Recovery of Adult Zebrafish Hearts for High-throughput Applications

Summary

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Abstract

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Introduction

Zebrafish are an excellent model for studying heart development and human disease^1,2. Specific advantages include the translucent nature of zebrafish embryos, the availability of many genetic mutants and transgenic reporter lines, and the availability of genome editing technologies. In addition to their advantages for studying early heart development, zebrafish are an ideal system for studying vertebrate heart regeneration³.

More recently, adult zebrafish are playing an important part in bioinformatics approaches to studying cardiovascular development and disease, due to their relatively large clutch size and relativel....

Protocol

NOTE: Always be sure that IACUC or ethics committee approval is in place before beginning any experimental procedure using zebrafish.

1. Prepare reagents and setup

1. Prepare the following solutions. Find the recipes for all of these solutions in standard zebrafish manuals¹⁰.
   • 500 ml of Egg Water
   • 200 ml of 0.03% Tricaine in Egg Water
   • 100 ml of 1x Phosphate-Buffered Saline (PBS)
   • RBC Lysis Buffer (optional)
   • Destination buffer of choice (fixative, Trizol, PBS, etc.) in a 1.5 ml microfuge tube on ice
     NOTE: Fixative and Trizol are toxic if they come into co....

Results

Using this method, an adult zebrafish heart can be dissected in less than 1 min, compared to over 5 min using traditional methods⁸. Hearts dissected using this method are reliably intact (Figure 2A), while traditional methods⁸ require cutting blindly into the pericardium and therefore commonly cause damage or loss of the atrium or bulbus arteriosus (Figure 2B). Hearts dissected maintain their structural integrity and are suitable for histology (Figure 2C

Discussion

While methods for dissecting the adult zebrafish heart have been described, these methods were time-consuming and commonly caused damage to the heart during dissection. To perform experiments where a large number of adult hearts may be needed, and/or when avoiding degradation of heart tissue is important for downstream applications, the time required using traditional dissection techniques is prohibitive. Similarly, reproducibly obtaining undamaged, intact hearts is important for study of heart structure and for immunohi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Small tank for transporting fish	Aquaneering	ZHCT100
Fish net	Petsmart	36-16731
250 ml glass beaker	Kimble	14005-250
9 cm polystyrene Petri dish	Nunc	172958
Razor blade	Personna American Safety Razor Company	94-120-71
2 Dumont #5SF forceps	Fine Science Tools	11252-00
Dissecting microscope	Olympus	SZX16
Tricaine	Sigma	A-5040
Plastic transfer pipette	Thermo Scientific	202-20S
Gooseneck light source	Dolan-Jenner Industries, Inc	Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System
Fluorescent light source	Lumen Dynamics	X-Cite 120Q	optional
Micro-scissors	Biomedical Research Instruments, Inc	11-1000	optional
RBC lysis buffer	eBioscience	00-4333-57	optional