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

National Institute On Aging

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Abstract

(1) First, we compared RNA expression of different PDE subtypes in rabbit SANC and ventricular myocytes (VM). Total RNA was reverse transcribed to generate complementary DNA (cDNA), and relative abundance of cDNA from 9 different PDE transcripts was measured with qPCR. PDE3A, PDE4B and PDE4D were the major PDE subtypes expressed in both rabbit SANC and VM. We verified expression of major PDE subtypes (PDE3A, PDE4A, PDE4B and PDE4D) at the protein level in the rabbit SA node and ventricle using western blot. Consistent with the qPCR data, PDE3A and PDE4A protein was more abundant in the rabbit ventricle than in SA node. There was comparable expression of PDE4B protein in the SA node and ventricle, while expression of PDE4D protein was more abundant in the SA node. The intracellular distributions of the most abundant isoforms of PDE3 (PDE3A), and PDE4 (PDE4B and PDE4D) in rabbit SANC were examined using immunostaining. PDE3A was detected both beneath sarcolemma and in a striated pattern within Z-lines of rabbit SANC, colocalized with the Z-line associated protein alfa-actinin. PDE3A co-localized with PDE4B beneath sarcolemma of SANC, while PDE4D co-localized with PDE3A in striated patterns inside SANC. Co-staining of PDE3A with SERCA or PLB antibodies showed that PDE3A co-localized with SERCA and PLB in SANC. Since PDE4D co-localized with PDE3A, PDE4D should be also in the proximity of major SR proteins SERCA and PLB. To test our first hypothesis, 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 into SR. Inhibition of PDE3 or PDE4 alone increased the amplitude of ICa,L by 60% and 4%, respectively, while dual PDE3+PDE4 inhibition or IBMX increased ICa,L by 100%. Inhibition of PDE3 alone increased spontaneous SANC firing was by 20%, while inhibition of PDE4 alone had no effect on spontaneous firing. Dual PDE3+PDE4 inhibition, however, similar to IBMX increased the spontaneous SANC firing by 50%. This 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 spontaneous SANC firing is regulated by dual PDE3+PDE4 activation, and a crucial role of PDE4 is unmasked only when PDE3 and PDE4 are concurrently inhibited. Thus, synergism of combined (PDE3+PDE4) activation in SANC 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. (2) Spontaneous firing of freshly isolated rabbit SANC was markedly suppressed by selective PKC inhibitors GF109203X or calphostin C, and this was accompanied by suppression of SR Ca2+ cycling. Eventually LCRs ceased and spontaneous beating of SANC stopped. Specifically, GF109203X decreased the LCR size and number per each spontaneous cycle and increased the LCR period, i.e. the time from the prior AP-induced Ca2+ transient to the subsequent LCR. The PKC inhibition produced increase in the LCR period correlated with the increase in the spontaneous SANC cycle length. All effects of GF109203X were reversible upon washout. Since Ca2+ cycling in SANC is critically dependent on L-type Ca2+ current (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 activation in rabbit SANC. PKC could be activated by ubiquitous enzyme phospholipase C (PLC), which plays a key role in Ca2+ signaling in numerous cell types. (3) The PKC superfamily consists of 3 major subgroups: conventional (alfa, beta, and gamma), novel (delta, epsilon, theta, eta) and atypical (zeta, lambda/iota). We detected all PKC subtypes at the RNA level (RT-qPCR) in both rabbit SA node and ventricle. Expression of PKC-beta was markedly higher in the rabbit SA node, compared to other PKC isoenzymes in either tissue. Expression of conventional PKC (alfa, beta) and novel PKC-delta was verified at the protein level in SANC and ventricular myocytes (VM). Western blot confirmed RNA results, showing a 6-fold higher PKC-beat protein abundance in SANC compared to VM; while expression of PKC-alfa was comparable in both cell types and PKC-delta protein was more abundant in VM. To verify whether PKC-beta regulates spontaneous beating of SANC we employed selective inhibitor of conventional PKC (alfa, beta, and gamma) isoforms Go6976 (10 mkmol/L), which had no effects on either LCR characteristics or spontaneous beating of freshly isolated rabbit SANC. Because selective PKC-delta inhibitors are not available, we explored effects of PKC-delta inhibition using two different PKC inhibitors: the inhibitor of conventional PKC (Go6976) and Go6983, which inhibits conventional PKC (alfa, beta, and gamma) and PKC-delta (but not PKC-epsilon). Go6983 (5 mkmol/L) markedly decreased the LCR size (from 7.10.4 to 4.50.3 mkm, n=4) and number per each spontaneous cycle (from 1.30.1 to 0.80.1, n=4). It also markedly increased the LCR period (time from the prior AP-induced Ca2+ transient to the subsequent LCR) which was paralleled by an increase in the spontaneous SANC cycle length. Rottlerin, another PKC-delta inhibitor, produced similar effects on LCR characteristics, and markedly and time-dependently decreased DD rate, leading to an increase in the spontaneous cycle length, and finally abrogated the spontaneous SANC firing. Thus, the basal activity of PKC-delta, but not that of PKC-beta, is essential for generation of LCRs and normal spontaneous firing of cardiac pacemaker cells.