Studies employing confocal Ca²⁺ imaging have unequivocally implicated intracellular Ca²⁺ release as a modulator of beating rate of cardiac pacemaker cells (Rigg & Terrar, 1996; Huser et al. 2000; Bogdanov et al. 2001). In sino-atrial nodal cell (SANC), local subsarcolemmal ryanodine receptor (RyR) Ca²⁺ release during the diastolic depolarization (CRDD) activates the electrogenic Na⁺/Ca²⁺ exchanger, producing an inward current that augments the rate of diastolic depolarization (DD), leading to an earlier occurrence of the subsequent action potential (AP), i.e. to an increase in the beating rate.

To test whether a concurrent voltage change, i.e. the DD, is actually required for the generation of CRDD, spontaneously beating single, SANC (n = 9) isolated from humanely killed rabbits were acutely voltage clamped (VC) at -60 mV (maximum diastolic potential) or -70 mV, and CRDD were imaged in the line scan mode using fluo-3 as Ca²⁺ indicator. CRDD, present during spontaneous beating, persisted during VC (Fig. 1). During the initial 400 ms of VC, a time corresponding to the next would-be AP if spontaneous firing were to have continued in the absence of VC, the total CRDD signal mass did not differ from that during DD during spontaneous beating (Fig. 1). With increasing time of VC, the total CRDD signal mass passed through a maximum (at 1.0 ± 0.2 s), then gradually decreased, and usually ceased at intervals > 5 s. We also observed that during VC the average level of cytosolic [Ca²⁺] gradually decreased to ~80 % of the level during spontaneous firing. When VC was removed, the cytosolic [Ca²⁺] and localized subsarcolemmal Ca²⁺ release returned to their pre-VC levels and spontaneous APs resumed. The addition of 50 µM Ni²⁺ to the bath prior to the voltage clamp did not affect the aforementioned results.

We conclude that the occurrence of localized RyR Ca²⁺ releases within a given DD during spontaneous beating does not require the concurrent membrane depolarization; its persistence, however, requires the regular occurrence of APs to provide Ca²⁺ influx sufficient to maintain cell and sarcoplasmic reticulum Ca²⁺ loading.