Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2018 Mar 13;114(5):1176–1189. doi: 10.1016/j.bpj.2017.12.043

Positive Feedback Mechanisms among Local Ca Releases, NCX, and ICaL Ignite Pacemaker Action Potentials

Alexey E Lyashkov 1, Joachim Beahr 2, Edward G Lakatta 1, Yael Yaniv 2,∗, Victor A Maltsev 1,∗∗
PMCID: PMC5883559  PMID: 29539403

Abstract

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 (ICaL). 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 ICaL activation threshold. During the ignition phase, ICaL-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 ICaL, as diastolic LCRs begin to occur below and continue within the voltage range of ICaL 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.

Introduction

The spontaneous diastolic depolarization (DD) is the signature of spontaneous automaticity of sinoatrial node cells (SANC), the primary heart pacemaker cells. The DD starts after the repolarization phase of the prior action potential (AP), i.e., at a maximum diastolic depolarization (MDP) of about −60 mV in rabbit SANC, a traditional cell type used in pacemaker research. The DD ends at the AP threshold of about −40 mV (1).

Early studies considered the DD rate to be mainly regulated by the decay of the delayed rectifier K+ current (IK) (2) or by a nonselective, hyperpolarization-activated current, the funny current (If) (3), i.e., an essentially cell surface membrane-limited, Hodgkin-Huxley type kinetics mechanism, a membrane clock (or M-clock, for short). More recent studies discovered that during DD the sarcoplasmic reticulum (SR), a major intracellular Ca store, generates rhythmic, spontaneous, locally propagating subsarcolemmal Ca releases (LCRs) via ryanodine receptors (4, 5, 6). The LCRs activate an inward Na/Ca exchanger (NCX) current (INCX) that contributes to DD (5). Because LCRs can be generated under voltage clamp (7), i.e., independent of the M-clock function, this mechanism was dubbed a Ca-clock (8). Additional studies have shown that the pacemaker cell is actually a coupled-clock system because both M- and Ca- clocks are functionally coupled by numerous voltage-, time-, and Ca-dependent mechanisms (9, 10). In addition to aforementioned Ca-clock/NCX mechanism, the coupling also includes Ca-clock “resetting” and “refueling” by Ca influx via L-type Ca current (ICaL) (9).

Although the coupled-clock system has been recently extensively studied both numerically (11, 12) and experimentally (13), many important questions remain unanswered, and the system complexity still remains underexplored. One such underexplored yet crucial aspect of the system operation is the interplay of key Ca-, time-, and voltage-dependent mechanisms during the entire DD. Indeed, although LCRs were reported by Vinogradova et al. (7) as genuine spontaneous events under voltage clamp, other studies showed that diastolic LCRs may be also activated by Ca influx of T-type Ca current (ICaT) (4) or ICaL (14), probably via Ca-induced Ca release (CICR) mechanism. The issue has become even more intriguing vis a vis the discovery that low-voltage threshold L-type channel isoform Cav1.3 contributes to the DD (at least in mouse) (15). A recent observation that activation of this channel during DD increases diastolic LCR activity (16), suggests that Cav1.3 component of ICaL may provide a functional bridge between Ca-clock and M-clock.

To gain further insights into the complex interactions between LCRs, NCX, and ICaL, we combined numerical model simulations and experimentally derived data of simultaneous AP recording and Ca imaging. We focused specifically on seeking to determine the onset and evolution of an AP “ignition” phase, during which all three mechanisms sought to dynamically interact to accelerate the DD to ignite the next AP. We hypothesized that the time and membrane potential (V) at which ignition is initiated could be uniquely identified within the fine structure of the DD, and that the initiation informs on the fidelity of clock coupling when the system function is perturbed.

In our model simulations the ignition phase begins when early (ICaL-independent) Ca release increases the magnitude of inward INCX. The early membrane depolarization via the INCX, If, and ICaT activates ICaL. The ICaL-mediated Ca influx, in turn, recruits more LCRs via CICR and hence, more INCX, creating a positive feedback, further accelerating DD. This new and complex DD mechanism is supported by our experimental observation of early LCRs. We also found that the LCR activity indeed increases as ICaL becomes progressively activated. The ignition mechanism is important not only for robust AP initiation, but also for effective regulation of the AP firing rate. Indeed, our experiments identified the ignition onset to occur when time derivative of membrane potential (dV/dt) = 0.15 V/s and further showed that it closely predicts the duration of each AP cycle in basal state AP firing, in the presence of autonomic receptor stimulation, and in response to partial inhibition of either the M-clock or Ca-clock mechanisms.

Materials and Methods

Cell preparation

Spontaneously beating SANCs were isolated from New Zealand White rabbit hearts as previously described (5). All animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication no. 85-23, revised 1996). Experimental protocols were approved by the Animal Care and Use Committee of the National Institutes of Health (protocol 034-LCS-2019). The dissociated cells were stored at 4°C and were used within 12 h of isolation. For all electrophysiology and Ca imaging experiments, the cells were superfused with Tyrode solution at 35 ± 0.5°C (Cell MicroControls) with the following composition (in mM): 140 NaCl, 5.4 KCl, 2 MgCl2, 5 HEPES, 1.8 CaCl2, and 5.5 glucose, and titrated to pH 7.4 with NaOH.

AP measurements and analysis

Spontaneous APs were recorded and the spontaneous AP characteristics were quantified as described previously (17). In short, spontaneous APs were recorded via a perforated patch-clamp with 35 μM β-escin (Sigma-Aldrich, St. Louis, Missouri) added to the pipette solution that contained (in mM) the following: 120 K-gluconate, 2.5 NaCl, 2.5 MgATP, 2.5 Na2ATP, 5 HEPES, and 20 KCl, and titrated to pH 7.2 with KOH. After a full cell access is established via patch perforation, SANC APs were recorded for at least 2 min under control conditions and 10 min after pharmacologic intervention. The AP recording was performed using an Axopatch-200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA). For electrophysiological recordings, cells from at least five rabbits were used for each intervention. The recorded data was originally filtered at 2 kHz by patch-clamp amplifier and sampled at 5 kHz by Digidata 1440A (Molecular Devices). An additional low-pass filter of 40 to 60 Hz was applied in our data analysis to identify the ignition onset from the recording noise. The original AP traces were analyzed with our custom-made computer program (18). The program calculated dV/dt and AP cycle length (APCL) as the duration between consecutive AP overshoot peaks. MDP was found as the lowest membrane potential between the peaks. We assumed that DD starts at the MDP. In addition to determining the ignition onset (Results), we also determined the take-off potential as previously reported (19) as V at the time point when DD rate reaches 0.5 V/s. Taking into account the high DD rate of 0.5 V/s, in our study we interpreted the take-off potential as the AP threshold, i.e., V that inevitably triggers the next AP.

Confocal imaging of LCRs

Ca cycling was measured with Fluo-4 AM (Molecular Probes, Eugene, OR) to assess spontaneous LCRs (20). SANC were loaded with 5 μM Fluo-4 AM for 20 min at room temperature, and superfused with Tyrode solution (as above) at 35 ± 0.5°C. For LCR measurements, all images were recorded with a scan line oriented along the long axis of the cell, in a proximity to the sarcolemmal membrane. The images were processed with IDL software (Exelis) or MATLAB (The MathWorks, Natick, MA). For Ca recording, cells from at least three rabbits were used.

Numerical model

We performed simulations using the Maltsev-Lakatta (ML) SANC numerical model that features a coupled-clock mechanism (9). This numerical model is freely available and can be downloaded and run in CellML format (http://models.cellml.org/workspace/maltsev_2009) using the Cellular Open Resource software developed by Alan Garny at Oxford University in the UK (21) (for recent development of this software see http://www.opencor.ws/). To simulate β-adrenergic receptor (β-AR) stimulation we used a modification of ML model specifically formulated and tested to numerically describe autonomic modulation of cardiac pacemaker cell automaticity (22).

Drugs

Isoproterenol, carbachol, and cyclopiazonic acid were purchased from Sigma. Ivabradine was obtained from TRC (Toronto, Canada). Ryanodine was obtained from Molecular Probes.

Statistical analyses

All data are presented as mean ± SE. ANOVA was used to determine statistical significance. We also used paired t-test to compare parameters in the presence of drugs versus control when such data were available in the same cells. p < 0.05 was considered significant.

Results

Analysis of AP ignition by numerical model simulations

We first explored the interplay of If, INCX, ICaT, and ICaL in silico and how this interplay is reflected in the fine structure of the DD with respect to an ignition phase (Fig. 1). According to the coupled-clock theory (9), a key event during DD is the increase of inward INCX due to the diastolic Ca release. Thus, we defined the ignition phase onset as a time point during DD at which the magnitude of the inward INCX becomes increasing. Our simulations showed that under basal conditions this occurs when V reaches −59.6 mV and dV/dt = 0.127 V/s (Fig. 1, A–C). At the ignition onset (illustrated by black vertical arrow) both activation of If and INCX contribute to the DD: when If is approaching its maximal value, but the magnitude of inward INCX only just begins to increase.

Figure 1.

Figure 1

Open in a new tab

Numerical model simulations reveal contributions of major ion currents and their interplay during the AP ignition phase of DD (shown in gray). Shown are (A) AP traces, (B) AP derivative dV/dt, (C) funny current (If) and Na/Ca exchange current (INCX), (D) T-type Ca current (ICaT), (E) L-type Ca current (ICaL), and (F) overlapped If, ICaT, and ICaL on a small scale of 4 pA.

The ignition phase (within gray shadow in Fig. 1) proceeds from the ignition onset to the take-off potential of −32.4 mV, i.e., when dV/dt = 0.5 V/s, considered to be the threshold for AP firing. Both ICaT and ICaL begin to increase during the ignition, but ICaT subsequently sharply decreases, whereas ICaL strongly increases (Fig. 1, D and E). Fig. 1 F shows further details of the interplay and contributions of If, ICaT, and ICaL on the same scale during the ignition. At the ignition onset (the left border of the gray shade), the respective values for INCX, If, ICaT, and ICaL are 8.3, 1.7, 1, and 0.4 pA, indicating that ICaL has a minor contribution to the ignition onset. During the ignition, the magnitudes of INCX and ICaL progressively and strongly increase, whereas both If and ICaT decline (after a small rise) (Fig. 1, C and F).

Fig. 2 illustrates model simulations of the Ca dynamics and ICaL during the ignition phase. It is important to note that INCX activation is determined by V and the submembrane Ca (Casub), rather than the bulk cytoplasmic Ca (Cacyt) (Fig. 2 A). The diastolic Ca release flux (Fig. 2 C, JSRCarel) that generates LCRs increases Casub reflecting the integrated LCR signal throughout the entire ignition phase, as the junctional SR becomes depleted of Ca (Fig. 2 B, CajSR). Progressive activation of ICaL during the ignition phase renders the ICaL-mediated Ca influx comparable to the Ca release flux (Fig. 2 C), indicating that, at least in part, Ca release is generated via CICR mechanism (Fig. 2 C, inset).

Figure 2.

Figure 2

Open in a new tab

Numerical model simulations show major components of intracellular Ca dynamics to the AP ignition phase of DD. Shown are (A) cytosolic Ca (Cacyt) and submembrane Ca (Casub), (B) junctional SR Ca (CajSR), and (C) SR Ca flux (JSRCarel) and absolute value of L-type current |ICaL| in the same units (pA) and same scale. The AP ignition phase is shown in gray. The inset illustrates ICaL amplitude that is comparable with that of JSRCarel, indicating the presence of diastolic Ca-induced Ca release.

To test the role of the diastolic CICR in AP firing in silico, we acutely, beginning at the MDP, excluded Ca influx generated by ICaL alone or together with ICaT, while keeping electrical components of these Ca currents intact. These simulations revealed that the diastolic CICR is crucially important in the model. In the absence of ICaL-mediated Ca influx, both the ignition and the occurrence of the next AP were substantially delayed: the cycle length increased from 331 to 411 ms (Fig. 3 A). The subsequent cycle increased further to 438 ms, and then ignition failed and AP firing ceased. In the absence of both ICaL and ICaT-mediated influxes, the ignition failed and AP generation ceased immediately (Fig. 3 B). In both cases, however, interference with CICR notably reduced the diastolic SR Ca releases (dotted and dashed curves of JSRCarel in Fig. 3 C). In case of the exclusion of Ca influxes via both ICaL and ICaT, the deviation of JSRCarel from its normal time course was larger and occurred much earlier (close to MDP, see inset in Fig. 3 C). The time course of diastolic INCX was also notably affected by the absence of Ca influx and related CICR (Fig. 3 E). That, in turn, abolished DD acceleration occurring with normal ignition (Fig. 3 F), indicative of clocks’ coupling during DD via CICR.

Figure 3.

Figure 3

Open in a new tab

Numerical model simulations illustrate the importance of diastolic CICR during AP ignition phase of DD. (A and B) Shown are Ca release flux and APs when Ca influx generated by Ca currents (ICaL or ICaL + ICaT, respectively) was excluded from model formulations, but respective electrical Ca currents remain unchanged. The exclusion of Ca influx was initiated from the MDP. (C–F) Shown are respective overlapped changes of JSRCarel, membrane potential (V), INCX, and dV/dt. Solid lines indicate normal ignition. Dotted lines indicate delayed ignition (Ca flux via ICaL was zero). Dashed lines indicate failed ignition (Ca flux via ICaL and ICaT was zero). See text for more details.

We next tested how the ignition phase becomes altered in the presence of β-AR simulation. Our simulations showed that in the presence of β-AR simulation the ignition phase onset occurs earlier and at a more hyperpolarized membrane potential (close to the MDP) (Fig. S1). We also noticed that the dV/dt level for the ignition phase onset remains almost unchanged (within 3%) in the model simulating β-AR stimulation effect (inset in Fig. S1). Another important observation was that the ignition phase duration becomes substantially shorter, with the magnitude of INCX substantially increasing.

The ignition phase onset in experimental AP recordings

To provide experimental evidence for the ignition mechanism predicted by our in silico simulations, we recorded and analyzed membrane potential (and its derivative, dV/dt) both separately and simultaneously with LCRs. It is important to note that under our experimental conditions we cannot directly record dynamics of INCX (or any other selective current) simultaneously with AP firing, and therefore we cannot assess the ignition phase onset as defined in our in silico simulations, i.e., reflecting the timing when the amplitude of inward INCX begins to increase in response to diastolic Ca release (LCRs in our experiments). Hence, we explored the ignition phase indirectly by monitoring the dV/dt with respect to the level of the ignition phase onset predicted by the simulations. To characterize the timing of the ignition onset within the pacemaker cycle, we introduced a new term, time-to-ignition-phase, that is defined as the time period from the AP peak to the ignition phase onset (Fig. 4 A).

Figure 4.

Figure 4

Open in a new tab

Ignition phase onset and ignition potential in experimental AP recordings. (A) A representative AP recording with its derivative dV/dt illustrating definitions of APCL and time-to-ignition-phase. (B and C) Graphs showing the relationships between AP firing cycle length and time-to-ignition-phase for different levels of sensitivity in finding the ignition onset in a representative SANC at dV/dt = 0.1 V/s and dV/dt = 0.15 V/s, respectively.

Specifically we tested whether 1) the ignition phase onset is detectable in the membrane potential recordings that are above the experimental noise, 2) the ignition phase onset is indeed linked to early LCR activity, 3) the ignition onset is below or very close to the previously reported initial part of ICaL activation (from −50 to −45 mV, see steady-state ICaL activation Boltzmann fit in Fig. 4 in Vinogradova et al. (23)), and 4) the timing of the ignition phase onset predicts the APCL in a variety of key perturbations of the system, including autonomic modulation or interference with either clock.

To address the noise issue, we analyzed our experimental AP recordings with respect to two levels of DD acceleration: 0.1 and 0.15 V/s; the first level is below, but the second level is slightly above the 0.127 V/s level predicted in silico for the ignition phase onset. An example of our analysis is shown in Fig. 4 A. Fig. 4 B illustrates the relationship between APCL and the time-to-ignition-phase. A relatively low value of the 0.1 V/s threshold rendered our search of early DD acceleration sensitive to noise, leading to false positive detections of the ignition onset (blue arrows in Fig. S2). As a result, APCL and the time-to-ignition-phase were poorly correlated (Fig. 4 B). This result indicated to us that a 0.1 V/s threshold for detection of the ignition onset is too low and too sensitive to noise (a combination of the instrumental noise and effect of early small, stochastic, unsynchronized LCRs). Therefore a higher threshold is required to robustly capture the moment when the LCR ensemble signal grows notably and can accelerate the DD.

Fig. 4 C illustrates the relationship between APCL and the time-to-ignition-phase when the ignition onset detection was set at a higher threshold dV/dt of 0.15 V/s, i.e., slightly higher than 0.127 V/s predicted by the model but, importantly, above the recording noise (Fig. S2). We found a tight correlation between APCL and the time-to-ignition-phase, indicating that the 0.15 V/s allows a faithful detection of the ignition phase onset separated from the noise. Thus, we report all our subsequent results using this ignition detection method. On average, the ignition phase onset occurred at the ignition potential of −52 mV (Table S1), i.e., near yet slightly below ICaL activation threshold that is between −50 and −40 mV.

The ignition phase onset reflects LCR onset

We explored the relationship between the LCR occurrence and ignition phase onset. Fig. 5 A illustrates simultaneous measurements of V and Ca signals. An LCR period is defined as a period of time from a preceding Ca transient peak to the time when an LCR begins to occur. The distributions of LCR periods and time-to-ignition-phase were tightly correlated (Fig. 5 B). The LCR periods and times-to-ignition-phase were also tightly correlated (Fig. 5 D), indicating that within a given cell the time to the ignition onset is linked to the timing of the LCR ensemble signal. Fig. 5 C illustrates the distribution of LCR periods compared with the times-to-ignition-phase. Although LCRs begin to occur close to ignition phase onset (±30 ms window), the majority of LCRs occur later during the ignition phase (60–90 ms after the ignition phase onset), i.e., near the membrane potential required for ICaL activation.

Figure 5.

Figure 5

Open in a new tab

The relationship between LCR period and time-to-ignition-phase. (A) A representative example of simultaneous recording of APs and confocal line scanning image of Ca signal (normalized Fluo-4 fluorescence signal) in single isolated rabbit SANC. LCRs are indicated by arrowheads. The LCR period was defined as the time from the peak of the prior AP-induced Ca transient to an LCR onset. (B) Distribution of LCR periods and time-to-ignition-phase in a representative cell. (C) Distribution of the time differences between LCR period and the time-to-ignition-phase. (D) The relationship between LCR period and time-to-ignition-phase (n = 4 cells from four rabbits). To see this figure in color, go online.

Changes in the onset of the ignition phase determine the APCL change in response to autonomic neurotransmitters

We next quantified the effects of the autonomic receptor stimulation on APCL in terms of the time-to-ignition-phase and ignition potential. An earlier ignition phase onset is expected to result in a reduction in DD duration (Fig. S1). Thus, we determined how these key parameters change in response to β-AR stimulation (by isoproterenol) or cholinergic receptor stimulation (by carbachol). Representative examples of isoproterenol and carbachol effects on the APs and AP ignition phase are shown in Figs. S3 and S4, respectively, and average data are illustrated in Fig. 6 A.

Figure 6.

Figure 6

Open in a new tab

Changes in the ignition phase in response to autonomic receptor stimulation. (A) Average changes of APCL, time-to-ignition-phase, MDP, and ignition potential in response to β-AR stimulation by isoproterenol (ISO, n = 7 from seven rabbits) or cholinergic receptor stimulation by carbachol (CCh, n = 6 from six rabbits). (B and C) The relationships between APCL and time-to-ignition-phase in controls and in response to isoproterenol or carbachol, respectively. ∗p < 0.05 versus drug control. See also Table S1.

Isoproterenol substantially and significantly shortened the average APCL, and also slightly hyperpolarized the MDP, but the latter trend was not statistically significant. Isoproterenol reduced time-to-ignition-phase and lowered the ignition potential (similar to the response predicted by our model simulations, Fig. S1). Carbachol (at the half maximal concentration of 0.1 μM (24)) did not affect the MDP, but prolonged the average APCL and delayed the ignition phase onset. Furthermore, the ignition potential became depolarized in response to carbachol. Changes in time-to-ignition-phase and concurrent changes in APCL were tightly correlated in response to isoproterenol or carbachol (Fig. 6, B and C).

Time-to-ignition-phase is a universal timing mechanism for APCL regulation by the coupled-clock system

Recent theoretical and experimental studies (10, 20, 25) have shown that a selective perturbation of only one clock (either M-clock or Ca-clock), namely by ivabradine, a direct If inhibitor, or by cyclopiazonic acid, a direct SERCA (SR Ca pump) inhibitor, affects the other clock indirectly. If the timing of the ignition phase onset is a universal timing mechanism of the coupled-clock system, an increase in steady-state APCL in response to either ivabradine or cyclopiazonic acid should be similarly linked to a change in the onset of the ignition phase.

We tested ivabradine at various concentrations, including a relatively small concentration of 3 μM ensuring its direct action to inhibit If and, also higher concentrations at which ivabradine also blocks ICaL and IK (17, 26). It is important to note, however, that at any concentration, ivabradine does not directly affect SR Ca cycling (27). In other terms, experiments performed with 10 and 30 μM of ivabradine can be considered as a nonselective perturbation of M-clock functions. Representative examples of ivabradine effects at different concentrations are shown in Fig. 7 C. Ivabradine, at all concentrations tested, led to an increase in both APCL and time-to-ignition-phase (Fig. 7, A and C). Although the MDP did not significantly change in response to ivabradine, the ignition potential became more depolarized (Fig. 7, B and C). Similar to the response to autonomic receptor stimulation, there was a tight correlation between APCL and time-to-ignition-phase in response to different concentrations of ivabradine (Fig. 7 D).

Figure 7.

Figure 7

Open in a new tab

Changes in the ignition phase in response to specific inhibition of M-clock by ivabradine (IVA). (A) Average changes of the APCL and time-to-ignition-phase. (B) Average changes of MDP and ignition potential. The respective changes are shown in response to different concentrations of ivabradine; n = 18 for control, n = 6 for 3 μM, n = 6 for 10 μM, and n = 6 for 30 μM each from at least five rabbits; ∗p < 0.05 versus drug control (see also Table S2). (C) Examples of original AP recordings and ignition analysis of ivabradine effect. The ignition phase onsets are shown by small circles. (D) The relationship between APCL and the time-to-ignition-phase. To see this figure in color, go online.

We tested the effect of direct perturbations of Ca-clock by application of cyclopiazonic acid, a direct and selective inhibitor of the SR Ca pump, devoid of direct actions on M-clock ion currents (20). Interestingly, despite a direct perturbation of the intracellular Ca cycling, the electrophysiological response and change in the ignition characteristics were similar to those obtained in response to ivabradine, making a direct perturbation of the M-clock. Cyclopiazonic acid did not significantly change MDP, but depolarized the ignition potential and prolonged APCL and time-to-ignition-phase (Fig. 8, A–C). There was also a tight correlation between APCL and time-to-ignition-phase in response to different concentrations of cyclopiazonic acid (Fig. 8 D).

Figure 8.

Figure 8

Open in a new tab

Changes in the ignition phase in response to specific inhibition of SR Ca pumping by cyclopiazonic acid (CPA). (A) Average changes of the APCL and time-to-ignition-phase. (B) Average changes of MDP and ignition potential. The respective changes are shown in response to different concentrations of cyclopiazonic acid; n = 13 for control, n = 7 for 0.5 μM, and n = 6 for 5 μM each from at least five rabbits; ∗p < 0.05 versus drug control (see also Table S3). (C) Examples of original AP recordings and ignition analysis of cyclopiazonic acid effect. The ignition onsets are shown by small circles. (D) The relationship between APCL and the time-to-ignition-phase. To see this figure in color, go online.

In addition to cyclopiazonic acid, we also directly perturbed Ca-clock performance by application of ryanodine. Ryanodine at nanomolar concentrations locks the release channel (ryanodine receptor) in an open subconductance state but inhibits the channel at high concentrations >100 μM (review (28)). When ryanodine receptors are locked in the subconductance state, the SR leaks Ca. We used ryanodine at a concentration of 3 μM, which, on average, increased APCL by 84% (Table S4). A representative example of the ryanodine effects on the AP and AP ignition is shown in Fig. S5. This degree of Ca-clock inhibition is optimal for study of the ignition process because it substantially affects AP firing, but does not stop AP firing.

Ryanodine substantially depolarized the ignition potential, prolonged APCL and the time-to-ignition-phase, but did not significantly affect the MDP (Fig. 9, A and B). To accurately interpret these ryanodine effects, we performed numerical model simulations of the effect of SR Ca leak (Fig. S7), mimicking a ryanodine-dependent lock of the release channels in the subconductance state. Our sensitivity analysis revealed that SR Ca leak can substantially increase APCL up to 47% (from 333 to 488 ms) that reproduces our experimental effect of 3 μM of ryanodine to increase APCL (Table S4). Additional model simulations of Ca dynamics (Fig. S7 B) showed that the SR Ca release continues in the presence of SR leak, but it becomes persistent. Although the Ca load of the leaky junctional SR substantially drops from ∼300 to ∼3 μM, the network SR (lacking release but featuring SERCA pumping) remains highly loaded with [Ca] > 1 mM (Fig. S7 C). This is the case because the diffusional resistance between the network SR and junctional SR limits the inter-SR flux and preserves the Ca load of the network SR from drainage into junctional SR. It is SR Ca pumping and the limited diffusion rate that keep SR Ca load and drives the persistent release flux in the leaky SR model.

Figure 9.

Figure 9

Open in a new tab

Changes in the ignition phase in response to specific inhibition of SR Ca release by ryanodine. (A) Average changes of the APCL and time-to-ignition-phase. (B) Average changes of MDP and ignition potential. The respective changes are shown in response to ryanodine (Ry, 3 μM); n = 8 from six rabbits; ∗p < 0.05 versus drug control (see also Table S4). (C) The relationships between APCL and time-to-ignition-phase.

Thus, the two direct Ca-clock perturbations we employed affect different targets: ryanodine makes Ca release channels leaky and generates a persistent Ca release flux, but CPA directly inhibits SERCA and reduces SR Ca pumping. However, the resultant functional deterioration of Ca-clock (independent of the type of perturbation) produces the same effect on ignition, i.e., the ignition phase onset becomes delayed and emanates from a more depolarized membrane potential. The tight correlation between APCL and time-to-ignition-phase was preserved in all perturbations of M-clock and Ca-clock tested here (Figs. 6, B and C, 7 D, 8 D, and 9 C). Our data thus support the idea of general importance of the time-to-ignition-phase for AP firing rate regulation, illustrated in Fig. S6, showing that the results of all perturbations are well fitted by a single linear function.

Parameter sensitivity analyses

To get further insights into how different factors affect the ignition, we performed sensitivity analyses of the respective model parameters of cell membrane currents ICaL, ICaT, INCX, and If (Fig. S8) and of the SR Ca pumping rate Pup (Fig. S9). To compare the model sensitivity to different system components, we varied each respective model parameter within the same range from 50 to 200% of its original value.

ICaL is a central player in the SANC coupled-clock system, as this current contributes to the late DD, forms the AP upstroke, and provides Ca influx to drive the Ca-clock. Intuitively, an increase in ICaL conductance (gCaL) is expected to accelerate Ca-clock kinetics and therefore shorten both DD and time-to-ignition-phase. However, our simulations showed that the time-to-ignition-phase increases, rather than decreases when gCaL is increased (red curve in Fig. S8 A) because the time-to-ignition-phase is also dependent upon the AP duration, which lengthens at larger gCaL values, as ICaL supports the AP plateau. At the same time DD indeed becomes shorter (data not shown) and the ignition potential shifts to more hyperpolarized values (blue curve in Fig. S8 A).

Our sensitivity analysis of INCX variations produced another interesting (and counterintuitive) effect on the ignition phase. INCX links Ca-clock and M clock and, intuitively, an increase in kNCX (i.e., INCX amplitude) is expected to enhance clock coupling and shorten both DD and time-to-ignition-phase. Our simulations showed, however, that the time-to-ignition-phase becomes longer (red curve in Fig. S8 B) because stronger clock coupling is achieved at the cost of cell Ca depletion by the enhanced NCX operation in the forward mode, ultimately hampering Ca-clock function. The ignition potential shifts toward more positive values (more depolarized membrane) at low kNCX, but remains hyperpolarized and rather stable at high kNCX (blue curve in Fig. S8 B), i.e., similar to the gCaL effect.

Decreasing ICaT prolongs time-to-ignition-phase, but has a little effect on the ignition potential (Fig. S8 C). Decreasing If prolongs time-to-ignition-phase and concurrently depolarizes the ignition potential (Document S1. Figs. S1–S9 and Tables S1–S4, Document S2. Article plus Supporting Material), similar to what we demonstrated experimentally using ivabradine to inhibit If (Fig. 8). SR Ca pumping is a critical component of Ca-clock (and the coupled-clock system), and its inhibition in the model (Fig. S9) and in our experiments (with cyclopiazonic acid, Fig. 9) resulted in prolongation of time-to-ignition-phase and depolarization of the ignition potential.

Discussion

Our combined numerical model simulations and experimental measurements in rabbit SANC uncovered a new, important, integrated regulatory pacemaker mechanism that includes close interactions of LCRs, INCX, and ICaL coupled by a powerful positive feedback loop via diastolic CICR and DD acceleration, ensuring fail-safe cardiac impulse initiation. We refer to this new mechanism as AP ignition. Early diastolic LCRs activate inward current INCX so that its amplitude becomes increasing. Thus, the increasing amplitude of the inward INCX during the DD (and its related DD acceleration) informs on the initiation of the ignition phase (Fig. 1). If and ICaT also contribute to the onset of the ignition phase, so that the increasing amplitudes of the inward INCX and the waning If and ICaT bring the membrane potential to ICaL threshold and the ignition phase begins.

In contrast to the prior perspectives that LCRs (and their attendant INCX) represent exclusively a late DD mechanism, we demonstrate here that the ignition begins rather early in diastole reflected by a relatively small increase in DD rate to only 0.127 V/s (in silico). A similar (slightly higher) DD rate increase to 0.15 V/s caused by LCRs was robustly identified above the noise in our experiential simultaneous recordings of Ca signals and APs. The experimentally determined ignition potential was also within the early DD phase, around −50 mV, i.e., below but yet very close to the ICaL activation threshold.

The early ignition in model simulations is initiated by INCX, If, and ICaT (Figs. 1 and 2). Indeed, the hyperpolarization-activated If increases in response to AP repolarization, and its If contribution to the early DD is consistent with numerous prior studies (29). The early INCX, in turn, is driven by the decaying AP-induced Ca transient (30) and also by the LCRs (6). It is important to note, however, that although the Ca transient-linked component of INCX amplitude decreases (as the Ca transient decays), the LCR-linked component of INCX amplitude increases, making net INCX amplitude to increase. Thus, the DD acceleration is linked to LCR-induced INCX, rather than to Ca transient-induced INCX.

In previous studies using two-dimensional camera recordings (6) LCRs have indeed been observed very early in the course of the DD, in fact, as early as at the MDP. In this study we detected and characterized the early LCRs using confocal line scanning technique that provides much better temporal resolution versus two-dimensional camera recordings. LCRs to begin to occur close to the ignition phase onset and the LCR ensemble Ca signal increases during the ignition phase, in line with our model prediction of the increasing Ca release flux enhanced by the diastolic CICR (Figs. 2 and 3). Indeed, after the ignition phase onset, ICaL becomes substantially activated even before the DD reaches the AP threshold (gray area in Fig. 1 E).

The ICaL-mediated Ca influx (Fig. 2 C) enhances Ca release from junctional SR (which remains still highly loaded with Ca, Fig. 2 B) via the diastolic CICR, thus forming a positive feedback. The diastolic CICR and the positive feedback are indeed important to control APCL because virtual inhibition of the ICaL-meditated Ca influx (but keeping electrical current intact) substantially decreases diastolic Ca release flux and diastolic INCX amplitude, resulting in an instant increase in cycle length followed by a complete halt of AP firing (Fig. 3 A). Thus, our model simulations suggest physiological importance of the diastolic CICR mechanism and specifies further biophysical details of dynamic functional interactions between LCRs and ICaL reported previously (15, 16). Because the ignition phase progression is linked to ICaL activation, the specific V value of the ICaL activation threshold becomes especially important for how early within the cycle the positive feedback progression begins. In these new terms, the physiological role of a low-voltage activation threshold L-type Ca channel isoform Cav1.3 is expected to be particularly important (versus a high-voltage activation threshold Cav1.2 isoform) to regulate the whole-cell ICaL activation threshold and, hence, the early ignition phase (15, 16). In these terms, ICaL generated by Cav1.2 isoform (in addition to Cav1.3) is important for later progression of the ignition phase culminating in AP take-off. Our simulations show that ICaT also has a contribution into this diastolic CICR mechanism (Fig. 3 B) that extends our understanding of the role of ICaT-induced LCRs reported previously (4).

Our simultaneous recordings of membrane potential and intracellular Ca indicate that the diastolic CICR process predicted by our model simulations is plausible: 1) the ignition phase proceeds over the voltage range from −50 to −40 mV, a range known to include both ICaT and ICaL activation (1); and 2) the histograms of LCR periods (Fig. 5, B and C) show that LCRs occur during ignition phase (when ICaL becomes supposedly activated).

Our experiments using a series of pharmacological perturbations further demonstrated the physiological importance of the ignition to regulate the APCL. We show that that the ignition phase onset always heralds the AP cycle in response to autonomic receptor stimulation or in the presence of direct M-clock or Ca-clock inhibition (Fig. S6). Specifically, both our experiments and model simulations (Figs. 6 and S1) revealed that shortening of APCL in response to stimulation of β-AR is linked to an earlier onset of the ignition phase and more hyperpolarized ignition potential. This effect can be interpreted as a reduction in the restitution time required for spontaneous ryanodine receptor activation and LCRs to occur simultaneously with an increase in ICaL. Prolongation of APCL in response to cholinergic receptor stimulation (Fig. 6) and more depolarized ignition potential is therefore can be interpreted as an increase in the restitution time required for spontaneous diastolic LCRs to occur (24) simultaneously with a decrease in ICaL. It has also been previously shown that the integrated LCR signal decreases in the presence of cholinergic receptor stimulation (24). Thus the delayed and weaker ignition causes APCL to increase.

Although the ignition phase is characterized by the aforementioned complex positive feedback interactions of LCRs, NCX, and ICaL, including CICR, we further interpret our results in more general terms of the SANC coupled-clock operation (9, 10). In these terms, the time-to-ignition-phase informs the timing and the status of M- and Ca-clock coupling. Two types of evidence support this idea:

  • 1.

    As defined in the model, the ignition phase begins when INCX becomes activated by the early diastolic Ca release (i.e., the integrated LCR signal). The average LCR period is a “master integrating function” of the SANC coupled-clock system (20, 27). Because there is a tight correlation between average LCR period and the average time-to-ignition-phase (Fig. 5 B), the latter also reflects the onset of a coupled-clock function during DD.

  • 2.

    A direct perturbation of either the M-clock (by ivabradine) or the Ca-clock (by cyclopiazonic acid or ryanodine) generates indirect effects on the other clock via clock-coupling mechanisms. As a result of this coupling, we found a similar response to these perturbations, i.e., ignition potential becomes more depolarized, and time-to-ignition-phase becomes longer (i.e., the clock coupling via CICR becomes delayed), culminating in APCL prolongation (Figs. 7, 8, and 9).

Our parametric sensitivity analyses of the model behavior (Figs. S8 and S9) are commensurate with the experimental effects of If inhibition (ivabradine) and SR Ca pumping inhibition (cyclopiazonic acid), i.e., delayed ignition onset at more depolarized membrane potentials. These analyses also highlight critical importance of coupled changes of the system parameters in AP rate regulation by both numerous ion currents and Ca cycling as it occurs naturally via autonomic receptor modulation. In our experiments (with isoproterenol, Fig. 6) and in model simulation of β-AR stimulation effect (Fig. S1), ignition begins earlier. However, a sole ICaL increase in the model (in the absence of respective K current changes) delays the ignition phase (Fig. S8 A) because ICaL increases AP duration. The ignition is also delayed when INCX amplitude alone is increased (in the absence of respective ICaL changes) (Fig. S8 B) because the forward mode NCX competes with SERCA for Ca, depleting SR and inhibiting Ca-clock (see also (31)).

In a prior analysis of DD fine structure (19) the major DD parameter was interpreted to be the take-off potential, occurring when dV/dt reaches 0.5 V/s. However, no Ca measurements were performed in that analysis and therefore the DD before the take-off potential was interpreted as the effect of If activation. In reality, as noted, LCRs begin to occur very early in diastole (6) and the ignition phase clearly begins remarkably earlier than the take-off potential (Tables S1, S2, S3, and S4), at much lower levels of clearly detectable dV/dt increase, as low as 0.15 V/s (Fig. 4). Thus, the take-off potential characterizes a very late diastolic state, basically representing a threshold of AP activation and therefore provides no biophysical insight into early LCR-NCX-ICaL interactions that are crucial for DD dynamics, as we demonstrate here in the new terms of the ignition phase.

Limitations

We have explored the interactions between LCRs, INCX, and ICaL coupled by a powerful positive feedback loop via diastolic CICR and DD. Although other specific mechanisms may indeed also contribute to the ignition phase and positive feedback, e.g. SK4 K+ channels (32), their parameters must be quantified in rabbit SANC to take them into account by a computational model. Formulations of ICaL in our numerical model (9) are based on whole-cell patch clamp measurements of ICaL in rabbit SANC by Vinogradova et al. (23) that includes currents of all L-type Ca channel isoforms. Specific contribution of Cav1.3 (15, 16) isoform to ICaL in rabbit SANC also remains unknown and also merits future study to help to clarify its specific role in the AP ignition phase. Another factor that might be important for the ignition phase, but not studied here, is store-operated Ca influx. Such Ca influx linked to expression of TRPC channels was reported in mouse sinoatrial node (33).

Conclusions

Our numerical model simulations and experimental studies revealed an integrated DD mechanism, which we refer to as AP ignition phase, featuring a powerful positive feedback relation among LCRs, INCX, and ICaL that grows during DD. The positive feedback is achieved via multiple time-, voltage-, and Ca- dependent mechanisms, including diastolic CICR. It is also important to note that the ignition phase onset is achieved by a combined action of LCRs (and their attendant INCX), If, and ICaT, preceding the onset; whereas powerful contribution of ICaL occurs next and drives (together with LCRs and INCX) the ignition phase itself. In terms of the coupled-clock system operation, AP ignition represents a new coupling mechanism that occurs during DD and is important for fail-safe system operation to generate an AP, something akin to a “rocket launch.”

Author Contributions

E.G.L., Y.Y., and V.A.M. designed the research and wrote the manuscript. A.E.L. and Y.Y. performed the experiments. J. B. and V.A.M. performed the simulations. A.E.L., J. B., Y.Y., and V.A.M. analyzed data. V.A.M. contributed analytic tools.

Acknowledgments

This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging. The work was also supported by the Joint Scientific Research Program Natural Science Foundation of China and Israel Science Foundation, no. 398/14 (Y.Y), the Israel Ministry of Science (Y.Y), an Aly-Kaufman Postdoctoral Fellowship (J.B.), and The Center for Absorption in Science, Ministry of Immigrant Absorption, State of Israel (J.B.).

Editor: Eric Sobie.

Footnotes

Yael Yaniv and Victor A. Maltsev contributed equally to this work.

Nine figures and four tables are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(18)30068-7.

Contributor Information

Yael Yaniv, Email: yaely@bm.technion.ac.il.

Victor A. Maltsev, Email: maltsevvi@mail.nih.gov.

Supporting Material

Document S1. Figs. S1–S9 and Tables S1–S4
mmc1.pdf (2MB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (5.6MB, pdf)

References

  • 1.Mangoni M.E., Nargeot J. Genesis and regulation of the heart automaticity. Physiol. Rev. 2008;88:919–982. doi: 10.1152/physrev.00018.2007. [DOI] [PubMed] [Google Scholar]
  • 2.Noble D. A modification of the Hodgkin--Huxley equations applicable to Purkinje fibre action and pace-maker potentials. J. Physiol. 1962;160:317–352. doi: 10.1113/jphysiol.1962.sp006849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.DiFrancesco D. The contribution of the ‘pacemaker’ current (if) to generation of spontaneous activity in rabbit sino-atrial node myocytes. J. Physiol. 1991;434:23–40. doi: 10.1113/jphysiol.1991.sp018457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Hüser J., Blatter L.A., Lipsius S.L. Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells. J. Physiol. 2000;524:415–422. doi: 10.1111/j.1469-7793.2000.00415.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bogdanov K.Y., Vinogradova T.M., Lakatta E.G. Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation. Circ. Res. 2001;88:1254–1258. doi: 10.1161/hh1201.092095. [DOI] [PubMed] [Google Scholar]
  • 6.Monfredi O., Maltseva L.A., Maltsev V.A. Beat-to-beat variation in periodicity of local calcium releases contributes to intrinsic variations of spontaneous cycle length in isolated single sinoatrial node cells. PLoS One. 2013;8:e67247. doi: 10.1371/journal.pone.0067247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Vinogradova T.M., Zhou Y.Y., Lakatta E.G. Rhythmic ryanodine receptor Ca2+ releases during diastolic depolarization of sinoatrial pacemaker cells do not require membrane depolarization. Circ. Res. 2004;94:802–809. doi: 10.1161/01.RES.0000122045.55331.0F. [DOI] [PubMed] [Google Scholar]
  • 8.Maltsev V.A., Vinogradova T.M., Lakatta E.G. The emergence of a general theory of the initiation and strength of the heartbeat. J. Pharmacol. Sci. 2006;100:338–369. doi: 10.1254/jphs.cr0060018. [DOI] [PubMed] [Google Scholar]
  • 9.Maltsev V.A., Lakatta E.G. Synergism of coupled subsarcolemmal Ca2+ clocks and sarcolemmal voltage clocks confers robust and flexible pacemaker function in a novel pacemaker cell model. Am. J. Physiol. Heart Circ. Physiol. 2009;296:H594–H615. doi: 10.1152/ajpheart.01118.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Lakatta E.G., Maltsev V.A., Vinogradova T.M. A coupled SYSTEM of intracellular Ca2+ clocks and surface membrane voltage clocks controls the timekeeping mechanism of the heart’s pacemaker. Circ. Res. 2010;106:659–673. doi: 10.1161/CIRCRESAHA.109.206078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Maltsev V.A., Yaniv Y., Lakatta E.G. Modern perspectives on numerical modeling of cardiac pacemaker cell. J. Pharmacol. Sci. 2014;125:6–38. doi: 10.1254/jphs.13r04cr. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Stern M.D., Maltseva L.A., Maltsev V.A. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells. J. Gen. Physiol. 2014;143:577–604. doi: 10.1085/jgp.201311123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Yaniv Y., Lakatta E.G., Maltsev V.A. From two competing oscillators to one coupled-clock pacemaker cell system. Front. Physiol. 2015;6:28. doi: 10.3389/fphys.2015.00028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chen B., Wu Y., Song L.S. Local control of Ca2+-induced Ca2+ release in mouse sinoatrial node cells. J. Mol. Cell. Cardiol. 2009;47:706–715. doi: 10.1016/j.yjmcc.2009.07.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Mangoni M.E., Couette B., Nargeot J. Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity. Proc. Natl. Acad. Sci. USA. 2003;100:5543–5548. doi: 10.1073/pnas.0935295100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Torrente A.G., Mesirca P., Mangoni M.E. L-type Cav1.3 channels regulate ryanodine receptor-dependent Ca2+ release during sino-atrial node pacemaker activity. Cardiovasc. Res. 2016;109:451–461. doi: 10.1093/cvr/cvw006. [DOI] [PubMed] [Google Scholar]
  • 17.Yaniv Y., Maltsev V.A., Lakatta E.G. The “funny” current (I(f)) inhibition by ivabradine at membrane potentials encompassing spontaneous depolarization in pacemaker cells. Molecules. 2012;17:8241–8254. doi: 10.3390/molecules17078241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lyashkov A.E., Juhaszova M., Lakatta E.G. Calcium cycling protein density and functional importance to automaticity of isolated sinoatrial nodal cells are independent of cell size. Circ. Res. 2007;100:1723–1731. doi: 10.1161/CIRCRESAHA.107.153676. [DOI] [PubMed] [Google Scholar]
  • 19.Bucchi A., Baruscotti M., DiFrancesco D. Modulation of rate by autonomic agonists in SAN cells involves changes in diastolic depolarization and the pacemaker current. J. Mol. Cell. Cardiol. 2007;43:39–48. doi: 10.1016/j.yjmcc.2007.04.017. [DOI] [PubMed] [Google Scholar]
  • 20.Yaniv Y., Sirenko S., Lakatta E.G. New evidence for coupled clock regulation of the normal automaticity of sinoatrial nodal pacemaker cells: bradycardic effects of ivabradine are linked to suppression of intracellular Ca2+ cycling. J. Mol. Cell. Cardiol. 2013;62:80–89. doi: 10.1016/j.yjmcc.2013.04.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Garny A., Noble D., Kohl P. CELLULAR OPEN RESOURCE (COR): current status and future directions. Philos Trans A Math Phys Eng Sci. 2009;367:1885–1905. doi: 10.1098/rsta.2008.0289. [DOI] [PubMed] [Google Scholar]
  • 22.Maltsev V.A., Lakatta E.G. A novel quantitative explanation for the autonomic modulation of cardiac pacemaker cell automaticity via a dynamic system of sarcolemmal and intracellular proteins. Am. J. Physiol. Heart Circ. Physiol. 2010;298:H2010–H2023. doi: 10.1152/ajpheart.00783.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Vinogradova T.M., Zhou Y.Y., Xiao R.P. Sinoatrial node pacemaker activity requires Ca(2+)/calmodulin-dependent protein kinase II activation. Circ. Res. 2000;87:760–767. doi: 10.1161/01.res.87.9.760. [DOI] [PubMed] [Google Scholar]
  • 24.Lyashkov A.E., Vinogradova T.M., Lakatta E.G. Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca(2+) cycling and I(KACh) Am. J. Physiol. Heart Circ. Physiol. 2009;297:H949–H959. doi: 10.1152/ajpheart.01340.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Nazarov I.B., Schofield C.J., Terrar D.A. Contributions of cardiac “funny” (f) channels and sarcoplasmic reticulum Ca2+ in regulating beating rate of mouse and guinea pig sinoatrial node. Physiol. Rep. 2015;3:e12561. doi: 10.14814/phy2.12561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Bois P., Bescond J., Lenfant J. Mode of action of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial node cells. Br. J. Pharmacol. 1996;118:1051–1057. doi: 10.1111/j.1476-5381.1996.tb15505.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Yaniv Y., Lyashkov A.E., Lakatta E.G. Stochasticity intrinsic to coupled-clock mechanisms underlies beat-to-beat variability of spontaneous action potential firing in sinoatrial node pacemaker cells. J. Mol. Cell. Cardiol. 2014;77:1–10. doi: 10.1016/j.yjmcc.2014.09.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lanner J.T., Georgiou D.K., Hamilton S.L. Ryanodine receptors: structure, expression, molecular details, and function in calcium release. Cold Spring Harb. Perspect. Biol. 2010;2:a003996. doi: 10.1101/cshperspect.a003996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.DiFrancesco D., Noble D. The funny current has a major pacemaking role in the sinus node. Heart Rhythm. 2012;9:299–301. doi: 10.1016/j.hrthm.2011.09.021. [DOI] [PubMed] [Google Scholar]
  • 30.Sanders L., Rakovic S., Terrar D.A. Fundamental importance of Na+-Ca2+ exchange for the pacemaking mechanism in guinea-pig sino-atrial node. J. Physiol. 2006;571:639–649. doi: 10.1113/jphysiol.2005.100305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Maltsev A.V., Yaniv Y., Maltsev V.A. RyR-NCX-SERCA local cross-talk ensures pacemaker cell function at rest and during the fight-or-flight reflex. Circ. Res. 2013;113:e94–e100. doi: 10.1161/CIRCRESAHA.113.302465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Haron-Khun S., Weisbrod D., Attali B. SK4 K+ channels are therapeutic targets for the treatment of cardiac arrhythmias. EMBO Mol. Med. 2017;9:415–429. doi: 10.15252/emmm.201606937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ju Y.K., Chu Y., Allen D.G. Store-operated Ca2+ influx and expression of TRPC genes in mouse sinoatrial node. Circ. Res. 2007;100:1605–1614. doi: 10.1161/CIRCRESAHA.107.152181. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figs. S1–S9 and Tables S1–S4
mmc1.pdf (2MB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (5.6MB, pdf)

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES