Cardiovascular Research

Cardiovascular Research 2000 48(2):254-264; doi:10.1016/S0008-6363(00)00153-X
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Localisation and functional significance of ryanodine receptors during β-adrenoceptor stimulation in the guinea-pig sino-atrial node

Lauren Rigg, Bronagh M Heath, Yi Cui¹ and Derek A Terrar^*

University of Oxford, Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK

^* Corresponding author. Tel.: +44-1865-271-615; fax: +44-1865-271-853

Received 4 November 1999; accepted 13 June 2000

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Recent evidence shows that calcium released from the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart rate. The aim of this study was to investigate the subcellular distribution of ryanodine receptors in the guinea-pig sino-atrial (SA) node and to determine their functional role in the regulation of pacemaker frequency in response to β-adrenoceptor stimulation. Methods: Monoclonal antibodies raised against the cardiac ryanodine receptor were used with confocal microscopy to investigate ryanodine receptor distribution in single guinea-pig SA node cells. The functional role of ryanodine receptors was investigated in both multicellular SA node/atrial preparations and in single SA node cells. Results: Ryanodine receptor labelling was observed in all SA node cells studied and showed both subsarcolemmal and intracellular staining. In the latter, labelling appeared as transverse bands with a regular periodicity of 2 µm. This interval resembled that of the expected sarcomere spacing but did not, however, depend on the presence of transverse tubules. The bands of ryanodine receptors appeared to be located in the region of the Z lines, based on co-distribution studies with antibodies to -actinin, myomesin and binding sites for phalloidin. Functional studies on single SA node cells showed that application of ryanodine (2 µmol/l) reduced the rate of firing of spontaneous action potentials (measured using the perforated patch clamp technique) and this was associated with changes in action potential characteristics. Ryanodine also significantly decreased the positive chronotropic actions of isoprenaline in both multicellular and single cell preparations. In single cells exposed to 100 nmol/l isoprenaline, ryanodine caused a decrease in the rate of firing and this was associated with a decrease in the amplitude of the measured calcium transients. Conclusions: These findings are the first to show immunocytochemical evidence for the presence and organisation of ryanodine receptor calcium release channels in mammalian SA node cells. This study also provides evidence of a role for ryanodine sensitive sites in the β-adrenergic modulation of heart rate in this species.

KEYWORDS SR (function); Calcium (cellular); Heart rate (variability); Receptors; Sinus node

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The sino-atrial (SA) node acts as the primary pacemaker of the heart and its regular spontaneous activity is thought to rely predominantly on the sequential activation of various ionic currents across the sarcolemma e.g. [1]. The possibility that calcium released from the sarcoplasmic reticulum (SR) might play an important role in regulating pacemaker activity in the mammalian SA node was raised by Rigg and Terrar [2], who showed that substances which interfere with the function of the SR (ryanodine to suppress calcium release and cyclopiazonic acid (CPA) to inhibit Ca²⁺ uptake into the SR) slowed the rate of spontaneous action potentials in the intact guinea-pig SA node. Other recent experiments also support a role for calcium released from the SR in regulating pacemaker activity in rabbit SA node [3–5], toad sinus venosus [6,7] and in other pacemaker regions of the heart [8,9].

The prevalence and distribution of the SR in the SA node has not been thoroughly investigated. Indeed, it is thought to be relatively sparse in SA node tissue compared to that observed in atrial or ventricular myocytes, where the SR plays a greater role in contraction. Early investigations described SA node cells as ‘pale’ or ‘empty’ when studied using conventional light or electron microscopy e.g. [10]. Masson-Pevet et al. [10] also reported that the SR in rabbit SA node cells was poorly developed, constituting only 1% of the total cell volume compared with the ventricle where the SR makes up approximately one third of the cell volume. Nevertheless, the negative chronotropic effect of substances which inhibit the function of the SR implies that the SR in SA node may be more extensive than previously estimated.

It is well known that in ventricular cells the contents of the SR are increased in the presence of β-adrenoceptor stimulation, mainly as a consequence of an increase in the L-type calcium current [11] and enhancement of Ca²⁺ uptake by the SR [12]. It is conceivable that filling and subsequent release of calcium from the SR may be enhanced during similar interventions in SA node tissue and may contribute significantly to the positive chronotropic effects of β-adrenoceptor stimulation. Indeed, it has been previously reported that this may be an important mechanism in amphibian pacemaker tissue [7,13] and in other cardiac conducting tissue. For example, Hancox et al. [9] have shown that isoprenaline increases the amplitude of calcium transients in isolated rabbit AV node cells. There are no studies reported to date investigating the SR-dependence of sympathetic modulation of beating rate in mammalian primary pacemaker tissue.

This study aims to determine the distribution of ryanodine receptor calcium release sites in single cells isolated from the guinea-pig SA node and to ascertain whether calcium release from the SR plays an important role in sympathetic modulation of heart rate.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Isolation of cardiac cells
SA node cells were isolated using a combination of previously described techniques [14,15]. Briefly, male guinea-pigs (weighing 600–650 g) were killed by cervical dislocation following stunning. The heart was rapidly removed and perfused by the Langendorff technique with a solution containing zero calcium for 3 min (composition (mmol/l): NaCl 137, KCl 5, NaHCO₃ 12, glucose 5, sodium pyruvate 1, NaH₂PO₄ 0.4, MgCl₂ 1, NaOH 1, EGTA 0.1, pH 7.4, 95% O₂–5% CO₂, 36°C). This was subsequently replaced with a solution of the same composition, lacking EGTA (referred to as solution A) but containing 25 mg collagenase (Type II, activity 172 U/mg, Worthington Biochemicals). Following collagenase perfusion for 12x the weight of the heart in grams (maximum exposure, 36 min), the atria were removed and further dissected to reveal the SA node region in solution A (room temperature, 22–23°C). The SA node region was identified as being bordered by the crista terminalis, superior and inferior vena cava and inter-atrial septum, cut into small strips (2x5 mm) perpendicular to the crista terminalis and dispersed in 2 ml of a solution high in potassium (composition (mmol/l): KCl 70, K₂ATP 5, MgSO₄ 5, K⁺ glutamate 5, taurine, 20, trisphosphocreatine 5, EGTA 0.04, succinic acid 5, KH₂PO₄ 20, glucose 10, HEPES 5, pH 7.2 with KOH) and stored at 4°C for at least 1 h before use. SA node cells used in the present study were dissociated from the entire guinea-pig SA node and therefore represent a mixed population of cells with corresponding heterogeneity in shape; most cells were characteristically spindle shaped but ‘spider’ shaped cells were also used in these experiments; see [16]. Irrespective of their shape, all healthy SA node cells showed faint striations, well defined membranes and displayed regular spontaneous activity when superfused with physiological saline solution (solution B), (mmol/l): NaCl 118.5, KCl 4.2, NaHCO₃ 14.5, KH₂PO₄ 1.18, MgSO₄·7H₂O 1.18, glucose 11.1 and CaCl₂ 2.5, pH 7.4, 95% O₂–5% CO₂, 36°C.

Ventricular cells were isolated using a previously described method [14].

2.2 Immunocytochemistry
Isolated cardiac cells were plated onto flamed glass coverslips (22x22 mm) and allowed to settle for at least 15 min. Cells were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline solution (PBS; Sigma-Aldrich) for 15 min, washed in fresh PBS (three changes, 15 min each), permeabilised using 0.2% Triton-X 100 (Sigma-Aldrich) for a maximum of 30 min, washed thoroughly in PBS before being incubated at room temperature with primary antibody. In some experiments a blocking medium was used prior to exposure to primary antibody (1% bovine serum albumin, 45 min exposure). After two washes in PBS (15 min each), cells were incubated in the dark with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibodies (obtained from Sigma-Aldrich, 1:40 dilution or Jackson Immunoresearch Laboratories, 1:100 dilution, both 1 h incubation). Finally, coverslips were washed thoroughly in PBS, mounted using Vectashield^® (Vector Laboratories) and permanently sealed with clear nail polish. Cells were stored in the dark at 4°C and visualised within 2 days. For control experiments, the primary antibody stage was omitted.

Mouse monoclonal antibodies to the canine cardiac ryanodine receptor [17] were obtained from Oncogene Research Products (Clone C3–33; 1:500 dilution, 2 h incubation). Mouse monoclonal anti--actinin antibodies were obtained from Sigma-Aldrich (Clone EA53, 34 µg/ml, 1 h incubation). Mouse monoclonal anti-myomesin antibodies [18] were a kind gift from Hans M. Eppenberger (Institute of Cell Biology, Zurich, Switzerland) and were used as the undiluted hybridoma supernatant (30 min incubation).

The cell surface membrane was investigated using the dye di-8-ANEPPS (Molecular Probes, 5 µmol/l, 45–50 min incubation before being washed in solution B at room temperature). Actin filaments were identified using Texas Red^®-X phalloidin (Molecular Probes 1.5 units/ml, 30 min incubation). Fixed cells were exposed to phalloidin after incubation with secondary antibodies.

Observations were carried out using a Leica DMIRB inverted microscope modified for confocal laser-scanning microscopy (x63 water immersion objective; 1.0–1.4 pinhole) and Leica TCS-NT analysis software. For measurement of FITC and di-8-ANEPPS, fluorescence excitation was at 488 nm and emission was collected >515 nm. An excitation filter at 568 nm and an emission filter at 600±15 nm were used to detect Texas Red fluorescence (TRITC).

2.3 Electrophysiology
Action potentials were recorded from single SA node cells using the perforated patch configuration and either an Axoclamp-2 amplifier in bridge mode or an Axopatch 200B amplifier in current-clamp mode (Axon Instruments). Patch pipettes (3–5 M) were filled with a solution containing (mmol/l): KCl 150, MgCl₂ 5, K₂ATP 1, HEPES 3, pH 7.2 with KOH. Amphotericin B (Sigma-Aldrich) was used at 240 µg/ml (1.25% DMSO). Voltage signals were filtered at 1 kHz, recorded onto video tape and analysed using PCLAMP software (Axon Instruments).

2.4 Calcium transient measurements
Calcium transients were measured using the membrane permeable fluorescent indicator, indo-1 AM (Sigma-Aldrich, prepared as a 10 mmol/l stock in DMSO). Cells were loaded for 15 min at room temperature with 3 µmol/l indo-1 AM (0.03% DMSO) and then washed for at least 15 min before making any recordings. The cell of interest was excited by light (355 nm) from a Xe arc lamp and reflected up through a x100 oil immersion objective (numerical aperture 1.2, Leica) to the specimen by a dichroic mirror (reflecting light shorter than 375 nm). Emitted light was collected by two photomultipliers at 410 and 495 nm. In some experiments a Cairn fluorescence system was used and emission was collected at 405 nm and 485 nm. Baseline fluorescence was recorded under the same conditions in the absence of a cell. Signals were filtered at 100 kHz, recorded onto video tape and analysed using WCP (whole cell programme, John Dempster, Strathclyde University) or PCLAMP software. Fluorescence ratios were calculated by subtracting baseline fluorescence from each trace and then dividing the 410 nm (or 405 nm) signal by the 495 nm (or 485 nm) signal using AXOGRAPH (Axon instruments) or PCLAMP software. In six cells, autofluorescence was less than 2% of the indo-1 signals and was considered negligible.

2.5 SA node/atrial preparation
This preparation has been described previously [2].

2.6 Drugs
Ryanodine (Calbiochem-Novabiochem) was prepared as a 2 mmol/l stock in distilled water. BAPTA-AM (Molecular Probes) was prepared as a 1 mmol/l stock in DMSO, stored at –20°C and protected from light. CPA (Sigma-Aldrich) was prepared as a 10 mmol/l stock in DMSO and stored at –20°C. Isoprenaline (Sigma-Aldrich) was freshly prepared before experiments as a 1 mmol/l stock (+ascorbic acid) and was kept on ice.

2.7 Statistics
Results are expressed as mean±S.E.M. Significance was determined using paired Student's t-test, unless otherwise stated (significance level, P<0.05).

The investigation was performed in accordance with the Home Office Guidance on the operation of the Animals (Scientific Procedures) Act 1986, published by HMSO, London, UK.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Localisation of ryanodine receptors in isolated guinea-pig SA node cells
Ryanodine receptor localisation was investigated in 30 SA node cells using monoclonal antibodies specific for RyR₂. Fig. 1A–D shows representative labelling that was observed in 90% of the cells studied (cell length, 129±6 µm and cell width, measured close to nucleus, 13±0.5 µm). RyR₂ labelling was observed as distinct bands, particularly at the poles of the cell and close to the nucleus, which displayed a regular periodicity of 1.85±0.03 µm (n = 27) and penetrated deep into the cell interior (Fig. 1Aiii). Labelling was also evident as distinct ‘spots’ located close to the sarcolemma, this is especially clear in Fig. 1C which represents the remaining 10% of SA node cells, in which the density of regular fluorescent banding was somewhat more punctate (cell length, 112±14 µm and cell width, 13±2 µm (n = 3)). These dimensions were not significantly different from those of the majority of the cells studied). In control experiments there was little or no fluorescence labelling (Fig. 1E). Fig. 1F shows that although there was a greater abundance of RyR₂ labelling in ventricular myocytes, the pattern was similar to that observed in SA node myocytes (inter-band interval 1.9±0.11 µm, n = 5).

View larger version (125K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Identification of ryanodine receptors (RyR2) in single cells isolated from the guinea-pig SA node. (Ai and B–D) single SA node cells labelled with anti-RyR2 antibody. (Aii) Transmission image of the cell shown in (Ai) (not to scale). (Aiii) XZ scan of the orientation in (Ai) highlighted by the arrows. (E) Control experiment. (F) Ventricular cell labelled with anti-RyR2 antibody. (G) SA node cell (left panel) and ventricular cell (right panel) labelled with di-8-ANEPPS (inset, XZ scan). Scale bars: 10 µm.

 
The arrangement of the surface membrane was investigated using the dye, di-8-ANEPPS. Fig. 1G shows an example of the staining observed in an SA node (left) and ventricular (right) cell. Surface staining was present in SA node cells and there appeared to be no invaginations characteristic of T-tubules, this was in contrast to the observations in ventricular cells. The absence of T-tubules in SA node cells, but not ventricular cells, is also illustrated in the transverse sections shown as insets in Fig. 1.

To determine the position of RyR₂ within the sarcomere the actin specific stain, Texas Red-phalloidin in combination with either anti-RyR₂ antibodies, antibodies raised against the Z-line associated protein -actinin or antibodies to the M-line protein, myomesin were used. Fig. 2A shows an SA node cell labelled with RyR₂ (i, green) and phalloidin (ii, red). Panel iii represents a merged image produced by superimposing i and ii. Areas giving rise to yellow show co-distribution of RyR₂ and actin. This is more clearly represented in the bottom panel which shows intensity plots for the two fluorescent agents. RyR₂ and phalloidin were regularly co-distributed with an interval of 1.83±0.1 µm between successive sites (n = 3). Fig. 2B shows a different SA node cell labelled with anti--actinin antibodies (i, green) and phalloidin (ii, red). -Actinin was localised in the centre of the actin band with an interval between bands of 1.73±0.2 µm (n = 4). Comparing the localisation of RyR₂ and -actinin with respect to phalloidin shows that both RyR₂ and -actinin are located in the region of the actin band centre which supports the hypothesis that RyR₂ is localised close to the Z-line in isolated SA node cells. This was confirmed using antibodies to the M-line protein, myomesin which was out of register with RyR₂ labelling (Fig. 2C).

View larger version (78K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Localisation of RyR2 with respect to sarcomeric structures in guinea-pig SA node cells. (A) i, Immunolocalisation of RyR2 (FITC, green); ii, labelling of actin filaments using Texas Red phalloidin (TRITC, red); iii, co-distribution of actin and RyR2; iv, fluorescence intensity analysis of RyR2 and phalloidin distribution. (B) i, Immunolocalisation of -actinin; ii, labelling of actin filaments using Texas Red phalloidin; iii, co-distribution of actin filaments and -actinin; iv, fluorescence intensity analysis of -actinin and phalloidin distribution. (C) i, Immunolocalisation of myomesin; ii, labelling of actin filaments using Texas Red phalloidin; iii, merged image of actin and myomesin distribution showing the different locations of these proteins; iv, fluorescence intensity analysis of myomesin and phalloidin distribution. Scale bars: 10 µm.

 
3.2 Effects of ryanodine on the isoprenaline log(concentration)–response curve
The effects of SR inhibition on β-adrenergic stimulation were initially investigated in the multicellular SA node/atrial preparation. Heart rate was determined from the interval between extracellularly recorded action potentials. Fig. 3 shows the mean cumulative log(concentration)–response curve to isoprenaline in control (open circles) and in the presence of ryanodine (filled circles).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effects of SR inhibition on the positive chronotropic actions of the β-adrenoceptor agonist, isoprenaline, in the multicellular guinea-pig SA node–atrial preparation. Cumulative log (concentration)–response curve for isoprenaline in the absence (closed circles) and presence (open circles) of 2 µmol/l ryanodine.

 
In the absence of ryanodine, 1–100 nmol/l isoprenaline caused a rapid increase in beating rate, a steady-state was attained within 3 min of exposure. At the maximum concentration of 100 nmol/l, isoprenaline increased heart rate by 35±6% from the control rate of 215±6 bpm to 294±5 bpm (P<0.05, n = 6). The preparation was washed thoroughly so as to return beat rate to control levels (at least 45 min) and then exposed to 2 µmol/l ryanodine for 30 min. In the presence of ryanodine, heart rate was significantly reduced by 30±3% (P<0.05, n = 6) to 151±20 bpm. Subsequent exposure to 1–100 nmol/l isoprenaline caused a suppression in the maxima of the log(concentration)–response curve. At the maximal dose of 100 nmol/l, isoprenaline increased heart rate by 23±8% to 184±23 bpm from its steady-state rate in the presence of ryanodine, representing a 34±5 bpm change in rate compared with a 79±4 bpm change in the absence of SR inhibition. Ryanodine therefore reduced the capacity of 100 nmol/l isoprenaline to increase rate by 56±7% (n = 6, P<0.05).

These results support the hypothesis that calcium release from the SR makes a significant contribution to the positive chronotropic effects of isoprenaline in the guinea-pig. This possibility was investigated further in single cells dissociated from the guinea-pig sino-atrial node.

3.3 Effects of ryanodine in single pacemaker cells
Sino-atrial node cells were dissociated from the entire node and, as has been previously reported for guinea-pig SA node cells [19], there was considerable variability in beat frequency between cells. The mean frequency however was not significantly different to that recorded in multicellular preparations (172±14 bpm in isolated cells, n = 26; 202±7 bpm in multicellular preparation, n = 25). The effects of ryanodine on rate of beating were investigated in five cells. Following a 5-min exposure to 2 µmol/l ryanodine, the rate was decreased by 75±17% from 145±52 to 18±11 bpm (n = 5, P<0.05). An example of the effects of ryanodine on action potentials is shown in Fig. 4 and Table 1 summarises the action potential characteristics. Of interest was a prominent shift in the MDP from –60±6 mV in control to –54±3 mV in the presence of ryanodine (n = 4, P<0.05). The direction and magnitude of this shift was similar to that observed previously in the multicellular preparation.

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Effects of ryanodine on rate and action potentials in guinea-pig SA node cells. Action potentials recorded from a single SA node cell using the perforated patch clamp configuration in control (left panel) and following a 5-min exposure to 2 µmol/l ryanodine. The arrow head indicates zero mV.

 

View this table: [in this window] [in a new window]   Table 1 Comparison of the effects of ryanodine on rate and action potential characteristics in single guinea-pig SA node cells and in guinea-pig multicellular preparationsa

 
3.4 Effects of β-adrenergic stimulation on action potentials and calcium transients in single SA node cells
Fig. 5 shows typical examples of the effect of 100 nmol/l isoprenaline on rate and characteristics of action potentials (A) and calcium transients (B) recorded from two separate SA node cells.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Effects of isoprenaline on action potentials and calcium transients in single cells isolated from the guinea-pig SA node. (A) Action potentials recorded from a single SA node cell in the absence (left panel) and presence of 100 nmol/l isoprenaline (right panel; 3 min exposure). (B) Calcium transients recorded in a different SA node cell in the absence (left panel) and presence (right panel) of 100 nmol/l isoprenaline. Calcium transients are expressed as a fluorescence ratio (F; 410/495 nm). (C) Action potentials and calcium transients (noisy trace) recorded simultaneously from another SA node cell in control (left panel) and in the presence of 100 nmol/l isoprenaline (right panel).

 
Isoprenaline (100 nmol/l) significantly increased the frequency of action potentials by 138±22% from a resting control rate of 132±19 bpm (P<0.05, n = 9). This was associated with prominent changes in action potential characteristics, in particular an increase in the rate of rise and in the overshoot of the action potential. The mean changes are summarised in Table 2A. Isoprenaline also increased the frequency of recorded calcium transients by 183±73% from a control rate of 89±15 bpm (P<0.05, n = 6, Fig. 5B shows a typical example). This was associated with a 74±5% increase in the amplitude of the transient and a 50±7% decrease in the time constant for decay of the transient from 154±19 ms in control to 74±3 ms in isoprenaline (n = 6, P<0.05).

View this table: [in this window] [in a new window]   Table 2 Effects of alterations in calcium handling on action potential characteristics in single SA node cells exposed to isoprenalinea

 
Fig. 5C shows an example of the temporal aspects of simultaneously recorded action potentials and calcium transients. In control (left panel), the calcium transient increased slowly during diastolic depolarisation, prior to the rapid upstroke of the action potential, and its decay extended beyond repolarisation. In the presence of isoprenaline (right panel) the decay of the transient was clearly quickened.

A consistent finding in this set of experiments was that the rate of spontaneous beating of SA node cells under control conditions and in the presence of 100 nmol/l isoprenaline was significantly less in indo-1 loaded cells than in non-indo-1 loaded cells. This phenomenon has been previously reported [6] and is likely to result from the inherent capacity of fluorescent indicators to buffer intracellular calcium [20]. The difference in rate in the presence of indo-1 appeared to be associated with alterations in the mean action potential characteristics recorded in the presence of isoprenaline. For instance, there was a significant, positive shift in the MDP and a reduction in the rate of rise and overshoot of the action potential (n = 6) compared to action potential characteristics measured in isoprenaline alone (unpaired data, P<0.05, n = 9) and the data are summarised in Table 2A. To test whether buffering of intracellular calcium might modulate both rate and characteristics of action potentials in single SA node cells the effects of the membrane permeable derivative of the calcium chelator, BAPTA (BAPTA-AM) were investigated. Exposure to 10 µmol/l BAPTA-AM in the presence of 100 nmol/l isoprenaline significantly reduced the beating frequency of cells from 309±41 bpm to 87±51 bpm (n = 6, P<0.05). Again, changes in rate were associated with changes in action potential characteristics, for example a positive shift in the MDP and a reduction in the rate of rise of the action potential (P<0.05), mean data are summarised in Table 2B.

3.5 Role of the calcium release from the stores during β-adrenergic stimulation
Fig. 6 shows an example of the effects of ryanodine on simultaneously recorded action potentials and calcium transients in a single SA node cell in the presence of 100 nmol/l isoprenaline (left panel) and following a 3 min (centre panel) and 10 min (right panel) exposure to 2 µmol/l ryanodine. Ryanodine consistently decreased the firing rate of action potentials and this was closely associated with changes in action potential characteristics (see Table 2A) and in the amplitude of the calcium transient. At 3 min exposure, the amplitude of the calcium transients was reduced by 66±12% (n = 6). Exposure to ryanodine for periods longer than 10 min (up to 15 min) was sufficient to completely suppress action potentials in the 6 SA node cells. At this point membrane potential settled around –40±4 mV. There were occasionally small fluctuations in voltage and in the fluorescence signal but these were difficult to measure with accuracy.

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Effects of SR inhibition on simultaneous action potentials and calcium transients in single guinea-pig SA node cells in the presence of isoprenaline. Left panel, action potentials and calcium transients (noisy trace) in the presence of 100 nmol/l isoprenaline. Centre panel, the effects of a 4-min exposure to ryanodine. Right panel, the effects of a 10 min exposure to ryanodine (note that the scale of the fluorescence ratio in the presence of ryanodine is different from that in the absence of ryanodine to facilitate a comparison between calcium transients and action potentials in these panels).

 
In separate experiments where only fluorescence ratios were measured, ryanodine gradually reduced the amplitude of the calcium transients by 53±6% with a corresponding decrease in rate of transients from 156±27 to 56±3 bpm (5 min exposure). Again, longer periods of exposure to ryanodine decreased the rate further (rate decreased to 12±12 bpm with a corresponding reduction in the amplitude of the transient, 93±7%, n = 6, P<0.05).

The effects of another SR inhibitor, CPA, on action potentials and calcium transients in single SA node cells were also investigated. In six experiments where calcium transients were measured alone and in three additional cells where calcium transients were recorded with simultaneous action potentials, a 10-min exposure to 30 µmol/l CPA reduced the frequency of calcium transients by 80±6% of control and this was associated with a reduction in the amplitude of the transients by 53±9% from control, n = 9 P<0.05. In a separate set of experiments where only action potentials were studied, 30 µmol/l CPA significantly reduced beating rate in the presence of 100 nmol/l isoprenaline by 52±10% from 289±24 to 134±27 bpm (P<0.05, n = 6) and caused a positive shift in the MDP from–68±3 to–63±4 mV.

These data support the hypothesis that calcium release from the SR makes a significant contribution to the calcium transient recorded in the presence of isoprenaline. It is possible that changes in the calcium transient underlie changes in action potential characteristics and thus beating rate.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
This study provides the first immunocytochemical evidence for the presence and organisation of ryanodine receptor calcium release channels in SA node cells. It appears from our observations that these calcium release channels are distributed in the cytoplasm as regular bands, localised at the Z-line and having a periodicity of 2 µm. RyR₂ labelling was also frequently observed close to the sarcolemma. The banding pattern did not however depend on the presence of transverse tubules as revealed by the experiments using di-8-ANEPPS, a marker of the surface membrane. This picture is in contrast to that seen in ventricular cells, where a 2-µm spacing for both transverse tubules and bands of RyR₂ receptors was observed.

The distribution of ryanodine receptors observed in SA node cells in the present study is similar to that reported for other myocytes which also possess little or no tubular system (e.g. avian ventricular myocytes, rabbit atrial cells) [21,22]. These cells have abundant subsarcolemmal SR and regularly arranged cytosolic stores, commonly referred to as corbular SR [21,23]. The rate of rise of the calcium transient in atrial cells is rather slow relative to that observed in ventricular muscle and is thought to rely on initial release of calcium from peripheral couplings [24]. It is possible that the spread of calcium induced calcium release from the periphery to the centre may occur in a similar fashion in the SA node.

Previous studies have suggested a functional role for calcium release from the SR in regulating pacemaker activity. In the present study, the observed reduction in firing frequency of pacemaker cells in the presence of ryanodine is consistent with earlier evidence from multicellular guinea-pig preparations [2] and from several other species [3–6]. In the present study, the reduction in beating rate of single cells in the presence of ryanodine was associated with distinct changes in action potential characteristics, in particular a prominent positive shift in the MDP, a reduction in the overshoot of the action potential and a decrease in the rate of rise of the action potential upstroke. In the rabbit, ryanodine shifted the take-off potential to more positive potentials and decreased the rate of rise [3,4]. However, unlike the observations presented here there was no shift in the MDP and an increase in the overall amplitude of the action potential which occurred as a result of an increase in the overshoot. In cat subsidiary pacemaker cells a positive shift in the MDP was observed whereas the rate of rise of the action potential became quicker which was also associated with an increase in the overshoot of the action potential [8]. It is likely that species variation in the expression of certain ion channels which underlie pacemaker activity and their differential regulation by intracellular calcium may account for these discrepancies. For instance, it is accepted that activity of delayed rectifier potassium channels is an important determinant of the MDP. Previous studies in ventricular cells have shown that the current through these channels (I_K) is sensitive to intracellular calcium [25], in particular the slowly activating component of I_K, I_Ks [26]. It has been shown that the predominant potassium current in guinea-pig pacemaker cells is I_Ks [27], whereas that in rabbit SA node cells is the rapidly activating component (I_Kr) [28]. Preliminary voltage clamp data from isolated guinea-pig SA node cells showed that I_K was sensitive to intracellular calcium [29]. It seems likely therefore that there may be significant differences in the extent to which calcium may modulate I_K and thus determine MDP in the two species. The shift in MDP observed in the presence of SR inhibition would influence the activation and amplitude of other pacemaker currents, in particular the hyperpolarisation activated current (I_f) which is thought to play a role in diastolic depolarisation but may itself be modulated by changes in intracellular calcium [30–32].

In the present study, a rise in intracellular calcium was observed prior to the rapid phase of the calcium transient and before the upstroke of the action potential. It remains to be established whether calcium release from the SR might be triggered by calcium entry through T-type calcium channels [33] or L-type calcium channels [34].

While investigating the effects of SR inhibition on calcium transients and action potentials in single SA node cells, it was consistently observed that the beat frequency of node cells loaded with indo-1 was significantly less than in non-indo loaded cells. The possibility that this resulted from the inherent buffering capacity of the fluorescent indicator was supported by the observation that another calcium chelator, BAPTA, reduced the rate of firing of action potentials and caused similar changes in action potential characteristics to those reported for indo-1. These data add further support to the hypothesis that intracellular calcium plays an important role in the regulation of pacemaker rate.

β-Adrenoceptors are coupled to an intracellular signalling pathway which promotes the formation of cAMP [35]. Direct actions of cAMP or subsequent activation of protein kinase A leads to the modulation of some of the ionic currents which underlie pacemaking (e.g. [1,36]). An increase in L-type calcium current would be expected to contribute to the observed increase in the rate of rise and overshoot of the action potential, and to increase calcium loading of the SR. β-Adrenoceptor stimulation with isoprenaline also increased the amplitude of the measured calcium transients and quickened their decay. The observation that SR inhibitors markedly attenuated the calcium transient in the presence of isoprenaline is consistent with the hypothesis that SR calcium contributes substantially to the calcium transient in SA node cells under these conditions. It was also observed that the effects of isoprenaline on rate of beating were markedly attenuated when the preparation was pretreated with ryanodine to suppress calcium release from the SR. These data therefore support the hypothesis that calcium release from the SR plays an important role in determining pacemaker frequency by influencing ionic currents in the presence of isoprenaline in mammalian primary pacemaker tissue.

It has been previously reported in the toad (Bufo marinus) which lacks the I_f current [37], that a large component of β-adrenoceptor regulation is dependent on the SR and that changes in rate are thought to be attributable to modulation of the amount of sodium calcium exchange current.

In summary, it appears that ryanodine receptor calcium release channels are more extensive and more organised in SA node cells than previously recognised. The results presented here are also consistent with a fundamental role for calcium released from the SR in regulating the beating frequency of the heart in the presence of β-adrenoceptor stimulation. The increase in calcium transients caused by β-adrenoceptor agonists, including the sympathetic transmitter noradrenaline, would be expected to contribute to their chronotropic effects. It seems likely that parasympathetic influences might reduce calcium transients and therefore exert opposite effects on rate, but future work is needed to test these possibilities.

Time for primary review 28 days.

	Acknowledgements

 
The authors wish to thank Professor Hans Eppenberger, Institute of Cell Biology, Zurich, Switzerland for supplying the anti-myomesin antibody used in this investigation and Dr. Gordon McMurray and Dr. Francisco Ciruela for their technical advice. Supported by the British Pharmacological Society and The Wellcome Trust.

	Notes

 
¹ Dr. Yi Cui has now moved to The Rayne Institute, University College London, London, UK.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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