Constitutive phosphodiesterase activity restricts spontaneous beating rate of cardiac pacemaker cells by suppressing
Tatiana M Vinogradova1, Syevda Sirenko, Alexey E Lyashkov
1Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr, Baltimore, MD 21224-6825, USA. vinogradovat@grc.nia.nih.gov
Abstract:
Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase A-dependent local subsarcolemmal ryanodine receptor Ca(2+) releases (LCRs). LCRs activated an inward Na(+)/Ca(2+) exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. 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 approximately 55%, indicating a high basal activity of PDEs in SANCs. A comparison of specific PDE1 to -5 inhibitors revealed that the specific PDE3 inhibitor, milrinone, accelerated spontaneous firing by approximately 47% (effects of others were minor) and increased amplitude of L-type Ca(2+) current (I(Ca,L)) by approximately 46%, indicating that PDE3 was the major constitutively active PDE in the basal state. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, ie, PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca(2+) release, Na(+)/Ca(2+) exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine, neither IBMX nor milrinone was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibition-induced augmentation of I(Ca,L) amplitude. 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 Ca(2+) release, and restrict the spontaneous SANC beating rate.
Insights
Phosphodiesterases (PDEs) in sinoatrial node cells (SANCs) normally limit heart rate by degrading cAMP. Inhibiting PDEs, particularly PDE3, increases cAMP, enhancing calcium release and accelerating spontaneous SANC beating. This reveals a novel PDE-dependent control mechanism for heart rate regulation.
Area of Science:
- Cardiovascular Physiology
- Cellular Electrophysiology
- Molecular Pharmacology
Background:
- Spontaneous beating of sinoatrial node cells (SANCs) is regulated by cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA).
- Local subsarcolemmal ryanodine receptor Ca(2+) releases (LCRs) activate Na(+)/Ca(2+) exchange current, influencing SANC firing rate.
- Elevated basal cAMP in SANCs suggested low phosphodiesterase (PDE) activity, but this was unexpectedly found to be high.
Purpose of the Study:
- To investigate the role of phosphodiesterases (PDEs) in regulating basal cAMP levels and spontaneous firing rate in rabbit SANCs.
- To identify the specific PDE isoforms responsible for basal PDE activity in SANCs.
- To elucidate the mechanism by which PDE activity influences SANC electrophysiology.
Main Methods:
- Utilized broad-spectrum and specific PDE inhibitors (e.g., IBMX, milrinone) on rabbit SANCs.
- Measured intracellular cAMP levels, phospholamban phosphorylation, and SANC firing rates.
- Assessed L-type Ca(2+) current (I(Ca,L)) and subsarcolemmal LCRs using electrophysiological techniques.
- Investigated the role of ryanodine receptors by using ryanodine to disable them.
Main Results:
- Total PDE inhibition (IBMX) increased cAMP levels 9-fold and SANC firing rate by ~55%.
- Specific PDE3 inhibition (milrinone) accelerated firing by ~47% and increased I(Ca,L) amplitude by ~46%, identifying PDE3 as the major basal PDE.
- PDE inhibition enhanced LCRs and accelerated diastolic depolarization, but this effect was abolished when ryanodine receptors were disabled.
Conclusions:
- Basal PDE activity in SANCs significantly limits cAMP levels and, consequently, PKA-mediated LCRs and SANC firing rate.
- PDE3 is the primary PDE isoform contributing to the regulation of basal SANC activity.
- PDE-dependent control of SANC beating rate is critically dependent on functional subsarcolemmal ryanodine receptor Ca(2+) release.
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