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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

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 references^{11,12,10,13-17}). However, our protocol for maintaining adult mouse SAMs in vitro preserves the characteristic morphology, spontan.......

Materials

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