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

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Abstract
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.