Effects of electrical stimulation and an intracellular calcium chelator on calcium movement in suspensions of isolated myocardial muscle cells

The procedure of Haworth RA, Hunter DR and Berkoff HA (J Mol Cell Cardiol, 1980;12:715–23) for the isolation of myocardial muscle cells from rat hearts has been modified by the addition of a step which involves centrifugation of the cells through a Percoll gradient. This increased the proportion of rod-shaped cells from 47 ± 2.2 to 80 ± 1.3% (mean ± SEM, n = 7). In the absence of electrical stimulation but in the presence of 1.3 mmol·litre−1 Ca2+ less than 2% of the cells beat spontaneously. This number was not increased by addition of the Ca2+-selective ionophore A23187. A method in which isolated myocytes suspended in a cylindrical incubation chamber are stimulated to beat by electrical impulses is described. At 1.3 mmol·litre−1 extracellular Ca2+, electrical stimulation increased by 30% the amount of 45Ca2+ exchanged in the period 0.25 to 3 min following addition of 45Ca2+. For myocytes subjected to electrical stimulation, the amount of 45Ca2+ exchanged increased as the concentration of extracellular Ca2+ increased. At 0.5 mmol·litre−1 extracellular Ca2+ verapamil reduced the amount of 45Ca2+ exchanged by 15% while La3+ reduced the amount of 45Ca2+ exchanged by 80%. Incubation of myocytes with the acetoxymethyl ester of the intracellular Ca2+ chelating agent bis (o-aminophenoxy)-ethane-N,N,N'N'–tetraacetic acid (BAPTA) for 45 min led to an inhibition of contraction. At 0.5 mmol·litre−1 extracellular Ca2+ half maximal inhibition was given by 200 μmol·litre−1 acetoxymethyl BAPTA. At 100 μmol·litre−1 acetoxymethyl BAPTA the degree of inhibition was reduced by decreasing the time of exposure of the cells to the Ca2+ chelating agent, but was not influenced by the presence or absence of Ca2+ in the medium during treatment of the cells with acetoxymethyl BAPTA. The inhibitory effect of BAPTA was almost completely reversed by increasing the concentration of extracellular Ca2+ from 0.5 to 5.0 mmol·litre−1 during measurement of cell contraction. Treatment of cells with acetoxymethyl BAPTA caused a small reduction in the amount of 45Ca2+ exchanged in 45Ca2+ exchange curves, and in the total amount of Ca2+ associated with the cells. It is proposed that BAPTA inhibits the contraction of myocardial muscle cells by decreasing the concentration of free Ca2+ in the myoplasm of the resting cell and preventing the transient increase in free Ca2+ in the myoplasm which follows depolarisation of the sarcolemma.