High-resolution Optical Mapping of the Mouse Sino-atrial Node

Summary

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

Abstract

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Introduction

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.^1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.^3-5 Such organization creates comp....

Protocol

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 80-23). All methods and protocols used in these studies have been approved by The University of Wisconsin Animal Care and Use Protocol Committee following the Guidelines for Care and Use of Laboratory Animals published by NIH (publication No. 85-23, revised 1996). All animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.

1. Heart Removal and Langendorff Perfusion

1. Prior to heart isolation, warm Tyrode solution to 37 °C by using a water....

Results

Optical Mapping of the Intact SAN from the Langendorff-perfused Heart
A typical example of an RA activation contour map reconstructed for spontaneous sinus rhythm is shown in Figure 3 for a Langendorff-perfused mouse heart. The early activation point is located within the intercaval region near the SVC where the SAN is anatomically defined.^3,16 Two RA activation contour maps acquired at 1.0 and 0.5 msec sampling rate are shown in Fi.......

Discussion

Here, we presented two types of mouse SAN preparations: 1) intact SAN in the Langendorff-perfused whole heart, and 2) SAN in the isolated, opened atrial preparation. These two types of preparation serve different experimental purposes. In the Langendorff-perfused whole heart preparation, the intact atrial structure is preserved which makes it possible to study complex atrial arrhythmias such as atrial fibrillation as well as interactions between the SAN and atrium during reentrant tachyarrhythmias.⁶ In contras.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
water jacket	Radnoti	1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures
water bath / circulator	Fisher Scientific	1016S
pressure amplifier	AD Instruments	MLT0670
EMD Millipore Nylon Net Filters	Fisher Scientific	NY1102500
Pressure transducer	AD Instruments	MLT0670
Stainless Steel Minutien Pins - 0.1 mm Diameter	Fine Science Tools	26002-10
Perfusion pump	World Precision Instruments	PERIPRO-4LS
Superfusion pump	World Precision Instruments	PERIPRO-4HS
Vannas Tubingen scissors	World Precision Instruments	503379
Dumont forceps	World Precision Instruments	501201, 500085
Mayo scissors	World Precision Instruments	501750
Kelly hemostatic forceps	World Precision Instruments	501241
Iris forceps	World Precision Instruments	15917
Iris scissors	World Precision Instruments	501263
ECG 12 mm needle (29-gauge) electrodes (monopolar)	AD Instruments	MLA1203
in-line Luer injection port	Ibidi	10820
Ultima-L CMOS camera	SciMedia	MiCAM-05
halogen lamp	Moritex USA Inc	MHAB-150W
NaCl	Fisher Scientific	S271-1
CaCl2 (2H2O)	Fisher Scientific	C79-500
KCl	Fisher Scientific	S217-500
MgCl2 (6H2O)	Fisher Scientific	M33-500
NaH2PO4 (H2O)	Fisher Scientific	S369-500
NaHCO3	Fisher Scientific	S233-3
D-Glucose	Fisher Scientific	D16-1
Blebbistatin	Tocris Bioscience	1760
RH237	ThermoFisher Scientific	S1109
Dimethyl sulphoxide (DMSO)	Sigma-Aldrich	D2650