The rabbit sinoatrial (SA) node is a heterogeneous structure. The cardiac impulse originates in a relatively small group of primary pacemaker cells (1,2). Typical primary pacemaker cells located in the center of the SA node are small (<8 μm in diameter and 25-30 μm in length) and have a very low density of poorly organized myofilaments (1,3). These cells are surrounded by latent or subsidiary pacemaker cells. The density, organization, and orientation of myofilaments in subsidiary pacemaker cells are intermediate between those of typical pacemaker cells and atrial cells (1,3). Under physiologic conditions, subsidiary pacemaker cells usually do not reach their activation threshold spontaneously and are excited by the dominant pacemaker. Proceeding from the center of the SA node to the periphery, the action potential (AP) evolves from a pacemaker type to an atrial type (1-4).
Both acetylcholine (ACh; 5) and vagal stimulation (6,7) shift the dominant pacemaker from the center to the periphery of the SA node. The reason for this shift is unknown, and it is the fundamental question we addressed. Several studies demonstrated that the central and periphery areas of the SA node consist of electrophysiologically heterogeneous cells with different membrane properties (8,9). Considering this finding, we would expect different responses to cholinergic action of cells from the central and peripheral areas of the SA node. We hypothesized (see also 5) a higher sensitivity to ACh action of typical pacemaker cells composing the central area of the SA node compared with subsidiary cells in the periphery. However, the fibers of the SA node are electrically coupled by low-resistance gap junctions (10,11). Because of mutual electrotonic influence, cells from different areas of the SA node can respond to cholinergic action in much the same way. At the same time, in the rabbit SA node, transmural vagal stimulation decreases intercellular coupling by a decrease in total membrane resistance (12). This way cholinergic action diminishes mutual electrotonic influence, and local differences in electrical activity may arise more easily. To clarify this uncertainty, we studied the effects of ACh and of vagal stimulation on APs recorded in the primary and subsidiary pacemaker areas of the rabbit SA node. Preliminary results of this work were published in abstract form (13).
MATERIALS AND METHODS
Experiments were performed in isolated right atrial preparations obtained from male rabbits (Shinshilla) weighting 2-3.5 kg. Rabbits were intravenously anesthetized with urethane (1.5 g/kg). After midsternal thoracotomy, the pericardium was opened and the heart quickly excised. The right atrium, with SA-node region intact, was dissected in the bath solution. The final preparation consisted of the crista terminalis (CT), the intercaval band in which the SA node is embedded, the orifice of the inferior vena cava, and small portions of the interatrial septum and pectinate muscles. The preparation was pinned on a thin coat of silicon on the bottom of a tissue bath (11 ml) with the endocardial side facing upward. The preparations were continuously superfused with oxygenated Tyrode's solution (15 ml/min). The temperature was kept constant at 36 ± 0.5°C. The solution contained (in mM): NaCl, 130; KCl, 4; CaCl2, 1.8; MgCl2, 1; NaHCO3, 24; NaH2PO4, 1.2; glucose, 5.6; and was saturated with a mixture of 95% O2 and 5% CO2. The pH was 7.3 ± 0.05. ACh was added to the perfusing solution at a concentration of 10−6-10−4M.
No measurements were performed during ≥40-60 min after the isolation procedure. ACh was infused continuously for 2 min with 15- to 20-min intervals between administrations.
Microelectrode recordings and experimental protocol
The experimental preparation is diagrammed in Fig. 1A. On the bottom of our recording chamber, a square network with line spacing of 1 mm was drawn. As the SA-node tissue is very thin, the network as well as position of each impalement was clearly seen through magnifying glass (×8) and depicted on the preparation image with respect to the network. Transmembrane potentials were recorded by using glass microelectrodes filled with 3.0 M KCl (tip resistance, 10-40 MΩ), and connected to high-input impedance amplifiers (WPI model KS-700, World Precision Instruments, New Haven, CT, U.S.A.). At the start of an experiment, the primary pacemaker site was found, and activation maps were made by recording intracellular potentials from 10 to 20 sites; the extracellular potential from the CT provided a reference signal. The pair of silver bipolar electrodes was used to record the extracellular potentials from the surface of the CT in an area activated earliest during normal spontaneous sinoatrial conduction (14). In some experiments, two simultaneous intracellular recordings were obtained; one electrode was inserted into a cell in the primary pacemaker area, and the other, into a cell in the subsidiary pacemaker area. The primary pacemaker area included those cells that during the normal spontaneous rhythm were activated earliest ∼20-30 ms before the activation of the reference site. In these cells, there was a smooth transition from diastolic depolarization to the upstroke of the AP. The subsidiary pacemaker cells were activated later and exhibited smooth or rapid transitions from diastolic depolarization to the upstroke of the AP.
Surface electrograms and transmembrane potentials were displayed on an oscilloscope (Tektronix 511, Tektronix, Inc., Beaverton, OR, U.S.A.) and stored on tape with a Hewlett Packard tape recorder (model 3964A, Hewlett Packard Company, Boise, ID, U.S.A.). After amplification, the analog records were digitized by an analog-digital converter with a sampling rate of 5,000 Hz. The spontaneous cycle length (intervals between successive APs) was recorded, as well as AP parameters: AP amplitude, AP duration at 50% repolarization, maximal diastolic potential, maximal upstroke velocity, and slope of diastolic depolarization were measured by using a computer program.
Postganglionic vagal stimulation was carried out by a technique described by Vincanzi and West (15). A pair of stimulating electrodes separated by 2 mm was placed directly on the endothelial surface of the cephalic portion of the SA node. Vagal stimuli were generated by a Harvard stimulator (Harvard Apparatus Ltd., South Natick, MA, U.S.A.). The vagal stimulation was delivered for 400 ms and consisted of 100-μs rectangular pulses 15-40 V in amplitude, and frequency of 100 Hz. The trains of pulses were triggered by the signal from the electrogram and delivered during the spontaneous AP of the SA node (7). These stimuli were subthreshold for SA or atrial cells but were of sufficient amplitude to stimulate post-ganglionic vagal nerve terminals. The intervals between successive trains were ≥5 min. Activation of β-adrenergic receptors was prevented by superfusing the preparation continuously with solution containing propranolol, 3 × 10−5M. All the effects of vagal stimulation and ACh superfusion reported here could be blocked by 1 μM atropine.
Analysis of variance (ANOVA) or a paired Student's test were performed where appropriate.
Comparison of the effects of ACh on cells from the central and subsidiary pacemaker areas of the SA node
In each of the five preparations studied, two APs were recorded simultaneously from the primary (P) and subsidiary (S) pacemaker areas (in Fig. 1A, these cells are shown by asterisks). Cell P was from the primary pacemaker area and was fired 30 ms before the CT; cell S was from subsidiary pacemaker area and was excited 15 ms before the CT. A high concentration of ACh (10−4M) was used so that appreciable amounts of agonist would rapidly reach the cells in the SA node. Figure 1B shows an atrial electrogram (EG) and continuous membrane potential recordings from P and S areas of the SA node obtained before and during ACh superfusion. Simultaneous recordings in Fig. 1B demonstrate that during ACh superfusion, the preparation retained a regular spontaneous rhythm. The cycle length progressively increased from 440 ms in control to 624 ms in ACh. The subsidiary pacemaker cell S persisted in initiating impulses with only slightly decreased amplitude (Fig. 1B, record S), whereas AP amplitude of the cell from the primary pacemaker area gradually decreased and then vanished (Fig. 1B, record P). The cell was considered inexcitable when its AP amplitude decreased >40% of the control value. The differences between the action of ACh on the electrical activity of P and S cells are further illustrated by using a faster display (Fig. 1C). In each case, five spontaneous APs taken from records B from the points underlined and labeled are shown superimposed: a control AP (Con; before ACh addition) and (1-4) at 10-s intervals after ACh superfusion began. The shapes of APs were markedly affected by ACh. In the left part of Fig. 1C, ACh superfusion suppressed P cell AP amplitude to <40% of control, and the membrane potential settled to a value near that of the control maximal diastolic potential. In the right part of Fig. 1C, ACh both shortened the duration of subsidiary pacemaker APs and caused a −8 mV increase in their maximal diastolic potential.
The time-course plots of the effects of ACh on AP configuration of cells from primary and subsidiary pacemaker areas for this experiment are shown in Fig. 2A-E. In these plots, change of AP amplitude, maximal diastolic potential, diastolic depolarization slope, AP duration at 50% repolarization, and time of cell activation with respect to the time of CT activation are plotted against the duration of ACh action. Zero time indicates the time when ACh entered the tissue bath. We assume that the gradual ACh action reflects the mixing time within the recording chamber and the time taken for ACh to diffuse to the pacemaker region. The summary plots of five experiments are shown in Fig. 2F-K. In these plots, the mean changes of the aforementioned parameters are plotted against percentage of maximal ACh suppression of the AP amplitude.
Figure 2A-F shows that there were marked differences in the response to ACh superfusion between cells from primary and subsidiary pacemaker areas. Statistical analysis of five experiments revealed significant (p < 0.001) reduction of P cell AP amplitude compared with both control and subsidiary cells. Whereas amplitude of S cell action APs only slightly (p > 0.05) decreased (see Fig. 2A, regular line), the amplitude of P cell APs decreased to several millivolts (see Fig. 2A, bold line). ACh also induced moderate hyperpolarization of S cells (see Fig. 2B and G, regular lines), whereas maximal diastolic potential of P cells returned to the control level (see Fig. 2B and G, bold lines).
After P cell AP amplitude was suppressed to ≤40% of the control value, AP configuration changed (see Fig. 1C, recordings 3 and 4). The most sensitive parameter for ACh action was the slope of diastolic depolarization of the P cell AP (see Fig. 2C, bold line). With ACh, there was a prominent reduction (p < 0.005) of the slope of depolarization in both groups, although in the primary pacemaker cells, it was greater (see Fig. 2H). ACh also induced pronounced shortening (p < 0.001) of AP duration, similar in both groups of cells (see Fig. 2I) and a shift of the primary pacemaker site (see Fig. 2E and K). In control, discharge of P cells preceded the atria time reference ∼30 ms (see Fig. 2E and K, bold lines). Under ACh the discharge of these cells came ∼30 ms after the atrial EG (see Fig. 2K, bold line). Activation time of subsidiary pacemaker cells also changed (p < 0.05; see Fig. 2K, regular line).
The effects of ACh were abolished by washout and prevented by the prior addition of atropine, 10−6M. The aforementioned effects of ACh in the primary pacemaker area were reproducible in the same cell by a second application of ACh after a 15-min washout.
Suppression of action-potential amplitude by ACh is predictable based on the maximal rate of action-potential upstroke
One hundred forty-one cells from 20 isolated rabbit preparations were impaled and subjected to ACh superfusion. APs of cells from different regions of the SA node ranged from those typical of pacemaker cells in the center of the SA node to those typical of atrial cells at the periphery of the node. During ACh superfusion, suppression of AP amplitude to ≤40% of control values was recorded in 35 cells. All these cells had control AP upstroke velocities <15 V/s. In Fig. 3, all cells with AP upstroke velocities <15 V/s (n = 66) were grouped by their dV/dt so that in each successive group, it increased by 3 V/s. All cells with AP upstrokes >15 V/s (n = 75) were assigned to a single group. Figure 3 shows the percentage of cells in which AP amplitude was ≤40% of control as a function of AP upstroke. The total number of cells in each group is shown at the bottom of the diagram. Incidence of reduction in AP amplitude was the highest (91%) among cells with AP upstrokes <3 V/s: during ACh superfusion of 22 cells in this group, 20 cells exceeded 40% reduction in amplitude; in the remaining two cells AP amplitude decreased to 45-70% of the control level. A summary of this group's control AP parameters is given in Table 1; they are typical of pacemaker cells and comparable to those reported by previous investigators (2,3).
We then determined which is the more important determinant of ACh-induced suppression of AP amplitude: the site where the cell is situated or the parameters of the cell AP. In five preparations, seven cells with typical pacemaker AP parameters located in the tail part of the SA node and excited after the CT were superfused with ACh. These cells were identified by Bournan et al. (6) and called "anomalous" pacemaker cells. A summary of this group's control AP parameters is given in Table 2. Summary plots of seven cells are shown in Fig. 4A-E. As in the case of primary cells, the mean changes of AP amplitude, maximal diastolic potential, diastolic depolarization slope, AP duration at 50% repolarization, and time of cell activation with respect to the time of CT activation are plotted against percentage of maximal suppressing effect of ACh. ACh induced the same changes in anomalous cell AP parameters as in the primary cells (see Fig. 2F-I and Fig. 4A-D), except for the activation time (compare Fig. 2K, bold line, and Fig. 4E).
To determine the concentration dependence of ACh action, the same seven typical pacemaker cells with AP upstrokes <3 V/s were superfused with ACh, 10−4M, 10−5M, and 10−6M. The effect of ACh on SA-node cells was dose dependent (see Fig. 5). Superfusion with ACh (10−4M) resulted in suppression of AP amplitude to ≤40% of control value in all seven calls; mean amplitude was reduced to 21% of control. After a 15-min washout and recovery of control AP parameters, the second concentration of ACh (10−5M) was tested. It induced suppression of AP amplitude to ≤40% of control in three of the seven cells. In the remaining four cells, AP amplitude decreased to 45-70% of the control value. An ACh concentration of 10−6M applied after 15-min washout decreased AP amplitude to ∼80% of the control value.
Effects of vagal stimulation on the cells from primary and subsidiary pacemaker areas of the SA node
To test whether vagal stimulation can induce inexcitability in cells from the SA node, 20 preparations were subjected to postganglionic vagal nerve stimulation. The effects of brief vagal stimulation (a train of stimuli was applied for 400 ms during the spontaneous AP in the SA node) on electrical activity in cells from primary and subsidiary pacemaker areas are shown in Fig. 6A and B, respectively. In Fig. 6A, the cell was excited 30 ms before the CT; vagal stimulation resulted in a 13-mV hyperpolarization of the membrane and slowing of spontaneous activity. Under control conditions, the cycle length was 445 ms; after vagal stimulation, the EG had a cycle length of 766 ms. Instead of a pacemaker AP, only a subthreshold depolarization was recorded. This subthreshold depolarization appeared 5 ms after the CT fired. The first SA-node AP occurred after a cycle length of 1,286 ms. Repeated vagal stimulations induced the same suppression of firing.
The effects of vagal stimulation on a cell from the subsidiary pacemaker area (the cell was excited 16 ms before the CT) is shown in Fig. 6B. In this case, vagal stimulation resulted in slowing of spontaneous activity (under control conditions, the cycle length was 440 ms; after vagal stimulation, the AP occurred after a cycle length of 616 ms), a slight hyperpolarization (∼2 mV) and decrease of AP amplitude.
Suppression of the action-potential amplitude by vagal stimulation is related to the maximal rate of action-potential upstroke
One hundred seventeen cells from 20 isolated right atrial preparations were impaled and subjected to post-ganglionic vagal nerve stimulation. During vagal stimulation, suppression of AP amplitude to ≤40% of control values was recorded in 63 cells. These cells had AP upstroke velocities <12 V/s. In Fig. 7, the cells with AP upstroke velocities <12 V/s (n = 99) were grouped by the dV/dt of the upstroke, so that in each successive group, AP upstroke velocity increased by 3 V/s. All cells with AP upstroke velocities >12 V/s (n = 18) were assigned to a single group. In Fig. 7, the percentage of cells in which AP amplitude was ≤40% of the control value are shown as function of AP upstroke (dV/dt). The total number of cells in each group is shown below the diagram. During ACh superfusion, incidence of reduction in AP amplitude was the highest (81%) among cells with AP upstroke velocities <3 V/s: during vagal stimulation of 47 cells in this group, 38 cells exceeded 40% reduction in amplitude. In the remaining nine cells, AP amplitude decreased to 45-70% of the control value. A summary of control parameters of this group's APs is given in Table 3. They are typical of pacemaker cells and comparable to those subjected to ACh action. With increasing upstroke velocity, occurrence of inexcitability decreased. Inexcitability did not occur in cells whose dV/dt exceeded 12 V/s (n = 18).
To compare the effect of vagal stimulation and ACh action, seven preparations were superfused with a solution containing propranolol. Each of eight impaled cells with a typical pacemaker AP was successively subjected to vagal stimulation and then to ACh (10−4M) action. ACh superfusion began within 15 min after vagal stimulation. Results of these experiments are shown in Fig. 8. Vagal stimulation and ACh action induced the same depression of pacemaker activity: there was ∼50-60% increase in mean cycle length in both cases (see Fig. 8A). Vagal stimulation as well as ACh action induced the same suppression of AP amplitude to ≤40% of control (see Fig. 8B).
Our data demonstrated that cholinergic stimulation suppressed excitation in >80% of cells with low AP upstroke velocities (dV/dt < 3 V/s). It is known that cells with low values of dV/dt < 3 V/s occupy rather large compact area (∼1 × 2 mm2) in the center of the SA node (2). Consequently, in our case during cholinergic stimulation, conduction into this area can be completely blocked. Previously it was shown that local depression of conduction induced by vagal stimulation in nodal tissue might lead to intranodal reentry (16). In our study, both vagal stimulation and ACh superfusion induced spontaneous tachyarrhythmias (see Fig. 9). Vagal stimulation induced spontaneous tachyarrhythmias in 9% of cases (10 cells; six preparations) from 117 cells (20 preparations) subjected to vagal stimulation. ACh superfusion induced spontaneous tachyarrhythmias in 18% of cases (25 cells; 13 preparations) from 141 cells (20 preparations) subjected to ACh action. It is possible that these tachyarrhythmias are result of local reentry around an inexcitable area within the SA node.
Cholinergic action and nonhomogeneity in the SA node
In our study, effects of ACh (10−4M) superfusion and vagal stimulation on primary and subsidiary areas of the SA node were investigated. We showed that in cells from the primary pacemaker area, ACh (10−4M) superfusion as well as vagal stimulation could suppress AP amplitude to <40% of control, whereas cells from the subsidiary pacemaker area as well as the atria continued to excite (see Figs. 1 and 6A). These cholinergic effects were antagonized completely by pretreatment with atropine (10−6M). It is well known that both vagal stimulation and ACh slow and arrest the generation of pacemaker potentials by activating M2-muscarinic receptors (17,18). In this study, we demonstrated that both ACh and vagal stimulation can produce arrest in local sites in the SA node while other parts of the node and atrial tissue continue to generate APs. It is believed that this suppression of amplitude was a result of ACh action. In the same cells, both vagal stimulation and ACh action produce the same suppression of AP amplitude (see Fig. 8B). A similar suppression of AP amplitude during local application of ACh (∼2 × 10−4M) in the rabbit SA node was previously observed by Paes de Carvalho et al (19). In preparations driven from the atrium, they reported full suppression by ACh of APs recorded in the SA node, as well as their recovery during washout.
We found that most frequently, cholinergically induced suppression of AP amplitude to ≤40% of control value was recorded in cells with typical pacemaker APs: a slow AP upstroke velocity (<3 V/s), a small overshoot, a rather long AP duration, and a steeply sloping diastolic depolarization with a smooth transition from the pacemaker depolarization to the upstroke phase (see Tables 1-3). Recent investigations demonstrated that different parameters of the SA node APs resulted from different membrane properties of the cells (8).
In this study, most cells in which suppression of AP amplitude to ≤40% of control value was the greatest (AP upstroke <3 V/s) were primary pacemaker cells and discharged 20-30 ms before the CT. Some cells with AP parameters typical for primary pacemaker cells were located in the tail part of the SA node and were excited after the CT (see Table 2). Such pacemaker activity was described by Bouman et al. (6) and Shibata et al. (20) and called anomalous pacemaker activity. ACh induced suppression of AP amplitude to ≤40% of control value in both primary and anomalous pacemaker cells (see Figs. 2 and 4). This suggests that ACh-induced suppression of the cell AP amplitude to ≤40% of control value depends primarily on the cell AP parameters. ACh suppressed the AP amplitude in typical pacemaker cells in a concentration-dependent manner (see Fig. 5).
A comparative study of the actions of ACh and vagal stimulation in both amphibian and mammalian heart demonstrated that the pathways activated by vagal stimulation and applied ACh have different kinetics of activation (21-23). Unfortunately, our experimental conditions could not reveal the difference between vagal stimulation and ACh superfusion. Although in our study, suppression of AP amplitude was many times slower in the case of ACh superfusion than during vagal stimulation; this effect is attributable to the slow increase of ACh concentration in the bath.
Ionic basis of cholinergically induced suppression of pacemaker action potentials
As for mechanisms underlying cholinergically induced suppression of typical pacemaker APs, the results reported here suggest that there is an inverse correlation between cholinergically induced inexcitability and AP upstroke velocity: the steeper upstroke is less associated with cholinergically induced reduction of the AP amplitude of this cell type. Electrophysiologic and morphologic studies of the rabbit SA-node tissue have shown that the rate of AP upstroke and the size of pacemaker cells gradually increases from the leading pacemaker site in the center of the SA node toward the periphery (1,2,24,25). Although the ionic nature of SA pacemaking is still a matter of debate (26,27), the recent investigations demonstrated that the rabbit SA node is composed of electrophysiologically heterogeneous pacemaker cells with different electrical membrane properties; L-type Ca current (iCa,L) plays a major role in pacemaking in the center of the SA node, whereas in pacemaking in the periphery, iNa current plays an essential role (8,9,28-31).
Vagal stimulation or added ACh is known to activate the muscarinic K+ current iK,ACh in cells of the SA node (32). Activation of iK,ACh results in hyperpolarization, increase in cycle length, and shortening of the SA-node cell AP. It has been reported also that ACh inhibits iCa,L current in isolated rabbit SA-node myocytes (33). This effect occurred in the absence of any previous adrenergic stimulation and was dose dependent; the maximal depression of iCa,L was obtained at 10−5M and reached 55.5 ± 6.5% (33). The suppression of AP amplitude demonstrated in our study may be a result of depression of iCa by ACh. Suppression of AP amplitude (≥60%) recorded in typical pacemaker cells in our study quantitatively is close to the depression of iCa,L (∼56%) reported by Petit-Jacque et al. (33). Previously DiFrancesco and Tromba (34) suggested that in SA-node cells, basal adenylate cyclase activity is extremely great. The depressant effect of ACh on iCa,L may occur via inhibition of the basal adenylate cyclase activity, leading to a decrease of cyclic adenosine monophosphate (cAMP)-dependent protein kinase stimulation and thus to dephosphorylation of calcium channels.
Changes in the cell electrical activity during ACh-induced suppression of action-potential amplitude
Because of the slow increase of ACh concentration in the recording chamber, it was possible to see consecutive changes in electrical activity during ACh-induced suppression of AP amplitude (see Figs. 1 and 2). At the beginning of ACh superfusion while ACh concentration was low, the most sensitive parameter to ACh action was the diastolic depolarization of P-cell APs (see Fig. 1C, left record, and Fig. 2C, bold line). The same reduction in the slope of the pacemaker depolarization without prominent hyperpolarization and significant change of other AP parameters was reported by Sibata et al. (20) in experiments with muscarine chloride (10−8M) action on primary pacemaker cells. They concluded that a reduction in an inward current iCa is responsible for the reduction of the pacemaker depolarization. Calculations made by Egan and Noble (35) support this conclusion. In SA-node cells, the hyperpolarization-activated current if, which is involved in the generation of pacemaker activity, also is strongly depressed by ACh (34). This effect is especially prominent at low ACh concentrations. ACh at nanomolar concentrations inhibits if and decreases pacemaker depolarization, whereas 22 times higher concentrations are required to activate iK,ACh(36). Thus in our experiments, at the start of ACh superfusion while ACh concentration was low, suppression of two currents if and iCa may induce a reduction in the slope of P-cell diastolic depolarization.
In our experiments, ACh induced a pacemaker shift from the center of the node to the periphery (see Fig. 2E and K). We did not study the site to which the primary pacemaker shifted. A pacemaker shift from the center of the node toward the upper part of the CT was recorded by Mackay et al. (5) during the addition of 1.4 × 10−5M ACh. We also observed evidence of a pacemaker shift from the center of the SA node toward the periphery after vagal stimulation (see Fig. 6A and B). Before vagal stimulation, the cells were excited 30 ms (Fig. 6A) and 16 ms (Fig. 6B) before the CT; after vagal stimulation, the subthreshold depolarization in Fig. 6A and AP in Fig. 6B were recorded 5 and 6 ms, respectively, after the CT. A pacemaker shift after vagal stimulation from the center of the node toward the periphery was observed previously by Opthof (37) and Kodama et al. (7). The exact mechanism of this shift is unknown. Our data demonstrate that vagal stimulation and ACh (10−4M) action suppress excitation in >80% of typical pacemaker cells that compose the center of the SA node. It is not improbable that inexcitable cells create inexcitable regions, such that during cholinergic action, the central area of the SA node is inexcitable. In this case, the cells that are more tolerant to cholinergic action-subsidiary pacemaker cells-should take the leading pacemaker role.
In the experiments made in this study, we observed spontaneous atrial tachyarrhythmias during both ACh superfusion and vagal stimulation (see Fig. 9). Previously we also demonstrated that in rabbit right atrium, ACh (10−4M) induced spontaneous atrial tachyarrhythmias (38). It is known that some atrial tachyarrhythmias can be caused by circus movement of the excitatory impulse, and cholinergic influence favors induction and perpetuation of such arrhythmias (39). Initiation of intraatrial circus movement is commonly considered to require unidirectional conduction block, conduction around the area of block, and reexcitation of the tissue proximal to the site of block (40,41). The circus movement in the frog heart model of Rosenshtraukh et al. (42) completely depended on vagally induced depression and block of atrial conduction. Cholinergically induced nonuniform depression of conduction in SA-nodal tissue might lead to intranodal microreentry (15) similar to that induced by extrastimulation (43). Our results demonstrated that vagal stimulation and ACh action induced suppression of excitation in >80% of typical pacemaker cells. It is possible that cholinergically induced inexcitable cells create inexcitable regions that might functionally be an obstacle for reentrant tachycardias or create unidirectional block and thus facilitate the initiation of reentry. The largest inexcitable region should occupy the primary pacemaker area; the other inexcitable regions may exist in the places where anomalous pacemaker cells are grouped. However, these assumptions need experimental support. Alings and Bouman (44) reported that in the rabbit, the area of the SA node with upstroke velocity <5 V/s increases 27 times with age (≤5 years). We suggest that this may help to explain the well-known age-related increase of atrial flutter and fibrillation.
Acknowledgment: We are deeply grateful to Professor Michael R. Rosen, College of Physicians and Surgeons of Columbia University, Department of Pharmacology, for the helpful discussions and critical review of the manuscript. The work was supported in part by Russian Foundation for Basic Research (grant 93-04-48-292).
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