Skip Navigation

Cardiovascular Research 2007 73(4):750-760; doi:10.1016/j.cardiores.2006.12.001
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ng, G. A.
Right arrow Articles by Coote, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ng, G. A.
Right arrow Articles by Coote, J. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2007, European Society of Cardiology

Autonomic modulation of electrical restitution, alternans and ventricular fibrillation initiation in the isolated heart

G. André Nga,*, Kieran E. Brackb, Vanlata H. Patela and John H. Cooteb

aCardiology group, Department of Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom
bDepartment of Pharmacology, Division of Neurosciences, University of Birmingham, Birmingham, United Kingdom

* Corresponding author. Department of Cardiovascular Sciences, Cardiology group, University of Leicester, Clinical Sciences Wing, Glenfield Hospital, Leicester LE3 9QP, U.K. Tel.: +44 116 250 2438; fax: +44 116 287 5792. Email address: gan1{at}le.ac.uk

Received 1 September 2006; revised 30 November 2006; accepted 4 December 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: Abnormal autonomic nerve activity is a strong prognostic marker for ventricular arrhythmias but the mechanisms underlying the autonomic modulation of ventricular fibrillation (VF) initiation are poorly understood. We examined the effects of direct sympathetic (SS) and vagus (VS) nerve stimulation on electrical restitution, alternans and VF threshold (VFT) in a novel isolated rabbit heart preparation with intact dual autonomic innervation.

Methods: Monophasic Action Potentials (MAPs) were recorded from a left ventricular epicardial site on innervated, isolated rabbit hearts (n=16). Standard restitution, effective refractory period (ERP), electrical alternans and VFT were measured at baseline and during SS and VS separately.

Results: The restitution curve was shifted downwards and made steeper with SS whilst VS caused an upward shift and a flattening of the curve. The maximum slope of restitution was increased from 1.30±0.10 at baseline to 1.86±0.17 (by 45±12%, P<0.01) with SS and decreased to 0.69±0.10 (by 51±6%, P<0.001) with VS. ERP was decreased from 127.3±2.5 ms to 111.8±1.8 ms with SS (by 12±2%, P<0.001) and increased to 144.0±2.2 ms with VS (by 13±2%, P<0.001). VFT was decreased from 4.7±0.6 mA to 1.9±0.5 mA with SS (by 64±5%, P<0.001) and increased to 8.7±1.1 mA with VS (by 89±14%, P<0.0005). There was a significant inverse relationship between the maximum slope of restitution and VFT (r=–0.63, P<0.0001). When compared with baseline, SS caused electrical alternans at longer pacing cycle lengths (139.0±8.4 vs. 123.0±7.8 ms, P<0.01) with greater degree of alternans (32.5±9.9 vs. 15.4±3.2%, P<0.05). It also caused a wider range of cycle lengths where alternans occurred (53.0±6.2 vs. 41.0±7.0 ms, P<0.05) whilst vagus nerve stimulation shortened this range (33.0±7.3 ms, P<0.001).

Conclusions: Sympathetic stimulation increased maximum slope of restitution and electrical alternans but decreased ERP and VF threshold whilst vagus nerve stimulation had opposite effects. The interaction between action potential duration and beat-to-beat interval may play an important role in the autonomic modulation of VF initiation.

KEYWORDS Ventricular fibrillation; Restitution; Autonomic nervous system; Sympathetic nerves; Vagus nerves; Electrical alternans


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Abnormal autonomic activity has been shown to be a strong prognostic factor in many cardiac patients especially in those with heart failure [1] or with previous myocardial infarctions [2]. There is strong evidence that the relationship between impaired cardiac autonomic control and mortality is the result of an increased susceptibility to lethal ventricular arrhythmias [3]. The mechanisms underlying the association between autonomic nervous system activity and ventricular arrhythmia initiation are poorly understood.

Historical work by Einbrodt with the inductorium suggests that vagus nerve stimulation may reduce the inducibility of ventricular fibrillation (VF) [4]. Recent clinical studies have shown that beta-blockers can reduce sudden death mortality in patients with heart failure [5]. These data suggest that activity of the autonomic nervous system is intimately associated with ventricular arrhythmogenesis with its disturbance leading to sudden arrhythmic death and, perhaps more importantly, that its manipulation may prevent such fatal arrhythmias.

It is believed that the onset of VF is associated with breakup of spiral waves or rotors into multiple wavelets and oscillations (reviewed in [6]). The "restitution hypothesis" states that oscillations are facilitated when the slope of the action potential duration (APD) restitution curve is greater than 1 [7]. In addition, there is recent evidence that drugs that reduce the slope of the restitution curve prevent the induction of VF [8], thus supporting the notion that electrical restitution may be a key determinant in the initiation of VF.

We have developed an isolated rabbit heart preparation with intact dual autonomic innervation [9] which allows the study of the effects of direct sympathetic and vagal stimulation on whole heart physiology in vitro without the interference from haemodynamic reflexes or humoral factors that occur in vivo. In this study, we applied this preparation to examine the ventricular electrophysiological effects of direct autonomic stimulation, especially investigating the effects on electrical restitution and correlating these changes with the effects of autonomic stimulation on VF initiation. The effects of autonomic stimulation on electrical alternans were also examined.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Isolated rabbit heart preparation with intact dual autonomic innervation
The isolated heart preparation with intact autonomic innervation has been described previously [9]. In brief, adult male New Zealand White rabbits (2.0–2.6 kg, n=16) were premedicated with Hypnorm (Janssen Pharmaceuticals Ltd, Oxford, UK; 0.1 mg kg–1 s.c.). General anaesthesia was induced with i.v. Hypnoval (Roche Products Ltd, Welwyn Garden City, UK; 1 mg kg–1) and maintained with pentobarbitone sodium (Sagital, Rhône Mérieux, Harlow, UK; 2 mg every 5 min i.v.). The rabbit was ventilated, after tracheotomy, at 60 breaths per min using a small-animal ventilator (Harvard Apparatus Ltd, Ednebridge, Kent, UK) with an O2/air mixture. The vagus nerves were isolated and cut and the blood vessels leading to and from the rib cage were ligated and dissected. The rabbit was killed with an overdose of Sagital (60 mg i.v.) together with 500 U heparin i.v. The anterior portion of the rib cage was removed and the descending aorta cannulated. The preparation extending from the neck to thorax was dissected as described before [9]. All procedures were undertaken in accordance with the Animals (Scientific Procedures) Act 1986 in the UK and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.2. Langendorff perfusion
The preparation was perfused via the aortic cannula in the modified Langendorff mode with Tyrode's solution of the following composition (mM): Na138, K4.0, Ca1.8, Mg1.0, HCO324.0, H2PO40.4, Cl121, glucose11. The pH of the solution was maintained at 7.4 by continuously bubbling with 95%O2/5%CO2 and temperature maintained at 37 °C. A perfusion rate of 100 ml min–1 was maintained using a Gilson Minipuls 3 peristaltic pump (Gilson Inc., Ohio, USA). Thebesian venous effluent was drained via a catheter placed at the left ventricular apex. Left ventricular pressure was monitored with a fluid-filled latex balloon connected to a pressure transducer (MTL0380, ADInstruments Ltd, Chalgrove, UK) with balloon volume adjusted to give zero end-diastolic pressure. Perfusion pressure was monitored with a second pressure transducer in series with the aortic cannula. A pair of platinum electrodes (Grass Instruments, Astro-Medical Inc., Slough, UK) was inserted into the right atrial appendage for recording atrial electrograms from which instantaneous intrinsic heart rate was derived.

2.3. Autonomic nerve stimulation
Each of the 2 vagus nerves was supported on separate custom-made bipolar silver electrodes (Advent Research Materials, UK: 0.5 mm diameter) for individual vagus nerve stimulation (VS). For sympathetic stimulation (SS), a quadripolar catheter (Biosense Webster Inc., Diamond Bar, USA, 2 mm electrode, 10 mm spacing) was inserted into the spinal canal at the 12th thoracic vertebra. The tip of the catheter was advanced to the level of the 2nd thoracic vertebra for stimulation of both sides of the cardiac sympathetic outflow, with the tip of the electrode acting as cathode and the remaining 3 electrodes acting as anodes. The sympathetic and vagal stimulation electrodes were connected to constant voltage square pulse stimulators (SD9, Grass Instruments, Astro-Med Inc., Slough, UK) which allowed stimulation over a range of frequencies (1 to 20 Hz) and strengths (1 to 20 V) at 2 ms pulse width.

The stimulation strength for both nerves, at a frequency of 5 Hz, that produced a heart rate 80% of the maximal response was used throughout the study. We then selected a frequency at this stimulus strength that would produce a steady state heart rate just less than 200 beats min–1 with SS and a heart rate just less than 100 beats min–1 with VS. This frequency for SS was chosen so heart rate did not override the cycle length (CL) used in the pacing protocols. The frequency for VS was chosen to ensure sinus rhythm was maintained during vagal stimulation i.e. no atrioventricular block or asystole [9]. We have shown previously that stimulation of the right vagus nerve produced a slower heart rate when compared with the left whilst the reverse was true in their effects on atrioventricular conduction [9]. However, in a previous preliminary study, we found no significant difference between right and left VS on ventricular electrophysiology [10]. Data presented in the current study on VS are with left vagus nerve alone. In the 11 hearts, SS was carried out at 5.1±0.9 Hz which increased heart rate from a baseline of 145.2±7.0 to 193.3±2.0 bpm. Left VS was carried out at 7.9±0.4 Hz which decreased heart rate from 144.0±7.0 to 85.5±5.3 bpm.

The vagus nerves had intermittently applied Tyrode's solution to maintain viability whilst perfusion of the preparation via the descending aorta ensured adequate perfusion of the sympathetic chain via spinal arteries. Viability of both SS and VS was confirmed by the presence of similar magnitude of heart rate responses toward the end of the protocols studied. Stable nerve stimulation effects were ascertained by the online monitoring of maintained heart rate response between pacing protocols and during the 5-second gap periods during VF threshold testing (see later).

2.4. Cardiac electrical recording and pacing
A custom-made suction electrode was used to record monophasic action potentials (MAP) from the epicardial surface of the left ventricular free wall using a DC-coupled high input impedance differential amplifier. A bipolar electrode was inserted into the right ventricular apex for pacing at twice the diastolic threshold with a constant current stimulator (DS7A, Digitimer, Welwyn Garden City, UK).

2.5. Electrical restitution
Standard restitution of MAP duration was obtained with right ventricular pacing using a 20-beat drive train (S1, 300 ms CL) followed by an extrastimulus (S2) (Fig. 1A, upper panel) and repeated with progressively shorter S1-S2 intervals [by 10 ms from 300 ms to 200 ms and by 5 ms from 200 ms to ventricular effective refractory period (ERP)]. ERP was defined as the longest coupling interval that failed to capture the ventricles. Timings of the beginning of the S1- and S2-MAP signals were noted and the delays from the pacing stimuli measured. MAP duration was measured, using a custom written program (Francis Burton, Glasgow University, UK) [11], from the beginning of the signal to 90% repolarisation (MAPD90) with the amplitude of the signal measured from the peak of the dome to the isoelectric line. Restitution was examined by analysis of the relationship between S2-MAPD90 and preceding diastolic intervals (DI=interval between the S1- and S2-MAP signals minus S1-MAPD90; Fig. 1A, lower panel). An exponential curve [MAPD90=MAPD90max (1–e–DI/{tau})] was fitted to this data using Microcal Origin (v 6.1, Origin, San Diego, CA, US) where MAPD90max was the maximum MAPD90 and {tau} was the time constant. The maximum slope of restitution was measured by analyzing the first derivative of the fitted curve. As the restitution hypothesis suggests that wavebreaks are more likely to occur at slope of restitution curve >1, this point was analyzed in the respective restitution curves.


Figure 1
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 A. Upper Panel: Ventricular monophasic action potentials (MAP) and right ventricular pacing stimulus used for the determination of ERP and standard electrical restitution with 20 stimuli delivered during the drive train (S1) at 300 ms cycle length and an extra stimulus (S2) delivered at programmed intervals. Lower Panel: Expanded section from Upper Panel showing the calculation of diastolic interval (DI) and measurement of MAP duration at 90% repolarization (MAPD90). B. Left ventricular (LV) pressure, MAP and ventricular pacing stimulus during the determination of ventricular fibrillation (VF) threshold. The first run of 20 beats at 300 ms cycle length followed by 30 stimuli (30 ms interval) did not produce VF whilst the second run with a higher pacing current (by 0.5 mA) did.

 
2.6. Ventricular fibrillation threshold
VFT was obtained with right ventricular pacing using a train of 30 stimuli (30 ms interval) spanning the refractory period at the end of a 20 beat drive train at 300 ms (Fig. 1B) and determined by progressively increasing the pacing current in 0.5 mA steps, with 5-second rest period before the next pacing train if no VF was induced. VFT was defined as the minimum current required to produce sustained VF.

VFT was also determined during constant pacing similar to the above protocol except that the 5-second rest periods were replaced with continuous ventricular pacing at 300 ms.

2.7. Experimental protocol 1 – restitution, ERP and VFT
After an equilibration period of 30 min, standard restitution and ERP were studied in 11 experiments. VFT was then determined using the VF induction protocol. A 2 ml bolus of Tyrode's solution containing concentrated KCl (50 mM) was given via the side arm of the aortic cannula for conversion back to sinus rhythm. An equilibration period of 15 min was allowed when heart rate, ventricular pressure and ERP returned to baseline values. VFT varied less than 3% on average with repeated measurements after conversion with KCl (data not shown).

Similar measurements of electrical restitution, ERP and VF threshold were made with SS or VS separately, when the new steady state heart rate was reached during nerve stimulation. After VF induction, nerve stimulation was stopped and the hearts were cardioverted with concentrated KCl bolus. The electrical parameters were measured during both SS and VS in all hearts but in random order. VFT was also determined during constant pacing in 8 of the 11 experiments.

2.8. Experimental protocol 2 – development of APD alternans
In 5 separate experiments, the development of APD alternans was examined with MAP duration measured at 50% repolarisation (MAPD50). This was chosen as it is a more reliably measured duration than, for instance 70% or 90% repolarisation at high pacing rates. Ventricular pacing was carried out with 50-beat trains and the MAPD50 of the last 2 paced beats were measured. This was repeated with progressively shorter CL in a stepwise fashion [by 10 ms from 300 to 200 ms; by 5 ms from 200 ms]. This protocol led to the development of alternans, defined as a variation of >5% in MAPD of sequential beats. Shorter CL caused increasing alternans and eventually VF. Peak alternans level was noted at the CL where the variation of APD between sequential beats was greatest as was the range of CL with alternans before VF (Alternans Range). Effects with SS and VS were compared with baseline.

2.9. Signal measurements and statistical analysis
All measured signals were recorded with a PowerLab 800/s system (ADInstruments Ltd, Chalgrove, UK) and digitised at 1 kHz using Chart software (ADInstruments Ltd) with the data stored and displayed on a Power Macintosh G3 personal computer (Apple).

All data are expressed as mean±SEM. The effect of SS and VS on restitution, ERP and VFT were analysed using ANOVA. Two-tailed P-value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Effect of sympathetic and vagal stimulation on electrical restitution of MAP duration
Ventricular pacing threshold was determined prior to each pacing protocol with and without nerve stimulation. Threshold was 0.138±0.010 mA without nerve stimulation. There was no significant difference during SS (0.148±0.011 mA, P>0.05) or VS (0.150±0.006 mA, P>0.05).

Fig. 2A shows the results of a typical experiment where MAPD90 was reduced during SS when compared to baseline, but lengthened during VS, at corresponding diastolic intervals. MAPD90max was reduced from a baseline of 144.1 ms to 129.9 ms with SS and increased to 151.9 ms with VS. It is also evident that the maximum slope of the restitution curve (dotted lines in Fig. 2A) was made steeper with SS but less steep with VS, when compared to baseline.


Figure 2
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 A. Plot of standard restitution curves [MAPD90 vs. preceding diastolic intervals (DI)] in a typical experiment at baseline and during sympathetic (SS) and vagus nerve stimulation (VS). The curves were fitted to the exponential curve [MAPD90=MAPD90max(1–e–DI/{tau})] (solid lines) and the maximum slopes (dashed lines) during restitution were calculated. B. Plot of first derivative of the fitted curves in A to calculate the slope of the restitution curves. A horizontal dotted line is plotted at slope=1.

 
Fig. 2B shows the first derivative of the fitted exponential restitution curves obtained in the experiment in Fig. 2A. It shows that the maximum slope of restitution was 1.87 at baseline occurring at a diastolic interval of 15.0 ms which was achieved at ERP. The maximum slope of restitution was increased to 2.9 with SS and occurred at a diastolic interval of 16.7 ms, whilst the maximum slope of restitution was decreased to 1.24 with VS and occurred at a diastolic interval of 12.3 ms. A horizontal dotted line is plotted at a slope of 1. The slope of 1 was reached at a diastolic interval of 21.2 ms at baseline, 24.9 ms with SS and 16.1 ms with VS. The range of diastolic intervals where the restitution slope was >1 was 6.2 ms at baseline, 8.2 ms with SS and 3.8 ms with VS.

Of the 11 hearts, MAPD90max was 133.7±2.8 ms and the maximum slope of restitution was >1 in 9 hearts at baseline, the mean value in the 11 hearts being 1.30±0.10. SS decreased MAPD90max to 121.9±3.4 ms and increased the maximum slope of restitution, that being >1 in all hearts, with the mean value increased to 1.86±0.17 (P<0.01, compared with baseline). VS increased MAPD90max to 141.7±4.0 ms and decreased the maximum slope of restitution in all hearts with the slope being >1 in 3 out of 11 hearts, the mean value decreased to 0.69±0.10 (P<0.001, compared with baseline). In the 9 hearts where the maximum slope of restitution was >1 at baseline, the range of diastolic intervals with restitution slope >1 was increased from 3.7±0.6 ms at baseline to 6.3±0.8 ms during SS (P<0.02).

3.2. Effect of sympathetic and vagal stimulation on VF threshold
Fig. 3A summarizes the mean data on maximum slope of restitution, ERP and VFT in the 11 hearts at baseline and during SS and VS. ERP was decreased from 127.3±2.5 ms at baseline to 111.8±1.8 ms with SS (P<0.001) and increased to 144.0±2.2 ms with VS (P<0.001). VFT was decreased from a baseline value of 4.7±0.6 mA to 1.9±0.5 mA with SS (P<0.001) and increased to 8.9±1.1 mA with VS (P<0.0005). The percentage changes in these parameters are summarized in Fig. 3B.


Figure 3
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 A. Maximum slope of standard restitution, effective refractory period (ERP) and ventricular fibrillation threshold (VFT) at baseline (BL) and during sympathetic (SS) and vagus nerve (VS) stimulation. (*P<0.02 vs. BL). B. Percentage change in maximum slope of standard restitution, ERP and VFT during SS and VS.

 
In 8 of the 11 hearts, VFT was measured with and without nerve stimulation during intrinsic heart rate and during constant ventricular pacing at 300 ms. This was to investigate whether the heart rate effects during autonomic nerve stimulation was the reason for the observed changes in VFT. SS and VS caused significant changes on VFT during constant pacing when compared to baseline, with no significant difference in VFT during intrinsic heart rate or with constant pacing although there was a trend towards a slightly lower VFT during constant pacing with VS (Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1 Ventricular fibrillation threshold measured at baseline (BL) and during sympathetic (SS) and vagus nerve (VS) stimulation when heart rate was allowed to vary (intrinsic HR) and when hearts were ventricularly paced at 300 ms

 
3.3. Correlation between autonomic modulation of restitution and VF threshold
In Fig. 4, the maximum slope of restitution in each of the 11 hearts is plotted against the corresponding VF threshold obtained – at baseline and with SS and VS. There was a significant inverse relationship between the 2 parameters (r=–0.63, P<0.0001).


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Relationship between ventricular fibrillation threshold (VFT) and maximum slope of standard restitution. Individual symbols represent values obtained at baseline (BL) and during sympathetic (SS) and vagus nerve stimulation (VS).

 
3.4. Effect of sympathetic and vagal stimulation on conduction time
During the extrastimulus protocol, delay from the S2-stimulus to the beginning of S2-MAP signal (S2-delay) increased with progressively shorter S1-S2 intervals. This prolongation in S2-delay suggests a slowing of conduction at short coupling intervals that would be consistent with restitution of conduction velocity [7]. Fig. 5 shows the effects of sympathetic and vagus nerve stimulation on S2-delay over the range of S1-S2 intervals in a typical experiment. For comparison, the mean S2-delay at S1-S2 interval of 150 ms was 40.6±3.4 ms without nerve stimulation – this was decreased by SS to 33.7±2.9 ms (P<0.05) and increased by VS to 46.4±3.2 ms (P<0.02). Although the exact conduction path of the paced beats were not mapped and hence conduction velocity not directly measured, these data suggested that conduction velocity was increased with SS and decreased with VS.


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Plot of the delay between pacing stimulus to the beginning of S2-MAP (S2-delay) vs. the range of S1-S2 intervals during extrastimulus protocol at baseline (BL) and during sympathetic (SS) and vagus nerve (VS) stimulation in a typical experiment.

 
3.5. Effect of sympathetic and vagal stimulation on APD alternans
In an attempt to gain further insight into the effects of autonomic stimulation on ventricular electrophysiology, the development of APD alternans was studied. Fig. 6A shows the representative traces in a typical experiment of the MAPs at the end of the 50-beat drive trains at progressively shorter pacing CLs (150 to 80 ms). The traces for pacing CLs 120 ms to 80 ms are shown with expanded timescale in Fig. 6B. At baseline (top trace), variation in the amplitude and duration of the MAPs (alternans) appeared at 110 ms pacing CL whereas it occurred at shorter CL (100 ms) with VS (middle trace) and longer CL (120 ms) with SS (bottom trace). Averaged data for the 5 experiments are summarised in Table 2. Alternans occurred at significantly longer CL and peak alternans level was greater with SS when compared with baseline. In all hearts, alternans level increased at progressively shorter pacing CL until VF occurred – CL at which VF occurred was not altered with SS but Alternans Range was greater than baseline. VS caused a decrease in Alternans Range, as a result of a small decrease in CL at which alternans occurred and a small increase in CL at which VF occurred. Peak alternans level was smaller with VS but this was not statistically significant.


Figure 6
View larger version (35K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 A. Representative traces in a typical experiment showing the last few MAPs during the 50-beat drive trains at different pacing cycle lengths (150 to 80 ms) at baseline (BL) and during left vagus nerve (LVS) and sympathetic stimulation (SS). B. Same traces as A showing 3 consecutive beats from each of the pacing cycle lengths of 120 to 80 ms at expanded timescale.

 

View this table:
[in this window]
[in a new window]

  		Table 2 Summary of data obtained for electrical alternans at baseline (BL) and during sympathetic (SS) and vagus nerve stimulation (VS)

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
This is the first study to demonstrate the effects of direct autonomic nerve stimulation on electrical restitution of ventricular myocardium in the isolated rabbit heart. It was shown that sympathetic stimulation steepens the slope of the standard APD restitution curve whilst vagal stimulation flattens it. Sympathetic stimulation increased the susceptibility of the heart to VF (by lowering VF threshold) whilst vagal stimulation offered a protective effect (by raising VF threshold). Autonomic modulation of standard restitution was associated with effects on VF threshold. Sympathetic stimulation caused alternans at longer pacing cycle lengths with greater degree of alternans. It also caused a wider range of cycle lengths where alternans occurred whilst vagus nerve stimulation shortened this range.

4.1. Electrical restitution and induction of VF in unstimulated hearts
According to the restitution hypothesis, if the slope of the APD restitution curve is >1, oscillations and wavebreaks can occur which facilitate fibrillation in the myocardium. A steep curve means that small changes in diastolic interval would lead to large changes in APD and hence dynamic instability. This has been supported by mathematical studies [12,13] and also recently described in biological studies [7,14]. Drugs which flatten the restitution curve make VF difficult to induce and can convert VF into "stable" ventricular tachycardia [8,15,16]. These data support the notion that the electrical restitution property of the myocardium plays an important role in determining the susceptibility of the heart to fibrillation.

Studies in canine hearts showed slopes of >1 in "dynamic" restitution curves obtained with rapid pacing whereas "standard" restitution studied using the extrastimulus protocol showed slopes of <1 in the same hearts [17]. This is in contrast with studies in smaller animals like rabbits [18,19] and guinea pigs [20,21] where dynamic restitution slopes were less than standard restitution slopes. This may be explained by differences in experimental protocols and species difference but, perhaps paradoxically, the latter studies are more in line with data obtained from human studies [22].

In the current study, the maximum slope of restitution averaged 1.35 in the 11 hearts, being >1 in 9, at baseline without any autonomic stimulation. VF was inducible in all hearts. The induction protocol used in this study employed constant current rapid pacing stimuli spanning most of the cardiac cycle. Control studies using this protocol demonstrated a variation of less than 6% in VF threshold determined with or without nerve stimulation (Table 1). There is suggestion that different induction protocols may induce different "types" of VF [23]. The use of the identical protocol at baseline and during nerve stimulation in the current study would make it more likely that the same "type" of VF was induced throughout the experiments.

4.2. Autonomic modulation of electrical restitution, ventricular conduction and refractoriness
4.2.1. Effects of sympathetic and vagus nerve stimulation
Sympathetic stimulation caused a downward shift in the APD restitution curve in addition to increasing the maximum slope of the curve. This is in agreement with previous studies using adrenergic agonists in an in vivo porcine model [24] and in humans [22]. The main advantage of the current study over in vivo studies is that the effects of direct nerve stimulation were observed rather than adrenergic analogues. In addition, the confounding influence of autonomic or haemodynamic reflexes and of interaction with parasympathetic activity or circulating humoral factors seen in vivo were avoided.

The shorter APD and ERP from sympathetic stimulation shown in this study, which agrees with previous in vivo data [25], are likely to involve the adrenergic modulation of several electrophysiological processes [26–28]. Although conduction velocity was not directly measured in the current study, data from the delay in activation time of the S2 stimulus suggest that sympathetic stimulation increase conduction velocity [29].

In contrast, vagus nerve stimulation caused an upward shift of the APD restitution curve and reduced the maximum slope. To our knowledge, there is no published data on the effects of vagus nerve stimulation or other modes of cholinergic activation on electrical restitution in ventricular myocardium. These changes are unlikely to represent interactions with sympathetic stimulation as there is no significant tonic background autonomic activity in the innervated isolated heart without nerve stimulation [9]. We have shown that vagal stimulation prolonged ERP in parallel with APD with, as shown in previous in vivo studies [25]. In addition, our data suggest that conduction velocity was decreased with vagal stimulation since excitability as indicated by pacing threshold did not change during vagal stimulation.

4.2.2. Possible electrophysiological mechanisms
Shortening of APD with premature stimuli in restitution involves a number of electrophysiological mechanisms. The effects of sympathetic stimulation without any background cholinergic influence are likely to involve an increase in ICa,L [30]. However, this increase in inward current would tend not only to lengthen APD but also prevent APD shortening with shorter diastolic intervals and hence unlikely to explain the changes in APD restitution and ERP. Incomplete recovery from inactivation of the ICa,L with an extrastimulus at short diastolic interval would lead to an abbreviation of APD (and hence the restitution curve) [31] but this is unlikely to be an underlying mechanism as inactivation of ICa,L is accelerated by adrenergic stimulation [32]. On the other hand, adrenergic stimulation increases outward currents including the delayed rectifier K+ current (IK), chloride current and transient outward current (Ito) [26–28,30]. Activation of these currents by sympathetic stimulation would shorten APD and possibly increase the steepness of the restitution curve. The relative contribution of these ion currents to the steepening of restitution slope warrants further study.

Flattening of the restitution curve with vagus nerve stimulation, in the absence of adrenergic influence, demonstrated in this study for the first time, would require either an enhancement of inward currents or a reduction in outward currents. Acetylcholine (ACh) mediates most actions from vagus nerve stimulation and is a strong inhibitor of ICa,L and contractile force in atrial cells [33]. Apart from a reduction of ICa,L by ACh in ferret [34] there are few studies to support a direct effect on unstimulated ICa,L in ventricular cells of most mammalian species [35]. The ACh-activated K+ current, IK.Ach, is increased with acetylcholine and vagal activity but is more abundant in atrial than ventricular cells [30]. Modulation of these currents would not explain the results in the current study as the inhibition of ICa,L and activation of IK.Ach with vagal stimulation would both lead to opposite effects on ventricular electrical restitution. Other electrophysiological mechanisms need to be explored.

4.3. Effects of autonomic activity on ventricular fibrillation and sudden death
Abnormal cardiac autonomic control including high levels of sympathetic activity and impaired parasympathetic control are associated with cardiac dysfunction [1,2] and adversely contributes to disease progression and mortality [36]. There is strong evidence that the relationship between impaired cardiac autonomic control and mortality is a result of an increased susceptibility to lethal ventricular arrhythmias [3].

In vivo animal studies have shown that sympathetic stimulation via the stellate ganglia increases the vulnerability to VF of the normal heart [37]. This is in line with the findings of the current study where both ERP and VF threshold are decreased with direct sympathetic stimulation in the isolated heart. High levels of vagal activity can exert powerful anti-arrhythmic effects which can counter the effects of acute ischaemia and sympathetic activation [38]. However, Kolman et al. [39] showed that vagal stimulation alone did not affect the VF threshold but prevented the decrease in VF threshold induced by simultaneous sympathetic stimulation. The conflicting nature of some of these data may be explained by the fact that the experiments were carried out in vivo and interference from circulating catecholamines and other neurotransmitters cannot be excluded. The current study demonstrated that VF threshold and ventricular ERP were both increased with vagus nerve stimulation in the absence of concomitant background sympathetic activity.

Further to this, data in Table 1 showed that the effects during autonomic nerve stimulation were not the result of chronotropic actions because the effects of SS and VS on VFT were still evident when heart rate was controlled.

4.4. Electrical restitution, APD alternans and VF initiation: An autonomic association?
The effects of sympathetic stimulation and opposite effects of vagal stimulation on electrical restitution and susceptibility to VF demonstrated within this study show an association between the maximum slope of restitution and the corresponding VF threshold. It is tempting to suggest that the autonomic modulation of VF initiation in the isolated heart may be mediated via electrical restitution. However, we do not know if this relationship is causal or coincidental as the underlying mechanisms may be different between sympathetic and vagus nerve stimulation. A strong supporting statement of a causal relationship comes form Weiss et al. [6] who suggested that "Dynamically induced heterogeneity is another mechanism that requires no preexisting heterogeneity of any kind, just an intervention eg, very rapid pacing or a large premature stimulus to create the first wavebreak. This type of wavebreak is determined primarily by electrical restitution i.e. dependence of APD and conduction velocity on the preceding DI".

4.5. Effects of direct sympathetic and vagus nerve stimulation on APD alternans
The lack of change of pacing threshold with sympathetic or vagal stimulation suggests that there was no major contribution from changes in excitability of ventricular myocytes. Our data on the effects of autonomic stimulation on electrical alternans provided some support for the restitution hypothesis but raised further questions. With sympathetic stimulation, alternans occurred over a wider range beginning at longer cycle lengths, with shorter cycle lengths causing VF, characteristics that are in agreement with the restitution hypothesis. Vagal stimulation reduced the range of cycle lengths where alternans occurred, this was produced by a small decrease in the cycle length where alternans occurred and a small increase in the cycle length where VF occurred. Hence, the effects are less than straightforward and cannot be directly related to the effects on the slope of the restitution curve.

APD alternans has been shown to be a key precursor to many serious arrhythmias. The cellular mechanisms underlying alternans are complex, with Ca2+ homeostasis being implicated together with changes in transmembrane ion channel activities [18,20,40]. This is further complicated by the effects of wavefronts and anisotropy on the interaction between APD and Ca2+ transient duration (concordance vs. discordance) [41]. Whilst the link between slope of the APD restitution curve and APD alternans had been well demonstrated mathematically [13], biological data that support this direct relationship are less well characterized [42,43]. Although it is attractive to suggest that the effects of sympathetic and vagus nerve stimulation on restitution directly related to those on APD alternans, other factors not examined in this study, such as cardiac memory [44,45] may be involved.

4.6. Limitations and implications of the study
In this study, MAPs were measured from only one epicardial site. The contribution of dispersion of repolarisation – either transmural or spatial over the epicardial surface – was not assessed. It was well demonstrated in classical experiments by Han and Moe [46] that adrenergic stimulation causes nonuniform recovery of excitability. Regional differences in electrical restitution may have an additional impact on arrhythmogenesis. The current study aimed at obtaining data on the effect of direct nerve stimulation on ventricular electrophysiology at one epicardial site to form a base from which studies on potential mechanisms and other electrophysiological effects may be developed. One such avenue of investigation is underway applying optical mapping techniques to the innervated heart preparation to study the spatial heterogeneity of electrophysiological parameters over the heart.

In the current study, data for left vagus nerve stimulation were presented although results were similar for right vagus nerve stimulation. Sympathetic outflow was stimulated bilaterally as the initial focus in developing the model centered on the cardiac effects of vagus nerve stimulation. Stimulation of either left or right stellate ganglion is possible and experiments are on-going to investigate the effects of possible regional innervation of unilateral sympathetic input to the heart [47].


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
This study is the first to demonstrate the effects of direct autonomic nerve stimulation on APD restitution in the ventricular myocardium of the whole heart. Sympathetic stimulation shifted the restitution curve downwards and made it steeper whilst vagus nerve stimulation had opposite effects. VF threshold was decreased with sympathetic stimulation and increased with vagal stimulation and there was a significant inverse relationship between the maximum slope of restitution and VF threshold. Electrical alternans was increased with sympathetic stimulation and reduced with vagal stimulation. The interaction between APD and beat-to-beat interval, i.e. APD restitution, is likely to play an important role in the autonomic modulation of VF initiation although the effects on other parameters may be involved.


   Acknowledgement
 
This study was supported by the British Heart Foundation (Project Grant PG/99008).


   Notes
 
Time for primary review 16 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

  1. Nolan J., Batin P.D., Andrews R., Lindsay S.J., Brooksby P., Mullen H., et al. Prospective study of heart rate variability and mortality in chronic heart failure – Results of the United Kingdom heart failure evaluation and assessment of risk trial (UK-Heart). Circulation (1998) 98:1510–1516.[Abstract/Free Full Text]
  2. La Rovere M.T., Bigger J.T., Marcus F.I., Mortara A., Schwartz P.J. Baroreflex sensitivity and heart rate variability in prediction of total cardiac mortality after myocardial infarction. Lancet (1998) 351:478–484.[CrossRef][ISI][Medline]
  3. Schwartz P.J. The autonomic nervous system and sudden death. Eur Heart J (1998) 19:F72–F80.[ISI][Medline]
  4. Einbrodt. Ueber Herzeizung und ihr Verhaeltnis zum Blutdruck. Akademie der Wissenschaften (Vienna). Sitzungsberichte (1859) 38:345–359.
  5. MERIT-HR Study Group. Effect of metoprolol CR XL in chronic heart failure: Metoprolol CR XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF). Lancet (1999) 353:2001–2007.[CrossRef][ISI][Medline]
  6. Weiss J.N., Chen P.S., Qu Z., Karagueuzian H.S., Garfinkel A. Ventricular fibrillation: how do we stop the waves from breaking? Circ Res (2000) 87:1103–1107.[Abstract/Free Full Text]
  7. Cao J.M., Qu Z.L., Kim Y.H., Wu T.J., Garfinkel A., Weiss J.N., et al. Spatiotemporal heterogeneity in the induction of ventricular fibrillation by rapid pacing importance of cardiac restitution properties. Circ Res (1999) 84:1318–1331.[Abstract/Free Full Text]
  8. Garfinkel A., Kim Y.H., Voroshilovsky O., Qu Z., Kil J.R., Lee M.H., et al. Preventing ventricular fibrillation by flattening cardiac restitution. Proc Natl Acad Sci U S A (2000) 97:6061–6066.[Abstract/Free Full Text]
  9. Ng G.A., Brack K.E., Coote J.H. Effects of direct sympathetic and vagal stimulation on the physiology of the whole heart – a novel model of isolated Langendorff perfused rabbit heart with intact dual autonomic innervation. Exp Physiol (2001) 86:319–329.[Abstract]
  10. Ng G.A., Brack K.E., Coote J.H. Differential effects of left and right vagus nerve stimulation on sinoatrial and atrioventricular nodes but not on ventricular electrophysiology – studies in the isolated rabbit heart with intact autonomic innervation. J Physiol (2001) 531P:182P. [Abstract].
  11. Kettlewell S., Walker N.L., Cobbe S.M., Burton F.L., Smith G.L. The electrophysiological and mechanical effects of 2,3-butane-dione monoxime and cytochalasin-D in the Langendorff perfused rabbit heart. Exp Physiol (2004) 89:163–172.[Abstract/Free Full Text]
  12. Nolasco J.B., Dahlen R.W. A graphic method for the study of alternation in cardiac action potentials. J Appl Physiol (1968) 25:191–196.[Free Full Text]
  13. Karma A. Electrical alternans and spiral wave breakup in cardiac tissue. Chaos (1994) 4:461–472.[CrossRef][ISI][Medline]
  14. Gilmour R.F., Chialvo D.R. Electrical restitution, critical mass, and the riddle of fibrillation. J Cardiovasc Electrophysiol (1999) 10:1087–1089.[ISI][Medline]
  15. Riccio M.L., Koller M.L., Gilmour R.F. Electrical restitution and spatiotemporal organization during ventricular fibrillation. Circ Res (1999) 84:955–963.[Abstract/Free Full Text]
  16. Omichi C., Zhou S., Lee M.H., Naik A., Chang C.M., Garfinkel A., et al. Effects of amiodarone on wave front dynamics during ventricular fibrillation in isolated swine right ventricle. Am J Physiol Heart Circ Physiol (2002) 282:H1063–H1070.[Abstract/Free Full Text]
  17. Koller M.L., Riccio M.L., Gilmour R.F. Dynamic restitution of action potential duration during electrical alternans and ventricular fibrillation. Am J Physiol Heart Circ Physiol (1998) 44:H1635–H1642.
  18. Goldhaber J.I., Xie L.H., Duong T., Motter C., Khuu K., Weiss J.N. Action potential duration restitution and alternans in rabbit ventricular myocytes: the key role of intracellular calcium cycling. Circ Res (2005) 96:459–466.[Abstract/Free Full Text]
  19. Banville I., Gray R.A. Effect of action potential duration and conduction velocity restitution and their spatial dispersion on alternans and the stability of arrhythmias. J Cardiovasc Electrophysiol (2002) 13:1141–1149.[CrossRef][ISI][Medline]
  20. Pruvot E.J., Katra R.P., Rosenbaum D.S., Laurita K.R. Role of calcium cycling versus restitution in the mechanism of repolarization alternans. Circ Res (2004) 94:1083–1090.[Abstract/Free Full Text]
  21. Choi B.R., Salama G. Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans. J Physiol - London (2000) 529:171–188.[CrossRef][Medline]
  22. Taggart P., Sutton P., Chalabi Z., Boyett M.R., Simon R., Elliott D., et al. Effect of adrenergic stimulation on action potential duration restitution in humans. Circulation (2003) 107:285–289.[Abstract/Free Full Text]
  23. Taneja T., Goldberger J., Johnson D., Kadish A. Is all ventricular fibrillation the same? Influence of mode of induction on characteristics of ventricular fibrillation. J Cardiovasc Electrophysiol (2000) 11:1355–1363.[CrossRef][ISI][Medline]
  24. Taggart P., Sutton P., Lab M., Dean J., Harrison F. Interplay between adrenaline and interbeat interval on ventricular repolarisation in intact heart in vivo. Cardiovasc Res (1990) 24:884–895.[ISI][Medline]
  25. Martins J.B., Zipes D.P. Effects of sympathetic and vagal nerves on recovery properties of the endocardium and epicardium of the canine left ventricle. Circ Res (1980) 46:100–110.[Free Full Text]
  26. Harvey R.D., Hume J.R. Autonomic regulation of a chloride current in heart. Science (1989) 244:983–985.[Abstract/Free Full Text]
  27. Volders P.G., Stengl M., van Opstal J.M., Gerlach U., Spatjens R.L., Beekman J.D., et al. Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation. Circulation (2003) 107:2753–2760.[Abstract/Free Full Text]
  28. Thomas D., Kiehn J., Katus H.A., Karle C.A. Adrenergic regulation of the rapid component of the cardiac delayed rectifier potassium current, I(Kr), and the underlying hERG ion channel. Basic Res Cardiol (2004) 99:279–287.[ISI][Medline]
  29. Munger T.M., Johnson S.B., Packer D.L. Voltage dependence of beta-adrenergic modulation of conduction in the canine Purkinje fiber. Circ Res (1994) 75:511–519.[Abstract/Free Full Text]
  30. Hartzell H.C. Regulation of cardiac ion channels by catecholamines, acetylcholine and 2nd messenger systems. Prog Biophys Mol Biol (1988) 52:165–247.[CrossRef][ISI][Medline]
  31. Gettes L.S., Reuter H. Slow recovery from inactivation of inward currents in mammalian myocardial fibres. J Physiol (1974) 240:703–724.[Abstract/Free Full Text]
  32. Yuan W., Bers D.M. Protein kinase inhibitor H-89 reverses forskolin stimulation of cardiac L-type calcium current. Am J Physiol (1995) 268:C651–C659.[ISI][Medline]
  33. Wang Y.G., Rechenmacher C.E., Lipsius S.L. Nitric oxide signaling mediates stimulation of L-type Ca2+ current elicited by withdrawal of acetylcholine in cat atrial myocytes. J Gen Physiol (1998) 111:113–125.[Abstract/Free Full Text]
  34. Boyett M.R., Kirby M.S., Orchard C.H., Roberts A. The negative inotropic effect of acetylcholine on ferret ventricular myocardium. J Physiol (1988) 404:613–635.[Abstract/Free Full Text]
  35. Bers D.M. Excitation-contraction coupling and cardiac contractile force, 1st Ed. (2001) Dordrecht: Kluwer Academic Publishers.
  36. Kleiger R.E., Miller J.P., Bigger J.T., Moss A.J. Multicenter Post-Infarction Research Group. Decreased heart rate variability and its association with increased mortality after acute myocardial infarction. Am J Cardiol (1987) 59:256–262.[CrossRef][ISI][Medline]
  37. Verrier R.L., Thompson P.L., Lown B. Ventricular vulnerability during sympathetic stimulation: role of heart rate and blood pressure. Cardiovasc Res (1974) 8:602–610.[Abstract/Free Full Text]
  38. Vanoli E., Schwartz P.J. Sympathetic parasympathetic interaction and sudden death. Basic Res Cardiol (1990) 85:305–321.[ISI][Medline]
  39. Kolman B.S., Verrier R.L., Lown B. The effect of vagus nerve stimulation upon vulnerability of the canine ventricle: Role of the sympathetic parasympathetic interactions. Circulation (1976) 52:578–585.[ISI]
  40. Walker M.L., Wan X., Kirsch G.E., Rosenbaum D.S. Hysteresis effect implicates calcium cycling as a mechanism of repolarization alternans. Circulation (2003) 108:2704–2709.[Abstract/Free Full Text]
  41. Weiss J.N., Karma A., Shiferaw Y., Chen P.S., Garfinkel A., Qu Z. From pulsus to pulseless: the saga of cardiac alternans. Circ Res (2006) 98:1244–1253.[Abstract/Free Full Text]
  42. Franz M.R. The electrical restitution curve revisited: steep or flat slope-which is better? J Cardiovasc Electrophysiol (2003) 14:S140–S147.[CrossRef][ISI][Medline]
  43. Ideker R.E., Rogers J.M., Gray R.A. Steepness of the restitution curve: a slippery slope? J Cardiovasc Electrophysiol (2002) 13:1173–1175.[CrossRef][ISI][Medline]
  44. Cherry E.M., Fenton F.H. Suppression of alternans and conduction blocks despite steep APD restitution: electrotonic, memory, and conduction velocity restitution effects. Am J Physiol Heart Circ Physiol (2004) 286:H2332–H2341.[Abstract/Free Full Text]
  45. Gilmour R.F. Jr., Otani N.F., Watanabe M.A. Memory and complex dynamics in canine cardiac Purkinje fibers. Am J Physiol (1997) 272:H1826–H1832.[ISI][Medline]
  46. Han J., Moe G.K. Nonuniform recovery of excitability in ventricular muscle. Circ Res (1963) 14:44–60.[Medline]
  47. Momose M., Tyndale-Hines L., Bengel F.M., Schwaiger M. How heterogeneous is the cardiac autonomic innervation? Basic Res Cardiol (2001) 96:539–546.[CrossRef][ISI][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ng, G. A.
Right arrow Articles by Coote, J. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ng, G. A.
Right arrow Articles by Coote, J. H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?