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Am J Physiol Heart Circ Physiol 291: H612-H623, 2006. First published March 24, 2006; doi:10.1152/ajpheart.00022.2006
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Postganglionic nerve stimulation induces temporal inhibition of excitability in rabbit sinoatrial node

Vadim V. Fedorov,1 William J. Hucker,1 Halina Dobrzynski,1,2 Leonid V. Rosenshtraukh,3 and Igor R. Efimov1

1Department of Biomedical Engineering, Washington University, St. Louis, Missouri; 2University of Manchester, United Kingdom; and 3Cardiology Research Center, Moscow, Russia

Submitted 5 January 2006 ; accepted in final form 10 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vagal stimulation results in complex changes of pacemaker excitability in the sinoatrial node (SAN). To investigate the vagal effects in the rabbit SAN, we used optical mapping, which is the only technology that allows resolving simultaneous changes in the activation pattern and action potentials morphologies. With the use of immunolabeling, we identified the SAN as a neurofilament 160-positive but connexin 43-negative region (n = 5). Normal excitation originated in the SAN center with a cycle length (CL) of 405 ± 14 ms (n = 14), spread anisotropically along the crista terminalis (CT), and failed to conduct toward the septum. Postganglionic nerve stimulation (PNS, 400–800 ms) reduced CL by 74 ± 7% transiently and shifted the leading pacemaker inferiorly (78%) or superiorly (22%) from the SAN center by 2–10 mm. In the intercaval region between the SAN center and the septal block zone, PNS produced an 8 ± 1-mm2 region of transient hyperpolarization and inexcitability. The first spontaneous or paced excitation following PNS could not enter this region for 500–1,500 ms. Immunolabeling revealed that the PNS-induced inexcitable region is located between the SAN center and the block zone and has a 2.5-fold higher density of choline acetyltransferase than CT but is threefold lower than the SAN center. The fact that the inexcitability region does not coincide with the most innervated area indicates that the properties of the myocytes themselves, as well as intercellular coupling, must play a role in the inexcitability induction. Optically mapping revealed that PNS resulted in transient loss of pacemaker cell excitability and unidirectional entrance conduction block in the periphery of SAN.

optical mapping; sinoatrial node; postganglionic vagal stimulation

IT IS WELL KNOWN that the parasympathetic nervous system can rapidly reduce the heart rate and shift a leading pacemaker site (6, 38). Moreover, vagal stimulation can precipitate atrial tachyarrhythmias (22). Meek and Eyster (28) demonstrated, by using string galvanometers, shift of the leading pacemaker site from sinoatrial node (SAN) region inferiorly, simultaneously with slowing of the heart rate during vagal stimulation in the dog. Many groups (6, 23, 27, 41, 44) then showed a shift of the leading pacemaker site in the rabbit during cholinergic and adrenergic stimulation. However, the mechanisms of these changes are not fully understood because of insufficient resolution of extracellular mapping systems and the impossibility to have simultaneous recordings from multiple microelectrodes. Currently, high resolution optical imaging is the only available technology that allows resolving simultaneous changes in the activation pattern and action potential (AP) morphology (18).

One of multiple theories suggests that cholinergic stimulation produced pacemaker shift from the center to periphery of the SAN because these areas consist of electrophysiologically heterogeneous cells (7, 30) with different sensitivity to cholinergic stimulation (23, 27, 44, 45). Recent computer simulations also demonstrated that the excitability of central SAN pacemaker cells is more sensitive to acetylcholine (ACh) than peripheral subsidiary pacemaker cells (1, 47). The study by Vinogradova et al. (44) that used microelectrode recordings in the isolated rabbit right atrium has shown that either ACh superfusion or postganglionic vagal stimulation hyperpolarizes cells with a low upstroke velocity in the SAN region and periphery and significantly reduces AP amplitude to <40% of control. Thus the hyperpolarized SAN cells remain inexcitable, and sinoatrial block prevails while cholinergic input is maintained. In contrast, cells from subsidiary pacemaker areas, as well as the atrium, remain fully excitable. Vinogradova et al. (44) hypothesized that cholinergic stimulation can induce local areas of transient inexcitable pacemaker cells in the intercaval region, thus shifting the leading pacemaker site from the center SAN to the periphery and cause atrial arrhythmia.

To test this hypothesis, we used high-resolution optical imaging techniques (18) to visualize the sequence of activation in isolated rabbit SAN under normal conditions and during postganglionic nerve stimulation (PNS). Immunohistochemistry was used to identify and characterize different areas of the SAN based on the levels of expression of {alpha}-actinin, connexin 43 (Cx43), neurofilament, and autonomic innervation and thus determine morphological criteria of pacemaker cells in which PNS can induce inexcitablity (15).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparations. The protocol was approved by the Washington University Animal Care and Use Committee. The isolated right atrial rabbit preparation as well as the experimental techniques have been previously described in detail (15, 17, 44) and thus will be outlined only briefly. New Zealand White rabbits (n = 17) of either sex weighing 2–3 kg were anesthetized. After a midsternal incision was made, the heart was removed and placed onto a Langendorff apparatus, where it was retrogradely perfused with oxygenated (95% O2-5% CO2) constant-temperature, modified Tyrode solution. The heart was stained with di-4-ANEPPS (D-1199, Molecular Probes), 1 µM, for 10–15 min. After staining was completed, the heart was removed from the Langendorff apparatus, and the right atrial SAN preparation (n = 12) or the right atrial SA and atrioventricular node (AVN) node preparations (n = 5) were dissected. The final preparations contained opened superior vena cava (SVC), the crista terminalis (CT), the intercaval band in which the SAN is located, the orifice of the inferior vena cava (IVC), and parts of the interatrial septum and pectinate muscles (see Fig. 1A). Five preparations also contained the triangle of Koch with the AVN (see Fig. 2). All preparations were pinned down on a thin silicon disk and superfused at 37°C, with a flow rate of 30 ml/min.


Figure 1
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  		Fig. 1. Quantification of innervation density. A: photograph of sinoatrial node (SAN) preparation with lines of sections indicated. Starting 4 mm below the superior vena cava (SVC), 3 levels of tissue sections were obtained and stained for both choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH). IVC, inferior vena cava. B: mosaic images were created to reconstruct immunostaining from the crista terminalis (CT) to the block zone. C: mosaic image was decomposed into 0.06-mm2 tiles. Each tile was thresholded for nerve staining (D), noise above threshold was removed, and holes of black pixels surrounded by white pixels were filled (E) (see insets of D and E for comparison). The area of white pixels in E was used as the nerve area for the image. The tile in C was also thresholded to show any tissue within the tile (F). The area of the white pixels in this image was used as the tissue area. Percentage of nerve densities were calculated as: density = [(nerve area)/(tissue area)] x 100.

 

Figure 2
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  		Fig. 2. Activation maps and optical action potentials (APs) recorded in the two rabbit SANs. A and C: photographs of SAN preparations with superimposed unifocal and bifocal activation sequences. Isochrones are separated by 3 and 2 ms. B: optical AP traces from photodiodes and electrogram (EG) recording from a bipolar electrode shown by 1–5 and EG in A: 1 from leading pacemaker site; 2 and 3 from superior and inferior part of SAN; 4 from CT; and 5 from the block zone region. D: optical AP trace shown by 5 in C. E: optical AP traces from photodiodes and an EG recording from bipolar electrode shown by 1–5 and EG in C. IAS, interatrial septum; RA, right atrium. Black line (A) and (black color in C) show the conduction block zone region near septal margin of SAN.

 
Two pairs of silver bipolar electrodes were used to record the extracellular potentials from the surface of the high and low right atrium and to pace the heart.

Optical mapping. During fluorescent imaging, optical signals were recorded from a 5 x 5- to 11 x 11-mm area of the SAN at a rate of 1,500 frames/s by using a 16 x 16 photodiode array as previously described (18). The excitation-contraction uncoupler 2,3-butanedione monoxime (BDM, 15 mmol/l) was added to suppress motion artifacts in optical signals caused by muscle contraction (10). Signals were low-pass filtered at 120 Hz, differentiated, normalized by the basic beat recordings, and plotted as two-dimensional intensity graphs, which were overlapped as frames with the image of the preparation to produce animations. Wavefronts of activation were visualized in these animations to identify the anatomical location of the pacemaker and conduction pathways as described (29). Each preparation was digitally photographed, and fields of view were identified with an accuracy of 200 µm. Activation time points were defined as change in fluoresence over time (18). Isochronal maps of activation were plotted by using the triangulation method (18).

The SAN preparations were restained by di-4-ANEPPS (1 µM/l) for 10–15 min during the experiment as needed. No measurements were performed until 5 min after the restaining procedure. Stability of the preparation was periodically verified by measuring the sinus rhythm cycle length. We considered the preparation stable if the cycle length remained within 30 ms from the control value taken in the beginning of the experiment.

Postganglionic nerve stimulation. PNS was carried out in nine preparations via a well-established technique first described by Vincenzi and West (43) and then validated and used by numerous investigators. A pair of PNS electrodes separated by ~2 mm was placed directly on the endocardial surface of the cephalic portion of the SAN. PNS was delivered for 200–2,000 ms and consisted of 100-µs rectangular pulses 2–15 V in amplitude and frequency of 200 Hz. The trains of pulses were triggered by the electrogram and delivered following the spontaneous AP of the SAN (44). These stimuli were subthreshold for sinoatrial or atrial cells but were of sufficient amplitude to stimulate postganglionic nerve terminals. The intervals between successive trains were at least 2 min. The contribution of beta-adrenergic system was explored in five preparations by administration of nadolol (2 µM).

Antibodies. We used the following antibodies: 1) mouse monoclonal anti-neurofilament-60 (60 kDa; catalog no. MAB5254; Chemicon, Harrow, UK) at dilution 1:100; 2) mouse monoclonal anti-{alpha}-actinin (catalog no. A7811, Sigma, St. Louis, MO) at dilution 1:500; and 3) mouse monoclonal anti-Cx43 (catalog no. MAB3068; Chemicon, Temecula, CA) at dilution 1:100; goat polyclonal anti-choline acetyltransferase (ChAT) (catalog no. AB144P; Chemicon) at dilution 1:50; and mouse anti-tyrosine hydroxylase (TH) (catalog no. MAB5280; Chemicon) at dilution 1:200.

Immunofluorescence labeling. After the optical mapping and PNS experiments, studied preparations (n = 5) were embedded in a freezing medium (Histo Prep; Fisher Scientific, Fairlawn, NJ), frozen in isopentane using liquid N2, and then stored at –80°C until immunohistochemistry was performed. For ChAT and TH immunolabeling, the heart was perfused with 3.7% paraformaldehyde after 5 min of Langendorff perfusion, left in paraformaldehyde overnight, and transferred to 20% sucrose for 2 days before the tissue was frozen.

Immunofluorescence experiments were carried out on frozen intact SAN preparations as previously described (15). Sections (16 µm) were cut perpendicular to the CT and collected on Superfrost Plus glass slides (Fisher Scientific). Tissue sections were fixed in 4% paraformaldehyde and washed three times with PBS. Tissue sections were permeabilized by incubating them in PBS containing 0.1% Triton X-100 for 30 min, washed in PBS, and then blocked in 1% bovine serum albumin (Sigma) in PBS for 1 h. After being washed three times with PBS, tissue sections and cells were incubated with the primary antibodies overnight at 4°C. The primary antibodies were diluted in 1% bovine serum albumin in PBS. The following day the slides were washed three times in PBS and incubated with secondary IgG (anti-mouse IgG or anti-goat IgG; 1:1,000) conjugated to either Alexa 488 or Alexa 555 (Molecular Probes; Eugene, OR) for 2 h. After being washed three times in PBS, tissue sections and cells were mounted with Vectashield (H-1,000; Vector, Burlingame, CA), coverslips were sealed with nail polish, and slides were stored in the dark at 4°C for subsequent viewing with a confocal microscope (Nikon C1; Melville, NY). No labeling above background was obtained when the primary or secondary antibodies were omitted (data not shown).

Immunolabeling quantification. Preparations (n = 3) stained for ChAT and TH were photographed at x40 magnification to create a mosaic of nerve immunolabeling throughout the entire tissue. For quantification, a custom MATLAB program was written to calculate innervation levels throughout the mosaic. Briefly, the mosaic image was randomly decomposed into tiles that were 0.06 mm2 on average. Within each of these tiles, the number of pixels representing the entire tissue was calculated as well as the number of pixels that represented only nerve staining. A three-step algorithm determined the area of an image, which corresponded to nerve staining as shown in Fig. 1. First, a threshold was determined for nerve staining in each mosaic image based on the histogram of the image as the gray value at which the number of pixels fell below ~0.2% of the total number of pixels in the image. This method was empirically determined to reproducibly and representatively select areas of nerve staining. Once the mosaic image was thresholded at this gray value, holes within areas above the threshold were filled. Finally, any area above threshold that consisted of <3 µm2 was discarded as noise. The amount of tissue within each tile was determined as the area of the image above a gray value of 10 for eight-bit images. With the use of this method, the density of innervation was calculated for each tile in the image as the nerve area divided by the tissue area, and the innervation density of tiles from the SAN center as well as the inexcitable region were compared with tiles from the CT. All images from the same preparation were photographed using identical settings.

Statistics. Group data are represented as means ± SE. Comparisons between groups of data were performed by using a Student's t-test and {chi}2-test analysis with Yates correction. Immunolabeling data were computed as densities and compared as ratios normalized to the density of the CT using a paired t-test. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activation pattern of right atrium under control conditions. Figure 2A shows the map of origin of AP in a representative SAN preparation. The white square shows the 9 x 9-mm field of view of the 16 x 16 photodiode array, and Fig. 2B presents typical optical recordings from the SAN and surrounding areas. The leading pacemaker site was 1.9 mm from the CT border. Activation began in the SAN center and rapidly spread along the CT inferiorly and superiorly. Slowly, propagation was observed perpendicular to CT, and block was observed in the septal margin of the SAN (black line in Fig. 2A), as described earlier (17).

We observed the same position of the leading pacemaker as in Fig. 2A in nine preparations (64%), which were considered "typical." SVC and IVC locations of the leading pacemaker were observed in three (21%) and two (14%) preparations, respectively. We measured conduction velocities near the center of SAN (within 1.5 mm from the leading pacemaker site) in the nine "typical" preparations toward the SVC (9.8 ± 0.8 cm/s) and IVC (6.7 ± 1.1 cm/s) and toward CT (3.5 ± 0.3 cm/s) and IAS (2.4 ± 0.6 cm/s). White arrows in Fig. 2A show direction of measurements of conduction velocity. In two experiments we observed bifocal activation of intercaval region (Fig. 2, C–E).

The cycle length in 14 preparations ranged from 336 to 578 ms (405 ± 14 ms) in the control conditions. Staining with voltage-sensitive dye di-4-ANEPPS induced a transient (15–20 min) increase of cycle length up to 456 ± 17 ms. During our experiments we had a relatively stable cycle length and the site of leading pacemaker for 2–5 h. Changes in cycle length were <15 ms/h in agreement with Bleeker et al. (3). Then the spontaneous cycle length could suddenly increase by 20–90 ms, and the leading pacemaker region could move inferiorly toward IVC (in 9 of 14 preparations). See Fig. 1 of the online data supplement.

Figure 3 shows a summary of leading pacemaker sites from all experiments (n = 14) recorded under all possible conditions. In three of five preparations containing AVN (60%), we observed a pacemaker shift to AVN (after more than 5 h of experiment). We observed the block zone in all 14 preparations, which spread from SVC to IVC in 13 of 14 preparations (93%). In a single preparation, we observed a conduction bridge between the intercaval region and septum. In this preparation, the block zone was responsible for a stimulation-induced reentry around the SAN and IVC region (see Fig. 2 of the online data supplement).


Figure 3
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  		Fig. 3. Sites of leading pacemaker in the RA region during different experiments. Photo of one right atrial preparation on which schematically shown all leading pacemaker sites that were recorded during 14 experiments is shown. Leading pacemaker sites from the different experiments are marked by black circles. Black dotted lines schematically show borders of block zone in septum region. CS, coronary sinus.

 
Effects of postganglionic nerve stimulation. Figure 4 illustrates a representative PNS experiment. The top left of Fig. 4 shows a photograph of the preparation. The field of view is shown in the left, and optical recordings from SAN region and atrial electrogram are illustrated in the right. During sinus rhythm (Fig. 4A), the leading pacemaker was located in the center of the intercaval region 2 mm from the CT border. PNS induced a transient (500–1,500 ms in different regions) hyperpolarization and significant suppression of AP amplitude in the pacemaker cells between the center of the SAN and the conduction block zone. It also caused an increase of spontaneous cycle length from 472 to 871 ms, and the pacemaker shifted inferiorly. The first wave of excitation following PNS propagated from IVC region but could not excite the pacemaker cells between the SAN center and the block zone (Fig. 4B). We observed only a subthreshold depolarization hypothetically due to electrotonic spread from nearby atrial cells. We defined cells as inexcitable when the AP amplitude decreases below 40% of control (44). The inexcitable region is shown in white. The second and third waves propagated from IVC region as well (not shown). Only the fourth wave originated from the central SAN region but did not excite border tissue near the block zone, which did not recover yet (Fig. 4C). The PNS-induced hyperpolarization was measured as a percentage of control AP amplitude (see optical signals). The value of hyperpolarization was increased from the SAN center (16%) to block zone (33%). The time course of recovery from hyperpolarization following PNS withdrawal was spatially nonuniform as well. The area of highest hyperpolarization recovered earlier, whereas areas of moderate hyperpolarization required a longer time for recovery. In addition, we observed a strong rebound effect in the area of highest hyperpolarization: spontaneous excitation could follow recovery from strong hyperpolarization. The depolarization following PNS was maximal between the center of SAN and block zone, small or absent between the SAN and CT, and absent in the atrial muscle. Similar observations were reproduced in all nine investigated preparations.


Figure 4
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  		Fig. 4. Effects of postganglionic nerve stimulation (PNS) on spread of spontaneous excitation and morphology of SAN region optical signals. Top left: beginning of activation sequence in control is shown. Field of view is shown with a square outline. Black dotted line shows the line of block zone. Top right: optical AP traces from photodiodes and an EG recording from bipolar electrode shown by 1–4 and EG. Vertical green lines on optical recordings show minimum and maximum levels of optical signals in control and maximum level of hyperpolarization induced by PNS. Bottom: activation sequences of spontaneous beats immediately before PNS (A), the first after PNS (B), and the fifth after PNS (C) with reestablishment of pacemaker dominance as before PNS. Isochrones are separated by 2 ms. White color indicates the temporarily unexcitable area induced by PNS.

 
We could not measure quantitatively the PNS effects on membrane potential because nonratiometric optical recordings cannot be precisely calibrated in terms of transmembrane voltage (18). However, PNS-induced changes of optical signals in different SAN regions correlated well with microelectrode data reported earlier (23, 44).

In all preparations (n = 9), PNS resulted in a transient initial slowing of SAN rate (74 ± 1%), which was followed by brief period of acceleration. However, the direction and distance of PNS-induced pacemaker shift were different from one preparation to another. During the initial slowing (the first beat after PNS), the leading pacemaker shifted inferiorly from the SAN center by 2–10 mm along the CT (Fig. 4) (n = 7) or superiorly (n = 2). During the second beat after PNS, the earliest excitation appeared from the same site such as the first beat (n = 7) or from the block zone border (n = 2), shown in Fig. 5. After PNS the first wave from IVC could not excite the region between SAN and the block zone (Fig. 5B). However, the second wave originated near the border of the block zone and propagated very slowly (Fig. 5C). This pacemaker site can be related to PNS-induced secondary depolarization of pacemaker cells (see above). The optical recordings of APs (Fig. 5, optical recordings 3 and 4, wave C) from this region had a two-component upstroke. Probably, the first component shows real excitation; the second shows electrotonic signal from the periphery. The observation of two-component upstroke in this region correlated well with microelectrode data reported earlier by Bleeker et al. (3, 4). Activation patterns in the subsequent three beats were similar to wave C in Fig. 5, and then pacemaker returned to the control site. In our experiments the leading pacemaker returned to the control site within 4–11 beats after 400–800 ms of PNS.


Figure 5
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  		Fig. 5. PNS induce pacemaker shift to border of block zone. Top left, photograph of SAN preparation with beginnings of control activation sequences (A–C). Top right, optical AP traces and EG recording from a bipolar electrode shown by 1–5 and EG. Bottom: activation sequences of spontaneous beat immediately before PNS (A) and the first (B) and second (C) beats following PNS. Isochrones are separated by 2 ms.

 
Figure 6 shows optical recordings during sinus rhythm and atrial pacing (cycle length = 300 ms), both before and after PNS. PNS induced hyperpolarization of SAN cells, slowing of rhythm, and the inferior shift of the pacemaker site (similar to Fig. 4, A and B). During atrial pacing, PNS also induced hyperpolarization of the SAN region and suppressed AP amplitude in the SAN center (Fig. 6, C and D). No significant difference was found between PNS-induced changes of optical recordings during both spontaneous rate and atrial pacing. The area of hyperpolarization-dependent inexcitability during spontaneous rhythm and during atrial pacing was practically the same (~8 mm2). Thus the size of PNS-induced inexcitable region does not depend on the direction of conduction.


Figure 6
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  		Fig. 6. PNS-induced loss of excitability during atrial pacing. Pattern of SAN excitation and morphology of optical APs during spontaneous rhythm and atrial pacing recorded before (A and C) and immediately after PNS (B and D). Third optical AP was recorded from leading pacemaker site in control. CT is shown with white dotted line on the maps.

 
We found that the average inexcitable area during the first spontaneous beat after PNS was 8.2 ± 1 mm2 (see example in Fig. 6B). However, the size of the inexcitable region was time dependent after PNS. During atrial pacing, only the first two waves could not excite the SAN region (see Fig. 6). When the first spontaneous AP after PNS (duration, 400–800 ms) occurred with a cycle length more than ~1,200 ms, we did not observe inexcitability in the SAN region. Therefore, we used the activation map of the first wave immediately after PNS (400 ms duration) during constant atrial pacing with cycle length equalling 300 ms (see example in Fig. 6D) to measure more precisely the average hyperpolarization-dependent inexcitable area, which was 8.9 ± 1 mm2 (n = 7).

The effects of PNS on SAN rhythm and activation pattern were reproducible for >1 h. After that, probably due to washout and/or photobleaching of the voltage-sensitive dye, PNS-induced changes were difficult to track in optical signals. Thus our pharmacological protocol was limited in time. In five experiments, we applied nadolol (2 µM) to prevent activation of beta-adrenergic receptors. Application of nadolol increased cycle length from 476 ± 17 to 510 ± 24 ms (P < 0.05). However, nadolol induced pacemaker shift only in two preparations (40%). In one preparation, the pacemaker shifted superiorly by 6 mm and in another by 6 mm inferiorly. Nadolol did not prevent PNS-induced hyperpolarization and pacemaker inexcitability in any of the experiments (n = 5). However, as expected, nadolol prevented secondary rhythm acceleration, which followed initial rhythm slowing after PNS in control.

Immunofluorescence labeling. We and others (15, 42) have previously identified by immunolabeling the SAN center as a neurofilament-positive region but Cx43-negative region. Therefore, we used these molecular markers to identify the SAN after optical mapping and PNS in four SAN preparations. From these preparations, sections were cut perpendicular to the CT through the SAN region (see Fig. 1A), its surrounding atrial muscle, and the block zone (which were identified functionally using optical mapping), and they were labeled by immunofluorescence for neurofilament, Cx43, {alpha}-actinin, ChAT, and TH (see Figs. 7 and 8, and Figs. 3 and 4 of the online data supplement).


Figure 7
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  		Fig. 7. Immunofluorescence labeling of connexin 43 (Cx43), neurofilament, and {alpha}-actinin in different regions of the SAN and its surrounding atrial muscle. High magnification views of Cx43, neurofilament, and {alpha}-actinin through the CT (A, B, and C), SAN center (D, E, and F), SAN periphery-inexcitable area (between SAN center and block zone), and block zone (* in J, K, and L).

 

Figure 8
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  		Fig. 8. Histology and immunofluorescence labeling of neurofilament 160, ChAT, and TH in different regions of the SAN and surrounding atrium in sister sections. A: Masson trichrome staining indicates an increase in connective tissue in the block zone compared with the rest of the myocardium. B: neurofilament was used as a marker of the SAN tissue. Both parasympathetic (ChAT, C) and sympathetic (TH, D) innervation was significantly higher in the SAN center than in the CT (E). See Table 1 and text for details.

 
We used neurofilament 160 as a marker of SAN tissue (15). SAN tissue was located in the intercaval region and ended at the endocardial surface of the CT (Fig. 7B). Cx43 labeling was absent in the SAN center but was present in the CT (see Fig. 7, and see Fig. 3 of the online data supplement). In the SAN periphery on the endocardial face of the CT, both neurofilament and Cx43 were present. Cx43-positive bundles were also found in the SAN periphery/inexcitable area toward the interatrial septum (Fig. 3C of the online data supplement).

Figure 7 shows high magnification views of representative tissue sections through the atrial muscle of the CT, SAN center, SAN periphery-PSN area (between SAN center and block zone), and block zone, each labeled for Cx43, neurofilament, and {alpha}-actinin. Atrial cells of the CT express Cx43 (Fig. 7A) but not neurofilament (Fig. 7B). In contrast, the cells of the SAN center do not express Cx43 (Fig. 7D) but express neurofilament (Fig. 7E). The cells of the SAN periphery-inexcitable area express neurofilament (Fig. 7F). In the periphery of the SAN there are cells that do and cells that do not express Cx43 (Fig. 7G). All atrial and nodal cells express {alpha}-actinin (Fig. 7, C, F, and I). The cells of the block zone express neither of the three proteins investigated (Fig. 7, J, K, and L), and this confirms the hypothesis, which was presented in our previous study, that the block zone may be simply explained by absence or reduced number of myocytes in this zone (15).

In three preparations, we also mapped autonomic innervation throughout the SAN and surrounding myocardium starting ~4 mm below SVC and moving ~4 mm toward IVC (Table 1, Fig. 8). Throughout this 4-mm region, we found that the density of both parasympathetic and sympathetic innervation was relatively constant in the SVC-IVC direction (Table 1). However, perpendicular to CT, we found significant differences in innervation density in the intercaval region. Density of parasympathetic innervation was assessed by immunolabeling of ChAT. ChAT density was 6.8-fold higher in the SAN center compared with CT (P < 0.0001). ChAT density in the inexcitable region near the block zone was 2.5-fold higher than in CT (P = 0.056). The ChAT density decreased from the SAN center to the block zone region (P < 0.001). Sympathetic innervation was assessed by TH immunolabeling. TH density was 4.5-fold higher in the SAN center compared with CT (P < 0.005) and 1.7-fold higher in the inexcitable region than in the CT (P < 0.05).


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  		Table 1. Average density values for both parasympathetic (ChAT) and sympathetic (TH) innervation from different intercaval regions (n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Activation pattern of SAN. The mammalian SAN is a very heterogeneous pacemaker structure (7, 30). Since Lewis et al. (25) and Eyster and Meek (19) revealed the site of earliest atrial activation over SAN, numerous studies have indicated that cardiac impulse originates in a relatively small group of primary pacemaker cells (~500–5,000) in the central part of SAN and is typically 0.5–2 mm from the CT in the intercaval region in the area of small interweaving cells (3, 30, 32, 37). However, animal and human studies have found leading pacemaker sites in different anatomical locations within and outside of SAN under physiological conditions (5).

We observed the "classic" position of the rabbit leading pacemaker site in only nine preparations (Fig. 2A); in another three preparations we observed the leading pacemaker site near SVC and in two preparations near IVC. The average cycle length in all SAN preparations was 405 ± 14 ms, which is consistent with data reported earlier by other groups, who did not use BDM and di-4ANEPPS in their studies (3, 14, 23, 41, 45).

Moreover, during our experiments after several hours, we consistently recorded a sudden pacemaker shift by 1–12 mm in the inferior direction. We also observed bifocal activation of the SAN region in two preparations (Fig. 2C). Therefore, we conclude that nearly all cells in the intercaval region ({alpha}-actinin-positive, neurofilament-positive, Cx43-negative region) located between SVC and IVC may play a role as leading pacemaker (Fig. 3). Our observations are in agreement with the concept of widely distributed atrial pacemaker complex presented by Boineau et al. (5). Based on functional observations in humans and canines, they suggested that initiation of the SAN impulse is dynamic and could be multicentric, with more than one focus initiating a single beat (5). To the best of our knowledge we present here the first observation of multicentric SAN activity in the rabbit heart. Our recent immunohistochemical investigation agreed with this theory and demonstrated that the rabbit SAN region extends from SVC to IVC (15).

Sano and Yamagishi (37) performed the first systematic mapping of the rabbit SAN region by using two microelectrodes. They observed highly anisotropic spread of activation from the SAN: starting in the leading pacemaker, AP propagated preferentially in an oblique cranial direction toward CT and appeared to block in the direction of the septum, an observation similar to ours made here and earlier (8, 17). They and Bleeker et al. (3) calculated the conduction velocity to be 2–8 cm/s or less around the leading pacemaker site. Our estimation of conduction velocities near the center of SAN in the nine preparations with "classic" position of leading pacemaker site shows very similar results: 2–12 cm/s depending on the direction (Fig. 2). The conduction is anisotropic with anisotropy ratio of 3.0 ± 0.3 (along vs. across CT).

Block zone between SAN and septum. We observed a block zone between SAN and septum in all 14 preparations (see Figs. 2–6), which spread from SVC to IVC in 13 preparations. In only one preparation we observed a conduction bridge between the intercaval region and septum. This is consistent with our earlier study, in which we saw one such bridge in eight studied preparations (17). We defined the border of the block zone as a region at which signal-to-noise ratio was below 2:1. Thus in 13 experiments (93%), septum was activated only after the wavefront originating in the SAN propagated around IVC, CT, and along the posterior nodal extension.

A block zone between the leading pacemaker site in the SAN center and the interatrial septum has been observed in several species (7). The conduction block could be the result of poor electrical coupling between cells (12) or poor excitability of the myocytes (4, 8, 31). In our recent study (15), we suggested that the block may be simply explained by the absence or reduced number of myocytes in this zone. In this study we investigated the region of functional block zone by immunofluorescence and confirmed that in the block zone there is a paucity of myocytes (see asterisk in Fig. 7, A and B), contrasted with the high density of myocytes in the SAN center, as well as an increase in fibrous tissue in this area, as seen in histological sections as shown in Fig. 8.

Comparison with previous studies and validation of optical mapping. Our activation maps of the rabbit SAN region qualitatively agree with maps obtained earlier using electrode-based mapping techniques (41). Unfortunately, the spatial resolution and dynamic range of existing extracellular electrical mapping systems is often insufficient to adequately characterize the dynamically changing leading pacemaker site (9). On the other hand, it is impossible to have high-quality simultaneous recordings from multiple intracellular microelectrodes during dynamic changes of pacemaker sites induced by different interventions, including PNS. At the present time, high resolution optical imaging appears to be the only available technology that allows resolving simultaneously changes in leading pacemaker sites and morphology of APs during dynamic changes in atrial pacemaker activity (18).

Numerous studies have demonstrated that cholinergic stimulation results in a pacemaker shift from the center of SAN to periphery in the rabbit and dog (6, 28, 38, 40, 41). Using extracellular recordings from the endocardial surface of SAN, Shibata et al. (41) showed that PNS in the presence of propranolol induced pacemaker slowing and shift by 1–6 mm along CT mostly toward SVC and occasionally toward IVC. We observed in our experiments the opposite; namely, PNS (400 ms, 200 Hz, before and after nadolol, 2 µM) induced pacemaker shift mostly toward IVC and only in a few cases toward SVC (Figs. 4–6). These differences could be explained by different size and probably age of preparations used by our groups. All our right atrial preparations included an intact IVC orifice, which were missing in the Shibata study. Moreover, our data agrees with a study conducted by Mackaay et al. (27) in the rabbit SAN and Schuessler et al. (38) in the canine heart in situ which demonstrated 1.2-mm and 10- to 20-mm inferior shifts of the earliest activation site induced by cholinergic stimulation.

Mechanisms of cholinergic-dependent local transient inexcitability. Vagally induced loss of excitability depends on the differential inhibitory effects of ACh on primary and subsidiary pacemaker cells (13, 44) and regional difference in either vagal innervations (26) or muscarinic receptor density in the SAN region (2). It was demonstrated that the rabbit SAN is composed of electrophysiologically heterogeneous pacemaker cells (7). Recently, we (44) and others (23, 45) observed a significant difference in excitability of primary pacemaker cells compared with subsidiary cells of the SAN during cholinergic stimulation.

The density of vagal innervation and M2 receptors in the SAN is greater than that of the atrial muscle (2, 26). Roberts et al. (34) showed significant differences in the pattern of nerves, ganglia, and fine nerve processes in the adult rabbit SAN. Beau et al. (2) demonstrated that muscarinic receptor density in the dominant pacemaker region was 18 ± 2% and 29 ± 7% higher than in adjacent superior and inferior regions, respectively. Our data agree with these findings, indicating that the level of ChAT expression is approximately seven times higher in the SAN center than in the atrial muscle (CT) and ~2.5-fold higher in the center of the PNS-induced inexcitable region than in the CT (Table 1, Fig. 8). The fact that PNS-induced inexcitability did not occur at the location of highest ChAT density, but rather near the block zone, indicates that parasympathetic innervation density alone cannot explain why inexcitability was induced here.

Thus we conclude that the nerves are necessary but not a sufficent factor to induce inexicitability. Opthof et al. (31) have shown that, in the rabbit SAN, little excitable transitional pacemaker cells exist in the zone of septal margin of the rabbit SAN (inexicatable region). Thus we hypothesize that the properties of myocytes (i.e., expression levels of M2 receptors and ionic channels), as well as the coupling between myocytes, therefore must also play a role in the induction of inexcitability.

Vagal stimulation or ACh added to perfusate activates the muscarinic K+ current (IK,ACh), shifts the activation curve of the hyperpolarization-activated inward current (If), and inhibits L-type Ca2+ current (ICa,L) in pacemaker cells of the SAN (14, 33, 36, 46). Activation of IK,ACh results in hyperpolarization, increase in cycle length, and shortening of the SAN cell AP (23). Kodama et al. (23) used microelectrode techniques to demonstrate that the amplitude of hyperpolarization was stronger in the center of the SAN and weaker in the surrounding SAN periphery after brief vagal stimulation. Here, we also reported that amplitude of PNS-induced pacemaker cell hyperpolarization increases in the lateral direction from CT to block zone, parallel to the direction of decreasing resting potential (Figs. 4–6). Thus strong IK,ACh-induced hyperpolarization could suppress excitation of pacemaker cells in this region, due to both automaticity and propagation.

Earlier, Vinogradova et al. (44) reported that cholinergically induced suppression of AP amplitude can be predicted based on the maximum rate of rise of AP upstroke. The upstroke velocity increased with distance from the SAN center toward CT but decreases toward the block zone (3, 8). It has been shown that the region of pacemaker cell with upstroke <10 V/s was ~2 x 6 mm (3, 8, 44). The size (3–12 mm2) and position of the inexcitable area in the present study are in agreement with microelectrode-based observations (44).

Another potentially important contributing fact is cell-cell coupling. Duivenvoorden et al. (16) presented evidence that suggests that PNS decreases intercellular coupling via a decrease in space constant. This cholinergic effect diminishes the mutual electrotonic influence of SAN cells and, therefore, can result in significant local differences in electrical activity of nearby pacemaker cells.

It is well known that increased parasympathetic tone can induce extrasystolic activity, which can precipitate atrial reentrant tachyarrhythmias (39). Kodama et al. (23) demonstrated that at the leading pacemaker site, brief PNS resulted in a large hyperpolarization followed by a depolarization. At other sites within the SAN and in the surrounding atrium, the hyperpolarization was smaller and the depolarization was small or absent. The depolarization in the center of the node was abolished by If block. We also observed that amplitude of secondary depolarization depends on the amplitude of previous hyperpolarization (Figs. 4–6). In two preparations, it induced spontaneous SAN excitation near the block zone, the region that was previously inexcitable (Fig. 5). We hypothesize that this phenomenon represents unidirectional entrance block in the latent pacemaker area, which may play an important role in cholinergic-induced extrasystolic activity and arrhythmogenesis (21, 35).

Limitations. Optical mapping technique has clear advantages over traditional micro- or macroelectrode-based techniques. Yet, it comes at a price of several disadvantages, which may have effect on observed results.

Optical mapping technique has limited duration of the experiment due to washout and/or photobleaching (18).

To eliminate movement artifact during optical recording, we used BDM, which has effect on many ion channels and could potentially affect the leading pacemaker site and conduction in the SAN region. However, previous studies of AVN conduction in the rabbit provided no evidence of such effects of BDM on SAN activation pattern (10). Moreover, our functional data agrees with that of previous groups that did not use BDM (23, 37).

Suppression of mechanical contractions by BDM prevented activation of stretch-activated channels, which can play an important role in pacemaker activity of SAN (11, 24), as well as in initiation of spontaneous vagally induced tachyarrhythmias (20). In this study we did not observe spontaneous PNS-induced tachyarrhythmias. However, previously on the same right atrial preparation, we observed a different type of cholinergic-induced atrial tachyarrhythmias (44). Thus the effects of BDM may explain our failure to observe spontaneous PNS-induced tachyarrhythmias in this study (see above).


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The project was supported by the Stanley and Lucy Lopata Endowment (Washington University) and National Heart, Lung, and Blood Institute Grant HL-58808.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. R. Efimov, Washington Univ., Campus Box 1097, One Brookings Drive, St. Louis, MO 63130 (e-mail: igor{at}wustl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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