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.
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.
Zebrafish are an excellent model for studying heart development and human disease1,2. 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 relatively quick and inexpensive breeding compared to other vertebrate models. Promising techniques include ribosome profiling, RNA-Seq, and cell dissociation and FACS sorting4-7. However, for these techniques the quality of the data can depend on obtaining a large number of samples in a rapid, efficient, and reproducible manner, before gene regulatory, metabolic, transcriptional, and other changes occur.
Dissection of adult zebrafish organs has been described in the past8,9. However, previous approaches to dissection of the heart were slow, ran the risk of damaging the heart during dissection, required special tools, and/or required fixation of the zebrafish prior to dissection; for these reasons, past approaches to zebrafish adult heart dissection were not optimized for high-throughput applications and/or applications requiring fresh tissue.
Here, we describe a method for adult zebrafish heart dissection that is simple, fast, efficient, and reproducible, while preserving cardiac morphology. This method does not include cutting into the pericardial space and therefore does not risk damaging the heart during dissection. Instead, this method relies on anatomical landmarks of the zebrafish, and therefore, it is highly reproducible. This dissection method is also versatile in that it can be used on fresh or fixed fish, and on zebrafish as young as one month old. Finally, this method results in minimal suffering to the zebrafish because after anesthesia and/or rapid cooling, the fish is additionally decapitated and pithed in the course of the dissection procedure.
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
2. Prepare Zebrafish
Figure 1. Zebrafish adult heart dissection utilizes zebrafish anatomical landmarks. (A) To decapitate the fish, lift the pectoral fin with a forceps and use a sharp clean razor blade along the red dotted line as shown. (B) To steady the fish head, place one tine of the forceps in the fish mouth while the other tine lies across the eye, and then turn the fish head so that the ventral surface is up and both tines of the forceps are stable against the bottom of the Petri dish. (C) Use the free forceps to cut the attachment of the operculum (arrow). (D) Lifting this, the dorsal aorta is visible as a white structure with a pink stripe denoting luminal blood (arrow). Please click here to view a larger version of this figure.
3. Dissect the Heart
4. Prepare the Heart for Downstream Applications
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), electron microscopy (Figure 2D), as well as for applications where the heart is dissociated or pulverized. In our hands, from a dissection of 50 hearts over the course of a week’s experiments, 50 hearts were obtained structurally intact. We also taught this technique to a colleague in the laboratory who had no experience with zebrafish dissection. 9 out of 9 hearts dissected by the beginner colleague were also structurally intact. This dissection method is suitable for juveniles older than 1 month old, and for adults of all ages.
Figure 2. Dissected hearts are intact and maintain architectural integrity. (A) In less than 1 min, an adult zebrafish heart with an intact atrium (A), ventricle (V), and bulbus arteriosus (BA) can be dissected from a euthanized fish. (B) Traditional adult zebrafish heart dissections take longer and even when care is taken, run the risk of damage. Here, only the ventricle is intact. (C) A heart dissected from a Tg(myl7dsRed) adult fish maintained its structural integrity for use in histology. The atrium (A), ventricle (V), and bulbus arteriosus (BA) are seen, and even smaller structures like the atrioventricular valve (AV, arrow) and the outflow tract (OFT, arrow) are intact. (D) Electron microscopy of a dissected wild-type adult heart shows that sarcomeres remain undamaged by the dissection process. Please click here to view a larger version of this figure.
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 immunohistochemical and microscopy methods. We found that even in the hands of beginners, 100% of hearts dissected were recovered structurally intact. Overall, the method for adult heart dissection presented here presents an advance in technique that expands the use of adult zebrafish for a variety of current and future applications, including use in heart injury and regeneration experiments.
There are several critical steps within this protocol. First, depending on the experiment for which the zebrafish hearts are required, it is important to rinse hearts as much as possible from blood cells. Examples of experiments that would benefit from removing blood cells include recovery of hearts for RNA-Seq, where presence of blood will alter gene expression profiles, or for FACS sorting, where blood cells may be difficult to sort from myocardial cells. After the fish is euthanized with Tricaine or ice, it is important to decapitate the fish quickly and allow bleeding to occur to help rid the heart of excess blood. If an intact heart is not required for the planned experiment, it can be helpful to cut the atrium and ventricle open when in PBS to further rinse out the blood. RBC lysis buffer can also be used instead of PBS.
Second, as mentioned above, occasionally the heart comes off with one of the pectoral fins rather than staying attached to the head. Therefore, pull the pectoral fins off under the microscope so that the heart can be identified. Finally, when dissecting the heart away from surrounding pericardium, try not to grasp the heart itself; instead, grasp two pieces of pericardium and pull them both away from the heart, which has more structural integrity than the friable pericardium. In this way, even minimal damage to the heart can be avoided.
The heart should be easily identifiable by its shape and contractions. As an additional aid in finding the heart, one can dissect a heart expressing a fluorescent protein under a fluorescence dissecting microscope. This is usually not needed but may be useful for beginners and/or for dissections of very small hearts.
One limitation of this technique is that dissecting hearts from small fish, i.e., younger than 3 weeks old, is difficult; other techniques have been designed for larval dissections9. Also, while it is easy to dissect the bulbus arteriosus, atrium, and ventricle from each other, it is not possible to dissect the atrioventricular valve on its own. The valve remains attached to the ventricle in most cases.
The authors have nothing to disclose.
The authors would like to thank Dr. Shaun Coughlin for hosting the filming of this procedure in his laboratory, and for general support. R.A. was supported by the NIH (F32HL110489) and the Sarnoff Cardiovascular Research Foundation. S.R. was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG) and the American Heart Association (AHA). D.Y.R.S was supported by the NIH (RO1HL54737), the Packard Foundation, and the Max Planck Society.
small tank for transporting fish | Aquaneering | ZHCT100 | |
fish net | Petsmart | 36-16731 | |
250mL glass beaker | Kimble | 14005-250 | |
9cm polystyrene petri dish | Nunc | 172958 | |
razor blade | Personna American Safety Razor Company | 94-120-71 | |
two 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 |