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) PKC inhibition by broad-spectrum PKC inhibitors Bis-I or calphostin-C suppressed or even arrested spontaneous firing of rabbit SANC, indicating increased basal PKC level which is obligatory for cardiac pacemaker function. Bis-I suppressed LCR parameters and increased the LCR period (time-interval from AP-induced Ca2+ transient to subsequent LCR) accompanied by prolongation of spontaneous SANC cycle length. Bis-I decreased L-type Ca2+current amplitude (whole-cell patch-clamp) and slowed its recovery from inactivation. Three classes of PKC isoforms (RT-qPCR) were detected in the sinoatrial node, including conventional-(alfa, beta, gamma), novel-(epsilon, delta, nu, theta) and atypical-(zeta, iota/lambda). Basal activity of PKCdelta was markedly elevated, as evidenced by high PKCdelta autophosphorylation at Thr505 site. Inhibition of conventional PKC isoforms had no effect on spontaneous SANC firing, while suppression of PKCdelta autophosphorylation decreased PLB phosphorylation, suppressed LCR parameters, concomitantly increased the LCR period and spontaneous SANC cycle length. Therefore, high basal PKCdelta activation potentiates efficacy of Ca2+ cycling proteins, including SERCA, L-type Ca2+channels (and likely others), regulates LCR period and characteristics, resultant Na+/Ca2+-exchanger current and is crucial for normal cardiac pacemaker function. (3) cAMP is a universal second messenger that can transmit various effects in the multitude of biological processes. Discovery of a family of exchange protein directly activated by cAMP (EPAC1 and EPAC2) presented novel aspects of cAMP action beyond well-established targets like PKA. High level of cAMP in SANC strongly suggests that in cardiac pacemaker cells EPAC could be activated in the basal state. We discovered that both EPAC1 and EPAC2 are expressed in rabbit SANC. EPAC activation by 8-pCPT-2-O-Me-cAMP augmented LCR characteristics, shortened the LCR period and, consistent with the coupled-clock theory, accelerated the spontaneous beating rate of SANC by 19%. Following inhibition of EPAC1 by CE3F4 or EPAC2 by HJC-0350 there were marked decrease in LCR parameters and increase in the LCR period accompanied by an increase in the spontaneous cycle length, indicating high basal activation of both EPAC1 and EPAC2. Overall Inhibition of EPAC1 and EPAC2 produced 30% or 25% decrease in the spontaneous SANC firing rate, respectively. The downstream target of EPAC is phospholipase C (PLC) which employs inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG) dependent signaling to regulate physiological responses. IP3 mediates the release of Ca2+ from intracellular Ca2+ stores, and DAG activates PKC. Inhibition of PLC activation by U-73122, but not its inactive analog U-73343, stopped spontaneous firing of intact rabbit SANC, indicating a key role of basal PLC activation for cardiac pacemaker function. To verify whether IP3R Ca2+ release represents a downstream target of EPAC-PLC pathway we employed membrane-permeable antagonist of IP3R Ca2+ release 2-APB, which produced no changes in the parameters of LCRs or amplitudes of AP-induced Ca2+ transients or spontaneous firing of rabbit SANC. These results strongly suggest that IP3R-mediated Ca2+ release is not a target of EPAC-PLC pathway, while PKC activity is critically important for cardiac pacemaker function. Therefore, EPAC-PLC pathway employ PKC (likely activity of PKCdelta) as a downstream target of EPAC-PLC-PKC pathway to regulate normal spontaneous beating of SANC.