To test the hypothesis that combined activity of (PDE3+PDE4) impacts on basal spontaneous SANC firing via regulation of cAMP/PKA signaling, we used phosphorylation of phospholamban (PLB) at Ser16 site as a marker of cAMP/PKA-dependent protein phosphorylation in SANC. Specific PDE3 inhibitor, cilostamide (Cil, 0.3 mkmol/L), or a PDE4 inhibitor, rolipram (Rol, 2 mkmol/L), increased PLB phosphorylation by 20%, but the combination of Cil+Rol increased PLB phosphorylation by 110%, an effect similar to that (140%) produced by broad spectrum PDE inhibitor IBMX. L-type Ca2+ current (ICa,L) ensures LCR existence, providing Ca2+ available for pumping in SR. Cil or Rol alone increased the amplitude of ICa,L by 60% and 4%, respectively, while (Cil+Rol) or IBMX increased ICa,L by 100%. Cilostamide increased the spontaneous SANC firing rate (perforated patch-clamp) by 20% (from 14813 to 17513 beat/min, n=8), while rolipram produced no acceleration of spontaneous firing at 2, 20 or 100 mkmol/L. Similar to IBMX, (Cil+Rol) increased the spontaneous SANC beating rate by 50% (from 1348 to 19711 beat/min, n=9), the effect was due to a marked increase in the LCR number, size and decrease in the LCR period that predicted the concomitant decrease in the spontaneous cycle length. When RyR were disabled by ryanodine and LCRs were abolished, both IBMX and (Cil+Rol) failed to accelerate DD rate or increase SANC firing rate indicating key role of Ca2+ cycling for PDE-dependent control of spontaneous beating. We conclude that both PDE3 and PDE4 regulate spontaneous SANC firing, and a crucial role of PDE4 is revealed only when PDE3 and PDE4 are concurrently inhibited. Thus, synergism of (PDE3+PDE4) suppresses basal cAMP/PKA-dependent PLB phosphorylation and reduces ICa,L amplitude to decrease RyR Ca2+ release, prolong the LCR period and limit the spontaneous SANC firing rate.

National Institute of Health (NIH)
National Institute on Aging (NIA)
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