Abstract 413: Constitutive Phosphodiesterase 3 Activity Controls cAMP-protein Kinase A Dependent Local Subsarcolemmal Ca2+ Releases and Spontaneous Beating Rate in Rabbit Sinoatrial Node Cells
We recently discovered that rabbit sinoatrial nodal cells (SANC) have more than a 10-fold higher basal level of cAMP than do ventricular myocytes, and that spontaneous, rhythmic PKA-dependent local subsarcolemmal Ca2+releases (LCR) from ryanodine receptors (RyR) occur in SANC during the late phase of diastolic depolarization (DD). LCR activate inward Na+-Ca2+ exchange current leading to an increase in DD rate and thus SANC spontaneous beating rate. We hypothesized that a low basal PDE activity could promote this high basal cAMP-PKA signaling. Contrary to our hypothesis a non selective PDE inhibitor, IBMX (100 μmol/L), induced 2-fold increase in cAMP level, indicating high basal activity of PDE in SANC. Using specific inhibitors against different PDE types, we found that suppression of PDE3 by milrinone (50 mol/L) produces 164% increase in the spontaneous beating rate (from 110 ±6 to 164 ±8 beat/min), similar to the effect of IBMX (170%), and exceeding a saturating concentration of μ-AR agonist isoproterenol (140%). Suppression of PDE3 markedly increased PKA-dependent phospholamban phosphorylation (Western blot) which was reversed by a specific peptide PKA inhibitor, PKI. Simultaneous recordings of action potential (patch-clamp) and LCR (Fluo-3, confocal linescan imaging) showed that milrinone induced an increase in the LCR amplitude (from 1.90 ±0.03 to 2.14 ±0.04 F/F0, P<0.01), size (from 6.1 ±0.4 to 8.5 ±0.6 μm, P<0.01) and decreased the LCR period (time from prior AP triggered SR Ca2+ release to LCR occurrence during DD) from 425.1 ±25.6 to 324.2 ±4.8 ms (P<0.01). This milrinone-induced reduction in the LCR period was highly correlated with the concomitant increase in SANC beating rate (R2 =0.98). When RyR were disabled by ryanodine, milrinone failed to amplify LCR, decrease their period, or to increase SANC spontaneous beating rate, even though the increase in the amplitude of L-type Ca2+ current in control (from 12.5 ±1.6 to 18.5 ±2.5 pA/pF with milrinone, P<0.05) and in the presence of ryanodine (from 11.3 ±2.8 to 16.4 ±3.8 pA/pF with milrinone, P<0.05) was the same. Thus, the basal cAMP-PKA dependent signaling that governs RyR LCR and regulates spontaneous beating of SANC is controlled in part, i.e. kept in check, by constitutive PDE3 activation.