Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca ...cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I
). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I
activation threshold. During the ignition phase, I
-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I
, as diastolic LCRs begin to occur below and continue within the voltage range of I
activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (∼0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.
The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca
and membrane ion current clocks driven by Ca
-calmodulin-activated ...adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A
, A
) that couple to G
protein-coupled receptors, reducing spontaneous AP firing rate
G
signaling that activates I
. Adenosine also inhibits adenylyl cyclase activity
G
signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to I
activation, adenosine impacts also Ca
G
signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca
and membrane clock coupling. To this end, we measured Ca
and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%,
= 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (
= 10) measured
membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (
= 40) measured
Ca
(assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca
releases (LCRs) (Ca
-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100 µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate G
and I
and to activate G
, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.
Cardiac pacemaker cells, including cells of the sinoatrial node, are heterogeneous in size, morphology, and electrophysiological characteristics. The exact extent to which these cells differ ...electrophysiologically is unclear yet is critical to understanding their functioning. We examined major ionic currents in individual intercaval pacemaker cells (IPCs) sampled from the paracristal, intercaval region (including the sinoatrial node) that were spontaneously beating after enzymatic isolation from rabbit hearts. The beating rate was measured at baseline and after inhibition of the Ca
pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured the density of funny current ( I
), delayed rectifier K
current ( I
) (a surrogate of repolarization capacity), and L-type Ca
current ( I
) using whole cell patch clamp
The ionic current densities varied to a greater extent than previously appreciated, with some IPCs demonstrating very small or zero I
. The density of none of the currents was correlated with cell size, while I
and I
densities were related to baseline beating rates. I
density was correlated with I
density but not with that of I
. Inhibition of Ca
cycling had a greater beating rate slowing effect in IPCs with lower I
densities. Our numerical model simulation indicated that 1) IPCs with small (or zero) I
or small I
can operate via a major contribution of Ca
clock, 2) I
-Ca
-clock interplay could be important for robust pacemaking function, and 3) coupled I
- I
function could regulate maximum diastolic potential. Thus, we have demonstrated marked electrophysiological heterogeneity of IPCs. This heterogeneity is manifested in basal beating rate and response to interference of Ca
cycling, which is linked to I
. NEW & NOTEWORTHY In the present study, a hitherto unrecognized range of heterogeneity of ion currents in pacemaker cells from the intercaval region is demonstrated. Relationships between basal beating rate and L-type Ca
current and funny current ( I
) density are uncovered, along with a positive relationship between I
and delayed rectifier K
current. Links are shown between the response to Ca
cycling blockade and I
density.
Uptake and release calcium from the sarcoplasmic reticulum (SR) (dubbed "calcium clock"), in the form of spontaneous, rhythmic, local diastolic calcium releases (LCRs), together with ...voltage-sensitive ion channels (membrane clock) form a coupled system that regulates the action potential (AP) firing rate. LCRs activate Sodium/Calcium exchanger (NCX) that accelerates diastolic depolarization and thus participating in regulation of the time at which the next AP will occur. Previous studies in rabbit SA node cells (SANC) demonstrated that the basal AP cycle length (APCL) is tightly coupled to the basal LCR period (time from the prior AP-induced Ca2+ transient to the diastolic LCR occurrence), and that this coupling is further modulated by autonomic receptor stimulation. Although spontaneous LCRs during diastolic depolarization have been reported in SANC of various species (rabbit, cat, mouse, toad), prior studies have failed to detect LCRs in spontaneously beating SANC of guinea-pig, a species that has been traditionally used in studies of cardiac pacemaker cell function. We performed a detailed investigation of whether guinea-pig SANC generate LCRs and whether they play a similar key role in regulation of the AP firing rate. We used two different approaches, 2D high-speed camera and classical line-scan confocal imaging. Positioning the scan-line beneath sarcolemma, parallel to the long axis of the cell, we found that rhythmically beating guinea-pig SANC do, indeed, generate spontaneous, diastolic LCRs beneath the surface membrane. The average key LCR characteristics measured in confocal images in guinea-pig SANC were comparable to rabbit SANC, both in the basal state and in the presence of β-adrenergic receptor stimulation. Moreover, the relationship between the LCR period and APCL was subtended by the same linear function. Thus, LCRs in guinea-pig SANC contribute to the diastolic depolarization and APCL regulation. Our findings indicate that coupled-clock system regulation of APCL is a general, species-independent, mechanism of pacemaker cell normal automaticity. Lack of LCRs in prior studies is likely explained by technical issues, as individual LCRs are small stochastic events occurring mainly near the cell border.
Background:
Late Na+ current (INaL) in human and dog hearts has been implicated in abnormal repolarization associated with heart failure (HF). HF slows inactivation gating of late Na+ channels, which ...could contribute to these abnormalities.
Aims:
To test how altered gating affects INaL time course, Na+ influx, and action potential (AP) repolarization.
Methods:
INaL and AP were measured by patch clamp in left ventricular cardiomyocytes from normal and failing hearts of humans and dogs. Canine HF was induced by coronary microembolization.
Results:
INaL decay was slower and INaL density was greater in failing hearts than in normal hearts at 24 °C (human hearts: τ659±16 vs. 529±21ms; n=16 and 4 hearts, respectively; mean±SEM; p<0.002; dog hearts: 561±13 vs. 420±17ms; and 0.307±0.014 vs. 0.235±0.019pA/pF; n=25 and 14 hearts, respectively; p<0.005) and at 37°C this difference tended to increase. These INaL changes resulted in much greater (53.6%) total Na+ influx in failing cardiomyocytes. INaL was sensitive to cadmium but not to cyanide and exhibited low sensitivity to saxitoxin (IC50=62nM) or tetrodotoxin (IC50=1.2 μM), tested in dogs. A 50% INaL inhibition by toxins or passing current opposite to INaL, decreased beat-to-beat AP variability and eliminated early afterdepolarizations in failing cardiomyocytes.
Conclusions:
Chronic HF leads to larger and slower INaL generated mainly by the cardiac-type Na+ channel isoform, contributing to larger Na+ influx and AP duration variability. Interventions designed to reduce/normalize INaL represent a potential cardioprotective mechanism in HF via reduction of related Na+ and Ca2+ overload and improvement of repolarization.
Beneficial clinical bradycardic effects of ivabradine (IVA) have been interpreted solely on the basis of If inhibition, because IVA specifically inhibits If in sinoatrial nodal pacemaker cells ...(SANC). However, it has been recently hypothesized that SANC normal automaticity is regulated by crosstalk between an “M clock,” the ensemble of surface membrane ion channels, and a “Ca2+ clock,” the sarcoplasmic reticulum (SR). We tested the hypothesis that crosstalk between the two clocks regulates SANC automaticity, and that indirect suppression of the Ca2+ clock further contributes to IVA-induced bradycardia. IVA (3μM) not only reduced If amplitude by 45±6% in isolated rabbit SANC, but the IVA-induced slowing of the action potential (AP) firing rate was accompanied by reduced SR Ca2+ load, slowed intracellular Ca2+ cycling kinetics, and prolonged the period of spontaneous local Ca2+ releases (LCRs) occurring during diastolic depolarization. Direct and specific inhibition of SERCA2 by cyclopiazonic acid (CPA) had effects similar to IVA on LCR period and AP cycle length. Specifically, the LCR period and AP cycle length shift toward longer times almost equally by either direct perturbations of the M clock (IVA) or the Ca2+ clock (CPA), indicating that the LCR period reports the crosstalk between the clocks. Our numerical model simulations predict that entrainment between the two clocks that involves a reduction in INCX during diastolic depolarization is required to explain the experimentally AP firing rate reduction by IVA. In summary, our study provides new evidence that a coupled-clock system regulates normal cardiac pacemaker cell automaticity. Thus, IVA-induced bradycardia includes a suppression of both clocks within this system.
•Bradycardic effects of ivabradine include reduction in intracellular Ca2+ cycling.•Local Ca2+ release period reports the crosstalk between membrane and Ca2+ clocks.•Definitive evidence for a coupled-clock function in heart pacemaker cells.•Possible clinical mechanism for ivabradine induced bradycardia.
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.
Action potential (AP) firing rate and rhythm of sinoatrial nodal cells (SANC) are controlled by synergy between intracellular rhythmic local Ca
releases (LCRs) ("Ca
clock") and sarcolemmal ...electrogenic mechanisms ("membrane clock"). However, some SANC do not fire APs (dormant SANC). Prior studies have shown that β-adrenoceptor stimulation can restore AP firing in these cells. Here we tested whether this relates to improvement of synchronization of clock coupling. We characterized membrane potential, ion currents, Ca
dynamics, and phospholamban (PLB) phosphorylation, regulating Ca
pump in enzymatically isolated single guinea pig SANC prior to, during, and following β-adrenoceptor stimulation (isoproterenol) or application of cell-permeant cAMP (CPT-cAMP). Phosphorylation of PLB (Serine 16) was quantified in the same cells following Ca
measurement. In dormant SANC LCRs were small and disorganized at baseline, membrane potential was depolarized (-38 ± 1 mV,
= 46), and I
, I
, and I
densities were smaller vs SANC firing APs. β-adrenoceptor stimulation or application of CPT-cAMP led to
spontaneous AP generation in 44 and 46% of dormant SANC, respectively. The initial response was an increase in size, rhythmicity and synchronization of LCRs, paralleled with membrane hyperpolarization and small amplitude APs (rate ∼1 Hz). During the transition to steady-state AP firing, LCR size further increased, while LCR period shortened. LCRs became more synchronized resulting in the growth of an ensemble LCR signal peaked in late diastole, culminating in AP ignition; the rate of diastolic depolarization, AP amplitude, and AP firing rate increased. I
, I
, and I
amplitudes in dormant SANC increased in response to β-adrenoceptor stimulation. During washout, all changes reversed in order. Total PLB was higher, but the ratio of phosphorylated PLB (Serine 16) to total PLB was lower in dormant SANC. β-adrenoceptor stimulation increased this ratio in AP-firing cells. Thus, transition of dormant SANC to AP firing is linked to the increased functional coupling of membrane and Ca
clock proteins. The transition occurs via (i) an increase in cAMP-mediated phosphorylation of PLB accelerating Ca
pumping, (ii) increased spatiotemporal LCR synchronization, yielding a larger diastolic LCR ensemble signal resulting in an earlier increase in diastolic I
; and (iii) increased current densities of I
, I
, and I
.
We reported an ultraslow late Na+ current (INaL) in ventricular cardiomyocytes of human hearts. INaL has been implicated in regulation of action potential duration in normal hearts and repolarization ...abnormalities in failing hearts. We have also identified sodium channel (NaCh) gating modes including bursts (BM) and late scattered openings (LSM) that together comprise INaL; however, the contribution of these gating modes to Na+ current (INa) remains unknown. In the present study, the late NaCh activity was recorded, analyzed, and modeled for heterologously expressed NaCh, Nav1.5, and for the native NaCh of ventricular mid-myocardial cardiomyocytes from normal and failing hearts.
We found that LSM gating was significantly slower in failing compared to normal myocytes and Nav1.5 (tau=474+/-10 vs. 299+/-9, and 229+/-12 ms, m+/-SEM; P<0.05, n=5-6). Total burst length of BM decreased with depolarization and was larger in failing compared to normal myocytes and Nav1.5. A complete INa decay was then numerically approximated as composed of NaCh populations operating in three gating modes described by separate Markov kinetic schemes: transient mode (TM), LSM, and BM. The populations of NaCh operating in each gating mode were estimated as 79.8% for TM, 20% for LSM, and 0.2% for BM, yielding an apparent four-exponential INa decay at -30 mV (maximum INa) (tau i approximately 0.4, 4, 50, and 500 ms). Whole-cell recordings confirmed the existence of all four predicted components. The model also predicted voltage and temperature dependence of INaL as well as INaL increase and slower decay in failing hearts and acceleration by amiodarone.
The early phase of Na+ current decay (<40 ms) involves all three NaCh gating modes, the intermediate phase (from 40 to 300 ms) is produced by BM+LSM, although the contribution of BM decreases with depolarization, and ultra-late decay (>300 ms) is determined solely by LSM. The concept of multi-mode composition for INaL provides a new rationale for INaL modulation by factors such as voltage, temperature, pharmacological agents, and pathological conditions.
Ca
2+
and
V
m
transitions occurring throughout action potential (AP) cycles in sinoatrial nodal (SAN) cells are cues that (1) not only regulate activation states of molecules operating within ...criticality (Ca
2+
domain) and limit-cycle (
V
m
domain) mechanisms of a coupled-clock system that underlies SAN cell automaticity, (2) but are also regulated by the activation states of the clock molecules they regulate. In other terms, these cues are both causes and effects of clock molecular activation (recursion). Recently, we demonstrated that Ca
2+
and
V
m
transitions during AP cycles in single SAN cells isolated from mice, guinea pigs, rabbits, and humans are self-similar (obey a power law) and are also self-similar to
trans
-species AP firing intervals (APFIs) of these cells
in vitro
, to heart rate
in vivo
, and to body mass. Neurotransmitter stimulation of β-adrenergic receptor or cholinergic receptor–initiated signaling in SAN cells modulates their AP firing rate and rhythm by impacting on the degree to which SAN clocks couple to each other, creating the broad physiologic range of SAN cell mean APFIs and firing interval variabilities. Here we show that Ca
2+
and
V
m
domain kinetic transitions (time to AP ignition in diastole and 90% AP recovery) occurring within given AP, the mean APFIs, and APFI variabilities within the time series of APs in 230 individual SAN cells are self-similar (obey power laws). In other terms, these long-range correlations inform on self-similar distributions of order among SAN cells across the entire broad physiologic range of SAN APFIs, regardless of whether autonomic receptors of these cells are stimulated or not and regardless of the type (adrenergic or cholinergic) of autonomic receptor stimulation. These long-range correlations among distributions of Ca
2+
and
V
m
kinetic functions that regulate SAN cell clock coupling during each AP cycle in different individual, isolated SAN cells not in contact with each other. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states. Thus, to ensure rapid flexibility of AP firing rates in response to different types and degrees of autonomic input, nature “did not reinvent molecular wheels within the coupled-clock system of pacemaker cells,” but differentially engaged or scaled the kinetics of gears that regulate the rate and rhythm at which the “wheels spin” in a given autonomic input context.
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