Isolating Primary Melanocyte-like Cells from the Mouse Heart

Summary

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. 

Abstract

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

Introduction

We recently identified a novel cell population in the heart of mice and humans that express melanin-synthesis enzymes and morphologically resemble cutaneous melanocytes. We called these cells 'cardiac melanocytes' and found they are present in anatomic locations from which atrial arrhythmia triggers often originate in humans. We also found cardiac melanocytes contribute to atrial arrhythmias in mice with germline deletion of Dct. Cardiac melanocytes are sparsely distributed throughout the mouse atrium where they comprise less than 0.1% of all atrial cells. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure t....

Protocol

1. Preparing the Necessary Solutions and Equipment

1. First, sterilize the surgical instruments (straight shape forceps, curved shape forceps, straight shape scissors, and curved shape scissors) by autoclaving them for 15 min. at 121 °C. Prepare 2 sets of instruments, where one set is used for dissecting out the hearts and the other set will be used for mincing up the tissue.
2. Dissolve 1 packet of MCDB153 culture media in 900 ml of sterilized, filtered water and stir slowly until the powder has dissolved. Add 1.2 g sodium bicarbonate, or 15.7 ml of sodium bicarbonate solution [7.5% w/v], for each liter of final volume of medium being prepared an....

Results

The protocol described in this article is an improved technique compared to the one we previously published for isolating cardiac melanocytes from the murine heart. This procedure employs the use of fluorescent markers driven by melanin synthesis enzyme-specific Cre-recombinase to label cardiac melanocyte-like cells. It is important to use a marker to label cardiac melanocytes when working with freshly isolated embryonic or neonatal cells, since the morphological differences that distinguish cardiac melanocytes from atri.......

Discussion

Cardiac melanocytes are present in anatomic locations within the heart that commonly give rise to atrial arrhythmia triggers. These regions include the pulmonary veins, the atrioventricular annulus, the posterior left atrium and the foramen ovale (Figure 4). To understand the role these cells play in arrhythmogenesis, we develop techniques to isolate them and study their physiology. However, we recognized that improving this technique to yield healthier and larger quantities of cardiac melanocytes would .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MCDB 153	Sigma-Aldrich	M7403	Dissolve in water
DPBS, no calcium no magnesium	Invitrogen	14190136	Stock conc.: 1x Working conc.: 1x
Penicillin-streptomycin	Invitrogen	15140163	Stock conc.: 100x Working conc.: 1x
Fetal Bovine Serum	Life Technology	Certified	Working conc.: 4%
TPA	Sigma-Aldrich	P1585	Dissolve in DMSO Stock conc.: 3.2 μM Working conc.: 32 nM
Transferrin	Sigma-Aldrich	T8158	Dissolve in DPBS Stock conc.: 0.1 mg/ml Working conc.: 1 μg/ml
Dibutyryl cyclic AMP	Sigma-Aldrich	D0627	Dissolve in DPBS Stock conc.: 0.5 mM Working conc.: 5 μM
Insulin	Sigma-Aldrich	I6634	Dissolve in 10 mM acetic acid Stock conc.: 1 mg/ml Working conc.: 5 μg/ml
Basic FGF	BD Biosciences	354060	Dissolve in water Stock conc.: 4 ng/μl Working conc.: 4 ng/ml