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(Circulation Research. 2004;94:802.)
© 2004 American Heart Association, Inc.

Cellular Biology

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

Tatiana M. Vinogradova, Ying-Ying Zhou, Victor Maltsev, Alexey Lyashkov, Michael Stern, Edward G. Lakatta

From the Gerontology Research Center, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, Md. Present address of Y.-Y.Z. is Schering-Plough Research Institute, Lafayette, NJ.

Correspondence to Dr Tatiana M. Vinogradova, National Institutes on Aging, Gerontology Research Center, Laboratory of Cardiovascular Science, 5600 Nathan Shock Dr, Baltimore, MD 21224-6825. E-mail vinogradovat{at}grc.nia.nih.gov

	Abstract

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

Key Words: sinoatrial node • ryanodine receptors • local Ca²⁺ release • permeabilized sinoatrial nodal cells

	Introduction

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Rhythmic intracellular Ca²⁺ cycling, not directly dependent on surface membrane signals, is an integral component of biological clocks that regulate vital functions throughout nature. The capacity to generate rhythmic changes of intracellular Ca²⁺ is imparted by organelles that sequester and release Ca²⁺, eg, endoplasmic or sarcoplasmic reticulum (SR), endowed with Ca²⁺ pumps and release channels. The periodicity of intracellular Ca²⁺ cycling is regulated by changes in the Ca²⁺ pump characteristics, Ca²⁺ release channel activation state, and the quantity of Ca²⁺ available for pumping and release.

The heart’s pacemaker cells generate spontaneous rhythmic changes of their membrane potential, leading to roughly periodic electrical depolarizations or action potentials (AP). Like other excitable cells, cardiac sinoatrial pacemaker cells are endowed with the apparatus to cycle Ca²⁺ into and out of SR, ie, a Ca²⁺ pump (SERCA2) and Ca²⁺ release channels (RyRs). Whereas recent studies have detected localized submembrane Ca²⁺ releases via RyRs occurring during diastolic depolarization of spontaneously beating sinoatrial pacemaker cells,^1,2 it has not been demonstrated whether such Ca²⁺ release require the concomitant diastolic membrane depolarization or whether it occurs rhythmically with characteristics sufficient to drive the rhythmic spontaneous beating of these cells. The aims of the present study were to determine the following: (1) whether the occurrence of submembrane LCRs during the diastolic depolarization of spontaneously beating rabbit sinoatrial nodal cells requires the concurrent surface membrane potential, or whether it is a manifestation of intracellular Ca²⁺ cycling not directly requiring depolarization; (2) if LCRs do reflect spontaneous Ca²⁺ cycling, whether this is rhythmic or roughly periodic; and (3) if LCRs are rhythmic, whether their periodicity is sufficient to modulate spontaneous beating and linked to the spontaneous cycle length.

	Materials and Methods

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SA Node Cell Preparations and Electrophysiological Recordings
Single spindle-shaped spontaneously beating sinoatrial nodal cells were isolated from the rabbit hearts as previously described³ and studied in Tyrode solution at 35±0.5°C. Perforated patch-clamp technique with 50 µmol/L ß-escin⁴ (Sigma) added to the pipette solution was used to record spontaneous APs with an Axopatch-200 B patch-clamp amplifier (Axon Instruments). The bath solution had the following composition (in mmol/L): NaCl 140; KCl 5.4; MgCl₂ 1; HEPES 5; CaCl₂ 1.8; and glucose 5.5; pH 7.4. The pipette solution contained (in mmol/L) K-gluconate 120, NaCl 10, MgATP 5, HEPES 5, and KCl 20; pH 7.2.

Cell Permeabilization
A subset of cells was permeabilized with 0.01% saponin⁵ in a solution containing (in mmol/L) K aspartate 100, KCl 20, MgATP 3, MgCl₂ 0.81, HEPES 10, phosphocreatine 10, and 5U/mL creatine phosphokinase. The control experimental solution was, as above, with 0.03 mmol/L fluo-4 K-salt. The free [Ca²⁺], at a given total Ca²⁺, Mg²⁺, ATP, and EGTA concentration was calculated, using a computer program (WinMAXC, Stanford University).

Confocal Imaging of Ca²⁺ Releases
Cells were placed on the stage of a Zeiss LSM-410 inverted confocal microscope (Carl Zeiss, Inc) and loaded with fluo-3 AM (Molecular Probes). All images were recorded in the linescan mode, with the scan line oriented along the long axis of the cell, close to sarcolemmal membrane (see Figure 1, inset) and processed with IDL software (5.4, Research Systems). In "skinned" sinoatrial nodal cells, 10 images were obtained from each cell, as the position of the scan line was moved from one side of the cell to the other. The procedure was repeated after every 2, 5, and 10 minutes. In order to identify LCRs and quantify their characteristics, customized software, which selected a level of fluorescence that was greater than the statistical deviation of the background noise, was used.⁶ The amplitude of each LCR was expressed as a peak value (F) normalized to minimal fluorescence (F₀), its spatial size was indexed as the full width at half maximum amplitude (FWHM), and its duration characterized as the full duration at half maximum amplitude (FDHM). The signal mass of an individual LCR was estimated as follows: M=FWHMxFDHMx1/2F/F₀ (where F/F₀=F/F₀–1). The total signal mass of LCRs in intact cells was calculated as the sum of signal masses of all LCRs between successive AP-induced Ca²⁺ transients during spontaneous beating, or during comparable time intervals during voltage clamp.

View larger version (28K): [in this window] [in a new window]   Figure 1. LCRs in the intact and permeabilized cells. A, Recordings of APs and confocal linescan images (see cell diagram) in a representative spontaneously beating sinoatrial nodal cell. B, Confocal linescan images in a "skinned" sinoatrial nodal cell recorded with Fluo-4 K salt, in a representative "skinned" cell bathed in 100, 150, and 250 nmol/L [Ca2+].

To estimate the absolute diastolic and systolic [Ca²⁺] in spontaneously beating cells fluo-3 fluorescence signals were calibrated using a procedure similar to previously reported⁷ (see expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org).

The sarcoplasmic reticulum Ca²⁺ content was estimated by rapid application of caffeine onto the cell (20 mmol/L, 1 second) by pressure-ejection through a pipette located 100 µm away from the cell.⁸ To prevent caffeine-induced increase in Na⁺-Ca²⁺ exchange current in the cell,⁹ caffeine was dissolved in Na⁺- and Ca²⁺-free solution (extracellular Na⁺ was replaced with N-methyl-D-glucamine⁹).

Noise Analyses
To describe and quantify periodicity of LCRs in voltage-clamped cells fast Fourier transform (FFT)¹⁰ was performed on Ca²⁺ waveforms obtained from linescan images. FFTs were also performed on recordings of current oscillations obtained during voltage clamp.

Numerical Modeling
A novel, primary sinoatrial node pacemaker cell model¹¹ featuring diastolic Ca²⁺ release was used to simulate effects of LCRs on ionic currents during voltage clamp.

Statistical Analysis
Data are presented as mean±SEM. The statistical significance of effects was evaluated by Student’s t test and analysis of variance (ANOVA) when appropriate. A value of P<0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Local RyR Ca²⁺ Releases Do Not Require Membrane Depolarization
LCR occurrence in a representative, spontaneously beating cell is depicted in Figure 1A. Calibration of fluo-3 fluorescence (see online data supplement) estimated an average diastolic and systolic [Ca²⁺] in intact cells of 160±13 and 1150±213 nmol/L, respectively (n=4). The average characteristics of LCRs in spontaneously beating cells are shown in Figure 2.

View larger version (36K): [in this window] [in a new window]   Figure 2. Comparison of spatiotemporal properties of LCRs in intact spontaneously beating and permeabilized cells. Average characteristics of subsarcolemmal LCRs in intact, spontaneously beating cells (n=11, 121 LCRs; white bars) and "skinned" cells bathed in 100 nmol/L Ca2+ (n=10, 413 LCRs; gray bars), 150 nmol/L Ca2+ (n=3, 100 LCRs; black bars), and 250 nmol/L Ca2+ (n=3, 103 LCRs; dark gray bars). FWHM is a spatial size, indexed as the full width at half-maximum amplitude; FDHM is a duration characterized as the full duration at half-maximum amplitude. [Ca2+]i value in sinoatrial nodal cells was calculated using pseudoratio24: [Ca2+]i=Kd(F/F0)/[Kd/[Ca2+]r+1–F/F0], where Kd is 1 µmol/L,31 and [Ca2+]i at rest ([Ca2+]r) for permeabilized cells was known and for intact cells was estimated as 160 nmol/L. For comparison with permeabilized cells, total LCR signal mass in spontaneously beating cells was normalized to a 100-µm length and a 1-second time interval. In permeabilized cells when free [Ca2+] was increased, spatiotemporal characteristics of LCRs changed, creating spatially and temporally clustered events. In this case, the number of LCRs that collectively produced the total signal mass was estimated, as previously described,32 by dividing the total signal mass by the signal mass of the averaged spontaneous solitary LCR at lowest Ca2+ concentration (100 nmol/L). *P<0.05, Newman-Keuls multiple-comparison test.

One method used to determine whether LCRs require a change in membrane potential was to permeabilize cells with saponin (Figure 1). LCRs occurred in permeabilized cells and their characteristics varied with the bathing [Ca²⁺] (Figures 1B and 2). Elevating free [Ca²⁺] from 100 to 250 nmol/L resulted in increases in the absolute LCR amplitude (Figure 2A), spatial width (Figure 2B), duration (Figure 2C), and frequency (Figure 2D). Figure 2 also shows that spatiotemporal properties of LCRs in permeabilized cells at 150 nmol/L Ca²⁺ most closely resemble those in spontaneously beating cells.

To determine whether ignition of LCRs during diastolic depolarization in spontaneously beating cells requires the concomitant depolarization, the membrane potential of these cells was acutely voltage-clamped. Figure 3A illustrates APs and confocal linescan images of subsarcolemmal Ca²⁺ in a representative, spontaneously beating cell prior to, during, and following acute voltage clamp at the maximum diastolic potential. The voltage clamp terminates spontaneous beating, which is restored on voltage clamp removal. The main point of the figure is that LCRs (arrows) not only occur during diastolic depolarization during spontaneous beating, but also occur during voltage clamp, in the absence of a change in membrane potential. Similar LCRs were recorded when cells were paced under voltage clamp conditions with an AP waveform (to ensure SR Ca²⁺ loading) before voltage clamping of the membrane at –60 mV (n=3, data not shown). We next compared the characteristics of LCRs occurring during diastolic depolarization during spontaneous beating to those occurring during voltage clamp. The time during voltage clamp of each cell was divided into "would-be" cycles, ie, time-intervals equal to the cycle length during spontaneous beating (Figure 4A). The first "would-be" cycle during voltage clamp is of particular significance, because at this time SR Ca²⁺ loading or RyR inactivation status would not be expected to differ from those during the prior spontaneous beating. Figure 4B demonstrates that the signal mass of local Ca²⁺ releases during the first "would-be" cycle does not differ from that during spontaneous beating.

View larger version (33K): [in this window] [in a new window]   Figure 3. LCR occurrence in the absence of changes in membrane potential in sinoatrial nodal cells with intact sarcolemma. A, Recordings of APs. B, Linescan image (see illustration). C, Normalized subsarcolemmal fluorescence averaged spatially over the band indicated by double-headed arrow in B, in a representative cell measured before, during, and after voltage clamp at the maximum diastolic potential.

View larger version (31K): [in this window] [in a new window]   Figure 4. LCR total signal mass within the cycle during spontaneous beating and during "would-be" inter-AP intervals during voltage clamp at the maximum diastolic potential. A, Linescan image of a representative sinoatrial nodal cell during spontaneous beating and during voltage clamp. Horizontal arrowheads indicate LCRs, and vertical arrows indicate "would-be" cycles during voltage clamp, ie, times corresponding to the inter-AP interval if spontaneous firing were to have continued in the absence of voltage clamp. Local change in Ca2+ resulting from each LCR event during diastolic depolarization or during voltage clamp was characterized by its signal mass (see Materials and Methods). B, Average total LCR signal mass in 9 cells in control during spontaneous beating, and during each "would-be" cycle of voltage clamp.

With increasing time during voltage clamp, in the absence of regularly occurring, spontaneous APs, the signal mass of Ca²⁺ release increased to a maximum during the second "would-be" cycle, then gradually decreased (see Figures 3 and 4), and finally ceased by the 14th "would-be" cycle and the damping of LCRs was accompanied by a decrease in the resting, submembrane [Ca²⁺] (Figure 3C). Figure 5D demonstrates that this decrease in diastolic [Ca²⁺] was significant and reached up to 80% of that during spontaneous firing. When voltage clamp was terminated and spontaneous excitations restored, submembrane diastolic [Ca²⁺] gradually returned to the control level.

View larger version (21K): [in this window] [in a new window]   Figure 5. Voltage clamp at the maximum diastolic potential decreases SR Ca2+ content and intracellular [Ca2+] in sinoatrial nodal cells. Spontaneous Ca2+ transients and caffeine-induced Ca2+ releases, indexed by F/F0, in a representative cell during spontaneous beating (A) and in a representative cell after several seconds of voltage clamp (B). C, Average response to caffeine in cells during spontaneous beating (n=9) and cells subjected to several seconds of voltage clamp (n=6). Initial rapid component of the caffeine-induced Ca2+ transient in each cell is normalized to the amplitude of Ca2+ transient during spontaneous beating. D, Averages of submembrane diastolic [Ca2+] during spontaneous beating before, during, and after voltage clamp in 9 cells. *P<0.05.

To determine whether a decrease in the SR Ca²⁺ load also occurs during voltage clamp, we applied a pulse of caffeine, which rapidly empties the SR Ca²⁺ store, during spontaneous beating (Figure 5A) and after several seconds of voltage clamp (Figure 5B). After several seconds of voltage clamp there was a significant reduction of the caffeine-induced Ca²⁺ release, suggesting a decrease in the SR Ca²⁺ load, to 54% of that during spontaneous beating (Figure 5C).

Although the experiments in Figures 3 and 4 demonstrate that LCRs in spontaneously beating cells do not require changes in membrane potential, there is still a possibility that they could be triggered by Ca²⁺ influx during spontaneous beating as well as during voltage clamp. It has been suggested that T-type Ca²⁺ current is a trigger for LCRs in latent cat atrial pacemaker cells.¹² To define whether this was the case in rabbit sinoatrial nodal cells, we compared number of LCRs during spontaneous beating before and after exposure to 50 µmol/L Ni²⁺. There was no difference in the number of LCRs per cycle during spontaneous beating before (1.3±0.2, n=6) and after (1.4±0.2, n=6) superfusion with 50 µmol/L Ni²⁺. Moreover, in all cells subjected to 50 µmol/L Ni²⁺, LCRs were not abolished during voltage clamp (n=4, see a representative example in online Figure 1, available in the online data supplement at http://circres.ahajournals.org).

Local RyR Ca²⁺ Releases Are Roughly Periodic
Insofar as LCRs during voltage clamp do not require a depolarizing trigger, it is possible that they are a manifestation of an intracellular rhythmic or roughly periodic Ca²⁺ oscillator. This periodicity is difficult to prove in cells voltage-clamped at –60 mV because at this potential LCRs become damped and ultimately cease (Figures 3 and 4), commensurate with the reduction in submembrane diastolic [Ca²⁺] and SR Ca²⁺ loading (Figures 3 and 5), likely mediated in part at least, by Ca²⁺ efflux via Na⁺-Ca²⁺ exchanger (NCX) during voltage clamp under these conditions. To reduce Ca²⁺ efflux during voltage clamp, the membrane was clamped in a subset of cells at –10 mV, ie, a potential closer to the reported reversal potential for NCX in rabbit myocytes (about –13 mV).¹³ Figure 6A demonstrates, in a representative cell, that at this membrane potential a decrease in the submembrane diastolic Ca²⁺ level does not occur during the voltage clamp, and that LCRs persist throughout the voltage clamp with little damping (lower trace). The FFT of the Ca²⁺ releases during voltage clamp (Figure 6B) exhibits periodicity, with a dominant power at 2.9 Hz (red lines in Figure 6B). Similar results were obtained in five cells.

View larger version (29K): [in this window] [in a new window]   Figure 6. Local Ca2+ releases exhibit periodicity during voltage clamp and are associated with current fluctuations of similar periodicity. A, Simultaneous recordings of AP (top) and linescan image (middle), normalized fluorescence (bottom) averaged over the image width in a representative cell before and during voltage clamp to –10 mV. Inset shows current oscillations recorded during voltage clamp. Because these oscillations were imposed on the total membrane current (online Figure 3), each data set was fit with a nonlinear regression line that was subtracted to give a difference signal to minimize frequency interference.33 B, Fast Fourier transform of Ca2+ waveform (red line) and current oscillations (green line) shown in A. Inset depicts smoothed curves obtained with Gaussian filter.

Our previous studies have demonstrated that subsarcolemmal LCRs occurring during diastolic depolarization in rabbit sinoatrial nodal cells activate inward NCX current.¹ Thus, the Ca²⁺ fluctuations observed during voltage clamp in Figure 6 might be expected to generate current fluctuations. We used a recently developed numerical model of primary sinoatrial nodal cells, which features LCRs during diastolic depolarization¹¹ to simulate the characteristics of putative membrane current oscillations that would be induced by the LCRs during voltage clamp. The model simulation predicted that when the membrane is clamped at –10 mV as in Figure 6, stable current oscillations, generated mostly by fluctuations in NCX current, occur with an amplitude about 10 pA (online Figure 3). The amplitude of experimentally measured current oscillations occurring concurrently with LCRs was about 14 pA (Figure 6, inset), close to the model prediction. Moreover, the FFT of these current oscillations exhibits similar periodicity as that of LCRs, with the dominant power at 2.9 Hz (green lines in Figure 6B). Importantly, the dominant period of 345 ms both in calcium and current oscillations during voltage clamp is about 20% shorter than the spontaneous cycle length of 435 ms in this cell before voltage clamp. Similar results were obtained in five cells.

Periodicity of Local RyR Ca²⁺ Releases During Steady-State Spontaneous Beating Is Linked to the Spontaneous Cycle Length
The spontaneous beating rate among isolated rabbit sinoatrial nodal cells varies over a substantial range of frequencies (from about 1.2 to 4 Hz). If submembrane Ca²⁺ releases during spontaneous beating are rhythmic and their occurrence does indeed modulate the spontaneous beating frequency, then cells that beat faster ought to have shorter spontaneous LCR periods than those that beat at a slower rate. To test this hypothesis, we compared the period of LCRs during spontaneous beating to the period of spontaneous cycle length in a large number of cells (n=23). The LCR period during spontaneous beating was characterized as the interval from the rapid upstroke of the Ca²⁺ transient caused by the prior AP to the upstroke of the subsequent LCRs (Figure 7).

View larger version (26K): [in this window] [in a new window]   Figure 7. Periodicity of LCRs during spontaneous beating and during the first "would-be" cycle of voltage clamp. A, Confocal linescan images (see cell illustration) and normalized subsarcolemmal fluorescence averaged over the image width in a representative sinoatrial nodal cell measured before and during voltage clamp. Double-headed arrows delineate the LCR period during spontaneous beating and during the first "would-be" cycle during voltage clamp. B, Red symbols, relationship between cycle length and the LCR period during spontaneous beating (10 cells, 86 LCRs); yellow symbols, the relationship between LCR period and the first "would-be" cycle during voltage clamp (13 cells, 17 LCRs). C, Red symbols, probability of LCR occurrence as a function of the relative cycle length in cells in B during spontaneous beating; yellow symbols, during the first "would-be" cycle during voltage clamp. Dashed line is the line of identity.

Figure 7B (red symbols) shows that the period of LCRs during spontaneous beating is variable among cells, and its variation directly correlates with the variation in the spontaneous cycle length. Importantly, regardless of the absolute spontaneous cycle length, which varies from cell to cell, LCRs predominantly occur at constant relative time, ie, at 80% to 90% of that cycle length. The yellow symbols in Figure 7 indicate the LCR periods during the first "would-be" cycle during voltage clamp. Note the same close correlation of LCR period with the first "would-be" cycle (Figure 7B) and the highest probability of LCR occurrence at 80% to 90% of the first "would-be" cycle during voltage clamp (Figure 7C).

Links Between LCR Period and the Spontaneous Beating Rate in the Transient State
Prior observations that ryanodine reduces the beating rate of pacemaker cells^1,14–18 have provided crucial clues regarding a role of RyR Ca²⁺ release in sinoatrial nodal cells. In the present study, the eventual decline in SR Ca²⁺ load and abolition of RyR Ca²⁺ release during voltage clamp mimics, in some ways, the effects of ryanodine in spontaneously beating sinoatrial nodal cells. Although such ryanodine effects are not reversible, removal of the voltage clamp permits spontaneous beating to resume and the cycle length gradually achieves the rate before voltage clamp. This transient state after removal of voltage clamp presents an additional opportunity to demonstrate a link between LCRs during the diastolic depolarization and the spontaneous beating rate as the cell Ca²⁺, SR Ca²⁺ load, and LCRs are restored on a beat-to-beat basis, to the pre–voltage clamp steady state. Figure 8 (representative example in Figure 8A, and average data in Figure 8B) shows that on removal of the voltage clamp, a gradual beat to beat recovery of LCR signal mass occurs concurrently with an increase in the diastolic depolarization rate and a reduction in the cycle length, until the pre–voltage clamp level of all parameters is achieved. The beat-to-beat recovery of total LCR signal mass and the increase in the rate of diastolic depolarization among cells were highly correlated, as were the recovery of LCR signal mass and recovery of spontaneous beating rate. Figure 8C demonstrates that the transient recovery of the LCR period after voltage clamp is highly correlated with recovery of spontaneous cycle length. Moreover, the relationship between LCR period and cycle length is the same as that observed during spontaneous beating in the steady state.

View larger version (30K): [in this window] [in a new window]   Figure 8. Simultaneous recovery of LCR period and cycle length after removal of voltage clamp. A, Simultaneous recordings of APs (top) and linescan image (bottom, see cell illustration) in a representative sinoatrial nodal cell during recovery from voltage clamp. Arrow indicates the moment of voltage-clamp removal. B, Average relative recovery of total signal mass of LCRs, cycle length, and diastolic depolarization (DD) rate after removal of voltage clamp (n=5). Recovery of total signal mass and diastolic depolarization rate of LCRs was highly correlated (R2=0.78), as was the recovery of total signal mass and spontaneous cycle length of LCRs (R2=0.92). C, Relationship of the recovery of the LCR period and that of the cycle length after removal of voltage clamp (green triangles) was highly correlated (R2=0.86). Relationship between LCR period and cycle length during spontaneous beating (red squares) is replotted from Figure 6B. Dashed line is the line of identity.

We next used our novel primary pacemaker cell numerical model to illustrate how the LCR period during diastolic depolarization affects the spontaneous steady-state cycle length of primary pacemaker cell when numerically approximated LCRs of different periods are introduced into the model. The simulated spontaneous cycle length closely followed the LCR period, demonstrating how LCRs are able to drive spontaneous beating rate of sinoatrial nodal cells over a wide physiological range (online Figure 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The advent of Ca²⁺ sensitive indicators, coupled to confocal imaging with simultaneous measurement of membrane potential, has permitted the detection of LCRs beneath the cell membrane during diastolic depolarization. Recent studies^1,2,12 have shown that LCRs during diastolic depolarization activate NCX, which produces a large inward current^1,9,19 in pacemaker cells, and enhances the rate of the later part of the diastolic depolarization.

The first novel finding of the present study is that the occurrence of LCRs during diastolic depolarization in rabbit sinoatrial nodal cells does not require the concomitant change in membrane potential. Thus, local LCRs occur in saponin "skinned" cells bathed at physiological [Ca²⁺] and during acute voltage clamp of spontaneously beating cells (Figures 1 and 3). During the initial stage of voltage clamp, ie, the first "would-be" cycle, when SR Ca²⁺ load and RyR inactivation are the same as during the prior spontaneous beating, the LCR characteristics are the same as those during the diastolic depolarization during spontaneous beating (Figure 4). The characteristics of LCRs in intact, spontaneously beating cells are strikingly similar to those in "skinned" cells bathed in 150 nmol/L Ca²⁺. As might be expected, calibration of the fluorescent Ca²⁺ probe estimated that diastolic [Ca²⁺] in spontaneously beating sinoatrial nodal cells averages about 160 nmol/L [Ca²⁺]_i. This [Ca²⁺]_i is comparable to that of a prior study in rabbit sinoatrial nodal cells,¹⁷ but lower than that in rabbit ventricular myocytes during stimulation (294 nmol/L).²⁰

A second novel finding of the present study, both measured experimentally and predicted by a numerical model simulations (see online data supplement), is that the occurrence of LCRs in sinoatrial nodal cells is rhythmic and generates rhythmic inward current fluctuations. Roughly periodic LCRs from the SR, eg, Ca²⁺ waves, or Ca²⁺ sparks that are not dependent on surface membrane depolarization have been demonstrated to occur in a variety of mammalian cell types.^21–24 The term "spontaneous" SR Ca²⁺ release has been applied to describe this phenomenon in other cell types, because no specific trigger external to the SR has been identified. Some evidence suggests that a Ca²⁺-dependent mechanism that is internal to the SR is involved in spontaneous local RyR Ca²⁺ release.²⁵

Although we have demonstrated that T-type Ca²⁺ current is not required for LCR occurrence in rabbit sinoatrial pacemaker cells, as it is in cat latent atrial pacemaker cells,¹² there is a possibility that endogenous messengers such as cADP-ribose, IP₃, or NAADP could trigger LCRs in rabbit pacemaker cells. Also, a Ca²⁺ triggers originating from other intracellular compartments cannot be ruled out. Regardless of the molecular release mechanism, LCRs occurrence in the absence of depolarization in rabbit sinoatrial nodal cells in the present study appears to be a manifestation of a general phenomenon of mammalian excitable cardiac cells. Sinoatrial nodal cells, however, utilize LCRs in the context of unique system of ionic channels²⁶ to modulate their spontaneous beating rate.

The fact that LCR occurrence during a given diastolic depolarization in spontaneously beating sinoatrial nodal cells does not require the concomitant depolarization does not mean that this Ca²⁺ release is unregulated or unstable. Quite the contrary. The timing and magnitude of this diastolic Ca²⁺ release is highly regulated by the occurrence of the prior AP, which, by triggering SR Ca²⁺ release, causes SR Ca²⁺ depletion and synchronizes inactivation states among RyRs. SR Ca²⁺ loading and RyR inactivation then restitute with time after AP, and during the late diastole, spontaneous Ca²⁺ release, via some RyRs begins to occur. The concurrence of a subsequent AP limits the extent of spontaneous diastolic release to that observed during each spontaneous cycle. If the next AP does not occur, as during voltage clamp, the spontaneous release grows in magnitude and becomes maximal, after about one second under the present conditions, before waning and ceasing over several seconds in the absence of APs (Figures 3 and 4). This transient increase in LCR total signal mass after cessation of spontaneous beating during voltage clamp and its subsequent decay strikingly resemble what has been referred to in rabbit cardiac muscle as rest potentiation^27,28 and decay of Ca²⁺ transients or Ca²⁺ spark frequency²⁹ or contractions after acute cessation of stimulation in ventricular myocytes. This transient overshoot and decay of Ca²⁺ release thought to result from time-dependent changes in SR Ca²⁺ loading and of the RyR activation status.³⁰

Regularly occurring APs also regulate diastolic Ca²⁺ release by activating sarcolemmal Ca²⁺ channels to cause Ca²⁺ influx, which when pumped into the SR, regulates its Ca²⁺ load, a major determinant of RyR Ca²⁺ release. The results of the present study (Figure 5) show that in pacemaker cells, diastolic [Ca²⁺] and caffeine releasable Ca²⁺ declines with time during voltage clamp. In other words, the Ca²⁺ load dissipates via efflux through NCX in the absence of regularly occurring APs. When diastolic [Ca²⁺], and presumably the SR Ca²⁺ load during voltage clamp, are maintained closer to that during spontaneous beating, by clamping the membrane at –10 mV to reduce Ca²⁺ loss via NCX, local RyR Ca²⁺ releases persist throughout the voltage clamp.

A third, and probably the most notable novel finding of the present study is that the LCR period during spontaneous beating of sinoatrial nodal cells is directly linked to the spontaneous cycle length. Both the period of LCRs during spontaneous beating and spontaneous steady-state cycle lengths varies among cells over a wide range (from about 250 to 900 ms). The LCR period is highly correlated with the cycle length during spontaneous beating (Figure 7B). In the transient state after recovery from voltage clamp, the spontaneous cycle length is also highly correlated with the recovery of LCRs (Figure 8C). In both transient and steady state, the LCR period is slightly shorter than the cycle length, suggesting that the latter is driven by the former. Simulations of a novel primary pacemaker cell numerical model show that experimentally derived LCRs of varying period is sufficient to drive the spontaneous cycle length over the wide physiological range.

In summary, rhythmic LCRs in rabbit sinoatrial nodal cells are linked to the spontaneous beating rate of these cells. Although the immediate cause of local submembrane Ca²⁺ release during diastolic depolarization is not dependent on the concomitant change in membrane potential, this Ca²⁺ release is highly and cyclically regulated by the occurrence of the prior and subsequent AP and is regulated by the ensemble of ion channels²⁶ that become activated and inactivated with the occurrence of APs during spontaneous beating of pacemaker cells. Thus, pacemaker function is dependent on intricate interactions between LCRs and APs: the LCR period modulates the occurrence of the next AP, which ensures an SR Ca²⁺ load sufficient to generate subsequent LCRs.

	Acknowledgments

 
This work was supported by an NIH National Institute on Aging Intramural Research Program. The authors are deeply grateful to Dr Harold A. Spurgeon and Bruce Ziman for their assistance.

	Footnotes

 
Original received June 23, 2003; resubmission received January 6, 2004; revised resubmission received January 30, 2004; accepted February 3, 2004.

	References

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