Phosphodiesterases Restrict Spontaneous Beating of Cardiac Pacemaker Cells

National Institute On Aging

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Abstract

Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase A-dependent LCRs from ryanodine receptors, which activate an inward Na+/Ca2+ exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. (1) Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase A-dependent phospholamban phosphorylation, and increased SANC firing rate by 55%, indicating a high basal activity of PDEs in SANCs. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, i.e., PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca2+ release, Na+/Ca2+ exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine IBMX inhibition was unable to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMP-mediated, protein kinase A-dependent increase of diastolic ryanodine receptor Ca2+ release and restrict the spontaneous SANC beating rate. (2) The PDE superfamily contains 11 distinct gene families (PDEs 111), and at least four PDE families (PDE1-PDE4) can hydrolyze cAMP in heart. At the messenger RNA level PDE3A, PDE4A, PDE4B and PDE4D are the major cAMP-degrading PDE subtypes expressed in both rabbit SANC and ventricular myocytes. Expressions of PDE3A and PDE4B mRNA in rabbit SANC are comparable and exceed expression of other PDE subtypes. Compared to PDE3 or PDE4, PDE1 mRNA in rabbit SANC is relatively low, but PDE1A transcript abundance in SANC surpasses that in VM by 4-fold. Nimodipine-sensitive activity of PDE1, measured in lysates of isolated rabbit SANC, accounted for 40% of total PDE activity, but PDE1 inhibition increased spontaneous firing of rabbit SANC by 15%. PDE1 activity might have a greater impact at higher cAMP levels; indeed, stimulation of ACs with forskolin markedly increases both cAMP level and PDE1 activity in paced mouse VM. Although average increases in the basal spontaneous beating rate of rabbit SANC by inhibition of single cAMP-degrading PDEs (PDE1-PDE4) is relatively small, concurrent inhibition of PDE3+PDE4 increases the spontaneous SANC beating rate by 48%, creating effect comparable to that of IBMX. An acceleration of spontaneous SANC firing by concomitant PDE3+PDE4 inhibition by 2-fold exceeds the summed increases in the spontaneous firing produced by inhibition of PDE3 (20%) and PDE4 (5%) alone, indicating that dual PDE3+PDE4 activation operates synergistically to suppress basal spontaneous firing of rabbit SANC. (3) L-type Ca2+ channels are essential for spontaneous firing of cardiac pacemaker cells: L-type Ca2+ current (ICa,L) generates AP upstroke in primary pacemaker cells and at the same time provides Ca2+ supply to replenish the SR Ca2+ store boosting LCR generation. Since basal PKA-dependent phosphorylation regulates L-type Ca2+ channels in SANC, PDE inhibition might affect ICa,L amplitude. Inhibition of PDE4 alone in rabbit SANC had no effect on ICa,L amplitude, while inhibition of PDE3 increased ICa,L by 60%. This effect was further amplified by dual PDE3+PDE4 inhibition which increased ICa,L amplitude in rabbit SANC by 100%. Therefore, dual PDE3+PDE4 activation regulates basal ICa,L amplitude in rabbit SANC in a synergistic manner, creating effect that markedly exceeds added effects of PDE3 or PDE4 activation alone. To study how PDE inhibition controls the SR Ca2+ refilling and LCR period, we compared kinetics of SR Ca2+ refilling in control and after PDE inhibition. Phosphorylation status of PLB at Ser16 site is a useful marker of PKA-dependent protein phosphorylation in SANC, since it relieves inhibition of SERCA, increasing SR pumping rate and shortening SR refilling time. Inhibition of either PDE3 or PDE4 alone produces only minor 20% increase in PLB phosphorylation at Ser16 site in SANC, but dual PDE3+PDE4 inhibition increased PLB phosphorylation by 110%, an effect comparable to that of IBMX. Therefore, basal PLB phosphorylation at Ser16 site in SANC is regulated by synergism of concurrent PDE3+PDE4 activation. This boost in basal PKA-dependent phosphorylation, produced by dual PDE3+PDE4 inhibition and reflected in PLB phosphorylation, likely affects multiple proteins involved in the regulation of cardiac pacemaker function. (4) Our studies established that PDE1A, PDE3A and PDE4B are localized beneath sarcolemma of rabbit SANC. Colocalization of PDE3A and PDE4B beneath sarcolemma of rabbit SANC suggests that these PDE subtypes could work together limiting Ca2+ influx through L-type Ca2+ channels in a synergistic manner. PDE3A is also detected in a striated pattern and colocalizes with the Z-line associated protein alpha-actinin in rabbit SANC. PDE3A is co-localized with SERCA, PLB and PDE4D in striated patterns inside SANC. Colocalization of PDE3A and PDE4D with SERCA and PLB suggests that these PDE isoforms could likely regulate cAMP-mediated PKA-dependent phosphorylation of major SR proteins in SANC. Indeed, physiologically relevant cAMP signals operate within the nanometer range, creating local cAMP nano-domains, while global cAMP is less involved in functional responses. Enhanced PDE activity as in rabbit SANC might facilitate local degradation of cAMP and create cAMP gradients forming nanodomain organization of cellular signaling cAMP gradients. Conclusion: The cAMP-degrading PDE1, PDE3, and PDE4 represent major PDE activities in rabbit SANC. Though inhibition of single PDE1-PDE4 only moderately increases spontaneous SANC firing, dual PDE3 + PDE4 inhibition produces a synergistic effect hastening the spontaneous SANC beating rate by 50%.