Heart Dissection: A Method to Observe Zebrafish Cardiac Development

- A zebrafish heart comprises four chambers, sinus venosus, atrium, ventricle, and bulbous arteriosis. It is located anteriorly on the ventral side between the operculum and the pectoral girdle, a part of the skeletal system that supports the pectoral fin. To dissect the heart, place a euthanized fixed fish into a Petri dish containing phosphate-buffered saline to maintain a constant pH of the fish tissues and orient it ventral side up.

Hold the fish head with forceps between the eyes and gills. Using a microscissor, cut across the gills, across the anterior belly, and then vertically, connecting the first two incisions. With a sharp tweezer, remove the pectoral fin and the underlying pectoral girdle to reveal the silver pericardium, the membrane that covers the heart. Remove the pericardium with forceps to make the heart visible. Now cut the artery connected to the bulbous arteriosis.

Next, place forceps under the atrium and scoop the heart out of the body cavity. Hold the heart with the forceps, and use another forceps to remove unwanted tissue present outside the heart. In the following protocol, we will dissect the heart from different sized fish to image its development over the fish's life span.

- Assign each fish a number-letter combination to allow an individual fish and the resulting dissected heart to be tracked for later analysis. Enter all data, including all measurements, into a spreadsheet and organize based on these labels. Before dissection, label PCR tube strips with assigned tracking number and fill tubes with PBT or other solution appropriate for further analysis. Orient the fish ventral side up in a Petri dish while stabilizing the body with forceps, holding the head between the eyes and gills. For fish shorter than 12 millimeters in standard length, using sharp forceps or a microneedle in a pinholder, remove the pectoral muscles and fins from the body to reveal the heart.

- The constant motion of the forceps during dissection can cause currents in the PBT that move the fish. This is particularly problematic for smaller fish. This, the microneedle needs to be used to remove pectoral muscles and fin and open the body cavity.

- Use the forceps to gently scoop the heart out of the cavity from under the atrium. If the fish contains a fluorescent cardiac marker, use a fluorescent scope for the dissection and verify removal of the heart by fluorescence. Once the heart is removed from the cavity, use the forceps to hold the heart and a microneedle to remove extra noncardiac tissue and the epicardial lining from the outside of the heart.

For fish longer than 12 millimeters in standard length, using spring-handled microscissors, make three incisions. First, make a transverse cut through the gills. Second, make another transverse cut at the anterior belly. And third, make a sagittal cut on the ventral side connecting both transverse cuts. Using sharp forceps, remove the pectoral muscles and fins from the body to open the body cavity and reveal the silvery tissue of the pericardium.

Remove this pericardial tissue. And the heart will become visible. Use the micro scissors to cut the artery connected to the bulbous arteriosis located superior to the heart. Once this artery is cut, place the tips of the forceps under the atrium and use the forceps to scoop the heart out of the cavity. If there is extra tissue that comes with the heart, that is fine, as it can be removed later. Alternatively, in some fish, it is possible to remove the heart by gently pulling the bulbous out, and the rest of the heart will follow.

- Be careful of the location of the atrium if using the alternative technique, as it can easily be damaged. If there's extra tissue that comes with the heart, this is fine and can be cleared after.

- Once the heart is removed from the cavity, use the two forceps to hold the heart and remove the extra tissue or one pair of forceps to hold the heart and a microneedle to remove extra noncardiac tissue.