Isolation of Adult Pig Ventricular Myocytes

Sedate adult pig (25 to 50 kg) with Telezol (500 mg, intramuscular). Administer atropine (1 mg, subcutaneous), to control salivary and bronchial secretions, and (200 mg, intravenous), to facilitate tracheal intubation. Anesthetize the animal with isoflurane (2-4%, inhalation).

Shave the chest area and wash with betadine antiseptic solution. Check for loss of corneal and pain reflexes to ensure that the animal is in a deep anesthetic plane. Open the chest cavity by peeling back the skin over the left side of the chest wall and then making an incision between the 4th and 5th ribs. Use rib spreaders to hold open the incision.

Open the pericardium and remove the heart, placing it in ice cold cardioplegia solution containing (in mM) NaCl 130, KCl 27, MgSO₄ 1.2, KH₂PO₄ 1.2, glucose 50, HEPES 6 (pH 7.4). Rinse the heart free of blood and transport to the laboratory in cardioplegia solution on ice.

Dissect the heart, removing a section of the left ventricular free wall supplied by the left anterior descending (LAD) coronary artery.

Attache the ventricular free wall to a Langendorff apparatus by cannulating the proximal end of the LAD coronary artery, and perfuse the tissue with Ca²⁺-free solution containing (in mM) NaCl 140, KCl 5.4, MgCl₂ 2.5, glucose 11, HEPES 5.5 (pH 7.4) maintained at 37^oC,.

Ligate any leaking arteries at the periphery of the free wall in order to maintain perfusion pressure throughout the bulk of the tissue. Continue perfusion using, constant pressure, until the effluent is free of any remaining blood (about 10 minutes).

Switch perfusion to Ca2+-free solution containing collagenase type 2 (~200 U/ml) and protease type XIV (~1 mg/ml) for 20 minutes.

Stop enzyme digestion of the tissue by perfusing with enzyme-free KB solution containing (in mM) K-glutamate 110, KH₂PO₄ 10, KCl 25, MgSO₄ 1.2, taurine 20, creatine 5, EGTA 0.5, glucose 20, HEPES 5 (pH 7.2) for 2 minutes.

Remove the tissue from the Langendorff apparatus and separate into different regions as necessary. The top half can be used to obtain myocytes from the base of the free wall, while the bottom half can be used to obtain myocytes from the apex.

Cut the tissue into small pieces, mince with scissors, and then filter through a 500 µm polyester mesh. Collect the filtrate containing isolated myocytes. Allow the cells to settle in KB solution maintained at 37°C for 25 minutes.

Aspirate the supernatant and then resuspend the pellet of isolated myocytes in Ca²⁺ containing solution for use in experiments.