A procedure is described for the routine and reproducible preparation of single myocytes from left ventricular tissue of the adult dog. Tissue dissociation was accomplished by repeated incubations of minced ventricle with a mixture of 0.05% trypsin and 0.05% collagenase. Studies by light microscopy and by transmission and scanning electron microscopy indicated that myocytes were cleanly separated at their lateral borders and at the intercalated discs. The intercalated discs retained structural characteristics observed in undissociated tissue and gave the appearance of hemidesmosomes. Approximately 60 to 90% of the cells were typical spindle-shaped myocytes and, of these, up to 80% initially exhibited contractile activity. However, since the cells do not depolarize spontaneously, contractile activity decreased within 1 h, and the myocytes remain quiescent unless stimulated by additions of Ca2+, ouabain or epinephrine. The specific activities of Mg2+-inhibited ATPase, creatine phosphokinase, hexokinase and isocitrate dehydrogenase in isolated myocytes were comparable to the levels in undissociated ventricular tissue. However, the level of glucose-6-phosphate dehydrogenase, which is low in intact tissue, was not measurable in myocytes. Canine myocytes metabolized glucose with kinetics for uptake and oxidation comparable to that obtained by intact organ perfusion. Furthermore, glucose oxidation was doubled when cells were incubated in the presence of insulin. Fatty acid oxidation was linear following an initial lag phase, and this was insensitive to extracellular carnitine. Canine myocytes demonstrated the ability to oxidize to extracellular leucine and linearly incorporated the amino acid into trichloroacetic acid-precipitable protein. © 1979.
