Spontaneous Action Potential Firing Rate Research Articles

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity Modulates Sinoatrial Nodal Pacemaker Cell Energetics

In rabbit sinoatrial node cells (SANC), high basal (i.e., without β-adrenergic receptor stimulation) Ca2+ activated adenylate cyclase (AC) via cAMP/PKA-CaMKII-dependent protein phosphorylation guarantees the occurrence of rhythmic intracellular Ca2+ release from sarcoplasmic-reticulum which drives spontaneous action potentials (APs). This high-throughput signaling consumes ATP. We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca2+ indirectly, not only consume ATP, but also regulate mitochondrial ATP production. A role for CaMKII in regulation of SANC ATP supply has not been explored. Based upon its involvement in the aforementioned phosphorylation signaling cascade we tested the hypotheses that, basal CaMKII activity not only regulates ATP consumption, but also ATP production. We superfused single, isolated rabbit SANC or SANC suspensions with CaMKII inhibitors (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)) and measured cytosolic Ca2+, cAMP, energetic indices and spontaneous AP firing rate. A partial reduction in basal CaMKII activity by KN-93 (0.5 μmol/L) or AIP (2 μmol/L) markedly slowed the kinetics of intracellular Ca2+ cycling and decreased the spontaneous AP firing rate. Further suppression of CaMKII activity (KN-93 3 μmol/L; AIP 10 μmol/L) to a level that eliminates all spontaneous APs, decreased the cAMP level and reduced O2 consumption and flavoprotein fluorescence. ATP was depleted,even though the ATP demand decreased. The structural inactive analog of KN-93, KN-92, neither decreased ATP demand (no effect on spontaneous AP firing rate) nor ATP turnover. Therefore,CaMKII signaling is required not only to drive normal automaticity in rabbit SANC, but is also tightly linked to SANC bioenergetics. Future studies are required to determine whether this link between CaMKII signaling and mitochondrial energetics occurs indirectly, via changes in Ca2+ release that affects activation of Ca2+-AC-cAMP/PKA signaling, or directly, via phosphorylation of mitochondrial proteins.

How Ca2+-Activated, cAMP/PKA-Dependent Phosphorylation Signaling Mediates Pacemaker Cell Activity: Experimental and In Silico Biochemical and Biophysics Perspectives

Ca2+-activated, adenylate cyclase (AC)-cAMP/PKA-dependent phosphorylation of both surface membrane electrogenic proteins of (“Membrane-clock”), and of intracellular proteins that generate rhythmic Ca2+ oscillations (“Ca2+-clock”), regulate the periodicity of each clock, and couple the two-clocks to regulate sinoatrial node cells (SANCs) normal automaticity.We developed a novel numerical model to simulate the coupling of SANC Ca2+-AC-cAMP/PKA signaling to functions of surface membrane and Ca2+ cycling molecules. The model incorporates experimentally measured a term for Ca2+-dependent AC-activity, and when Ca2+ changes predicts resulting changes in downstream cAMP/PKA phosphorylation-dependent signaling that produce changes in ion channels conductance and intracellular Ca2+ kinetics that ultimately change the spontaneous action potential (AP) firing rate.Model predictions of Ca2+-dependent changes in the cAMP/PKA phosphorylation cascade, of spontaneous AP firing rate and of the stoichiometry-relationships between cAMP, phospholamban phosphorylation and AP firing rate (line in the figure) faithfully reproduced the experimentally measured variables and their stoichiometry (points in the figure).The simulations of this novel integrative model of biochemical and biophysical signaling within the coupled-clock model further support the importance of high throughput signaling via Ca2+-AC-cAMP-PKA phosphorylation cascade in normal SANC automaticity.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Mitochondrial-SR Ca 2+ Cycling Modulates Normal Automaticity of Rabbit Cardiac Sinoatrial Nodal Pacemaker Cells

A coupled-clock system within sinoatrial node cells (SANC) confers robustness and regulates their normal automaticity: basal cAMP-mediated, protein kinase A-dependent phosphorylation of Ca2+ cycling-proteins enables sarcoplasmic reticulum (SR) to generate spontaneous rhythmic Local, subsarcolemmal Ca2+Releases (LCRs) (“Ca2+ clock”). LCRs activate an inward Na+-Ca2+ exchange current that accelerates the diastolic depolarization promoting the ensemble of surface membrane ion channels (“membrane clock”) to generate the next action potential (AP). Intracellular Ca2+ enters mitochondria through the mitochondrial uniporter and is extruded by the mitochondrial Na+-Ca2+ exchanger. We hypothesized that mitochondrial Ca2+ cycling is coupled to SANC-clocks via its impact on the intracellular Ca2+ cycling. Specific inhibition of Ca influx into and efflux from mitochondria in intact single isolated SANC was effected by Ru360 and CGP-37157, respectively. Changes in mitochondrial Ca2+ content (Cam) were indexed by selective quenching of the fluorescent Ca2+ probe, Indo-1 in the cytosol by Mn2+. Ru360 decreased Cam to 80±8% control and increased the spontaneous SANC AP firing rate to 111±1%. Conversely, CGP-37157 increased Cam to 119±7% control and reduced the spontaneous AP firing rate to 89±2%. Blocking Ca influx into mitochondria increased the average LCR size (measured via confocal linescans images of fluo-4) from 4.2±0.1 to 6.1±0.2µm and reduced the normal LCR period from 317±5 to 274±6ms. In contrast, inhibition of Ca efflux from mitochondria reduced LCR size to 3.6±0.1µm and increased LCR period to 389±7ms. Changes in LCR period by specific inhibition of Ca2+ influx or efflux into and from the mitochondria from 274±6 to 389±7ms predicted (R2=0.84) the concomitant changes in the spontaneous SANC AP cycle length from 349±5 to 462±6ms. We conclude that Ca2+ cycling into and out of mitochondria interact with the SANC coupled-clock system to modulate normal automaticity.

A novel quantitative explanation for the autonomic modulation of cardiac pacemaker cell automaticity via a dynamic system of sarcolemmal and intracellular proteins

Classical numerical models have attributed the regulation of normal cardiac automaticity in sinoatrial node cells (SANCs) largely to G protein-coupled receptor (GPCR) modulation of sarcolemmal ion currents. More recent experimental evidence, however, has indicated that GPCR modulation of SANCs automaticity involves spontaneous, rhythmic, local Ca(2+) releases (LCRs) from the sarcoplasmic reticulum (SR). We explored the GPCR rate modulation of SANCs using a unique and novel numerical model of SANCs in which Ca(2+)-release characteristics are graded by variations in the SR Ca(2+) pumping capability, mimicking the modulation by phospholamban regulated by cAMP-mediated, PKA-activated signaling. The model faithfully predicted the entire range of physiological chronotropic modulation of SANCs by the activation of beta-adrenergic receptors or cholinergic receptors only when experimentally documented changes of sarcolemmal ion channels are combined with a simultaneous increase/decrease in SR Ca(2+) pumping capability. The novel numerical mechanism of GPCR rate modulation is based on numerous complex synergistic interactions between sarcolemmal and intracellular processes via membrane voltage and Ca(2+). Major interactions include changes of diastolic Na(+)/Ca(2+) exchanger current that couple earlier/later diastolic Ca(2+) releases (predicting the experimentally defined LCR period shift) of increased/decreased amplitude (predicting changes in LCR signal mass, i.e., the product of LCR spatial size, amplitude, and number per cycle) to the diastolic depolarization and ultimately to the spontaneous action potential firing rate. Concomitantly, larger/smaller and more/less frequent activation of L-type Ca(2+) current shifts the cellular Ca(2+) balance to support the respective Ca(2+) cycling changes. In conclusion, our model simulations corroborate recent experimental results in rabbit SANCs pointing to a new paradigm for GPCR heart rate modulation by a complex system of dynamically coupled sarcolemmal and intracellular proteins.

Cardiac Glycoside Chronotropic and Arrhythmogenic Effects in Sinoatrial Nodal Pacemaker Cells (SANC) Occur Along a Continuum of Electrochemical Gradients of Na+ (ENa) and Ca2+ (ECa)

Cardiac glycosides reduce ENa and ECa (due to an increase of Na+i via Na-K pump inhibition, and of Ca2+i, due to a secondary reduction in Ca efflux for Na influx via NaCa exchange). Here we show that exposure of single rabbit SANC to the cardiac glycoside, digoxigenin (10-20μM) results in a continuum of time-dependent effects. Within 30s to 1 min, the rate of rhythmic spontaneous action potentials (AP) increases by 20% (n=3) and this is associated with an earlier occurrence (reduced period) of local sub-membrane Ca2+ releases (LCR's) during diastolic depolarization, detected by confocal Ca2+ imaging. Approximately 1-3 minutes following AP rate acceleration, LCR period lengthens by 40%, accompanied by a similar reduction in the rhythmic AP rate. The changes in LCR period during the biphsic changes in rhythmic AP firing rate increase are highly correlated with the changes in AP cycle length (R2=0.98). A progressive increase in the steady level of diastolic Ca2+ beneath the surface membrane then ensues usually within 4 to 6 additional minutes, LCR's became undetectable, and dysrhythmic and chaotic AP firing occurs. Numerical model simulations (Maltsev-Lakatta model, AJP 2009) in which Nai was increased progressively 5-15mM during glycoside exposure reproduced the experimental results. That rate and rhythm regulation of SANC AP firing during cardiac glycoside exposure occurs along a continuum of ENa/ECa is in agreement with repeated observations over the last decade, showing that the SANC spontaneous AP firing rate is critically dependent on the timing of acute changes in sub-membrane ECa during DD caused by LCR occurrence (LCR period) that accelerates DD by activation of an inward Na/Ca exchange current.

What keeps us ticking: a funny current, a calcium clock, or both?

The processes underlying the initiation of the heartbeat, whether due to intracellular metabolism or surface membrane events, have always been a major focus of cardiac research. About 50 years ago, a pioneering work initiated by Silvio Weidmann and others applied the Hodgkin–Huxley formalism of membrane excitation to interpret the cardiac electrical activity, including the pacemaker depolarization (see D. Noble, 1979. The initiation of the heartbeat, Clarendon Press). The underlying idea was that voltageand time-dependent gating of various surface membrane channels not only generated the cardiac pacemaker action potential (AP), but also controlled the spontaneous depolarization between AP's and thus determinedwhen thenextAPwouldoccur. According to this description, the ensemble of surface membrane ion channels works as a clock that regulates the rate and rhythm of spontaneous AP firing, otherwise known as normal automaticity. A formidable research effort then concentrated in attempting to target which of the surface membrane ion channels had an important role in controlling the spontaneous diastolic depolarization (DD). Originally, a major role was attributed to the “IK-decay theory”. This was strongly influenced by the previous Hodgkin–Huxley model of nerve AP, which described the slow depolarization following a nerve AP as due to the decay of a K current. This model of pacemaker depolarization lasted some 20 years, until it was turned upside-down by a full re-interpretation based on the discovery of the If current. Other ionic currents gated by membrane depolarization, i.e. ICaL, ICaT, IST, non-gated and non-specific background leak currents, and also a current generated by the Na–Ca exchange (NCX) carrier, were also proposed to be involved in pacemaking. Based on a wealth of experimental evidence, If is today considered as the most important ion channel involved in the rate regulation of cardiac pacemaker cells, and is sometimes referred to as “the pacemaker channel.” Several studies, some of which recent, have also shown that in addition to voltage and time, surface membrane electrogenic molecules are strongly modulated by Ca and phosphorylation. The studies of a sub-group of pacemaker cell researchers focusing Journal of Molecular and Cellular Cardiology 47 (2009) 157–170

Circadian Control of Membrane Excitability in Drosophila melanogaster Lateral Ventral Clock Neurons

Drosophila circadian rhythms are controlled by a neural circuit containing approximately 150 clock neurons. Although much is known about mechanisms of autonomous cellular oscillation, the connection between cellular oscillation and functional outputs that control physiological and behavioral rhythms is poorly understood. To address this issue, we performed whole-cell patch-clamp recordings on lateral ventral clock neurons (LN(v)s), including large (lLN(v)s) and small LN(v)s (sLN(v)s), in situ in adult fly whole-brain explants. We found two distinct sizes of action potentials (APs) in >50% of lLN(v)s that fire APs spontaneously, and determined that large APs originate in the ipsilateral optic lobe and small APs in the contralateral. lLN(v) resting membrane potential (RMP), spontaneous AP firing rate, and membrane resistance are cyclically regulated as a function of time of day in 12 h light/dark conditions (LD). lLN(v) RMP becomes more hyperpolarized as time progresses from dawn to dusk with a concomitant decrease in spontaneous AP firing rate and membrane resistance. From dusk to dawn, lLN(v) RMP becomes more depolarized, with spontaneous AP firing rate and membrane resistance remaining stable. In contrast, circadian defective per(0) null mutant lLN(v) membrane excitability is nearly constant in LD. Over 24 h in constant darkness (DD), wild-type lLN(v) membrane excitability is not cyclically regulated, although RMP gradually becomes slightly more depolarized. sLN(v) RMP is most depolarized around lights-on, with substantial variability centered around lights-off in LD. Our results indicate that LN(v) membrane excitability encodes time of day via a circadian clock-dependent mechanism, and likely plays a critical role in regulating Drosophila circadian behavior.

Circadian- and Light-Dependent Regulation of Resting Membrane Potential and Spontaneous Action Potential Firing of Drosophila Circadian Pacemaker Neurons

The ventral lateral neurons (LNvs) of adult Drosophila brain express oscillating clock proteins and regulate circadian behavior. Whole cell current-clamp recordings of large LNvs in freshly dissected Drosophila whole brain preparations reveal two spontaneous activity patterns that correlate with two underlying patterns of oscillating membrane potential: tonic and burst firing of sodium-dependent action potentials. Resting membrane potential and spontaneous action potential firing are rapidly and reversibly regulated by acute changes in light intensity. The LNv electrophysiological light response is attenuated, but not abolished, in cry(b) mutant flies hypomorphic for the cell-autonomous light-sensing protein CRYPTOCHROME. The electrical activity of the large LNv is circadian regulated, as shown by significantly higher resting membrane potential and frequency of spontaneous action potential firing rate and burst firing pattern during circadian subjective day relative to subjective night. The circadian regulation of membrane potential, spontaneous action potential firing frequency, and pattern of Drosophila large LNvs closely resemble mammalian circadian neuron electrical characteristics, suggesting a general evolutionary conservation of both physiological and molecular oscillator mechanisms in pacemaker neurons.

Presynaptic modulation of cortical synaptic activity by calcineurin.

Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism.

Gentamicin causes the fast depression of action potential and contraction in cultured cardiocytes

The short-term cardiotoxic effects of gentamicin were studied on electrical and mechanical activities in cultures of ventricular confluent cardiocytes of rats. Application of gentamicin to the bath lowered the spontaneous action potential firing rate which was restored when the external calcium was elevated. Fast local application and withdrawal of drugs to non-pacemaker cells allowed effect/dose curves to be made. Such application of 200 nmol gentamicin (approximated concentration: 0.8 × 10 −3-8.0 × 10 −3 M) lowered the contraction amplitude by 50% and the action potential overshoot by 20% while the action potential duration and maximum diastolic potential remained unchanged. Gentamicin withdrawal was followed by a rapid (half time = 5 s) and complete restoration of contraction. Verapamil, Mn 2+, EDTA and La 3+ also diminished contraction and overshoot but their effects were reversed 28, 25, 6 and 1.5 times more slowly, respectively. Gentamicin, like La 3+, could act extracellularly, probably by competing with calcium ions for some external membrane site.

Neuronal membrane sensitivity to a salmon calcitonin analogue with negligible ability to lower serum calcium

The effects of [Gly 8-des-Leu 16- d-Arg 24]-salmon calcitonin (CTA) on spontaneous extracellular activity and the passive membrane properties of rat forebrain neurons were studied in vivo and in vitro. This analogue had negligible ability to lower serum calcium relative to salmon calcitonin (SCT), however, the pattern of neuronal membrane sensitivity to CTA was similar to that produced by SCT and calcitonin gene-related peptide. Depression of extracellularly recorded spontaneous action potential firing rate was the predominant response of neurons to microiontophoretic application of each peptide in vivo. Intracellular recordings from septal neurons suggest that the extracellularly observed inhibitory effects may be due to changes in membrane potential. We conclude that different structure-activity relationships may apply to the pharmacological effects of calcitonin in brain and its serum calcium-lowering action.

Actions of phencyclidine on rat locus coeruleus neurones in vitro

Intracellular recordings were made from neurones in the rat locus coeruleus in a brain slice maintained in vitro. Phencyclidine and related psychotomimetic drugs, applied in known concentrations in the fluid bathing the slice, depressed responses to N-methyl- D-aspartic acid noradrenaline (in the presence of the uptake inhibitor desmethylimipramine) and [ D-Ala 2,MePhe 4,Gly-ol 5]enkephalin and also prolonged the action potential. The sensitivities of these responses to depression by phencyclidine was N-methyl- D-aspartic acid (IC 50 0.4 μM) > noradrenaline (IC 50 3.9 μM) > [ D-Ala 2,MePhe 4,Gly-ol 5]enkephalin (IC 50 8.5μM) > prolongation of the action potential (41% increase by 30 μM). Stereoselectivity was observed only in the depression of responses to N-methyl- D-aspartic acid where (+)-l-(l-phenylcyclohexyl)-3-methyl piperidine was 3.3-fold more potent in suppressing TV-methylD-aspartic acid depolarisations than its (—) isomer. The responses to JV-methyl-D-aspartic acid were also depressed by the structurally unrelated psychotomimetic (+)-JV-allyK/V-normetazocine (IC50 0.9 /jM). All of the effects of the psychotomimetic drugs examined were slow in onset and difficult to reverse following washout. No effect of phencyclidine (0.03–100 ??) or related drugs was observed on membrane potential, input resistance or spontaneous action potential firing rate of locus coeruleus neurones. The depression of responses to JV-methyl-D-aspartic acid by phencyclidine was the most potent and the only stereoselective effect of those studied. The importance of this effect and of those not showing stereoselectivity in relation to the phencyclidine behavioural syndrome is discussed.