Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

[Narrator] The overall goal of this procedure is to yield a consistent preparation for the culture of functional adult mouse cardiomyocytes. First remove the mouse heart under anesthesia and cannulation. Perfuse the heart and digest with collagenase. Then dissociate the cells and reintroduce the calcium. Next seed the cells and culture the primary cardiomyocytes. Results from western blots and tritiated leucine incorporation assays can demonstrate function such as the activation of PI3 kinase-dependent AKT phosphorylation and the increase of protein synthesis rate induced by Ouabain.

- After isolation of the heart, quickly fan the aorta from many other vessels, then handle the aorta very carefully and hook it up quickly on the first try. With practice you will be able to determine when to stop the collagenase digestion of the heart by accurately monitoring the color and the softness change.

- Demonstrating the procedure will be Dr Daxiang Li, a postdoctoral fellow in my laboratory.

- [Narrator] Just prior to the perfusion, prepare fresh digestion buffer containing type II collagenase and warm the buffer to 37 degrees Celsius when ready for isolation. Soak scissors and forceps in 70% ethanol and set them to dry. Now weigh the mouse to the nearest 10th of a gram. Record its body weight, strain, sex and date of birth. Inject 200 microliters of Heparin intraperitoneally to prevent coagulation of blood in the coronary arteries. After 10 minutes, anesthetize the mouse. After five to 10 minutes, check that the mouse is non-responsive to a tail or toe pinch, then secure the mouse in a supine position by gently fixing the fore paws and hind paws to a work surface. Wipe the chest and abdomen with 70% ethanol, then make a midline skin incision from mid abdomen to the diaphragm. Hold the sternum and cut bilaterally. Next, cut the diaphragm and retroflect the thoracic cage to expose the heart. Now lift the heart slightly and dissect the heart out of the thoracic cavity as close as possible to the dorsal thoracic wall. Transfer the heart to cold profusion buffer in a 100 millimeter dish. Carefully trim away the connective tissues such as lungs, thymus, bronchi and esophagus. Now identify the aorta and it's cranial branches which are hidden by the thymus and fat pad. Cut the aorta below its first branch, grasp the aortic wall and lift the heart. Check that the profusion fluid is dripping, then slightly slip the heart onto the perfusion fluid-filled aortic cannula keeping the tip of the cannula just above the aortic valve. Clamp the aorta to the cannula and ligate the aorta to the cannula with 6-0 surgical silk. Perfuse the heart with calcium-free profusion buffer at a flow rate of four milliliters per minute for about four to five minutes until the effluents become clear, then switch to digestion buffer containing 50 micromolar calcium chloride. If applicable, increase the after load pressure to 70 to 80 millimeters of mercury when digesting. When the heart becomes slightly pale and flaccid, check that it is spongy when gently pinched, then stop the digestion. With 70% ethanol-soaked forceps, pull the aorta from the cannula and put the heart into a sterile 60 millimeter dish containing 2.5 milliliters of digestion buffer. Now move into the cell culture hood and use sterile supplies and sterile techniques. Remove the aorta, atria and great vessels with fine surgical scissors. Then gently tease the ventricle into 10 to 12 small pieces. With a 10 milliliter transfer pipette, gently pipette the heart pieces and cells and transfer the sample to a 15 milliliter polypropylene conical tube. Rinse the dish with 7.5 milliliters of calcium solution I and transfer the wash to the conical tube, resulting in a final volume of 10 milliliters. Next, disperse the large pieces of heart tissue by gently pipetting with a fine tip transfer pipette. Then centrifuge for three minutes at 20 G's to separate out small non-myocyte cells such as endothelial cells and fibroblasts. Aspirate off the supernatant. Resuspend the myocyte pellet in 10 milliliters of calcium solution I supplemented with ATP and equilibrate for three to five minutes. Next transfer duplicate 10 microliter aliquots to a hemocytometer. Count the rod-shaped and the round myocytes, then calculate the total number of myocytes and determine the percentage of rod-shaped myocytes. Pellet the myocytes by centrifugation. Remove the supernatants and wash the pellet successively in 10 milliliters of increasing concentrations of calcium. For mouse myocyte primary culture, resuspend the final myocyte pellet in five milliliters of plating medium to get calcium-tolerant cardiomyocytes. Count the total number of myocytes and calculate the percentage of rod-shaped cyocytes. Adjust the concentration of rod-shaped myocytes to 25,000 rod-shaped myocytes per milliliter. Gently pipette the myocytes to ensure equal cell density for each dish or plate. After aspirating off the laminen coating solution from the culture plates, seed the cardiomyocytes and evenly disperse the cells by gently sliding the dishes forward and backward and side to side in a cross-like pattern on the surface of the culture hood. Place the cultures in a 2% carbon dioxide incubator at 37 degrees Celsius to allow myocyte attachment at a rate of about 80%. Now gently aspirate off the medium containing unattached myocytes and cell debris. Replenish the culture medium by aiming at the sides of the dishes one plate at a time. Immediately return the cells to the culture incubator. In a successful preparation, the isolated myocytes normally have a distinct rode shape with rectangular ends and clear cross striations. As a functional assay, adult C57 black six mouse cardiomyocytes were cultured and treated with Ouabain and ET1. These data show that Ouabain and ET1 can increase AKT phosphorylation in a dose-dependent manner and also induce tritiated leucine incorporation during protein synthesis. This technique paved the way for researchers in the field of heart disease to explore the mechanism of cardiac regulation in genetic-modified mice.