The Ca²⁺ content of the sarcoplasmic reticulum (SR) is central to excitation-contraction coupling. To date, there is little information concerning SR Ca²⁺ content obtained at physiological temperatures and rates of stimulation. In this study we have used the perforated patch technique to elicit action potentials at physiological rates (4-8 Hz) in rat ventricular myocytes.

Adult male rats were humanely killed by cervical dislocation and ventricular myocytes were isolated by collagenase digestion. Changes in [Ca²⁺]_i were measured using Fluo-3 AM. Following steady-state stimulation, the SR, Ca²⁺ content was measured by switching to voltage clamp mode and rapidly applying 10 mM caffeine (Varro et al. 1993). All experiments were performed at 37 °C. Data are presented as means ± S.E.M. from n experiments. Statistical analysis was carried out using repeated measures ANOVA.

Action potential duration, measured at 90 % repolarisation, was increased from 4 to 6 Hz (P < 0.05) but unchanged between 6 and 8 Hz (48 ± 3, 63 ± 5 and 59 ± 8 ms respectively; n = 5-14). The amplitude of the systolic Ca²⁺ transient was greater at 6 Hz (P < 0.01) than at 4 and 8 Hz (327 ± 28, 261 ± 22 and 266 ± 28 nM, respectively; n = 12-15). Despite this biphasic effect on the amplitude of the systolic Ca²⁺ transient, the SR Ca²⁺ content increased with stimulation frequency (72 ± 6.4, 101 ± 8.1 and 121 ± 7.9 µM at 4, 6 and 8 Hz, respectively; P < 0.05 between all frequencies, n = 8-9).

The decrease in the amplitude of the systolic Ca²⁺ transient on increasing frequency from 6 to 8 Hz arose through the combined effects of systolic [Ca²⁺]_i remaining unchanged (545 ± 51 and 535 ± 51 nM) and an increase in diastolic [Ca²⁺]_i (219 ± 33 and 269 ± 38 nM; P < 0.01). Thus, under the conditions used in these experiments, which have been designed to be as physiological as possible, Ca²⁺ transient amplitude and SR Ca²⁺ content appear to dissociate at high stimulation frequencies. This may arise through frequency-dependent effects on recovery from inactivation of the L-type Ca²⁺ channel and/or SR Ca²⁺ release through the ryanodine receptor. Furthermore, endogenous chronotropic effectors, e.g. adrenergic stimulation, may additionally act on intracellular Ca²⁺ handling such that this relationship may be restored.

This work was supported by the British Heart Foundation.