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Am J Physiol Heart Circ Physiol 279: H15-H25, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 1, H15-H25, July 2000

Modulation of arrhythmias by isoproterenol in a rabbit heart model of d-sotalol-induced long Q-T intervals

Joseph F. Spear and E. Neil Moore

Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sympathetic influences have been implicated in arrhythmias associated with both congenital and acquired long Q-T intervals. We recorded epicardial electrograms, a left ventricular endocardial monophasic action potential (MAP), and a bipolar electrocardiogram in 23 isolated rabbit hearts. Spontaneous focal arrhythmias appeared within 8-18 min following 92 µM d-sotalol in 15 of 23 hearts. The epicardial activation-recovery interval was shorter at baseline and increased to a significantly greater degree after d-sotalol administration in the hearts that developed focal activity. The standard deviation of the activation-recovery interval of the epicardial sites also increased. With the addition of 0.01 µM isoproterenol, the incidence of focal activity increased, and its mean cycle length was shortened by 7%. Also, myocardial recovery time in the epicardium was shortened to a greater degree than the endocardial MAP duration. It did not alter local epicardial heterogeneity of recovery but did increase the regional dispersion between epicardial recovery times, and the endocardial MAP duration. Therefore, beta -adrenergic stimulation in the presence of d-sotalol favors the appearance of arrhythmias by increasing the propensity for closely coupled focal activity and the temporal dispersion of recovery.

early afterdepolarization; myocardial refractoriness; triggered activity; reentry; beta -adrenergic receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CONGENITAL LONG-Q-T SYNDROME has a diverse genetic basis with several mutations of ion channel genes contributing to the phenotype (35). One form, the LQT2, is associated with a defect in the rapidly activating delayed rectifier potassium current (IKr) due to a chromosome-7-linked mutation in the human ether à go-go-related (HERG) gene, which encodes the channel protein (37). Because some antiarrhythmic agents that block potassium channels (class III) such as d-sotalol target the IKr channel (9-11, 24), this acquired alteration in channel function has been used to model the long-Q-T syndrome (8, 42, 45).

A similarity in both the congenital and acquired forms of the long-Q-T syndrome is that the condition predisposes to the potentially lethal arrhythmia torsade de pointes (4, 8, 22, 42, 45). This is a serious proarrhythmic side effect of class III antiarrhythmic therapy (21). Recent findings indicate that the characteristic shifting polarity of the R waves seen on the body-surface electrocardiogram (ECG) during the tachycardia can be due to both multifocal, early afterdepolarization (EAD)-induced triggered beats and triggered beats precipitating reentry (4, 13, 14, 35, 40).

It is recognized that sympathetic drive plays an important facilitative role in the appearance of arrhythmias in the acquired LQT2 form (8, 35, 43). Both alpha - and beta -receptors have been implicated (8, 35). Also, the lack of beta -adrenergic blocking potency by d-sotalol probably played a role in the increased mortality found in a recent clinical trial (43); however, the mechanistic details have yet to be worked out. Among its other effects, beta -adrenergic receptor simulation counters the prolongation of myocardial recovery due to IKr block with d-sotalol via a protein kinase A-mediated increase in IKs, the slowly activating delayed rectifier potassium current (38). It also increases ICaL, the L-type calcium current (30). The former would tend to favor reentry (20, 31, 34), and the latter favor EAD-induced triggered activity (23).

The present study was designed to evaluate those factors contributing to spontaneous arrhythmias in a d-sotalol-induced model of LQT2 and to investigate how beta -receptor stimulation with isoproterenol modifies the arrhythmias and their electrophysiological substrate.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Langendorff perfused rabbit heart. All experiments conformed to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals [DHHS publication (NIH) 86-23, Revised 1985] and were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. The general characteristics of our preparation have been previously described (39, 40).

Twenty-three adult New Zealand White rabbits of either sex (3.0-4.0 kg) were sedated with a combination of ketamine hydrochloride (40 mg/kg) and xylazine (4.4 mg/kg) administered intramuscularly. Sedated rabbits were heparinized (500 units iv) and euthanized with pentobarbital sodium (25 mg/kg iv). The heart was removed via a thoracotomy and rinsed in oxygenated Tyrode solution. The aorta was cannulated and the heart hung on a Langendorff perfusion apparatus in which the coronary arteries were perfused with oxygenated temperature-controlled Tyrode solution at a constant pressure of 80 mmHg. The constant pressure was provided by a column of perfusate of fixed height above the aortic cannula and was surrounded by a water jacket. The heart was surrounded by a temperature-controlled, water-jacketed 50-ml chamber with an overflow such that it was submerged in its effluent. Myocardial temperature was monitored with a thermistor probe placed within the right ventricle. After 15-20 min of constant pressure perfusion in which the coronary flow was verified to be at least 30 ml/min (range of 35-50 ml/min), the perfusion was switched to a constant flow of 40 ml/min for the duration of the experiment. In preliminary experiments, coronary flows of as low as 10 ml/min were able to maintain the electrophysiological parameters stable for the duration of our experiments. The Tyrode solution was recirculated from a 2-liter reservoir. Its millimolar composition was 137.0 NaCl, 24.0 NaHCO3, 5.5 dextrose, 2.7 KCl, 2.7 CaCl2, 0.9 NaH2PO4, and 0.5 MgCl2. The concentrations of calcium (2.7 mM) and potassium (2.7 mM) were chosen to facilitate EAD-induced triggered activity (12, 23). Experiments commenced after an equilibration period of ~30 min. Temperature changes were accomplished by switching the water-jacket supplies among three separate constant-temperature circulators.

d-Sotalol (a gift from Bristol-Myers Squibb) was dissolved in Tyrode solution and introduced directly into the perfusate reservoir to produce a concentration of 25 mg/l (92 µM), which permits maximal prolongation of recovery and triggered activity (29, 32, 45). Isoproterenol (Sigma Chemical) at a concentration of 0.01 µM was added in some experiments.

Electrophysiological methods. A bipolar ECG was monitored using two 1-cm-diameter gold-plated electrodes fixed to the wall of the heart chamber. The ECG was used to monitor the general condition of the heart. If S-T segment abnormality (indicating regional injury) was observed, the heart was not used. To slow the heart rate for pacing, atrioventricular block was induced by ablating the bundle of His through a right atriotomy using a portable battery-powered cautery. A flexible silicone rubber plaque of 27 bipolar (2.0-mm separation) 0.5-mm-diameter silver electrodes arranged in a 3 × 9 array with a 7.5-mm interelectrode spacing was wrapped around the heart. The left anterior descending coronary artery was used as a landmark to facilitate orienting the electrodes similarly for each heart. The three rows of nine bipolar electrodes covered both right and left ventricles at their bases, midregions, and apices. A monophasic action potential (MAP) recording catheter (16, 27, 40) was positioned on the left ventricular endocardium at its apex. The bipolar electrograms, ECG, and MAP were recorded in two groups of 16 simultaneous channels using a switching circuit.

The heart was paced by constant-current, 2.0-ms duration pulses delivered at twice diastolic threshold intensity through a pair of close bipolar electrodes embedded in the plaque and positioned over the epicardium of the posterior left ventricle.

The ECG, the epicardial electrograms, and the MAP were amplified using custom-designed amplifiers with a bandwidth of 0.1-1,000 Hz. The amplified electrograms were recorded on a strip-chart recorder at a paper speed of 100 mm/s. Local epicardial activation time was determined by measuring the time of the occurrence of the intrinsic deflection of the electrogram relative to the pacing artifact using a manual digitizer tablet (GTCO, Rockville, MD) interfaced with a computer (Hewlett-Packard, Sunnyvale, CA). The left ventricular endocardial MAP duration was measured from the upstroke to 95% repolarization. The resolution of the system was 0.1 ms. The intrinsic deflection of an electrogram was taken as the time of the most rapid deflection. The recovery time for each electrogram was taken as the time from the stimulus artifact to the most rapidly changing late part of the T wave. It has been shown that this provides a measure of local action potential repolarization time and can be used to track changes induced by interventions (18, 25). The activation-recovery interval was defined as the difference between the recovery time and activation time of an electrogram. This provides an index of local recovery that is independent of activation time. The standard deviation of the mean activation-recovery interval of all 27 electrograms was used as an index of the intrinsic heterogeneity of epicardial recovery time in the heart. A more regional measure of heterogeneity was determined by grouping electrograms into those recorded from the base, midregion, or apex of the ventricles.

Experimental protocols. Hearts were separated into two groups. Group I consisted of 19 hearts given d-sotalol. Group II hearts (n = 13) were given isoproterenol as well. Of the 13 group II hearts, 9 were also included in group I. After the equilibration period, baseline measurements were made: the ECG, epicardial electrograms, and MAP were recorded during pacing at basic cycle lengths of 600, 700, 1,000, and 2,000 ms (100-30 beats/min), and in some cases at perfusion temperatures of 25°C and 30°C as well as 36°C. The range of the pacing basic cycle lengths that we used was chosen to allow us to pace without interference from spontaneous automatic beats at the long cycle lengths or by being limited by refractoriness with the short cycle lengths.

After baseline measurements, the hearts were perfused with Tyrode solution at 36°C containing d-sotalol. In most hearts, within 8-18 min spontaneous closely coupled beats appeared following both paced and spontaneous beats occurring at long basic cycle lengths. At 20 min after d-sotalol was given, the heart was paced at a basic cycle length of 2,000 ms or greater, and ~40-60 sequential paced beats (test beats) were recorded together with any resultant ectopy. These test periods were used to quantify arrhythmia severity during a given experimental condition.

Data analysis. During baseline measurements and following d-sotalol or d-sotalol and isoproterenol administration at each temperature and pacing basic cycle length, the local activation times as indicated by the intrinsic deflections and times of recovery as indicated by the local T waves were digitized for each electrogram and tabulated. Because all activation and recovery data were obtained during pacing from the same relative left ventricular epicardial site, and because the activation sequence was not changed by our interventions, we were able to use changes in mean activation time as a direct index of changes in mean conduction velocity (40).

The epicardial electrograms were also used to generate activation sequence maps, which together with additional criteria allowed us to characterize the spontaneous rhythms. Our criteria have been previously described (40). Non-reentrant focal arrhythmias were characterized by the following criteria. The activation sequence maps showed a focal origin with a radial spread of activity from that region. The extra beat was closely coupled to the recovery time of the previous beat. (We distinguished spontaneous automatic ventricular beats as showing radial spread from their origin but occurring with long coupling intervals usually >2,000 ms.) The extra beats followed beats having long coupling intervals (>= 2,000 ms). Though not conclusive, these characteristics are consistent with the arrhythmias being due to triggered activity (4, 12, 13, 40).

We characterized reentrant arrhythmias using the following criteria. The activation sequence maps showed a circuitous activation sequence. The circuitous conducting beats followed after a locally blocked beat. The extra beats followed beats with short coupling intervals (<= 400 ms). These characteristics are consistent with the arrhythmias being due to reentry (4, 13, 40).

To quantify the incidence and coupling intervals of the d-sotalol-induced arrhythmias, we analyzed 40-60 sequential paced beats (test beats) that were delivered at cycle lengths of 2,000 ms or greater during a given experimental condition in each heart. Paced beats showing no close-coupled ectopy were noted, as well as the number and coupling intervals of close-coupled ectopic activity. Their mean cycle lengths and whether they fit the criteria for focal or reentrant beats were also noted. The ectopy incidence was defined as the number of episodes of ectopy divided by the number of test beats. An ectopy index was calculated to quantify the severity of the arrhythmias associated with a given experimental condition. The ectopy index was defined as the ectopy incidence multiplied by the fraction of hearts showing ectopy. The ectopy index ranges from 0.0 for no hearts showing ectopy to 1.0 for all hearts showing every test beat followed by ectopy.

Results are presented as means ± SD. Differences in measured variables between baseline and d-sotalol or d-sotalol plus isoproterenol administration were compared by a two-tailed Student's t-test for paired data. Differences among two or more nonpaired variables were compared using a single-factor ANOVA. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characteristics of spontaneous arrhythmias induced by d-sotalol. Spontaneous arrhythmias appeared in 15 of 23 hearts perfused with 25 mg/l d-sotalol. These arrhythmias, although their incidence was lower and they were less severe, were similar in characteristics to those we previously reported for quinidine (40). EADs were observed in the endocardial MAP recordings. The mean cycle length of 385 episodes of focal activity measured 20 min after d-sotalol at a temperature of 36°C was 373.2 ± 54.71 ms. The overall ectopy index of focal beats for d-sotalol was 0.62, and for quinidine it was 0.88 (40). The number of beats in individual episodes ranged from 1 to 20 sequential beats with 331 of the 385 episodes showing only 1 spontaneous closely coupled beat. In our experience with quinidine (40), 12% of the episodes of focal activity precipitated reentry in 10 of 12 hearts (ectopy index for reentry of 0.100). With d-sotalol, reentry occurred less frequently. Only 20 episodes of reentry were precipitated by the 331 episodes of focal activity (6%) in 5 of 23 hearts. The ectopy index for reentry was only 0.015.

Figure 1A presents epicardial electrograms (1-27) and a left ventricular endocardial MAP recorded after the addition of d-sotalol. The left group of recordings shows a spontaneous automatic beat not followed by a triggered beat. However, the MAP recording exhibited a prominent EAD (indicated by the asterisk). The right group of recordings in Fig. 1A shows a stimulated beat that was followed by a closely coupled triggered beat (indicated by the arrows). In this case, an intrinsic deflection can be seen interrupting the EAD in the MAP recording.


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  		Fig. 1.   An example of d-sotalol-induced early afterdepolarizations (EADs) and triggered activity. A: selected records obtained from 27 epicardial sites together with a left ventricular (LV) endocardial monophasic action potential (MAP) during a spontaneous automatic beat (left) exhibiting an EAD (*) and during a stimulated beat (S in right) which was followed by a spontaneous triggered beat (arrows). Dissociated atrial activity (a) can be seen in some of the records that were located near the atria at the upper margin of the electrode array (sites 3, 6, 9, 12, and 24). B: activation sequence maps for the paced beat (top) and the spontaneous triggered beat (bottom). The numbers are activation times in milliseconds, referenced to the pacing stimulus artifact (S in A). The recording sites correspond to the 27 electrograms in A and are in sequence in columns from 1 at the bottom right of the posterior right ventricle (Post RV) to 27 at the upper left of the anterior right ventricle (Ant RV). LAD, left anterior descending coronary artery.

Figure 1B shows epicardial activation sequence maps of the stimulated beat and the resulting spontaneous triggered beat. All activation times shown in the maps are in milliseconds relative to the stimulus artifact of the stimulated beat. For the stimulated beat (Fig. 1B, top map) the activation sequence exhibited uniform and rapid conduction radiating from the pacing site on the posterior left ventricle. In contrast, the epicardial activation sequence for the triggered beat (Fig. 1B, bottom map), which was closely coupled to the stimulated beat, showed slow conduction (indicated by the closely spaced isochrones) emanating from the earliest epicardial site on the posterior left ventricular apex.

Figure 2 shows an example of closely coupled triggered activity leading to reentry in a heart that repeated multiple similar episodes. In Fig. 2A, after a pause a spontaneous automatic beat (the first beat in the sequence) precipitated a close-coupled focal beat (second beat), which in turn precipitated five reentrant beats.


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  		Fig. 2.   An example of closely coupled focal activity precipitating reentry after the addition of d-sotalol. A: selected epicardial records and a bipolar electrocardiogram (ECG) obtained during a spontaneous tachycardia of seven beats. Numbers at left indicate the map sites from which the epicardial electrograms were recorded. Dissociated atrial activity (a) can be seen in some of the records obtained from the ventricular base (sites 6, 12, and 24). B: activation sequence maps for the first three beats. Recording sites are numbered as in Fig. 1. The sites corresponding to the electrograms in A are therefore from alternate columns. Isochrones were drawn at 10 ms for beat 1, 20 ms for beat 2, and 50 ms for beat 3.

Figure 2B shows the activation sequence maps for the automatic beat (beat 1), the closely coupled triggered beat (beat 2), and the first reentrant beat (beat 3). All activation times shown in the maps are in milliseconds relative to the earliest epicardial site for the automatic beat. The earliest epicardial site for the automatic beat was in the posterior right ventricular apex, and it rapidly spread to both ventricles. The left ventricular base near the left anterior descending coronary artery was the last site activated. The closely coupled triggered beat first activated the posterior left ventricular apex at 265 ms and spread slowly, exhibiting multiple sites of block in the left ventricle (dashes in the map). Reentry occurred in a figure-eight form near a site of block of the previous beat and spread from the left ventricular base to the apex. It then radiated both anteriorly and posteriorly over the right and left ventricles and returned back toward the base of the heart. Although the ECG in Fig. 2A exhibited multiple morphologies during the transition from focal activity to reentry, it did not show the modulated twisting of points that is typical of torsade de pointes. A more typical electrocardiographic pattern has been demonstrated by others using a similar model during a wandering vortex form of reentry (4).

Differences in the characteristics of hearts exhibiting ectopy with d-sotalol. We compared the electrophysiological characteristics for the 15 hearts with ectopy and 8 hearts without ectopy 20 min following d-sotalol at 36°C during constant ventricular pacing at a basic cycle length of 700 ms (86 beats/min). d-Sotalol had no effect on epicardial conduction velocity, as indicated by no change in the mean activation times. At baseline, the mean MAP duration was significantly longer than the epicardial mean activation-recovery interval (250.0 ms vs. 223.4 ms, P = 0.003). Both the mean activation-recovery interval and the MAP duration were significantly (P < 0.001) prolonged by d-sotalol by approximately the same proportionate amount. The hearts with ectopy tended to have somewhat shorter activation-recovery intervals and MAP durations at baseline, although these did not reach significance (P = 0.124 and P = 0.074, respectively). However, in the hearts showing ectopy, both parameters were prolonged to a greater degree by d-sotalol than they were in those without ectopy. This was significantly so for the mean activation-recovery interval (P = 0.048). The mean standard deviation of the activation-recovery interval was also increased significantly in the ectopy group (P = 0.001).

Figure 3 shows data from a subset of 19 hearts in which the parameters were measured over the full range of pacing rates. The mean activation time (Fig. 3A) was not significantly different at any heart rate, at baseline, or after d-sotalol administration in either the ectopy or no-ectopy groups. In addition, Fig. 3 shows that d-sotalol's greater prolongation of the mean activation-recovery interval (Fig. 3B) and the MAP duration (Fig. 3D) in the hearts developing ectopy was amplified at faster heart rates. Interestingly, this produced a greater prolongation of recovery at the faster heart rates, which is contrary to the expected reverse-rate dependence. Reverse-rate dependence has been suggested to be due to incomplete deactivation and accumulation of IKs at the faster rates, thus causing earlier repolarization (24). Our shortest pacing cycle length of 600 ms in these studies may not have been sufficiently short for this to occur. The standard deviation of the activation-recovery interval (Fig. 3C) increased with d-sotalol to a significantly greater degree in the hearts with ectopy at the faster heart rates.


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  		Fig. 3.   Effects of pacing heart rate (abscissas) on the mean epicardial activation time (A), the mean epicardial activation-recovery interval (B), the mean standard deviation of the activation-recovery interval (C), and the mean left ventricular endocardial MAP duration (D) during baseline (left) and 20 min after administration of 25 mg/l d-sotalol (right). Data are separated into those hearts having spontaneous ectopy and those not having ectopy. Vertical bars are means ± SD. *Significant difference by nonpaired Student's t-test between ectopy and no ectopy. n, Number of hearts.

Facilitation of arrhythmias by isoproterenol. In preliminary experiments, we determined that 0.01 µM isoproterenol alone did not precipitate spontaneous ventricular arrhythmias. This dose of isoproterenol shortened the atrial spontaneous cycle length in our atrioventricular-blocked preparation by an average of 33%. Of the 13 group II hearts, 8 exhibited ectopy following d-sotalol. The addition of 0.01 µM isoproterenol precipitated the appearance of ectopy in one of the five hearts that did not show it with d-sotalol alone. We followed seven of the hearts with d-sotalol-induced arrhythmias over a period of 70 min before adding isoproterenol. The arrhythmia severity with d-sotalol alone tended to decline with time. However, isoproterenol added to the perfusate reversed this effect. Figure 4 shows pooled data obtained from the seven hearts over a period of 70 min after administration of d-sotalol and after the addition of isoproterenol at three temperatures. The mean activation-recovery interval (Fig. 4A) and mean MAP duration (Fig. 4B) obtained during ventricular pacing at a basic cycle length of 700 ms are plotted together with the characteristics of the spontaneous focal arrhythmias. The relative arrhythmia severity is indicated in Fig. 4C by the ectopy index (fraction of hearts with arrhythmias times the arrhythmia incidence per heart) and in Fig. 4D by the mean number of extra beats in the episodes of ectopy. The sequence of the interventions in the protocol is shown in the bars below the graphs. There were no arrhythmias at baseline. Arrhythmias first appeared at an average of 12.4 min after d-sotalol adminstration (range of 8-18 min for all seven hearts). Peak severity occurred at around 20 min and decreased thereafter. After 31-50 min, six of seven hearts still showed arrhythmias; after 51-70 min, only five of seven hearts continued to show arrhythmias with a reduced incidence per heart. This occurred despite the fact that the mean activation-recovery interval (Fig. 4A) and the MAP duration (Fig. 4B) remained prolonged over this period. Within 2-3 min following the addition of 0.01 µM isoproterenol, the arrhythmias returned in all seven hearts. This dose of isoproterenol reduced the mean activation-recovery interval and mean MAP duration from 354.5 ms to 254.7 ms (P = 0.005) and from 390.0 ms to 320.0 ms (P = 0.033), respectively. As occurred in our previous study with quinidine-induced arrhythmias (40), lowering the perfusion temperature suppressed the closely coupled focal activity. At 25°C, even in the presence of isoproterenol, only one of seven hearts exhibited ectopy with an incidence of 0.58 (ectopy index = 0.08). Ectopy returned when the temperature was returned to 36°C.


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  		Fig. 4.   Pooled data obtained from seven hearts during baseline (BASE) over a 70-min period after administration of 25 mg/l d-sotalol and after the addition of 0.01 µM isoproterenol at temperatures of 36, 30, 25, and 36°C. Data are plotted on a common abscissa. Interventions used in the protocol are shown at the bottom and are separated by vertical dashed lines. Columns represent the mean of data from seven hearts. Vertical bars indicate the standard deviation. Mean epicardial activation-recovery interval (A) and mean endocardial MAP duration (B) are plotted together with the characteristics of the focal arrhythmias. The relative arrhythmia severity is indicated (C) by the ectopy index (fraction of hearts with arrhythmias times the arrhythmia incidence per heart) and the mean number of extra beats in the episodes of ectopy (D). In C, the ratio of the number of hearts with ectopy to the total is shown at the top of each column.

The electrophysiological characteristics of the group II hearts during ventricular pacing at a basic cycle length of 700 ms are presented in Table 1. The one heart that did not show ectopy with d-sotalol but did after isoproterenol is included in the no-ectopy group. In the hearts with ectopy, isoproterenol increased the conduction velocity as indicated by a significant 12% decrease in the mean activation time. Isoproterenol also significantly reduced the mean activation-recovery interval by 20% and the MAP duration by 12%. The greater percentage reduction of the activation-recovery interval versus the MAP duration was significant (P = 0.031). Thus although isoproterenol was facilitative of ectopy, it significantly reduced myocardial recovery times. The mean cycle length of the ectopy in the group II hearts was 368.0 ± 56.2 ms with d-sotalol alone and was decreased by 7% to 341.8 ± 81.8 ms with the addition of isoproterenol (P < 0.001). Thus the shortening of the mean cycle length of the ectopy with isoproterenol correlated better with the reduction in mean MAP duration than the reduction in mean epicardial activation-recovery interval. The mean standard deviation of the activation-recovery interval was not influenced by isoproterenol.

                              
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  		Table 1.   Electrophysiological parameters in hearts with ectopy or no ectopy after addition of isoproterenol

Effects of d-sotalol and isoproterenol on the heterogeneity of regional recovery. The larger decrease in the epicardial mean activation-recovery interval relative to the left apical endocardial MAP duration induced by isoproterenol (Table 1) was not due to regional differences in recovery between apical and basal regions of the ventricles. We did not find any regional differences among epicardial mean activation-recovery intervals in recordings from the base, midregion, and apex of the ventricles during baseline, in the presence of d-sotalol, or with the addition of isoproterenol. However, others have reported regional differences in isolated perfused guinea pig hearts (26).

Although d-sotalol prolonged recovery proportionately in the epicardial regions and in the endocardial MAP, the recovery times in the epicardial regions were shortened to a greater degree by isoproterenol than in the endocardial MAP region. This is shown in Fig. 5. The group II data are separated into those with ectopy (eight hearts) and those with no ectopy (five hearts). In the positive range of the y-axis, the effects of d-sotalol on the regional mean recovery times are presented as percent changes from baseline values. In the negative range, the effect on these parameters of adding isoproterenol is presented as the percent change from d-sotalol values. For the ectopy hearts, the percent change from baseline induced by d-sotalol was similar for all regions. However, the epicardial regions shortened to a greater degree than the endocardial MAP when isoproterenol was added (P = 0.047 by ANOVA). Thus isoproterenol increased the dispersion of recovery between endocardium and epicardium. For the no-ectopy hearts, the responses to d-sotalol and isoproterenol were of a smaller magnitude. For the epicardial sites after the addition of isoproterenol, this difference was significant.


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  		Fig. 5.   Relative effects of 25 mg/l d-sotalol and d-sotalol with 0.01 µM isoproterenol on regional recovery during ventricular pacing at a basic cycle length of 700 ms. Data are from eight hearts with ectopy (A) and five with no ectopy (B). The four columns are pooled data obtained from the apex, midregion, or base of the ventricles, and the endocardial MAP. Vertical bars are means ± SD. In the positive range of the ordinate, the effect of d-sotalol on the regional mean activation-recovery intervals is presented as percent change from baseline values. In the negative range, the effect of adding isoproterenol on these parameters is presented as the percent change from d-sotalol values. P value is by ANOVA. *Significant difference between ectopy and no ectopy by nonpaired Student's t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of our study are the following. Hearts exhibiting d-sotalol-induced ectopy had shorter ventricular recovery times at baseline before the addition of d-sotalol. After the addition of d-sotalol, the ventricular recovery times increased to a greater degree in the hearts developing ectopy. This suggests a difference in the density and/or regulation of ion channels targeted by d-sotalol.

d-Sotalol increased the local heterogeneity of epicardial recovery (standard deviation of the activation-recovery intervals at the 27 epicardial sites) but not the regional recovery among the apex, midregion, and base region of the epicardium and left endocardial apex. The addition of 0.01 µM isoproterenol facilitated the appearance and shortened the mean cycle length of the ectopic activity. It also shortened the regional epicardial recovery times to a greater degree than endocardial MAP duration, yet did not decrease the standard deviation of the local epicardial mean activation-recovery intervals. Thus isoproterenol increased the overall heterogeneity of myocardial recovery.

Differences in repolarization between hearts with and without d-sotalol-induced arrhythmias. d-Sotalol at a concentration of 25 mg/l (92 µM) induced spontaneous bradycardia-dependent arrhythmias in 15 of 23 perfused hearts (65%). That there were differences in recovery times at baseline between hearts susceptible to and hearts not susceptible to d-sotalol-induced arrhythmias (Fig. 3) implies a difference in the ion channels carrying outward current mediating repolarization in the two populations. d-Sotalol is a relatively selective blocker of IKr, the rapid component of the delayed rectifier potassium current (9), but is also reported to block ITO, the transient outward current (5), which is prominent in rabbit ventricular epicardium (15). The greater prolongation of repolarization by d-sotalol in susceptible hearts suggests that the channels that were prominent in causing earlier repolarization at baseline were also responsible for the greater prolongation when blocked by d-sotalol.

beta -Receptor stimulation and reentry. The available evidence indicates that experimentally induced torsade de pointes involves both triggered beats and reentrant activity (4, 13, 14, 35, 40). Previous work has shown that d-sotalol both induces spontaneous close-coupled triggered activity (4, 8), a trigger for reentry, and increases local heterogeneity of myocardial recovery (45, 42), the substrate for reentry. Our findings are consistent with these studies (Figs. 1-4). In addition, we have shown that isoproterenol has electrophysiological effects that make reentry more likely by exacerbating both its trigger and substrate. With isoproterenol, the incidence of closely coupled focal activity increased (Fig. 4), and its mean cycle length was shortened by 7% from 368.0 to 341.8 ms. Isoproterenol shortened myocardial repolarization (Table 1). It not only did not attenuate the increased local epicardial heterogeneity (standard deviation of the activation-recovery interval) induced by d-sotalol (Table 1), but it also increased the regional temporal dispersion between epicardial apex, midregion, and base, and the endocardial MAP duration (Fig. 5). The decrease in mean activation time (increase in conduction velocity) that we observed with isoproterenol (Table 1) could antagonize reentry by increasing its wavelength, thus decreasing an excitable gap in the circuit, or by eliminating unidirectional block, depending on the circumstances (36). Thus our findings indicate that isoproterenol in the presence of d-sotalol increases the propensity for reentry by increasing the incidence of spontaneous closely coupled beats, shortening their cycle length, shortening the overall myocardial recovery time, and increasing the heterogeneity of recovery (20, 31, 34).

beta -Receptor stimulation and triggered activity. The majority of the arrhythmias in our model were due to focally arising closely coupled activity, which occasionally also caused reentry following local block of the focal beat (Figs. 1 and 2). There is strong evidence obtained clinically and in animal models that triggered activity is due to EADs (1, 6, 8, 12, 14, 19, 23, 40, 45). Increased intracellular calcium due to increased transmembrane calcium flux appears to be the common denominator in the generation of EADs, although multiple precipitating mechanisms probably contribute (6, 19, 23, 32). Increased intracellular calcium can occur by increasing current through the ICaL channel or via slowing or reversing the sodium/calcium exchanger (6, 32). Prolonging action potential duration by blocking IK or ITO increases intracellular calcium via the ICaL window current (23).

Most often EADs are bradycardia dependent. Presumably this is because slow rates further prolong action potential duration, although short coupling intervals have also been reported to facilitate their appearance (6, 8).

Prolongation of myocardial recovery time alone was not sufficient for inducing closely coupled focal activity in our studies. Low temperature, which greatly prolonged myocardial recovery time, suppressed the focal activity (Fig. 4). The mechanism for low-temperature suppression of the focal activity in our studies is not entirely clear. However, studies on isolated ventricular tissues have shown that EADs that precipitate triggered activity are suppressed by low temperature (7).

Isoproterenol that shortened myocardial recovery time facilitated closely coupled focal activity (Fig. 4). This can be explained by the fact that isoproterenol increases IKs (38) and a cAMP-sensitive chloride current (33) that favors shortening of recovery time. Isoproterenol increases ICaL, which though it would tend to increase recovery time, also increases intracellular calcium (30). The net effect of isoproterenol in the presence of d-sotalol is to shorten recovery time but increase intracellular calcium flow through the L-type calcium channels. The reactivation of inward calcium current during the action potential plateau and/or the increase in intracellular calcium contribute to EAD formation (6, 32, 41).

Both the endocardial Purkinje system and M cells, which have long action potential durations and are found in the midmyocardium, have been implicated as sites for the origin of EADs (1, 2, 12, 13). The usual site of origin of triggered beats in the intact heart remains somewhat controversial. Recent studies suggest that they probably arise from the endocardium (3, 13). We did not have intramural ventricular recordings, and we used only one endocardial MAP recording site; therefore, we could not determine definitively whether the focal beats originated in the midmyocardium or endocardium.

A transmural gradient in the expression of IK channels is reported to contribute to the transmural gradient in action potential duration seen in feline and canine ventricular muscle (17, 28). Studies in perfused canine ventricular wedges indicate that in the absence of abnormal cellular electrical coupling, the electrotonic influences in the functional syncytium of the myocardium tend to blur the transmural gradient in action potential duration seen prominently in isolated tissues and myocytes. In addition, the selective prolongation of M cell repolarization by d-sotalol is attenuated (2, 44). In our study, d-sotalol prolonged epicardial and endocardial recovery times proportionately by the same amount (Fig. 5). However, our finding that isoproterenol shortened the recovery time in the endocardial sites to a lesser degree than the epicardial sites is consistent with a transmural variation in the density of IKs channels.

Limitations. In our studies we utilized 27 bipolar epicardial recording sites. The resolution was far less than could have been provided using high-density optical mapping techniques. The disadvantage of optical mapping for our studies was that, although it provides high recording resolution, it also provides a view of only one surface of the total right and left ventricular area. Because we needed to distinguish focally originating activity from reentry following a blocked beat, a global ventricular recording array was necessary. The 27-site resolution was sufficient to allow us to distinguish focal beats from reentry (40) (Figs. 1 and 2).

Interpretation of EADs in MAP recordings requires caution. Artifacts such as baseline instability, catheter motion, and offset saturation of signals can confound the recording. Although reliable signals as we presented in Fig. 1 are usually obtainable, there were cases where EADs could not be reliably confirmed.

We used close bipolar epicardial recordings to measure activation-recovery intervals, because we found that "far-field" effects from activity at distant regions of the heart were confounding our electrogram recordings in the unipolar configuration. However, the measurement of activation-recovery intervals using bipolar recordings has limitations in that the averaging effect on the local activity tends to underestimate the degree of transmural heterogeneity. Activation-recovery intervals using unipolar electrograms have been shown to better approximate recovery times under conditions of heterogeneity of recovery (13).

Although the small rabbit heart is ideal for studying focal arrhythmias, establishing reentrant arrhythmias in a normal rabbit heart is problematic. Because the activation and repolarization characteristics are similar to those seen in larger mammalian hearts, the reentrant circuits tend to require a large portion of the total ventricular area (Fig. 2) and are therefore more difficult to establish. We did not observe reentry under baseline conditions, and the incidence of spontaneous reentry with d-sotalol in our preparation was low. Also, we could detect no change following the addition of isoproterenol. Because we did not attempt to provide a baseline substrate facilitative for reentry or directly evaluate the susceptibility of the hearts to reentry by provocative measures, we were unable to quantify the impact of the changes in myocardial conduction and recovery on the vulnerability to reentry in our preparation. However, in larger hearts, and in settings where there are preexisting conditions, these changes would be expected to increase vulnerability to reentry.


    ACKNOWLEDGEMENTS

The authors thank Kenneth J. Fitzgerald for expert technical assistance.


    FOOTNOTES

This work was supported by a grant from the American Heart Association.

Address for reprint requests and other correspondence: J. F. Spear, Suite 201E, Department of Animal biology, School of Veterinary Medicine, 3800 Spruce St., Philadelphia, PA 19104-6046 (E-mail: spearj{at}vet.upenn.edu).

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

Received 9 March 1999; accepted in final form 13 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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