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Biology

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

Published: October 23rd, 2016

DOI:

10.3791/54555

1Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, 2Department of Bioengineering, University of Colorado Anschutz Medical Campus, 3Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus
* These authors contributed equally

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). 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.

Pacemaker myocytes in the sinoatrial node of the heart (sinoatrial myocytes, "SAMs") 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 m....

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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.

  1. Prepare 1 L each of the followi.......

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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.......

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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.......

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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.

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

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