Isolated myocytes from adult canine left ventricle: Ca2+ tolerance, electrophysiology, and ultrastructure

Abstract

We have developed a method for isolating single cardiac muscle cells in high yield (greater than 5 X 10(7) cells) from the canine left ventricle. Most of the myocytes are single cells with ultrastructural detail indistinguishable from intact ventricular myocardium, and more than 50% of the isolated cells remain elongated for at least 7 h in 0.5 mM calcium. Electrophysiological studies demonstrate that external potassium has a strong influence on repolarization in the isolated ventricular cells. Action potentials in [K+]o = 3.78 mM exhibit a positive over-shoot (greater than zero potential), but repolarization often arrests at congruent to -35 mV unless driven to more negative potentials by hyperpolarizing current. This phenomenon of two levels of resting potential is not observed at [K+]o = 5.78 mM. At the higher potassium concentration, values for maximum diastolic potential, amplitude, maximum rate of rise of phase 0, and action potential duration all are similar to those of intact ventricular muscle. However, the potential at the peak of the action potential plateau (phase 2) in the isolated myocyte is considerably more negative than that of intact myocardium. In addition, there is a conspicuous notch between phases 1 and 2 of the action potential in the isolated myocyte, whereas the notch is small or absent in intact myocardial action potentials. In summary, our method results in a preparation of stable, ultrastructurally and electrophysiologically intact cells, which should prove useful in studies requiring a large and homogeneous population of myocardial cells.