Name: isolation of cardiac and vascular smooth muscle cells from adult, juvenile, larval and embryonic zebrafish for electrophysiological studies
Uploaded: 2022-02-09T14:30:00
Description: Watch this Scientific Journal Video about Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies at JoVE.com

This work was supported by NIH grants HL140024 to CGN and HL150277 to CMC. Figure 1 and Figure 2 were created with BioRender.com.

All zebrafish (wild type strain AB, both male and female) were raised, maintained, and handled for the experiments following the guidelines of the Washington University Institutional Animal Care and Use Committee (IACUC).
1. Isolation of atrium, ventricle, and bulbous arteriosus from adult, juvenile, and larval zebrafish
<ol>
	<li>Euthanize fish using cold-shock, i.e., by immersing in 4 &#176;C water, for ~10 s.</li>
	<li>Using curved forceps, transfer fish into a large Petr...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. (55), e3165 (2011).</li><li>Brette, 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=Characterization+of+isolated+ventricular+myocytes+from+adult+zebrafish+(Danio+rerio).">Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio).</a> Biochemical and Biophysical Research Communications. 374 (1), 143-146 (2008).</li><li>Sander, V., Sune, G., Jopling, C., Morera, C., Izpisua Belmonte, J. 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An electrophysiological study.</a> Journal of Molecular and Cellular Cardiology. 48 (1), 161-171 (2010).</li><li>Huang, 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=Cardiovascular+consequences+of+KATP+overactivity+in+Cantu+syndrome.">Cardiovascular consequences of KATP overactivity in Cantu syndrome.</a> JCI insight. 3 (15), 137799 (2018).</li><li>Singareddy, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-sensitive+potassium+channels+in+zebrafish+cardiac+and+vascular+smooth+muscle.">ATP-sensitive potassium channels in zebrafish cardiac and vascular smooth muscle.</a> The Journal of Physiology. , (2021).</li><li>Burns, C. G., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+hearts+from+zebrafish+embryos.">Purification of hearts from zebrafish embryos.</a> BioTechniques. 40 (3), 274-282 (2006).</li><li>Seiler, C., Abrams, J., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+intestinal+smooth+muscle+development+using+a+novel+sm22&#945;-b+promoter.">Characterization of zebrafish intestinal smooth muscle development using a novel sm22&#945;-b promoter.</a> Developmental Dynamics. 239, 2806-2812 (2010).</li><li>Yang, X. 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=Whole+amount+in+situ+hybridization+and+transgene+via+microinjection+in+zebrafish.">Whole amount in situ hybridization and transgene via microinjection in zebrafish.</a> Shi Yan Sheng Wu Xue Bao. 36 (3), 243-247 (2003).</li><li>Kompella, S. N., Brette, F., Hancox, J. C., Shiels, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenanthrene+impacts+zebrafish+cardiomyocyte+excitability+by+inhibiting+IKr+and+shortening+action+potential+duration.">Phenanthrene impacts zebrafish cardiomyocyte excitability by inhibiting IKr and shortening action potential duration.</a> The Journal of General Physiology. 153 (2), 202012733 (2021).</li><li>Jou, C. J., Spitzer, K. W., Tristani-Firouzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+effectively+uncouples+the+excitation-contraction+process+in+zebrafish+embryonic+heart.">Blebbistatin effectively uncouples the excitation-contraction process in zebrafish embryonic heart.</a> Cellular Physiology and Biochemistry. 25 (4-5), 419-424 (2010).</li></ol>

Previous methods for isolating zebrafish ventricular myocytes5,6, aimed at generating myocytes for culture or electrophysiological studies, provided cells of lower yield and involved lengthy steps of multiple centrifugations that adversely affected the cell quality and viability. The protocols described here are reliable, cover each of the significant cardiovascular tissues (ventricle, atria, and VSM), and importantly are quite practical for acute isolation of li...

Zebrafish are small teleost fish that have long been used as a model vertebrate organism1 and have recently come to prominence as a viable vertebrate system for high throughput screening of genes and drugs2,3. However, physiological analysis of zebrafish tissues is not well developed. In the cardiovascular system, methods have been described for dissection of zebrafish hearts4 and isolation of ventricular cardiac myocytes5,6,7. There are few detailed descriptions of the effective isolation of atrial myocytes and no reports of vascular smooth muscle (VSM) preparations for patch-clamp studies.
The current work describes methodology for the isolation of zebrafish cardiac and vascular myocytes, viable for electrophysiological and functional studies. This approach includes modifications of previously reported protocols for zebrafish ventricular myocyte isolation5,6 and adapts methods from mammalian VSM cell isolations8, allowing for the isolation of zebrafish vascular smooth muscle cells from the bulbous arteriosus (BA). The protocols result in efficient yields of isolated atrial, ventricular, and VSM cells from zebrafish that can be reliably used in patch-clamp studies for up to 8 h9.
Despite their nearly transparent larvae that develop entirely outside the parental organism, exploring their promised ontogenetic potential in studying cardiovascular development has been limited by challenges in extracting and analyzing tissues at a young&#160;age. The current article addresses this limitation by demonstrating patch-clamp experiments on zebrafish hearts isolated as early as 3 days post-fertilization (dpf), using an adapted, published extraction method10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Centrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>Fisher Scientific</td>
			<td>14-955-459</td>
			<td></td>
		</tr>
		<tr>
			<td>19 Guage Needle</td>
			<td>BD</td>
			<td>305187</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione Monoxime (BDM)</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Centrifuge Tubes</td>
			<td>Sigma-Aldrich</td>
			<td>EP0030119479</td>
			<td>For embryonic heart isolation</td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier and Digidata 1200 digitizer</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Used for action potential recordings</td>
		</tr>
		<tr>
			<td>Benchtop Mini Centrifuge</td>
			<td>Southern Labware</td>
			<td>MLX-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Sigma-Aldrich</td>
			<td>203390</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Strainer Sieve</td>
			<td>Cole-Parmer</td>
			<td>EW-06336-71</td>
			<td>100 &#956;m sieve for embryonic heart isolation</td>
		</tr>
		<tr>
			<td>Collagenase Type H</td>
			<td>Sigma-Aldrich</td>
			<td>C8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>324626</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastase</td>
			<td>Worthington</td>
			<td>LS003118</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Dumont</td>
			<td>Style #5</td>
			<td>Ceramic-coated forceps for adult and juvenile CV tissue isolation (Need two)</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>K2ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Petri Dish</td>
			<td>Sigma-Aldrich</td>
			<td>P5981</td>
			<td>For dissociation</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Hematocrit Capillary Tubes</td>
			<td>Kimble Chase</td>
			<td>41A2502</td>
			<td>Soda lime glass for patch pipettes</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003118</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-6A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Sigma-Aldrich</td>
			<td>P5606</td>
			<td>100 mm x 20 mm, for embryonic heart isolation</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>806552</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td>For adult and juvenile zebrafish decapitation</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Fine Forceps</td>
			<td>Dumont</td>
			<td>Style #SF</td>
			<td>For isolating larval CV tissues (Need two)</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoshaker</td>
			<td>ThermoFisher Scientific</td>
			<td>13687711</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine Methanesulfonate (MS222)</td>
			<td></td>
			<td></td>
			<td>For anaesthetizing zebrafish larvae</td>
		</tr>
		<tr>
			<td>Trypsin Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>T6522</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cardiac and vascular smooth muscle cells from adult, juvenile, larval and embryonic zebrafish for electrophysiological studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The above protocols reliably and routinely provide sufficient cardiac and vascular myocytes of consistent quality amenable for patch-clamp studies as recently reported in extensive studies of ATP-sensitive potassium (KATP) channels in wild-type and mutant zebrafish cardiovasculature9. Representative traces of recordings of such KATP channel activity from isolated cardiomyocytes are shown in Figure 3A-C. In the case of cells isola...

Watch this Scientific Journal Video about Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies at JoVE.com

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Studying native ion channel properties using patch clamp electrophysiology requires access to acutely isolated cells. This protocol describes methods to access individual cardiovascular myocytes from zebrafish at different ages. This protocol is robust, simple, easily reproducible, and isolates cardiovascular myocytes from zebrafish in their native state with relatively high yield and viability.To begin, transfer the fish into a large Petri dish partially filled with perfusion buffer and place it under a dissecting microscope. Hold the fish in the non-dominant hand with the ventral side facing the dissection scope. With the fine forceps in the dominant hand, gently tear open the pectoral muscles and fins of the fish to reveal the cardiovascular tissues including atrium, ventricle, and bulbus arteriosus.Gently pull the bulbus arteriosus at the intersecting tip of the bulbus arteriosus and the ventral aorta. Carefully separate the bulbus arteriosus from the aorta by pinching the forceps, locating the atrium's tip into the multiple venous branches that converge. Then, pluck the tip of the atrium off the sinus venosus to isolate the cardiovascular tissues from the rest of the body.To dissect cardiomyocytes, pluck the atrium and ventricle out of the cardiovascular tissue. Make a gentle tear into the ventricle using fine forceps to drain excess blood, which can be ensured when the cardiac tissue turns from bright red to a pale salmon color. Pool the isolated atrium and ventricle into a separate 1.5-milliliter centrifuge tube containing perfusion buffer.Replace the perfusion buffer in the tubes with 750 microliters of digestion buffer. Place the tubes on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute. Allow digestion of the tissues until they become translucent.End the digestion by gently spinning down the tissues in a bench-top minicentrifuge at 2, 000 times G for three to five seconds and replace the supernatant digestion buffer with 750 microliters of stopping buffer. Gently replace the stopping buffer with 500 to 750 microliters of perfusion buffer and triturate the tissues 30 times using a flame-polished Pasteur pipette to disperse the cells. Then, gently triturate the ventricles.For uniform sampling, gently pipette the cells up and down a couple of times to resuspend, before adding a drop of them for corresponding studies. Pluck the adult bulbus arteriosus from the cardiovascular tissues and pool five bulbus arteriosus into a 1.5-milliliter centrifuge tube containing S1 buffer. Replace S1 with 400 microliters of S2 buffer and allow papain digestion of the bulbus arteriosus on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute for 20 minutes.Allow the partially digested bulbus arteriosus to settle down for one minute and replace the S2 supernatant with 500 microliters of S3 buffer containing collagenase. Digest the tissues on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute for three to five minutes. End the digestion by gently spinning down the tissues in a bench-top minicentrifuge at 2, 000 times G for three to five seconds, and replace the S3 supernatant with 500 microliters of S1.Gently triturate the pelleted tissues to disperse vascular smooth muscle cells.Plate dispersed vascular smooth muscle cells onto glass cover slips of appropriate size. Keep the cover slips at room temperature for 30 minutes for the cells to attach, and use them within the next six hours. Anesthetize the embryos.Then, concentrate the embryos by transferring them to a five-milliliter centrifuge tube, then remove excess media. Wash the embryos with three milliliters of cold perfusion buffer twice, then resuspend them in two milliliters of perfusion buffer. Using the embryonic heart isolation apparatus, draw one milliliter of the embryos into the needle and immediately expel them back into the tube.Pass the fragmented embryos through a 100-micrometer cell strainer sieve placed in a funnel. Collect the filtered hearts in another five-milliliter centrifuge tube. Mix the filtrate well and separate one-milliliter aliquots into 1.5-milliliter centrifuge tubes.Centrifuge all the tubes on a bench-top minicentrifuge for five seconds and discard the supernatant. Carry out serial resuspension of the pellets in the tubes using one milliliter of perfusion buffer. Gently pipette the hearts up and down twice before adding a drop for corresponding studies For cells isolated from bulbus arteriosus, the vascular smooth muscle cells were confirmed by tagln:EGFP expression.Representative traces of recordings of ATP-sensitive potassium channel activity from isolated cardiomyocytes are shown here. Successful single-channel recordings were obtained in patches excised from the embryonic hearts. Ventricular myocytes exhibited stable hyperpolarized membrane potentials, and action potentials were stimulated via current injection through the patch pipette.Atrial myocytes exhibited spontaneous action potential firing. Action potential properties are summarized here. While replacing the buffers during cardiomyocyte dissociation, care should be taken that the digestive tissues are not disturbed.Tissue fragmentation should be accomplished only by pipette trituration. During the collagenous digestion of the bulbus tissues, examine for floating, dilated, and translucent tissues every minute. Once the tissue fragmentation is apparent, cease the digestion.Once the cells are isolated, they can be kept alive for hours and used for electrophysiological analysis using patch clamp techniques. This has allowed us, for instance, to show that ATP-sensitive potassium channels in these zebrafish tissues are essentially identical to those in mammals.

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