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) We discovered that PDE3A and PDE4B are colocalized beneath sarcolemma of rabbit SANC, which allow these PDE subtypes work in concert and limit Ca2+ influx through L-type Ca2+ channels in a synergistic manner. PDE3A was also detected in the striated pattern and colocalizes with PDE4D and Z-line protein alpha-actinin. At Z-lines PDE3A and PDE4D are colocalized with SERCA and PLB adjusting cAMP-mediated PKA-dependent phosphorylation of major the sarcoplasmic reticulum (SR) Ca2+ cycling proteins in SANC. Rapid degradation of cAMP by concurrent synergistic activation of PDE3+PDE4 provides functional barriers to cAMP diffusion, and therefore creates discrete intracellular pools of cAMP, functionally and spatially compartmentalizing signaling pathways within SANC. (4) 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 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 phospholamban (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. (5) In addition to PDEs cardiac pacemaker cells have additional protective mechanism which regulates the feed-forward basal cAMP-activated phosphorylation-dependent signaling via protein phosphatase 1 (PP1) which limits phosphorylation level of key proteins, involved in the regulation of Ca2+ clock mechanisms. Specifically, inhibition of PP1 significantly increased PLB phosphorylation (by 23-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites; increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; substantially increased sarcoplasmic reticulum Ca2+ load; augmented L-type Ca2+ current amplitude; augmented LCRs characteristics and decreased LCR period in intact and permeabilized SANC, and increased the basal spontaneous SANC firing rate.