An efficient isolation procedure of Ca-tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies

Abstract

A simple procedure which provides a large yield of isolated ferret ventricular myocytes is described. The enzymatic dissociation was performed by perfusion of the whole heart with the "Langendorff method" at 37 degrees C, without an incubation period. Special attention was given to the period of perfusion with Ca-free or low-calcium containing solutions and to the proportion of both collagenase and elastase used. The viability and calcium tolerance of the isolated cells were tested by ultrastructural and electrophysiological studies. Photo-microscopy showed that 60 to 80% of the isolated cells had an elongated shape (18 microns in diameter, 150 microns in length) and did not beat spontaneously in normal Tyrode solution. The morphological and ultrastructural integrity of these cells was shown in SEM by their smooth surface with regularly spaced T-tubule openings and in TEM by the regular distribution of the transverse tubular system, mitochondrium and sarcomeres. Using the whole-cell patch-clamp technique, they had a resting membrane potential of -72 mV, two types ("Purkinje like" and "ventricular like") of action potentials could be elicited and they were correctly affected by well-known modulators of calcium channels. This technique was successfully applied to the rat heart and could be used for heart dissociation of small mammals. It can simultaneously provide isolated cells of different regions of the heart and can be easily and routinely used by any investigator.