Isolation of calcium tolerant myocytes from adult rat hearts: Review of the literature and description of a method

Myocytes have been isolated from adult rat hearts since 1969. The early preparations exhibited the Ca 2+ paradox. Over the ensuing years, numerous groups have reported the isolation of Ca 2+ tolerant cardiac myocytes. In the present review, detailed comparisons have been made of the yields, viability, and relative Ca 2+ tolerance of these different myocyte preparations. The factors to which these investigators attributed the increased Ca 2+ tolerance are considered and the current information regarding the mechanism of the Ca 2+ paradox is reviewed. A method is given which incorporates several of the modifications described. By this method 40–60% of the ventricular weight was disaggregated into single myocytes within 45 min after the sacrifice of the rats. Viability without further purification was 82 ± 0.7% (n = 35) and Na i +/K i + ratios were normal. Upon incubation with 2 mM Ca 2+ for 1 hr at 37°C, viability decreased by 6% and ATP and creatine phosphate remained at physiological levels. The preparation is very stable since upon incubation in culture medium containing fetal bovine serum and 1.25 mM free Ca 2+ at 25°C for 20 hr, viability decreased only 13% (rod-shaped and trypan blue criteria). The factors which contribute to the quality and Ca 2+ tolerance of this preparation are discussed.