Abstract
The cardiac conduction system allows the synchronized propagation of electrical activity through heart muscle. This is initiated by the spontaneous activity of the specialized pacemaker cells of the sino-atrial node (SAN). The SAN region underlies automaticity in mammals and therefore has a crucial role in the pathogenesis of cardiac disorders such as arrhythmia. Isolation of SAN tissue and SAN cells is critical to advance our understanding of SAN structure and function in health and disease. Initially, isolation of SAN tissue and SAN cells was carried out in the rabbit owing to its larger size and similar electrical properties to human. This protocol was optimized by Mangoni and Nargeot (2001) for use in mice to take advantage of advancements in transgenic models. Here, we provide a step-by-step guide to dissecting the SAN tissue and isolating pacemaker cardiomyocytes from mouse hearts using an enzyme digestion approach.
Keywords: Conduction system, SAN, Mouse SAN cells, Primary cell isolation, Pacemaker cells
Background
Cardiovascular diseases and abnormalities are a major cause of morbidity and mortality and are major burden on healthcare systems. Arrhythmias as result of changes in the structure, rate and rhythm of the cardiac conduction system underlie many cardiac disorders. In the past, a lack of availability of homogenous conduction system cell lines has proven to be a hindrance to investigators wishing to study the function of conduction system cells in health and disease. The emergence of transgenic animal models of cardiac abnormalities provides an even greater impetus to reliably isolate healthy conduction system cells. Intact nodes allow investigations into expression profiles of important signaling proteins and genes using immunohistochemistry and qPCR approaches. More detailed functional analysis is possible on isolated single nodal cells using patch-clamp for example. The SAN tissue and the cells it consists of were first isolated many decades ago (Noble and Tsien, 1968). However, the cell population isolated was generally a mixed population. SAN specific cells were first isolated from rabbit SAN by DiFranscesco et al. (1986) in the mid-1980s to allow study of ion channel expression. More recently, the protocol was adapted for isolation of SAN cells from mice by Mangoni and Nargeot (2001). This has allowed transgenic mouse models specific to the SAN to be studied in greater detail. We describe the procedure for isolating the SAN tissue and single SAN cells. The intact node can be used for investigation of gene and protein expression as well as for tissue histology, whilst acutely isolated cells can be interrogated in detail for their electrophysiological properties.
Materials and Reagents
Equipment
Procedure
The procedure is illustrated in Figure 1. Note: Stock solutions for dissection and isolation of the SAN can be prepared the day before isolation (Recipes 1, 2, 4, 5 and 7) as it can take ~1 h to make them. Solutions should be kept in the fridge (~4-8 °C) and can be used for isolation for up to a week. For working solutions, such as the enzyme solution it is advisable to make on the day. All working solutions need to be pre-warmed (in a water bath) at 37 °C before use (~15-20 min). It is critical that CaCl2 (10 µl of 100 mM stock to 2.5 ml-final concentration 400 µM) is added to the enzyme solution, otherwise tissue digestion will be compromised. Figure 1. Schematic of the protocol for the isolation of SAN cells from a mouse heart
Recipes
Acknowledgments
We would like to thank the BHF for funding (RG/15/15/311742). The work was facilitated by the NIHR Biomedical Research Centre at Barts. Special thanks to Dr Mangoni for allowing Dr Nobles to visit his laboratory to learn the procedure for isolation of murine SAN cardiomyocytes. This protocol was adapted/modified from the protocol of Mangoni and Nargeot (2001).
Competing interests
We have no conflicts of interest.
Ethics
All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the British Home Office regulations (covered by project license PE9055EAD) and by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
References
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