Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, 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. 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. A comparison of specific PDE1 to -5 inhibitors revealed that the specific PDE3 inhibitor, milrinone, accelerated spontaneous firing by 47% (effects of others were minor) and increased amplitude of L-type Ca2+ current (ICa,L) by 46%, indicating that PDE3 was the major constitutively active PDE in the basal state. 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, neither IBMX nor milrinone was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibition-induced augmentation of ICa,L amplitude. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMP-mediated, protein kinase A-dependent increase of diastolic ryanodine receptor Ca2+ release, and restrict the spontaneous SANC beating rate. 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), specific phosphodiesterase-3 inhibitor (milrinone), or by 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 mouse isolated sinoatrial node (SAN). Suppression of PDE activity by the broad spectrum PDE inhibitor IBMX (100 micro mol/L) increased the spontaneous beating rate of mouse SAN from 37518 bpm to 6237 bpm (n=6, P<0.05). The positive chronotropic effects of IBMX indicate that an increase in intracellular cAMP level activates PKA and accelerates SAN automaticity, independent of external receptor stimulation. Stated in other terms, these data indicate constitutively active PDE in mouse SAN tissue, which degrades cAMP, leads to suppression of downstream cAMP-mediated PKA-dependent signaling. When PDE activity is blunted, PKA dependent signaling and the spontaneous beating rate are markedly enhanced in mouse SAN. This idea was tested using anti-PS16 PLB antibody and PLB total antibody. On average, IBMX increased the ratio of PS-16 PLB to total PLB by 51.238.87% comparing to its basal level (n=10 cells, P<0.05). Similar to isolated rabbit SANC, PDE inhibition in mouse SANC increased LCR occurrence, size and duration. In permeabilized mouse SANC, in conditions of tight control of cytoplasmic Ca2+, IBMX (5M), significantly (p<0.03) increased LCRs size (4.4 to 70.5m), duration (38 to 563.6ms), the Ca2+ signal of LCR ensemble (more than 2 fold), the percent of cells that produce periodic LCRs (from 72% in control to 100 % during IBMX application), the rhythmicity index (RI, assessed via autocorrelation function). 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. To avoid human bias and quantify nano-scale LCR, we reanalyzed IBMX data measured via confocal microscopy in skinned mouse SANC with the novel auto spark detection program, which was recently developed in our laboratory. We found that indeed LCR Ca2+ signal was increased in in response to PDE inhibition in young. Specifically, during 2 min incubation of SANC with IBMX (5M), LCR amplitude increased by 10%, LCR size by 64%, LCR duration by 56% vs that in Control ( P<0.05, n=9). The average Ca2+ signal of individual LCR increased by 2.2 fold (P<0.05, n=9). and the Ca2+ signal of LCR ensemble by 1.5 fold. Similar to 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, similar to pacemaker cells. This ensemble of rhythmic local Ca2+ wavelets generated a periodic high-amplitude Ca2+ signal. When we measured RyR2 phosphorylation by phospho-imaging of permeabilized VM, we found significant enhancement of RyR2 phosphorylation at Ser2809 in response to PDE inhibition by IBMX compared to control. Addition of IBMX in the presence of the protein phosphatase (PP) inhibitor CyA further enhanced PKA-dependent phosphorylation, which resulted in further increase of the amplitude, width, duration and number of LCRs, which not only further amplified the Ca2+ signal of individual LCRs but also amplified the Ca2+ signal of the LCR ensemble by 8-fold over control in permeabilized ventricular myocytes (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. Inhibition of PDE3 alone with 0.3 M cilostomide (n=8) or PDE4 alone with 2 M rolipram (n=7) had minor effect on LCR number or size in permeabilized SANC. In contrast, dual inhibition of PDE3 and PDE4 by 0.3 M cilostomide + 2 M rolipram produced marked increase in LCR number (n=6) and size (n=6), and caffeine- induced SR Ca2+ realize in permeabilized SANC. *P < 0.05. Conclusion: spontaneous LCRs are regulated by dual PDE3 and PDE4 activation.
