Local Ca²⁺ releases during the diastolic depolarization (CRDD) in sinoatrial nodal cells (SANC) activate Na⁺/Ca²⁺ exchange current and modulate the membrane potential. Since CRDD is a stochastic process we hypothesized that it produces spontaneous, beat-to-beat pacemaker potential fluctuations that characterize the SANC chronotropic state.

All procedures accorded with local guidelines. Rabbits were humanely killed with an overdose of anaesthetic and isolated SANCs loaded with fluo-3 AM were studied using confocal microscopy combined with a perforated patch-clamp technique. Pipettes were filled with (mM): 120 potassium gluconate, 20 KCl, 5 NaCl, 5 Hepes and 5 MgATP (pH 7.2, 34 °C).

The diastolic depolarization (DD), i.e. spontaneous depolarization between action potentials (AP) measured by whole-cell current clamp, displays two components: an initial linear one followed by a non-linear one that extends to the next AP upstroke. DD fluctuations, extracted by subtracting the DD averaged over several beats (bold curve in Fig. 1) from the DD of a given beat increased with time following a prior AP, achieving a maximum at 20-60 ms before the subsequent AP upstroke (Fig. 2). The evolution of CRDD occurrence, measured by confocal Ca²⁺ imaging, exhibited a similar time course, indicating a close link between CRDDs and DD fluctuations (Fig. 2). Strong positive correlations were observed among the changes in amplitudes of DD fluctuations, the average non-linear DD component amplitude, and beating rate in response interventions that alter CRDD magnitude (Bogdanov et al. 2001; Vinogradova et al. 2002). Ryanodine receptor blockade, intracellular Ca²⁺ chelation (BAPTA-AM), inhibition of sarcolemmal L-type Ca²⁺ channels (nifedipine), or β-adrenergic receptor stimulation (isoprenaline) that produced a 3-fold range of beating rates (70-200 b.p.m.), paralleled by a 3-fold variation of the average amplitudes of DD fluctuations (0.5-1.5 mV). In contrast, these perturbations did not significantly affect the initial, linear DD slope.

These observations support the idea that variations in CRDD magnitude produce fluctuations in the later, non-linear part of the DD, which modulate its amplitude and thus the time at which the subsequent AP fires. Thus spontaneous Ca²⁺-dependent DD fluctuations modulate the SANC chronotropic state.