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+ (composition in mM): NaCl 136, KCl 5.4, NaH2PO4 0.33, MgCl2 1, glucose 10, HEPES 5, BDM 30, CaCl2 0.9. Adjust pH to 7.4 with NaOH at 4 &#176;C.</li>
	<li>Prepare 4% low-melt agarose: Put 0.6 g low-melt agarose in 15 mL Tyrode&#39;s solution without Ca2+. Heat the mixture in a microwave oven two times at 750 W for 10-15 s until the agarose is dissolved. Keep the solution at a constant temperature of 37 &#176;C and stir continuously.</li>
	<li>Keep Dulbecco&#39;s Modified Eagle Medium (DMEM) without serum at 37 &#176;C bubbled with carbogen (5% CO2, 95% O2).</li>
</ol>
2. Prepare the Microtome
<ol>
	<li>Switch on the microtome.</li>
	<li>Fill the outer microtome chamber with ice.</li>
	<li>Fill the inner microtome chamber with ice cold Tyrode&#39;s solution without Ca2+ and continuously oxygenate the solution with 100% O2.</li>
	<li>Place a steel blade into the blade-holder of the microtome.</li>
</ol>
3. Mouse Heart Isolation
<ol>
	<li>Inject 2,500 U Heparin subcutaneously. Wait 15 min.</li>
	<li>Sacrifice the animal by cervical dislocation.</li>
	<li>Open the chest by sternotomy.</li>
	<li>Carefully dissect the pericardium using small scissors and a forceps #5.</li>
	<li>Insert a cannula into the ascending aorta and perfuse the coronary arteries in situ with ice cold Tyrode&#39;s solution without Ca2+ until remaining blood is removed.</li>
	<li>Gently resect the heart with a forceps and scissors and transfer the heart in ice cold Tyrode&#39;s solution rode without Ca2+.</li>
	<li>Separate the atria from the ventricles with a scalpel or scissors.</li>
</ol>
4. Embedding of the Ventricles in 4% Low-melt Agarose
<ol>
	<li>Place the ventricles with the apex facing upwards in the agarose mold (Figure 1). Place the pin in the middle of the mold in the left ventricular chamber.</li>
	<li>Fill the mold with 4% low-melt agarose at 37 &#176;C, until the heart is completely covered.</li>
	<li>Place the mold on ice for faster hardening of the agarose preventing floating of the tissue.</li>
	<li>Remove the agarose block containing the ventricles from the mold with a scalpel.</li>
	<li>Turn the block upside down and fill ventricular chambers and the gap on the backside of the agarose block, which is left by the pin of the molt, with 4% low-melt agarose using a syringe with a 20 G needle.</li>
	<li>Trim the agarose block with a scalpel to achieve a flat bottom of the block and upright position of the cardiac apex.</li>
</ol>
5. Slicing the Ventricular Tissue
<ol>
	<li>Fix the block on the specimen holder of the microtome with a drop of cyanoacrylate glue. Face the cardiac apex upwards.</li>
	<li>Place the specimen holder into the inner specimen chamber of the microtome, which is filled with ice cold Tyrode without Ca2+. Completely cover the agarose block with Tyrode&#39;s solution.</li>
	<li>Prepare short-axis slices at a thickness of 150-400 &#181;m, depending on the further application (for sharp electrode recordings 150-200 &#181;m), keep vibration frequency of the blade at 60-70 Hz and move the blade forward as slowly as possible.</li>
	<li>Use a fine brush to carefully remove the remaining agarose from the slices.</li>
	<li>Gently transfer slices with a Pasteur pipette into the Tyrode&#39;s solution with 0.9 mM Ca2+ aerated with 100% O2 and store them for at least 30 min on ice to recover from the slicing procedure.</li>
	<li>Afterwards, keep slices for 30 min in DMEM at 37 &#176;C, aerated with carbogen, to wash out BDM before further use.</li>
</ol>
6. Preparing the Sharp Electrode Setup
<ol>
	<li>For pre-heating, switch on all electric devices 30 min before the recordings start.</li>
	<li>Put a 3 cm cell culture dish on the heating plate placed on the inverted microscope.</li>
	<li>Place the custom-made ring electrode (Figure 2) in the dish and connect grounding wires of the pre-amplifier and the stimulation electrode.</li>
	<li>Connect the flexible tubes of the perfusion system to the dish.</li>
	<li>Fill the reservoir of the perfusion system with DMEM, aerated with carbogen.</li>
	<li>Switch on the perfusion pump and set the perfusion rate to 2-3 mL/min.</li>
	<li>Adjust the temperature of DMEM in the dish to 37 &#176;C by regulating the flow heater and the heating plate.</li>
</ol>
7. Action Potential Recordings
<ol>
	<li>Place a ventricular slice into the DMEM filled dish.</li>
	<li>Check the structural integrity and viability (based on contractile function) of the tissue with the inverted microscope.</li>
	<li>Fill a recording glass electrode with 3 M KCL.</li>
	<li>Fill a stimulation electrode with DMEM.</li>
	<li>Place the recording electrode and the stimulation electrode on the electrode holders.</li>
	<li>Place the stimulation electrode carefully on the slice and switch on the electric stimulator. Start with a stimulation frequency of 1-2 Hz.</li>
	<li>Move the recording electrode with the micromanipulator over the intended recording position.</li>
	<li>Slowly lower the recording electrode until the tip touches the tissue.</li>
	<li>Apply a short rectangular electric pulse through the recording electrode to penetrate the cell membrane.</li>
	<li>Carefully reposition the recording electrode until a stable signal is ensured.</li>
	<li>Start recording of action potentials.</li>
</ol>

<ol><li>Yamamoto, C., McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrical+activities+in+thin+sections+from+the+mammalian+brain+maintained+in+chemically-defined+media+in+vitro%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">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/pubmed?term=((Preparation+of+cortical+brain+slices+for+electrophysiological+recording%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Precision-cut+tissue+slices+as+a+tool+to+predict+metabolism+of+novel+drugs%5BTitle%5D)+AND+%22Expert+Opin+Drug+Metab+Toxicol%22%5BJournal%5D)">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/pubmed?term=((Cardiopulmonary+toxicity+of+peat+wildfire+particulate+matter+and+the+predictive+utility+of+precision+cut+lung+slices%5BTitle%5D)+AND+%22Part+Fibre+Toxicol%22%5BJournal%5D)">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/pubmed?term=((In+vitro+chronotropic+effects+of+Erythrina+senegalensis+DC+%28Fabaceae%29+aqueous+extract+on+mouse+heart+slice+and+pluripotent+stem+cell-derived+cardiomyocytes%5BTitle%5D)+AND+%22J+Ethnopharmacol%22%5BJournal%5D)">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/pubmed?term=((Cardiac+tissue+slices%3A+preparation%2C+handling%2C+and+successful+optical+mapping%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">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/pubmed?term=((Tissue+slices+from+adult+mammalian+hearts+as+a+model+for+pharmacological+drug+testing%5BTitle%5D)+AND+%22Cell+Physiol+Biochem%22%5BJournal%5D)">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/pubmed?term=((Patch-clamp+recordings+on+rat+cardiac+muscle+slices%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">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/pubmed?term=((Establishment+and+characterization+of+a+mouse+embryonic+heart+slice+preparation%5BTitle%5D)+AND+%22Cell+Physiol+Biochem%22%5BJournal%5D)">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/pubmed?term=((Neonatal+murine+heart+slices.+A+robust+model+to+study+ventricular+isometric+contractions%5BTitle%5D)+AND+%22Cell+Physiol+Biochem%22%5BJournal%5D)">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/pubmed?term=((Ventricular+slices+of+adult+mouse+hearts--a+new+multicellular+in+vitro+model+for+electrophysiological+studies%5BTitle%5D)+AND+%22Cell+Physiol+Biochem%22%5BJournal%5D)">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/pubmed?term=((Electrophysiological+maturation+and+integration+of+murine+fetal+cardiomyocytes+after+transplantation%5BTitle%5D)+AND+%22Circ.+Res%22%5BJournal%5D)">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/pubmed?term=((Time-course+of+the+electrophysiological+maturation+and+integration+of+transplanted+cardiomyocytes%5BTitle%5D)+AND+%22J.+Mol.+Cell+Cardiol%22%5BJournal%5D)">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/pubmed?term=((Cell+persistence+and+electrical+integration+of+transplanted+fetal+cardiomyocytes+from+different+developmental+stages%5BTitle%5D)+AND+%22Int.+J.+Cardiol%22%5BJournal%5D)">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/pubmed?term=((Electrophysiological+integration+and+action+potential+properties+of+transplanted+cardiomyocytes+derived+from+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cardiovasc.+Res%22%5BJournal%5D)">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/pubmed?term=((Reversible+blockade+of+gap+junctional+communication+by+2%2C3-butanedione+monoxime+in+rat+cardiac+myocytes%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">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/pubmed?term=((Inhibitory+effect+of+2%2C3-butanedione+monoxime+%28BDM%29+on+Na%28%2B%29%2FCa%282%2B%29+exchange+current+in+guinea-pig+cardiac+ventricular+myocytes%5BTitle%5D)+AND+%22Br+J+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Differential+subunit+composition+of+the+G+protein-activated+inward-rectifier+potassium+channel+during+cardiac+development%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Differential subunit composition of the G protein-activated inward-rectifier potassium channel during cardiac development.</a> J Clin Invest. 114, 994-1001 (2004).</li>
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</ol>

The authors have nothing to declare.

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).

<table><tbody><tr><td>Leica VT 1000s</td><td>Leica Microsystems, Wetzlar, Germany</td><td></td><td>Microtome with vibrating blade.</td></tr><tr><td>Stainless Steel Blades</td><td>Campden Instruments, Loughborough, England</td><td>7550-1-SS</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Sigma-Aldrich, St. Louise, USA</td><td>Z627992</td><td></td></tr><tr><td>Fine brush, &lt;em&gt;e.g.&lt;/em&gt; size 6 (4/32&quot;)</td><td>VWR, International, Radnor, USA</td><td>149-2125</td><td></td></tr><tr><td>Preparation table</td><td></td><td></td><td>self made</td></tr><tr><td>Molt for embedding ventricles in agarose</td><td></td><td></td><td>self made</td></tr><tr><td>1 mL Syringe</td><td>Becton, Dickinson; Franklin Lakes, USA</td><td>300013</td><td></td></tr><tr><td>27 G x 3/4`` Needles</td><td>Braun, Melsungen, Germany</td><td>4657705</td><td></td></tr><tr><td>20 G 11/2`` Needles</td><td>4657519</td><td></td><td></td></tr><tr><td>Small scissor</td><td>WPI, Sarasota, USA</td><td>501263</td><td></td></tr><tr><td>Tweezers #5, 0.1 x 0.06 mm tip</td><td>WPI, Sarasota, USA</td><td>500342</td><td></td></tr><tr><td>Oxygen gas (medical grade O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>Carbogen gas (95 % O&lt;sub&gt;2&lt;/sub&gt;, 5 % CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>7647-14-5</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746436</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746495</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>NIST200B</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, St. Louise, USA</td><td>51558</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S5761</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Sigma-Aldrich, St. Louise, USA</td><td>G8270</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>M7506</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S8045</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Henkel, D&amp;uuml;sseldorf, Germany</td><td></td><td></td></tr><tr><td>Low-melt Agarose</td><td>Roth, Karlsruhe, Germany</td><td>6351.2</td><td></td></tr><tr><td>Heparin-sodium-25000 I.E./5 mL</td><td>Ratiopharm, Ulm, Germany</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX</td><td>ThermoScientific, Waltham, USA</td><td>10566016</td><td></td></tr><tr><td>SEC-10LX Amplifier</td><td>npi electronic GmbH, Tamm, Germany</td><td>SEC-10LX</td><td></td></tr><tr><td>EPC 9</td><td>HEKA Elektronik GmbH, Lambrecht, Germany</td><td></td><td></td></tr><tr><td>Zeiss Axiovert 200</td><td>Zeiss, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Low magnification Micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>Nm-3</td><td></td></tr><tr><td>High magnification, three-axis micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>MHW-3</td><td></td></tr><tr><td>Peristaltic perfusion pump</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>PPS2</td><td></td></tr><tr><td>2-channel temperature controller</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>TCO02</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus Europe GmbH, Planegg, Germany</td><td>Grass SD9</td><td></td></tr><tr><td>Glass capillaries</td><td>WPI, Sarasota, USA</td><td>1B150F-1</td><td></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 ...

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7.3K Views.  University of Cologne. 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.

Video: Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Neuroscience

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Bioengineering

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Biology

Generation of Porcine Ventricular Slices: A Vibratome-based Technique to Prepare Ultra-thin Slices of Pig Heart Tissue

Source: Ou, Q. et. al., <a href="https://www.jove.com/v/60913">Slicing and Culturing Pig Hearts under Physiological Conditions.</a> J. Vis. Exp. (2020)
This video demonstrates a vibratome-based procedure to generate viable, ultra-thin sections of pig heart. The prepared tissue slices can be cultured and used as model systems for pharmacological testing.

Source: Ou, Q. et. al., Slicing and Culturing Pig Hearts under Physiological Conditions. J. Vis. Exp. (2020)
This video demonstrates a vibratome-based ...

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Summary

Explore More Videos

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Slicing and Culturing Pig Hearts under Physiological Conditions

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

Generation of Porcine Ventricular Slices: A Vibratome-based Technique to Prepare Ultra-thin Slices of Pig Heart Tissue