Relative Densities of Muscarinic Cholinergic and β-Adrenergic Receptors in the Canine Sinoatrial Node and Their Relation to Sites of Pacemaker Activity
Abstract The site of earliest extracellular electrical activation in the sinoatrial node (SAN) is known to shift in response to autonomic stimuli, but the mechanisms underlying this phenomenon and the determinants of the location of dominant pacemaker activity have not been elucidated. The present study was designed to characterize the spatial distribution of muscarinic cholinergic and β-adrenergic receptors in the canine SAN and to determine whether a consistent relationship exists between autonomic receptor densities and the site of dominant pacemaker activity. We used quantitative light-microscopic autoradiography of radioligand binding sites to characterize the spatial distribution of muscarinic cholinergic and β-adrenergic receptor subtypes in tissue sections containing the SAN and adjacent right atrial muscle from 18 canine hearts. Muscarinic receptor density was 5.4 times greater in SAN cells than in atrial myocytes (P<.01). Total β-adrenergic receptor density was more than 3 times greater in SAN cells than in atrial myocytes (P<.0001), due entirely to the significantly greater number of β1-adrenergic receptors in the SAN. The region of dominant pacemaker activity, localized in 4 hearts with in vitro mapping, consistently exhibited greater densities of muscarinic and β1-adrenergic receptors than other SAN regions. Muscarinic receptor density in the dominant pacemaker region was 18±2% and 29±7% higher than in adjacent superior and inferior regions, respectively. β1-Receptor density in the dominant site was 53±5% and 26±4% higher than in adjacent superior and inferior SAN regions, respectively. Thus, the SAN is richly endowed with both muscarinic cholinergic and β1-adrenergic receptors. Differential responsiveness to autonomic inputs, related perhaps to regional differences in receptor density, may help determine the spatial localization of pacemaker activity.
- β-adrenergic receptor
- muscarinic cholinergic receptor
- sinoatrial node
- atrial pacemaker complex
- electrophysiological mapping
The atrial pacemaker complex has been described as a group of widely distributed pacemakers in the sinoatrial node (SAN) and may also include extranodal atrial pacemaker sites.1 2 3 4 5 It has been hypothesized that this atrial pacemaker complex consists of a group of functionally differentiated cells that generate specific ranges of heart rates.6 Shifts in the site of earliest extracellular activation have been observed in numerous preparations,7 8 9 10 11 12 13 14 15 16 17 particularly in response to direct nerve stimulation, or infusion of autonomic drugs or the naturally occurring neurotransmitters acetylcholine and norepinephrine.7 8 9 Multiple, spatially dispersed pacemaker sites also function spontaneously in the isolated, perfused canine right atrium preparation, in which the influences of neural input, circulating chronotropic substances, and reflexes are eliminated.18 As in the open-chest preparation, the location of earliest extracellular activation in the isolated perfused atrial preparation shifts in response to administration of acetylcholine and norepinephrine. With infusion of acetylcholine, the site of earliest activation shifts inferiorly in some preparations, associated with a decrease in rate, whereas in others a superior shift has been seen.18 With infusion of norepinephrine, the site of earliest activation is consistently found in the superior region of the SAN and exhibits an increased rate. These observations suggest that the superior region of the SAN may be more responsive to β-adrenergic stimulation, possibly because of differences in receptor density or in receptor coupling to effector molecules, whereas there may be no consistent regional difference in SAN responsiveness to acetylcholine.
The SAN is richly endowed with β-adrenergic receptors, as demonstrated by autoradiographic methods that permit direct measurement of receptor densities in small or complex structures.19 20 21 However, there has been no direct assessment of the relative distributions of muscarinic cholinergic receptor densities in the SAN and adjacent atrial muscle nor is it known whether regional receptor density differences within the SAN might correspond to the distribution of areas of pacemaker dominance. Accordingly, we used quantitative light-microscopic autoradiography to characterize the spatial distribution of muscarinic cholinergic receptors and β-adrenergic receptor subtypes in the sinoatrial node and adjacent right atrial myocardium in the canine heart. We also characterized receptor densities in regions of dominant pacemaker activity, identified with electrophysiological mapping in vitro, to determine whether any consistent relationship exists between cholinergic or adrenergic receptor density and the location of dominant pacemaker activity.
Materials and Methods
Right Atrial Isolation and Tissue Acquisition
Normal adult mongrel dogs (n=18) weighing between 20 and 25 kg were anesthetized with 30 mg/kg of intravenous pentobarbital sodium. The dogs were intubated and ventilated with a positive-pressure Harvard respirator. A median sternotomy was performed and the heart was cradled in the pericardium. The azygous vein was ligated and divided and 100 μg/kg of intravenous heparin was administered. The right atrium was excised as previously described22 by ligating and dividing the inferior and superior venae cavae, cross-clamping the aorta, infusing cold cardioplegia solution into the aortic root, and covering the heart with iced saline. Tissue was acquired for quantitative autoradiographic studies by dissecting the SAN and the adjacent crista terminalis and right atrial free wall from the remainder of the atrium. The tissue was frozen rapidly in liquid nitrogen and stored in sealed containers at −70°C until used in experiments.
Tissue that was to be analyzed electrophysiologically was dissected in a similar fashion, except that the right coronary artery was first cannulated and perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2. The SAN and adjacent structures were excised and superfused in the same solution at 37°C.
Activation maps (n=4) were constructed as previously described.22 Briefly, the endocardial surface of the dissected SAN and adjacent crista terminalis and right atrial free wall was placed on an electrode template containing 250 unipolar electrodes (interelectrode distance, 3 mm), and unipolar electrograms were acquired with a custom-designed VAX (Digital Equipment Corp) recording system. The time of local activation for each electrogram was determined by the maximum negative rate of voltage change (−dV/dtmax). To facilitate recording of transmembrane potentials, the fibrous layer of epicardium was carefully dissected away to permit penetration by microelectrodes into the underlying cells. No change was observed in activation sequence maps recorded before and after this dissection. Transmembrane potentials were recorded with a floating microelectrode to maintain a satisfactory impalement. The microelectrode tip was pulled in the conventional manner to a resistance of 20 to 40 mΩ.22 Microelectrode signals were recorded with a conventional electrometer (WPIDUO773) and displayed on a sweep oscilloscope (Tektronix 511).
The location of the dominant pacemaker was determined by identifying the earliest depolarizing cells within the sinus node. Multiple microelectrode impalements were performed from the epicardial side while unipolar extracellular electrograms were recorded simultaneously from the endocardial surface. The moment of activation for the transmembrane potentials was defined at the time of one half amplitude. Transmembrane potentials were referenced to the earliest electrogram, and cells were rejected as dominant pacemakers if their moment of activation failed to precede this electrogram by at least 20 milliseconds.22
Frozen, unfixed tissue blocks containing the SAN and adjacent atrial myocardium were cut into sections 12 μm thick in a plane parallel to the long axis of the node and the crista terminalis. To define radioligand binding and rinsing conditions suitable for identification of muscarinic cholinergic receptor densities in myocardial tissue sections, preliminary experiments were performed using transmural sections of unfixed canine left ventricular myocardium. Once these conditions were validated, muscarinic cholinergic receptor distributions were characterized in sections containing the SAN and adjacent atrial myocardium. Slide-mounted frozen tissue sections were incubated at 25°C in a buffer composed of (in mmol/L) NaCl 120, KCl 4.8, MgSO4 0.12, CaCl2 1.3, NaH2PO4 20.3, dextrose 10, pH 7.4, and containing 0.5 nmol/L l-[benzilic-4,4′-3H]quinuclidinyl benzilate ([3H]QNB) (45.4 Ci/mmol; New England Nuclear). Nonspecific binding was defined as binding of [3H]QNB in the presence of 1 μmol/L atropine. Nonspecifically bound radioactivity was removed by incubating tissue sections in the same buffer without radioligand or unlabeled displacer for 15 minutes at 22°C.
To measure the densities of total β-adrenergic receptors and the β1- and β2-receptor subtypes in selected regions of the SAN and neighboring structures, sections were mounted on gelatin-coated slides and incubated for 1 hour at 37°C in a buffer composed of (in mmol/L) NaCl 154, MgCl2 10, Tris-HCl 10, pH 7.4, and containing 25 pmol/L (−)[125Iodo]cyanopindolol (ICYP) (2200 Ci/mmol; New England Nuclear). Nonspecific binding was defined as binding of radioligand in the presence of 1 μmol/L 1-propranolol. These autoradiographic conditions have been extensively validated in previous studies.23 24 25 Selective binding of radioligand to β2-adrenergic receptors was achieved by incubating sections with 25 pmol/L ICYP in the presence of 0.1 μmol/L CGP-20712A, a β1-selective displacer. In previous studies,23 we have shown that under these conditions, CGP-20712A maximally inhibits binding of ICYP to β1-adrenergic receptors (93%) while minimally inhibiting binding to the β2-subtype (0.8%). In all experiments, sections were incubated with radioligand in large volumes of buffer (50 mL) so that the concentration of free radioligand did not change measurably during incubation intervals. Nonspecifically bound radioactivity was removed by incubating tissue sections in the same buffer without radioligand or unlabeled displacer for 1 hour at 22°C. Previous studies have shown that this rinsing procedure removes the majority of nonspecifically bound radioactivity without removing any specifically bound radioligand.24 25 After being rinsed in buffer, sections were dipped briefly in distilled water to remove buffer solutes, dried under a gentle stream of air, and prepared for autoradiography as described below.
Muscarinic and adrenergic radioligand binding sites were localized autoradiographically in tissue sections using the emulsion-coated coverslip method, as previously described.26 Briefly, acid-washed, gelatin-coated coverslips coated with Kodak NTB2 nuclear track emulsion (Eastman Kodak) were glued at one end to slides containing radiolabeled sections. After exposure of the emulsion for either 6 weeks ([3H]QNB) or 24 hours (ICYP), the unglued edge of each coverslip was gently lifted from the slides, and the emulsion was developed with Kodak D19 developer diluted 1:1 with water for 4 minutes and fixed with Kodak fixer for 4 minutes at 25°C. After photographic processing, the tissue sections were stained with hematoxylin and eosin, and the coverslips were sealed permanently to the slides. The tissue and overlying developed grains in the emulsion layer were examined by light microscopy.
In all experiments, grain densities were determined by counting grains per unit tissue area of SAN or atrial muscle. Analysis of the distribution of muscarinic and total β-adrenergic receptors as well as β1- and β2-receptor subtypes was performed as described in detail in previous publications.23 24 25 Receptor densities were determined by counting the number of autoradiographic grains in 10 to 15 separate test areas from at least three separate sections of each sample. The β-receptor subtype grain-density values were corrected as previously described23 to account for the modest amount of binding of ICYP to β1-receptors and the small degree of inhibition of ICYP binding to β2-receptors that occurred under the experimental conditions.
The proportion of total tissue area occupied by SAN cells or atrial myocytes was assessed morphometrically by computer-assisted planimetry in selected sections stained with Masson’s trichrome stain, in which atrial myocytes are clearly distinguished from SAN cells and in which cells and extracellular matrix components are also clearly delineated. Groups of SAN and atrial myocytes corresponding to the same groups analyzed by receptor autoradiography in adjacent sections were displayed on a video monitor, and the aggregate cellular area in each region was measured. Grain-density measurements in corresponding regions of adjacent sections were normalized to account for variations in cell density.
In selected experiments, sections containing the SAN and adjacent atrial muscle were incubated with different concentrations of [3H]QNB (0.5 and 1.8 nmol/L) and ICYP (25 and 50 pmol/L), and after rinsing to remove nonspecifically bound radioactivity, the labeled sections were prepared for autoradiography. The ratio of specific grains per unit area of SAN and atrial muscle was measured for each concentration to determine whether the relative specific grain densities in those two compartments remained constant under different radioligand binding conditions.
Radioligand grain-density data are expressed as mean±SD. The statistical significance of differences in grain density and morphometric parameters was determined with Student’s t test.
Muscarinic Receptor Density in SAN and Surrounding Atrial Muscle
Sections of canine left ventricle were used in initial studies to establish radioligand binding conditions suitable for autoradiographic quantification of muscarinic cholinergic receptors in myocardial tissue sections. It was determined that after incubation of slide-mounted sections in buffer containing radioligand, rinsing in the incubation buffer (not containing radioligand or unlabeled displacers) for 15 minutes at 22°C resulted in removal of >75% of nonspecifically bound radioactivity, whereas specifically bound radioligand was not removed (data not shown). However, more prolonged rinsing caused displacement of specifically bound radioligand. Analysis of radioligand association kinetics indicated that incubation of sections for 1 hour at 25°C was sufficient to achieve steady state binding over a broad range of radioligand concentrations (data not shown). Using these radioligand binding and rinsing conditions, we performed binding isotherms and Scatchard analysis with left ventricular sections. As shown in Fig 1⇓, [3H]QNB binding was rapid, saturable, and of high affinity and specificity. A Kd of ≈0.2 nmol/L was observed. In subsequent autoradiography studies involving the SAN and atrial muscle, sections were incubated with 0.5 nmol/L [3H]QNB, a concentration that leads to near-saturation binding but with an excellent specific-binding ratio (mean specific binding under these conditions in SAN sections was >80% of total binding).
Sections of right atrium from 17 dogs containing the SAN and adjacent atrial myocytes were incubated under equilibrium binding conditions with 0.5 nmol/L [3H]QNB alone or in the presence of 1 μmol/L atropine. The distribution of muscarinic receptors in the SAN and surrounding atrial muscle regions was determined autoradiographically. Representative preparations are shown in Figs 2⇓ and 3⇓, and the combined results are shown in Fig 4⇓. Muscarinic receptor density was greater in the SAN (446±69 grains/104 μm2) than in atrial myocytes (83±15 grains/104 μm2, P<.01). When muscarinic receptor density in the SAN was normalized to the muscarinic density of atrial muscle within each individual preparation, thus controlling for modest variations in actual measured radioligand concentrations based on counts and emulsion exposure intervals between experiments, the amount of specific binding was 6.1±0.8 times greater in SAN regions than in atrial myocytes (P<.01). Analysis of sections incubated with [3H]QNB concentrations of 0.5 or 1.8 nmol/L revealed no significant differences in the ratio of specific grain densities in the SAN and atrial muscle at each concentration. Thus, the greater specific radioligand binding localized to the SAN compared with atrial muscle was due to differences in muscarinic receptor densities in these two tissues rather than to differences in radioligand affinity by receptors in SAN and atrial muscle.
β-Adrenergic Receptor Densities in SAN and Surrounding Atrial Muscle
The relative proportions of total β-receptors and of β1- and β2-receptor subtypes in the SAN and atrial muscle regions were determined autoradiographically. Fig 5⇓ summarizes data from 15 individual SAN–atrial muscle tissue preparations. Specific binding ratios were ≥90% in both the SAN and atrial myocytes. The total β-receptor density was more than three times greater in SAN cells than in atrial myocytes (P<.0001). This difference in the total β-receptor densities of the two tissues (173 grains/104 μm2 section area) was due entirely to the significantly greater number of β1-receptors in the SAN (mean difference, 173 grains/104 μm2, P<.0001). β1-Receptors were 6.4-fold more abundant per unit cell area in the SAN than in atrial muscle (P<.01). In contrast, there were no significant differences in β2 density in the SAN and atrial muscle. The SAN contained 86±2% β1-receptors, whereas atrial myocytes contained only 59±6% β1-receptor subtypes (P=.0003). Thus, the SAN region contains a very high concentration of β1-receptors. However, the SAN does not express β1-adrenergic receptors exclusively. It has a β2-receptor density equivalent to the atrial content of β2-adrenergic receptors. No differences in the SAN:atrial muscle ratio of total β-adrenergic receptors or receptor subtypes were observed in sections incubated with 25 or 50 pmol/L ICYP. Thus, there were no apparent differences in affinity for ICYP by receptors in SAN versus atrial muscle.
Heterogeneous Distribution of Muscarinic and β-Adrenergic Receptors in the SAN
To determine whether regions of dominant pacemaker activity are characterized by differences in muscarinic cholinergic and/or β-adrenergic receptor density compared with other SAN regions, we measured receptor densities in the dominant pacemaker region and adjacent SAN regions in four separate preparations. All four preparations had a spontaneous rhythm in vitro with an average rate of 110±10 beats per minute. In each preparation, the dominant pacemaker site was mapped as previously described and its position was marked with a fine prolene suture. The location of the suture was readily identified in frozen sections, thereby facilitating analysis of autoradiographic grain densities in the immediate vicinity of the dominant pacemaker. In addition, grain densities were measured in selected regions of compact SAN that were located from 1 to 4 mm superior or inferior to the dominant pacemaker site. In all four preparations, the dominant pacemaker site was consistently located at the junction of the lower and middle thirds of the SAN. A diagram of the location of the dominant pacemaker is shown in Fig 6⇓.
Grain-density analysis revealed that muscarinic, total β-adrenergic, and β1-adrenergic receptor densities were significantly greater in the dominant pacemaker region than in adjacent superior and inferior regions. Muscarinic receptor density in the dominant pacemaker region was 18±2% greater than in adjacent superior (rostral) and 29±7% greater than in adjacent inferior (caudal) SAN regions (P<.05 for both) (Fig 7⇓). The dominant pacemaker region also had 50±5% more total β-receptors than adjacent superior and 21±3% more than inferior SAN regions (P<.03 for both) (Fig 8⇓). The differences were due entirely to increased β1-receptor density in the dominant pacemaker region (53±5% more than in superior and 26±4% more than in inferior regions, P<.03 for both). β2-Receptor densities were equal in all SAN regions. When the muscarinic or β-adrenergic receptor densities of these SAN regions were normalized to the receptor density of atrial muscle (data not shown), a similar pattern of receptor distribution was observed.
The purposes of this study were to characterize the spatial distribution of muscarinic cholinergic receptors and β-adrenergic receptor subtypes in the SAN and adjacent right atrial myocardium and to determine whether regional receptor density differences within the SAN might correspond to the distribution of areas of pacemaker dominance in the canine heart.
Using quantitative light-microscopic autoradiography we observed that the canine SAN contained approximately six times more muscarinic receptors than the surrounding atrial muscle. We also showed that the SAN contained more than three times as many total β-adrenergic receptors per unit tissue area than surrounding atrial muscle, due entirely to a greater β1-adrenergic receptor density in the SAN. Approximately equal numbers of β2-adrenergic receptors occurred in the SAN and surrounding atrial muscle. Therefore, the sinus node is richly endowed with both muscarinic cholinergic and β-adrenergic receptors, consistent with its marked sensitivity to and functional dependence on parasympathetic and sympathetic stimuli.
Our observations concerning the relative proportions of muscarinic receptors in the SAN and atrial muscle are novel and were based on development and validation of methods for muscarinic receptor autoradiography. Our results on β-adrenergic receptors are similar to those reported by Muntz.19 However, unlike Muntz, we observed that the canine SAN contained a significantly greater proportion of β1-adrenergic receptors than that found in the adjacent atrial muscle. Muntz used subtype-selective displacers (betaxolol and ICI-118,551) that are <100-fold selective for their respective receptors, leading to some degree of radioligand binding to the inappropriate receptor subtype. This may have explained the modest differences reported by Muntz in the values for the percentage of β1-receptors determined with betaxolol and ICI-118,551. In the present study, we used CGP-20712A, a displacer that is ≈1000-fold selective for the β1-adrenergic receptor subtype, thus minimizing uncertainty caused by limited subtype selectivity of displacing ligands. Other than this limited discrepancy between our data and Muntz’s findings, however, the results of the two studies are highly consistent.
By measuring the muscarinic and β-adrenergic receptor densities at sites of dominant pacemaker activity in the isolated perfused atrial preparation, we were able to determine whether further levels of heterogeneity exist within the SAN. The current finding that muscarinic cholinergic receptor density was highest in the region of the dominant pacemaker may explain recent observations showing that the location of dominant pacemaker activity shifts in response to cholinergic stimulation.27 Thus, a direct negative effect of acetylcholine, mediated by muscarinic cholinergic receptors, may underlie a shift in dominant pacemaker activity away from the site of highest muscarinic cholinergic receptor density. The finding that the area of greatest β-adrenergic receptor density consistently coincided with the location of dominant pacemaker activity in the absence of circulating catecholamines or autonomic nerve inputs suggests a central role for the β-adrenergic receptor in the modulation of dominant pacemaker location and rate. The 26% to 53% differences in β-adrenergic receptor density between the dominant pacemaker region and adjacent SAN regions are quantitatively similar to the physiologically important differences in β-receptor density found by Bristow et al28 29 in patients with congestive heart failure compared with normal subjects. Although our data cannot fully explain the underlying mechanism, it is interesting to note that the β2-receptor has recently been shown to exert an inotropic influence even in the absence of epinephrine.30 Independent functioning of the β1-adrenergic receptor could similarly influence chronotropic activity in the SAN.
Results of previous studies18 have shown that infusion of norepinephrine reproducibly shifts the site of earliest extracellular activation superiorly within the SAN. Our present data would predict that the β-adrenergic receptor density in the shifted sites should be lower than that found in the dominant pacemaker site. Therefore, if the β-receptor is a primary modulator of the site of pacemaker dominance, our data would predict that the location of dominant pacemaker activity under basal conditions and during adrenergic stimulation would be the same. Although no data exist in canine preparations, results of rabbit SAN studies support this conclusion31 and are consistent with the temporal and spatial dissociation observed between extracellular electrograms and pacemakers in the SAN.22 Future experiments to determine muscarinic cholinergic and β-adrenergic receptor density in the dominant pacemaker regions identified in response to autonomic stimuli will be required to confirm this prediction.
This work was supported by National Institutes of Health grants HL-32257, HL-33722, and HL-17646 SCOR in Coronary and Vascular Diseases. Dr Beau was supported by an ACC/Merck Adult Cardiology Fellowship Award. We thank Timothy Tolley for technical assistance and Susan Johnson for secretarial assistance.
- Received February 21, 1995.
- Accepted August 8, 1995.
- © 1995 American Heart Association, Inc.
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