Concurrent PDE3 and PDE4 Activation Suppresses Local Ca2+ Releases (LCR) to Regulate Normal Spontaneous Firing of Sinoatrial Node Cells (SANC)

Abstract

The spontaneous SANC beating rate is controlled by cAMP-mediated, PKA-dependent (cAMP/PKA) LCRs from ryanodine receptors (RyR). LCRs activate an inward Na+/Ca2+ exchange current increasing diastolic depolarization (DD) rate and spontaneous SANC firing. High basal level of cAMP in SANC is regulated by high phosphodiesterase (PDE) activity. In rat ventricular myocytes (VM) dual inhibition of both PDE3 and PDE4 synergistically increased the contraction amplitude. We studied whether PDE3 alone or concurrent (PDE3+PDE4) activation regulated spontaneous firing of rabbit SANC. Specific PDE3 inhibitor (cilostomide) or PDE4 inhibitor (rolipram) alone increased spontaneous firing (perforated patch-clamp) by only∼20% and ∼5%, respectively. But concurrent (PDE3+PDE4) inhibition increased spontaneous firing by∼48% accompanied by increases in LCR number, size and decrease in the LCR period that predicted concomitant reduction in SANC cycle length. In permeabilized SANC inhibition of PDE3 or PDE4 alone produced minor changes in LCR characteristics, while concurrent (PDE3+PDE4) inhibition markedly increased LCR size and number. Inhibition of PDE3 or PDE4 alone increased phospholamban (PLB) phosphorylation at Ser16site (marker of cAMP/PKA-dependent phosphorylation) by∼20%; inhibition of (PDE3+PDE4), however, increased PLB phosphorylation by∼110%. L-type Ca2+ current (ICa,L) provides Ca2+available for pumping into sarcoplasmic reticulum. PDE3 or PDE4 inhibition alone increased ICa,L amplitude by∼60% and∼5%, respectively, while concurrent (PDE3+PDE4) inhibition increased ICa,L by∼100%. When RyR were disabled by ryanodine (PDE3+PDE4) inhibition failed to increase SANC firing rate. Western blots revealed diverse expression of PDE subtypes in SA node and ventricle: more PDE4D was expressed in former, but more PDE3A in the latter; PDE4B was similarly expressed in both tissues. Thus, concurrent (PDE3+PDE4) activation regulates spontaneous SANC firing in a synergistic manner, by suppressing basal cAMP/PKA-dependent phosphorylation, reducing RyR Ca2+release and prolonging the LCR period and spontaneous cycle length.