Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMPmediated, protein kinase A-dependent local subsarcolemmal ryanodine receptor Ca2+ releases (LCRs). LCRs activate an inward Na+/Ca2+ exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. (1) Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase A-dependent phospholamban phosphorylation, and increased SANC firing rate by 55%, indicating a high basal activity of PDEs in SANCs. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, i.e., PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca2+ release, Na+/Ca2+ exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine IBMX inhibition was unable to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMPmediated, protein kinase A-dependent increase of diastolic ryanodine receptor Ca2+ release and restrict the spontaneous SANC beating rate. (2) Expression of PDE1A protein was 5-fold higher in rabbit sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. Nimodipine-sensitive activity of PDE1, measured in lysates of isolated rabbit SANCs, accounted for 40% of total PDE activity, but PDE1 inhibition increased spontaneous firing of rabbit SANCs by only 15%. Combined activities of PDE3 and PDE4 represent the major basal PDE activities in the rabbit SA node, accounting for 50% in cytosolic and 90% in SR fractions. Average increases in the basal spontaneous beating rate of rabbit SANCs by inhibition of PDE3 or PDE4 alone were relatively small, but concurrent inhibition of PDE3+PDE4 increased the spontaneous SANC beating rate by 48%, creating an effect comparable with that of IBMX. Similar to intact SANC, inhibition of PDE3 alone (n=8) or PDE4 alone had minor effect on LCR number or size in permeabilized SANC. In contrast, dual inhibition of PDE3 and PDE4 produced marked increase in LCR number, size and caffeine- induced SR Ca2+ content in permeabilized SANC. (3) To study how PDE inhibition controls the SR Ca2+ refilling and LCR period, we compared kinetics of SR Ca2+ refilling in control and after PDE inhibition. Phosphorylation of phospholamban (PLB) has been used as index of SR pumping rate and SR refilling was estimated by the time to 90% decay of the AP-initiated global cytosolic Ca2+ transient (T-90). Graded PLB phosphorylation by a broad-spectrum phosphodiesterase inhibitor (IBMX), by selective PDE3 or PDE4 inhibition or specific PKA inhibitor peptide (PKI) were paralleled by proportional changes in T-90. Concomitant changes in T-90 and LCR period were highly correlated with changes in the spontaneous cycle length. Results obtained in isolated rabbit SANC were recently confirmed in the isolated mouse sinoatrial node (SAN). Suppression of PDE activity by broad spectrum PDE inhibitor IBMX increased the spontaneous beating rate of mouse SAN by 66%, confirming existence of constitutively active PDE. IBMX also increased the ratio of PS-16 PLB to total PLB by 51% comparing to its basal level. Like in rabbit SANC, PDE inhibition in mouse SANC increased LCR occurrence, size and duration. In permeabilized mouse SANC IBMX also significantly increased LCRs size, duration, the Ca2+ signal of LCR ensemble (more than 2-fold). Thus, suppression of PDE activity increases cAMP-mediated PKA-dependent phosphorylation, accelerates the SR Ca2+ refilling rate, decreases the LCR period, stimulates the Ca2+ clock, and increases the spontaneous beating rate of isolated SANC and SAN tissue. (4) Like in isolated rabbit and mouse SANC, PDE inhibition with IBMX in permeabilized rabbit ventricular myocytes (VM) increased the number of LCR events and LCR periodicity measured by Fast Fourier Transforms (FFT), and clock-like local Ca2+ releases, partially synchronized in space and time emerged. This ensemble of rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. RyR2 phosphorylation at Ser2809 site in permeabilized VM was markedly increased by IBMX, and further increased in the presence of the protein phosphatase (PP) inhibitor CyA which enhanced PKA-dependent phosphorylation. The latter resulted in further increase of the amplitude, width, duration and number of LCRs, which not only amplified the Ca2+ signal of individual LCRs but also amplified the Ca2+ signal of the LCR ensemble by 8-fold over control in permeabilized VM. Thus, suppression of PDE activity in VM has a similar effect on the Ca2+ clock as in pacemaker cells. Specifically, it accelerates the SR Ca2+ cycling via phosphorylation dependent increase in the rate of SR Ca2+ pumping or kinetics of RyR activation, or by the reduction in the threshold for spontaneous RyR activation. Conclusion: The cAMP-degrading PDE1, PDE3, and PDE4 represent major PDE activities in rabbit SANC, and PDE inhibition by IBMX increases spontaneous firing of SANC by 50%. Though inhibition of single PDE1-PDE4 only moderately increases spontaneous SANC firing, dual PDE3 + PDE4 inhibition produces a synergistic effect hastening the spontaneous SANC beating rate by 50%.
