PDE3, PDE4 and PKC regulate local Ca2+ releases and cardiac pacemaker firing

Lakatta, Edward

Abstract

To test our first 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 the 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 combination of (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 combined (PDE3+PDE4) inhibition 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. To test our second hypothesis, we used specific PKC inhibitors GF109203X (10 mol/L) or calphostin C (1 mol/L). We have discovered that SANC pacemaker activity is critically dependent on PKC activity. When PKC activity was suppressed by GF109203X or calphostin C, it markedly suppressed SR Ca2+ cycling and stopped spontaneous beating of freshly isolated rabbit SANC. Specifically, GF109203X decreased the LCR size (from 5.80.3 to 2.80.3 m) and number per each spontaneous cycle (from 1.40.2 to 0.70.1);increased the LCR period, i.e. the time from the prior AP-induced Ca2+ transient to the subsequent LCR. The increase in LCR period during PKC inhibition predicted an increase in the spontaneous cycle length. All effects of GF109203X were reversed upon washout. Since Ca2+ cycling in SANC is critically dependent on ICa,L, which contributes to the AP upstroke and modulates the SR Ca2+ content, we studied effects of GF109203X and calphostin C on ICa,L. PKC inhibition by both inhibitors markedly suppressed ICa,L amplitude, strongly suggesting that modulation of ICa,L could be one of the major targets of basal PKC activity in rabbit SANC.