Mammalian cardiac impulse is generated in sinoatrial node (SAN) cells, and its automaticity is sustained by a complex interplay of different ionic currents. Evidence in cardiac and neuronal tissues points to the ‘funny’ (I_f) current as a major player both in pacemaker generation and its modulation by autonomic transmitters (DiFrancesco, 1993). However, the relative importance of individual ionic components in pacemaking is still debated. Recent data indicate a role for Ca²⁺ release from the sarcoplasmic reticulum (SR) in pacemaker activity (Terrar & Rigg, 2000). Based on evidence that disruption of ryanodine receptor (RyR) function abolishes Ca²⁺ transients and β-receptor (β-R)-induced modulation of heart rate, Vinogradova et al. (2002) proposed a novel Ca²⁺-dependent, I_f-independent mechanism of pacemaker regulation by β receptors relying on the activation of subsarcolemmal RyRs.

To check if the I_f-dependent rate-regulation mechanism is still operating after impairment of RyR function, we measured the spontaneous rate from single rabbit SAN cells (or small groups of cells) before and during perfusion with ryanodine (3 mM). In agreement with previous data, the spontaneous rate was decreased by ryanodine (31.0 ± 3.3 %, n = 5; mean ± S.E.M.). In the presence of ryanodine (2-3 min exposure), the action of isoprenaline (ISO, 1 mM) on rate was strongly reduced, although not fully abolished (11.4 ± 2.0 vs. 26.4 ± 2.0 % in control, n = 4). To test if the reduction of ISO effect involved a decreased cAMP availability, we used a membrane-permeable cAMP analogue (pCPT-cAMP, 100 mM) and found that the mean frequency increase induced by pCPT-cAMP in ryanodine-treated cells (20.0 ± 3.6 %) did not change significantly with respect to control (22.5 ± 6.8 %) (n = 3). This suggests that the I_f-dependent rate-modulation pathway is still operating in ryanodine-treated cells. However, as well as activating I_f channels directly (DiFrancesco, 1986), pCPT-cAMP could also activate phosphorylation-dependent mechanisms by activation of protein kinase A (PKA). In an attempt to discriminate between phosphorylation-dependent and phosphorylation-independent processes, preliminary experiments using Rp-cAMPs, a cAMP analogue unable to activate PKA and known to activate I_f channels (Bois et al. 1997), suggest that ryanodine does not reduce the rate increase caused by Rp-cAMPs: 50 mM Rp-cAMPs increased the rate by 18 % in control conditions, and by 22 % after ryanodine treatment in two cells.

Our data support the view that ryanodine treatment does not impair the cAMP-dependent mechanism of rate modulation mediated by I_f channels. Disruption of RyR function and depletion of Ca²⁺ stores by ryanodine may impair the β-R modulation of rate by reducing the ability of β-R stimulation to induce cAMP synthesis.

This work was supported by Cofin 2000 to D.D. R.B.R. was supported by NIH grant HL-28958.

All procedures accord with current National guidelines.