Alexey E. Lyashkov

High Basal Protein Kinase A–Dependent Phosphorylation Drives Rhythmic Internal Ca2+ Store Oscillations and Spontaneous Beating of Cardiac Pacemaker Cells

Local, rhythmic, subsarcolemmal Ca2+ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na+-Ca2+ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of β-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca2+ cycling and beating rate is also present in vivo, as the regulation of beating rate by local β-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, phospholamban and L-type Ca2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na+-Ca2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.

Rhythmic Ryanodine Receptor Ca2+ Releases During Diastolic Depolarization of Sinoatrial Pacemaker Cells Do Not Require Membrane Depolarization

Abstract— Localized, subsarcolemmal Ca2+ release (LCR) via ryanodine receptors (RyRs) during diastolic depolarization of sinoatrial nodal cells augments the terminal depolarization rate. We determined whether LCRs in rabbit sinoatrial nodal cells require the concurrent membrane depolarization, or are intrinsically rhythmic, and whether rhythmicity is linked to the spontaneous cycle length. Confocal linescan images revealed persistent LCRs both in saponin-permeabilized cells and in spontaneously beating cells acutely voltage-clamped at the maximum diastolic potential. During the initial stage of voltage clamp, the LCR spatiotemporal characteristics did not differ from those in spontaneously beating cells, or in permeabilized cells bathed in 150 nmol/L Ca2+. The period of persistent rhythmic LCRs during voltage clamp was slightly less than the spontaneous cycle length before voltage clamp. In spontaneously beating cells, in both transient and steady states, LCR period was highly correlated with the spontaneous cycle length; and regardless of the cycle length, LCRs occurred predominantly at a constant time, ie, 80% to 90% of the cycle length. Numerical model simulations incorporating LCRs reproduce the experimental results. We conclude that diastolic LCRs reflect rhythmic intracellular Ca2+ cycling that does not require the concomitant membrane depolarization, and that LCR periodicity is closely linked to the spontaneous cycle length. Thus, the biological clock of sinoatrial nodal pacemaker cells, like that of many other rhythmic functions occurring throughout nature, involves an intracellular Ca2+ rhythm.

Biophysical Characterization of the Underappreciated and Important Relationship Between Heart Rate Variability and Heart Rate

Heart rate (HR) variability (HRV; beat-to-beat changes in the R-wave to R-wave interval) has attracted considerable attention during the past 30+ years (PubMed currently lists >17 000 publications). Clinically, a decrease in HRV is correlated to higher morbidity and mortality in diverse conditions, from heart disease to fetal distress. It is usually attributed to fluctuation in cardiac autonomic nerve activity. We calculated HRV parameters from a variety of cardiac preparations (including humans, living animals, Langendorff-perfused heart, and single sinoatrial nodal cell) in diverse species, combining this with data from previously published articles. We show that regardless of conditions, there is a universal exponential decay-like relationship between HRV and HR. Using 2 biophysical models, we develop a theory for this and confirm that HRV is primarily dependent on HR and cannot be used in any simple way to assess autonomic nerve activity to the heart. We suggest that the correlation between a change in HRV and altered morbidity and mortality is substantially attributable to the concurrent change in HR. This calls for re-evaluation of the findings from many articles that have not adjusted properly or at all for HR differences when comparing HRV in multiple circumstances.

Constitutive Phosphodiesterase Activity Restricts Spontaneous Beating Rate of Cardiac Pacemaker Cells by Suppressing Local Ca2+ Releases

Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase A–dependent local subsarcolemmal ryanodine receptor Ca2+ releases (LCRs). LCRs activated an inward Na+/Ca2+ 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 ≈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 ≈47% (effects of others were minor) and increased amplitude of L-type Ca2+ current (ICa,L) by ≈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 Ca2+ release, Na+/Ca2+ 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 ICa,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 Ca2+ release, and restrict the spontaneous SANC beating rate.

Membrane Potential Fluctuations Resulting From Submembrane Ca2+ Releases in Rabbit Sinoatrial Nodal Cells Impart an Exponential Phase to the Late Diastolic Depolarization That Controls Their Chronotropic State

Stochastic but roughly periodic LCRs (Local subsarcolemmal ryanodine receptor–mediated Ca2+Releases) during the late phase of diastolic depolarization (DD) in rabbit sinoatrial nodal pacemaker cells (SANCs) generate an inward current (INCX) via the Na+/Ca2+ exchanger. Although LCR characteristics have been correlated with spontaneous beating, the specific link between LCR characteristics and SANC spontaneous beating rate, ie, impact of LCRs on the fine structure of the DD, have not been explicitly defined. Here we determined how LCRs and resultant INCX impact on the DD fine structure to control the spontaneous SANC firing rate. Membrane potential (Vm) recordings combined with confocal Ca2+ measurements showed that LCRs impart a nonlinear, exponentially rising phase to the DD later part, which exhibited beat-to-beat Vm fluctuations with an amplitude of approximately 2 mV. Maneuvers that altered LCR timing or amplitude of the nonlinear DD (ryanodine, BAPTA, nifedipine or isoproterenol) produced corresponding changes in Vm fluctuations during the nonlinear DD component, and the Vm fluctuation response evoked by these maneuvers was tightly correlated with the concurrent changes in spontaneous beating rate induced by these perturbations. Numerical modeling, using measured LCR characteristics under these perturbations, predicted a family of local INCX that reproduced Vm fluctuations measured experimentally and determined the onset and amplitude of the nonlinear DD component and the beating rate. Thus, beat-to-beat Vm fluctuations during late DD phase reflect the underlying LCR/INCX events, and the ensemble of these events forms the nonlinear DD component that ultimately controls the SANC chronotropic state in tight cooperation with surface membrane ion channels.

Ca2+-stimulated basal adenylyl cyclase activity localization in membrane lipid microdomains of cardiac sinoatrial nodal pacemaker cells

Spontaneous, rhythmic subsarcolemmal local Ca2+ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca2+ releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca2+ and cAMP/PKA regulatory components of automaticity are coupled via Ca2+ activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca2+-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca2+ over the entire physiological Ca2+ range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca2+ cycling proteins drive sarcoplasmic reticulum Ca2+ releases, which, in turn, activate ACs. This feed forward “fail safe” system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

Rhythmic Ca2+ Oscillations Drive Sinoatrial Nodal Cell Pacemaker Function to Make the Heart Tick

Abstract: Excitation‐induced Ca2+ cycling into and out of the cytosol via the sarcoplasmic reticulum (SR) Ca2+ pump, ryanodine receptor (RyR) and Na+‐Ca2+ exchanger (NCX) proteins, and modulation of this Ca2+cycling by β‐adrenergic receptor (β‐AR) stimulation, governs the strength of ventricular myocyte contraction and the cardiac contractile reserve. Recent evidence indicates that heart rate modulation and chronotropic reserve via β‐ARs also involve intracellular Ca2+ cycling by these very same molecules. Specifically, sinoatrial nodal pacemaker cells (SANC), even in the absence of surface membrane depolarization, generate localized rhythmic, submembrane Ca2+ oscillations via SR Ca2+ pumping‐RyR Ca2+ release. During spontaneous SANC beating, these rhythmic, spontaneous Ca2+ oscillations are interrupted by the occurrence of an action potential (AP), which activates L‐type Ca2+ channels to trigger SR Ca2+ release, unloading the SR Ca2+ content and inactivating RyRs. During the later part of the subsequent diastolic depolarization (DD), when Ca2+ pumped back into the SR sufficiently replenishes the SR Ca2+ content, and Ca2+‐dependent RyR inactivation wanes, the spontaneous release of Ca2+ via RyRs again begins to occur. The local increase in submembrane [Ca2+] generates an inward current via NCX, enhancing the DD slope, modulating the occurrence of the next AP, and thus the beating rate. β‐AR stimulation increases the submembrane Ca2+ oscillation amplitude and reduces the period (the time from the prior AP triggered SR Ca2+ release to the onset of the local Ca2+ release during the subsequent DD). This increased amplitude and phase shift causes the NCX current to occur at earlier times following a prior beat, promoting the earlier arrival of the next beat and thus an increase in the spontaneous firing rate. Ca2+ cycling via the SR Ca2+ pump, RyR and NCX, and its modulation by β‐AR stimulation is, therefore, a general mechanism of cardiac chronotropy and inotropy.

The Integration of Spontaneous Intracellular Ca2+ Cycling and Surface Membrane Ion Channel Activation Entrains Normal Automaticity in Cells of the Heart's Pacemaker

Abstract:  Although the ensemble of voltage‐ and time‐dependent rhythms of surface membrane ion channels, the membrane “Clock”, is the immediate cause of a sinoatrial nodal cell (SANC) action potential (AP), it does not necessarily follow that this ion channel ensemble is the formal cause of spontaneous, rhythmic APs. SANC also generates intracellular oscillatory spontaneous Ca2+ releases that ignite excitation (SCaRIE) of the surface membrane via Na+/Ca2+ exchanger activation. The idea that a rhythmic intracellular Ca2+ Clock might keep time for normal automaticity of SANC, however, has not been assimilated into mainstream pacemaker dogma. Recent experimental evidence, derived from simultaneous, confocal imaging of submembrane Ca2+ and membrane potential of SANC, and supported by numerical modeling, indicates that normal automaticity of SANC is entrained and stabilized by the tight integration of the SR Ca2+ Clock that generates rhythmic SCaRIE, and the surface membrane Clock that responds to SCaRIE to immediately produce APs of an adequate shape. Thus, tightly controlled, rhythmic SCaRIE does not merely fine tune SANC AP firing, but is the formal cause of the basal and reserve rhythms, insuring pacemaker stability by rhythmically integrating multiple Ca2+‐dependent functions, and effects normal automaticity by rhythmic ignition of the surface membrane Clock.

Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca2+ cycling and IKACh

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a G(i)-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current (I(f)) or L-type Ca(2+) currents (I(Ca,L)), respectively; and 2) direct G(i)-coupled activation of ACh-activated potassium current (I(KACh)). More recent studies, however, have indicated that Ca(2+) cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca(2+) clock) generates rhythmic, spontaneous local Ca(2+) releases (LCR) that are AC-PKA dependent. LCRs activate Na(+)-Ca(2+) exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was approximately 10 nM, half maximal inhibition (IC(50)) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. G(i) inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that I(f) blockade did not affect BRR at any [CCh] and that I(KACh) activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC(50), CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by approximately 50%. The dose response of BRR to CCh in the presence of I(KACh) blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC(50), CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC(50) CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca(2+) cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on G(i) activation and the extent of G(i) coupling to Ca(2+) cycling via PKA signaling or to I(KACh): at low [CCh], I(KACh) activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca(2+) signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca(2+) signaling and I(KACh) activation underlies a more pronounced BRR.

Crosstalk between Mitochondrial and Sarcoplasmic Reticulum Ca2+ Cycling Modulates Cardiac Pacemaker Cell Automaticity

Background Mitochondria dynamically buffer cytosolic Ca2+ in cardiac ventricular cells and this affects the Ca2+ load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca2+ releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na+-Ca2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP). Objective To determine if mitochondrial Ca2+ (Ca2+ m), cytosolic Ca2+ (Ca2+ c)-SR-Ca2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity. Results Inhibition of mitochondrial Ca2+ influx into (Ru360) or Ca2+ efflux from (CGP-37157) decreased [Ca2+]m to 80±8% control or increased [Ca2+]m to 119±7% control, respectively. Concurrent with inhibition of mitochondrial Ca2+ influx or efflux, the SR Ca2+ load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca2+ signal were highly correlated with the change in the SR Ca2+ load (r2 = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111±1% control; CGP-37157, 89±2% control) in response to changes in [Ca2+]m were predicted by concurrent changes in LCR period (r2 = 0.84). Conclusion A change in SANC Ca2+ m flux translates into a change in the AP firing rate by effecting changes in Ca2+ c and SR Ca2+ loading, which affects the characteristics of spontaneous SR Ca2+ release.