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(Circulation Research. 2003;92:e41.)
© 2003 American Heart Association, Inc.

Research Commentary

Sarcoplasmic Reticulum Ca²⁺ Release Is Not a Dominating Factor in Sinoatrial Node Pacemaker Activity

H. Honjo, S. Inada, M.K. Lancaster, M. Yamamoto, R. Niwa, S.A. Jones, N. Shibata, K. Mitsui, T. Horiuchi, K. Kamiya, I. Kodama, M.R. Boyett

From the Departments of Humoral Regulation and Circulation (H.H., R.N., K.K., I.K.), Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; School of Engineering (S.I., K.M., T.H.), Tokyo Denki University, Tokyo, Japan; School of Biomedical Sciences (M.K.L., M.Y., S.A.J., M.R.B.), University of Leeds, Leeds, UK; and Tokyo Women’s Medical College (N.S.), Tokyo, Japan.

Correspondence to Professor M.R. Boyett, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. E-mail m.r.boyett{at}leeds.ac.uk

Abstract

Recent work on isolated sinoatrial node cells from rabbit has suggested that sarcoplasmic reticulum Ca²⁺ release plays a dominant role in the pacemaker potential, and ryanodine at a high concentration (30 µmol/L blocks sarcoplasmic reticulum Ca²⁺ release) abolishes pacemaking and at a lower concentration abolishes the chronotropic effect of ß-adrenergic stimulation. The aim of the present study was to test this hypothesis in the intact sinoatrial node of the rabbit. Spontaneous activity and the pattern of activation were recorded using a grid of 120 pairs of extracellular electrodes. Ryanodine 30 µmol/L did not abolish spontaneous activity or shift the position of the leading pacemaker site, although it slowed the spontaneous rate by 18.9±2.5% (n=6). After ryanodine treatment, ß-adrenergic stimulation still resulted in a substantial chronotropic effect (0.3 µmol/L isoproterenol increased spontaneous rate by 52.6±10.5%, n=5). In isolated sinoatrial node cells from rabbit, 30 µmol/L ryanodine slowed spontaneous rate by 21.5±2.6% (n=13). It is concluded that sarcoplasmic reticulum Ca²⁺ release does not play a dominating role in pacemaking in the sinoatrial node. The full text of this article is available at http://www.circresaha.org.

Key Words: sinoatrial node • automaticity • ryanodine receptor • ß-adrenergic stimulation • Ca²⁺ transient

Sarcoplasmic reticulum (SR) Ca²⁺ release has been implicated in sinoatrial (SA) node pacemaking. It is assumed that the intracellular Ca²⁺ transient resulting from SR Ca²⁺ release activates inward Na⁺-Ca²⁺ exchange current, and this helps to generate the pacemaker depolarization. Two studies by Lakatta and coinvestigators^1,2 published recently in Circulation Research have placed the spotlight on this mechanism: Bogdanov et al¹ suggested that SR Ca²⁺ release may be obligatory for pacemaking, because they observed that a high concentration (30 µmol/L) of ryanodine, which blocks the SR Ca²⁺ release channel, abolishes the spontaneous activity of isolated SA node cells from rabbit. Vinogradova et al² boldly suggested that the positive chronotropic effect of ß-adrenergic stimulation is the result of the increase in the Ca²⁺ transient caused by ß-adrenergic stimulation, because they observed that the chronotropic effect in isolated SA node cells from rabbit is abolished or greatly reduced after the suppression of the Ca²⁺ transient by a submaximal concentration of ryanodine (3 µmol/L). These recent reports are surprising. Previously, the role of SR Ca²⁺ release was thought to be more minor, because in isolated SA node cells from rabbit and guinea pig, suppression of the Ca²⁺ transient by a variety of interventions (including up to 10 µmol/L ryanodine) did not abolish pacemaking and just decreased spontaneous rate by 21% to 37%.^3–5 In this scenario, it is assumed that multiple ionic currents (I_Na, I_Ca,L, I_Ca,T, I_K,r, I_b,Na, and I_f as well as I_NaCa) are involved in the generation of the pacemaker potential.⁶ As highlighted by DiFrancesco and Robinson,⁷ the conclusion that the chronotropic effect of ß-adrenergic stimulation is the result of an increase in the Ca²⁺ transient is also surprising, because the chronotropic effect of ß-adrenergic stimulation has been previously attributed to actions on ionic currents such as I_Ca,L, I_K,r, and I_f. The discrepancy between the results of Bogdanov et al¹ and others could be due to either (1) the use of a maximal concentration (30 µmol/L) of ryanodine by Bogdanov et al¹ and a submaximal concentration (10 µmol/L) of ryanodine by others or (2) the well-known heterogeneity of the SA node⁸ and the possibility that different investigators have used different SA node cell types. The aim of the present study was to investigate the effect of a maximal concentration of ryanodine on the intact SA node of the rabbit.

Materials and Methods

The SA node and some surrounding atrial muscle were dissected from the rabbit heart as described previously⁹ and then superperfused with modified Krebs-Ringer solution at 32°C. The Krebs-Ringer solution contained (in mmol/L) NaCl 120.3, KCl 4.0, CaCl₂ 1.2, MgSO₄ 1.3, NaHPO₄ 1.2, NaHCO₃ 25.2, and glucose 11.0 (pH 7.4) (gassed with 95% O₂/5% CO₂). Ryanodine or isoproterenol was added when required. Extracellular potential recordings were made from the epicardial surface of the preparation using a grid of 120 pairs of modified bipolar electrodes arranged in a 10x12 matrix with an interelectrode distance of 1 mm.¹⁰ Signals were amplified (80 dB), filtered (0.5 to 30 Hz), acquired, and stored on a personal computer. The point of initial negative deflection in each electrogram was taken as the time of local activation.¹¹ From these measurements, activation maps were constructed by plotting isochrones at 5-ms intervals. Single cells were isolated from the rabbit SA node using a collagenase-elastase digestion technique as described previously.¹² Cells were loaded with the Ca²⁺ indicator fluo-3 (5 µmol/L fluo-3 AM for 5 minutes at room temperature). Cells were superfused with Tyrode solution at 37°C. The Tyrode solution contained (in mmol/L) NaCl 134, KCl 4, CaCl₂ 2, MgCl₂ 1, glucose 11, and HEPES 10 (pH 7.4) (titrated with NaOH). Ryanodine was added when required. Experiments were conducted on spontaneously active single cells only. Fluo-3 fluorescence was excited using light with a wavelength of 488 nm and measured between 520 and 560 nm. Movement artifacts were avoided by measuring fluorescence from the entire cell, which was subject to uniform illumination intensity. Fluorescence is expressed as the ratio of F (fluorescence intensity) to F₀ (lowest diastolic fluorescence intensity during course of an experiment). Data are expressed as mean±SEM (number of preparations or cells). Statistical significance was evaluated by Student’s t test, and a value of P<0.05 was considered significant.

New Zealand White rabbits were purchased from Harlan Ltd (Leicestershire, UK). All animal procedures were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.

Results

Under control conditions, the right atrial preparations showed regular spontaneous activity and the spontaneous rate was 124.5±7.7 bpm (n=6). Figure 1A shows a typical example of an activation map. As expected, the action potential was first initiated (asterisk in Figure 1A) in the center of the SA node. From here, it propagated to the crista terminalis (CT). In the opposite direction (toward the interatrial septum), conduction was blocked (as shown by the clustering of the isochrones) and conduction occurred around the top of the block zone to reach the interatrial septum (SEP). The application of 30 µmol/L ryanodine resulted in a significant decrease of the spontaneous rate of 18.9±2.5% to 100.9±6.8 bpm (P<0.001) (Figure 2B). In no preparation did ryanodine cause spontaneous activity to cease. The ryanodine was applied for 60 minutes. This was sufficient for access of ryanodine to the tissue, because after this time spontaneous rate had settled at a new steady state and the mechanical beating of the preparation was very weak. Figure 1B shows the activation map from the same preparation as Figure 1A after the treatment with 30 µmol/L ryanodine. The position of the leading pacemaker site (asterisk) and the pattern of activation were unchanged. Similar results were obtained from the other preparations studied. It is well known that effects of ryanodine are irreversible,¹³ and therefore after ryanodine application, ryanodine was washed off. The subsequent application of 0.3 µmol/L isoproterenol for 10 or 15 minutes resulted in a substantial increase of the spontaneous rate in all preparations studied. Isoproterenol significantly increased the spontaneous rate by 52.6±10.5% to 160.3±13.7 bpm (P<0.005; n=5) (Figure 2B). Figure 1C shows the activation map from the same preparation as in Figure 1A after the treatment with 0.3 µmol/L isoproterenol. The leading pacemaker site (asterisk) was shifted toward the superior vena cava and the pattern of activation was altered.

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of 30 µmol/L ryanodine and 0.3 µmol/L isoproterenol on pacemaking. A through C, Activation maps of a right atrial preparation under control conditions (A), after the application of ryanodine for 60 minutes (B), and after the washoff of ryanodine and the application of isoproterenol for 15 minutes (C). Dots indicate position of recording electrodes; asterisk, position of the leading pacemaker site; lines, isochrones at 5-ms intervals; CT, crista terminalis; SVC, superior vena cava; SEP, interatrial septum; IVC, inferior vena cava; and RA, right atrial appendage.

View larger version (20K): [in this window] [in a new window]   Figure 2. A, Effect of 30 µmol/L ryanodine on spontaneous Ca2+ transients recorded from an isolated SA node cell. Averaged traces are shown before (Control) and after application of ryanodine. B, Mean percentage change in spontaneous rate (±SEM) on application of ryanodine and isoproterenol to the intact sinoatrial node and on application of ryanodine to isolated SA node cells. The number of cells (n) is shown below the bars.

The present findings from the intact SA node of the rabbit differ from those of Bogdanov et al¹ and Vinogradova et al² from isolated SA node cells from rabbit. To test whether this difference is due to the use of a multicellular preparation rather than isolated cells, the effect of 30 µmol/L ryanodine on the spontaneous Ca²⁺ transients of cells isolated from the rabbit SA node was investigated. In 13 cells, application of ryanodine for 5 to 10 minutes (sufficient for attainment of steady-state effect) increased the time to peak of the Ca²⁺ transient (time from minimum diastolic Ca²⁺ to peak systolic Ca²⁺) from 62±8 to 114±11 ms (a slowing of 89±1%; P<0.0001) and reduced the amplitude of the Ca²⁺ transient by 51.3±8.4% (P<10^-4) as expected (Figure 2A shows an example). It also slowed their rate by 21.5±2.6% from 216±12 to 168±12 bpm (P<5x10^-6) (Figure 2). Under control conditions, there was a rise in diastolic Ca²⁺ during late diastole in all cells (n=11) in which the signal-to-noise ratio of the fluorescence signal was sufficiently good to detect changes during diastole. The rise was abolished after the application of ryanodine.

Discussion

On the basis of these results, it is concluded that block of SR Ca²⁺ release by 30 µmol/L ryanodine does not abolish spontaneous activity in the intact SA node of the rabbit, although it does reduce the spontaneous rate. In the present study, the decrease in rate caused by 30 µmol/L ryanodine (18.9±2.5%) is similar to that reported by others^3–5 (21% to 37%) on reducing the Ca²⁺ transient by a variety of interventions (including ryanodine). The continued spontaneous activity in the presence of 30 µmol/L ryanodine observed in the present study differs from the cessation of spontaneous activity reported by Bogdanov et al¹ in isolated SA node cells from rabbit. The difference is not the result of a difference in ryanodine concentration and it is unlikely to be due to the difference in preparation used, because in the present study in isolated SA node cells from rabbit continued spontaneous activity in the presence of 30 µmol/L ryanodine was again observed. It is possible that the heterogeneity of cell types in the rabbit SA node is responsible for the difference in the results obtained in the present study and the study of Bogdanov et al.¹ We have recently reported that immunolabeling of Ca²⁺ handling proteins, including the SR Ca²⁺ release channel (ryanodine receptor), Na⁺-Ca²⁺ exchanger, and L-type Ca²⁺ channel is significantly sparser and more poorly organized in the center of the rabbit SA node compared with its periphery.¹⁴ This could lead to a regional difference in the role of SR Ca²⁺ release and this may explain the difference between the two studies. The action potentials shown in the studies of Bogdanov et al¹ and Vinogradova et al² resemble those from transitional or peripheral cells rather than central (ie, leading pacemaker) cells. Another possibility is that the differences are the result of the method of cell isolation or cell dialysis in the study of Bogdanov et al.¹ The differences are unlikely to be the result of the measurement of spontaneous rate from extracellular potential or fluorescence recordings in the present study and from intracellular action potential recordings in the study of Bogdanov et al.¹ The results from the present study support the view that multiple ionic currents are involved in the generation of the pacemaker potential in the intact SA node. This is consistent with the known slowing or abolition of spontaneous activity of rabbit SA node (isolated cells or intact tissue) on block of I_Na, I_Ca,L, I_Ca,T, I_K,r, and I_f.⁸

In the present study, 0.3 µmol/L isoproterenol still had a marked chronotropic effect on the intact SA node of the rabbit after treatment with 30 µmol/L ryanodine. Isoproterenol 0.3 µmol/L increased the spontaneous rate by 52.6±10.5% in the present study after ryanodine treatment. In comparison, in the study of Vinogradova et al² 0.3 µmol/L isoproterenol increased the spontaneous rate by 32% under control conditions, whereas after treatment with 3 µmol/L ryanodine it had no effect on spontaneous rate. It is possible that in the cells studied by Vinogradova et al² the effect of isoproterenol on SR Ca²⁺ release was a dominating factor in the chronotropic effect of isoproterenol, whereas in the intact SA node it is not, because in the intact SA node pacemaking is primarily controlled by cells in which SR Ca²⁺ release plays a minor role. In rabbit SA node, isoproterenol also increases I_Ca,L, I_K, and I_st, shifts the I_K,r and I_f activation curves, and accelerates deactivation of I_K,r.^15–17 Using mathematical models of rabbit SA node cells, we concluded that all of these actions contribute to the chronotropic effect of ß-stimulation, although the acceleration of deactivation of I_K,r may be the most important factor.¹⁸

Although the present study suggests that SR Ca²⁺ release is not a dominating factor during pacemaking under normal conditions or after ß-stimulation in the mammalian SA node, it is possible that it may be in the amphibian sinus venosus. A low concentration (2 µmol/L) of ryanodine as well as BAPTA and TBQ (blocker of SR Ca²⁺ pump) abolishes pacemaking of isolated sinus venosus cells from the toad.^19,20 However, these experiments should be confirmed using the intact sinus venosus.

Acknowledgments

This work was supported by the British Heart Foundation.

Received August 20, 2002; revision received September 20, 2002; accepted September 25, 2002.

References

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This Article Abstract Full Text (PDF) All Versions of this Article: 92/3/e41    most recent 01.RES.0000055904.21974.BEv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Honjo, H. Articles by Boyett, M.R. Search for Related Content PubMed PubMed Citation Articles by Honjo, H. Articles by Boyett, M.R. Related Collections Calcium cycling/excitation-contraction coupling Ion channels/membrane transport Autonomic, reflex, and neurohumoral control of circulation