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Spontaneous stellate ganglion nerve activity and ventricular arrhythmia in a canine model of sudden death
Spontaneous stellate ganglion nerve activity and ventricular arrhythmia in a canine model of sudden death Shengmei Zhou, MD,* Byung-Chun Jung, MD, PhD,* Alex Y. Tan, MD, FHRS,* Vinh Quang Trang,* Ghassan Gholmieh, MD, PhD,† Seong-Wook Han, MD, PhD,‡ Shien-Fong Lin, PhD,*‡ Michael C. Fishbein, MD,储 Peng-Sheng Chen, MD,*‡ Lan S. Chen, MD†¶ From the *Division of Cardiology, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, † Childrens Hospital Los Angeles and Keck School of Medicine, University of Southern California, Los Angeles, California, ‡Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, Indiana, 储Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, Los Angeles, California, and ¶Pediatric Neurology, Riley Hospital for Children and Indiana University School of Medicine, Indianapolis, Indiana. BACKGROUND Little information is available on the temporal relationship between instantaneous sympathetic nerve activity and ventricular arrhythmia in ambulatory animals. OBJECTIVE The purpose of this study was to determine if increased sympathetic nerve activity precedes the onset of ventricular arrhythmia. METHODS Simultaneous continuous long-term recording of left stellate ganglion (LSG) nerve activity and electrocardiography was performed in eight dogs with nerve growth factor infusion to the LSG, atrioventricular block, and myocardial infarction (experimental group) and in six normal dogs (control group). RESULTS LSG nerve activity included low-amplitude burst discharge activity (LABDA) and high-amplitude spike discharge activity (HASDA). Both LABDA and HASDA accelerated heart rate. In the experimental group, most ventricular tachycardia (86.3%) and sudden cardiac death were preceded within 15 seconds by either LABDA or HASDA. The closer to onset of ventricular tachycardia, the higher the nerve activity. The majority of HASDA was followed
Introduction Ventricular arrhythmia and sudden cardiac death (SCD) are major causes of mortality in patients with heart disease. However, the immediate triggers of ventricular arrhythmia and SCD remain unclear. Clinical studies show a circadian variation of SCD in patients.1 Beta-blockade is associated with a reduction in SCD mortality after myocardial infarction (MI) in randomized clinical trials.2 Increased cardiac sympathetic nerve density is seen in patients with a clinical history of ventricular arrhythmia.3 Cardiac sympathetic hyThis study was supported by American Heart Association (AHA) Grant 0435135N; a Heart Rhythm Society Postdoctoral Fellowship Award; National Institutes of Health (NIH) Grants HL78932, HL58533, HL66389, and HL71140; AHA Established Investigator Award 0540093N; and Price, Piansky, and Medtronic–Zipes endowments. Address reprint requests and correspondence: Dr. Lan S. Chen, 575 West Drive, XE 040, Indianapolis, Indiana 46202. E-mail address: [email protected]. (Received April 18, 2007; accepted September 6, 2007.)
immediately by either ventricular arrhythmia (21%) or QRS morphology changes (65%). HASDA occurred in a circadian pattern. HASDA occurred twice as often in the experimental group than in the control group. Electrical stimulation of LSG increased transmural heterogeneity of repolarization (Tpeak-end intervals) and induced either ventricular tachycardia or fibrillation in the experimental group but not in the control group. Immunohistochemical studies revealed increased synaptogenesis and nerve sprouting in the LSG in the experimental group. CONCLUSION Two distinct types of LSG nerve activity (HASDA and LABDA) are present in the LSG of ambulatory dogs. The majority of malignant ventricular arrhythmias are preceded by either HASDA or LABDA, with HASDA particularly arrhythmogenic. KEYWORDS Arrhythmia; Cardiac electrophysiology; Sympathetic nerve activity (Heart Rhythm 2008;5:131–139) © 2008 Heart Rhythm Society. All rights reserved.
perinnervation in dogs significantly increases the incidence of SCD.4 The left stellate ganglion (LSG) is known to be important in cardiac arrhythmogenesis.5 Left cardiac sympathetic denervation, including LSG resection, decreases the incidence of ventricular arrhythmia in patients with MI.6 These data suggest that increased sympathetic tone is important in the generation of ventricular arrhythmia and SCD.7 However, except for a case report of a sheep with SCD during acute MI,8 little direct evidence supports a temporal relationship between spontaneously increased sympathetic discharges and cardiac arrhythmia and SCD in ambulatory humans or animals. To fill this gap of knowledge, we have developed methods for performing long-term continuous (24 hours per day, 7 days per week) recording of electrocardiogram (ECG) and stellate ganglion nerve activity (SGNA) in normal dogs and in dogs with heart failure.9,10 We also developed a high-yield canine model of ventricular arrhythmia and SCD.4 The first aim of the study
1547-5271/$ -see front matter © 2008 Heart Rhythm Society. All rights reserved.
doi:10.1016/j.hrthm.2007.09.007
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was to identify and characterize sympathetic nerve activity immediately preceding spontaneous paroxysmal ventricular arrhythmias by performing long-term continuous ambulatory nerve recording in this canine model. The second aim was to perform LSG stimulation before euthanasia in these dogs to confirm the hypothesis that LSG discharges preceded ventricular arrhythmias.
munostained using a modified immunocytochemical ABCperoxidase method as described previously.11 Growth-associated protein-43 and synaptophysin immunoreactivity was quantified using a computer-assisted image analysis system (Image-Pro Plus 4.0, MediaCybernetics, Carlsbad, CA, USA). Immunoreactivity was expressed as the total area of positive staining per unit area (m2/mm2).
Methods
Data analysis
This study consisted of eight dogs in the experimental group and six normal dogs from a previous study.9 All dogs underwent left thoracotomy under general anesthesia for radiotransmitter implantation and ambulatory nerve recordings. The protocol was approved by the Institutional Animal Care and Use Committee.
Surgery for the experimental group General anesthesia was maintained by isoflurane inhalation. Complete atrioventricular (AV) block was produced by radiofrequency ablation of the AV junction. Left lateral thoracotomy was performed and MI created by ligating the left anterior descending coronary artery below the first diagonal branch. An osmotic pump (Durect Co., Cupertino, CA, USA) was implanted near the LSG for continuous infusion of nerve growth factor (Sigma Chemical Co., St. Louis, MO, USA). A pacemaker was implanted for backup ventricular pacing at 40 bpm. A radiotransmitter (D70-EEE, Data Sciences International, St. Paul, MN, USA) was inserted into a subcutaneous pocket. One pair of electrodes was used to record from the LSG; another pair was implanted in subcutaneous tissue for ECG recording. One of the two electrodes for ECG recording was placed in the upper right quadrant of the chest and the other in the lower left quadrant. Simultaneous left SGNA and ECG were continuously recorded 24 hours per day, 7 days per week.
Direct electrical stimulation of LSG At the end of the monitoring period, all surviving dogs were anesthetized with isoflurane for in vivo study. SGNA, surface ECG, and blood pressure were recorded simultaneously using a CardioLab 4.0 system (Prucka Engineering Inc., Houston, TX, USA) before and after electrical stimulation of LSG (5-ms pulse width at 20 Hz) for 15 seconds. To test the afterdischarge threshold, the stimulation current was ramped up from 1 mA, 2 mA, and 5 mA, followed by 5-mA increments of each stimulation until afterdischarges occurred. QT interval, Tpeak-end interval (Tpe), corrected QT interval (QTc), and corrected Tpeak-end interval (Tpec) were measured before and after stimulation. QTc and Tpec were QT and Tpe, respectively, divided by the square root of the preceding R-R interval. The dogs were euthanized by exsanguination during general anesthesia.
Immunohistochemistry staining LSGs were removed for immunostaining of synaptophysin (Chemicon, Temecula, CA, USA) and growth-associated protein-43 (Chemicon). Five-micrometer sections were im-
Entire datasets were manually analyzed for the occurrence and frequencies of high-amplitude spike discharge activity (HASDA) and ventricular tachycardia (VT), which was defined as ⱖ3 consecutive beats with heart rate exceeding 100 bpm. Episodes of VT were randomly selected to determine integrated sympathetic nerve activity12 20 seconds before onset of VT and the association between HASDA and arrhythmia. High-pass (30-Hz) filtering was used first to eliminate ECG signals from SGNA recording channels. Integrated nerve activity (V) was calculated by averaging the amplitude of SGNA over each 100-ms segment. Integrated nerve activity preceding the onset of VT or ventricular fibrillation (VF) was measured and compared with the results of integrated nerve activity from a randomly selected 1-minute arrhythmia-free period during the same hour of VT occurrence. The recordings from six normal control dogs9 were analyzed using the same methods for comparison.
Statistical analysis All quantitative data were presented as mean ⫾ SD. Student’s t-tests were used to compare the means between two groups. Cosinor test was used to determine the significance of circadian variation. Fisher exact test was used to determine the correlation between elevated SGNA and VT. P ⱕ.05 was considered significant.
Results The simultaneous recording lasted 55 ⫾ 40 days in the experimental group and 43 ⫾ 7 days in the control group.
Stellate ganglia nerve activity Two distinctly different patterns of SGNA were observed in both groups: low-amplitude burst discharge activity (LABDA, Figure 1A) and HASDA (Figure 1B). LABDA accounted for the majority of LSG nerve activity. HASDA was uncommon compared with LABDA. The amplitude of LABDA varied from 0.05 to 0.8 mV; most were in the range from 0.1 to 0.4 mV. The duration of each LABDA episode varied considerably from 0.5 second to 6 minutes; most episodes lasted 2 to 10 seconds. The occurrence of LABDA changed from time to time, from 0 to more than 200 episodes per hour. HASDA had 6.9 ⫾ 1.5 (range 3–12) spikes per episode, with spike amplitude of 0.91 ⫾ 0.16 mV. The spikes had a roughly regular interval and low frequency (cycle length 152 ⫾ 17.7 ms). The average duration of HASDA was 1,059 ⫾ 254 ms. Most HASDA was associated with downward baseline shift, which were morphologically similar to the depolarization shift during the epileptiform discharges.
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Figure 1 Patterns of left stellate ganglion nerve activity (SGNA) in normal control dog. A: Low-amplitude burst discharge activity (LABDA). B: High-amplitude spike discharge activity (HASDA). Both LABDA and HASDA were associated with heart rate acceleration shown on simultaneously recorded ECG. Units for SGNA and ECG are given in millivolts in this and all other figures.
SGNA and heart rate in the control group LABDA and HASDA induced immediate heart rate acceleration in normal dogs (Figures 1A and 1B). In 50 randomly selected runs of HASDA, R-R intervals were shortened from 395 ⫾ 39 ms before discharge to 347 ⫾ 26 ms immediately after discharge (P ⬍.01). SGNA was not associated with arrhythmia in normal dogs.
SGNA and ventricular arrhythmias in the experimental group Three dogs in the experimental group died suddenly. The recordings of a witnessed SCD event when one of the authors (SZ) entered the dog run for routine examination are shown in Figure 2. Accelerated idioventricular rhythm occurred soon after onset of LABDA (Figure 2A, arrow).
Figure 2 Example of increased left stellate ganglion nerve activity (SGNA) preceding ventricular fibrillation (VF) and sudden cardiac death. A: Increased low-amplitude burst discharge activity (LABDA) resulted in accelerated idioventricular rhythm. B: VF occurred approximately 40 seconds later. Panels A and B are continuous. C: A 6-second recording from panel B. INA ⫽ integrated nerve activity; P ⫽ P wave, which is dissociated from ventricular activation due to complete AV block. Units for INA are given in millivolts in this and all other figures.
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Figure 3 Examples of increased stellate ganglion nerve activity (SGNA) preceding ventricular tachycardia (VT). A: One episode of low-amplitude burst discharge activity (LABDA) before onset of VT. B: Two episodes of LABDA prior to onset of VT. C: LABDA followed by increased atrial rate (arrow, p wave) and accelerated idioventricular rhythm. Panels A, B, and C were recorded from three different dogs from the experimental group. INA ⫽ integrated nerve activity.
Integrated nerve activity showed an abrupt increase of SGNA. VF occurred 40 seconds later. Approximately 10 seconds after VF onset, a burst of an abrupt eightfold in-
crease of LABDA occurred (Figure 2B, arrow). This large increase of SGNA is consistent with sympathetic activation in response to hypotension. Another death occurred unwit-
Figure 4 Quantitative analyses of stellate ganglion nerve activity (SGNA) prior to the onset of ventricular tachycardia (VT). A: SGNA averaged over each 10-second segment for 1 minute before onset of 70 VT episodes. The closer to the onset of VT, the higher the average SGNA became. B–E: Selected examples of integrated nerve activity 60 seconds before onset of VT, showing variable patterns of increased SGNA before onset of VT. *P ⬍.05; ** P ⬍.01 vs – 60 seconds; ††P ⬍.01 vs –10 seconds; ‡P ⬍.05 vs –10 seconds.
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nessed at 2:32 AM. VF was also preceded by LABDA, which continued for 150 seconds before VF occurred. A third sudden death occurred spontaneously after the dog was being anesthetized with isoflurane in preparation for a second surgery; no recordings were available. In the experimental group, all dogs had phase 1 VT during the first 3 days after surgery and phase 2 VT that commenced 5 to 7 days later.4 The frequency of phase 2 VT was 2.3 ⫾ 1.2 episodes per day in the experimental group. Two hundred five episodes of phase 2 VT from six dogs in experimental group were randomly selected, and the SGNA of a 20-second period immediately before onset of VT was analyzed. A total of 177 episodes (86.3%) of VT were preceded within 10 to 15 seconds by increased SGNA, either LABDA (96.6%) or HASDA (3.4%). In contrast, the SGNA exceeded three times the baseline noise level only 36% ⫾ 10% of the time among all the dogs studied. Fisher exact test showed a significant positive association between VT and sympathetic discharge (P ⬍.0001). The latency between onset of HASDA and VT was 1.7 ⫾ 1.3 seconds, and the latency between LABDA and VT was 14.2 ⫾ 8.8 seconds (P ⬍.001). Among the VT episodes, 97.1% were monomorphic and 2.9 % were polymorphic. Average VT duration was 15.4 ⫾ 8.1 seconds, and VT rate was 159 ⫾ 39.8 bpm. The amplitude and duration of LABDA that preceded VT varied from episode to episode. For example, the onset of VT shown in Figure 3A was preceded by continuous LABDA over a 10-second period, whereas two separate bursts of LABDA preceded the onset of VT in Figure 3B. LABDA also increased the atrial rate and ventricular escape rhythm in the experimental group, which had complete AV block (Figure 3C). To determine whether a crescendo increase of SGNA occurred prior to onset of VT, integrated nerve activity of SGNA was measured 1 minute before onset of 70 randomly selected VT episodes. The results showed that the closer to the onset of VT, the higher integrated nerve activity became (Figure 4). The time-averaged integrated SGNA at 0 to 10 seconds immediately preceding VT was significantly higher than that at 50 to 60 seconds before onset of VT (18 ⫾ 8.7 V vs 12 ⫾ 5.6 V, n ⫽ 70, P ⬍.01; Figure 4A). The amplitude of increased SGNA and the duration elapsed between onset of increased SGNA and VT varied considerably among VT episodes. Figures 4B to 4E show selected examples of integrated nerve activity 60 seconds before onset of VT episodes. Integrated nerve activity also was measured from a randomly selected 1-minute arrhythmia-free period during the same hour of each selected VT episode. The timeaveraged integrated SGNA 1 minute preceding VT episodes was significantly higher than that of randomly selected arrhythmia-free 1-minute segments (15 ⫾ 5.8 V vs 11 ⫾ 3.7 V, n ⫽ 70 for both, P ⬍.01). Because the average VT occurrence was 2.3 ⫾ 1.2 episodes per day while intermittent LABDA occurred throughout the day, less than 1% of LABDA episodes were followed by VT.
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Figure 5 Frequency and circadian variation of stellate ganglion nerve activity (SGNA) in the experimental group. A: Average number of highamplitude spike discharge activity (HASDA) per day from weeks 2 to 8. B: Average number of HASDA in each hour of the day. The number of HASDA shown in panel B is the average HASDA recorded from five dogs in experimental group over a 10-day period. C: Integrated SGNA (almost all low-amplitude burst discharge activity) on 8 different days in four dogs. We used the midnight value as 1 and obtained the ratio of each hour against that value. Cosinor test showed P ⬍.0001 for the presence of circadian variation in panels B and C. NGF ⫽ nerve growth factor.
HASDA and cardiac electrical instability HASDA appeared approximately 2 to 5 days after the surgery, reached a peak around the second week, and persisted until the end of the study (Figure 5A). Figure 5B shows the 24-hour distribution of HASDA episodes in the experimental group. The vivarium staff turned on the light around 6 to 7 AM and began cage cleaning and feeding afterward. These activities most likely account for the increased HASDA in the early morning. The frequency of HASDA in the experimental group was twice that in the control group (14.1 ⫾ 3.1 per day vs 7.2 ⫾ 5.2 per day, P ⬍.05) in the third week after the first surgery. Total integrated SGNA was analyzed and showed significant circadian variation (Figure 5C). To investigate the effects of HASDA on cardiac arrhythmia, 502 runs of HASDA from the experimental group were randomly selected. In contrast to LABDA, which usually was not followed by ventricular arrhythmia, 21% of HASDA induced ventricular arrhythmia, including short runs of VT (Figure 6A) and single premature ventricular contraction or couplets (Figure 6B). In addition, 65% of HASDA was followed by changes of QRST morphology
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Figure 6 High-amplitude spike discharge activity (HASDA) induced ventricular tachycardia (A), premature ventricular contraction (PVC; B), and QRST morphology change (C) in the experimental group. SGNA ⫽ stellate ganglion nerve activity.
(Figure 6C). These morphology changes could be due to altered conduction velocity from the same ectopic focus, fusion with a different ectopic focus, or both. These findings suggest that HASDA has effects on cardiac excitability and conduction in diseased ventricles.
Electrical stimulation of LSG Direct electrical stimulation was administered to LSG of all surviving dogs before euthanasia. The threshold to induce LSG afterdischarges (Figure 7) in the experimental group was significantly lower than in the control group (3 ⫾ 1.9 mA vs 9.4 ⫾ 6.8 mA, P ⬍.05). In the normal group, electrical stimulation of LSG induced sinus tachycardia and shortened QT (before vs after, 294 ⫾ 21 ms vs 249 ⫾ 29 ms, n ⫽ 6, P ⬍.01), QTc (396 ⫾ 17 ms vs 378 ⫾ 16 ms, n ⫽ 6, P ⬍.05), Tpe (58⫾ 15 ms vs 45 ⫾ 9.6 ms, n ⫽ 6, P ⬍.05), and Tpec (78 ⫾ 16.2 ms vs 69 ⫾ 15 ms, n ⫽ 6, P ⬍.05). In the experimental group, electrical stimulation of LSG increased ventricular rate from 44 ⫾ 3.7 bpm to 57 ⫾ 7.4 bpm (n ⫽ 4, P ⬍.05); prolonged QTc (322 ⫾ 19.8 ms vs 372 ⫾ 37 ms, n ⫽ 4, P ⬍.05), Tpe (77 ⫾ 8.3 ms vs 102⫾ 12.3 ms, n ⫽ 4, P ⬍.05), and Tpec (66 ⫾ 8.6 ms vs 95 ⫾ 11.0 ms, n ⫽ 4, P ⬍.05; Figure 7A); and induced VT in 4 of 4 dogs and VF in 2 of 4 dogs (Figures 7B and 7C). In both
groups, stimulation of LSG resulted in an immediate increase of blood pressure, and the increased blood pressure returned to baseline seconds after stimulation was discontinued.
Synaptogenesis and nerve sprouting in LSG To explore the possible mechanisms of the lower threshold inducing LSG afterdischarges in the experimental group, immunohistochemical staining of LSG with antibodies to growth-associated protein-43 (marker for nerve sprouting) and to synaptophysin (marker for synapses) was performed. Dense synaptophysin-immunopositive ringlike structures were observed around neurons in the experimental group (Figure 8A, white arrow). Quantitative analyses showed that the immunoreactivity of synaptophysin was higher in the experimental group than in the control group (14,807 ⫾ 4,463 m2/mm2 vs 9,321 ⫾ 3,518 m2/mm2, P ⬍.05), indicating increased synaptogenesis (Figures 8A and 8B). The immunoreactivity of growth-associated protein-43 also was significantly higher in the experimental group compared with the control group (52,978 ⫾ 12,613 m2/mm2 vs 41,116 ⫾ 4,121m2/mm2, P ⬍.05), indicating increased nerve sprouting activity (Figures 8C and 8D). Of note, no fibrosis in LSG was observed in either group, suggesting that long-term electrode recording did not cause significant neuronal injury to LSG.
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Figure 7 A: Electrical stimulation of left stellate ganglion (LSG) lengthened the corrected QT (QTc) from 325 to 402 ms and the corrected Tpe (Tpec) from 45 to 82 ms (at 10 mA). LSG stimulation induced ventricular tachycardia (VT; B) and ventricular fibrillation (VF; C) in the experimental group. Afterdischarges were present in LSG recordings after electrical stimulation. BP ⫽ blood pressure (scale 0 –200 mmHg); SGNA ⫽ stellate ganglion nerve activity.
Discussion The majority of malignant ventricular arrhythmias in the experimental group were preceded within 10 to 15 seconds by two distinct types of LSG nerve activity: LABDA or HASDA. HASDA was uncommon but was more likely to be followed immediately by ventricular arrhythmia. LSG stimulation induced LSG afterdischarges and increased Tpec intervals. These findings suggest that LSG nerve activity is associated with ventricular arrhythmia and SCD, probably by increasing transmural heterogeneity of repolarization.
Cardiac sympathetic activity in conscious animals 13,14
Jardine et al successfully recorded cardiac sympathetic nerve activity in sheep using exteriorized wires. The recording was obtained for 2-hour fixed time segments daily in conscious sheep. However, short-duration recordings do not allow detection of relatively unusual nerve activity, such as HASDA. LSG is a major source of cardiac sympathetic innervation. With long-term recordings in ambulatory dogs, two distinct types of augmented SGNA (LABDA and
HASDA) were detected. The presence of HASDA in LSG is an unexpected finding. The osmotic pump used for nerve growth factor infusion emptied its content within 4 to 5 weeks from the time of implantation. There was no significant difference in HASDA occurrence between weeks 4 and 8 after surgery. Furthermore, HASDA was seen in normal dogs without osmotic pump implantation and in a group of dogs with pacing-induced heart failure.10 Therefore, the occurrence of HASDA was not due to nerve growth factor infusion by osmotic pumps.
Two distinct types of SGNA and ventricular arrhythmia Augmented SGNA in the form of HASDA and LABDA preceded ventricular arrhythmia in diseased ventricles. However, neither HASDA nor LABDA by itself was pathological, as in normal dogs they were associated only with accelerated heart rates. Most of the LABDA did not induce ventricular arrhythmia, and short bursts of LABDA were less likely to induce ventricular arrhythmias than short
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Figure 8 Synaptophysin (SYN) and growth-associated protein-43 (GAP43) immunostaining of left stellate ganglion in the experimental group and the control group. Both SYN and GAP43 immunoreactivity (brown granular structures, arrows) were increased in the experimental group compared with the control group. An example of the perineuronal ring structure (white arrow) is shown in panel A. Scale bar ⫽ 50 m.
bursts of HASDA. We hypothesize that short bursts of LABDA may not have sufficient instantaneous strength to reach the arrhythmogenic threshold. However, persistent LABDA discharges could sufficiently elevate local (myocardial) or circulating catecholamine levels to arrhythmogenic threshold and even cause SCD. Compatible with the latter hypothesis, we found that persistently increased LABDA preceded the occurrence of two SCD episodes, and the integrated SGNA of the 0 to 10 seconds before onset of VT was higher than that of the 50 to 60 seconds before onset of VT. We also found that the integrated SGNA of 1-minute segments prior to VT was higher than randomly selected 1-minute segments without VT. Furthermore, we found that high-frequency electrical stimulation of LSG induced LSG afterdischarges and increased Tpe interval, a marker of transmural15 or global16 heterogeneity of repolarization. These data demonstrate that a crescendo increase of SGNA precedes VT/VF in dogs of the experimental group, probably by inducing increased heterogeneity of repolarization.
HASDA versus LABDA Unlike LABDA, most of HASDA induced either ventricular arrhythmia or QRST morphology changes in the experimental group. We speculate that HASDA represents synchronized massive postganglionic neuronal discharges. This hypothesis is based on the fact that HASDA had much higher amplitude than LABDA, and HASDA was associated with depolarization shifts, similar to epileptiform discharges.17 However, different from epileptiform discharges, HASDA is also observed in normal controls. HASDA usually is of short duration, which probably does not induce sufficient catecholamine release to cause prolonged VT or SCD.
Circadian variation of sympathetic nerve activity The increased sympathetic tone in the morning to early afternoon could be related in part to behavioral arousal. Corbalan et al18 showed that conscious canines with MI experience ventricular tachyarrhythmias in response to arousal. Two of four SCD episodes in a previous study4 and
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one of three in this study were witnessed, suggesting that human activity and behavioral arousal might have played a role in triggering SCD. However, not all SCDs occurred during the daytime. One of the dogs in our study died at 2:32 AM, presumably during sleep. Previous studies have documented that rapid eye movement (REM) sleep is associated with surges of heart rate and sympathetic activity.19 –21 In this study, intermittent sympathetic surges occurred at night. Because 20% of SCDs occur at night,22 it is possible that sympathetic surges during REM sleep are responsible for SCD episodes.
Increased neuronal excitability and synaptogenesis of LSG Increased neuronal excitability of LSG occurred in the experimental group, as evidenced by a decrease of afterdischarge threshold and an increase of HASDA occurrence. Immunostaining showed a significant increase of structures positive for synaptophysin and growth-associated protein-43 in the LSG of the experimental group compared with the control group. Growth-associated protein-43 is a marker for axonal growth and nerve sprouting.23 Synaptophysin is concentrated mainly in synaptic terminals. These histologic data suggest that nerve sprouting and synaptogenesis of LSG in the experimental group is a likely mechanism for the increased sympathetic neuronal excitability. However, this hypothesis cannot be proven with data reported in this study.
Clinical implications The trigger mechanism of increased SGNA for ventricular arrhythmias concluded from this study may exist in patients with MI. We previously demonstrated increased nerve growth factor protein levels in dogs with MI, without either AV block or nerve growth factor infusion.24 These data indicate that synaptogenesis and neuronal hyperexcitability of LSG may occur after MI even without exogenous nerve growth factor infusion. Use of nerve growth factor infusion and creation of AV block aggravated the existing neural and electrophysiologic remodeling and increased arrhythmogenesis. The characteristics of increased SGNA before ventricular arrhythmias, especially HASDA, may help develop new devices for detecting and preventing the impending onset of malignant ventricular arrhythmias.
Acknowledgments We thank Juan Song, PhD, Angela Lei, Hongmei Li, Avile McCullen, Lei Lin, and Elaine Lebowitz for assistance.
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References 1. Muller JE, Ludmer PL, Willich SN, et al. Circadian variation in the frequency of sudden cardiac death. Circulation 1987;75:131–138. 2. The Norwegian Multicenter Study Group. Timolol-induced reduction in mortality and reinfarction in patients surviving acute myocardial infarction. N Engl J Med 1981;304:801– 807. 3. Cao J-M, Fishbein MC, Han JB, et al. Relationship between regional cardiac hyperinnervation and ventricular arrhythmia. Circulation 2000;101:1960 –1969. 4. Cao J-M, Chen LS, KenKnight BH, et al. Nerve sprouting and sudden cardiac death. Circ Res 2000;86:816 – 821. 5. Schwartz PJ, Stone HL. Left stellectomy in the prevention of ventricular fibrillation caused by acute myocardial ischemia in conscious dogs with anterior myocardial infarction. Circulation 1980;62:1256 –1265. 6. Schwartz PJ, Motolese M, Pollavini G, et al.Italian Sudden Death Prevention Group. Prevention of sudden cardiac death after a first myocardial infarction by pharmacologic or surgical antiadrenergic interventions. J Cardiovasc Electrophysiol 1992;3:2–16. 7. Rubart M, Zipes DP. Mechanisms of sudden cardiac death. J Clin Invest 2005;115:2305–2315. 8. Jardine DL, Charles CJ, Forrester MD, et al. A neural mechanism for sudden death after myocardial infarction. Clin Auton Res 2003;13:339 –341. 9. Jung B-C, Dave AS, Tan AY, et al. Circadian variations of stellate ganglion nerve activity in ambulatory dogs. Heart Rhythm 2005;3:78 – 85. 10. Ogawa M, Zhou S, Tan AY, et al. Left stellate ganglion and vagal nerve activity and cardiac arrhythmias in ambulatory dogs with pacing-induced congestive heart failure. J Am Coll Cardiol 2007;50:335–343. 11. Zhou S, Paz O, Cao J-M, et al. Differential -adrenoceptor expression induced by nerve growth factor infusion into the canine right and left stellate ganglia. Heart Rhythm 2005;2:1347–1355. 12. Gholmieh G, Courellis S, Dimoka A, et al. An algorithm for real-time extraction of population EPSP and population spike amplitudes from hippocampal field potential recordings. J Neurosci Methods 2004;136:111–121. 13. Jardine DL, Charles CJ, Melton IC, et al. Continual recordings of cardiac sympathetic nerve activity in conscious sheep. Am J Physiol Heart Circ Physiol 2002;282:H93–H99. 14. Jardine DL, Charles CJ, Ashton RK, et al. Increased cardiac sympathetic nerve activity following acute myocardial infarction in a sheep model. J Physiol 2005;565:325–333. 15. Antzelevitch C. T peak-Tend interval as an index of transmural dispersion of repolarization. Eur J Clin Invest 2001;31:555–557. 16. Opthof T, Coronel R, Wilms-Schopman FJ, et al. Dispersion of repolarization in canine ventricle and the electrocardiographic T wave: Tp-e interval does not reflect transmural dispersion. Heart Rhythm 2007;4:341–348. 17. Westmoreland BF. Epileptiform electroencephalographic patterns. Mayo Clin Proc 1996;71:501–511. 18. Corbalan R, Verrier R, Lown B. Psychological stress and ventricular arrhythmias during myocardial infarction in the conscious dog. Am J Cardiol 1974;34: 692– 696. 19. Somers VK, Dyken ME, Mark AL, et al. Sympathetic-nerve activity during sleep in normal subjects. N Engl J Med 1993;328:303–307. 20. Kirby DA, Verrier RL. Differential effects of sleep stage on coronary hemodynamic function during stenosis. Physiol Behav 1989;45:1017–1020. 21. Rowe K, Moreno R, Lau TR, et al. Heart rate surges during REM sleep are associated with theta rhythm and PGO activity in cats. Am J Physiol 1999;277(3 Pt 2):R843–R849. 22. Lavery CE, Mittleman MA, Cohen MC, et al. Nonuniform nighttime distribution of acute cardiac events: a possible effect of sleep states. Circulation 1997;96: 3321–3327. 23. Skene JH, Jacobson RD, Snipes GJ, et al. A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes. Science 1986;233: 783–786. 24. Zhou S, Chen LS, Miyauchi Y, et al. Mechanisms of cardiac nerve sprouting after myocardial infarction in dogs. Circ Res 2004;95:76 – 83.
Introduction Ventricular arrhythmia and sudden cardiac death (SCD) are major causes of mortality in patients with heart disease. However, the immediate triggers of ventricular arrhythmia and SCD remain unclear. Clinical studies show a circadian variation of SCD in patients.1 Beta-blockade is associated with a reduction in SCD mortality after myocardial infarction (MI) in randomized clinical trials.2 Increased cardiac sympathetic nerve density is seen in patients with a clinical history of ventricular arrhythmia.3 Cardiac sympathetic hyThis study was supported by American Heart Association (AHA) Grant 0435135N; a Heart Rhythm Society Postdoctoral Fellowship Award; National Institutes of Health (NIH) Grants HL78932, HL58533, HL66389, and HL71140; AHA Established Investigator Award 0540093N; and Price, Piansky, and Medtronic–Zipes endowments. Address reprint requests and correspondence: Dr. Lan S. Chen, 575 West Drive, XE 040, Indianapolis, Indiana 46202. E-mail address: [email protected]. (Received April 18, 2007; accepted September 6, 2007.)
immediately by either ventricular arrhythmia (21%) or QRS morphology changes (65%). HASDA occurred in a circadian pattern. HASDA occurred twice as often in the experimental group than in the control group. Electrical stimulation of LSG increased transmural heterogeneity of repolarization (Tpeak-end intervals) and induced either ventricular tachycardia or fibrillation in the experimental group but not in the control group. Immunohistochemical studies revealed increased synaptogenesis and nerve sprouting in the LSG in the experimental group. CONCLUSION Two distinct types of LSG nerve activity (HASDA and LABDA) are present in the LSG of ambulatory dogs. The majority of malignant ventricular arrhythmias are preceded by either HASDA or LABDA, with HASDA particularly arrhythmogenic. KEYWORDS Arrhythmia; Cardiac electrophysiology; Sympathetic nerve activity (Heart Rhythm 2008;5:131–139) © 2008 Heart Rhythm Society. All rights reserved.
perinnervation in dogs significantly increases the incidence of SCD.4 The left stellate ganglion (LSG) is known to be important in cardiac arrhythmogenesis.5 Left cardiac sympathetic denervation, including LSG resection, decreases the incidence of ventricular arrhythmia in patients with MI.6 These data suggest that increased sympathetic tone is important in the generation of ventricular arrhythmia and SCD.7 However, except for a case report of a sheep with SCD during acute MI,8 little direct evidence supports a temporal relationship between spontaneously increased sympathetic discharges and cardiac arrhythmia and SCD in ambulatory humans or animals. To fill this gap of knowledge, we have developed methods for performing long-term continuous (24 hours per day, 7 days per week) recording of electrocardiogram (ECG) and stellate ganglion nerve activity (SGNA) in normal dogs and in dogs with heart failure.9,10 We also developed a high-yield canine model of ventricular arrhythmia and SCD.4 The first aim of the study
1547-5271/$ -see front matter © 2008 Heart Rhythm Society. All rights reserved.
doi:10.1016/j.hrthm.2007.09.007
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was to identify and characterize sympathetic nerve activity immediately preceding spontaneous paroxysmal ventricular arrhythmias by performing long-term continuous ambulatory nerve recording in this canine model. The second aim was to perform LSG stimulation before euthanasia in these dogs to confirm the hypothesis that LSG discharges preceded ventricular arrhythmias.
munostained using a modified immunocytochemical ABCperoxidase method as described previously.11 Growth-associated protein-43 and synaptophysin immunoreactivity was quantified using a computer-assisted image analysis system (Image-Pro Plus 4.0, MediaCybernetics, Carlsbad, CA, USA). Immunoreactivity was expressed as the total area of positive staining per unit area (m2/mm2).
Methods
Data analysis
This study consisted of eight dogs in the experimental group and six normal dogs from a previous study.9 All dogs underwent left thoracotomy under general anesthesia for radiotransmitter implantation and ambulatory nerve recordings. The protocol was approved by the Institutional Animal Care and Use Committee.
Surgery for the experimental group General anesthesia was maintained by isoflurane inhalation. Complete atrioventricular (AV) block was produced by radiofrequency ablation of the AV junction. Left lateral thoracotomy was performed and MI created by ligating the left anterior descending coronary artery below the first diagonal branch. An osmotic pump (Durect Co., Cupertino, CA, USA) was implanted near the LSG for continuous infusion of nerve growth factor (Sigma Chemical Co., St. Louis, MO, USA). A pacemaker was implanted for backup ventricular pacing at 40 bpm. A radiotransmitter (D70-EEE, Data Sciences International, St. Paul, MN, USA) was inserted into a subcutaneous pocket. One pair of electrodes was used to record from the LSG; another pair was implanted in subcutaneous tissue for ECG recording. One of the two electrodes for ECG recording was placed in the upper right quadrant of the chest and the other in the lower left quadrant. Simultaneous left SGNA and ECG were continuously recorded 24 hours per day, 7 days per week.
Direct electrical stimulation of LSG At the end of the monitoring period, all surviving dogs were anesthetized with isoflurane for in vivo study. SGNA, surface ECG, and blood pressure were recorded simultaneously using a CardioLab 4.0 system (Prucka Engineering Inc., Houston, TX, USA) before and after electrical stimulation of LSG (5-ms pulse width at 20 Hz) for 15 seconds. To test the afterdischarge threshold, the stimulation current was ramped up from 1 mA, 2 mA, and 5 mA, followed by 5-mA increments of each stimulation until afterdischarges occurred. QT interval, Tpeak-end interval (Tpe), corrected QT interval (QTc), and corrected Tpeak-end interval (Tpec) were measured before and after stimulation. QTc and Tpec were QT and Tpe, respectively, divided by the square root of the preceding R-R interval. The dogs were euthanized by exsanguination during general anesthesia.
Immunohistochemistry staining LSGs were removed for immunostaining of synaptophysin (Chemicon, Temecula, CA, USA) and growth-associated protein-43 (Chemicon). Five-micrometer sections were im-
Entire datasets were manually analyzed for the occurrence and frequencies of high-amplitude spike discharge activity (HASDA) and ventricular tachycardia (VT), which was defined as ⱖ3 consecutive beats with heart rate exceeding 100 bpm. Episodes of VT were randomly selected to determine integrated sympathetic nerve activity12 20 seconds before onset of VT and the association between HASDA and arrhythmia. High-pass (30-Hz) filtering was used first to eliminate ECG signals from SGNA recording channels. Integrated nerve activity (V) was calculated by averaging the amplitude of SGNA over each 100-ms segment. Integrated nerve activity preceding the onset of VT or ventricular fibrillation (VF) was measured and compared with the results of integrated nerve activity from a randomly selected 1-minute arrhythmia-free period during the same hour of VT occurrence. The recordings from six normal control dogs9 were analyzed using the same methods for comparison.
Statistical analysis All quantitative data were presented as mean ⫾ SD. Student’s t-tests were used to compare the means between two groups. Cosinor test was used to determine the significance of circadian variation. Fisher exact test was used to determine the correlation between elevated SGNA and VT. P ⱕ.05 was considered significant.
Results The simultaneous recording lasted 55 ⫾ 40 days in the experimental group and 43 ⫾ 7 days in the control group.
Stellate ganglia nerve activity Two distinctly different patterns of SGNA were observed in both groups: low-amplitude burst discharge activity (LABDA, Figure 1A) and HASDA (Figure 1B). LABDA accounted for the majority of LSG nerve activity. HASDA was uncommon compared with LABDA. The amplitude of LABDA varied from 0.05 to 0.8 mV; most were in the range from 0.1 to 0.4 mV. The duration of each LABDA episode varied considerably from 0.5 second to 6 minutes; most episodes lasted 2 to 10 seconds. The occurrence of LABDA changed from time to time, from 0 to more than 200 episodes per hour. HASDA had 6.9 ⫾ 1.5 (range 3–12) spikes per episode, with spike amplitude of 0.91 ⫾ 0.16 mV. The spikes had a roughly regular interval and low frequency (cycle length 152 ⫾ 17.7 ms). The average duration of HASDA was 1,059 ⫾ 254 ms. Most HASDA was associated with downward baseline shift, which were morphologically similar to the depolarization shift during the epileptiform discharges.
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Figure 1 Patterns of left stellate ganglion nerve activity (SGNA) in normal control dog. A: Low-amplitude burst discharge activity (LABDA). B: High-amplitude spike discharge activity (HASDA). Both LABDA and HASDA were associated with heart rate acceleration shown on simultaneously recorded ECG. Units for SGNA and ECG are given in millivolts in this and all other figures.
SGNA and heart rate in the control group LABDA and HASDA induced immediate heart rate acceleration in normal dogs (Figures 1A and 1B). In 50 randomly selected runs of HASDA, R-R intervals were shortened from 395 ⫾ 39 ms before discharge to 347 ⫾ 26 ms immediately after discharge (P ⬍.01). SGNA was not associated with arrhythmia in normal dogs.
SGNA and ventricular arrhythmias in the experimental group Three dogs in the experimental group died suddenly. The recordings of a witnessed SCD event when one of the authors (SZ) entered the dog run for routine examination are shown in Figure 2. Accelerated idioventricular rhythm occurred soon after onset of LABDA (Figure 2A, arrow).
Figure 2 Example of increased left stellate ganglion nerve activity (SGNA) preceding ventricular fibrillation (VF) and sudden cardiac death. A: Increased low-amplitude burst discharge activity (LABDA) resulted in accelerated idioventricular rhythm. B: VF occurred approximately 40 seconds later. Panels A and B are continuous. C: A 6-second recording from panel B. INA ⫽ integrated nerve activity; P ⫽ P wave, which is dissociated from ventricular activation due to complete AV block. Units for INA are given in millivolts in this and all other figures.
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Figure 3 Examples of increased stellate ganglion nerve activity (SGNA) preceding ventricular tachycardia (VT). A: One episode of low-amplitude burst discharge activity (LABDA) before onset of VT. B: Two episodes of LABDA prior to onset of VT. C: LABDA followed by increased atrial rate (arrow, p wave) and accelerated idioventricular rhythm. Panels A, B, and C were recorded from three different dogs from the experimental group. INA ⫽ integrated nerve activity.
Integrated nerve activity showed an abrupt increase of SGNA. VF occurred 40 seconds later. Approximately 10 seconds after VF onset, a burst of an abrupt eightfold in-
crease of LABDA occurred (Figure 2B, arrow). This large increase of SGNA is consistent with sympathetic activation in response to hypotension. Another death occurred unwit-
Figure 4 Quantitative analyses of stellate ganglion nerve activity (SGNA) prior to the onset of ventricular tachycardia (VT). A: SGNA averaged over each 10-second segment for 1 minute before onset of 70 VT episodes. The closer to the onset of VT, the higher the average SGNA became. B–E: Selected examples of integrated nerve activity 60 seconds before onset of VT, showing variable patterns of increased SGNA before onset of VT. *P ⬍.05; ** P ⬍.01 vs – 60 seconds; ††P ⬍.01 vs –10 seconds; ‡P ⬍.05 vs –10 seconds.
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nessed at 2:32 AM. VF was also preceded by LABDA, which continued for 150 seconds before VF occurred. A third sudden death occurred spontaneously after the dog was being anesthetized with isoflurane in preparation for a second surgery; no recordings were available. In the experimental group, all dogs had phase 1 VT during the first 3 days after surgery and phase 2 VT that commenced 5 to 7 days later.4 The frequency of phase 2 VT was 2.3 ⫾ 1.2 episodes per day in the experimental group. Two hundred five episodes of phase 2 VT from six dogs in experimental group were randomly selected, and the SGNA of a 20-second period immediately before onset of VT was analyzed. A total of 177 episodes (86.3%) of VT were preceded within 10 to 15 seconds by increased SGNA, either LABDA (96.6%) or HASDA (3.4%). In contrast, the SGNA exceeded three times the baseline noise level only 36% ⫾ 10% of the time among all the dogs studied. Fisher exact test showed a significant positive association between VT and sympathetic discharge (P ⬍.0001). The latency between onset of HASDA and VT was 1.7 ⫾ 1.3 seconds, and the latency between LABDA and VT was 14.2 ⫾ 8.8 seconds (P ⬍.001). Among the VT episodes, 97.1% were monomorphic and 2.9 % were polymorphic. Average VT duration was 15.4 ⫾ 8.1 seconds, and VT rate was 159 ⫾ 39.8 bpm. The amplitude and duration of LABDA that preceded VT varied from episode to episode. For example, the onset of VT shown in Figure 3A was preceded by continuous LABDA over a 10-second period, whereas two separate bursts of LABDA preceded the onset of VT in Figure 3B. LABDA also increased the atrial rate and ventricular escape rhythm in the experimental group, which had complete AV block (Figure 3C). To determine whether a crescendo increase of SGNA occurred prior to onset of VT, integrated nerve activity of SGNA was measured 1 minute before onset of 70 randomly selected VT episodes. The results showed that the closer to the onset of VT, the higher integrated nerve activity became (Figure 4). The time-averaged integrated SGNA at 0 to 10 seconds immediately preceding VT was significantly higher than that at 50 to 60 seconds before onset of VT (18 ⫾ 8.7 V vs 12 ⫾ 5.6 V, n ⫽ 70, P ⬍.01; Figure 4A). The amplitude of increased SGNA and the duration elapsed between onset of increased SGNA and VT varied considerably among VT episodes. Figures 4B to 4E show selected examples of integrated nerve activity 60 seconds before onset of VT episodes. Integrated nerve activity also was measured from a randomly selected 1-minute arrhythmia-free period during the same hour of each selected VT episode. The timeaveraged integrated SGNA 1 minute preceding VT episodes was significantly higher than that of randomly selected arrhythmia-free 1-minute segments (15 ⫾ 5.8 V vs 11 ⫾ 3.7 V, n ⫽ 70 for both, P ⬍.01). Because the average VT occurrence was 2.3 ⫾ 1.2 episodes per day while intermittent LABDA occurred throughout the day, less than 1% of LABDA episodes were followed by VT.
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Figure 5 Frequency and circadian variation of stellate ganglion nerve activity (SGNA) in the experimental group. A: Average number of highamplitude spike discharge activity (HASDA) per day from weeks 2 to 8. B: Average number of HASDA in each hour of the day. The number of HASDA shown in panel B is the average HASDA recorded from five dogs in experimental group over a 10-day period. C: Integrated SGNA (almost all low-amplitude burst discharge activity) on 8 different days in four dogs. We used the midnight value as 1 and obtained the ratio of each hour against that value. Cosinor test showed P ⬍.0001 for the presence of circadian variation in panels B and C. NGF ⫽ nerve growth factor.
HASDA and cardiac electrical instability HASDA appeared approximately 2 to 5 days after the surgery, reached a peak around the second week, and persisted until the end of the study (Figure 5A). Figure 5B shows the 24-hour distribution of HASDA episodes in the experimental group. The vivarium staff turned on the light around 6 to 7 AM and began cage cleaning and feeding afterward. These activities most likely account for the increased HASDA in the early morning. The frequency of HASDA in the experimental group was twice that in the control group (14.1 ⫾ 3.1 per day vs 7.2 ⫾ 5.2 per day, P ⬍.05) in the third week after the first surgery. Total integrated SGNA was analyzed and showed significant circadian variation (Figure 5C). To investigate the effects of HASDA on cardiac arrhythmia, 502 runs of HASDA from the experimental group were randomly selected. In contrast to LABDA, which usually was not followed by ventricular arrhythmia, 21% of HASDA induced ventricular arrhythmia, including short runs of VT (Figure 6A) and single premature ventricular contraction or couplets (Figure 6B). In addition, 65% of HASDA was followed by changes of QRST morphology
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Figure 6 High-amplitude spike discharge activity (HASDA) induced ventricular tachycardia (A), premature ventricular contraction (PVC; B), and QRST morphology change (C) in the experimental group. SGNA ⫽ stellate ganglion nerve activity.
(Figure 6C). These morphology changes could be due to altered conduction velocity from the same ectopic focus, fusion with a different ectopic focus, or both. These findings suggest that HASDA has effects on cardiac excitability and conduction in diseased ventricles.
Electrical stimulation of LSG Direct electrical stimulation was administered to LSG of all surviving dogs before euthanasia. The threshold to induce LSG afterdischarges (Figure 7) in the experimental group was significantly lower than in the control group (3 ⫾ 1.9 mA vs 9.4 ⫾ 6.8 mA, P ⬍.05). In the normal group, electrical stimulation of LSG induced sinus tachycardia and shortened QT (before vs after, 294 ⫾ 21 ms vs 249 ⫾ 29 ms, n ⫽ 6, P ⬍.01), QTc (396 ⫾ 17 ms vs 378 ⫾ 16 ms, n ⫽ 6, P ⬍.05), Tpe (58⫾ 15 ms vs 45 ⫾ 9.6 ms, n ⫽ 6, P ⬍.05), and Tpec (78 ⫾ 16.2 ms vs 69 ⫾ 15 ms, n ⫽ 6, P ⬍.05). In the experimental group, electrical stimulation of LSG increased ventricular rate from 44 ⫾ 3.7 bpm to 57 ⫾ 7.4 bpm (n ⫽ 4, P ⬍.05); prolonged QTc (322 ⫾ 19.8 ms vs 372 ⫾ 37 ms, n ⫽ 4, P ⬍.05), Tpe (77 ⫾ 8.3 ms vs 102⫾ 12.3 ms, n ⫽ 4, P ⬍.05), and Tpec (66 ⫾ 8.6 ms vs 95 ⫾ 11.0 ms, n ⫽ 4, P ⬍.05; Figure 7A); and induced VT in 4 of 4 dogs and VF in 2 of 4 dogs (Figures 7B and 7C). In both
groups, stimulation of LSG resulted in an immediate increase of blood pressure, and the increased blood pressure returned to baseline seconds after stimulation was discontinued.
Synaptogenesis and nerve sprouting in LSG To explore the possible mechanisms of the lower threshold inducing LSG afterdischarges in the experimental group, immunohistochemical staining of LSG with antibodies to growth-associated protein-43 (marker for nerve sprouting) and to synaptophysin (marker for synapses) was performed. Dense synaptophysin-immunopositive ringlike structures were observed around neurons in the experimental group (Figure 8A, white arrow). Quantitative analyses showed that the immunoreactivity of synaptophysin was higher in the experimental group than in the control group (14,807 ⫾ 4,463 m2/mm2 vs 9,321 ⫾ 3,518 m2/mm2, P ⬍.05), indicating increased synaptogenesis (Figures 8A and 8B). The immunoreactivity of growth-associated protein-43 also was significantly higher in the experimental group compared with the control group (52,978 ⫾ 12,613 m2/mm2 vs 41,116 ⫾ 4,121m2/mm2, P ⬍.05), indicating increased nerve sprouting activity (Figures 8C and 8D). Of note, no fibrosis in LSG was observed in either group, suggesting that long-term electrode recording did not cause significant neuronal injury to LSG.
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Figure 7 A: Electrical stimulation of left stellate ganglion (LSG) lengthened the corrected QT (QTc) from 325 to 402 ms and the corrected Tpe (Tpec) from 45 to 82 ms (at 10 mA). LSG stimulation induced ventricular tachycardia (VT; B) and ventricular fibrillation (VF; C) in the experimental group. Afterdischarges were present in LSG recordings after electrical stimulation. BP ⫽ blood pressure (scale 0 –200 mmHg); SGNA ⫽ stellate ganglion nerve activity.
Discussion The majority of malignant ventricular arrhythmias in the experimental group were preceded within 10 to 15 seconds by two distinct types of LSG nerve activity: LABDA or HASDA. HASDA was uncommon but was more likely to be followed immediately by ventricular arrhythmia. LSG stimulation induced LSG afterdischarges and increased Tpec intervals. These findings suggest that LSG nerve activity is associated with ventricular arrhythmia and SCD, probably by increasing transmural heterogeneity of repolarization.
Cardiac sympathetic activity in conscious animals 13,14
Jardine et al successfully recorded cardiac sympathetic nerve activity in sheep using exteriorized wires. The recording was obtained for 2-hour fixed time segments daily in conscious sheep. However, short-duration recordings do not allow detection of relatively unusual nerve activity, such as HASDA. LSG is a major source of cardiac sympathetic innervation. With long-term recordings in ambulatory dogs, two distinct types of augmented SGNA (LABDA and
HASDA) were detected. The presence of HASDA in LSG is an unexpected finding. The osmotic pump used for nerve growth factor infusion emptied its content within 4 to 5 weeks from the time of implantation. There was no significant difference in HASDA occurrence between weeks 4 and 8 after surgery. Furthermore, HASDA was seen in normal dogs without osmotic pump implantation and in a group of dogs with pacing-induced heart failure.10 Therefore, the occurrence of HASDA was not due to nerve growth factor infusion by osmotic pumps.
Two distinct types of SGNA and ventricular arrhythmia Augmented SGNA in the form of HASDA and LABDA preceded ventricular arrhythmia in diseased ventricles. However, neither HASDA nor LABDA by itself was pathological, as in normal dogs they were associated only with accelerated heart rates. Most of the LABDA did not induce ventricular arrhythmia, and short bursts of LABDA were less likely to induce ventricular arrhythmias than short
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Figure 8 Synaptophysin (SYN) and growth-associated protein-43 (GAP43) immunostaining of left stellate ganglion in the experimental group and the control group. Both SYN and GAP43 immunoreactivity (brown granular structures, arrows) were increased in the experimental group compared with the control group. An example of the perineuronal ring structure (white arrow) is shown in panel A. Scale bar ⫽ 50 m.
bursts of HASDA. We hypothesize that short bursts of LABDA may not have sufficient instantaneous strength to reach the arrhythmogenic threshold. However, persistent LABDA discharges could sufficiently elevate local (myocardial) or circulating catecholamine levels to arrhythmogenic threshold and even cause SCD. Compatible with the latter hypothesis, we found that persistently increased LABDA preceded the occurrence of two SCD episodes, and the integrated SGNA of the 0 to 10 seconds before onset of VT was higher than that of the 50 to 60 seconds before onset of VT. We also found that the integrated SGNA of 1-minute segments prior to VT was higher than randomly selected 1-minute segments without VT. Furthermore, we found that high-frequency electrical stimulation of LSG induced LSG afterdischarges and increased Tpe interval, a marker of transmural15 or global16 heterogeneity of repolarization. These data demonstrate that a crescendo increase of SGNA precedes VT/VF in dogs of the experimental group, probably by inducing increased heterogeneity of repolarization.
HASDA versus LABDA Unlike LABDA, most of HASDA induced either ventricular arrhythmia or QRST morphology changes in the experimental group. We speculate that HASDA represents synchronized massive postganglionic neuronal discharges. This hypothesis is based on the fact that HASDA had much higher amplitude than LABDA, and HASDA was associated with depolarization shifts, similar to epileptiform discharges.17 However, different from epileptiform discharges, HASDA is also observed in normal controls. HASDA usually is of short duration, which probably does not induce sufficient catecholamine release to cause prolonged VT or SCD.
Circadian variation of sympathetic nerve activity The increased sympathetic tone in the morning to early afternoon could be related in part to behavioral arousal. Corbalan et al18 showed that conscious canines with MI experience ventricular tachyarrhythmias in response to arousal. Two of four SCD episodes in a previous study4 and
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one of three in this study were witnessed, suggesting that human activity and behavioral arousal might have played a role in triggering SCD. However, not all SCDs occurred during the daytime. One of the dogs in our study died at 2:32 AM, presumably during sleep. Previous studies have documented that rapid eye movement (REM) sleep is associated with surges of heart rate and sympathetic activity.19 –21 In this study, intermittent sympathetic surges occurred at night. Because 20% of SCDs occur at night,22 it is possible that sympathetic surges during REM sleep are responsible for SCD episodes.
Increased neuronal excitability and synaptogenesis of LSG Increased neuronal excitability of LSG occurred in the experimental group, as evidenced by a decrease of afterdischarge threshold and an increase of HASDA occurrence. Immunostaining showed a significant increase of structures positive for synaptophysin and growth-associated protein-43 in the LSG of the experimental group compared with the control group. Growth-associated protein-43 is a marker for axonal growth and nerve sprouting.23 Synaptophysin is concentrated mainly in synaptic terminals. These histologic data suggest that nerve sprouting and synaptogenesis of LSG in the experimental group is a likely mechanism for the increased sympathetic neuronal excitability. However, this hypothesis cannot be proven with data reported in this study.
Clinical implications The trigger mechanism of increased SGNA for ventricular arrhythmias concluded from this study may exist in patients with MI. We previously demonstrated increased nerve growth factor protein levels in dogs with MI, without either AV block or nerve growth factor infusion.24 These data indicate that synaptogenesis and neuronal hyperexcitability of LSG may occur after MI even without exogenous nerve growth factor infusion. Use of nerve growth factor infusion and creation of AV block aggravated the existing neural and electrophysiologic remodeling and increased arrhythmogenesis. The characteristics of increased SGNA before ventricular arrhythmias, especially HASDA, may help develop new devices for detecting and preventing the impending onset of malignant ventricular arrhythmias.
Acknowledgments We thank Juan Song, PhD, Angela Lei, Hongmei Li, Avile McCullen, Lei Lin, and Elaine Lebowitz for assistance.
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