The Effects of Nitric Oxide Synthase Inhibition on Epinephrine-
Induced Arrhythmia and Myocardial Damage
OMAR M.E. ABDEL-SALAM1, MARAWAN ABD EL BASET MOHAMED SAYED2,
ENAYAT A. OMARA3, AMANY A. SLEEM2
1Department of Toxicology and Narcotics,
Medical Research and Clinical Studies Institute, National Research Centre,
Tahrir Street, Dokki, Cairo,
EGYPT
2Department of Pharmacology,
Medical Research and Clinical Studies Institute, National Research Centre,
Tahrir Street, Dokki, Cairo,
EGYPT
3Department of Pathology,
Medical Research and Clinical Studies Institute, National Research Centre,
Tahrir Street, Dokki, Cairo,
EGYPT
Abstract: - We have recently reported that methylene blue (MethyB) was able to inhibit epinephrine-induced
arrhythmias and cardiac muscle injury. In this study, we investigated the effect of nitric oxide synthase
inhibition by NG-nitro-L-arginine methyl ester (L-NAME) on cardiac arrhythmias, and myocardial damage
induced by epinephrine in rats. Whether nitric oxide inhibition would affect the antiarrhythmic and cardiac
protective actions of MethyB was also examined. L-NAME (40 mg/kg), L-arginine (200 mg/kg) + L-NAME,
or MethyB (100 mg/kg) + L-NAME were given intraperitoneally (i.p.). Cardiac arrhythmia was then induced
with intravenous (i.v.) injection of 10 μg/kg epinephrine. Results showed that epinephrine injection caused
marked bradycardia (221.0 ± 1.37 vs. 409.4 ± 3.18 beats/min), shortened QTc interval (0.096 ± 0.0093 vs.
0.177 ± 0.0008 s), increased QRS duration (0.040 ± 0.0035 vs. 0.0185 ± 0.0002 s), decreased R wave amplitude
(0.176 ± 0.0051 vs. 0.21 ± 0.0009 mv), ST segment height (-0.026 ± 0.007 vs. -0.002 ± 0.0005 mv), and
induced ventricular extrasystoles. L-NAME given to untreated control rats resulted in a decrease in heart rate
(288.2 ± 0.88 vs. 409.4 ± 3.18 beats/min), and increased R wave amplitude (0.436 ± 0.004 vs. 0.21 ± 0.0009
mv) compared to controls. L-NAME did not cause extrasystoles in untreated control rats but significantly
increased the number of extrasystoles and duration of arrhythmia in the epinephrine-treated group. The
administration of L-arginine (200 mg/kg, i.p.) to epinephrine plus L-NAME-treated rats resulted in increased
heart rate and markedly decreased the number of extrasystoles and duration of arrhythmia. Methylene blue
given at 100 mg/kg to rats treated with epinephrine and L-NAME caused a marked increase in heart rate. It also
normalized QRS duration, prevented ST segment depression, markedly suppressed ventricular extrasystoles,
and decreased the duration of arrhythmia compared with either epinephrine or L-NAME plus epinephrine-
treated groups. Epinephrine injection caused disorganization, and necrosis of cardiac cells, interstitial
hemorrhage, and cellular infiltrations. These changes were markedly improved by treatment with either L-
NAME or L-NAME/MethyB. These results suggest that (i) inhibiting nitric oxide synthase by L-NAME
increases epinephrine-induced arrhythmia which is inhibited by L-arginine or MethyB; (ii) either L-NAME
alone or in combination with MethyB prevented cardiac muscle injury induced by epinephrine; (iii) L-NAME
did not prevent the cardiac protective and antiarrhythmic actions of MethyB.
Key-Words: - cardiac arrhythmia; epinephrine; L-arginine; L-NAME; antiarrhythmic; cardioprotection;
metylene blue; nitric oxide synthase
Received: June 11, 2022. Revised: September 4, 2023. Accepted: September 27, 2023. Published: October 10, 2023.
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1 Introduction
The development of cardiac arrhythmias is common
during anesthesia and surgery. The intra-operative
use of the vasopressor drug epinephrine to increase
cardiac inotropy and maintain blood pressure can
cause clinically relevant cardiac arrhythmias, [1],
[2]. The administration of this catecholamine also
carries the risk of causing direct cardiac muscle
toxicity, apoptosis of cardiac myocytes, [3], [4], and
focal necrosis of the myocardium, [3], [5]. These
effects of catecholamines involve mainly ß-
adrenergic receptors, [4], [5], [6], in addition to an
α-adrenergic receptor-mediated vascular spasm and
myocardial ischemia, [7], and are attributable to the
oxidation products of catecholamines such as
adrenochrome and other reactive oxygen species,
[7], [8].
Nitric oxide plays an important role in cardiac
physiology in the regulation of cardiac excitability
and contractility and in the control of vascular tone
and coronary blood flow, [9]. It is also involved in
pathological disease states e.g., heart failure and
coronary artery disease, [10]. Nitric oxide is
produced from L-arginine by the action of nitric
oxide synthases in endothelial cells, cardiac
myocytes (endothelial nitric oxide synthase), and
nerves (neuronal nitric oxide synthase). A third
isoform (inducible nitric oxide synthase) is not
constitutively expressed, but is induced by
inflammatory signals, [11]. There is evidence that
endogenous nitric oxide may have a cardiac
protective role in suppressing cardiac arrhythmias
caused by ischemia-reperfusion injury, by its ability
to maintain synchronous beating and conductivity,
coronary vasodilatation, a decrease in oxidative
stress, [12], and suppression of sympathetic nerve
activity, [9].
The pre- or intra-operative use of the synthetic
dye methylene blue (MethyB) is an effective rescue
therapy in paraplegic shock, occurring during or
after cardiac surgery requiring cardiopulmonary
bypass and characterized by decreased systemic
vascular resistance, and severe hypotension
unresponsive to intravenous fluids and vasopressor
drugs, [13], [14]. The vasoplegic syndrome is
attributed to a systemic inflammatory response with
excessive production of nitric oxide and
inflammatory cytokines, [15]. Methylene blue, by
virtue of its ability to inhibit nitric oxide synthases,
[16] and inhibit soluble guanylate cyclase, thereby,
preventing cyclic guanosine 3’5’-monophosphate–
dependent vasorelaxant action of nitric oxide, [17],
is thought to counteract the effect of the excessively
released nitric oxide on vascular smooth muscle
cells, and increase the response to vasopressors in
vasoplegic shock, [15]. MethyB has been shown to
exhibit cardioprotective effects, [18], [19], and to
inhibit epinephrine-induced arrhythmias and cardiac
muscle injury, [20].
The aims of this study were therefore to: (i)
investigate the effects of inhibiting nitric oxide
synthases by NG-nitro-L-arginine methyl ester (L-
NAME) on the epinephrine-induced cardiac
arrhythmias and muscle injury; (ii) examine the role
of nitric oxide in the antiarrhythmic and cardiac
protective actions of MethyB.
2 Materials and Methods
2.1 Animals
Male Sprague-Dawley rats weighing 170-180 g
were used in the study. Rats were obtained from the
Animal House Colony of the National Research
Centre. Animals were kept under temperature- and
light-controlled conditions (20–22 C and a 12-hour
light/dark cycle) and given free access to tap water
and standard laboratory rodent chow. Animal
procedures followed the guidelines of the Institute's
ethics committee for the use of animals in
experimental studies and the Guide for Care and
Use of Laboratory Animals by the U.S. National
Institutes of Health.
2.2 Drugs and Chemicals
NG-nitro-L-arginine methyl ester (L-NAME),
methylene blue (Sigma Chemical Co., St. Louis,
MO, U.S.A), and epinephrine (Nile Co., Egypt)
were used in the study and freshly dissolved in
saline before the experiments to obtain the
necessary doses.
2.3 Experimental Groups
Rats were randomly divided into six equal groups (n
= 8 per group) and treated as follows:
Group 1 was given intraperitoneal (i.p.) saline
(served as a negative control).
Group 2 was given i.p. saline before induction of
cardiac arrhythmia by i.v. injection of 10 μg/kg of
epinephrine (served as a positive control).
Group 3 was given L-NAME (40 mg/kg, i.p.) only
Group 4 was given L-NAME (40 mg/kg, i.p.), 30
min before the arrhythmia was induced by an i.v.
injection of epinephrine.
Group 5 was given L-NAME (40 mg/kg, i.p.) 30
min before administering L-arginine (200 mg/kg),
and followed 30 min later by an i.v. injection of
epinephrine.
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Group 6 was given L-NAME (40 mg/kg, i.p.) 30
min before administering MethyB (100 mg/kg), and
followed 30 min later by an i.v. injection of
epinephrine.
2.4 Electrocardiography
After 30 minutes of drug or saline administration,
rats were anesthetized with an intraperitoneal
injection of thiopental sodium (45 mg/kg). The ECG
was then recorded with the ECG Powerlab module.
The latter consisted of Powerlab/8sp and Animal
Bio-Amplifier (Australia), in addition to Lab Chart
7 software with an ECG analyzer. After the
establishment of a steady state, arrhythmia was
induced by the i.v. injection of 10 µg/kg
epinephrine. ECG recording was continued until the
termination of the arrhythmia, [21]. The average
heart rate, RR interval, PR interval, QRS interval,
QTc (corrected QT interval), R wave amplitude, ST
height, number of extrasystoles, and duration of
arrhythmia after different treatments were
determined over 15 minutes.
2.5 Cardiac Histopathology
Cardiac specimens were immediately fixed in 10%
formalin at room temperature, treated with a
conventional grade of alcohol and xylol, embedded
in paraffin, and sectioned at 5 µm thicknesses. The
sections were stained with haematoxylin and eosin
(H&E) to study the histopathological changes using
a light microscope (Olympus CX 41 with DP12
Olympus digital camera).
2.6 Statistical Analysis
Data are presented as mean ± SE for measurement
variables over 15 minutes. Comparison between
groups was performed with a one-way analysis of
variance (ANOVA) followed by Tukey’s multiple
comparison test. GraphPad Prism 6 for Windows
(GraphPad Prism Software Inc., San Diego, CA,
USA) was used, and differences were considered
statistically significant when probability values were
less than 0.05.
3 Results
3.1 Electrocardiographic Recordings
A representative electrocardiographic (ECG) trace
of the saline control is shown in Figure 1. ECG
recordings in the L-NAME-only group showing ST
segment depression are presented in Figure 2. ECG
recordings in the epinephrine control group showed
the presence of bradycardia and ventricular
extrasystoles (Figure 3). The group treated with L-
NAME plus epinephrine showed ventricular
extrasystoles and ventricular tachycardia (Figure 4
and Figure 5). ECG changes in the L-NAME plus
epinephrine-treated group were prevented by prior
treatment with MethyB (Figure 6).
Fig. 1: Representative ECG tracing in the saline
control group.
Fig. 2: Representative ECG changes in L-NAME-
only-treated group.
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Fig. 3: Representative ECG tracings of the changes
induced by intravenous epinephrine injection.
Bradycardia, ventricular premature beats, and
ventricular tachycardia.
Fig. 4: Representative ECG tracings of the changes
in the epinephrine and L-NAME-treated group.
Ventricular premature beats.
Fig. 5: Representative ECG tracings of the changes
in the epinephrine and L-NAME-treated group
Ventricular premature beats and ventricular
tachycardia.
Fig. 6: Representative ECG tracings of the
epinephrine, L-NAME, and MethyB-treated group.
3.2 Electrocardiographic Parameters
3.2.1 Effects of Epinephrine
The heart rate in the saline control group was 409.4
± 3.18 beats/min. The i.v. administration of
epinephrine caused marked bradycardia (221.0 ±
1.37 vs. 409.4 ± 3.18 beats/min), increased PR
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interval (0.057 ± 0.001 vs. 0.043 ± 0.0002 s), RR
interval (0.548 ± 0.016 vs. 0.148 ± 0.001 s),
shortened QTc interval (0.096 ± 0.0093 vs. 0.177 ±
0.0008 s), increased QRS duration (0.040 ±0.0035
vs. 0.0185 ± 0.0002 s), decreased R wave amplitude
(0.176 ± 0.0051 vs. 0.21 ± 0.0009 mv), ST segment
height (-0.075 ± 0.007 vs. 0.002 ± 0.0005 mv), and
induced ventricular extrasystoles (Table 1, Figure 7
and Figure 8).
3.2.2 Effects of L-NAME
L-NAME given to untreated control rats resulted in
sinus bradycardia (288.2 ± 0.88 vs. 409.4 ± 3.18
beats/min), increased RR interval (0.20 ± 0.001 vs.
0.148 ± 0.001 s), increased PR interval (0.057 ±
0.001 vs. 0.043 ± 0.0002 s), increased QRS duration
(0.030± 0.0009 vs. 0.0185 ± 0.0002 s), shortened
Qtc interval (0.092 ± 0.0024 vs. 0.177 ± 0.0008 s)
and increased R wave amplitude (0.436 ± 0.004 vs.
0.21 ± 0.0009 mv) and decreased ST segment height
(-0.075 ± 0.0009 vs. 0.0023 ± 0.0005 mv) (Table 1,
Figure 7 and Figure 8).
3.2.3 Effects of Epinephrine and L-NAME
In epinephrine-treated rats, L-NAME decreased
heart rate (155.9 ± 1.42 vs. 221.0 ± 1.37 beats/min),
increased R wave amplitude (0.36 ± 0.0018 ±
0.0018 vs. 0.176 ± 0.0051 mv) and decreased ST
segment height (-0.054 ± 0.0011 vs. -0.026 ± 0.007
mv) compared to epinephrine control. On the other
hand, the number of extrasystoles and duration of
arrhythmia induced by epinephrine injection was
significantly increased by administering L-NAME
(Table 1, Figure 7, and Figure 8).
3.2.4 Effects of epinephrine, L-NAME and L-
arginine
The administration of L-arginine to epinephrine and
L-NAME-treated rats resulted in increased heart rate
(307.3 ± 2.10 vs. 155.9 ± 1.42 beats/min), increased
R wave amplitude (0.480 ± 0.0015 vs. 0.36 ± 0.0018
mv) and markedly decreased the number of
extrasystoles and duration of arrhythmia compared
to epinephrine + L-NAME group (Table 1, Figure 7
and Figure 8).
3.2.5 Effects of epinephrine, L-NAME, and
MethyB
The ECG changes induced by epinephrine and L-
NAME in heart rate, QRS duration, and ST segment
height were markedly ameliorated by prior
treatment with MethyB which also caused marked
inhibition of extrasystoles and markedly shortened
the duration of arrhythmia compared to rats treated
with epinephrine or L-NAME + epinephrine (Table
1, Figure 7 and Figure 8).
Table 1. Effect of L-NAME or L-NAME + MethyB on epinephrine-induced electrocardiogram changes and
arrhythmia.
Parameter/ Group
Normal
control
L-NAME
Epinephrine
+
L-NAME
Epinephrine +
L-NAME +
L-arginine
Epinephrine +
L-NAME+
MethyB
Heart rate/min
409.4 ± 3.18
288.2 ± 0.88*+
155.9 ± 1.42*+
307.3 ± 2.10*+#
320.3 ± 1.72*+#
RR interval
(s)
0.148 ± 0.001
0.20 ± 0.001*+
0.446 ± 0.01*+
0.198 ± 0.004*+#
0.178 ± 0.003*+#
PR interval
(s)
0.043 ± 0.0002
0.057±
0.0018*
0.056 ± 0.0001*
0.054 ± 0.007*
0.052 ±0.0011*
QTc interval
(s)
0.177 ± 0.0008
0.092 ±
0.0024*
0.102 ± 0.009*
0.106 ± 0.003*
0.115± 0.010*+
QRS duration
(s)
0.0185 ±
0.0002
0.030±
0.0009*+
0.031± 0.001*+
0.024 ±0.002*+#
0.017± 0.002*+#
R wave amplitude
(mv)
0.21 ± 0.0009
0.436 ±
0.004*+
0.36 ± 0.0018*+
0.480 ±
0.0015*+#
0.32 ± 0.0063*+#
ST segment height
(mv)
-0.0023 ±
0.0005
-0.075 ±
0.0009+
-0.054 ±
0.0011*+
-0.035 ±
0.0051*#
-0.003 ± 0.0012+#
Duration of arrhythmia
(s)
0.0 ± 0.0
0.0 ± 0.0
3191 ± 75.46*+
249 ± 10.3*+#
100.1 ± 12.39*+#
Number of
extrasystoles/15 min
0.0 ± 0.0
0.0 ± 0.0
1101 ± 40.14*+
252.5 ± 8.1*+#
130.1 ± 10.01*+#
MethyB: methylene blue. Data were expressed as mean ± SE (n = 8). Data were analyzed by one-way ANOVA followed by
Tukey’s multiple comparison test. *: p<0.05: significantly different from the normal control group. +: p<0.05:
significantly different from the epinephrine control group. #: p<0.05: significantly different from the epinephrine + L-
NAME group.
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C o n t r o l
L - N A M E ( 4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0
100
200
300
400
500
H e a r t r a t e (b e a t s / m i n )
*
*
*
+
+
+
*
*+
##
C o n t r o l
L - N A M E (4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0 .0
0 .2
0 .4
0 .6
R R i n t e r v a l ( s e c )
*
*
*
*+
+
++
*#
#
C o n t r o l
L - N A M E ( 4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0 .0 0
0 .0 2
0 .0 4
0 .0 6
0 .0 8
P R in t e r v a l ( s e c )
*****
C o n t r o l
L - N A M E ( 4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0 .0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
Q T c in t e r v a l )
**
*
**
+
C o n t r o l
L - N A M E (4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0 .0 0
0 .0 1
0 .0 2
0 .0 3
0 .0 4
0 .0 5
Q R S d u r a t i o n
+
+
*
*
*
*
*
+
+
#
#
C o n t r o l
L - N A M E (4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0 .0
0 .2
0 .4
0 .6
R w a v e a m p l i tu d e ( m v )
*
+
+
*
*
*
+
#
+
*
#
Fig. 7: Effects of treatment with L-NAME, L-
NAME + L-arginine, or L-NAME plus methylene
blue (MethyB) on the epinephrine-induced changes
in heart rate, RR interval, PR interval, QTc, QRS
duration, and R wave amplitude. *: p<0.05:
significantly different from the normal control
group. +: p<0.05: significantly different from the
epinephrine control group. #: p<0.05: significantly
different from the epinephrine + L-NAME group.
C o n t r o l
L - N A M E ( 4 0 m g / k g )
E p i n (1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
- 0 . 0 5
0 .0 0
S T s e g m e n t (m v )
*
*
+
+
*
+
+
#
#
C o n t r o l
L - N A M E ( 4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0
1000
2000
3000
4000
D u r a t io n o f a r r h y t h m i a ( s e c )
+
*
*
*+
*+#
#
C o n t r o l
L - N A M E ( 4 0 m g / k g )
E p i n ( 1 0 / k g )
E p i n + L - N A M E
E p i n + L - N A M E + L - a r g i n i n e
E p i n + L - N A M E + M e t h y B
0
500
1000
1500
N u m b e r o f e x t r a s y s t o l e s
+
*
*
+
*
*
+
#
#
Fig. 8: Effects of treatment with L-NAME, L-
NAME plus L-arginine or L-NAME and methylene
blue (MethyB) on the epinephrine-induced changes
in ST wave height, duration of arrhythmia, and
number of ventricular extrasystoles. *: p<0.05:
significantly different from the normal control
group. +: p<0.05: significantly different from the
epinephrine control group. #: p<0.05: significantly
different from the epinephrine + L-NAME group.
3.2 Cardiac Histopathology
Sections of the saline control group showed normal
myocardial architecture. The myofibers were intact,
branching, and cylindrical with acidophilic
cytoplasm and exhibited vesicular nuclei, transverse
striations, and obvious intercalated discs. They were
separated by scanty connective tissue containing
fibroblasts that were identified by their flat nuclei
(Figure 9A). Cardiac tissues of rats treated with
epinephrine showed many histopathological
changes, especially in the cardiac cells. There were
disorganized cardiac cells, necrotic cardiac cells
with focal areas of degeneration, widening of the
intercellular spaces, and deeply stained (pyknotic)
nuclei. Congestion and dilatation of blood vessels
with interstitial haemorrhage were seen. Marked
cellular infiltrations were common in many sections
(Figure 9B).
The group treated with epinephrine and L-
NAME showed nearly normal myocardial
architecture, with mild interstitial haemorrhage and
congestion of blood vessels. Most myofibers were
intact, while others had mild widening of the
intercellular spaces and few pyknotic nuclei (Figure
9C). Meanwhile, rats treated with epinephrine and
L-NAME with MethyB showed almost normal
myocardium and minimal interstitial haemorrhage.
Most myofibers were intact, while others had mild
widening of the intercellular spaces and few
pyknotic nuclei (Figure 9D).
Fig. 9: Representative photomicrographs of Hx & E
stained heart tissue sections. (A) Saline control
shows the normal histological architecture of
cardiac myocytes (M). Most appear longitudinally
with rounded vesicular centrally located nuclei (N).
In between the cardiac myocytes, there was a
delicate layer of connective tissue. (B) Epinephrine
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showing disorganized cardiac cells, necrotic cardiac
cells with focal areas of degeneration (arrowhead),
widening of the intercellular spaces (Ct) and deeply
stained (pyknotic) nuclei (P), congestion and
dilatation of blood vessels (Bv) with interstitial
haemorrhage (star) and marked cellular infiltrations
(arrow). (C) Epinephrine and L-NAME showing
nearly normal myocardial architecture, with mild
interstitial haemorrhage (star) congestion blood
vessels (Bv). Most myofibers were intact while
others had mild widening of the intercellular spaces
(Ct) and few pyknotic nuclei (P). (D) Epinephrine +
L-NAME + MethyB showing almost normal
myocardium and minimal interstitial haemorrhage
(star). Most myofibers were intact while others had
mild widening of the intercellular spaces (Ct) and
few pyknotic nuclei (P).
4 Discussion
In this study, we investigated whether nitric oxide
synthase blockade with L-NAME has a modulating
effect on the development of arrhythmias and
cardiac muscle injury after epinephrine infusion in
rats. Because MethyB, an inhibitor of nitric oxide
synthase exerts anti-arrhythmic and cardiac
protective effects, we also investigated whether
these actions of MethyB could be affected by NOS
inhibition with L-NAME. The results of this study
showed that L-NAME itself induced ECG changes
in the form of sinus bradycardia, shortened Qtc
interval, and an increase in QRS duration and R
wave amplitude. On the other hand, in epinephrine-
treated rats, L-NAME caused an increase in the
bradycardiac response but counteracted the increase
in QRS duration and R wave amplitude induced by
epinephrine. However, L-NAME increased the
number of ventricular extrasystles and the duration
of the arrhythmia induced by epinephrine. L-
arginine, the precursor of nitric oxide was found to
counteract the bradycardiac response and suppress
the number of ventricular extrasystles and duration
of arrhythmia compared with either epinephrine
alone or epinephrine plus L-NAME. These findings
may suggest that endogenous nitric oxide is endued
with an anti-arrhythmic function. Other researchers
reported an increase in blood pressure and a
bradycardiac response to i.v. L-NAME (7.5 mg/kg)
in intact anesthetized rats, [22]. Our results are also
supported by the study of Pabla and Curtis, [23], in
the rat-isolated heart where L-NAME caused
significant bradycardia. In their study, L-NAME
exacerbated ventricular fibrillation in hearts
subjected to 60 minutes of ischaemia. Because these
L-NAME effects were counteracted by L-arginine,
the substrate for nitric oxide synthase, they were
attributed to a decrease in nitric oxide
bioavailability.
Nitric oxide produced from the cardiac
endothelial cells, cardiac myocytes, and nerves
participates in the regulation of cardiac excitability
and contractility, [9]. Studies have suggested that
the provision of nitric oxide protection, whereas
inhibition of nitric oxide synthesis increased
ischemia-reperfusion injury, [12]. Moreover, a study
by [21], has suggested that the release of nitric oxide
and prostaglandins may account for the protection
against epinephrine-induced arrhythmia by
bradykinin. Several mechanisms have been
postulated to account for the anti-arrhythmogenic
action of nitric oxide, including effects on ion
channels and gap junctions, increase in intracellular
cyclic GMP via guanylyl cyclase, and a decrease in
oxidative stress-mediated arrhythmogenesis, [12].
Moreover, the basal tone of nitric oxide was found
to suppress the stimulatory action of sympathetic
nerve activity in the heart, [9]. Accordingly,
blockade of nitric oxide synthesis by L-NAME
would be expected to enhance epinephrine-mediated
stimulation of the myocardium. Nitric oxide is also
important in the maintenance of vascular tone and
thus coronary blood flow, [11], and it is possible
that blockade of nitric oxide synthesis by L-NAME
with a consequent decrease in cardiac muscle
perfusion accounted for the exacerbation of
ventricular arrhythmias following epinephrine
infusion in the present study.
Excessive amounts of epinephrine can cause
direct cardiac toxicity, band necrosis, and
stimulation of myocyte apoptosis, [3], [4], [5].
Myocardial cellular damage caused by epinephrine
is due to the direct stimulation of adrenergic beta 1
receptors in cardiac myocytes, which involves
increases in cAMP and Ca2+ influx, [4], [6]. In
addition, there is the effect of stimulation of α-
adrenergic receptors in coronary arteries, which
results in a decrease in coronary perfusion, and thus
myocardial ischemia, [7]. There is also evidence for
the involvement of adrenochrome, an oxidation
metabolite of epinephrine, [8], and reactive oxygen
metabolites, [7], in causing myocardial cell damage.
In the present study, we found that inhibition of
nitric oxide synthesis with L-NAME conferred
protection against the severe cardiac muscle injury
caused by epinephrine. The mechanism underlying
this effect of L-NAME is not clear but may be
related to decreased formation of oxyradicals such
as peroxynitrite and other reactive species that are
generated during myocardial ischemia and/or
oxidation of catecholamines, [7], [8]. In support of
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.15
Omar M. E. Abdel-Salam,
Marawan Abd El Baset Mohamed Sayed,
Enayat A. Omara, Amany A. Sleem
E-ISSN: 2224-2902
151
Volume 20, 2023
this notion are studies that have reported an
antiarrhythmic effect for antioxidants in
epinephrine-induced arrhythmia in rats, which could
be caused by the antioxidant's ability to decrease the
circulating levels of aminochromes produced by the
oxidative metabolism of the catecholamine, [24],
[25], [26].
MethyB protects against epinephrine-induced
arrhythmia and cardiac muscle damage, [20].
Because MethyB inhibits nitric oxide synthesis,
[16], [17], we thought that the cardiac protective
effects of MethyB would be affected by decreasing
nitric oxide bioavailability using L-NAME.
Interestingly, we found that after inhibiting nitrite
oxide synthases by L-NAME, MethyB was capable
of suppressing ventricular extrasystoles and almost
restoring the normal cardiac rhythm. Moreover, it
afforded almost complete protection of the
myocardium in rats treated with epinephrine or L-
NAME plus epinephrine. These findings may
suggest that mechanisms other than lowering nitric
oxide levels underlie the beneficial effects of the
dye in the epinephrine model of arrhythmia and
cardiac damage. It is also suggested that while
cardiac nitric oxide dyshomeostasis after L-NAME
is involved in the exacerbation of arrhythmia, the
development of myocardial injury after epinephrine
infusion is likely to be largely mediated by other
pathways, including the adrenergic stimulation of
the myocardium (
1), coronary vasospasm and
decreased myocardial perfusion (
1) and the release
of the oxidation products of epinephrine. Hence,
blocking nitric oxide synthesis with L-NAME did
not affect the cardioprotective action of MethyB.
MethyB may exert its cardioprotective action via an
antioxidant mechanism. In biological systems,
MethyB cycles between its oxidized and reduced
forms. It is reduced by the enzyme NADPH or
thioredoxin to the colorless leucoMethyB, to be re-
oxidized by reacting with O2, [27]. The redox
cycling property of MethyB may help block the
production of reactive oxygen metabolites by the
mitochondria, [28]. The antioxidant action of
MethyB has been shown both in vitro, [29], and in
vivo, [30], [31]. MethyB has been shown to inhibit
the production of superoxide radicals (O2–
) by
xanthine oxidase, and thus prevent free radical-
mediated tissue injury, [29]. The dye was also
shown to improve mitochondrial respiratory
function in cardiac mitochondria, [18], restore the
depleted energy stores in cardiomyocytes following
hydrogen sulfide toxicity, [19], and preserve
intracellular Ca++ homeostasis and excitation-
contraction coupling in mouse myocytes treated
with sodium cyanide, [32].
5 Conclusion
The present results indicate that pretreatment with
the nitric oxide synthase inhibitor L-NAME caused
a significant increase in epinephrine arrhythmias,
which could be ameliorated with L-arginine or
MethyB. L-NAME, however, like MethyB, afforded
histological protection against the deleterious
cardiac muscle damage evoked by the
catecholamine. Our results also indicate that L-
NAME did not inhibit the cardiac protective and
antiarrhythmic actions of MethyB. This finding is of
particular clinical relevance as it suggests that these
cardiac beneficial effects of MethyB may not be
mediated by inhibition of nitric oxide release.
Further research is, therefore, required to delineate
the mechanism (s) underlying the cardiac protective
properties of MethyB.
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WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
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Omar M. E. Abdel-Salam,
Marawan Abd El Baset Mohamed Sayed,
Enayat A. Omara, Amany A. Sleem
E-ISSN: 2224-2902
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Contribution of Individual Authors to the
Creation of a Scientific Article (Ghostwriting
Policy)
Omar Abdel-Salam, Marawan Abd El Baset, and
Amany Sleem designed the study. Marwan Abd El
Baset conducted the experiments. Enayat Omara
performed the histological studies and their
interpretation. Omar Abdel-Salam prepared the
manuscript. Omar Abdel-Salam, Marawan Abd El
Baset, Amany Sleem, and Enayat Omara approved
the final version of the manuscript.
Sources of Funding for Research Presented in a
Scientific Article or Scientific Article Itself
No funding was received for conducting this study.
Conflict of Interest
The authors have no conflict of interest to declare.
Creative Commons Attribution License 4.0
(Attribution 4.0 International, CC BY 4.0)
This article is published under the terms of the
Creative Commons Attribution License 4.0
https://creativecommons.org/licenses/by/4.0/deed.en
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WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.15
Omar M. E. Abdel-Salam,
Marawan Abd El Baset Mohamed Sayed,
Enayat A. Omara, Amany A. Sleem
E-ISSN: 2224-2902
154
Volume 20, 2023