This protocol could help to improve the atrial yield from a perfused mouse heart. The main advantage of this method is that the atrial and ventricular myocyte can be isolated simultaneously under the same digestion condition. This method could provide insight into the mechanisms of heart disease at the cellular and subcellular level.
After euthanizing an eight to 10-week-old male C57 black six mouse, use tissue forceps to lift the skin of the xiphoid. Then using tissue scissors, make a minor lateral incision through the skin. Perform a blunt dissection between the skin and fascia.
Extend the skin incision toward the axillae in a V shape on both sides and continue the incision through the ribcage. Then using tissue forceps, clamp the sternum and deflect the ribcage upward to fully expose the heart and lungs. Using curved forceps, peel off the pericardium.
If the thymus gland covers the great vessels, use two curved forceps to tear the thymus gland toward both sides, then gently pull the base of the heart toward the tail until the aorta and its branch arteries are visible as a Y-shaped blood vessel. Transect the aorta at the left common carotid artery, then cut the brachiocephalic artery. Excise the heart and immediately immerse it in a Petri dish containing Tyrode's solution to wash and pump out the residual blood.
Then transfer the heart to another Petri dish containing solution one. Under a stereo microscope, using fine Iris scissors, trim any surplus tissue. Expel air bubbles from the syringe.
Then with the assistance of two straight tying forceps, perform retrograde aortic cannulation, taking care that the whole cannulation process is performed under the liquid surface. Adjust the cannulation depth such that the cannula tip is in the ascending aorta, taking care not to penetrate the aortic valves. Then with a pre-knot 3-O suture, ligate the aorta to the cannula notch.
Gently inject solution one from the syringe to flush out the residual blood. Then connect the cannulated heart to the Langendorff apparatus, taking care not to introduce any air bubbles into the heart. After connecting the cannulated heart to the Langendorff apparatus, perfuse the heart with solution one for approximately two minutes.
Using a single-use sterile polyethylene pipette, draw up about 2.5 milliliters of solution three and prewarm it in the water bath for later use. Then to digest the tissues, perfuse the heart with the remaining solution three for approximately 11 to 12 minutes. After the first two minutes of perfusion with solution three, recycle the perfused solution to the perfusate reservoirs by the peristaltic pump for reuse until digestion is completed.
When the heart becomes swollen, turning slightly pale and flacid, using toothed forceps, gently pinch the myocardium. If an imprint is visible, terminate the digestion and detach the heart from the apparatus. To isolate the arterial and ventricular myocytes, use forceps to remove ventricles and the atria and place them in different Petri dishes.
Then add the pre-warmed solution three to both dishes. Using blunt forceps, triturate the tissues into a turbid texture, then gently pipette the tissue for even digestion without introducing air bubbles. To arrest the remaining enzyme activity, using a pipette, transfer the turbid digested tissue into solution four, then centrifuge for 20 seconds at 192 times G.After removing the supernatant, resuspend the cell sediment in solution five.
To avoid calcium paradox and calcium overload, reintroduce the calcium in a stepwise manner by gradually adding a total of 50 microliters of 100 millimolar per liter calcium chloride to the cell suspension. Once all the calcium chloride has been added, store the cells in Tyrode's solution for the patch clamp study. Cannulation depth is associated with the perfusion of the atria and its appendages.
When the cannula tip is at the ascending aorta, both atrial appendages are inflated indicating sufficient atria perfusion. However, when the cannula tip is at the aortic root, both atrial appendages are wizened, indicating insufficient perfusion. Atrial myocytes isolated by cannulation at the ascending aorta had higher survival rates than those isolated by cannulation at the aortic root both before and after calcium reintroduction.
In contrast, there was no difference in the survival rates of ventricular myocytes isolated by cannulation at either the ascending aorta or the aortic root both before and after calcium reintroduction. The whole-cell patch clamp recording of the sodium current in the isolated atrial and ventricular myocytes, as well as the current densities confirm that the quality of the isolated cells meets the requirements for electrophysiological experiments. When transecting the aorta, make sure to retain a sufficient gland for ligation.
Otherwise, the cannula may penetrate the aortic valve after ligation.
