This method can help answer key questions in the field of individual cardiomyocyte function and allows studying excitation-contraction-coupling. The main advantage of this technique is that a high yield of viable atrial cardiomyocytes from hearts with atrial cardiomyopathy can be isolated and used for subsequent experiments. Begin by placing a cannula with the long, sharp tip removed on top of the Langendorff apparatus and start the flow.
Turn on the heating module and adjust so that the perfusion buffer at the tip of the needle maintains the desired temperature for perfusion. Once the temperature calibration is complete, transfer the cannula to a 10 ml syringe filled with ice-cold cannulation buffer. Place two ready-prepared double overhand knots over the cannula for rapid subsequent cannulation of the aorta.
Remove the heart from the euthanized, heparinized rat by pinching the base of the heart with forceps and gently pulling down towards the tail of the animal. Cut across the aorta while maintaining a pull on the heart, leaving a 5-mm-long segment of the aorta attached to the heart. Quickly transfer the heart into 50 ml of ice-cold cannulation buffer in a 50 ml beaker.
Wait approximately 10 s for the heart to cool and cease contractions. Then, transfer the heart into a Petri dish, containing 50 ml of fresh, ice-cold cannulation buffer and carefully remove the fatty tissues surrounding the aorta using forceps and scissors. Insert the modified cannula 3 mm into the aorta without damaging the aortic valve.
Fix the aorta onto the cannula by tying one of the two cannulation knots at the indentation proximal to the tip of the cannula. Tie the second cannulation knot at the indentation distal to the tip of the cannula. Gently flush the aorta with 5 ml of ice-cold cannulation buffer using the syringe attached to the cannula until no blood is visible in the coronary arteries.
Then, use forceps to gently massage the left atrium while injecting the remaining 5 ml of cannulation buffer to flush any remaining blood. Unmount the cannula with the attached heart from the syringe. Mount the cannula with the attached heart to the Langendorff, using an appropriate adapter.
Start the peristaltic pump of the Langendorff apparatus to initiate the perfusion of the cardiac tissue. Quickly tie a double overhand knot around the base of the heart excluding the aorta. When the right and left atrium and coronary sinus inflate, puncture the atrium with the butterfly needle of the pressure control device and allow the atrium to deflate.
Manipulate the intraluminal pressure of the atrium by adjusting the elevation of the hose attached to the butterfly needle. Keep the atrium slightly inflated throughout the rest of the procedure. It is critical that the step is performed swiftly, as a prolonged inflation of the atrium will result in cardiomyocyte death.
Position a temperature probe between the left atrium and the left ventricle to measure the approximate temperature of the left atrium. Adjust the temperature of the Langendorff heating module accordingly, targeting an approximate temperature of 37 C of the left atrium. After 3 min of perfusion with perfusion buffer, switch to digestion buffer and perfuse for approximately 14-18 min.
When the left atrial structure collapses and the tissue acquires a milky texture, pinch the atrium using forceps and an act of slight pull. Remove the left atrium using fine scissors and transfer the atrium into a large weighing boat containing 2 ml of stopping buffer, fully submerging the tissue. Use fine scissors to mince the atrial tissue into small pieces of roughly 2 sq mm.
Disperse the tissue by gently triturating the tissue with a transfer pipette for approximately 5 min until a macroscopic dissociation of the tissue can be observed. Avoid generating air bubbles, as exposing the cells to air will result in cardiomyocyte death. Then, transfer the cell suspension to a 15 ml conical tube.
After allowing the tissue chunks to settle for 30 s, remove the supernatant and transfer to another 15 ml conical tube. Then allow the cells to settle for 15 min. After removing and discarding the supernatant, add 2 ml of Step 1 Buffer and allow the cardiomyocytes to settle for 10 min.
After 10 min, remove and discard the final supernatant. Add 250 microliters of normal Tyrode, containing 1 mM calcium. Transfer 50 microliters of the atrial cardiomyocyte suspension onto a laminin-coated glass bottom dish and allow the cardiomyocytes to settle at room temperature for 10 min.
After 10 min, add 500 microliters of normal Tyrode containing 1 mM calcium chloride to the cells. Evaluate cell morphology and viability under a light microscope. Randomly select approximately 100 cells and classify them as either viable or unviable to estimate the viability of the cell isolation procedure.
Viable cells are characterized by symmetric sarcomere structure, the absence of membrane blebs and a rod shape. Add 10 micromolar Fluo-4-AM to 500 microliters of normal Tyrode and use this to replace the normal Tyrode containing 1 mM calcium chloride in the glass bottom dish. After 20 min, remove the Fluo-4-AM solution and wash the cells twice with 500 microliters of normal Tyrode.
Transfer the dish to a confocal microscope and visualize the calcium excitation, using 40 x magnification. Then, perfuse the cells with normal Tyrode heated to 37 C using an appropriate superfusion device. Stimulate the cardiomyocytes in an electrical field with commercially available, microscope-mounted stimulator electrodes at a frequency of 1 Hz and an electrical current of 24 Amps.
After waiting for 1 min to allow the cells to reach a steady state of calcium ion handling, acquire line scan images by repetitive scanning. This image shows the transverse line scan of a single cardiomyocyte. The change of cytosolic calcium concentration is shown by excitation of the calcium-sensitive fluorescent dye, Fluo-4-AM.
The arrows indicate the electrical stimulation conducted at 1 Hz.This image shows the transverse calcium transients derived from this single cell. Once mastered, this technique can be performed in 3 h. While attempting this protocol, it is important to remember the time sensitivity of certain steps in the isolation procedure.
Following this procedure, other methods of sub-cellular imaging can be performed in order to answer additional questions regarding atrial cardiomyocyte remodeling.
