A subscription to JoVE is required to view this content. Sign in or start your free trial.
The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.
Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.
Zebrafish are small teleost fish that have long been used as a model vertebrate organism1 and have recently come to prominence as a viable vertebrate system for high throughput screening of genes and drugs2,3. However, physiological analysis of zebrafish tissues is not well developed. In the cardiovascular system, methods have been described for dissection of zebrafish hearts4 and isolation of ventricular cardiac myocytes5,6,7. There are few detailed descriptions of the ....
All zebrafish (wild type strain AB, both male and female) were raised, maintained, and handled for the experiments following the guidelines of the Washington University Institutional Animal Care and Use Committee (IACUC).
1. Isolation of atrium, ventricle, and bulbous arteriosus from adult, juvenile, and larval zebrafish
The above protocols reliably and routinely provide sufficient cardiac and vascular myocytes of consistent quality amenable for patch-clamp studies as recently reported in extensive studies of ATP-sensitive potassium (KATP) channels in wild-type and mutant zebrafish cardiovasculature9. Representative traces of recordings of such KATP channel activity from isolated cardiomyocytes are shown in Figure 3A-C. In the case of cells isola.......
Previous methods for isolating zebrafish ventricular myocytes5,6, aimed at generating myocytes for culture or electrophysiological studies, provided cells of lower yield and involved lengthy steps of multiple centrifugations that adversely affected the cell quality and viability. The protocols described here are reliable, cover each of the significant cardiovascular tissues (ventricle, atria, and VSM), and importantly are quite practical for acute isolation of li.......
This work was supported by NIH grants HL140024 to CGN and HL150277 to CMC. Figure 1 and Figure 2 were created with BioRender.com.
....Name | Company | Catalog Number | Comments |
1.5 mL Centrifuge Tubes | Eppendorf | 22364111 | |
10 mL Syringe | Fisher Scientific | 14-955-459 | |
19 Guage Needle | BD | 305187 | |
2,3-Butanedione Monoxime (BDM) | Sigma-Aldrich | B0753 | |
5 mL Centrifuge Tubes | Sigma-Aldrich | EP0030119479 | For embryonic heart isolation |
Axopatch 200B amplifier and Digidata 1200 digitizer | Molecular Devices | Used for action potential recordings | |
Benchtop Mini Centrifuge | Southern Labware | MLX-106 | |
Blebbistatin | Sigma-Aldrich | 203390 | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A9418 | |
Calcium Chloride | Sigma-Aldrich | C4901 | |
Cell-Strainer Sieve | Cole-Parmer | EW-06336-71 | 100 μm sieve for embryonic heart isolation |
Collagenase Type H | Sigma-Aldrich | C8051 | |
Collagenase Type II | Worthington | LS004176 | |
Collagenase Type IV | Worthington | LS004188 | |
Curved Forceps | Fisher Scientific | 16-100-110 | |
DTT | Sigma-Aldrich | D0632 | |
EGTA | Sigma-Aldrich | 324626 | |
Elastase | Worthington | LS003118 | |
Fetal Bovine Serum (FBS) | Sigma-Aldrich | F2442 | |
Fine Forceps | Dumont | Style #5 | Ceramic-coated forceps for adult and juvenile CV tissue isolation (Need two) |
Glucose | Sigma-Aldrich | G8270 | |
HEPES | Sigma-Aldrich | H3375 | |
Insulin | Sigma-Aldrich | I2643 | |
K2ATP | Sigma-Aldrich | A8937 | |
Large Petri Dish | Sigma-Aldrich | P5981 | For dissociation |
Magnesium Chloride | Sigma-Aldrich | M8266 | |
Micro-Hematocrit Capillary Tubes | Kimble Chase | 41A2502 | Soda lime glass for patch pipettes |
Papain | Worthington | LS003118 | |
Pasteur Pipettes | Fisher Scientific | 13-678-6A | |
Petri Dish | Sigma-Aldrich | P5606 | 100 mm x 20 mm, for embryonic heart isolation |
Phosphate-Buffered Saline (PBS) | Sigma-Aldrich | 806552 | |
Potassium Chloride | Sigma-Aldrich | P3911 | |
Scissors | Fine Science Tools | 14090-09 | For adult and juvenile zebrafish decapitation |
Sodium Chloride | Sigma-Aldrich | S9888 | |
Sodium Hydroxide | Sigma-Aldrich | S8045 | |
Super Fine Forceps | Dumont | Style #SF | For isolating larval CV tissues (Need two) |
Taurine | Sigma-Aldrich | T0625 | |
Thermoshaker | ThermoFisher Scientific | 13687711 | |
Tricaine Methanesulfonate (MS222) | For anaesthetizing zebrafish larvae | ||
Trypsin Inhibitor | Sigma-Aldrich | T6522 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved