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ORIGINAL CONTRIBUTION
Endothelin-B receptors and ventricular arrhythmogenesisin the rat model of acute myocardial infarction
Dimitrios L. Oikonomidis • Dimitrios G. Tsalikakis • Giannis G. Baltogiannis • Alexandros T. Tzallas •
Xanthi Xourgia • Maria G. Agelaki • Aikaterini J. Megalou • Andreas Fotopoulos • Apostolos Papalois •
Zenon S. Kyriakides • Theofilos M. Kolettis
Received: 25 March 2009 / Revised: 10 September 2009 / Accepted: 21 September 2009 / Published online: 17 October 2009
� Springer-Verlag 2009
Abstract The arrhythmogenic effects of endothelin-1
(ET-1) are mediated via ETA-receptors, but the role of
ETB-receptors is unclear. We examined the pathophysio-
logic role of ETB-receptors on ventricular tachyarrhythmias
(VT/VF) during myocardial infarction (MI). MI was induced
by coronary ligation in two animal groups, namely in wild-
type (n = 63) and in ETB-receptor-deficient (n = 61) rats.
Using a telemetry recorder, VT/VF episodes were evaluated
during phase I (the 1st hour) and phase II (2–24 h) post-MI,
with and without prior b-blockade. Action potential duration
at 90% repolarization (APD90) was measured from mono-
phasic epicardial recordings and indices of sympathetic
activation were assessed using fast-Fourier analysis of heart
rate variability. Serum epinephrine and norepinephrine were
measured with radioimmunoassay. MI size was similar in the
two groups. There was a marked temporal variation in
VT/VF duration; during phase I, it was higher (p = 0.0087)
in ETB-deficient (1,519 ± 421 s) than in wild-type (190 ±
34 s) rats, but tended (p = 0.086) to be lower in ETB-defi-
cient (4.2 ± 2.0 s) than in wild-type (27.7 ± 8.0 s) rats
during phase II. Overall, the severity of VT/VF was greater
in ETB-deficient rats, evidenced by higher (p = 0.0058)
mortality (72.0% vs. 32.1%). There was a temporal variation
in heart rate and in the ratio of low- to high-frequency
spectra, being higher (\0.001) during phase I, but lower
(p \ 0.05) during phase II in ETB-deficient rats. Likewise,
1 h post-MI, serum epinephrine (p = 0.025) and norepi-
nephrine (p \ 0.0001) were higher in ETB-deficient (4.20 ±
0.54, 14.24 ± 1.39 ng/ml) than in wild-type (2.30 ± 0.59,
5.26 ± 0.67 ng/ml) rats, respectively. After b-blockade,
VT/VF episodes and mortality were similar in the two
groups. The ETB-receptor decreases sympathetic activation
and arrhythmogenesis during the early phase of MI, but these
effects diminish during evolving MI.
Keywords Endothelin � B-receptors �Myocardial infarction � Ventricular arrhythmias
Introduction
Ventricular tachycardia (VT) and ventricular fibrillation
(VF) during acute myocardial infarction (MI) are a leading
D. L. Oikonomidis � G. G. Baltogiannis �A. T. Tzallas � M. G. Agelaki � A. J. Megalou �T. M. Kolettis (&)
Department of Cardiology, University of Ioannina,
1 Stavrou Niarxou Avenue, 45110 Ioannina, Greece
e-mail: [email protected]
D. G. Tsalikakis
Engineering Informatics and Telecommunications,
University of Western Macedonia,
Kozani, Greece
A. T. Tzallas
Department of Computer Sciences,
University of Ioannina, Ioannina, Greece
X. Xourgia � A. Fotopoulos
Department of Nuclear Medicine,
University of Ioannina, Ioannina, Greece
A. Papalois
ELPEN Research Laboratory, Pikermi,
Athens, Greece
Z. S. Kyriakides
Department of Cardiology,
Athens Red Cross Hospital,
Athens, Greece
A. Papalois � Z. S. Kyriakides � T. M. Kolettis
Cardiovascular Research Institute,
Ioannina, Greece
123
Basic Res Cardiol (2010) 105:235–245
DOI 10.1007/s00395-009-0066-7
cause of death worldwide. Despite the intense research
during the past decades, the complex pathophysiology
of ischemia-induced VT/VF is incompletely understood.
Experimental [22] and clinical [28] studies have dem-
onstrated that the production of endothelin-1 (ET-1), a
21-amino acid peptide, rises markedly during MI. In
addition to its direct electrophysiologic actions [24],
ET-1 regulates sympathetic stimulation [34], a well-
described arrhythmogenic mechanism [31]. Thus, ET-1
is implicated in arrhythmogenesis during acute MI, an
observation carrying potential therapeutic implications
[10].
Although the role of ET-1 during acute MI is entren-
ched, the underlying mechanisms remain poorly defined.
The effects of ET-1 are mediated via two (ETA and ETB)
G-protein-coupled receptors [26], which are widely dis-
tributed in mammalian organs, including the heart, the
adrenal gland and peripheral neurons. The effects of ET-1
during MI appear to be mediated mainly via activation of
the ETA-receptor [10]. On the other hand, the role of the
ETB-receptor remains controversial, as previous studies
indicated protective [6], neutral [23] or detrimental [32]
effects on ischemic VT/VF. This issue is further com-
plicated by the fact that most previous studies [6, 23]
were performed in the isolated heart model that can
address ventricular arrhythmogenesis only in part. In fact,
since in vitro preparations are devoid of sympathetic
innervation, ventricular arrhythmias are absent during the
delayed phase of MI [5]. Furthermore, in vivo studies [6,
32] were performed in anesthetized animals and have,
therefore, limited the observation time window to 1–2 h
post-infarction. Finally, another source of discrepancy in
the hitherto published results is the variability in the
ischemic models. In this regard, the presence or absence
of reperfusion affects ventricular arrhythmogenesis
post-MI, since the mechanisms of reperfusion-induced
arrhythmias differ from those responsible for ischemic
arrhythmias [7].
The purpose of the present study was to examine the
pathophysiologic role of ETB-receptors on ventricular
arrhythmogenesis during acute MI. We used the in vivo rat
model, which offers several advantages, as the rat exhibits
a high frequency of VT/VF after MI, with a time course
corresponding to human findings [9, 16]. In the present
work, we investigated ischemic VT/VF in the absence of
reperfusion and we extended the observation time to 24 h
post-MI. Two animal groups were included, namely wild-
type rats and a previously characterized homozygous rat
strain [14, 15], carrying a naturally occurring deletion of
the ETB-receptor gene. To provide further insight into the
underlying mechanisms, we evaluated sympathetic stimu-
lation comprehensively, by measuring electrocardiographic
(ECG) indices, as well as serum catecholamines.
Moreover, we assessed the incidence of VT/VF with or
without acute b-blockade prior to MI generation. Finally,
we examined the alterations of the left ventricular (LV)
monophasic action potential (MAP), produced by ische-
mia/infarction.
Methods
Experimental animal population
Of the 145 rats initially included in the study, 13 died
during intubation or during the surgical procedure. Two
further animals were excluded, because the knot of the
suture was incorrectly tied and no detectable infarct
was present 24 h post-ligation. Thus, the final study
population consisted of 130 rats, of similar age
(22 ± 0.1 weeks) and weight (249 ± 2 g). Of these, 66
(20–23 weeks, 240 ± 2 g) were wild-type and 64 (20–
24 weeks, 259 ± 3 g) were ETB-deficient. The animals
were housed in individual cages, under optimal laboratory
conditions (controlled humidity, temperature and light/
dark cycles). The guidelines stated in the ‘Principle of
laboratory animal care’ (NIH publication No. 86-23,
revised 1985) were followed, as well as national legis-
lation. The study protocol was approved by the local state
authority (approval reference number: 694/11-02-08).
Two animal populations were used, namely wild-type and
homozygous ETB-receptor-deficient rats. The latter rat
strain, referred to as spotting lethal (sl) strain, carries a
naturally occurring deletion in the ETB-receptor gene that
abrogates the expression of functional ETB-receptors.
This rat model has been characterized previously [14, 15]
and a colony has been bred in our animal facilities, kindly
provided by Professor M. Yanagisawa (University of
Texas Southwestern Medical Center, Dallas, Texas,
USA). Rats homozygous (sl/sl) for this mutation exhibit
coat color spotting and intestinal aganglionosis and
die of intestinal obstruction approximately at the age of
1 month. Dopamine b-hydroxylase promoter has been
used to direct ETB transgene expression in sl/sl rats to
support normal enteric nervous system development [14].
These transgenic sl/sl rats live into adulthood, but are
ETB-deficient in the cardiovascular system, rendering
this rat strain a useful tool in the study of the patho-
physiological role of ETB-receptors [14, 15]. In addition,
this rat model can be used in the study of the sympathetic
response to various stimuli, since a low level of expres-
sion of ETB-receptors driven by the endogenous pro-
moter in the adrenal medulla has been reported [15].
Nonetheless, it should be noted that the exact level of
expression of functional ETB-receptors in adrenergic
tissues of these rats remains uncertain.
236 Basic Res Cardiol (2010) 105:235–245
123
Study protocol
Myocardial infarction (MI) was induced in wild-type and
ETB-deficient rats. Subsequently, continuous ECG
recordings were performed for 24 h, or until animal death.
MAP signals were recorded at baseline, as well as 5 min
and 24 h after MI. Infarct size was measured in all survi-
vors after the 24-h observation period. Indices of sympa-
thetic activation were assessed from ECG recordings, using
fast-Fourier transform power spectrum calculations in the
frequency domain. Moreover, serum epinephrine and nor-
epinephrine were measured using radioimmunoassay. To
further explore the importance of sympathetic activation,
the incidence of VT/VF was evaluated after acute
b-adrenergic blockade in a separate set of experiments. The
study protocol is depicted in Fig. 1.
The 24-h post-MI observation period was divided in two
phases. Phase I was defined as the first hour after ligation
and phase II was defined as the time period between the
second and 24th hour post-ligation. This separation is
useful not only because it corresponds with observations in
patients [9], but also because it provides some indications
on the arrhythmogenic mechanisms post-MI [5].
Implantation of telemetry transmitter
One day prior to MI induction, a continuous ECG telemetry
transmitter (Dataquest, Data Sciences International, Tran-
soma Medical, Arden Hills, MN, USA) was implanted in
the abdominal cavity, using a previously described method
[1, 29]. The animals were intubated, mechanically venti-
lated (ventilator model 7025, Ugo Basile, Comerio, VA,
Italy) and anesthetized with isoflurane. The leads were
tunneled under the skin and the transmitter was secured in
the abdominal cavity. The rats were housed in individual
cages, placed on a receiver that continuously captured the
signal, independently of animal activity. The ECG signal
was displayed with the use of a computer program (A.R.T.
2.2, Dataquest, Data Sciences International, Transoma
Medical, Arden Hills, MN, USA) and was stored for
analysis.
Generation of acute MI
As much as 24 h after the implantation of the telemetry
transmitter, the animals were re-anesthetized, as outlined
above. Through a left thoracotomy, the heart was exposed
and the left coronary artery was ligated with a 6-0 suture,
placed between the pulmonary artery cone and the left
atrial appendage, as previously described [1, 11, 29]. A six-
lead ECG was obtained immediately after the procedure
and ST-segment elevation was considered proof of induced
MI. Upon cessation of anesthesia, the animals regained
consciousness within 2 to 3 min. No resuscitation attempts
were allowed at any time during the study.
Mortality
Total mortality is reported for the 24-h observation period
and separately for phases I and II. The mode of death was
classified as either tachyarrhythmic or bradyarrhythmic,
according to prior definitions [1, 11, 29]. Specifically,
tachyarrhythmic death was defined as ventricular asystole,
immediately preceded by an episode of VT/VF and brady-
arrhythmic death as an abrupt onset of complete atrio-
ventricular block.
Infarct sizing
At the end of the 24-h observation period, the animals were
killed with potassium chloride. The heart was then excised,
frozen (in -20�C for 1 h), hand-cut in five 2-mm slices,
incubated (in triphenyltetrazolium chloride for 15 min at
37�C) and fixed (in 10% formalin for 20 min), as previ-
ously outlined in detail [1, 11]. The slices were scanned
and the areas of infarcted and non-infarcted myocardium
were measured from both sides of each slice. The average
was multiplied by slice thickness and values were summed.
Fig. 1 Study protocol
Basic Res Cardiol (2010) 105:235–245 237
123
Infarct size was defined as the ratio between the infarcted
and total LV volume. In rats displaying infarct size below
20%, the ligation procedure was considered unsatisfactory
and these rats were excluded from the study.
Heart rate
Sinus heart rate (HR) was measured from continuous 10-s
ECG recordings, from which non-sinus beats were exclu-
ded. The mean value of these RR intervals was used to
determine HR at each time point. HR was calculated at
baseline, at the 5th and 30th minutes post-ligation and
hourly thereafter.
Arrhythmia analysis
All stored ECG tracings were analyzed off-line indepen-
dently by three blinded operators (D.O., G.G.B., M.G.A.).
VT and VF episodes were recorded and the duration of
each episode was measured using the time-scale provided
by the software. VT was defined as 4 or more consecutive
premature ventricular contractions and VF as a signal with
indistinguishable QRS deflections [33]. However, separa-
tion of VT and VF is often difficult [1, 11, 29], hence we
report VT/VF collectively.
We used two previously established methods for
arrhythmia analysis [8, 29].
In the first method [29], we calculated the duration of
VT/VF for each hourly interval. To overcome differences
in mortality rates and timing, VT/VF duration was nor-
malized to survival time (i.e. the time at risk of tachy-
arrhythmia occurrence) [29]. The duration of VT/VF
episodes is also reported separately for phases I and II.
In the second method, a simpler quantification provided
by the ‘arrhythmia score’ [8] was used. This method esti-
mates the severity of VT/VF episodes based on their tim-
ing, duration and their impact on mortality [8], assigning
score values from 2 to 9. This method also accounts for
differences in mortality rates and timing, by giving a score
of 9 for the time period(s) following death.
ECG indices of sympathetic activation
Heart rate variability (HRV) for the assessment of cardiac
autonomic status in conscious rats has been described
previously in detail [21]. We used 60-s ECG segments after
exclusion of non-sinus beats. In the frequency domain, the
fast-Fourier transform power spectrum was calculated
using the Welch periodogram, by dividing the time series
into a constant number of segments, overlapping by 50%.
After application of a Hanning window and subtracting the
mean value, the segment periodogram was calculated and
the power spectra of all segments were averaged. Low-
frequency (between 0.5 and 0.8 Hz) and high-frequency
([0.8 Hz) bands were calculated. As indices of sympa-
thetic activity, we report the percentage of peak power in
the low-frequency band and the ratio of low-frequency to
high-frequency bands.
Serum catecholamine levels
In a separate set of experiments, catecholamine measure-
ments were performed in wild-type (n = 15) and ETB-
deficient (n = 15) rats. For reference, 6 additional rats
from the two groups (n = 3 from each group) were sham-
operated. Blood was collected by internal jugular venous
puncture; it was centrifuged immediately and the serum
was stored at -20�C. Catecholamine measurements were
performed 1 h after coronary ligation. If profound brady-
cardia was observed prior to this time point, indicative of
imminent death, venous puncture was performed immedi-
ately. Serum levels of epinephrine and norepinephrine were
measured using radioimmunoassay kits, obtained from
BioSource Europe S.A., Nivelles, Belgium.
Ventricular tachyarrhythmias after b-blockade
To further clarify the pathophysiologic role of the sympa-
thetic system during MI, we performed a separate set
of experiments, in which rats were pre-treated with a
b-adrenergic blocker. In these experiments, wild-type
(n = 20) and ETB-deficient rats (n = 21) were treated
with intravenous propranolol 15 min prior to MI genera-
tion. After surgical exposure, the internal jugular vein was
cannulated with a 21G venous cannula and propranolol was
administered at a dose of 0.2 mg/kg as a slow injection,
according to a previously reported regimen [4]. Subse-
quently, MI was induced, as explained above.
Monophasic action potential recordings
The method used in our laboratory for MAP recordings has
been described previously [11]. In brief, a MAP probe
(model 200, EP Technologies, San Jose, CA, USA) was
placed on the lateral LV wall. The signal was amplified
with the use of a pre-amplifier (model 300, EP Technolo-
gies, San Jose, CA, USA) and filtered at 50 Hz, using a
digital notch filter. The signal was further filtered using a
band pass filter, allowing a signal range of 0.05–500 Hz.
Two-minute recordings were stored into a personal com-
puter, equipped with an analog-to-digital converter (model
BNC 2110, National Instruments Corporation, Dallas, TX,
USA). As much as 50 consecutive sinus beats per recording
were analyzed and the LV action potential duration at 90%
of repolarization (APD90) was measured at baseline, as
well as 5 min and 24 h post-ligation.
238 Basic Res Cardiol (2010) 105:235–245
123
Statistical analysis
All values are given as mean ± standard error of the mean
(S.E.M.). Differences between continuous variables in the
two rat groups were compared using Student’s unpaired t-
test. Changes in continuous variables over time were
compared using the analysis of variance for repeated
measures, followed by the post-hoc Duncan’s multi-stage
test. The continuous variables describing the arrhythmia
frequencies were not normally distributed and were com-
pared using the non-parametric Wald-Wolfowitz multiple-
runs test. Pair-wise comparisons between categorical
variables were performed with Fisher’s exact two-tailed
test. Kaplan–Meier survival curves were constructed for
the two groups and were compared using the Peto-and-Peto
Wilcoxon test. Statistical significance was defined at an
alpha level of 0.05.
Results
MI without prior b-blockade
This part of the study was conducted in 83 rats (43 wild-
type and 40 ETB-receptor-deficient).
Heart rate
There was a significant variance (F = 19.3, p \ 0.0001) in
HR over time between the two groups. HR was higher in
ETB-deficient rats during the first hour and in wild-type
rats after the 8th hour post-ligation, as shown in Fig. 2a.
Mortality
Total mortality after the 24-h observation period was
higher (p = 0.0058) in ETB-deficient (18/25; 72.0%) than
in wild-type rats (9/28; 32.1%). This difference was pro-
duced during phase I, as mortality was higher (p = 0.0009)
in ETB-deficient (14/25; 56%) than in wild type rats (3/28;
10.7%). During phase II, total mortality was comparable
(p = 0.45) in the two animal groups. The respective figures
were 4/11 (36.3%) in ETB-deficient and 6/25 (24%) in
wild-type rats. Tachyarrhythmic death was observed in
16/18 (88.8%) of fatalities in ETB-deficient rats, as
opposed to 5/9 (55.5%) in wild-type rats, but this differ-
ence was not statistically significant (p = 0.13). Bradyar-
rhythmic death was comparable (p = 0.67) in the two rat
groups. The Kaplan–Meier curves for total mortality for
both animal groups are shown in Fig. 3. Comparison of the
two curves revealed higher (p = 0.00076) mortality in
ETB-deficient rats.
Infarct size
Infarct size was calculated for the 25 survivors. Mean
infarct size was almost identical in the two groups.
Respective values were 31.5 ± 0.8% in ETB-deficient and
32.4 ± 1.1% in wild-type rats (p = 0.66).
Fig. 2 Heart rate. Heart rate in
the two groups without (a) or
with (b) prior b-blockade
Fig. 3 Kaplan–Meier survival curves. 24-h survival was higher in
wild-type than in ETB-deficient rats
Basic Res Cardiol (2010) 105:235–245 239
123
Ventricular tachyarrhythmias
There was a marked temporal variation in the duration of
VT/VF episodes between the two groups. The hourly dis-
tribution of VT/VF was higher in ETB-deficient rats during
phase I but lower during phase II (Fig. 4a).
Phase I
During phase I, the duration of VT/VF was higher
(p = 0.0087) in ETB-deficient (381.5 ± 84.2 s) than in
wild-type rats (147.1 ± 12.3 s). This difference remained,
when corrected for the actual survival time. The duration of
VT/VF episodes (per hour alive) in ETB-deficient rats was
1,519.7 ± 421.3 s vs. 190.8 ± 34.6 s in wild-type rats.
Phase II
During phase II, the incidence of VT/VF was lower
(p = 0.00091) in ETB-deficient than in wild-type rats. The
respective values were 47.4 ± 19.1 s vs. 376.4 ± 87.1 s.
However, when the duration of VT/VF was corrected for
the actual survival time, this difference was of marginal
statistical significance (p = 0.086). The respective values
were 4.2 ± 2.0 s and 27.7 ± 8.0 s.
Arrhythmia score
At the 2–6-h time interval, the arrhythmia score was
marginally (p = 0.073) higher in ETB-deficient rats
(6.12 ± 0.73) than wild-type rats (4.62 ± 0.38). During
the remaining time intervals, it was significantly (p \ 0.01)
higher in ETB-deficient than in wild-type rats. All values
are depicted in Fig. 5a.
ECG indices of sympathetic activation
There was a temporal variation in the ECG indices of sym-
pathetic activation between the two groups. Sympathetic
activation was higher in ETB-deficient rats during phase I, but
lower during phase II. Values are shown in Fig. 6a, b.
Serum catecholamine levels
Serum epinephrine levels 1 h post-ligation were higher
(p = 0.025) in ETB-deficient (4.20 ± 0.54 ng/ml) than in
wild-type rats (2.30 ± 0.59 ng/ml). Similarly, serum nor-
epinephrine levels 1 h post-ligation were higher (p \ 0.0001)
in ETB-deficient (14.24 ± 1.39 ng/ml) than in wild-type rats
(5.26 ± 0.67 ng/ml). Values are depicted in Fig. 6c.
MAP recordings
Changes in APD90 over time are shown in Fig. 7. Five
minutes post-ligation, APD90 did not change in wild-type
rats, but shortened (p \ 0.0001) in ETB-deficient rats. As
much as 24 h after ligation, APD90 was comparable in the
two groups.
MI with prior b-blockade
This part of the study was conducted in 41 rats (20 wild-
type and 21 ETB-receptor-deficient).
Fig. 4 Hourly distribution of
ventricular tachyarrhythmias.
Without prior b-blockade (a),
ventricular tachyarrhythmia
(VT/VF) duration was higher in
ETB-deficient rats during phase
I, but lower during phase II.
These differences disappeared
after b-blockade (b)
Fig. 5 Arrhythmia score. Without prior b-blockade (a), arrhythmia
score was higher in ETB-deficient rats (asterisk). Differences
disappeared after b-blockade (b)
240 Basic Res Cardiol (2010) 105:235–245
123
Heart rate
Heart rate (HR) changes over time showed a similar pattern
(F = 1.01, p = 0.45) between the two groups. However,
HR tended to be lower in ETB-deficient rats the first hour
post-ligation onwards, as shown in Fig. 2b.
Mortality after b-blockade
After acute propranolol administration, total mortality after
the 24-h observation period was almost identical in the two
groups, being 12/21 (57.1%) in ETB-deficient and 11/20
(55.0%) in wild-type rats. Equivalent mortality rates
between the two groups were observed during both phases.
During phase I, total mortality was 10/21 (47.6%) in ETB-
deficient and 10/20 (50.0%) in wild-type rats. During phase
II, total mortality was 2/11 (18.1%) in ETB-deficient and
1/10 (10.0%) in wild-type rats. Tachyarrhythmic death was
observed in 8/12 (66.6%) of fatalities in ETB-deficient rats
and in 8/11 (72.7%) in wild-type rats; again, this difference
was not statistically significant (p = 0.55).
When total mortality rates were compared in experi-
ments with and without b-blockade prior to MI induction,
no significant differences were found in either group.
Specifically, total mortality in ETB-deficient rats was
comparable (p = 0.35) without (18/25; 72.0%) and with
(12/21; 57.1%) prior b-blockade. Similarly, total mortality
in wild-type rats was comparable (p = 0.14) without (9/28;
32.1%) and with (11/20; 55.0%) prior b-blockade.
Ventricular tachyarrhythmias after b-blockade
Acute propranolol administration decreased the incidence
of VT/VF in the whole animal study cohort; nevertheless,
this effect was much more prominent in ETB-deficient
rats during phase I. As a result, the temporal variation in
the duration of VT/VF episodes between the two groups
disappeared completely (Fig. 4b).
Phase I
During phase I, there was a trend (p = 0.056) toward lower
VT/VF duration in ETB-deficient (30.09 ± 6.11 s) than in
Fig. 6 Indices of sympathetic activation. Significant differences
(asterisk) between the two groups in the low-frequency spectrum
(a) and the ratio of low- to high-frequency spectra (b) of the fast-
Fourier analysis of heart rate variability, as well as in serum
catecholamines (c)
Fig. 7 Action potential duration. Significant shortening (asterisk) of
APD90 5 min post-ligation in ETB-deficient rats
Basic Res Cardiol (2010) 105:235–245 241
123
wild-type rats (40.94 ± 17.42 s). However, this difference
became insignificant (p = 0.18), when corrected for the
actual survival time. The duration of VT/VF episodes (per
hour alive) in ETB-deficient rats was 53.38 ± 13.12 s vs.
94.15 ± 37.95 s in wild-type rats.
Phase II
During phase II, the incidence of VT/VF was comparable
(p = 0.49) in ETB-deficient and in wild-type rats. The
respective values were 37.30 ± 37.22 s and 22.70 ±
20.09 s. The lack of significant difference between the two
groups remained when corrected for the actual survival
time. The respective values were 1.62 ± 1.61 s and
0.98 ± 0.87 s.
Arrhythmia score after b-blockade
After acute propranolol administration, arrhythmia score
was similar in the two rat groups during the whole obser-
vation period (Fig. 5b).
Discussion
The arrhythmogenic potential of ET-1 during acute MI has
received increasing attention during the past years [10], but
the underlying mechanisms remain incompletely under-
stood. The present study examined the significance of
ETB-receptors on ventricular arrhythmogenesis in the rat
model of permanent coronary ligation, i.e. without the
confounding effects of reperfusion. Despite the widespread
application of reperfusion strategies, our results are clini-
cally relevant, given the substantial proportion of acute MI
patients (at the range of 20–30%), currently not being
treated with reperfusion [30].
A bimodal distribution of ischemic VT/VF has been
demonstrated in animal models [16] and available infor-
mation indicates that a similar curve may apply to patients
[9]. This distinction is clinically useful, because different
treatment strategies pertain after MI. During phase II,
patients are invariably hospitalized and are likely to receive
appropriate antiarrhythmic therapy, but phase I ventricular
tachyarrhythmias usually occur prior to medical attention
and account for the vast majority of out-of-hospital sudden
cardiac deaths.
The results of the present study indicate that the ETB-
receptor is involved in ventricular arrhythmogenesis during
acute MI, but its pathophysiologic role may differ,
depending on the time period after the onset of ischemia.
We report a discernible temporal dispersion in the inci-
dence of VT/VF in the two rat groups, with preponderance
in ETB-deficient rats during phase I and in wild-type rats
during phase II. Sympathetic stimulation showed a similar
pattern, implicating this as the primary arrhythmogenic
mechanism in the setting of MI. Overall, the severity of
VT/VF was greater in ETB-deficient rats, as evidenced by
higher arrhythmia scores and increased mortality.
Phase I arrhythmogenesis
We report a higher incidence of VT/VF in ETB-deficient
rats during phase I, indicating a beneficial role of ETB-
receptors. This conclusion is in accordance with the results
of Crockett et al. [6], who found an antiarrhythmic effect
after ETB-receptor stimulation, shortly after myocardial
ischemia. Moreover, in isolated working rabbit hearts [23],
administration of an ETB-receptor antagonist augmented
the electrophysiologic effects of ET-1. Earlier work from
our laboratory [2, 19] lends further support to these find-
ings. Using an identical experimental setting with the
present study, we reported only a modest antiarrhythmic
effect after dual ET-receptor blockade during phase I [19],
as opposed to a marked reduction in VT/VF after selective
ETA-blockade [2]. Taken together, these findings suggest
that the ETB-receptor may partially counteract the
arrhythmogenic effects of ET-1 in the early phase of MI.
Mechanism of antiarrhythmic action of ETB receptors
during phase I
Here, we report higher ECG indices of sympathetic activity
and markedly higher catecholamine levels during phase I in
ETB-deficient than in wild-type rats. Thus, in the absence
of ETB-receptors, the increased arrhythmogenesis during
phase I is likely secondary to enhanced sympathetic acti-
vation. This conclusion is reinforced by the pronounced
effect of b-blockade on phase I arrhythmogenesis,
observed in our experiments. Specifically, the marked
difference in arrhythmogenesis, due to increased incidence
of VT/VF in ETB-deficient rats, disappeared completely
after acute propranolol administration.
It has been known for years that ET-1 regulates sym-
pathetic stimulation [34], but the precise pathophysiologic
mechanisms and the relative importance of ETA- and ETB-
receptors under ischemic conditions is poorly character-
ized. In our experiments, HR and HRV-indices as well as
serum norepinephrine levels were higher in ETB-deficient
rats during phase I, resulting in pronounced shortening of
the APD90. Several lines of evidence suggest that sym-
pathetic stimulation is an important arrhythmogenic
mechanism during MI, as it increases resting membrane
potential and shortens APD90, leading to dispersion of
repolarization, functional re-entry and VT/VF [16, 18, 31].
Sympathetic activation during MI consists of increased
central nervous sympathetic tone, increased catecholamine
242 Basic Res Cardiol (2010) 105:235–245
123
release from the adrenal medulla and increased myocardial
norepinephrine release. Norepinephrine release from the
sympathetic nerve terminals in the ventricular myocardium
is complex and incompletely understood. During phase I,
norepinephrine release occurs first by exocytosis and sub-
sequently by a reverse function of the norepinephrine
transporter, which facilitates the release of the accumulated
free axoplasmic norepinephrine. ET-1 promotes non-exo-
cytotic norepinephrine release through the ETA-receptor,
which is linked to the Na?/H?-exchanger [13]; ETA-
receptor stimulation results in axoplasmic sodium accu-
mulation and reversal of norepinephrine transporter [13].
Despite the available information on the ETA-receptor,
very few data exist on the role of the ETB-receptor on
sympathetic stimulation during myocardial ischemia. In
our experiments, the excessive sympathetic stimulation,
observed in ETB-deficient rats early post-MI, indicates a
protective role of the ETB-receptor and concurs with pre-
vious findings in isolated, Langendorff-perfused rat hearts
[36]. In this study [36], ETB-deficient rats displayed
exaggerated norepinephrine overflow after global ischemia
and this effect was abolished by selective ETA-receptor
blockade. Recently, Isaka et al. [17] demonstrated that both
ETA- and ETB-receptors are located in the LV sympathetic
nerve varicosities and modulate norepinephrine release. In
their experiments in isolated ischemic guinea pig hearts
[17], exogenously applied ET-1 increased norepinephrine
release and ventricular tachyarrhythmias in a dose-depen-
dent manner, mediated by stimulation of ETA-receptors.
Moreover, in agreement with our findings, Isaka et al.
[17] reported marked elevation of norepinephrine release
after selective ETB-receptor blockade. Whether the ETB-
receptor decreases sympathetic activity directly by inhibiting
the Na?/H?-exchanger [17], or indirectly by decreasing ET-1
release [3] is unknown and constitutes subject for future
research.
The regulatory effects of ET-1 on sympathetic stimu-
lation are exerted not only in the myocardium, but also in
the adrenal gland; there is strong evidence suggesting that
ET-1 is involved in the regulation of adrenal catecholamine
secretion [35]. In line with the aforementioned observa-
tions, different effects of ETA- and ETB-receptors appear
to exist also in the adrenal gland [35]. In this regard,
activation of ETA-receptors under ischemic conditions
augments the secretion of catecholamines, while ETB-
receptors exert an inhibitory action [27].
In summary, our data, examined in context with previ-
ous findings, suggest a protective effect of ETB-receptors
on ventricular arrhythmogenesis during phase I post-MI.
This effect appears to be exerted mainly by a decrease in
ETA-mediated sympathetic stimulation, both at the myo-
cardial and at the adrenal gland level. To this end, the
markedly elevated norepinephrine levels, found in our
ETB-deficient rat cohort, are secondary to enhanced sym-
pathetic stimulation at both levels, reflecting the dual
source of serum norepinephrine. In contrast, the less pro-
nounced difference in serum epinephrine levels between
the two rat groups can be explained by the inhibitory action
of ETB-receptors only in the adrenal gland.
Phase II arrhythmogenesis
A rather unexpected result in our experiments was the
decreased incidence of VT/VF in ETB-deficient rats during
phase II. This finding was accompanied by lower HR and
decreased sympathetic activity, as shown by fast-Fourier
ECG analysis. Surprisingly, relatively scant information
exists on phase II sympathetic activation in vivo. In the
present study, the use of an implantable ECG recording
system permitted the long-term evaluation of autonomic
function in conscious, untethered animals. In the control
group, sympathetic activation increased during phase I and
remained elevated during phase II. This pattern of sym-
pathetic stimulation was consistent with previous findings
from our laboratory [1, 11, 19]. In contrast, ETB-deficient
rats displayed pronounced sympathetic activation during
phase I, which subsequently decreased during phase II.
Nonetheless, this was of marginal importance, since mor-
tality was comparable in the two rat groups during this
phase.
The explanation for our finding is unclear, but norepi-
nephrine depletion [12] appears likely. The precise dura-
tion of ischemia required to cause this effect is unknown,
but it varies between 30 min and 60 min, depending on
species and preparation [12]. Thus, catecholamine deple-
tion during phase II might have been more pronounced in
ETB-deficient rats, secondary to increased sympathetic
activation during phase I.
In addition to catecholamines, phase II tachyarrhythmias
are thought to represent the balance between several pro-
and anti-arrhythmic substances [5]. ET-1 is involved in
several elements of the cytokine cascade elicited post-MI
and may indirectly interfere with phase II arrhythmogene-
sis. The decreased arrhythmogenesis in ETB-deficient rats
during phase II in our experiments underscores the complex
role of ET-1 during evolving MI and the need for future
research on this issue.
Strengths and limitations of the study
The present work addressed the role of ETB-receptors
on sympathetic activation and arrhythmogenesis during
an extended period post-MI, a topic that has not been
previously examined in vivo. Although data from animal
experiments should be extrapolated to humans with cau-
tion [25], we feel that our findings improve current
Basic Res Cardiol (2010) 105:235–245 243
123
understanding on the mechanisms of ischemic VT/VF.
However, three limitations should be acknowledged. First,
measurements of MAP recordings at several time intervals
post-ligation would have permitted more detailed assess-
ment of the underlying mechanisms. On the other hand,
such measurements would have interfered with the
arrhythmia recording process and were omitted. Second,
we did not include measurements of the ventricular
refractory period, a key element of arrhythmogenesis.
Nonetheless, no significant effects of ETA-receptor
blockade were found from our group in a previous study in
patients with coronary artery disease [20]. Finally, in the
present work, we did not assess LV function in the two rat
groups. Since LV dysfunction constitutes an important
cause of sympathetic activation, differences in post-MI LV
function between the two rat groups may have accounted
for the observed differences in sympathetic activation. The
effects of the ETB-receptor on LV function during MI
merit further study.
Conclusions
The present study indicates a prominent role of the ETB-
receptor on ventricular arrhythmogenesis during MI. Dur-
ing the early phase, the ETB-receptor attenuates ventricular
arrhythmogenesis by decreasing sympathetic activation.
During subsequent stages of acute MI, the pathophysio-
logic importance of the ETB-receptor appears diminished.
Further studies on the electrophysiologic properties of
ET-1 during MI in the presence or absence of reperfusion
are required.
Acknowledgments Agapi Vilaeti, MD, and Eleftheria Karambela,
RN, assisted during the experiments. Eleni Goga, MSc, offered
invaluable help as a research coordinator. This work was supported by
the Cardiovascular Research Institute, Ioannina and Athens, Greece.
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