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European Journal of Pharmacology 580 (2008) 241 – 249


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Effects of dual endothelin receptor blockade on sympathetic activation and


arrhythmogenesis during acute myocardial infarction in rats
Theofilos M. Kolettis a,⁎, Giannis G. Baltogiannis a,1 , Dimitrios G. Tsalikakis b,2 ,
Alexandros T. Tzallas b , Maria G. Agelaki a,1 , Andreas Fotopoulos c ,
Dimitrios I. Fotiadis b , Zenon S. Kyriakides d
a
Departments of Cardiology, University of Ioannina, 1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece
b
Departments of Computer Sciences, University of Ioannina, 1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece
c
Departments of Nuclear Medicine, University of Ioannina, 1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece
d
Red Cross Hospital, 1 Erythrou Stavrou Street, 115 26 Athens, Greece

Received 28 June 2007; received in revised form 11 October 2007; accepted 3 November 2007
Available online 13 November 2007

Abstract

The effects of dual (ETA and ETB) endothelin receptor blockade on ventricular arrhythmogenesis during acute myocardial infarction are not
well defined. We randomly allocated Wistar rats to bosentan (100 mg/kg daily, n = 24), a dual endothelin receptor antagonist, or vehicle (n = 23).
After 7 days of treatment, myocardial infarction was induced by permanent coronary ligation. Ventricular tachyarrhythmias were evaluated for
24 h following ligation, using a miniature telemetry electrocardiogram recorder. Action potential duration was measured from monophasic
epicardial recordings and sympathetic activation was assessed by heart rate variability and catecholamine serum level measurements. Compared to
controls (1012 ± 185 s), bosentan (59 ± 24 s) markedly decreased (P b 0.00001) the total duration of ventricular tachyarrhythmias during the
delayed (1–24 h) phase post-ligation, with a modest effect during the early (0–1 h) phase (132 ± 38 s, versus 43 ± 18 s, respectively, P = 0.053).
Treatment did not affect infarct size or total mortality. Action potential duration at 90% repolarization prolonged in controls (from 93.1 ± 4.7 ms to
117.6 ± 6.9 ms), displaying increased temporal dispersion (from 4.14 ± 0.45 ms to 10.42 ± 2.51 ms, both P b 0.001), but was preserved in treated
animals. Bosentan decreased norepinephrine, but increased epinephrine levels 24 h post-ligation. Low frequency spectra of heart rate variability,
an index of net sympathetic tone, were lower in bosentan-treated rats. Dual endothelin-1 receptor blockade decreases ventricular tachyarrhythmias
during myocardial infarction without reperfusion, by preventing repolarization inhomogeneity. Diverse treatment effects on sympathetic activation
may ameliorate the antiarrhythmic action.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Autonomic nervous system; Catecholamines; Endothelin-1; Myocardial infarction; Ventricular tachyarrhythmias

1. Introduction proportion of acute mortality, with rates remaining stable over


the past years (Zheng et al., 2002). Several mechanisms are
Myocardial infarction remains a leading cause of death operative in the genesis of ischemic ventricular tachyarrhyth-
worldwide. Ventricular tachyarrhythmias, namely ventricular mias, including the accumulation of neurohumoral substances
tachycardia and ventricular fibrillation, account for a substantial in the myocardium (Clements-Jewery et al., 2005).
Endothelin-1 rises markedly during acute myocardial infarc-
tion (Loennechen et al., 2001; Stewart et al., 1991) and the
magnitude of this rise correlates with prognosis (Yasuda et al.,
⁎ Corresponding author. Tel.: +30 265 1097227; fax: +30 265 1097053. 1990). Endothelin-1 plays a role in arrhythmogenesis, as indi-
E-mail address: thkolet@cc.uoi.gr (T.M. Kolettis). cated by a decrease in the early incidence of post-infarction
1
Supported, in part, by a grant from the Cardiovascular Research Institute,
Ioannina and Athens, Greece. ventricular tachyarrhythmias after endothelin receptor blockade
2
Supported, in part, by a grant from the national General Council for Research (Raschack et al., 1998; Baltogiannis et al., 2005). The effects of
and Technology, Athens, Greece. endothelin-1 are mediated via activation of two (ETA and ETB)
0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2007.11.002
242 T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249

different G-protein coupled receptors (Molenaar et al., 1993), Declaration of Helsinki and with the Guide for the Care and Use
but the role of ETB receptors on arrhythmogenesis during acute of Laboratory Animals as adopted and promulgated by the United
myocardial infarction remains controversial. Antiarrhythmic States National Institutes of Health (Publication No. 85-23,
actions of dual receptor blockade were found in some studies revised 1996). The study protocol was approved by the local state
(Douglas et al., 1998; Geshi et al., 1999), but not in others authority.
(Richard et al., 1994). Moreover, the selective endothelin ETB
receptor agonist sarafotoxin was reported to decrease ischemic 2.2. Drug administration
ventricular tachyarrhythmias in rats (Crockett et al., 2000), but
in isolated rabbit hearts ETB receptor stimulation had no de- Bosentan was a kind gift from Actelion Pharmaceuticals Ltd.,
tectable electrophysiological effects either during normal, or Allschwil, Switzerland. Fresh preparations were made every day
during ischemic conditions (McCabe et al., 2005). as a micro-suspension in 5% arabic gum, containing bosentan
Given the important pathophysiologic role of endothelin-1 in (100 mg/kg body weight) and were administered by gavage once
ischemic heart disease, there is a growing pharmacological daily for 7 days. This dosage has been shown to produce
research on the effects of endothelin receptor blockade in this significant and sustained pharmacologic effects (Fraccarollo
setting. To this end, both selective and dual endothelin receptor et al., 1997; Mulder et al., 1997). The control group received the
blockade have been advocated. Although most of the detri- same quantity of vehicle for the same time period.
mental effects of endothelin-1 appear to be mediated by the
ETA receptor, selective antagonists increase endothelin-1 levels 2.3. Implantation of telemetry transmitter
and may adversely affect left ventricular function and pulmo-
nary vascular remodeling (Hu et al., 1998; Nguyen et al., 1998; Six days after randomization, a continuous electrocardio-
Fraccarollo et al., 2002). In contrast, dual endothelin receptor gram telemetry transmitter (Dataquest, Data Sciences Interna-
blockade was reported to ameliorate ventricular dilatation and tional, Transoma Medical, Arden Hills, Minnesota, USA) was
dysfunction and to decrease endothelin-1 expression (Clozel implanted in the abdominal cavity, after slight modification of
et al., 2002; Fraccarollo et al., 1997; Oie et al., 1998). Moreover, the previously described technique (Opitz et al., 1995). Under
in contrast to selective ETA receptor blockade, dual endothelin brief ether anesthesia, the animals were intubated, mechanically
receptor blockade has been shown to decrease mortality in the ventilated (ventilator model 7025, Ugo Basile, Comerio, Italy)
chronic phase of experimental myocardial infarction in rats and anesthetized with 2% isoflurane. The use of this agent
(Mulder et al., 1997). Therefore, the purpose of the present allows the rats to regain consciousness rapidly (within 2–3 min)
study was to examine the effects of bosentan, a dual endothelin after cessation of anesthesia. All animals were housed in cages,
receptor peptide antagonist, (a) on the infarct size and (b) on placed on a receiver continuously capturing the electrocardio-
ventricular tachyarrhythmias. All previously available data on gram signal, independent of animal activity.
the effects of endothelin-1 receptor blockade on ventricular
tachyarrhythmias are limited to the 60–120 min following 2.4. Monophasic action potential recordings
myocardial infarction generation. An important novelty of the
present work is the extension of the observation time window; A monophasic action potential probe (model 200, EP
in this study, we examined the incidence of ventricular tachyar- Technologies, Sunnyvale, California, USA) was placed on the
rhythmias, occurring during a 24-h observation period after epicardium, exerting mild, constant pressure, as previously
permanent coronary occlusion in the conscious rat. Moreover, described (Franz, 1999). The signal was amplified (preamplifier
the effects of endothelin-1 receptor blockade on catecholamine model 300, EP Technologies, Sunnyvale, California, USA),
release are unclear. To provide further insight into possible filtered at 50 Hz with a digital notch filter and for ranges
pathophysiologic mechanisms, we examined the effects of b0.05 Hz and N 500 Hz with a band pass filter. The recordings
bosentan on the monophasic action potential and on indices of were stored in a personal computer, equipped with an analog-to-
sympathetic activation. digital converter (BNC 2110, National Instruments Corporation,
Dallas, Texas, USA). Monophasic action potential signals were
2. Materials and methods recorded from the lateral left and right ventricular walls imme-
diately prior to and 24 h after myocardial infarction generation.
2.1. Experimental design
2.5. Generation of myocardial infarction
The study was conducted in 56 Wistar rats of similar age and
weight (20 ± 1 weeks old, 200–250 g, respectively). The rats Seven days after randomization, myocardial infarction was
were randomized in 1:1 fashion to receive either bosentan or generated as previously described (Pfeffer et al., 1979), by an
vehicle. This sample size enables an approximately 90% power operator blinded to treatment assignment. The left coronary
to detect a 50% decrease in ventricular tachyarrhythmias. artery was ligated using a 6–0 suture, placed between the
The animals were housed in individual cages, under optimal pulmonary artery cone and the left atrial appendage. A six-lead
laboratory conditions (controlled temperature, humidity and light/ electrocardiogram was obtained and ST-segment elevation was
dark cycles) and were given water and standard rat chow ad considered proof of induced MI. In a further group of 6 animals,
libitum. The research was conducted in accordance with the a sham operation was performed, by encircling but without
T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249 243

ligating the left coronary artery. After the procedure, electro- ment. The significance of premature ventricular contractions,
cardiogram recording was continued for 24 h or until spon- couplets and triplets is debated (Opitz et al., 1995), hence
taneous death. No resuscitation attempts were allowed at any such count was omitted. Ventricular tachycardia and ventricular
time during the study. fibrillation episodes were recorded and the duration of each
episode was measured using the time-scale provided by the
2.6. Catecholamine level measurements software. According to the guidelines provided by the Lambeth
Conventions for determination of experimental arrhythmias,
Twenty-four h post-ligation, the animals were re-anesthetized ventricular tachycardia was defined as 4 or more consecutive
and the internal jugular vein was exposed. Blood was collected premature ventricular contractions and ventricular fibrillation as
by venous puncture, centrifuged and the serum was stored at a signal with indistinguishable QRS deflections (Walker et al.,
− 20 °C. Levels of epinephrine and norepinephrine were 1988). Despite these guidelines, separation of ventricular tachy-
measured using radioimmunoassay (BioSource Europe S.A., cardia and ventricular fibrillation is often difficult (Opitz et al.,
Nivelles, Belgium). Subsequently, the site of previous thoracot- 1995), hence we report these two arrhythmias collectively, as
omy was re-opened for monophasic action potential recordings. ‘ventricular tachyarrhythmias’. Since different mechanisms are
thought to be operative for ventricular tachyarrhythmias oc-
2.7. Infarct size curring during various time periods post-infarction (Clements-
Jewery et al., 2005), we recorded the number and duration of
The rats were sacrificed using a lethal dose of potassium ventricular tachyarrhythmia episodes for each hourly interval
chloride and infarct size was measured as described previously post-ligation.
(Ytrehus et al., 1994). The heart was excised, frozen (in − 20 °C We used two previously established methods for arrhythmia
for one hour), hand-cut in five 2 mm-slices that were incubated analysis (Curtis and Walker, 1988; Opitz et al., 1995).
(in triphenyltetrazolium chloride for 15 min at 37 °C) and fixed In the first method (Opitz et al., 1995), we calculated the
(in 10% formalin for 20 min). After scanning (Scanjet 4570c/ duration of ventricular tachyarrhythmias for each hourly inter-
5500c, Hewlett-Packard, Palo Alto, California, USA), the areas val, as the sum of durations of each episode recorded during this
of infarcted and non-infarcted myocardium were measured from interval. The distribution of the hourly duration of ventricular
both slice sides and averaged. Planimetry was performed using tachyarrhythmias is reported for the entire (24-h) observation
a previously validated software program (Image Tool, Uni- period. To provide insight into possible mechanisms, ventri-
versity of Texas, USA). The volumes of infarcted and non- cular tachyarrhythmias episodes were separated in those
infarcted myocardium (defined as measured areas multiplied by occurring in the early post-ligation period (phase I, i.e. during
slice thickness) were calculated for each slice and summed. the first h) and in the late post-ligation period (phase II, i.e. from
Infarct size (expressed as a percentage) was defined as the ratio the second h to the end of the recording or to spontaneous death)
between infarcted and total left ventricular volume. (Clements-Jewery et al., 2005). Censoring effects, due to differ-
ences in mortality rates and timing, may confound the results
2.8. Analysis of monophasic action potentials (Opitz et al., 1995), hence the hourly duration of ventricular
tachyarrhythmias was normalized to survival time (i.e. the time
Arrhythmic recordings with unstable resting potential or with at risk of tachyarrhythmia occurrence).
electrical artifacts were excluded. The software utilized for In the second method, a simpler quantification provided by
monophasic action potential analysis, developed and validated at the arrhythmia score was used (Curtis and Walker, 1988). A
our Institution (Tsalikakis et al., 2003), permits signal acquisition score of 2 was given for one spontaneously reverting ventricular
and off-line analysis. Fifty sinus beats per tracing were analyzed tachyarrhythmia episode and 3 for two or more episodes, with a
and the action potential duration at 90% of repolarization was total combined duration of b60 s. A score of 4 was given for
measured at baseline and 24 h post-ligation. The standard episodes with a combined total duration of 60–119 s, 5 for
deviation of the action potential duration at 90% of repolarization ventricular tachyarrhythmias of a combined duration of N 119 s,
was calculated for each tracing, as a measure of beat-to-beat 6 for fatal ventricular tachyarrhythmias starting at b 15 min
variation, indicating electrical alternans (Franz, 1999). post-ligation, 7 for fatal ventricular tachyarrhythmias starting
between 4 min and 14 min, 8 for fatal ventricular tachyar-
2.9. Sinus heart rate rhythmias between 1 and 3 min, and 9 for fatal episodes starting
b1 min post-ligation. The arrhythmia score was calculated for
We analyzed 5-min continuous electrocardiogram recordings, four post-ligation time periods, i.e., 0–1 h, 1–6 h, 6–12 h, and
from which non-sinus beats were excluded and the mean value of 12–24 h. This method also accounts for differences in mortality
RR intervals was used. Heart rate was calculated at baseline, rates and timing, by giving a score of 9 for the time period(s)
at the 5th, 30th and 60th min post-ligation and hourly thereafter. following death.

2.10. Arrhythmia analysis 2.11. Mortality

The stored electrocardiographic tracings were analyzed inde- Tachyarrhythmic death was defined as ventricular asy-
pendently by two of the authors, blinded to treatment assign- stole, immediately preceded by an episode of ventricular
244 T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249

and were excluded. Thus, the final animal study population


consisted of 52 rats, of which 24 (223 ± 3 g) were allocated
bosentan and 23 (224 ± 3 g) were allocated vehicle, while 5 rats
(224 ± 4 g) were sham-operated. During the 24-h period post-
ligation, 3 animals in the bosentan group (12.5%, all during
phase I), and 8 (34.7%, 3 during phase I and 5 during phase II)
in the control group had fatal ventricular tachyarrhythmias
(P = 0.093). Two (8.3%, 1 during phase I and 1 during phase II)
further rats in the bosentan group and 1 (4.3%, during phase II)
further rat in the control group died of bradyarrhythmia. The
Fig. 1. Study protocol. Footnote: ECG = electrocardiogram, HRV = heart rate
variability, MAP = monophasic action potential, MI = myocardial infarction.
overall mortality did not differ significantly between the two
groups (P = 0.21).

tachyarrhythmia and bradyarrhythmic death as ventricular asy- 3.2. Infarct size and sinus heart rate
stole, immediately preceded by bradycardia (b200 bpm)
associated with complete atrioventricular block. Infarct size was comparable between groups (37.9 ± 1.5% in
bosentan-treated and 39.1 ± 1.4% in control rats, P = 0.57).
2.12. Heart rate variability Sinus heart rate increased significantly over time (F = 5.93,
P b 0.0001, Fig. 2), but without differences between groups
Heart rate variability for the assessment of cardiac autonomic (F = 0.91, P = 0.38). An increase was also present in sham-
status in conscious rats has been described previously in detail operated animals (F = 2.0, P = 0.007), probably attributable to
(Kruger et al., 1997). We used 5-min electrocardiographic the procedure, with sinus heart rate returning to baseline values
segments after exclusion of non-sinus beats. Time domain after the third h of recording.
parameters included the S.D. of RR intervals, the root mean
square of S.D. and the coefficient of variance (S.D./RR). In the 3.3. Number and duration of ventricular tachyarrhythmia
frequency domain, the fast Fourier transform power spectrum episodes
was calculated using the Welch periodogram, by dividing the
time series into a constant number of segments, overlapping by Apart from scarce premature ventricular contractions, no
50%. After application of a Hanning window and subtracting the ventricular tachyarrhythmias were recorded in sham-operated
mean value, the segment periodogram was calculated and the animals. Significantly (P = 0.0017) fewer ventricular tachyar-
power spectra of all segments were averaged. Low-frequency rhythmia episodes were found during phase II in bosentan-
(N0.5 Hz b 0.8 Hz) and high-frequency (N 0.8 Hz) bands were treated rats (46 ± 20) than in controls (248 ± 92). In contrast, the
calculated. The percentage of peak power in each band and their number of phase I ventricular tachyarrhythmia episodes did not
ratio are reported. The study protocol is depicted in Fig. 1. differ (P = 0.178) between the bosentan-treated (7 ± 2) and the
control (19 ± 4) groups. The mean duration of each episode was
2.13. Statistical analysis almost identical in the two groups (14.1 ± 7.6 s and 14.1 ± 4.8 s,
respectively).
All values are given as mean ± S.E.M. Mortality rates were
compared using two-tailed Fisher's exact test, while infarct size 3.4. Hourly duration of ventricular tachyarrhythmias during
was compared using Student's t-test. Differences in heart rate phases I and II
and action potential duration were compared using the analysis
of variance for repeated measures, followed by Duncan's multi- When both phases were considered together, the hour-
range test. The non-parametric Mann–Whitney U-test was used ly duration of ventricular tachyarrhythmias was shorter
for comparisons of catecholamine concentrations, as well as for
ventricular tachyarrhythmias frequencies and durations. Differ-
ences in the hourly incidence of ventricular tachyarrhythmias
were assessed using the Kruskal–Wallis analysis of variance by
ranks. Statistical significance was defined at an alpha level of
0.05.

3. Results

3.1. Animal population and mortality

Fifty animals were chosen, of which 26 were randomized


to bosentan and 24 to vehicle; 6 further animals were sham- Fig. 2. Sinus heart rate. Footnote: No differences in sinus heart rate (in beats per
operated. Of the 56 animals, 4 died during the procedure minute, bpm) were present between bosentan-treated rats and controls.
T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249 245

Table 1
Changes in action potential duration and temporal variability over time
Bosentan Control
LV a APD90 b baseline (ms) 94.1 ± 3.6 93.1 ± 4.7
LV a APV90 b 24 h post-MI c (ms) 97.6 ± 7.2 117.6 ± 6.9 d
Beat-to-beat variability LV a APD b baseline (ms) 3.46 ± 0.32 4.14 ± 0.45
Beat-to-beat variability LV a APD b 24 h post-MI c 4.46 ± 0.54 10.42 ± 2.51 d
(ms)
a
LV: left ventricular.
b
APD90: action potential duration at 90% of repolarization.
c
MI: myocardial infarction.
Fig. 3. Hourly distribution of ventricular tachyarrhythmias post-coronary d
P b 0.05 versus baseline.
ligation. Footnote: The horizontal arrow on the x axis indicates significant
differences between bosentan and controls. MI = myocardial infarction, VT =
ventricular tachycardia, VF = ventricular fibrillation.
3.7. Monophasic action potential duration

(P b 0.00001) in the bosentan group (92 ± 28 s), compared Of the 33 survivors, analyzable signals were available from
to controls (1013 ± 179 s). This was due to a decrease 23 animals. No variance (F = 2.12, P = 0.17) was present in right
(P b 0.00001) in the hourly duration of phase II ventricular ventricular action potential duration, or in the beat-to-beat
tachyarrhythmias in the bosentan group (59 ± 24 s), compared to variability of the action potential duration (F = 1.52, P = 0.23).
controls (1012 ± 185 s). During phase I, there was a trend There was a significant (F = 8.58, P = 0.0137) variance in left
(P = 0.053) towards a decrease in bosentan-treated rats (43 ± ventricular action potential duration, due to an increase (P =
18 s), compared to controls (132 ± 38 s). Similar results were 0.00063) in values in the control group 24 h post-ligation, while
obtained after normalization for survival times. For phase I, the values remained constant (P = 0.44) in the bosentan group
trend (P = 0.082) persisted, in favor of bosentan (168 ± 120 s) (Table 1). Furthermore, a statistical variance (F = 6.61, P =
compared to controls (253 ± 101 s). For phase II, values re- 0.018) was found in beat-to-beat variability of left ventricular
mained significantly (P b 0.00001) shorter in bosentan-treated action potential duration, due to an increase (P = 0.00051) from
rats (3 ± 1 s), than in controls (104 ± 28 s). baseline values in the control group. Beat-to-beat variability
remained unchanged (P = 0.52) in the bosentan group (Table 1).
3.5. Distribution of the hourly duration of ventricular
tachyarrhythmias 3.8. Catecholamine levels

The results over the entire 24-h period are shown in Fig. 3. Norepinephrine levels 24 h post-ligation were lower
Values of ventricular tachyarrhythmia hourly duration were (P b 0.00001) in bosentan-treated rats (10.2 ± 5.4 μg/l), com-
significantly shorter in the bosentan group between the second pared to controls (74.0 ± 1.8 μg/l). However, an opposite effect
and eleventh h post-ligation. was found in epinephrine levels, being higher (P = 0.00631) in
the bosentan (20.0 ± 4.9 μg/l), compared to the control group
3.6. Arrhythmia score (5.2 ± 0.6 μg/l).

Arrhythmia score was similar between 0–1 h (phase I,


P = 0.26) and 12–24 h (P = 0.23) post-ligation in the two groups. Table 2
Differences in favor of bosentan were found between 1–6 h Frequency domain parameters of heart rate variability post-ligation
(P = 0.000495) and 6–12 h (P = 0.001073), as shown in Fig. 4. Control Bosentan Sham
Low frequency (% of peak)
3 min 21.8 ± 3.5 11.9 ± 1.7 a 6.3 ± 2.9 a
30 min 35.2 ± 8.4 36.5 ± 7.1 b 10.3 ± 3.4
90 min 23.3 ± 4.8 18.0 ± 3.4 15.3 ± 2.5
4.5 h 32.4 ± 6.3 18.6 ± 2.3 a 11.3 ± 1.4 a
8.5 h 28.2 ± 5.3 22.9 ± 3.9 3.9 ± 0.8
24 h 33.3 ± 6.1 18.1 ± 3.4 a 11.5 ± 2.0 a

High frequency (% of peak)


3 min 42.5 ± 5.3 38.9 ± 4.7 29.2 ± 5.9
30 min 41.9 ± 5.3 41.2 ± 3.5 26.4 ± 4.2
90 min 41.0 ± 3.9 46.5 ± 3.7 42.5 ± 4.7
4.5 h 44.6 ± 7.5 41.3 ± 3.7 33.7 ± 3.4
8.5 h 54.0 ± 10.3 46.0 ± 3.9 17.9 ± 7.1
24 h 39.8 ± 9.9 55.2 ± 4.5 29.5 ± 2.8
Fig. 4. Arrhythmia score. Footnote: The horizontal arrow on the x axis indicates a
P b 0.05 versus controls.
significant differences between bosentan and controls. b
P b 0.05 versus 3 min.
246 T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249

clinically relevant. Third and foremost, all previously published


studies reported only on early (phase I) ventricular tachyar-
rhythmias in anesthetized animals. Indeed, in our experiments,
we observed only a modest reduction in ventricular tachyar-
rhythmias in bosentan-treated rats during this time frame. A
major strength of our study is the utilization of the telemetry
recording system, which facilitates the prolongation of the ob-
servation period without the confounding effects of anesthesia.
We report a prominent antiarrhythmic effect of bosentan
during phase II (between the second and eleventh h post-
Fig. 5. Heart rate variability. Footnote: Cardiac autonomic tone expressed by the ligation). The difference in the antiarrhythmic effect of bosentan
ratio of low frequency (LF) to high frequency (HF) spectra in heart rate between the two phases is further underscored by the fact that
variability analysis. Small asterisk indicates a trend (P = 0.07) and bigger asterisk all tachyarrhythmic deaths were observed during phase I in
a significant difference between bosentan and controls.
treated rats. During myocardial infarction, endothelin-1 produc-
tion is increased in rats (Loennechen et al., 2001) and humans
3.9. Heart rate variability (Yasuda et al., 1990), but its exact time course is not well
defined (Stewart et al., 1991). Progressive rises in endothelin-1
No significant differences were found in the time domain may explain the more pronounced antiarrhythmic effect of
(data not shown). In the frequency domain, the low frequency bosentan observed during phase II in our experiments.
spectrum and the ratio low frequency to high frequency spectra
were lower in bosentan-treated animals, at 3 min, 4.5 h and 24 h 4.2. Antiarrhythmic mechanism of bosentan
post-ligation (Table 2, Fig. 5).
An ongoing controversy exists, whether the arrhythmogenic
4. Discussion effect of endothelin-1 is direct, a consequence of ischemia, or a
combination of both (Duru et al., 2001). Previous studies dem-
4.1. Main findings and comparison with previous studies onstrating a reduction in infarct size after bosentan administra-
tion have utilized the ischemia/reperfusion model (Wang et al.,
We examined the effects of bosentan, a dual endothelin 1995). In our setting of permanent coronary ligation, no dif-
receptor peptide antagonist, on arrhythmogenesis in the rat ference in infarct size was found, and similar results were
model of permanent coronary ligation. This model is well reported in an identical model, after acute administration of the
characterized, as the rat exhibits a high frequency of ventricular dual endothelin receptor antagonist tezosentan (Clozel et al.,
tachyarrhythmias post-ligation (Opitz et al., 1995), with a time 2002). The decrease in the incidence of ventricular tachyar-
course resembling that observed in humans post-myocardial rhythmias without a decrease in infarct size, points towards a
infarction (Clements-Jewery et al., 2005; Fenoglio et al., 1979). direct antiarrhythmic effect of bosentan.
We report a reduction in ventricular tachyarrhythmias in Although direct arrhythmogenic actions of endothelin-1
bosentan-treated animals. Previous relevant studies have were demonstrated long ago (Yorikane et al., 1991), the under-
yielded conflicting results (Douglas et al., 1998; Geshi et al., lying mechanisms remain unclear (Duru et al., 2001). In vitro,
1999; Richard et al., 1994). Apart from species differences, endothelin-1 progressively increased the occurrence of sponta-
diversity in pharmacological agents used and protocol designs, neous calcium transients, via stimulation of the inositol 1,4,5-
the explanation for the diversity in the previously reported triphosphate pathway, leading into trains of events (Proven
results may be threefold: First, the presence or absence of et al., 2006). In vivo, early afterdepolarizations were increas-
reperfusion. Several differences exist with respect to the mech- ingly observed after low dose endothelin-1 intracoronary
anisms and temporal distribution between ischemia- and infusion (Merkely et al., 1998). These findings provide addi-
reperfusion-induced ventricular tachyarrhythmias. To our tional explanation for the pronounced antiarrhythmic effects
knowledge, the present study is the first to investigate the of bosentan during phase II in our study and allow the gen-
antiarrhythmic potential of dual endothelin receptor blockade in eration of hypotheses on the underlying electrophysiological
an in vivo model of permanent coronary ligation. Our results are mechanisms.
clinically relevant, given recent reports emphasizing the Endothelin-1 prolongs the action potential duration (Yor-
substantial proportion of acute myocardial infarction patients ikane et al., 1991) and may produce focal ventricular tachyar-
currently not being treated with reperfusion (Rosengren et al., rhythmias via triggered activity (Merkely et al., 1998). On the
2004). Second, the arrhythmogenic potential of endothelin-1 other hand, the ionic changes on cell membrane of ischemic
was previously examined after exogenously administered endo- cardiomyocytes display spatial inhomogeneity (Fenoglio et al.,
thelin-1 in some studies (Szokodi et al., 1998) and from the 1979), facilitating reentry (Clements-Jewery et al., 2005).
actions of endogenous endothelin-1 in others (Alberola Aguilar Although the differences between endothelin-1- and ischemia-
et al., 2000; Raschack et al., 1998). However, it is been shown induced ventricular tachyarrhythmias have been clearly deli-
that these actions are substantially different (Sharif et al., 1998), neated, an additive or synergistic effect is likely (Becker et al.,
suggesting that only the effects of endogenous endothelin-1 are 2000). Our study suggests that the preservation of action
T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249 247

potential duration after bosentan treatment may have aborted II, the role of sympathetic activation becomes more complex,
triggered activity, as well as ischemic reentrant mechanisms. due to norepinephrine accumulation into the axoplasm and in
non-exocytotic local metabolic release (Schomig et al., 1991).
4.3. Are endothelin ETB receptors involved in These changes, occurring to a different extent in various myo-
arrhythmogenesis? cardial regions (Li et al., 2004), lead to increased norepinephr-
ine in the interstitium of ischemic myocardium and in the
Most studies previously reporting an antiarrhythmic action plasma due to spillover (Lameris et al., 2000; Li et al., 2004).
after endothelin receptor blockade have used selective ETA In our experiments, we found a marked decrease in serum
receptor agents (Raschack et al., 1998; Duru et al., 2001). In the norepinephrine levels 24 h post-ligation in treated rats, in-
present study, the reduction in ventricular tachyarrhythmias in dicating another antiarrhythmic mechanism of bosentan. More-
bosentan-treated rats was similar to previous results after selec- over, heart rate variability indices of sympathetic activation
tive ETA receptor blockade in an identical experimental setting were decreased in treated rats, 4.5 and 24 h post-ligation.
(Baltogiannis et al., 2005). Taken together, these findings sug- These findings are in accordance with recent evidence that both
gest that the antiarrhythmic effects of endothelin-1 receptor endothelin receptors are located in the left ventricular sym-
blockade are mainly attributed to ETA receptor blockade and pathetic nerve varicosities and modulate norepinephrine release
ETB receptors are not substantially involved in ventricular (Isaka et al., 2007); in isolated ischemic guinea pig hearts,
arrhythmogenesis. This conclusion gains importance, given the exogenously applied endothelin-1 increased norepinephrine
scarcity of in vivo studies and concurs with data from in vitro release and ventricular tachyarrhythmias in a dose dependent
experiments (Alexiou et al., 1998; Kelso et al., 1998; McCabe manner. These effects were attenuated by selective ETA, as well
et al., 2005). In isolated working rabbit hearts, the endothelin as by dual receptor blockade (Isaka et al., 2007). These findings,
ETB receptor agonist sarafotoxin had a neutral effect on ven- when examined together with our results, indicate that the link
tricular tachyarrhythmias, while ETA receptor activation de- between endothelin-1 and the sympathetic system is via activa-
creased the ventricular effective refractory period and facilitated tion of the ETA receptor, but dual endothelin receptor blockade
the induction of ventricular fibrillation (McCabe et al., 2005). In confers a decrease in sympathetic activity during the delayed
a similar setting, endogenous endothelin-1 displayed a direct phase post-coronary occlusion.
arrhythmogenic action that was partially suppressed by ETA We report increased epinephrine levels 24 h post-ligation in
receptor antagonists, while ETB receptor blockade was in- bosentan-treated rats. The explanation for this diverse effect of
effective (Alexiou et al., 1998). bosentan on catecholamines may be twofold: First, endothelin-
1 has positive inotropic effects providing short-term support
4.4. Endothelin-1 and sympathetic activation during myocardial infarction; inhibition of this support might
have caused additional sympathetic activation. Nonetheless, the
The importance of sympathetic activation in modulating the hemodynamic response after endothelin-1 receptor blockade is
occurrence of ischemic ventricular tachyarrhythmias is well complex and conflicting results have been reported (Clozel
established (Corr and Gillis, 1978). Similarly, the link between et al., 2002; Nguyen et al., 2001). Second, bosentan, at the
endothelin-1 and the sympathetic system was reported long ago dosage used in the present study, produces prominent and sus-
(Wennmalm et al., 1989), but their interactions during myo- tained vasodilatory effects (Fraccarollo et al., 1997; Mulder
cardial infarction and the resultant effects on arrhythmogenesis et al., 1997). Thus, a reflex sympathetic activation may occur
remain unclear. Early catecholamine release contributes to the (Gelzer et al., 2004) and may account for the comparable sinus
genesis of early and delayed after depolarizations, triggered heart rate seen in our experiments. However, the net balance
activity and ventricular tachyarrhythmias (Schomig et al., 1991). after the diverse effects of bosentan on endogenous catechola-
Endothelin-1 is involved in the adrenal gland function, but ETA mines appears to be a decrease in sympathetic activation, as
and ETB receptors may have diverse actions in this regard evidenced by heart rate variability indices. Importantly, the dose
(Nagayama et al., 2000). In an in vitro model, catecholamine of bosentan used in the present study is high when compared to
output was inhibited by selective ETA receptor blockade, while doses used clinically. Such high dosage was selected to com-
the ETB receptor antagonist BQ-788 was ineffective. However, pensate for the higher metabolism in rats and has been pre-
the inhibitory effect of ETA receptor blockade was abolished by viously shown to produce maximal pharmacologic effects
pretreatment with BQ-788 (Nagayama et al., 2000). (Fraccarollo et al., 1997; Mulder et al., 1997). Nonetheless, our
These observations may explain some of our findings. results indicate that future studies should evaluate the effects of
Although bosentan blunted the very early sympathetic response, lower pharmacological dosages of endothelin receptor blockade
ETB receptor blockade might have negated the expected in- in the post-myocardial infarction setting. Such a strategy may
hibition of the main catecholamine surge, caused by ETA maintain the antiarrhythmic properties locally on ventricular
receptor blockade. In other words, during phase I, bosentan had myocardium, but may avoid the untoward peripheral effects.
a rather neutral effect on central catecholamine release, resulting
in almost identical heart rate variability indices of sympathetic 4.5. Limitations of the study
activation 30 min post-ligation. In keeping with previous
findings (Crockett et al., 2000), this may explain the modest We feel that our work improves current understanding on the
antiarrhythmic effect observed during this phase. During phase mechanisms of arrhythmogenesis during myocardial infarction.
248 T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249

However, three limitations may be apparent. First, the assess- antiarrhythmic drug development and for safety pharmacology evaluation.
ment of sympathetic activation would have been more accurate Br. J. Pharmacol. 145, 551–564.
Clozel, M., Qiu, C., Qiu, C.S., Hess, P., Clozel, J.P., 2002. Short-term endothelin
if catecholamine measurements prior to coronary ligation were receptor blockade with tezosentan has both immediate and long-term
included. Second, although our sample size was optimal for the beneficial effects in rats with myocardial infarction. J. Am. Coll. Cardiol. 39,
assessment of the antiarrhythmic effects of bosentan, our study 142–147.
was underpowered to detect differences in mortality. Third, Corr, P.B., Gillis, R.A., 1978. Autonomic neural influences on the dysrhythmias
resulting from myocardial infarction. Circ. Res. 43, 1–9.
ventricular remodeling, as well as the development of collateral
Crockett, T.R., Sharif, I., Kane, K.A., Wainwright, C.L., 2000. Sarafotoxin 6c
circulation, both correlating with ventricular tachyarrhythmias, protects against ischaemia-induced cardiac arrhythmias in vivo and in vitro
were not examined in our study; however, we feel that such in the rat. J. Cardiovasc. Pharmacol. 36, S297–S299.
indices as early as 24 h post-ligation, would have added little Curtis, M.J., Walker, M.J.A., 1988. Quantification of arrhythmias using scoring
value to our study. systems: an examination of seven scores in an in vivo model of regional
myocardial ischemia. Cardiovasc. Res. 22, 656–665.
Douglas, S.A., Nichols, A.J., Feuerstein, G.Z., Elliott, J.D., Ohlstein, E.H.,
4.6. Conclusions 1998. SB 209670 inhibits the arrhythmogenic actions of endothelin-1 in the
anesthetized dog. J. Cardiovasc. Pharmacol. 31 (Suppl 1), S99–S102.
In the rat model of permanent coronary ligation, bosentan Duru, F., Barton, M., Luescher, T.F., Candinas, R., 2001. Endothelin and cardiac
decreases phase II ventricular tachyarrhythmias. Prevention of arrhythmias: do endothelin antagonists have a therapeutic potential as
action potential prolongation and its temporal variability as well antiarrhythmic drugs? Cardiovasc. Res. 49, 272–280.
Fenoglio Jr., J.J., Karagueuzian, H.S., Friedman, P.L., Albala, A., Wit, A.L.,
as decreased norepinephrine levels are potential mechanisms. 1979. Time course of infarct growth toward the endocardium after coronary
Further studies should examine whether these effects are main- occlusion. Am. J. Physiol. 236, H350–H356.
tained with lower dosage, avoiding systemic effects. The inter- Fraccarollo, D., Hu, K., Galuppo, P., Gaudron, P., Ertl, G., 1997. Chronic
play between endothelin-1 and sympathetic activation during endothelin receptor blockade attenuates progressive ventricular dilation and
ischemia/reperfusion and their effects on ventricular arrhyth- improves cardiac function in rats with myocardial infarction: possible
involvement of myocardial endothelin system in ventricular remodeling.
mogenesis is also subject for future research. Circulation 96, 3963–3973.
Fraccarollo, D., Galuppo, P., Bauersachs, J., Ertl, G., 2002. Collagen
Acknowledgements accumulation after myocardial infarction: effects of ETA receptor blockade
and implications for early remodeling. Cardiovasc. Res. 54, 559–567.
This work was supported, in part, by the national General Franz, M.R., 1999. Current status of monophasic action potential recording:
theories, measurements and interpretations. Cardiovasc. Res. 41, 25–40.
Council for Research and Technology (project: PENED- Gelzer, A.R., Attmann, T., Radicke, D., Nydam, D., Candinas, R., Lutter, G.,
01ΕD511), Athens, Greece and by the Cardiovascular Research 2004. Effects of acute systemic endothelin receptor blockade on cardiac
Institute, Ioannina and Athens, Greece. electrophysiology in vivo. J. Cardiovasc. Pharmacol. 44, 564–570.
We wish to thank Actelion Pharmaceuticals Ltd., Allschwil, Geshi, E., Nomizo, A., Arata, Y., Nakatani, M., Katagiri, T., 1999. Effect of non-
selective endothelin blockade, TAK-044, on the ischemic cellular injury of
Switzerland, for providing bosentan and Boston Scientific
rat heart. Basic Res. Cardiol. 94, 94–101.
Hellas for providing the monophasic action potential probe and Hu, K., Gaudron, P., Schmidt, T.J., Hoffmann, K.D., Ertl, G., 1998. Aggravation
preamplifier. of left ventricular remodeling by a novel specific endothelin ET(A)
Tzihad Albouharali, MD, performed all radioimmunoassay antagonist EMD94246 in rats with experimental myocardial infarction.
measurements. Panagiotis Lekkas and Anastasia Alevizatou J. Cardiovasc. Pharmacol. 32, 505–508.
assisted during the experiments. Eleni Goga, MSc offered in- Isaka, M., Kudo, A., Imamura, M., Kawakami, H., Yasuda, K., 2007. Endothelin
receptors, localized in sympathetic nerve terminals of the heart, modulate
valuable help as a research coordinator. norepinephrine release and reperfusion arrhythmias. Basic Res. Cardiol.
102, 154–162.
References Kelso, E.J., Spiers, J.P., McDermott, B.J., Scholfield, C.N., Silke, B., 1998.
Receptor-mediated effects of endothelin on the L-type Ca+ current in
Alberola Aguilar, A.M., Revert, F., Moya, A., Beltran, J., Garcia, J., San Martin, ventricular cardiomyocytes. J. Pharmacol. Exp. Ther. 286, 662–669.
E., Sancho, S., Such, L., 2000. Intravenous BQ-123 and phosphoramidon Kruger, C., Kalenka, A., Haunstetter, A., Schweizer, M., Maier, C., Ruhle, U.,
reduce ventricular ectopic beats and myocardial infarct size in dogs submitted Ehmke, H., Kubler, W., Haass, M., 1997. Baroreflex sensitivity and heart
to coronary occlusion and reperfusion. Gen. Pharmacol. 35, 143–147. rate variability in conscious rats with myocardial infarction. Am. J. Physiol.
Alexiou, K., Dschietzig, T., Simsch, O., Laule, M., Hundertmark, J., Baumann, 273, H2240–H2247.
G., Stangl, K., 1998. Arrhythmogenic effects induced by coronary Lameris, T.W., de Zeeuw, S., Alberts, G., Boomsma, F., Duncker, D.J.,
conversion of pulmonary big endothelin to endothelin: aggravation of this Verdouw, P.D., Veld, A.J., van den Meiracker, A.H., 2000. Time course and
phenomenon in heritable hyperlipidemia. J. Am. Coll. Cardiol. 32, mechanism of myocardial catecholamine release during transient ischemia
1773–1778. in vivo. Circulation 101, 2645–2650.
Baltogiannis, G.G., Tsalikakis, D.G., Mitsi, A.C., Hatzistergos, K.E., Elaio- Li, W., Knowlton, D., Van Winkle, D.M., Habecker, B.A., 2004. Infarction alters
poulos, D., Fotiadis, D.I., Kyriakides, Z.S., Kolettis, T.M., 2005. Endothelin both the distribution and noradrenergic properties of cardiac sympathetic
receptor-A blockade decreases ventricular arrhythmias after myocardial neurons. Am. J. Physiol, Heart Circ. Physiol. 286, H2229–H2236.
infarction in rats. Cardiovasc. Res. 67, 647–654. Loennechen, J.P., Stoylen, A., Beisvag, V., Wisloff, U., Ellingsen, O., 2001.
Becker, R., Merkely, B., Bauer, A., Geller, L., Fazekas, L., Freigang, K.D., Voss, Regional expression of endothelin-1, ANP, IGF-1, and LV wall stress in the
F., Senges, J.C., Kuebler, W., Schoels, W., 2000. Ventricular arrhythmias infarcted rat heart. Am. J. Physiol, Heart Circ. Physiol. 280, H2902–H2910.
induced by endothelin-1 or by acute ischemia: a comparative analysis using McCabe, C., Hicks, M.N., Kane, K.A., Wainwright, C.L., 2005. Electro-
three-dimensional mapping. Cardiovasc. Res. 45, 310–320. physiological and haemodynamic effects of endothelin ETA and ETB
Clements-Jewery, H., Hearse, D.J., Curtis, M.J., 2005. Phase 2 ventricular receptors in normal and ischaemic working rabbit hearts. Br. J. Pharmacol.
arrhythmias in acute myocardial infarction: a neglected target for therapeutic 146, 118–128.
T.M. Kolettis et al. / European Journal of Pharmacology 580 (2008) 241–249 249

Merkely, B., Geller, L., Toth, M., Kiss, O., Kekesi, V., Solti, F., Vecsey, T., after ischaemia and reperfusion in rats: studies with bosentan, a mixed ETA–
Horkay, F., Tenczer, J., Juhasz-Nagy, A., 1998. Mechanism of endothelin- ETB antagonist. Br. J. Pharmacol. 113, 869–876.
induced malignant ventricular arrhythmias in dogs. J. Cardiovasc. Rosengren, A., Wallentin, L., Gitt, K., Behar, S., Battler, A., Hasdai, D., 2004.
Pharmacol. 31 (Suppl 1), S437–S439. Sex, age, and clinical presentation of acute coronary syndromes. Eur. Heart
Molenaar, P., O'Reilly, G., Sharkey, A., Kuc, R.E., Harding, D.P., Plumpton, C., J. 25, 663–670.
Gresham, G.A., Davenport, A.P., 1993. Characterization and localization of Schomig, A., Haass, M., Richardt, G., 1991. Catecholamine release and
endothelin receptor subtypes in the human atrioventricular conducting arrhythmias in acute myocardial ischaemia. Eur. Heart J. 12, 38–47 (Suppl F).
system and myocardium. Circ. Res. 72, 526–538. Sharif, I., Kane, K.A., Wainwright, C.L., 1998. Endothelin and ischaemic
Mulder, P., Richard, V., Derumeaux, G., Hogie, M., Henry, J.P., Lallemand, F., arrhythmias-antiarrhythmic or arrhythmogenic? Cardiovasc. Res. 39,
Compagnon, P., Macé, B., Comoy, E., Letac, B., Thuillez, C., 1997. Role of 625–632.
endogenous endothelin in chronic heart failure: effect of long-term treatment Stewart, D.J., Kubac, K.B., Costello, K.B., Cernacek, P., 1991. Increased plasma
with an endothelin antagonist on survival, hemodynamics, and cardiac endothelin-1 in the early hours of acute myocardial infarction. J. Am. Coll.
remodeling. Circulation 96, 1976–1982. Cardiol. 18, 38–43.
Nagayama, T., Kuwakubo, F., Matsumoto, T., Fukushima, Y., Yoshida, M., Szokodi, I., Horkay, F., Merkely, B., Solti, F., Geller, L., Kiss, P., Selmeci, L.,
Suzuki-Kusaba, M., Hisa, H., Matsumura, Y., Kimura, T., Satoh, S., 2000. Kekesi, V., Vuolteenaho, O., Ruskoaho, H., Juhasz-Nagy, A., Toth, M.,
Role of endogenous endothelins in catecholamine secretion in the rat adrenal 1998. Intraepicardial infusion of endothelin-1 induces ventricular arrhyth-
gland. Eur. J. Pharmacol. 406, 69–74. mias in dogs. Cardiovasc. Res. 38, 356–364.
Nguyen, Q.T., Cernacek, P., Calderoni, A., Stewart, D.J., Picard, P., Sirois, P., Tsalikakis, D.G., Fotiadis, D.I., Kolettis, T., Michalis, L.K., 2003. Automated
White, M., Rouleau, J.L., 1998. Endothelin A receptor blockade causes system for the analysis of heart monophasic action potentials. Comput.
adverse left ventricular remodeling but improves pulmonary artery pressure Cardiol. 30, 339–342.
after infarction in the rat. Circulation 98, 2323–2330. Walker, M.J., Curtis, M.J., Hearse, D.J., Campbell, R.W., Janse, M.J., Yellon,
Nguyen, Q.T., Cernacek, P., Sirois, M.G., Calderone, A., Lapointe, N., Stewart, D.M., Cobbe, S.M., Coker, S.J., Harness, J.B., Harron, D.W., 1988. The
D.J., Rouleau, J.-L., 2001. Long-term effects of nonselective endothelin A Lambeth conventions: guidelines for the study of arrhythmias in ischaemia
and B receptor antagonism in postinfarction rat. Importance of timing. infarction, and reperfusion. Cardiovasc. Res. 22, 447–455.
Circulation 104, 2075–2081. Wang, Q.D., Li, X.S., Lundberg, J.M., Pernow, J., 1995. Protective effects of
Oie, E., Bjonerheim, R., Grogaard, H.K., Kongshaug, H., Smiseth, O.A., non-peptide endothelin receptor antagonist bosentan on myocardial
Attramadal, H., 1998. ET-receptor antagonism, myocardial gene expression, ischaemic and reperfusion injury in the pig. Cardiovasc. Res. 29, 805–812.
and ventricular remodeling during CHF in rats. Am. J. Physiol. 275, Wennmalm, A., Karwatowska-Prokopczuk, E., Wennmalm, M., 1989. Role of
H868–H877. the coronary endothelium in the regulation of sympathetic transmitter release
Opitz, C.F., Mitchell, G.F., Pfeffer, M.A., Pfeffer, J.M., 1995. Arrhythmias and in isolated rabbit hearts. Acta Physiol. Scand. 136, 81–87.
death after coronary artery occlusion in the rat. Continuous telemetric ECG Yasuda, M., Kohno, M., Tahara, A., Itagane, H., Toda, I., Akioka, K., Teragaki,
monitoring in conscious, untethered rats. Circulation 92, 253–261. M., Oku, H., Takeuchi, K., Takeda, T., 1990. Circulating immunoreactive
Pfeffer, M.A., Pfeffer, J.M., Fishbein, M.C., Fletcher, P.J., Spadaro, J., Kloner, endothelin in ischemic heart disease. Am. Heart J. 119, 801–806.
R.A., Braunwald, E., 1979. Myocardial infarct size and ventricular function Yorikane, R., Koike, H., Miyake, S., 1991. Electrophysiological effects of
in rats. Circ. Res. 44, 503–512. endothelin-1 on canine myocardial cells. J. Cardiovasc. Pharmacol. 17
Proven, A., Roderick, H.L., Conway, S.J., Berridge, M.J., Horton, J.K., Capper, (Suppl 7), S159–S162.
S.J., Bootman, M.D., 2006. Inositol 1,4,5-trisphosphate supports the Ytrehus, K., Liu, Y., Tsuchida, A., Miura, T., Liu, G.S., Yang, X.M., Herbert, D.,
arrhythmogenic action of endothelin-1 on ventricular cardiac myocytes. Cohen, M.V., Downey, J.M., 1994. Rat and rabbit heart infarction: effects of
J. Cell Sci. 119, 3363–3375. anesthesia, perfusate, risk zone, and method of infarct sizing. Am. J. Physiol
Raschack, M., Juchelka, F., Rozek-Schaefer, G., 1998. The endothelin-A 267, H2383–H2390.
antagonist LU 135 252 supresses ischemic ventricular extrasystoles and Zheng, Z.J., Croft, J.B., Giles, W.H., Mensah, G.A., 2002. Sudden cardiac death
fibrillation in pigs and prevents hypoxic cellular decoupling. J. Cardiovasc. in the United States, 1989 to 1998. Circulation 104, 2158–2163.
Pharmacol. 31 (Suppl 1), S145–S148.
Richard, V., Kaeffer, N., Hogie, M., Tron, C., Blanc, T., Thuillez, C., 1994. Role
of endogenous endothelin in myocardial and coronary endothelial injury