Ventricular activation during sympathetic imbalance and its computational reconstruction

Ventricular activation during sympathetic imbalanceand its computational reconstruction

MARTYN P. NASH,1 JUDITH M. THORNTON,1 CLAIRE E. SEARS,2

ANTHONY VARGHESE,3 MARK O’NEILL,1 AND DAVID J. PATERSON1

1University Laboratory of Physiology, Oxford OX1 3PT; and 2Department of CardiovascularMedicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; and 3Institute forMathematics and its Applications, University of Minnesota, Minneapolis, Minnesota 55455Received 20 June 2000; accepted in final form 11 August 2000

Nash, Martyn P., Judith M. Thornton, Claire E.Sears, Anthony Varghese, Mark O’Neill, and David J.Paterson. Ventricular activation during sympathetic imbal-ance and its computational reconstruction. J Appl Physiol 90:287–298, 2001.—We characterized the epicardial activationsequence during a norepinephrine (NE)-induced ventriculararrhythmia in anesthetized pigs and studied factors thatmodulated it. Subepicardial NE infusion caused the QRScomplex to invert within a single beat (n 5 35 animals, 101observations), and the earliest epicardial activation consis-tently shifted to the randomly located infusion site (n 5 14).This preceded right atrial activation, whereas the total ven-tricular epicardial activation time increased from 20 6 4 to50 6 9 ms (P , 0.01). These events were accompanied by aventricular tachycardia and a drop in left ventricular pres-sure, which were fully reversed after the infusion wasstopped. Epicardial pacing at the infusion site mimicked allelectrical and hemodynamic changes induced by NE. Thearrhythmia was prevented by propranolol and abolished bycardiac sympathetic or vagal nerve stimulation. Focal auto-maticity was computationally reconstructed using a two-dimensional sheet of 256 3 256 resistively coupled ventricu-lar cells, where calcium handling was abnormally high in thecentral region. We conclude that adrenergic stimulation to asmall region of the ventricle elicits triggered automaticityand that computational reconstruction implicates calciumoverload. Interventions that reduce spatial inhomogeneitiesof intracellular calcium may prevent this type of arrhythmia.

catecholamines; cardiac mapping; porcine heart; ventriculararrhythmia

IT IS WELL ESTABLISHED THAT cardiac sympathetic imbal-ance can lead to ventricular arrhythmias, although theunderlying cellular electrophysiological mechanismsare not fully understood. Phase 4 depolarization (ab-normal automaticity) and afterdepolarizations (trig-gered automaticity) (22) have been implicated becauseboth can be provoked by catecholamines (8, 27) orexercise (8) and prevented or terminated by b-adren-ergic blockade (8) and acetylcholine (27), all of whichmodulate the levels of intracellular calcium in ventric-ular myocytes. Calcium-channel antagonists also pre-vent triggered automaticity (8, 27), whereas they are

ineffective in terminating abnormal automaticity (27).Previous studies (18, 19) have shown that, in the invivo nonischemic pig heart, a localized subepicardialinfusion of norepinephrine (NE), the permeant cyclic-AMP analog dibutyryl-cAMP, or inhibitors of cyclicnucleotide phosphodiesterase were effective in causinga sustained ventricular arrhythmia, resulting in de-pressed hemodynamics. In the clinical situation, high-adrenergic tone is known to occur around regions ofischemia (11) or during right ventricular (RV) outflowtract dysfunction (16), both of which may facilitatespontaneous activity in the ventricular myocardium(10).

Our aims were threefold: 1) to test whether thehemodynamic changes were a direct consequence of theformation of a new pacemaker at the NE infusion site;2) to examine whether changing global cardiac auto-nomic input, through stimulation of the cardiac sym-pathetic or vagal nerves, modulated the electrical ac-tivation sequence during the arrhythmia and whetherelectrical pacing at the infusion site mimicked thepattern of activation caused by the infusion of NE; and3) to elucidate the electrophysiological basis underly-ing this arrhythmia, we attempted to reconstruct it intwo dimensions using a network model of 256 3 256resistively coupled ventricular cells, for which the cen-tral region had high-adrenergic tone. Some of the re-sults have been communicated in abstract form (12,13).

MATERIALS AND METHODS

The investigation conforms to the “Guide for the Care andUse of Laboratory Animals” published by the National Insti-tutes of Health [DHEW publication no. (NIH) 85–23, revised1996, Office of Science and Health Reports, DRR/NIH, Be-thesda, MD 20205] and under a British Home Office ProjectLicense (no. PPL 30/1133).

Anesthesia

Thirty-five domestic pigs of either sex [mean weight, 24 65 (SD) kg] were premedicated with azaperone (5 mg/kg im;

Address for reprint requests and other correspondence: M. P.Nash, Univ. Laboratory of Physiology, Parks Road, Oxford OX1 3PT,UK (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

J Appl Physiol90: 287–298, 2001.

8750-7587/01 $5.00 Copyright © 2001 the American Physiological Societyhttp://www.jap.org 287

Stresnil, Janssen), and a surgical plane of anesthesia wasinduced with 2–3% halothane (Fluothane, ICI) in 100% oxy-gen via an Ayre’s T piece with a mask and bag. After thesurgery, halothane was replaced with a bolus infusion ofa-chloralose (100 mg/kg iv; Sigma Chemical) and repeatedapproximately every 2 h as required. Stainless steel needleelectrodes were then inserted subcutaneously into each limband into the chest wall to monitor the electrocardiograph(ECG).

Surgery and Instrumentation

A tracheotomy was performed, and a cuffed endotrachealtube (9 mm, Vygon) was introduced ;80 mm into the tra-chea. The cuff was inflated to provide an airtight seal. Fluid-filled catheters (Portex) were inserted into two femoral ar-teries and one femoral vein for sampling of arterial blood,monitoring of arterial blood pressure (ABP), and intravenousinfusion of anesthetic and fluids, respectively. Artificial ven-tilation (Oxford Mark II ventilator, Penlon) was used tomaintain arterial carbon dioxide tension between 35 and 45Torr by adjusting tidal volume and frequency as required.Animals were ventilated with a hyperoxic gas mixturethroughout the experiment. A midline thoracotomy was per-formed, and the chest was retracted laterally. After incisionof the pericardium, the heart was suspended in a pericardialcradle. For all experiments, the cranial projections of the vagiwere cut.

In the first group of experiments (group A, n 5 21), acatheter (Wallace Y-can, 19 gauge) was inserted into the leftventricle (LV) via the apical dimple and secured. The cranialprojections of the cardiac sympathetic nerves were ligatedand cut. Communicating branches to the thoracic sympa-thetic chain were also cut.

In the second group of animals (group B, n 5 14), a Millarcatheter (Mikro-Tip, TC-510 control unit; Millar Instru-ments, Houston, TX) pressure sensor was inserted into theLV via the apical dimple. An elasticized sock containing 127unipolar stainless steel electrodes (Biomedical InstrumentsDesigners, Montreal, Quebec) was slipped over the ventri-cles. In addition, one custom-built silver electrode was at-tached to the right atrium with the use of continuous suction.

Intensive Care

After completion of surgery, at least 1 h elapsed beforeexperiments were begun. The thoracic cavity was covered byplastic film to reduce cooling. Core body temperature wasmeasured by a rectal thermistor and maintained at 38 6 1°Cby heating lamps beneath the operating table. Fluid wasreplaced by an intravenous sterile saline drip (;100 ml/h).Arterial blood samples (0.3 ml) were regularly withdrawninto preheparinized syringes and analyzed for pH, bloodgases, and electrolytes (Na1, K1, Ca21) (RadiometerABL505, Copenhagen, Denmark). For all animals (n 5 35),mean values for arterial pH, arterial CO2 tension, and arte-rial O2 tension were 7.44 6 0.07 (SD), 41 6 6 Torr, and 319 686 Torr, respectively. Respiratory acidosis was corrected byadjusting the ventilator. Metabolic acidosis was corrected byintravenous infusion of 4.2% sodium bicarbonate solution asrequired.

Hemodynamic Measurements

ABP was measured by using a saline-filled pressure trans-ducer (SensoNor 840) calibrated in the midaxillary line. Forgroup A, left ventricular pressure (LVP) was also measuredin this way, and, for group B, LVP was measured directly by

using the Millar catheter. All signals were recorded onto apenwriter (MT8P, Lectromed). The analog inputs werepassed to a real-time data-acquisition system (MP 100,Biopac Systems) employing Acqknowledge 3.0 software forthe Macintosh (Macintosh Quadra 950). Heart rate (HR) andthe rate of change of LVP (LVdP/dt) were computed usingthis software.

Epicardial Electropotential Mapping

For the experiments in group B, 63 of the sock electrodesand the right atrial electrode were connected to a 64-channelUnemap cardiac mapping system (Uniservices, Auckland,New Zealand). The selected sock electrodes spanned thewhole of the ventricular epicardium, with an interelectrodespacing of ;15 mm. The animal and all cable shields wereearthed to the system ground (computer earth potential).Unipolar epicardial electropotentials (referred to the systemground) were continuously sampled at 900 Hz with a rollingbuffer of 8 s and could be digitally stored for subsequentanalysis. During three of these experiments, two-dimen-sional echocardiography (HP SONOS 5500) was used to es-timate LV ejection fraction.

Protocols

In all animals (n 5 35), ventricular arrhythmias wereinduced by regional subepicardial infusions of NE bitartrate(10 mmol/l in 0.9% saline, 150 ml/min). The concentration andrate of infusion were chosen because they elicited reproduc-ible arrhythmias within 1–5 min after the onset of theinfusion. Infusion was through a 25-gauge hypodermic nee-dle to which was attached a 3FG polyvinyl chloride catheter(;150 mm long) and a three-way tap. The needle was in-serted 1–5 mm at randomly selected locations throughoutthe ventricular subepicardium. Solutions were infused byusing a three-syringe microinjection pump (CMA/100). Incases in which subepicardial infusion of NE did not elicit aventricular arrhythmia within 5 min (;20% of infusions), theneedle was removed, and infusion was begun in another partof the ventricle. Susceptibility to the arrhythmia did notdepend on the choice of infusion site.

Group A (n 5 21). The vehicle (saline) was infused (150ml/min) subepicardially in all animals for ;10–15 min.

To test whether the arrhythmia was due to b-adrenergicstimulation alone, propranolol (0.1–0.25 mg/kg iv; Inderal,ICI) was administered during the NE-induced ventriculararrhythmia (n 5 8). The specific b-agonist isoproterenol(1 mmol/l, 150 ml/min, n 5 2) or the a-agonist phenylephrine(1 and 10 mmol/l, 150 ml/min, n 5 2) was also infused sub-epicardially in the absence of b-blockade.

To test whether the arrhythmia could be modulated byglobal autonomic input, cardiac sympathetic nerve stimula-tion (5 Hz, 5 V, 3-ms width, 30-s duration; caudal limb of theright ansa subclavia, n 5 6) or vagal nerve stimulation (10Hz, 10 V, 3-ms width, 30-s duration; left vagus, n 5 2; rightvagus, n 5 4) was undertaken during the NE-induced ven-tricular arrhythmia. Each period of stimulation lasted for30 s and was followed by a recovery period, while the infusionof NE was maintained. Pre- and postarrhythmia stimula-tions were also performed to test the stability of the nerves.In addition, NE was infused intravenously (1 mg zkg21 zmin21

for 1 min) during stable arrhythmias (n 5 3). Control intra-venous infusions of NE were also performed before subepi-cardial NE infusion.

Group B (n 5 14). Ventricular electropotential mappingwas performed for control, NE-induced ventricular arrhyth-

288 VENTRICULAR ACTIVATION DURING SYMPATHETIC IMBALANCE

mia, and recovery states. In addition, a single electrode wasused to detect right atrial activation (n 5 10).

A custom-built roving bipolar electrode was used to pacehearts (0.5–1.5 V, 2-ms width, 30-s duration, n 5 3) at thesame site in which NE had previously been infused. Thepacing needle was inserted ;2 mm subepicardially, andthe pacing rate was matched to the HR observed duringthe previous NE-induced arrhythmia.

Epicardial surface (n 5 2) or transesophageal (n 5 1)echocardiography was used to estimate LV ejection fractionduring the ventricular arrhythmias.

Electropotential Signal Analysis

With reference to Fig. 1, epicardial electropotential sig-nal analysis was performed as follows. After epicardialsignal acquisition using the elasticized electrode sock (Fig.1A), the electropotentials for a single heart cycle wereidentified from the 8 s of recorded data, as shown in Fig.1B. The most negative electropotential slope was computedfor each signal (Fig. 1C), and this was chosen to mark theinstant at which the associated sock electrode detectedexcitation of its adjacent portion of epicardium. An eventmarker was used to identify the time of excitation for eachsignal.

The set of event markers was then used to order thesignals, such that the signal that detected earliest activationwas listed first (Fig. 1C). For this earliest signal, the marker

time was referred to as the datum (red dashed line in Fig.1C). Activation times were then computed for all other sig-nals by taking the difference between their marker times andthe datum.

Two-dimensional Activation Maps

From the known topographical organization of the sockelectrodes, estimated epicardial electrode locations wereplotted by using the two-dimensional Hammer projection(20), for which the LV and RV free walls constitute thecentral and lateral portions, respectively (see Fig. 1D). Theleft anterior and posterior descending coronary arterieswere superimposed for anatomic clarity. Nodal activationtimes were computed from the set of spatially arrangedelectrode activation times using a two-dimensional finiteelement-fitting technique, which incorporated linear leastsquares (6). Red portions of this smoothly continuous two-dimensional activation map highlight regions of earliestepicardial excitation, whereas blue denotes regions of lat-est epicardial excitation. The spatial resolution of theelectrodes was considered sufficient during control andarrhythmic states, because doubling the electrode density(i.e., halving the interelectrode spacing) over one-half ofthe epicardial surface produced a similar two-dimensionalactivation pattern and range of times for that region (notshown).

Fig. 1. Schematic representation of cardiac electropotential mapping and signal analysis. Epicardial signals wererecorded by using an elasticized sock with 63 epicardial electrodes (A) connected to a cardiac mapping system (B).C: a single heart cycle was identified, and the epicardial activation time for each electrode was determined by usingthe most negative electropotential slope. Dt, Time difference. D: an epicardial activation map was fitted to theelectrode activation times and displayed by using the Hammer projection. This 2-dimensional (2D) topographyrepresents the ventricular epicardial surface, where the left ventricle (LV) makes up the central portion of theprojection, the right ventricle (RV) comprises the border regions, but the apex is retained as a single point. E: theactivation time field was also fitted on the epicardial surface of an anatomically accurate 3-dimensional (3D)mathematical model of the ventricles, for which anterior (left) and posterior (right) views are illustrated andcontours of simultaneous epicardial activation have been superimposed. LAD and PDA (thick lines), left anteriorand posterior descending coronary arteries, respectively.

289VENTRICULAR ACTIVATION DURING SYMPATHETIC IMBALANCE

Epicardial Activation Sequence Renderedin Three Dimensions

A further technique was developed to produce epicardialactivation maps with anatomic realism. The epicardial acti-vation sequence was reconstructed by fitting a scalar field ofactivation times onto the epicardial surface of an anatomi-cally accurate mathematical model of the canine ventricles(14) rendered in three dimensions, as shown in Fig. 1E. Theestimated three-dimensional locations of the electrodes wereprojected onto the epicardial surface of the anatomic model,and the epicardial activation time field was approximated byusing linear interpolation of a set of parameters defined atspatial locations that coincided with the finite element nodesused to define the geometry. As for the two-dimensionalactivation map, nodal epicardial activation times were com-puted by using a standard finite element-fitting technique.

Computer Modeling

Abnormal intracellular calcium handling has been impli-cated in the onset of this arrhythmia (18). In an attempt tosimulate this, we modified a recent model of the electrical

activity in ventricular myocytes (15) to include effects of NEon cell membrane properties. The effect of NE was modeledby using a fivefold elevation in the conductances of the L-type calcium channel of the cell sarcolemmal membrane(from 0.25 to 1.25 mA z l zmmol21) and the sarcoplasmic retic-ulum (SR) calcium-ATPase uptake pump (from 1 to 5mmol z l21 zs21). This is consistent with changes in calciumcurrents due to isoproterenol that have been reported in theliterature (1). Action potential propagation was studied byusing a two-dimensional sheet of 256 3 256 cells isotropicallyinterconnected via linear resistors to represent the intercel-lular gap junctions. To simulate the spatial decrease in NEconcentration due to ligand diffusion and metabolism, theupregulation factor for the L-type and SR uptake calciumconductances was chosen to decrease according to a two-dimensional normal distribution with a variance of 60 celllengths. In this way, conductance changes were negligible ata distance of 90 cell lengths from the center, consistent withthe reported zone of increased cAMP levels surroundingintramyocardial NE infusion sites (18). Action potentialswere initiated by stimulating one edge of the sheet at a rateof 1 Hz with a twice-threshold stimulus for 2 ms. A control

Fig. 2. Induction of a ventricular arrhythmia by subepicardial infusion (150 ml/min) of 10 mmol/l norepinephrine(NE). Needle, insertion point of needle; NE im, start of infusion. Traces are as follows (from the top down): arterialblood pressure (ABP), LV pressure (LVP), electrocardiograph (ECG), and rate of change of LVP (LVdP/dt). Bottomtrace illustrates the ECG at the point marked X, but on an expanded time scale. Approximately 90 s after the NEinfusion was begun, there was a dramatic change in the configuration of the ECG, which was accompanied by amarked hemodynamic depression.


simulation with normal intracellular calcium handling wasundertaken for the same number of cycles. The ionic currentsof a single-model myocyte were also investigated to elucidatea possible cellular mechanism for the NE-induced arrhyth-mia.

Cellular simulations were initially performed by using aSilicon Graphics Power Challenge XL (12 symmetric MIPS90-MHz R8000 processors; 1-GB physical DRAM) and subse-quently by using a Silicon Graphics Cray Origin2000 (84symmetric MIPS 195-MHz R10000 processors; 21-GB physi-cal DRAM; Silicon Graphics, Mountain View, CA). Theparallel computations incorporated a variable-step, fourth-order, Runge-Kutta numerical integration method, as de-scribed previously (26).

Statistical Analysis

All data are expressed as means 6 SD. Mean arterial bloodpressure (MAP), LVP, HR, LVdP/dt, and activation times forcontrol, arrhythmic, and recovery states were compared byusing a one-way analysis of variance with repeated mea-sures. Post hoc comparisons were performed by using Dun-nett’s method, and statistical significance was accepted atP , 0.05.

RESULTS

NE-induced Ventricular Arrhythmia

Infusion of NE into the ventricular subepicardiumelicited a reproducible ventricular arrhythmia on 101separate occasions in 35 animals. Within 1–5 min afterthe start of the infusion, there was a marked change inthe amplitude and direction of the QRS complex, whichmasked the P wave (Fig. 2). The abnormal electricalactivity was accompanied by a ventricular tachycardia(control, 126 6 18 beats/min; arrhythmia, 153 6 20beats/min; P , 0.01, n 5 35) and a profound hemody-namic depression (see Figs. 2 and 3). Restoration ofnormal sinus rhythm and hemodynamic performanceoccurred within 10 min after the subepicardial infusionof NE was stopped. Recovery was accompanied by asmall, transient overshoot of all hemodynamic vari-ables (Fig. 3), which was significant compared with

control (P , 0.05; n 5 35). A subsequent ventriculararrhythmia could be established by using the same NEinfusion site.

Sustained ventricular arrhythmia was also elicitedby subepicardial infusion of the b-agonist isoproterenol(n 5 2), but could not be provoked with subepicardialinfusions of the a-agonist phenylephrine (n 5 2) or thesaline vehicle (n 5 21), or by similar infusions of NEadministered intravenously. After a stable arrhythmiawas established, intravenous administration of a bolusof propranolol (n 5 8) abolished it within ;2 min. Afterthis, the hemodynamic performance quickly returnedto its prearrhythmic state. It was not possible to elicitthe arrhythmia in the b-blocked animal.

Sympathetic nerve stimulation. Stimulation of theright cardiac sympathetic nerve abolished the ventric-ular arrhythmia within 10.6 6 7.5 s (17 observations in6 animals). Sympathetic nerve stimulation caused theQRS complex to revert to its control form and the HR toincrease by 90 6 16 beats/min above the arrhythmicrate. No recurrence of the arrhythmia was seen duringany 30-s period of sympathetic stimulation. However,the arrhythmia and hemodynamic depression were re-established 47 6 16 s after stimulation was stopped.Infusion of NE (1 mg zkg21 zmin21 iv) for 1 min reversedthe arrhythmia within 35 6 21 s (5 observations in 3animals). As observed during sympathetic stimulation,the QRS complex reverted, and all hemodynamic vari-ables were elevated (HR increased by 42 6 34 beats/min above the arrhythmic rate). Recurrence of thearrhythmia took place 30 6 28 s after the infusion wasstopped.

Vagal nerve stimulation. Stimulation of the left (5observations in 2 animals) or right (13 observations in4 animals) vagus nerves reversed the ventricular ar-rhythmia after 7.4 6 4.9 s. Although control vagalstimulation reduced all hemodynamic variables, vagalstimulation during a NE-induced arrhythmia causedsmall increases in ABP, LVP, and LVdP/dt (see Fig. 4),whereas the HR decreased by 46 6 11 beats/min after

Fig. 3. Effect of subepicardial infusion (150 ml/min) of 10 mmol/l NE on hemodynamics for allexperiments (n 5 35 animals, 101 observations).Values correspond to those before infusion (con-trol), the maximum hemodynamic depressionduring NE infusion (arrhythmia), and the valuesreached after termination of the arrhythmia (re-covery). A: mean arterial blood pressure (MAP);B: maximum systolic LVP; C: heart rate (HR); D:maximum LVdP/dt (1LVdP/dt). bpm, Beats/min.wStatistically significant (P , 0.01) changes inthe hemodynamic variables compared with thecontrol state.


the termination of the arrhythmia. After the stimula-tion was stopped, hemodynamics continued to increasein the presence of normal electrical activity (Fig. 4) for31 6 9 s until the arrhythmia was reestablished.

Epicardial Electropotential Mapping

During the NE-induced arrhythmia for the group Bexperiments, right atrial activation followed the earli-est ventricular epicardial activation (20 observationsin 10 animals). Figure 5 illustrates the onset of onearrhythmia. The first two cycles show control condi-tions, in which right atrial excitation preceded ventric-ular activation. After the ventricular repolarization ofthe second cycle, a spontaneous ventricular depolariza-tion was observed. Subsequently, earliest ventricularepicardial activation preceded right atrial activationon a one-to-one basis until after the NE infusion wasstopped.

Figure 6 illustrates epicardial activation maps dur-ing one experiment, for which a ventricular arrhyth-mia was elicited after ;90 s of NE infusion. Undercontrol conditions, the earliest epicardial activationoccurred near the equatorial region of the RV and thelatest epicardial activation occurred in the postero-basal region. In this experiment, NE was infused nearthe apex of the posterior LV wall, and, during theensuing arrhythmia, the location of earliest epicardialactivation closely matched the NE infusion site. Thischange in the cardiac activation sequence caused theQRS complex to invert within a single beat and re-sulted in a marked depression of the ABP. Within 2min of the stop of the NE infusion, the cardiac activa-tion sequence, ECG, and hemodynamics were restoredto their control states.

The events described above were observed consis-tently for all group B experiments (27 observations in14 animals). The group data (see Fig. 7) showed thatthe total ventricular epicardial activation time (definedas the time taken for the wave of excitation to spreadfrom earliest to latest epicardial activation sites) sig-nificantly increased from 20 6 4 ms under controlconditions to 50 6 9 ms (P , 0.01, n 5 14) during theNE-induced ventricular arrhythmia. During all 27 NE-induced arrhythmias, earliest epicardial activationwas detected within one electrode spacing (;15 mm) ofthe needle site. The MAP significantly decreased from62 6 13 mmHg (control) to 43 6 10 mmHg during thearrhythmia (P , 0.01, n 5 14). The total ventricularepicardial activation time and MAP recovered to con-trol values after the NE infusion was stopped.

Epicardial electrical stimulation. Ventricular epi-cardial pacing at the NE infusion site mimicked allelectromechanical changes observed during the NE-induced ventricular arrhythmia. Figure 8 shows acti-vation maps obtained during one experiment, for which

Fig. 4. Effects of right and left cardiac vagalnerve stimulation [10 Hz, 10 V, 3-ms width, 30-sduration from stimulation (stim on)] during asubepicardial NE-induced ventricular arrhyth-mia. The sustained arrhythmia was elicited;180 s after the onset of NE infusion (notshown). Traces are as follows (from top down):ABP, ECG, and HR. Both right and left vagalstimulation reversed the ECG inversion, but thearrhythmia recurred shortly after stimulationwas stopped (stim off). The lack of recovery of theABP during right vagal stimulation was likelydue to the greater decrease in HR, comparedwith the effects of left vagal stimulation.

Fig. 5. Right atrial (RA) vs. ventricular epicardial (Vepi) electropo-tential recordings at the onset of a subepicardial NE-induced ven-tricular arrhythmia. Under control conditions (first 2 cycles), RAactivation preceded Vepi activation. A spontaneous ventricular acti-vation marks the onset of the arrhythmia, after which RA activationfollowed Vepi activation on a one-to-one basis.


NE was infused into a LV equatorial site. The subse-quent arrhythmia resulted in an increase in the totalventricular epicardial activation time from 21 ms (con-trol) to 49 ms. After a full recovery, the needle waswithdrawn, and the epicardium was electrically stim-ulated at the same location with a similar rate to thatobserved during the NE-induced arrhythmia. The totalventricular epicardial activation time for the pacing-induced arrhythmia increased from 21 ms (control, notshown) to 54 ms, and the activation maps for the NE-and pacing-induced arrhythmias were qualitativelysimilar (see Fig. 8). In two subsequent experiments,epicardial activation times increased from 14 and 18ms (control) to 39 and 51 ms for the NE-induced ar-rhythmias and to 39 and 46 ms for the pacing-inducedarrhythmias. The corresponding decreases in MAPwere 15, 36, and 28 mmHg, respectively, for the NE-induced arrhythmias and 16, 31, and 27 mmHg, re-spectively, for the pacing-induced arrhythmias. Themorphologies of the lead II ECG QRS complex weresimilar during corresponding NE- and pacing-inducedventricular arrhythmias.

Fig. 6. Epicardial activation sequences superimposed on an anatomically accurate 3D mathematical model of theventricles during normal sinus rhythm (A), NE-induced ventricular arrhythmia (B), and recovery (C). Earliestepicardial activation (o) shifted from the apical regions of the RV free wall during control conditions to the NEinfusion site (thick white arrow) near the apex of the LV free wall during the arrhythmia. Latest epicardialactivation (q) occurred adjacent to the posterobasal portions of the interventricular septum under controlconditions but moved to the posterobasal region of the LV free wall during the arrhythmia. At the onset of thearrhythmia, the QRS complex of the ECG inverted, and this was immediately followed by a dramatic decrease inthe ABP. All changes were fully reversed after the infusion was stopped. Thick black lines denote the LAD and PDAcoronary arteries.

Fig. 7. Total ventricular epicardial activation time (TVEAT; A) andMAP (B) during control, ventricular arrhythmia, and recovery statesfor group B experiments (n 5 14 animals, 29 observations). Duringthe NE-induced ventricular arrhythmia, TVEAT significantly in-creased and MAP significantly decreased compared with control,*P , 0.01. The epicardial activation sequence and hemodynamicsfully recovered after the NE infusion was stopped.


Echocardiographic studies. Pacing-induced arrhyth-mias resulted in estimated ejection fraction decreasesfrom 64, 52, and 50% (controls) to 20, 38, and 33%,respectively. Echocardiographic recordings were takenduring two NE-induced arrhythmias, for which theestimated ejection fractions decreased from 60 and55% (controls) to 30 and 30%, respectively. In all cases,mitral and aortic valve function remained normal.

Computer Modeling

At the beginning of the two-dimensional cellularnetwork model simulation, the stimulated wave of elec-trical excitation proceeded “normally” from left toright, as illustrated in Fig. 9A. Several cycles later, thecentral region of NE-affected cells spontaneously depo-larized before the normal wave of activation (Fig. 9B).A further seven cycles later, triggered automaticitywas fully established, and the activation sequence wascompletely dominated by the central zone, which actedas an abnormal pacemaker with a rate of ;2 Hz (Fig.9C). Spontaneous depolarization did not occur duringthe control simulation (not shown), for which calciumhandling was unchanged.

Alternative simulations with 1) just the L-typecalcium conductance increased and not the SR up-

take calcium conductance, 2) the L-type and SRuptake calcium conductances upregulated by a factorof two instead of five, and 3) the normal pacemakerreduced from 1 to 0.5 Hz also reproduced triggeredautomaticity, although the time to onset was greaterin all three cases (simulation results not shown). Thesize of the NE-affected zone in the network modelwas varied to determine whether this altered ar-rhythmogenesis. NE-affected zones smaller thanthat described above did not result in arrhythmia,whereas larger zones did during the same simulatedtime period. Moreover, when the overall size of thenetwork was increased (while the size of the centralzone was kept constant), characteristics of the sim-ulated arrhythmias remained unchanged, ruling outany mathematical edge effects on the required size ofthe NE-affected zone to elicit arrhythmia. To testwhether other effects of NE could produce triggeredautomaticity, an alternative model simulation thatincorporated modulation of the slow component ofthe delayed rectifier current and the sodium/potas-sium exchanger current instead of the calcium con-ductances was also performed (not shown). Thismodel did not result in arrhythmia over the samesimulated time period.

Fig. 8. Epicardial activation sequences during normalsinus rhythm (A) and ventricular arrhythmias inducedchemically by subepicardial NE infusion (B) and elec-trically using epicardial electrical stimulation (STIM;C). Activation maps are superimposed on a 3D mathe-matical model of ventricular anatomy. During the ven-tricular arrhythmias, the location of earliest epicardialactivation (o) shifted to the infusion/stimulation sites(thick white arrows), whereas latest epicardial activa-tion (q) remained in the posterobasal region. All eventswere fully reversible. Thick black lines represent theLAD and PDA coronary arteries.


Analysis of the ionic behavior for a single-model cellwith upregulated calcium conductances revealed a sig-nificant increase in the peak cytosolic calcium concen-tration and SR calcium release flux compared with thecontrol case, as illustrated in Fig. 10. This led to spon-taneous calcium release from the SR, which caused thesodium-calcium exchanger to admit sodium in an at-tempt to remove the excess calcium. The effect of thisinward sodium current was to depolarize the cell be-yond threshold. Spontaneous beating was sustained aslong as intracellular calcium remained high.

DISCUSSION

The new findings of this work are as follows. 1)Characterization of the epicardial electrical eventsduring a ventricular arrhythmia, induced by a unifocalinfusion of NE directly into the subepicardium, re-vealed activation patterns consistent with a region oftriggered automaticity near the infusion site, which

could be comprehensively mimicked by epicardial pac-ing. 2) Stimulation of the right cardiac sympathetic orleft or right cardiac vagal nerves abolished the NE-induced ventricular arrhythmia. 3) Two-dimensionalcomputational reconstruction of the NE-induced ar-rhythmia replicated triggered automaticity due to cal-cium overload, which caused the sodium-calcium ex-changer to act as an abnormal pacemaker.

Electromechanical Characterization of theNE-induced Ventricular Arrhythmia

We elicited a stable ventricular arrhythmia throughsubepicardial infusion of NE or by using epicardialelectrical stimulation and compared the observed epi-cardial activation sequence with that obtained duringcontrol conditions. The location of NE infusion wasrandomly chosen throughout the exposed ventricles.During all sustained NE-induced arrhythmias, thezone of earliest ventricular epicardial activation con-

Fig. 9. Simulation of the activation sequence using a 2D mathematical model of ventricular myocardium for whichthe central region had high-adrenergic tone. Cellular transmembrane ionic currents described by a recent model(15) were incorporated into an arrangement of 256 3 256 resistively coupled myocytes. The L-type calcium-channelconductance and the sarcoplasmic reticulum (SR) calcium-ATPase uptake conductance were elevated by a factorof 5 at the center of the tissue sheet (white vertical marker). A: the control spread of activation proceeded from leftto right, where red and indigo represent regions of depolarized and repolarized tissue, respectively. The centralcells were last to repolarize, because the plateau phase of their action potentials was extended because of theupregulated calcium currents. B: triggered automaticity became apparent several cycles later, when the centralregion spontaneously depolarized just before the arrival of the control activation sequence. C: a further 7 cycleslater, the central tissue was the dominant pacemaker and completely captured the activation sequence. The fullanimation may be viewed at the following internet address: http://www.physiol.ox.ac.uk/;djp/NEarrhythmia/2Dnetsim.mpg.


sistently matched the NE infusion site, and there wasa significant increase in the total ventricular epicardialactivation time compared with the control state. Thiswas associated with a drop in cardiac output and ABP.Epicardial electrical stimulation near NE infusionsites that had previously been used to elicit ventriculararrhythmias closely mimicked the electrical events andhemodynamic changes associated with the NE-inducedarrhythmias. Epicardial activation sequences andtimes were similar for the NE- vs. pacing-induced ar-rhythmias, as were the decreases in ABP, LVP, LVdP/dt, and LV ejection fraction, consistent with the notionthat the region of tissue adjacent to the infusion sitebecame the dominant pacemaker.

We chose a high concentration of NE (10 mmol/l insaline at 150 ml/min) because it elicited an arrhythmiawithin 1–5 min from the start of the infusion. Podzu-weit et al. (18, 19) used the same concentration of NEin normal Tyrode infused at 10–20 ml/min and estab-lished arrhythmias within 2 min. They also reportedarrhythmias with 1 mmol/l NE. We could not elicitreproducible arrhythmias using the reduced infusionrate, and this was most likely due to the absence ofcalcium in our infusate. To determine the effectiveregion of influence of NE on the ventricular myocar-dium, Podzuweit (18) measured levels of cAMP insmall ventricular tissue cores (;4-mm diameter and6-mm length) at the onset of the arrhythmias andfound that myocardial cAMP was ;45% greater in the4-mm core at the infusion site compared with the

adjacent control tissue cores. The localized inhomoge-neous nature of the NE infusion was critical in elicitingthis arrhythmia because similar concentrations ad-ministered intravenously did not elicit the sustainedventricular arrhythmia. Furthermore, reversal of thearrhythmia by intravenous NE administration (1mg zkg21 zmin21) is most likely due to the overdrivepacing effect of the sinoatrial node. This illustrates therequirement for localization of the effects of NE to theventricular myocardium to elicit the sustained ventric-ular arrhythmia. The subepicardial infusion of NE wasunlikely to have permanently damaged the affectedcells, because the arrhythmia was fully reversible andcould be elicited by using the same needle site onsuccessive occasions with an interim period of fullrecovery of the activation sequence and hemodynam-ics.

Modulation of the NE-inducedVentricular Arrhythmia

The arrhythmia was also reproduced by subepicar-dial infusion of the specific b-agonist isoproterenol butnot the a-agonist phenylephrine, whereas intravenousinfusion of propranolol abolished the ectopic activity,consistent with a role for modulation of cAMP andcalcium currents in arrhythmogenesis. Indeed, Podzu-weit (18) elicited similar ventricular arrhythmias inthe pig using intramyocardial infusion of a membrane-permeable analog of cAMP, inhibitors of cyclic nucleo-

Fig. 10. A recent computational model (15) was used toquantify the changes in ventricular transmembranepotential (Vm) and ionic behavior between the controlstate, for which the model cell was continuously pacedat 2 Hz with a twice-threshold stimulus for 2 ms (A),and under high-adrenergic tone (simulated by a 5-foldelevation of the L-type calcium-channel and SR calci-um-ATPase uptake conductances; B), while paced asabove for 50 s, after which time the stimulus wasswitched off and the cell exhibited automaticity. Up-regulating these conductances caused the peak cytoso-lic calcium concentration ([Ca]i) and SR calcium releaseflux (jrel) to increase markedly, compared with the con-trol state, whereas the potassium current (IK 5 IKr1 1IKr2 1 IKs, where IKr1 and IKr2 are the rapid componentsand IKs is the slow component) remained similar. Thisintracellular calcium overload caused the sodium-cal-cium exchanger current (INaCa) to reverse and depolar-ize the cell, thus acting as an abnormal pacemaker witha spontaneous rate of ;2.2 Hz.


tide phosphodiesterases, or NE. In that study, theNE-induced ectopic activity was abolished by the coin-fusion of choline esters or calcium antagonists, but notby tetrodotoxin. These observations strongly implicatea b-adrenergic, cAMP modulation of intracellular cal-cium that leads to spontaneous depolarization of theventricular myocardium. Catecholamines are known toenhance automaticity in Purkinje fibers (17), althoughthis is not a unique property of specialized conductingfibers, because automaticity can also occur in isolated,perfused ventricular tissue (7). Moreover, catecholaminescan induce afterdepolarizations in normal canine ven-tricular myocytes (21) and reentry in the ventricle (9).Therefore, we cannot exclude the possibility that spon-taneous discharge may have elicited a premature stim-ulus to initiate ventricular reentry, although our re-sults showed a regular cycle length and stationary siteof earliest epicardial activation during the NE-inducedarrhythmias.

The results of the present study show that stimula-tion of the right cardiac sympathetic nerves and intra-venous infusion of NE can abolish the adrenergic-induced ventricular arrhythmia. Both interventionsincreased HR and facilitated the reestablishment ofthe sinoatrial node as the dominant pacemaker. Left-and right-sided vagal stimulation also abolished thearrhythmia (see the ECG trace in Fig. 4); however,ABP did not fully recover because of the decrease in HRcaused by vagal stimulation itself. Vagal stimulationtook ;7 s to terminate the arrhythmia, which is morecharacteristic of responses involving the indirect mod-ulation of ion channels by intracellular messengersrather than the rapid activation of the acetylcholine-stimulated outward potassium current that can de-crease HR within one or two heartbeats (4). The indi-rect effect of cholinergic activation, which is known tocause a decrease in the L-type calcium current inventricular cells via the inhibition of adenylate cyclase-cAMP system (5) and via the nitric oxide-cGMPpathway (3, 25), is implicated in the antiarrhythmicproperty of vagal stimulation. Therefore, the antiar-rhythmic effects of sympathetic and vagal nerve stim-ulation may act via two different mechanisms: theformer directly restoring the natural site of pacemak-ing, and the latter involving a reduction in calciumcurrents and diastolic intracellular calcium in the ven-tricle.

Computational Reconstruction of theNE-induced Arrhythmia

Abnormal modulation of intracellular calcium isthought to be central to the NE-induced arrhythmia.First, the arrhythmia is abolished by b-adrenergicblockers (Ref. 18, see also RESULTS), calcium-channelantagonists, and choline esters (18). Second, it is initi-ated by permeant cAMP analogs (18). Therefore, weattempted to mimic aspects of this arrhythmia by al-tering calcium handling in a two-dimensional compu-tational network of excitable cardiac myocytes. Thesechanges led to intracellular calcium overload, which in

turn caused an abnormal influx of sodium through thesodium-calcium exchanger. This inward current depo-larized the model cells beyond threshold and acted asan abnormal pacemaker (8). This suggests a possiblecellular mechanism for the focal automaticity observedduring subepicardial NE infusion.

Study Limitations

The preparation used for the present study was anopen-chest, anesthetized pig. This altered the globalECG and could, therefore, have affected the magni-tudes of the recorded epicardial electropotentials com-pared with the closed-chest animal. However, we re-cently found that the pattern of epicardial activationwas not significantly altered after the chest had beenreclosed (unpublished observation).

Perhaps the biggest limitation of this study is thatthe activation sequence was deduced from just epicar-dial electropotential recordings. For this reason, wecannot identify which specific cell types are responsiblefor the abnormal pacemaking during the NE infusion.Indeed, intracellular iontophoresis of cAMP enhancesphase 4 depolarization and increases the rate of con-traction in spontaneously active Purkinje fibers (24). Inaddition, the pig heart is known to host Purkinje tissuethroughout the ventricular wall (23). Therefore, it ispossible that the NE infusion enhanced Purkinje cellautomaticity to elicit the arrhythmia. However, this isunlikely in the present preparation, because Purkinjefiber abnormal automaticity is unaffected by calcium-channel antagonism (8), whereas triggered automatic-ity is terminated by verapamil (8, 18, 27). Endocardial-to-epicardial ventricular cell heterogeneity may alsoplay a role in the genesis of this arrhythmia, althoughthis is difficult to determine because the electrophysi-ology of the various ventricular cell types in the pig isnot well characterized.

Whereas our computational reconstruction impli-cates abnormal calcium handling in the central zone ofthe cellular network model, it is possible that automa-ticity could have arisen from a time-dependent decay ofthe potassium current (17), which has been shown to bemodulated by catecholamines (2), coupled with a pro-gressive activation of the L-type calcium-channel cur-rent. However, model simulations that incorporatedmodulation of the slow component of delayed rectifiercurrent and the sodium/potassium exchanger currentinstead of the calcium conductances showed compara-tively smaller effects. Other limitations of the com-putational model that prevent a direct comparisonbetween the simulation results and in vivo measure-ments include species differences (guinea pig compu-tational model vs. in vivo pig), topography (2-dimen-sional cellular network model vs. 3-dimensional in vivoventricles), and effects of anisotropy (isotropic intercel-lular resistances vs. anisotropic in vivo conductances).Nevertheless, the computational model demonstratesthat triggered automaticity can be reproduced by con-sidering a small region of calcium-overloaded ventric-ular tissue.


The clinical correlates of this arrhythmia remain tobe determined, although pathological circumstancescan lead to high-sympathetic drive to a localized regionof myocardium (10, 11, 16). We conclude that the ven-tricular arrhythmia described in this study is a likelyconsequence of spatial inhomogeneities in the concen-tration of NE in the ventricular myocardium that leadto triggered automaticity and result in depressed he-modynamics. Interventions that reestablish the sino-atrial node as the dominant pacemaker or that reduceregional inhomogeneities in NE may prevent or termi-nate this type of arrhythmia.

We thank Chris Hirst and Vivienne Harris for technical assis-tance and Dr. A. Banning for performing the echocardiography.

This study was supported by British Heart Foundation Pro-gramme Grant BG 95003. Computational simulations were madepossible by support from the Oxford Supercomputing Centre ofOxford University and the Wellcome Trust, which provided access totwo Silicon Graphics high-performance parallel computers.

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Ventricular activation during sympathetic imbalance and its computational reconstruction