Effects of Ca2+ deficiency, collagenase, and mechanical dispersion on the ultrastructure of cardiac myocytes of adult rats

A new perfusion medium for isolating cardiac myocytes from adult rats was developed, thereby yielding numerous viable cells with few morphological changes. The main factors in the isolation procedure are Ca2+ deficiency, collagenase, and mechanical dispersion. Their effects on the ultrastructure of cardiac myocytes were separately tested. In isolated hearts, perfusion with a medium containing a physiological Ca2+ concentration (2.5 mM, controls) preserved the cellular fine structure well, whereas perfusion with a medium containing 2.5 mM Ca2+ plus 0.05% collagenase caused swelling and disruption of most cells. Perfusion with a Ca2+-deficient medium followed by a medium with a low Ca2+ concentration (25 microM) either containing or lacking collagenase resulted in widening of the T-tubules, reduced electron density of the external lamina and occasional separation, or even dissolution of this layer. Some cells were damaged and hypercontracted. These appeared more numerous in suspensions, that means after mechanical dispersion of the myocardium. However, most of the isolated cells were regularly shaped (up to 30-60 min as shown in another study) and their ultrastructure was only slightly altered. This corresponds to an adequate preservation of the cell membranes proven in earlier membrane transfer studies.