Name: isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of ca2+ transients and l-type calcium current
Uploaded: 2020-11-03T15:00:00
Description: Watch this Scientific Journal Video about Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current at JoVE.com

This work was supported by German Research Foundation (DFG; Clinician Scientist Program In Vascular Medicine (PRIME), MA 2186/14-1 to P. Tomsits and D. Sch&#252;ttler; VO1568/3-1, IRTG1816, and SFB1002 project A13 to N. Voigt), German Research Foundation under Germany&#8217;s Excellence Strategy (EXC 2067/1- 390729940 to N. Voigt), German Centre for Cardiovascular Research (DZHK; 81X2600255 to S. Clauss and N. Voigt; 81Z0600206 to S. K&#228;&#228;b), the Corona Foundation (S199/10079/2019 to S. Clauss), the ERA-NET on Cardiovascular Diseases (ERA-CVD; 01KL1910 to S. Clauss), the Heinrich-and-Lotte-M&#252;hlfenzl Stiftung (to S. Clauss) and the Else-Kr&#246;ner-Fresenius Foundation (EKFS 2016_A20 to N. Voigt). The funders had no role in manuscript preparation.

All animal procedures were approved by the Lower Saxony Animal Review Board (LAVES, AZ-18/2900) and were conducted in accordance with all institutional, national, and international guidelines for animal welfare.
1. Prearrangements
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	<li>Prepare 1 L of 10x perfusion buffer (Table 1), 500 mL of 1x perfusion buffer (Table 2), 50 mL of digestion buffer (Table 3), 10 mL of stop buffer (Table 4), 1 L of Tyrode solution (<stron...

<ol>
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This article provides an easy and functional way to obtain high quality atrial and ventricular myocytes from the same mouse for patch-clamp studies with simultaneous calcium transient recordings. The quality of the obtained data highly depends on the quality of the cell isolation. As mentioned above, many methods to isolate murine cardiomyocytes have been described previously9,10,11,12. The iso...

Cardiac arrhythmias are common and one of the current major healthcare challenges since they affect millions of people worldwide. Arrhythmias are associated with high morbidity and mortality1,2 and represent the underlying cause for the majority of sudden cardiac deaths3. Up to date treatment options have improved patient survival but are still mainly symptomatic treatments rather than targeting the underlying mechanisms. Thus, these treatments have limited efficacy and may frequently cause severe side effects4,5,6. An improvement of current treatment options requires insight into the underlying pathophysiology, creating the need for suitable models to study. Small animal models - and specifically mouse models - play a crucial role in arrhythmia research as they allow to study key mechanisms of arrhythmogenesis, for example the genetic impact on cellular electrophysiology, ion channel function or calcium handling7,8.
For this purpose, isolated atrial and ventricular cardiomyocytes of sufficient quantity and viability are required. A broad spectrum of different isolation approaches to obtain atrial and ventricular myocytes has been previously described9,10,11,12,13 and some groups have presented data from simultaneous measurements of L-type current and calcium current induced calcium transients from either atrial14 or ventricular15 murine cardiomyocytes. However, to our best knowledge there is no data available of atrial as well as ventricular measurements from one animal. Researchers focus on a broad variety of topics ranging from electrophysiology to proteomics, functional studies as cell contractility or protein interactions, mitochondrial function, or genetics &#8211; all in need of isolated cardiomyocytes. Many of the published protocols thus have not been specifically developed for patch clamp studies, leading to limited yields and insufficient cell quality for patch clamp studies. Thus, simultaneous patch clamp and calcium transient measurements of atrial and ventricular cells isolated from one animal cannot be performed with established protocols.
Isolation of murine &#8211; especially atrial &#8211; myocytes for patch clamp experiments remains challenging. This article provides a simple and fast method for the isolation of high-quality murine atrial and ventricular myocytes via retrograde enzyme based Langendorff perfusion, which subsequently allows simultaneous measurements of both net membrane current and current induced calcium transients from one animal. This article elaborates a protocol for the isolation of atrial and ventricular myocytes derived from wild type mice and mice carrying genetic mutations. This protocol can be used for male and female mice alike. The myocyte isolation, images, and representative results described below were obtained from wild type C57Bl/6 mice at the age of 6 (&#177; 1) months. Nevertheless, this protocol has successfully been used for mice at various ages ranging from 2 to 24 months with different genotypes. Figure 1 shows the isolation setup and a close-up of a cannulated heart during enzyme perfusion.

<table>
	<tbody>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>31550</td>
			<td></td>
		</tr>
		<tr>
			<td>27G cannula</td>
			<td>Servoprax</td>
			<td>L10220</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Aminopyridine</td>
			<td>Sigma-Aldrich</td>
			<td>A78403</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Aortic cannula</td>
			<td>Radnoti</td>
			<td>130163-20</td>
			<td></td>
		</tr>
		<tr>
			<td>BaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>342920</td>
			<td></td>
		</tr>
		<tr>
			<td>blunt surgical forceps</td>
			<td>Kent Scientific</td>
			<td>INS650915-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Calf Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12133C</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma-Aldrich</td>
			<td>C0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulating heated water bath</td>
			<td>Julabo</td>
			<td>ME</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington</td>
			<td>LS994177</td>
			<td></td>
		</tr>
		<tr>
			<td>disscetion scissors</td>
			<td>Kent Scientific</td>
			<td>INS600124</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-aspartat K+-salt</td>
			<td>Sigma-Aldrich</td>
			<td>A2025</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-3</td>
			<td>Invitrogen</td>
			<td>F3715</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-3 AM</td>
			<td>Invitrogen</td>
			<td>F1242</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-triphosphate tris salt</td>
			<td>Sigma-Aldrich</td>
			<td>G9002</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating coil</td>
			<td>Radnoti</td>
			<td>158821</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Ratiopharm</td>
			<td>25.000 IE/5ml</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H9136</td>
			<td></td>
		</tr>
		<tr>
			<td>induction chamber</td>
			<td>CWE incorporated</td>
			<td>13-40020</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Cp-pharma</td>
			<td>1214</td>
			<td></td>
		</tr>
		<tr>
			<td>Jacketed heart chamber</td>
			<td>Radnoti</td>
			<td>130160</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1049360250</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl x 6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 x 7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M9397</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 x 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh (200 &#181;m)</td>
			<td>VWR-Germany</td>
			<td>510-9527</td>
			<td></td>
		</tr>
		<tr>
			<td>pasteur pipette</td>
			<td>Sigma Aldrich</td>
			<td>Z331759</td>
			<td></td>
		</tr>
		<tr>
			<td>petri-dishes</td>
			<td>Thermo Fisher</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic Acid F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>Sigma-Aldrich</td>
			<td>P8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Ismatec</td>
			<td>ISM597D</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical forceps</td>
			<td>Kent Scientific</td>
			<td>INS650908-4</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical scissors</td>
			<td>Kent Scientific</td>
			<td>INS700540</td>
			<td></td>
		</tr>
		<tr>
			<td>suturing silk</td>
			<td>Fine Science Tools</td>
			<td>NC9416241</td>
			<td></td>
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		<tr>
			<td>syringe</td>
			<td>Merck</td>
			<td>Z683531-100EA</td>
			<td></td>
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		<tr>
			<td>Taurin</td>
			<td>Sigma-Aldrich</td>
			<td>86330</td>
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isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of ca2+ transients and l-type calcium current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation yield is determined after calcium reintroduction by pipetting 10 &#181;L of cell suspension onto a microscope slide. More than 100 viable, rod-shaped, non-contracting cells/10 &#181;L for atrial cell isolation and more than 1,000 viable, rod-shaped, non-contracting cells/10 &#181;L for ventricular cell isolation are considered as sufficient yield and are commonly obtained using this protocol. Atrial cells obtained with this protocol showed a variety of different cell types containing cells of the cardiac conduc...

Watch this Scientific Journal Video about Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current at JoVE.com

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Patch clamp and calcium imaging experiments are labor intensive, time consuming and challenging. This protocol provides a convenient method to isolate high quality murine atrial and ventricular cardiomyocytes, suitable for patch clamp experiments and simultaneously performed calcium imaging. The main advantage of this protocol is that we can obtain atrial and ventricular cardiomyocytes from the same animal.The isolation of murine atrial cardiomyocytes is especially challenging and has been a limiting factor for previous experiments. Begin by pre-filling the Langendorff apparatus with perfusion buffer and ensuring it is air free. Fix an aortic cannula under the dissection microscope and connect it with a one-milliliter syringe filled with perfusion buffer.After removing the heart from the euthanized mouse, put the heart into room temperature perfusion buffer and cannulate the aorta with a blunt-end needle under the microscope as quickly as possible. Firmly tie the heart to the needle with a piece of suturing silk, and disconnect the syringe. After aortic cannulation, immediately connect the cannulated heart to the Langendorff apparatus, avoiding any air entering the system.Perfuse the heart with perfusion buffer for one minute at exactly 37 degrees Celsius and a perfusion rate of four milliliters per minute. Switch from perfusion to digestion buffer and perfuse for exactly nine minutes at a temperature of 37 degrees Celsius and a perfusion rate of four milliliters per minute. When finished, transfer the digested heart to a petri dish with enough digestion buffer to keep it fully covered.Then carefully dissect the atria and ventricles under the microscope. Transfer the atria into a petri dish with 1.5 milliliters of digestion buffer and the ventricles into another petri dish with three milliliters of digestion buffer. Carefully but quickly pull the atria apart into tiny pieces Using blunt forceps.Dissolve the tissue by carefully pipetting up and down with a 1000-microliter pipette tip which was previously cut to widen the tip opening. Transfer the solution to a 15-milliliter centrifuge tube and add an equivalent amount of stop buffer by pipetting down the side of the tube. Pass all three milliliters of the solution through a 200-micrometer nylon mesh to remove the remaining larger tissue pieces that have not been fully digested.To perform ventricular dissection, chop the ventricular tissue into tiny pieces using dissection scissors or forceps, and pipette up and down with another 1000-microliter pipette tip to dissolve. Transfer the cell and tissue solution into a 15-milliliter centrifuge tube and add an equivalent amount of stop buffer by carefully pipetting it down the side of the tube to end the reaction. Pass all six milliliters of cell and tissue solution through a 200-micrometer nylon mesh to remove larger pieces that have not been fully digested.Leave both atrial and ventricular cell suspensions on the bench at room temperature for six minutes to settle. Then centrifuge the tubes at 5G for two minutes. Discard the supernatants using a plastic Pasteur pipette and carefully resuspend the cell pellets in 10 milliliters of calcium-free Tyrode's solution.Leave the atrial and ventricular cells for eight minutes for sedimentation. Centrifuge the atrial cells at 5G for one minute. Then discard the supernatants from both cell samples and carefully resuspend the pellets in 10 milliliters of Tyrode's solution with 100 micromolar calcium.Leave the cells for eight minutes for sedimentation, then repeat the centrifugation of atrial cells. Discard the supernatants and carefully resuspend the cell pellets in 10 milliliters of Tyrode's solution with 400 micromolar calcium. Repeat this process once more, resuspending the cells in Tyrode's solution with one millimolar calcium.All atrial cells are small with cell capacitances ranging from approximately 35 to 100 picofarad. A typical cell from the atrial working myocardium is shown here. Ventricular myocytes are more rod-shaped and larger, with cell capacitances ranging from 100 to around 400 picofarads.Examples of L-type calcium current measurements with simultaneous cytosolic calcium transients from one atrial myocyte and one ventricular myocyte are shown here. Before attempting this technique, Make sure to practice the organ harvest. It is crucial that the step is performed fast and all tissue cuts are made at the correct locations.When organ harvest is performed at reproducible quality, take some time to perfect cannulation. Cells isolated with this protocol are suitable for patch clamp measurements. In addition, they can be loaded with fluorescent dyes, allowing for a wide range of imaging experiments.

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