Studying native ion channel properties using patch clamp electrophysiology requires access to acutely isolated cells. This protocol describes methods to access individual cardiovascular myocytes from zebrafish at different ages. This protocol is robust, simple, easily reproducible, and isolates cardiovascular myocytes from zebrafish in their native state with relatively high yield and viability.
To begin, transfer the fish into a large Petri dish partially filled with perfusion buffer and place it under a dissecting microscope. Hold the fish in the non-dominant hand with the ventral side facing the dissection scope. With the fine forceps in the dominant hand, gently tear open the pectoral muscles and fins of the fish to reveal the cardiovascular tissues including atrium, ventricle, and bulbus arteriosus.
Gently pull the bulbus arteriosus at the intersecting tip of the bulbus arteriosus and the ventral aorta. Carefully separate the bulbus arteriosus from the aorta by pinching the forceps, locating the atrium's tip into the multiple venous branches that converge. Then, pluck the tip of the atrium off the sinus venosus to isolate the cardiovascular tissues from the rest of the body.
To dissect cardiomyocytes, pluck the atrium and ventricle out of the cardiovascular tissue. Make a gentle tear into the ventricle using fine forceps to drain excess blood, which can be ensured when the cardiac tissue turns from bright red to a pale salmon color. Pool the isolated atrium and ventricle into a separate 1.5-milliliter centrifuge tube containing perfusion buffer.
Replace the perfusion buffer in the tubes with 750 microliters of digestion buffer. Place the tubes on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute. Allow digestion of the tissues until they become translucent.
End the digestion by gently spinning down the tissues in a bench-top minicentrifuge at 2, 000 times G for three to five seconds and replace the supernatant digestion buffer with 750 microliters of stopping buffer. Gently replace the stopping buffer with 500 to 750 microliters of perfusion buffer and triturate the tissues 30 times using a flame-polished Pasteur pipette to disperse the cells. Then, gently triturate the ventricles.
For uniform sampling, gently pipette the cells up and down a couple of times to resuspend, before adding a drop of them for corresponding studies. Pluck the adult bulbus arteriosus from the cardiovascular tissues and pool five bulbus arteriosus into a 1.5-milliliter centrifuge tube containing S1 buffer. Replace S1 with 400 microliters of S2 buffer and allow papain digestion of the bulbus arteriosus on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute for 20 minutes.
Allow the partially digested bulbus arteriosus to settle down for one minute and replace the S2 supernatant with 500 microliters of S3 buffer containing collagenase. Digest the tissues on a thermoshaker at 37 degrees Celsius and 800 revolutions per minute for three to five minutes. End the digestion by gently spinning down the tissues in a bench-top minicentrifuge at 2, 000 times G for three to five seconds, and replace the S3 supernatant with 500 microliters of S1.Gently triturate the pelleted tissues to disperse vascular smooth muscle cells.
Plate dispersed vascular smooth muscle cells onto glass cover slips of appropriate size. Keep the cover slips at room temperature for 30 minutes for the cells to attach, and use them within the next six hours. Anesthetize the embryos.
Then, concentrate the embryos by transferring them to a five-milliliter centrifuge tube, then remove excess media. Wash the embryos with three milliliters of cold perfusion buffer twice, then resuspend them in two milliliters of perfusion buffer. Using the embryonic heart isolation apparatus, draw one milliliter of the embryos into the needle and immediately expel them back into the tube.
Pass the fragmented embryos through a 100-micrometer cell strainer sieve placed in a funnel. Collect the filtered hearts in another five-milliliter centrifuge tube. Mix the filtrate well and separate one-milliliter aliquots into 1.5-milliliter centrifuge tubes.
Centrifuge all the tubes on a bench-top minicentrifuge for five seconds and discard the supernatant. Carry out serial resuspension of the pellets in the tubes using one milliliter of perfusion buffer. Gently pipette the hearts up and down twice before adding a drop for corresponding studies For cells isolated from bulbus arteriosus, the vascular smooth muscle cells were confirmed by tagln:EGFP expression.
Representative traces of recordings of ATP-sensitive potassium channel activity from isolated cardiomyocytes are shown here. Successful single-channel recordings were obtained in patches excised from the embryonic hearts. Ventricular myocytes exhibited stable hyperpolarized membrane potentials, and action potentials were stimulated via current injection through the patch pipette.
Atrial myocytes exhibited spontaneous action potential firing. Action potential properties are summarized here. While replacing the buffers during cardiomyocyte dissociation, care should be taken that the digestive tissues are not disturbed.
Tissue fragmentation should be accomplished only by pipette trituration. During the collagenous digestion of the bulbus tissues, examine for floating, dilated, and translucent tissues every minute. Once the tissue fragmentation is apparent, cease the digestion.
Once the cells are isolated, they can be kept alive for hours and used for electrophysiological analysis using patch clamp techniques. This has allowed us, for instance, to show that ATP-sensitive potassium channels in these zebrafish tissues are essentially identical to those in mammals.
