halbach, 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=Cell+persistence+and+electrical+integration+of+transplanted+fetal+cardiomyocytes+from+different+developmental+stages.">Cell persistence and electrical integration of transplanted fetal cardiomyocytes from different developmental stages.</a> Int. J. Cardiol. 171, e122-e124 (2014).</li><li>Halbach, 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=Electrophysiological+integration+and+action+potential+properties+of+transplanted+cardiomyocytes+derived+from+induced+pluripotent+stem+cells.">Electrophysiological integration and action potential properties of transplanted cardiomyocytes derived from induced pluripotent stem cells.</a> Cardiovasc. Res. 100, 432-440 (2013).</li><li>Verrecchia, F., Herve, 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=Reversible+blockade+of+gap+junctional+communication+by+2,3-butanedione+monoxime+in+rat+cardiac+myocytes.">Reversible blockade of gap junctional communication by 2,3-butanedione monoxime in rat cardiac myocytes.</a> Am J Physiol. 272, C875-C885 (1997).</li><li>Watanabe, 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=Inhibitory+effect+of+2,3-butanedione+monoxime+(BDM)+on+Na(+)/Ca(2+)+exchange+current+in+guinea-pig+cardiac+ventricular+myocytes.">Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes.</a> Br J Pharmacol. 132, 1317-1325 (2001).</li><li>Fleischmann, B. 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=Differential+subunit+composition+of+the+G+protein-activated+inward-rectifier+potassium+channel+during+cardiac+development.">Differential subunit composition of the G protein-activated inward-rectifier potassium channel during cardiac development.</a> J Clin Invest. 114, 994-1001 (2004).</li><li>Peinkofer, G., 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=From+Early+Embryonic+to+Adult+Stage:+Comparative+Study+of+Action+Potentials+of+Native+and+Pluripotent+Stem+Cell-Derived+Cardiomyocytes.">From Early Embryonic to Adult Stage: Comparative Study of Action Potentials of Native and Pluripotent Stem Cell-Derived Cardiomyocytes.</a> Stem Cells Dev. 25, 1397-1406 (2016).</li></ol>

Ventricular slices enable electrophysiological, pharmacological and mechanical studies with a preserved in vivo like tissue structure and direct access of the measurement technology to all regions of the heart. Physiological action potential properties have been demonstrated in embryonic, neonatal and adult slices 9,10,11. Vitality of the slices, except for the surface layers directly damaged by the slicing procedure, h...

Thin tissue slices have been used frequently in basic science since Yamamot and Mcllwain showed in 1966 that electrical activity of brain slices is maintained in vitro1. Since then, electrophysiological and pharmacological studies have been conducted on slices from brain 2, liver 3, lung 4 and myocardial tissue 5,6,7. First patch-clamp recordings in ventricular slices from neonatal rat hearts were described in 1990 8, but this technique fell into oblivion for some time. More than one decade later, our group established a new method to prepare murine embryonic 9, neonatal 10 and adult 11 heart slices. These viable tissue slices can be used for acute experiments (adult slices can be cultivated for several hours) or short-term culture experiments (embryonic and neonatal slices can be cultivated for a few days). Slices show in vivo like electrophysiological characteristics and a homogenous excitation spread as assessed by sharp electrode action potential and micro electrode array recordings 11. Due to their &#34;two-dimensional&#34; morphology, they allow direct access of recording electrodes to all regions of the ventricle, which makes them an interesting tool for electrophysiological investigations and raises new experimental options in comparison to Langendorff-perfused whole hearts. Drug response of the slices to ion channel blockers like verapamil (L-type Ca2+-channel blocker), lidocain (Na+-channel blocker), 4-aminopyridine (unselective voltage dependent K+-channel blocker) and linopirdine (KCNQ K+-channel blocker) 9,11 corresponded to known effects on dissociated cardiomyocytes. Isometric force measurements revealed a positive force frequency relationship and strongly suggested intact contractile function 10. These findings demonstrated that murine ventricular slices are suitable as an in vitro tissue model for physiological and pharmacological studies. Furthermore, ventricular slices of recipient hearts in combination with sharp electrode recordings have proven to be a very helpful tool to characterize electrical and mechanical integration as well as maturation of transplanted fetal 12,13,14 and stem cell-derived 15 cardiomyocytes.
In summary, ventricular slices are a valuable and well-establish multicellular tissue model and should be considered complementary to dissociated cardiomyocytes and Langendorff-perfused hearts in cardiovascular research, with the major advantage of providing an in vivo like tissue structure (in contrast to dissociated cells) as well as direct access of measurement technologies like sharp electrode recordings to all regions of the heart (in contrast to whole heart preparations).

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			<td height="42" style="height:42px;width:248px;">Leica VT 1000s</td>
			<td style="width:340px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;">Microtome with vibrating blade.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Stainless Steel Blades</td>
			<td style="width:340px;">Campden Instruments, Loughborough, England</td>
			<td>7550-1-SS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes&#160;</td>
			<td>Sigma-Aldrich, St. Louise, USA</td>
			<td>Z627992&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Fine brush, e.g. size 6 (4/32&#34;)</td>
			<td style="width:340px;">VWR, International, Radnor, USA</td>
			<td>149-2125</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Preparation table</td>
			<td style="width:340px;"></td>
			<td style="width:140px;"></td>
			<td style="width:167px;">self made</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Molt for embedding ventricles in agarose</td>
			<td style="width:340px;"></td>
			<td style="width:140px;"></td>
			<td style="width:167px;">self made</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:248px;">1 ml Syringe</td>
			<td style="width:340px;">Becton, Dickinson;&#160; Franklin Lakes, USA</td>
			<td>300013</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">27Gx3/4`` Needles</td>
			<td rowspan="2" style="width:340px;">Braun, Melsungen, Germany</td>
			<td>4657705</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">20G 11/2`` Needles</td>
			<td>4657519</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Small scissor</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td>501263</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Tweezers #5, 0.1 x 0.06 mm tip</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td>500342</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen gas (medical grade O2)</td>
			<td style="width:340px;">Linde, Munich, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Carbogen gas (95 % O2, 5 % CO2)&#160;</td>
			<td style="width:340px;">Linde, Munich, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">NaCL</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">7647-14-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">KCL</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">746436</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">CaCl2</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">746495</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">KH2PO4</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">NIST200B&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">HEPES</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">51558</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">NaHCO3</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">S5761&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">D(+)-Glucose</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">G8270&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">MgSO4</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">M7506&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">NaOH</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">S8045&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Cyanoacrylate glue&#160;</td>
			<td style="width:340px;">Henkel, D&#252;sseldorf, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Low-melt Agarose&#160;</td>
			<td style="width:340px;">Roth, Karlsruhe, Germany</td>
			<td style="width:140px;">6351.2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Heparin-sodium-25000 I.E./5mL</td>
			<td style="width:340px;">Ratiopharm, Ulm, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Dulbecco&#39;s Modified Eagle Medium&#160; (DMEM), high glucose, GlutaMAX</td>
			<td style="width:340px;">ThermoScientific, Waltham, USA</td>
			<td style="width:140px;">10566016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">SEC-10LX Amplifier</td>
			<td style="width:340px;">npi electronic GmbH, Tamm, Germany</td>
			<td>SEC-10LX</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">EPC 9</td>
			<td style="width:340px;">HEKA Elektronik GmbH, Lambrecht, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Zeiss Axiovert 200</td>
			<td style="width:340px;">Zeiss, Oberkochen, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">&#160;Low magnification Micromanipulator</td>
			<td style="width:340px;">Narashige, Tokyo, Japan</td>
			<td style="width:140px;">Nm-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">High magnification, three-axis micromanipulator</td>
			<td style="width:340px;">Narashige, Tokyo, Japan</td>
			<td style="width:140px;">MHW-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Peristaltic perfusion pump</td>
			<td style="width:340px;">Multi Channel Systems, Reutlingen, Germany</td>
			<td style="width:140px;">PPS2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">2-channel temperature controller</td>
			<td style="width:340px;">Multi Channel Systems, Reutlingen, Germany</td>
			<td style="width:140px;">TCO02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Square pulse stimulator</td>
			<td style="width:340px;">Natus Europe GmbH, Planegg, Germany</td>
			<td style="width:140px;">Grass SD9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Glass capillaries</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td style="width:140px;">1B150F-1</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

murine short axis ventricular heart slices for electrophysiological studies

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca2+-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 &#181;m are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode&#39;s solution with 0.9 mM Ca2+ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 &#176;C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode.

Myocardial infarction leads to a virtually irreversible loss of cardiomyocytes. Cell replacement therapy using stem cell-derived cardiomyocytes for exogenous cardiac regeneration is a promising therapeutic approach. Electrical integration and maturation of the transplanted cells are crucial for safety and efficiency of cell replacement therapy.
To assess integration and maturation, we transplanted cardiomyocytes derived from ind...

Watch this Scientific Journal Video about Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies at JoVE.com

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro.

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

The overall goal of this technique is to obtain viable ventricular heart slices, which are suitable for electrophysiological recordings. This method can help answer key questions in the field of cardiac electrophysiology on the tissue level, such as on electrical integration of transplanted cardiomyocytes or effects of cardioactive drugs. The main advantage of this technique is that viable ventricular tissue slices have a preserved, in vivo-like tissue structure, and it enables direct measurements in all regions of the heart.Though this method can provide insight into murine heart electrophysiology, it can also be applied to other animals, like rabbit or guinea pig. To begin this procedure, first wipe the skin of the animal with 70%ethanol. After opening the thorax, carefully dissect the heart's pericardium, using small scissors and forceps.Insert a cannula into the left ventricle and perfuse the heart in situ with ice cold Tyrode solution without calcium, until the remaining blood is removed. Then, gently resect the heart and transfer it to the ice cold Tyrode solution without calcium. Separate the atria from the ventricles with a scalpel or scissors.Next, place the ventricles with the apex, facing upwards, in the agarose mold. Subsequently, place the pin in the middle of the mold and in the left ventricular chamber. Fill the mold with 4%low-melt agarose at 37 degrees Celsius until the heart is completely covered.Then, place the mold on ice for faster agarose hardening to prevent the tissue from floating. After one minute, remove the agarose block, containing the ventricles, from the mold with a scalpel. After that, turn the block upside down and fill the ventricular chambers and the gap on the backside of the agarose block with 4%low-melt agarose using a syringe.Then, trim the agarose block with a scalpel to achieve a flat bottom and upright position of the cardiac apex. In this procedure, fix the block on the specimen holder of the microtome with a drop of cyanoacrylate glue, with the cardiac apex facing upward. Next, place the specimen holder into the inner specimen chamber of the microtome, which is filled with ice cold Tyrode without calcium.Ensure that the agarose block is completely covered with Tyrode solution. Prepare the slices at a thickness of 150 to 400 micrometers, depending on further application. Keep the vibration frequency of the blade, at 60 to 70 Hertz, and move the blade forward, as slowly as possible.Afterward, use a fine brush to carefully remove the remaining agarose from the slices. Then, gently transfer the slices with a Pasteur pipette into the Tyrode solution, with 0.9 millimolar calcium, aerated with 100%oxygen. Store them for at least 30 minutes on ice to recover from the slicing procedure.Placing the slices on a net helps keep them flat. Afterwards, use a filter paper and a second net to transfer the slices in DMEM at 37 degrees Celsius, bubbled with carbogen, to wash out BDM before further use. For preheating, switch on all the electric devices 30 minutes before the recordings.Place a three centimeter cell culture dish with the heating plate on the inverted microscope. Then, place the custom-made ring electrode in the dish, and connect the grounding wires of the preamplifier and the stimulation electrode. After that, connect the flexible tubes of the perfusion system to the dish.Fill the reservoir of the perfusion system with DMEM and aerate it with carbogen. Then, switch on the perfusion pump and set the perfusion rate to two to three milliliters per minute. Adjust the temperature of DMEM in the dish to 37 degrees Celsius, by regulating the flow heater and the heating plate.Now, place a ventricular slice into the DMEM-filled dish. With the inverted microscope, check the structural integrity and viability of the tissue. Then, fill a recording glass electrode with three molar potassium chloride, and a stimulation electrode with DMEM.Next, place the recording electrode in the stimulation electrode on the electrode holders. Subsequently, place the stimulation electrode carefully on the slice. Switch on the electric stimulator and start with a stimulation frequency of one to two Hertz.Then, move the recording electrode with a micromanipulator to the intended recording position. Slowly lower the recording electrode until the tip touches the tissue. Once the recording detects the contact, apply a short rectangular electric pulse through the recording electrode to penetrate the cell membrane.Next, carefully reposition the recording electrode until a stable signal is ensured. Following that, begin the recording of action potentials. To assess integration and maturation, cardiomyocytes, derived from iPSCM expressing eGFP, were transplanted into the healthy hearts of adult mice.Shown here is a ventricular heart slice, containing the transplanted iPSCM, which was focally stimulated by a unipolar electrode, placed in the host tissue. Intracellular action potentials were recorded with sharp glass microelectrodes filled with three molar potassium chloride and eGFP-positive transplanted iPSCM, and neighboring host tissue within the slices. Here are the action potential recordings, in the healthy host tissue and the transplanted iPSCM.The slice was focally stimulated with a stimulation electrode placed in the host tissue, at around two Hertz and five Hertz. Once mastered, this technique can be done in one to two hours, if it is performed properly. While attempting this procedure, it is important to remember to keep the forward speed of the microtome blade as slow as possible.Following this procedure, other methods like first measurements can be performed in order to answer additional questions. After its development, this technique paved the way for researchers in the field of cell therapy to explore electric and integration, and maturation of transplanted cardiomyocytes in recipient hearts. After watching this video, you should have a good understanding of how to prepare viable ventricular short axis slices, using a microtome with a vibrating blade.These slices are particularly suitable for electrophysiological recordings sharp glass electrodes.

murine-short-axis-ventricular-heart-slices-for-electrophysiological

Research

JoVE Journal

Medicine

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Use of Two Intracorporeal Ventricular Assist Devices As a Total Artificial Heart

Mechanical circulatory support (MCS) has been introduced as a viable alternative to heart transplantation primarily through the use of intracorporeal ventricular assist devices (VADs) for support of the left ventricle. However, certain clinical scenarios warrant biventricular mechanical support. One strategy for some patients is the excision of both ventricles and the implantation of two VAD pumps as a total artificial heart (TAH). This has recently been made possible by the improvements in device design and the small profile of centrifugal devices. This TAH approach remains experimental with many important challenges such as the device settings to balance the right and left circulation, the orientation of the devices and the outflow graft with their influence on hemolysis and stability, and the outcome of chronic support using such an orientation. This protocol aims to provide a reproducible approach for total artificial heart replacement with two intracorporeal centrifugal VADs in a cow model.

Here, we present a protocol using two centrifugal pumps as a total artificial heart replacement.

Mechanical circulatory support (MCS) has been introduced as a viable alternative to heart transplantation primarily through the use of intracorporeal ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Use of a Percutaneous Ventricular Assist Device/Left Atrium to Femoral Artery Bypass System for Cardiogenic Shock

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the left ventricle by draining blood from the left atrium (LA) and returning it to the systemic arterial circulation via the femoral artery. It can provide flows ranging from 2.5-5 L/min depending on the size of the cannula. Here, we discuss the mechanism of action of LAFAB, available clinical data, indications for its use in cardiogenic shock, steps of implantation, post-procedural care, and complications associated with the use of this device and their management.
We also provide a brief video of the procedural component of device therapy, including the pre-placement preparation, percutaneous placement of the device via transseptal puncture under echocardiographic guidance and the post-operative management of device parameters.

The following article describes the stepwise procedure for placement of a device (e.g., Tandemheart) in cardiogenic shock (CS) that is a percutaneous left ventricular assist device (pLVAD) and a left atrial to femoral artery bypass (LAFAB) system that bypasses and supports the left ventricle (LV) in CS.

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the ...

Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of neurological disorders. Despite strong pre-clinical evidence, TH has not shown efficacy in clinical trials of most neurological disorders. The only successful trials employing therapeutic hypothermia were related to cardiac arrest in adults and hypoxic ischemic injury in neonates. Further investigations into the parameters of its use, and study design comparisons between pre-clinical and clinical studies, are warranted. This article demonstrates two methods of short-duration hypothermia induction. The first method allows for rapid hypothermia induction in rats using ethanol spray and fans. This method works by cooling the skin, which has been less commonly used in clinical trials and may have different physiological effects. Cooling is much more rapid with this technique than is achievable in human patients due to differences in surface area to volume ratio. Along with this, a second method is also presented, which allows for a clinically achievable cooling rate for short-duration hypothermia. This method is easy to implement, reproducible and does not require active skin cooling.

This article describes two methods of whole-body short-duration hypothermia induction in rats. The first, rapid induction method, employs active cooling using fans and ethanol spray for a rapid decrease in temperature. The second method is a gradual cooling method. This is achieved using the combination of isoflurane anesthesia and the reduction of temperature settings on the homeothermic heat mat. This results in a gradual decrease in core body temperature without the use of any external cooling devices.&#160;

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of ...