Cytosolic free calcium concentration in individual cardiac myocytes in primary culture

Cytosolic free Ca²⁺ concentration, [Ca²⁺](i), of single isolatd Ca²⁺-tolerant rat ventricular myocytes in primary culture was determined by digital video imaging of intracellular fura-2 fluorescence. In deenergized myocytes in which contractile elements were uncoupled by 2,3-butanedione monoxime, the maximum and minimum fluorescence intensity ratio values of fura-2 in the cell were similar when compared with those of fura-2 solutions observed in the microscope. Through the use of in vitro calibration [Ca²⁺](i) in quiescent, rod-shaped myocytes was 90 ± 6 nM. There was no detectable spatial heterogeneity in [Ca²⁺](i) in resting myocytes. Localized regions of [Ca²⁺](i) elevation were observed in cells undergoing spontaneous rhythmic contractions or when subjected to mild depolarization by KCl. Additions of gramicidin or veratridine resulted in massive increases in [Ca²⁺](i) (>1 μM) and immediate cell hypercontracture. Ruthenium red elicited a modest increase in [Ca²⁺](i) but extracellular ATP or epinephrine had no effect. We conclude the following: 1) digital video imaging of resting cardiac cells did not reveal any subcellular Ca²⁺ gradients; 2) the fluorescence properties of intracellular fura-2 were similar to that in free solution; and 3) subcellular heterogeneity of [Ca²⁺](i) in isolated myocytes was observed in cells undergoing spontaneous rhythmic contraction.