Membrane Potential Fluctuations Resulting From Submembrane Ca2+ Releases in Rabbit Sinoatrial Nodal Cells Impart an Exponential Phase to the Late Diastolic Depolarization That Controls Their Chronotropic State
Stochastic but roughly periodic LCRs (Local subsarcolemmal ryanodine receptor–mediated Ca2+ Releases) during the late phase of diastolic depolarization (DD) in rabbit sinoatrial nodal pacemaker cells (SANCs) generate an inward current (INCX) via the Na+/Ca2+ exchanger. Although LCR characteristics have been correlated with spontaneous beating, the specific link between LCR characteristics and SANC spontaneous beating rate, ie, impact of LCRs on the fine structure of the DD, have not been explicitly defined. Here we determined how LCRs and resultant INCX impact on the DD fine structure to control the spontaneous SANC firing rate. Membrane potential (Vm) recordings combined with confocal Ca2+ measurements showed that LCRs impart a nonlinear, exponentially rising phase to the DD later part, which exhibited beat-to-beat Vm fluctuations with an amplitude of approximately 2 mV. Maneuvers that altered LCR timing or amplitude of the nonlinear DD (ryanodine, BAPTA, nifedipine or isoproterenol) produced corresponding changes in Vm fluctuations during the nonlinear DD component, and the Vm fluctuation response evoked by these maneuvers was tightly correlated with the concurrent changes in spontaneous beating rate induced by these perturbations. Numerical modeling, using measured LCR characteristics under these perturbations, predicted a family of local INCX that reproduced Vm fluctuations measured experimentally and determined the onset and amplitude of the nonlinear DD component and the beating rate. Thus, beat-to-beat Vm fluctuations during late DD phase reflect the underlying LCR/INCX events, and the ensemble of these events forms the nonlinear DD component that ultimately controls the SANC chronotropic state in tight cooperation with surface membrane ion channels.
The spontaneous diastolic depolarization (DD) of sinoatrial nodal cells underlies their rhythmic activity. It results from the instantaneous change in the ensemble of ionic currents expressed by sinoatrial nodal pacemaker cells (SANCs), which have been studied extensively using the voltage-clamp technique.1,2 The major currents thought to be involved in diastolic depolarization during normal spontaneous activity in the SANCs are as follows: rapid and slow delayed rectifier K+ currents; L- and T-type Ca2+ currents; If; a background current; and an Na+/Ca2+ exchange (NCX) current (INCX).
Recent studies have demonstrated the occurrence3 and relevance4–7 of submembrane LCRs (Local subsarcolemmal Ca2+ Releases) via ryanodine receptors (RyRs) to the spontaneous firing rate of SANCs. Specifically, spontaneous, voltage-independent,7 roughly periodic LCRs in SANCs are generated because of an enhanced Ca2+ cycling by sarcoplasmic reticulum (SR) proteins, which, in contrast to ventricular myocytes, are highly phosphorylated at the basal state.8 The Ca2+ release activation phase of the cycling occurs during the later, nonlinear part of diastolic depolarization and produce an inward current via activation of NCX.5,9 Whereas the resultant depolarization from the local NCX activation is likely the event that links individual LCR to DD acceleration, quantitative characterization of how LCRs, in cooperation with the established membrane-delimited pacemaker mechanisms, specifically control the fine structure of the DD has not been reported.
The present study used a combination of experimental and numerical approaches to identify depolarizations induced by the LCR-activated, local NCX currents and to determine how these depolarizations impact on the fine structure of the DD to control the spontaneous firing rate under a variety of conditions that we had previously shown to alter LCR timing and/or amplitude (ie, ryanodine, BAPTA, nifedipine, isoproterenol [ISO]). The results of our study show that LCR-induced NCX currents link LCRs to the spontaneous beat-to-beat membrane potential (Vm) fluctuations, the ensemble of which imparts an exponentially rising phase to the late nonlinear DD fine structure, followed by ICaL activation and action potential (AP) upstroke, thus linking LCRs to the cycle length and hence spontaneous SANC beating rate.
Materials and Methods
Preparation and Imaging of Cells
Single rabbit SANCs were isolated as described previously,10 using protocols approved by the Animal Care and Use Committee of our institution. Cells were loaded with fluo-3 AM (Molecular Probes), and a LSM-410 microscope (Carl Zeiss Inc) was used to image Ca2+ in the line scan mode. Image processing was performed with IDL software (Research Systems, Boulder, Co), and Ca2+ release was expressed as fluo-3 fluorescence (F) normalized to its minimal value (F0). Cells chosen for the study had a spindle-like or spider-like shape.
A perforated patch clamp was used to record membrane potential by an Axopatch 2D amplifier (Axon Instruments, Foster City, Calif), operating in the current-clamp mode. Pipettes were filled with (in mmol/L) potassium gluconate 120, KCl 20, NaCl 5, HEPES 5, MgATP 5, and (in μmol/L) β-escin 50 (pH 7.2). The extracellular bathing solution contained (in mmol/L) NaCl 140, KCl 5.4, MgCl2 1, HEPES 5, CaCl2 1.8, and glucose 5.5 (pH 7.4). All experiments were performed at 35°C.
Data were acquired using pClamp software (Axon Instruments). Spontaneous beating of SANCs was continuously recorded for at least 7 to 10 minutes in control conditions as well as during drug (ISO, BAPTA-AM, nifedipine, cesium, and ryanodine) application. Drugs, dissolved in the bath solution, were usually applied for 4 to 5 minutes before recordings of APs demonstrating effect of drug were taken for the analysis.
Analysis of DD Shape and Its Beat-to-Beat Fluctuations
We analyzed the fine structure DD by calculating the mean DD slope, the amplitude of nonlinear DD component (NDDC), and the AP take-off potential as well as beat-to-beat fluctuations, by calculating the mean DD and variance estimates (Figure 1A; also see the online data supplement for details, available at http://circres.ahajournals.org).
A numerical primary SANC model9 was used to simulate SANC electrophysiology and LCR stochastics (see the online data supplement for details).
Data are expressed as mean±SEM. Statistical significance was determined using Students t test (significance level, P<0.05).
Beat-to-Beat Fluctuations in Pacemaker Potential
Spontaneous APs of rabbit SANCs recorded continuously in control conditions (Figure 1B; Table II in the online data supplement) were similar to those reported in previous studies.1,10,11 The DD consists of 2 components: an initial linear component, and a later, nonlinear component (Figure 1A and the online data supplement). Under control conditions, the time from the prior maximum diastolic potential (MDP) to the onset of the nonlinear component in 31 SANCs averaged 145±8 ms (NDDC delay time in supplemental Table II). We observed beat-to-beat membrane potential fluctuations in the NDDC (Figure 1C) and quantified them during the 100-ms segment preceding AP upstroke as the residual variance in each beat after the group mean DD had been subtracted (Figure 1D and the online data supplement).
The superimposition of 7 individual DDs of the beats in the representative cell (Figure 1C) and the mean of DD of 7 consecutive beats (bold curve) reveals that beat-to-beat fluctuations in DD begin to occur approximately 70 ms before the next AP upstroke and increase with time. Figure 1D shows in more detail the membrane potential fluctuations about the mean DD preceding each of the 7 APs. Figure 1E shows the time course of the total ensemble variance (see the online data supplement for calculation details) in the cell during control conditions. For the 50-ms segment of the NDDC analyzed, the mean value of DD fluctuations was 1.07±0.06 mV (n=31).
Local Ca2+ Releases Are Linked to the Nonlinear Diastolic Depolarization Phase
Figure 2A shows the membrane potential recording of a representative SANCs superimposed on a line-scan image of [Ca2+]i with the scanned line positioned close to the sarcolemmal membrane parallel to the longitudinal axis of the cell (inset in top left). Note, that subsarcolemmal LCRs occur during the nonlinear DD, peaking before the next AP upstroke. The Ca2+ releases have widths of 2 to 8 μm. Therefore, some are small wavelets, but some appear like sparks (ie, events with no evident Ca2+ propagation from one release site to another); however, they have a larger size (2 to 5 times) compared with sparks in ventricular myocytes.5
In Figure 2B, the average time course of the LCR occurrence is superimposed on that of DD variance. The novel finding here is that the time course of LCR evolution during DD and that of DD variance are virtually superimposable. The DD variance peaks at 1.51±0.15 mV2 (n=31) at −40 ms, ie, when the nonlinear component contributes significantly to DD (see Figure 2C).
DD Fine Structure and Cycle Length Responses to Maneuvers Known to Alter LCRs
Figure 3A, left, shows an averaged time course of DD variance (n=6); in control, DD variance increases between 80 and 40 ms before the subsequent AP and is markedly decreased by ryanodine. On average (Figure 3A, right), ryanodine at this concentration reduced the amplitude of the DD fluctuations for the 50-ms segment preceding AP upstroke by 60% (n=6). The profound inhibition of DD variance during ryanodine exposure strongly suggests that the DD fluctuations are caused by stochastic Ca2+ releases via the RyRs. It is important to note that in response to ryanodine the amplitude of the NDDC also decreased by approximately 50%, the onset of the nonlinear DD was delayed by approximately 2-fold, and the cycle length increased nearly 2-fold (supplemental Table II).
To provide additional evidence that DD variance is caused by LCRs, we exposed SANCs to a fast Ca2+ chelator, BAPTA-AM (5 μmol/L). Figure 3B shows that BAPTA strikingly decreases DD variance. On average (Figure 3B, right), the DD fluctuations for the 50-ms segment preceding AP upstroke were decreased by approximately 50%. Of note, BAPTA also reduced the amplitude of the NDDC by approximately 50%, delayed its onset by 3.5-fold, and increased the cycle length by nearly 3-fold (supplemental Table II).
We have previously shown that LCR characteristics vary with the extent of the SR Ca2+ load and that voltage-dependent Ca2+ current is required for Ca2+ influx to load the SR to fuel LCRs.7 Thus, DD variance caused by stochastic LCRs would be expected to vary with changes in voltage-dependent Ca2+ influx during the prior AP plateau. To reduce Ca2+ influx, we used the L-type Ca2+ channel blocker nifedipine (supplemental Table II). As was the case for ryanodine and BAPTA, 100 nmol/L nifedipine suppressed DD fluctuations (by ≈50%, from 1.11±0.07 mV to 0.59±0.04 mV), reduced the amplitude of the NDDC (by ≈25%), delayed its onset (by 85%), and prolonged the cycle length by 80% (supplemental Table II).
The β-adrenergic receptor agonist, ISO increases Ca2+ influx and has previously been shown to increase LCR frequency and size in rabbit SANCs.6 Figure 3C, left, shows that ISO increases the DD variance. On average (Figure 3C, right), ISO increased the DD fluctuations over a 50-ms interval preceding the AP upstroke by 40%. Notably, ISO also increased the amplitude of the NDDC by 20%, reduced its time of onset by 30%, and reduced the cycle length by 30% (supplemental Table II).
It has been proposed that the hyperpolarization-activated current If plays an important role in the control of automaticity,12 but the physiological impact of If in the generation of the DD has been and continues to be a matter of debate.13,14 The contribution of If to the amplitude of the NDDC and its variance in SANCs was assessed by exposure to 2 mmol/L cesium, an If blocker.15 As shown in supplemental Table II, on average, Cs+ had a significant, but small (5%), negative chronotropic effect under the present conditions,16 and, in contrast to the other potent chronotropic interventions tested above (BAPTA, ryanodine, nifedipine, and ISO), Cs+ had no substantive effect on nonlinear DD amplitude, delay (supplemental Table II), or on DD fluctuations.
Nonlinear DD Fine Structure Predicts the Chronotropic State
Figure 4A schematically summarizes the average effect of the aforementioned chronotropic interventions on the onset and amplitude of the NDDC and spontaneous cycle length. Because the DD noise may include a “background” noise related to the recording apparatus, as well as to some electrogenic processes other than of LCR origin, we characterized the response to chronotropic interventions as the change in noise parameters from the control state. Figure 4B demonstrates the relationship of changes in the NDDCs to changes in the spontaneous beating rate in response to the aforementioned perturbations. Note the strong positive correlations between changes in the beating rate, changes in the mean amplitude of the NDDC, and changes in DD fluctuations.
Numerical Estimation of NCX Current and DD Disturbance Evoked by an Individual LCR
A central hypothesis of the origin of DD fluctuations is that they are caused by INCX fluctuations evoked by stochastically occurring LCRs (Figure 4C). These INCX fluctuations and related membrane depolarizations were evaluated numerically. Our novel numerical model (online data supplement) predicted that an LCR within submembrane space during DD produces INCX of approximately −0.27 pA, which results in a membrane potential response of approximately 0.17 mV at the LCR (temporal) center (Figure 5). Also note that a single LCR occurrence results in earlier action potential firing with a phase shift being approximately 1 ms.
INCX Links LCRs to DD Fine Structure and SANC Chronotropic State
We simulate INCX generated by an ensemble of stochastic LCRs to determine whether INCX could provide a mechanistic link between LCRs and the observed DD noise, NDDC, and ultimately the spontaneous beating rate. Figure 6A shows that LCRs indeed control the simulated spontaneous beating rate of our SANC model throughout the entire physiological range. When LCRs enhance with ISO, the beating rate substantially increases from 134 bpm to 194 bpm (“ISO” panel). When LCRs cease, the beating rate substantially drops from 134 bpm to 91 bpm (“No LCRs” panel), thus simulating the ryanodine effect.
The chronotropic effects were mainly mediated by significant (≈2-fold) changes in diastolic INCX (Figure 6B), which were accompanied by respective DD fluctuation changes (Figure 7A and 7B). In case of ISO, this was attributable to an increase in the amplitude of an LCR-induced NCX current (0.71 pA versus 0.27 pA; Figure 6B, inset). This increase, in turn, resulted from an increase in LCR spatial size (more NCX within each fragment) and a larger LCR amplitude. The simulated changes in DD noise and nonlinear DD amplitude for the chronotropic interventions were consistent with our experimental findings (Figure 7C and 7D), thus predicting the importance of the nonlinear DD amplitude (originating from LCRs) with respect to beating rate regulation.
We specifically examined the mechanistic basis of the fluctuations of the exponentially rising DD phase by varying the number of LCRs in the model. It turned out that the larger LCR numbers resulted in greater nonlinear DD fluctuations and NDDC amplitudes (supplemental Figure I), thus providing direct evidence for our concept.
Finally, we observed experimentally the cycle length fluctuations of approximately ±5% of the mean value in a typical isolated SANC, and these are predicted, in part, by the model with LCR parameters of the basal state (Figure 7E). The fluctuations occur because of a great impact of stochastic ensemble of individual LCR/INCX events on DD of each cycle.
The present study, using a combination of experimental and numerical approaches, characterized the role of LCR-activated NCX current fluctuations to Vm fluctuations during the DD, and demonstrated how LCR occurrence was linked to specific changes in the fine structure of the DD and how the DD fine structure controls the SANC firing rate.
The DD is characterized by its start, the MDP, slope, and its end, ie, the take-off potential of the next AP. The DD slope is an important characteristic of DD because it provides an estimate of the rate at which the membrane potential moves to AP threshold. Unfortunately, a uniform method to calculate the DD slope has not been applied in prior studies. In some studies,17,18 a mean DD slope was measured between MDP and the take-off potential. In others, the mean DD slope was characterized during the first 50 to 200 ms,19,20 or the first two-thirds of the DD21 excluding the last, curved DD part from analysis. These discrepancies could be an additional source of differences among the DD slope values obtained in the literature. For most SANCs, the DD slope gradually increases from MDP to very high values near AP threshold. In contrast, in subsidiary pacemaker cells22 and in atrial fibers during abnormal automaticity,23 DD exhibits 2 phases: an initial steeper slope followed by a more gradual slope. We observed a similar plateauing of DD in isolated rabbit SANCs in the presence of negative chronotropic interventions, when their spontaneous beating rate was reduced to that of subsidiary or abnormal pacemakers.
Based on perspectives from our previous studies5–9 demonstrating that LCRs occur during the later part of the DD and that, in most rabbit SANCs, the approximate last third of DD rises exponentially, we fit the DD by the sum of a linear function of time and an exponential term (see Figure 1A and the online data supplement). We discovered that Vm noise occurred mostly during the later exponential DD component and demonstrated that the LCR occurrence directly correlated with these membrane potential fluctuations.
NDDC Controls SANC Chronotropic State
Our experimental results and model simulations predict how the fine structure of the DD is affected by LCR-initiated NCX-mediated currents that contribute to DD noise, and how the ensemble of these currents affects the onset and amplitude of the NDDC. Our results also demonstrate that changes in the DD fine structure are linked to changes in the spontaneous firing rate. We observed that all negative chronotropic interventions, which, in prior studies,5,6 have been shown to suppress LCRs during DD, ie, ryanodine, buffering of intracellular Ca2+ by BAPTA, or inhibition of Ca2+ influx and SR Ca2+ loading by nifedipine, delay the onset of the NDDC, reduce the amplitude of beat-to-beat fluctuations about its mean, and reduce its amplitude. In contrast, β-adrenergic stimulation increases the number, amplitude, and size of LCRs and effects their earlier occurrence. This, in turn, shifts the onset of the NDDC to an earlier time following the prior AP (supplemental Table II), enhances the amplitude of DD fluctuations, and increases the amplitude of the NDDC. Thus, SANC chronotropic state is controlled by the timing and abundance (including synchronicity) of LCR emergence after the previous AP. Because LCR emergence critically depends on SR Ca2+ load, which, in turn, depends on Ca2+ influx via ICaL and pumping Ca2+ into SR by SERCA, the SANC operational paradigm involves a tight cooperation of both membrane and internal Ca2+ cycling proteins and can be schematically presented by 2 interacting loops of membrane channel function cycling and Ca2+ cycling (Figure 4C). Also, an almost synchronous emergence of LCRs (resulting in the powerful NDCC) during the late DD phase requires resetting of the phases of individual LCRs. This is achieved by ICaL initiation of CICR and depletion of the SR Ca2+ load. The regulation of SANC chronotropic state via LCR interaction with the membrane ion channel cycling thus has multiple control points, some of which are affected by the perturbations used in the present study. All perturbations that affect cell or SR Ca2+ loading and SR Ca2+ release affect spontaneous RyR activation and cause changes in the LCR characteristics. Changes in spontaneous SR Ca2+ release during the later DD caused by nifedipine or ISO (via their affect on Ca2+ influx and thus Ca2+ available for SR Ca2+ pumping) are distinct from their effect on the trigger function of ICaL on CICR. Thus, changes in the beat-to-beat fluctuations in the Vm caused by nifedipine or ISO during the later DD report the chronotropic state, at least in part, because of their effect on LCRs. ISO, in addition, may alter LCRs via its effect on RyR phosphorylation and phospholamban8 and indirectly on L-type Ca2+ channels, supplying more Ca2+ for SR Ca2+ loading. ISO also enhances inwardly directed ICaL and other ion channels and thus may directly accelerate SANC cycle (ie, via its effect on the external loop in Figure 4C). However, our previous experiments6 and computer simulations8 indicated that the chronotropic effect of protein kinase A-dependent modulation of ion channels only (ie, without enhancement of intracellular Ca2+ cycling proteins) is markedly dampened.
Because the variations in timing of the onset of the NDDC or of its amplitude reflect variations in the timing and amplitude of underlying LCR-activated NCX currents, and because direct experimental evidence and numerical simulations confirm that the timing and amplitude of LCRs are tightly linked to the spontaneous SANC firing rate,7,9 we reasoned that the LCR-induced changes in the DD fine structure ought to predict changes in the firing rate. Indeed, maneuvers that affect LCR characteristics effected changes in the fine structure of the DD that were accompanied by changes in the spontaneous firing rate, and both were highly correlated (Figure 4B). Our numerical model simulations of pacemaker currents, membrane potential, and beating rates (Figures 6 and 7⇑) support this interpretation of the experimental correlations in Figures 3 and 4⇑. The strong correlations between changes in the beating rate and changes in the onset of the nonlinear DD (supplemental Table II), or changes in DD fluctuations or amplitude of the later NDDC, demonstrated by the present results also further support the idea developed above, ie, that depolarizations induced by stochastic LCRs driving the NCX are a crucial component of nonlinear phase of DD in rabbit SANCs5 (Figure 4C).
Computer Modeling Provides a Mechanistic Insight Into NDDC and Rate Control
Our primary SANC model, featuring stochastic LCR characteristics (see online data supplement for detail), shows that an individual LCR produces an inward current of 0.27 pA that generates a 0.17 mV DD disturbance (Figure 5). Despite the fact that the NCX current fluctuations cannot be measured experimentally during spontaneous beating, the model simulation is validated (1) by similarity of the timing and amplitude of DD noise arising from the superposition of membrane responses to individual LCRs in simulations and that observed experimentally (compare Figure 1 with Figure 7) and (2) the simulated beat-to-beat cycle length fluctuations caused by stochastic behavior of LCRs predict (at least in part) those observed experimentally (Figure 7E). To our knowledge, the latter is a novel finding and merits further study, especially as it relates to stability of pacemaker function24 (together with other known factors influencing the SANC cycle length variations such as stochastic opening and closing of individual ion channels25).
By grading the LCR number, the model has directly tested the mechanistic basis for the exponential rising DD phase. An increase in LCR number resulted in respective increases in the NDDC amplitude and the mean DD fluctuation (supplemental Figure I). The model thus supports the hypothesis that the fluctuating nonlinear late DD phase observed experimentally is caused by current fluctuations elicited by LCRs (Figure 6B, insets). We interpret these results to indicate that the superimposition/synchronization of nonlinear DD responses to individual LCRs provides a mechanism by which LCRs change the DD time course from a linear to nonlinear fashion, thus modulating the timing to reach the DD take-off-potential (Figure 4C). Interestingly, this novel regulatory mechanism does not require a highly developed SR or abundant RyR expression,26 because the RyR Ca2+ release signaling requires that only those RyRs that are close to the plasma membrane produce LCRs; the initial signaling is subsequently greatly amplified by the (voltage-gated) ion channel mechanisms.
The Initial Linear DD Component
Our experiments also showed that the slope of the initial linear DD component decreased significantly under BAPTA, and increased under ISO (supplemental Table II), suggesting an additional important role of If, delayed rectifier potassium, and other currents during the initial part of diastolic depolarization. Although a recent study suggests that If, passing only Na+ and K+,27 is implicated in cAMP modulation of SANC firing rate, it would appear to contribute mostly to the linear DD component, which, under control conditions, might be considered as its Ca2+-independent part, because most (>80%) LCRs occur during the later exponential DD phase, ie, during the last third of the DD7 (Figure 2B). Other Ca2+-independent mechanisms, eg, modulation of delayed rectifier potassium current, may also affect a beating rate via regulating MDP and the slope of the linear DD component. Although Cs+, used in our study to block If, is not specific If blocker, 2 mmol/L Cs+ produces the same effect on the AP parameters and the spontaneous beating rate as more specific If blockers, UL-FS-49 and ZD-7288.16
Several potassium currents in the SAN could be affected by Cs+. However, IK1 is absent in the rabbit SA node; Ito is present only in small amount of cells in the periphery; and IKr plays the most prominent role in the rabbit SA node. However, effect of Cs+ on the IKr does not offset its effect on If, because deactivation of IKr is a major determinant of the linear part of the DD,1 and its suppression by Cs+ would induce an additional decrease in the slope of diastolic depolarization and, as a result, in the spontaneous beating rate. The very modest effect of Cs+ on the DD slope and spontaneous beating rate observed in our experiments suggests IKr is not significantly affected by Cs+.
In summary, spontaneous, stochastic but roughly periodic LCRs occurring during the later NDDC activate local NCX currents that produce depolarizations of sufficient magnitude to produce membrane noise, detectable as beat-to-beat fluctuations in membrane potential during this part of the DD. The timing and magnitude of these Ca2+-dependent DD membrane potential fluctuations reflects the timing and magnitude of the underlying LCR. Thus, the status of LCR characteristics are reflected in the fine structure of the DD and via its control of the nonlinear DD characteristics, LCR control the SANC firing rate. This influence of intracellular Ca2+ release on SANC automaticity has a counterpart in other cardiac cell types (see online data supplement and28).
Sources of Funding
This research was supported by the Intramural Research Program of the NIH, National Institute on Aging.
↵*Both authors contributed equally to this work.
Original received November 17, 2004; resubmission received July 24, 2006; accepted September 18, 2006.
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