Name: methods for the isolation, culture, and functional characterization of sinoatrial node myocytes from adult mice
Uploaded: 2016-10-23T14:00:00
Description: Watch this Scientific Journal Video about Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice at JoVE.com

We thank Dr. Christian Rickert for critical reading of the manuscript. This work was supported by a grant from the National Heart Lung and Blood Institute (R01-HL088427) to CP. EJS was supported by 5T32-AG000279 from the National Institute on Aging. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus. The standard protocol below has been optimized using male C57BL/6J mice of 2-3 months of age.
1. Prepare Solution Stocks and Supplies in Advance of Experiments
NOTE: Refer to Materials Table for necessary equipment and supplies.
<ol>
	<li>Prepare 1 L each of the followi...

<ol>
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D., 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=Complete+atrial-specific+knockout+of+sodium-calcium+exchange+eliminates+sinoatrial+node+pacemaker+activity.">Complete atrial-specific knockout of sodium-calcium exchange eliminates sinoatrial node pacemaker activity.</a> PloS ONE. 8 (11), e81633 (2013).</li><li>Torrente, A. G., Zhang, R., 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=Burst+pacemaker+activity+of+the+sinoatrial+node+in+sodium-calcium+exchanger+knockout+mice.">Burst pacemaker activity of the sinoatrial node in sodium-calcium exchanger knockout mice.</a> Proc Nat Acad Sci USA. 112 (31), 9769-9774 (2015).</li><li>Denyer, J. C., Brown, H. 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This paper presents detailed protocols for the isolation and culture of fully differentiated sinoatrial node myocytes from adult mice. The isolation protocol reliably produces spontaneously active mouse SAMs suitable for either immediate electrophysiological analysis or subsequent culture. Similar protocols have been reported by many other groups (for example, see references11,12,10,13-17). However, our protocol for maintaining adult mouse SAMs in vitro preserves the characteristic morphology, spontan...

Pacemaker myocytes in the sinoatrial node of the heart (sinoatrial myocytes, &#34;SAMs&#34;) generate spontaneous, rhythmic action potentials (APs) that propagate through the myocardium to initiate each heartbeat. Experiments using acutely isolated SAMs from many species have been essential for elucidation of mechanisms that contribute to the generation of pacemaker activity. SAMs are highly specialized cardiomyocytes that differ substantially from their counterparts in the atrial and ventricular myocardium in terms of morphology, function, and protein expression. The hallmark of spontaneous APs in SAMs is a spontaneous depolarization during diastole that drives the membrane potential to threshold to trigger the next AP1,2. This &#34;pacemaker potential&#34; depends on the coordinated activity of many different membrane currents including the &#34;funny current&#34; (If), T- and L-type calcium currents, and the sodium-calcium exchanger current (INCX), which is driven by Ca2+ release from the sarcoplasmic reticulum3,4.
While acutely isolated mouse SAMs are an essential experimental preparation for the study of pacemaking, the isolation of SAMs from mice can be a challenging method to adopt because the indistinct anatomy and small size of the mouse SAN requires a nuanced microdissection and the combined enzymatic and mechanical dissociation of the cells requires careful optimization.
We provide here a detailed video demonstration of a protocol that has been successfully used to isolate SAMs from adult mice for patch clamp recordings5-8. To our knowledge, there is no such visual demonstration available from any other source. In addition, a new method is demonstrated in which isolated SAMs from adult mice can be maintained in vitro for several days, thereby permitting the introduction of proteins, genetically encoded reporter molecules or RNAi via adenoviral infection9.

<table border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Sylgruard/Elastomer Kit</td>
			<td style="width:167px;">Dow Corning</td>
			<td colspan="2" style="width:237px;">184 SIL ELAST KIT 0.5KG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Borosilicate 9&#34; pasteur pipettes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Small, round bottomed culture tubes</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">352059</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Large, round bottomed culture tubes</td>
			<td style="width:167px;">Corning</td>
			<td style="width:175px;">14-959-11B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Elastase</td>
			<td style="width:167px;">Worthington Biochemical</td>
			<td style="width:175px;">LS002279</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Liberase TM</td>
			<td style="width:167px;">Roche</td>
			<td style="width:175px;">5401119001</td>
			<td>&#160;Tissue dissociation solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Heparin</td>
			<td style="width:167px;">SAGENT Pharmaceuticals&#160;</td>
			<td style="width:175px;">NDC 25021-400-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Mouse Laminin</td>
			<td style="width:167px;">Corning</td>
			<td style="width:175px;">CB-354232</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">12 mm round glass coverslips</td>
			<td style="width:167px;">Fisher&#160;</td>
			<td style="width:175px;">12-545-80</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">24-well culture plate</td>
			<td style="width:167px;">Fisher</td>
			<td style="width:175px;">08-772-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Ad-mCherry</td>
			<td style="width:167px;">Vector Biolabs</td>
			<td style="width:175px;">1767</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Ad-eGFP</td>
			<td style="width:167px;">Vector Biolabs</td>
			<td style="width:175px;">1060</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Plastic, disposable transfer pipette</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Micro scissors</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">17-467-496</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dumont #4 Forceps</td>
			<td style="width:167px;">Roboz Instruments</td>
			<td style="width:175px;">RS-4904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Tissue Forceps</td>
			<td style="width:167px;">Roboz Instruments</td>
			<td style="width:175px;">RS-8164</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dissecting Iris Scissors</td>
			<td style="width:167px;">WPI, Inc.</td>
			<td style="width:175px;">501264</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Dissecting Pins</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:175px;">26002-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Sigma</td>
			<td>60128</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">KH2PO4</td>
			<td>Sigma</td>
			<td>60353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>54457</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glucose</td>
			<td>Sigma</td>
			<td>G0350500</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">taurine</td>
			<td>Sigma</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-glutamate</td>
			<td>Sigma</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-aspartate</td>
			<td>Sigma</td>
			<td>A6558</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgSO4</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">creatine</td>
			<td>Sigma</td>
			<td>C0780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Amphotericin-B</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:175px;">1397-89-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Isoproterenol</td>
			<td style="width:167px;">Calbiochem</td>
			<td style="width:175px;">420355</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Media199</td>
			<td>Sigma</td>
			<td style="width:175px;">M4530</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">2,3-butanedione monoxime (BDM)</td>
			<td>Sigma</td>
			<td style="width:175px;">B0753</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td style="width:175px;">SH30071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td style="width:175px;">A5611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Insulin&#160;&#160;</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Transferrin</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Selenium</td>
			<td>Sigma</td>
			<td style="width:175px;">I3146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin</td>
			<td>GE Healthcare</td>
			<td style="width:175px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin</td>
			<td>Hyclone</td>
			<td style="width:175px;">SV30010</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for the isolation, culture, and functional characterization of sinoatrial node myocytes from adult mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

The protocols described here have been previously employed to isolate spontaneously active SAMs from adult mice that are suitable for a variety of different patch clamp studies5-8. In addition, the protocols allow for isolated SAMs that can be maintained in culture for up to one week. Gene transfer into the cultured cells can be accomplished via adenoviral infection9. The results presented in this section derive from our previous work and are shown here as examples o...

Watch this Scientific Journal Video about Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice at JoVE.com

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

The overall goal of this procedure is to isolate spontaneously active sinoatrial node myocytes from mice for short or medium-term electrophysiological or imaging experiments. Isolated sinoatrial node myocytes are important for the study of cardiac pacemaking mechanisms, in particular the study of specific ion currents or timing of intracellular calcium release. But relatively few labs use isolated sinoatrial myocytes from mice in part because it is very difficult to learn the procedure from written methods alone.After preparing all the required solutions for the isolation, remove the fur from the animal's chest with scissors. Then transect the ribcage to expose the chest cavity using external tissue forceps and dissection scissors. Rinse the chest cavity with two milliliters of warmed complete Tyrode's with heparin.Under a dissecting microscope, carefully remove the lungs and thymus using internal scissors and dissection forceps. While gently holding the apex of the heart, carefully cut the inferior vena cava and the aorta to remove the heart from the chest cavity. Then transfer the heart to one of the silicone dissection dishes and then rinse it with two to four milliliters of warmed heparinized complete Tyrode's.Orient the heart such that the posterior vessels are visible and facing up. Next, immobilize the heart by pinning it through the apex into the silicone dissection dish. Afterward, locate the groove between the ventricles and the atria.Using the internal dissection scissors, make an incision at the groove while keeping it close to the ventricles. Flush the groove and the incision with additional warmed heparinized complete Tyrode's as needed to allow a clear view of the atria and the valves. Then continue to cut along the groove to separate the atria from the ventricles.Subsequently, transfer the atrial tissue to the second silicone dissection dish and rinse it with three milliliters of warmed heparinized complete Tyrode's. Afterward, while stretching the preparation gently, pin the tissue through the inferior vena cava. Then flip the tissue so that the animal's right atrium is now on the experimenter's left side and the left atrium is on the right side of the experimenter.It's very important to obtain and recognize orientation in order to isolate the correct tissue. Proceed to pin the tissue through the superior vena cava, then the right and left atrial appendages. Remove any remaining fat or other tissue to allow a clear view of the preparation.Subsequently, open the anterior wall of the atria by cutting through the venae cavae. Rotate the dish as necessary to dissect both the superior and inferior venae cavae. Reposition the pins as necessary to visualize the inter-atrial septum.Then cut along the inter-atrial septum to remove the left atrium. Re-pin the preparation, stretch it gently, and remove the fat from behind the sinoatrial node. After that, remove the right atrial appendage then free the sinoatrial node by cutting along the crista terminalis which appears as a dark orange streak bordering the atrial appendage.Re-pin or unpin the nodal tissue and cut it laterally to produce three equally-sized strips. In this procedure, using the narrow fire polished pipette, transfer three strips of sinoatrial node tissue to the first of the three small round bottom tubes containing 2.5 milliliters of low calcium magnesium Tyrode's and incubate for five minutes. After five minutes, transfer the tissue strips to the second small round bottom tube containing 2.5 milliliters of low calcium magnesium Tyrode's.Wash the tissue strips by gently swirling the tube or by gently pipetting with the narrow pipette. Do not invert the tube. Then transfer the tissue strips to the third small round bottom tube containing 2.5 milliliters of low calcium magnesium Tyrode's and repeat the washing step.Afterward, transfer the strips to the large round bottom tube containing 2.5 milliliters of low calcium magnesium Tyrode's with enzymes. Mix every five minutes by gently swirling the tube. Following enzyme digestion, use a narrow fire polished pipette to gently transfer the tissue strips to the first of the three small round bottom tubes containing modified KB solution.Then transfer the strips through the second and third small round bottom tubes to wash the tissue before transferring to the large round bottom tube that contains KB solution. Using the larger fire polished pipette, dissociate the cells in the large round bottom tube by constant trituration, taking care to keep the dissociation tube submerged in the 35 degree Celsius water bath and to avoid introducing bubbles into the solution. We have found that a forceful expulsion of the solution at a frequency of approximately one per second or one Hertz works best.Now add 75 microliters of sodium chloride calcium chloride adaptation solution to the tissues. Swirl gently to mix and incubate them for five minutes. Continue to add calcium in increments as outlined in the written protocol.Following calcium re-adaptation, collect the sinoatrial myocytes by allowing them to settle for about 10 minutes or by centrifugation at 2, 000 times g for three minutes. For acutely isolated cells, gently remove and discard about five milliliters of the supernatant using a transfer pipette. Then store the cells at room temperature for up to eight hours for patch clamp experiments.Subsequently, functional assessments of the isolated sinoatrial myocytes can be done using patch clamp recordings for spontaneous action potentials and membrane currents. Spontaneously active sinoatrial myocytes can be maintained in culture for up to six days. The cultured cells retain an overall morphology that is very similar to that of the acutely isolated sinoatrial myocytes with no significant changes in the average length, width, cross-section area, or membrane capacitance of cultured cells compared to the acutely isolated cells.The cells are suitable for a range of experimental studies such as current and voltage clamp recordings. Shown here as examples are spontaneous action potentials and the funny current. Once mastered, the sinoatrial node dissection, isolation, and calcium re-introduction can be completed in about two hours so that you can have cells ready to go for your experiments.While attempting this procedure, it's important to remember that the orientation of the tissue is critical for a good dissection and that the enzymatic and mechanical dissociation steps should be optimized as outlined in the text. Following this procedure, the isolated sinoatrial node myocytes can be used for a variety of experiments including single-cell electrophysiology, calcium imaging and immunocytochemistry. The cells can also be maintained in culture for experiments that require gene manipulation or the expression of reporter molecules.After watching this video, you should have a good understanding of how to isolate sinoatrial myocytes from adult mice. Best of luck with your experiments.

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