Patch clamp and calcium imaging experiments are labor intensive, time consuming and challenging. This protocol provides a convenient method to isolate high quality murine atrial and ventricular cardiomyocytes, suitable for patch clamp experiments and simultaneously performed calcium imaging. The main advantage of this protocol is that we can obtain atrial and ventricular cardiomyocytes from the same animal.
The isolation of murine atrial cardiomyocytes is especially challenging and has been a limiting factor for previous experiments. Begin by pre-filling the Langendorff apparatus with perfusion buffer and ensuring it is air free. Fix an aortic cannula under the dissection microscope and connect it with a one-milliliter syringe filled with perfusion buffer.
After removing the heart from the euthanized mouse, put the heart into room temperature perfusion buffer and cannulate the aorta with a blunt-end needle under the microscope as quickly as possible. Firmly tie the heart to the needle with a piece of suturing silk, and disconnect the syringe. After aortic cannulation, immediately connect the cannulated heart to the Langendorff apparatus, avoiding any air entering the system.
Perfuse the heart with perfusion buffer for one minute at exactly 37 degrees Celsius and a perfusion rate of four milliliters per minute. Switch from perfusion to digestion buffer and perfuse for exactly nine minutes at a temperature of 37 degrees Celsius and a perfusion rate of four milliliters per minute. When finished, transfer the digested heart to a petri dish with enough digestion buffer to keep it fully covered.
Then carefully dissect the atria and ventricles under the microscope. Transfer the atria into a petri dish with 1.5 milliliters of digestion buffer and the ventricles into another petri dish with three milliliters of digestion buffer. Carefully but quickly pull the atria apart into tiny pieces Using blunt forceps.
Dissolve the tissue by carefully pipetting up and down with a 1000-microliter pipette tip which was previously cut to widen the tip opening. Transfer the solution to a 15-milliliter centrifuge tube and add an equivalent amount of stop buffer by pipetting down the side of the tube. Pass all three milliliters of the solution through a 200-micrometer nylon mesh to remove the remaining larger tissue pieces that have not been fully digested.
To perform ventricular dissection, chop the ventricular tissue into tiny pieces using dissection scissors or forceps, and pipette up and down with another 1000-microliter pipette tip to dissolve. Transfer the cell and tissue solution into a 15-milliliter centrifuge tube and add an equivalent amount of stop buffer by carefully pipetting it down the side of the tube to end the reaction. Pass all six milliliters of cell and tissue solution through a 200-micrometer nylon mesh to remove larger pieces that have not been fully digested.
Leave both atrial and ventricular cell suspensions on the bench at room temperature for six minutes to settle. Then centrifuge the tubes at 5G for two minutes. Discard the supernatants using a plastic Pasteur pipette and carefully resuspend the cell pellets in 10 milliliters of calcium-free Tyrode's solution.
Leave the atrial and ventricular cells for eight minutes for sedimentation. Centrifuge the atrial cells at 5G for one minute. Then discard the supernatants from both cell samples and carefully resuspend the pellets in 10 milliliters of Tyrode's solution with 100 micromolar calcium.
Leave the cells for eight minutes for sedimentation, then repeat the centrifugation of atrial cells. Discard the supernatants and carefully resuspend the cell pellets in 10 milliliters of Tyrode's solution with 400 micromolar calcium. Repeat this process once more, resuspending the cells in Tyrode's solution with one millimolar calcium.
All atrial cells are small with cell capacitances ranging from approximately 35 to 100 picofarad. A typical cell from the atrial working myocardium is shown here. Ventricular myocytes are more rod-shaped and larger, with cell capacitances ranging from 100 to around 400 picofarads.
Examples of L-type calcium current measurements with simultaneous cytosolic calcium transients from one atrial myocyte and one ventricular myocyte are shown here. Before attempting this technique, Make sure to practice the organ harvest. It is crucial that the step is performed fast and all tissue cuts are made at the correct locations.
When organ harvest is performed at reproducible quality, take some time to perfect cannulation. Cells isolated with this protocol are suitable for patch clamp measurements. In addition, they can be loaded with fluorescent dyes, allowing for a wide range of imaging experiments.
