Intracellular Ca²⁺ buffering in cardiac muscle is normally attributed to either fixed cytosolic buffers or active Ca²⁺ extrusion, but a slower passive exchange with intracellular organelles may occur. Rabbit cardiac myocytes were enzymatically isolated from New Zealand White male rabbits killed with an intraveneous injection of sodium pentobarbitone (100 mg kg^-1). Isolated cells were incubated with either of the indicators fura-2 AM or fluo-3 AM for 1 h (37°C) followed by a further 1 h indicator-free incubation. Calcium dynamics were studied in the absence of ATP on populations of fura-2 AM-loaded cells using a cuvette-based system and on single cells using confocal microscopy. For the cuvette studies, aliquots of 2 Ω 10⁶ cells were permeabilised by brief exposure to 0.01 mg β-escin and the cells placed in a cuvette containing a mock intracellular solution of the following composition (mM): 0.05 EGTA, 25 Hepes, 0.9 Mg²⁺, 100 KCl, 0.01 Na⁺, pH 7.0 (20-22°C). Free [Ca²⁺] was monitored with the addition of 1.3 µM fluo-3 (free acid).

Addition of 50 nmol Ca²⁺ caused Ca²⁺ influx into a sub-cellular compartment (s) shown by the rise in fura-2 340/380 nm fluorescence ratio and the corresponding decline in fluo-3 fluorescence. Addition of 5 mM EGTA (and consequent lowering of free [Ca²⁺]) caused Ca²⁺ efflux from of this compartment. The rate of decline is speeded up by the addition of the ionopore A23187 4-bromo (10 µM). This supports the notion that a component of the fura-2 signal is from within organelle(s). The fura-2 signal from the permeabilised myocytes showed two distinct components. A rapid component had a rise time that was similar to the bulk solution (fluo-3). R_min and R_max of this component were similar to those of the free acid, but the K_d of the signal was higher (960 ± 25 vs. 465 ± 12 nM; means ± S.E.M., P < 0.05; n = 5, Student’s t test). The second slower component of the fura-2 signal had a half-time of 136 ± 19 s (n = 5). This signal was unaffected by further addition of β-escin, but 1 µM A23187 4-bromo significantly increased the rate of change of the signal (similar results were observed with ionomycin). Ruthenium Red, CCCP and thapsigargin did not inhibit this slow component. In parallel experiments on single fluo-3 AM-loaded cells, the remaining fluorescence after β-escin treatment showed a biphasic rise in fluorescence in response to a rapid increase of [Ca²⁺] from < 1 nM to 0.38 µM Ca²⁺. This was not observed in cells not loaded with fluo-3 AM but in the presence of fluo-3 free acid (10 µM). These results suggest there is passive exchange of cytosolic [Ca²⁺] with an intracellular compartment/ organelle in isolated rabbit cardiac myocytes.