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1Division of Cardiology, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California; 2Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana; 3Division of Neurology, Department of Pediatrics, Childrens Hospital and University of Southern California Keck School of Medicine, Los Angeles, California; and 4Division of Anatomical Pathology and Laboratory Medicine, Department of Pathology, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California
Submitted 11 November 2007 ; accepted in final form 2 June 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSDISCLOSURESREFERENCES |
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atrial tachycardia; electrophysiology; autonomic nervous system; sympathetic nerves; computerized mapping
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METHODS |
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Surgery
Under isoflurane anesthesia, the chest was opened via a median sternotomy. The left (LSG) and right SG (RSG) were visually identified. A pair of bipolar hook electrodes was attached to each SG to record SG nerve activity. Signal was filtered with a 30- to 500-Hz band pass and acquired at 979 samples/s using Prucka Labsys (Austin, TX) along with surface ECG.
High-density epicardial mapping of thoracic veins, including PVs and vein of Marshall (VOM) and adjoining left atrium (LA) and right atrium (RA), was performed using a 1,792-bipolar channel mapping system. Three or four flexible electrode plaques, each with 496 bipolar electrodes with a 1-mm resolution, were used to map the RA, LA, left PVs, and right PVs, according to methods published in a previous report (18).
SG Stimulation and Electrophysiological Studies
We stimulated the SG using three protocols.
Bilateral SG stimulation and blood sampling for catecholamine. We sought to demonstrate that SG stimulation increases cardiac sympathetic tone by comparing transcardiac (coronary sinus minus aorta) levels of catecholamines before and after bilateral SG stimulation. We sampled blood simultaneously from the coronary sinus, aorta, and cephalic vein. Both SG were then maximally simultaneously stimulated at a level at least thrice their thresholds (30 mA, 20 Hz, 2-ms pulse width) for a period of 30 s. The threshold is defined as current needed to produce a 20% or more rise in systolic blood pressure (SBP) or heart rate. Immediately thereafter, blood was resampled from the same sites. The samples were stored in ice and later processed for ELISA catecholamine assays.
Hemodynamic effects of unilateral SG stimulation. To determine whether there is functional asymmetry between the hemodynamic effects of LSG and RSG activation, we performed graded stimulation of each SG (2–35 mA) and recorded their differential effects on heart rate, blood pressure, and SG nerve activity.
Arrhythmic effects of unilateral SG stimulation. The aim of this protocol is to determine whether SG stimulation can induce arrhythmias and to determine whether there is functional asymmetry in the arrhythmic effects of left vs. right stellate stimulation. Each SG was stimulated separately at 20–40 mA. High-density mapping was performed 1) at baseline sinus rhythm without SG stimulation; 2) at baseline after SG stimulation; 3) after mechanical crushing of the sinus node but without SG stimulation; and 4) after sinus node crushing and after SG stimulation. When SG stimulation was performed, mapping was acquired immediately after the end of the stimulation train. We avoided atrial or PV electrical stimulation throughout the experiment, as these maneuvers are known to induce arrhythmias even without sympathetic stimulation.
Histology
During in vivo studies, the ectopic sites were identified on playback of high-density maps and marked on the back surface of mapping plaques. At the end of the experiment, the mapping plaques were removed from the tissue, and the sites were marked on the tissue using indelible Indian ink. Digital photos of the experimental setup with mapping plaques in situ aid in ensuring accurate electrical-to-anatomical correlation of the ectopic sites. The heart-PV preparation was fixed in a nonaqueous fixative, Carnoy's solution, to optimize staining for glycogen with PAS reagent. PAS staining is used to identify glycogen-rich specialized conduction cells, such as Purkinje cells (3). Multiple tissue blocks were sampled from the sites of ectopic focus, as well as other sites, including the LA and RA appendages, the PVs, VOM, and atrioventricular (AV) junction. The slides were stained with routine hematoxylin and eosin and Masson's trichrome for structural examination. The slides were also immunostained with antibodies to tyrosine hydroxylase (TH) for sympathetic nerves, according to methods described previously (4). Light microscopy was used to examine the slides. Nerve density and PAS staining were analyzed using computerized morphometry (Image Pro) (4). We quantified and compared the densities of sympathetic nerves and PAS staining at multiple locations. These included the ectopic atrial or venous sites, and other nonectopic atrial or venous sites that are at least 10 mm away from the focus. We defined the ectopic site histologically as any area within a 5-mm circle containing the Indian ink-marked spot. The Indian ink spot remains visible on the paraffin tissue block.
Catecholamine Assay
Serum epinephrine and norepinephrine concentrations were assayed by using a competitive enzyme immunoassay kit per manufacturer's protocol (Alpco Diagnostics, Salem, NH). Transcardiac catecholamine levels (pg/ml) were defined by coronary sinus minus aorta concentrations.
Electrophysiological Data Analyses
The absolute maximum time derivative of volume was selected as the time of local activation for bipolar electrograms. Dynamic display and isochronal maps were used to analyze the activation sequence. A focal activation was defined as an activation pattern with wavefronts spreading outward in all directions. An activation wavefront that originates from the edge of the mapped region was not considered focal, unless originating from the distal PV.
Statistical Analyses
Data are expressed as means ± SD. Values between two groups were compared with Student's two-tailed t-tests, and multiple-group comparisons were made with ANOVA, followed by Newman-Keuls post hoc analysis. A 2 x 2 
2 test was used to test the association between two categorical data. A P value of 
0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESREFERENCES |
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The SG was easily identified in all dogs (Fig. 1A). Histological section (Fig. 1B) showed the presence of TH-positive ganglion cells and nerve fibers, confirming the correct identification of this structure. Figure 1C shows the effects of bilateral SG stimulation on blood catecholamine levels sampled from the coronary sinus, aorta, and peripheral vein in all dogs studied. SG stimulation resulted in a rise of norepinephrine levels in the coronary sinus (P < 0.01), aorta (P < 0.01), and peripheral vein (P < 0.01), without a concomitant rise of epinephrine levels [P = nonsignificant (NS)]. Transcardiac (coronary sinus minus aorta) norepinephrine levels (P < 0.01), but not epinephrine levels (P = NS), were significantly elevated after SG stimulation, consistent with the fact that norepinephrine is the catecholamine released from nerve endings during SG stimulation.
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Figure 2 illustrates the results of graded stimulation of LSG vs. RSG. There was no difference in the threshold to induce a 20% rise in SBP and heart rate during LSG vs. RSG stimulation (6.8 ± 3.1 vs. 5.8 ± 3.3 mA, P = NS). At weak stimulation strengths (10–15 mA) (Fig. 2A), RSG stimulation induced a greater change in heart rate than LSG stimulation (P < 0.05 at both 10 and 15 mA), whereas LSG stimulation (Fig. 2B) produced a greater change in SBP than RSG stimulation (P < 0.01 at 15 mA). At stronger stimulation strengths (20–35 mA), there were no significant differences between the effects of LSG vs. RSG stimulation on either heart rate or SBP. Graded stimulation with increasing current strengths produced a progressive rise of heart rate and SBP until 30 mA. Beyond that, the rise in SBP after 35-mA SG stimulation was less compared with 30 mA, suggesting that fatigue had set in. Weak unilateral SG stimulation (15 mA) elicited neural afterdischarges from the ipsilateral ganglion only (Fig. 2B). On the other hand, stronger unilateral SG stimulation (20–40 mA) elicited bilateral SG afterdischarges (Fig. 2C). The threshold to elicit bilateral SG afterdischarges (the afterdischarge threshold) was 20.8 ± 8.6 mA for LSG and 17.0 ± 9.7 mA for RSG (P = NS).
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Table 1 summarizes the results of high-density mapping.
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After sinus node crushing. After sinus node crushing (but without SG stimulation), a slow junctional rhythm or ectopic atrial rhythm (CL 1,204 ± 152 ms) was observed in all dogs. Subsequent SG stimulation induced 20 episodes of ectopic beats in seven out of eight dogs. Among these 20 episodes, the earliest activity was observed to arise from the LA in 2 episodes, the RA in 5 episodes, the PVs in 11 episodes, and VOM in 2 episodes. SG stimulation after sinus node crushing also induced PV tachycardia (7 episodes) in three dogs (CL 273 ± 35 ms, duration 16 ± 4 s).
Figure 3 shows an episode of PV tachycardia originating from the right superior PV (RSPV). Figure 3A shows baseline sinus rhythm. The earliest site (arrow) was from the sinus node direction. The blue color on the third panel shows the delayed activation of VOM. After sinus node crushing, RSG stimulation induced an RSPV tachycardia (Fig. 3B, beat b). The earliest activation site was within the RSPV, and the propagation was centrifugal from that early site (beat b, arrow). After its termination, an ectopic atrial rhythm resumed (Fig. 3B, beat c). Electrograms 1–4 are taken from sites adjacent to the site of initiation of tachycardia.
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Histological Examinations
We performed histological analyses of a total of 38 tissue blocks from eight dogs. Table 1 also presents a summary of the histological examinations performed. Among the eight dogs, seven dogs developed ectopic foci after sinus node crushing and SG stimulation. Six of these seven dogs had thoracic vein ectopies, whereas one dog had only atrial ectopies (Table 1). For the six dogs with thoracic vein ectopies, five blocks were obtained from each dog (one each from LA, RA, AV junction, ectopic thoracic vein, and nonectopic vein). For the remaining two dogs without thoracic vein ectopies, four rather than five blocks per dog were obtained (one instead of two thoracic vein blocks per dog). Among the 38 blocks, 14 were from thoracic veins (6 ectopic veins and 8 nonectopic veins), 8 from LA, 8 from RA, and 8 from the AV junction. A total of 38 slides were stained for both PAS and TH (14 from PV, 8 each from LA, RA, and AV junction).
Purkinje-like cells in the PV. Figure 6 shows the results of PAS staining. The PAS-positive cells, which stain entirely deep magenta, are cells with a greater amount of glycogen compared with regular myocardial cells (light purple). Figure 6A shows PAS staining of the LSPV. Arrows point to PAS-positive cells clustering on the endocardial surface as a thin layer up to three cells thick. Figure 6B shows NKX2.5 staining of the same PV, showing that PAS-positive cells also stain positive for NKX2.5. NKX2.5 is a myogenic transcription factor expressed in significantly higher levels by Purkinje fibers compared with normal myocardial cells (16). Its higher expression in PAS-positive cells than normal myocardial cells suggests that these cells are specialized conduction cells akin to Purkinje cells. We used canine AV junction (Fig. 6, C and D) and ventricular Purkinje cells (Fig. 6E) as internal positive controls. Figure 6C shows the bundle of His at the AV junction, which stained deep magenta. In contrast, the surrounding ventricular myocardium stained light purple. Figure 6D is a higher power view of the bundle of His, showing PAS-positive cells (arrows). Figure 6G shows Purkinje cells (arrows) on the subendocardial side of the ventricular myocardium in the same dog. Figure 6, E and F, show connexin 43 and 40 staining, respectively, in PV. We found that PV and atria stain positive for connexin 40 and 43, but negative for connexin 45. The subendocardial portion of the PV myocardium, where PAS-positive cells are clustered, have relatively less connexin staining compared with the midmyocardial portion. As opposed to canine sinus node, PV and atria were immunonegative for hyperpolarization-activated cation channels HCN2 and HCN4.
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DISCUSSION |
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Ectopic Pacemakers After Sinus Node Crushing
Sympathetic nerve activity regulates intracellular calcium dynamics (2). Because intracellular calcium release plays important roles in atrial pacemaker activity (7, 12), sympathetic stimulation can induce focal discharges from the VOM and the PVs in dogs (1, 9). In this study of normal dogs, we demonstrated that, after sinus node crushing, sympathetic nerve stimulation can induce focal ectopic atrial beats and tachycardia arising most commonly from thoracic veins. However, the arrhythmias may also arise from the LA and RA. These findings suggest that latent ectopic pacemakers are constitutively present in normal dogs and are unmasked by the elimination of the dominant sinus node influence and with sufficient adrenergic stimulation. It is possible that, in patients with sick sinus node, these latent ectopic pacemakers may take over and serve as either the subsidiary pacemakers or sources of rapid activations that help trigger or sustain atrial arrhythmia.
Histological Findings of the Ectopic Pacemakers
We found high densities of PAS-positive cells at the sites serving as ectopic pacemakers. The high NKX2.5 expression further suggests that these cells are specialized conduction cells. Because of the large differences of densities between the ectopic pacemaker site and other sites, these PAS-positive cells most likely played a role in the generation of the ectopic activity. This conclusion is further strengthened by the finding that, within the same dog, the pacemaker sites tended to be fixed, rather than randomly distributed throughout the thoracic veins and atria. We also found that there is significantly increased sympathetic innervation at the ectopic sites. We hypothesize that the higher density of sympathetic innervation at the ectopic sites translates to exposure of the pacemaker cells at those sites to more intense adrenergic stimuli during each burst of sympathetic nerve activity. This, in turn, promotes both automaticity and triggered activity at those sites. Kwong et al. (11) observed that connexin 43 staining clustered around the edges of the canine sinus node. The authors hypothesized that this distribution allows connexins to act as specialized conduction bundles directing propagation out of the sinus node. In our study, we observed relatively less connexin 43 staining on the subendocardial layer of PV, where PAS-positive cells are clustered.
Functional Asymmetry of LSG and RSG stimulation
Crampton et al. (8) reported that excessive or unopposed activity of the LSG, or subnormal activity of the RSG, account for the pathophysiological manifestations of the long QT syndrome. Schwartz et al. (15) reported that LSG ablation was an effective treatment for ventricular arrhythmias in patients with long QT. These data suggest that there is functional asymmetry between the effects of LSG and RSG stimulation in terms of ventricular arrhythmogenesis. However, less is known about the effects of LSG vs. RSG activation on atrial electrophysiology. One aim of the present study was to determine whether there is functional asymmetry of RSG and LSG stimulation on atrial arrhythmias. LSG stimulation had a greater effect on blood pressure than heart rate, whereas the opposite was true for RSG at lower stimulation strengths. However, there was no significant functional asymmetry in the hemodynamic effects of right and left stellate stimulation at higher stimulation strengths. There was also no difference in arrhythmogenic potential of left- vs. right-sided stellate stimulation. One potential explanation for this observation is that, at higher stimulation levels, stimulation of one SG activates both SG.
Limitations
This study did not perform intracellular recordings directly from the ectopic PAS-positive cells. Therefore, we cannot determine the exact origin of the ectopic rhythms. Other investigators have performed intracellular recordings from isolated PV cell preparations and demonstrated the presence of nodal action potentials (5). Their data and ours strongly support the hypothesis that PVs contain specialized conduction cells, and that these cells play an arrhythmogenic role under autonomic stimuli.
Conclusions
We conclude that thoracic veins can serve as subsidiary pacemakers and dominant sources of ectopic atrial tachycardias under hyperadrenergic stimuli, once the sinus node is eliminated, because of the presence of abundant sympathetic innervation and PAS-positive specialized conduction cells.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESREFERENCES |
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESREFERENCES |
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