Cardioprotection by methylene Blue Against Epinephrine-Induced
Cardiac Arrhythmias and Myocardial Injury
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,
Cairo,
EGYPT
2Department of Pharmacology,
Medical Research and Clinical Studies Institute,
National Research Centre,
Cairo,
EGYPT
3Department of Pathology,
Medical Research and Clinical Studies Institute,
National Research Centre,
Cairo,
EGYPT
*Corresponding Author
Abstract: - Methylene blue is used in the treatment of vasoplegic syndrome after cardiac surgery, anaphylaxis,
and septic shock refractory to epinephrine and fluid resuscitation. In this study, we investigated the potential
protective effect of methylene blue on the development of cardiac arrhythmias after injection of epinephrine in
rats. Methylene blue was given intraperitoneally at doses of 50 or 100 mg/kg. Cardiac arrhythmia was then
induced with 10 μg/kg of epinephrine intravenously. In untreated, control rats, epinephrine caused bradycardia
(96.48 ± 1.06 vs. 365.03 ± 0.68 beats/min), increased PR interval (0.54 ± 0.04 vs. 0.039 ± 0.004), RR interval
(0.64 ± 0.003 vs. 0.16 ± 0.004 sec), shortened QTc interval (0.067 ± 0.05 vs. 0.1 ± 0.004 sec), increased QRS
duration (0.048 ± 0.005 vs. 0.028 ± 0.002 sec), decreased R wave amplitude (0.3 ± 0.03 vs. 0.49 ± 0.04 mv),
decreased the height of the ST segment (-0.0696 ± 0.004 vs. -0.0054 ± 0.003 mv), and caused ventricular
extrasystoles (7.92 ± 0.56 vs. 0.5 ± 0.5). Methylene blue given at 50 or 100 mg/kg increased the heart rate,
decreased RR interval, QRS duration and the drop in the ST height, increased duration of QTc interval and R
wave amplitude and decreased the number of extrasystoles. The histological study showed that methylene blue
protected against myocardial structural disorganization, cellular damage, necrosis, and haemorrhage between
muscle fibres induced by epinephrine injection. We conclude that methylene blue dose-dependently prevented
epinephrine-induced arrhythmias and cardiac muscle injury.
Key-Words: - methylene blue; epinephrine; cardiac arrhythmia; cardioprotection
Received: April 29, 2022. Revised: February 17, 2023. Accepted: March 14, 2023. Published: April 28, 2023.
1 Introduction
Methylene blue is an autoxidizable synthetic
phenothiazine dye with a wide range of clinical
applications. The dye has been used in treatment of
methemoglobinaemia by virtue of its redox-cycling
between its blue oxidized and colorless reduced
(leuco-methylene blue) states [1]. It is also
administered to treat malaria [2] and for the
prevention and treatment of encephalopathy
associated with the alkylating drug ifosfamide in
patients with cancer [3]. One of the most important
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DOI: 10.37394/23208.2023.20.7
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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clinical uses of methylene blue, however, is its
intravenous infusion in patients with vasoplegic
shock occurring during cardiopulmonary bypass
surgery. The vasoplegic syndrome is characterized
by decreased systemic vascular resistance and
severe hypotension which is refractory to therapy
with vasopressors eg., catecholamines and
vasopressin and intravenous administration of fluids
[4,5]. This state of vasodilatory shock is caused by
the development of a systemic inflammatory
response and the over production of nitric oxide
[6,7]. Methylene blue is an inhibitor of nitric oxide
synthases and guanylyl cyclase enzymes thereby
block the production of nitric oxide and antagonize
its vasorelaxant effects [8,9]. This property of
methylene blue is largely thought to mediate its
beneficial effects in increasing the systemic vascular
resistance and mean arterial blood pressure when
administered in cardiosugery, obviating the need for
high doses of catecholamine drugs and vasopressin
[10].
The intraoperative use of epinephrine to treat
hypotension associated with vasoplegic shock or
other clinical states carries the risk of inducing
ventricular arrhythmia and damage to the
myocardium [11,12]. Epinephrine mediates its
cardiovascular effects by stimulating cardiac β1-
and β2- adrenergic receptors causing an increase in
heart rate, myocardial contractility and increased
excitability of pacemaker tissue that may result in
ventricular arrhythmias. It also acts on α1-
adrenergic in cardiac and vascular smooth muscle in
high doses causing cardiac stimulation and
increased peripheral vascular resistance [13,14].
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 12 h/12 h
light/dark cycle) and given free access to tap water
and standard laboratory rodent chow. Animal
procedures followed the guidelines of the Institute
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 (Publication No. 85-23, revised
1996).
2.2 Drugs and Chemicals
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 four equal groups
(n=8/group) and treated as follows:
Group 1: received intraperitoneal (i.p.) saline and
served as negative control.
Group 2: were given i.p. saline prior to induction of
cardiac arrhythmia by intravenous injection of 10
μg/kg of epinephrine (positive control).
Group 3: were treated with i.p. methylene blue at 50
mg/kg, 30 min before the induction of arrhythmia.
Group 4: received i.p. methylene blue at 100 mg/kg,
30 min before the induction of arrhythmia.
2.4 Electrocardiography
After 30 min of drug or saline administration, rats
were anesthetized with i.p injection of 45 mg/kg
thiopental. ECG was recorded using the ECG
Powerlab module, which consists of Powerlab/8sp
and Animal Bio-Amplifier (Australia), in addition to
Lab Chart 7 software with ECG analyzer.
After a steady state was established, arrhythmia was
induced by intravenous injection of epinephrine at
10 μg/kg and ECG recording continued until the
termination of the arrhythmia [15]. The heart rate,
RR interval, PR interval, QRS interval, QT Interval,
QTc, R wave amplitude, ST height, number of
extrasystoles, and duration of heart block after
epinephrine injection were determined.
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) in order to study the histopathological
changes using a light microscope (Olympus Cx 41
with DP12 Olympous digital camera).
2.6 Statistical Analysis
Data are presented as mean ± SE for measurement
variables. Comparison between groups was
performed with 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
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statistically significant when probability values were
less than 0.05.
3 Results
3.1 Electrocardiographic Results
Following epinephrine injection, ECG recordings
showed variable degrees of bradycardia and
ventricular extrasystoles. Representative ECG
tracings in saline control and after epinephrine
injection are shown in figures 1 & 2. The ECG
changes induced by epinephrine were ameliorated
by prior treatment with methylene blue in a dose-
dependent manner (Figure 3 & Figure 4).
Fig. 1: Representative ECG tracing in saline control.
Fig. 2: Two representative ECG tracings of the
changes induced by epinephrine injection.
Ventricular premature beats and bradycardia.
Fig. 3: Two representative ECG tracings of the
effect of methylene blue at 50 mg/kg on the changes
induced by epinephrine injection.
Fig. 4: Two representative ECG tracings of the
effect of methylene blue at 100 mg/kg in
epinephrine-treated rats.
The heart rate of saline control rats was 365.0 ± 0.68
beats/min. Epinephrine control had significantly
lower heart rate by 73.6% (96.5 ± 1.06 beats/min).
Methylene blue given at 50 or 100 mg/kg reversed
the epinephrine-induced bradyarrhythmia and
increased the heart rate by 124.5% and 130.5%,
respectively, as compared to the epinephrine control
group. Epinephrine injection significantly increased
RR interval by 300% from 0.16 ± 0.004 sec in the
saline control group to 0.64 ± 0.003 sec. Treatment
with methylene blue 50 or 100 mg/kg reduced the
RR interval after epinephrine-induced arrhythmia by
57.8% and 60.9%, respectively, compared to the
epinephrine control group.
Rats treated with epinephrine exhibited significantly
longer PR interval by 38.5% as compared to the
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saline group. Methylene blue had no significant
effect on PR interval in the epinephrine-treated rats.
Epinephrine significantly shortened QTc interval by
35.6% as compared to the saline control. Methylene
blue given at 50 or 100 mg/kg prevented the
epinephrine-induced shortening of QTc interval.
Epinephrine control rats exhibited longer QRS
duration compared with the saline control by 71.4%.
The higher dose of methylene blue brought QRS
duration almost to the normal saline group value.
R wave amplitude of the epinephrine group was
significantly decreased by 38.8% compared with the
saline control value. However, methylene blue given
at 50 or 100 mg/kg, increased R wave amplitude by
153.3% and 76.7%, respectively, compared to the
epinephrine control value. It was also noted that R
wave amplitude in rats treated with methylene blue
at 50 mg/kg was significantly higher than in the 100
mg/kg group.
The ST height is different in rodents than humans as
it is not iso-electric and appears as a shoulder
emerging from QRS complex. In the epinephrine
group, ST height decreased to -0.0696 ± 0.004 mv
from normal saline control value of -0.0054 ± 0.003
mv. Methylene blue administered at 50 mg/kg,
significantly increased the drop in ST height. In
contrast, treatment with 100 mg/kg methylene blue
returned the ST height to comparable value to that
of the saline group value.
Methylene blue (50 or 100 mg/kg) reduced the
duration of epinephrine-induced bradycardia by
60.3% and 92.0%, respectively. It also significantly
reduced the number of epinephrine-induced
extrasystoles by 71.6% and 89.4%. The effect of
methylene blue on ECG tracings and ECG
parameters is shown in Table 1, Figure 5 & Figure
6.
Table 1. Effect of methylene blue on epinephrine-induced electrocardiogram parameters and arrhythmia.
Parameter/ Group
Normal
control
Epinephrine
Epinephrine + MB 100 mg/kg
Heart rate (bpm)
365.0 ± 0.68
96.5 ± 1.06*
222.4 ± 1.64*+#
RR interval (s)
0.16 ± 0.004
0.64 ± 0.003*
0.25 ± 0.002*+#
PR interval (s)
0.039 ± 0.004
0.054 ±
0.004*
0.049 ± 0.003
QT interval (s)
0.042 ± 0.003
0.053 ± 0.005
0.049 ± 0.006
QTc interval (s)
0.104 ± 0.004
0.067 ±
0.006*
0.09 ± 0.005*+#
QRS duration (s)
0.028 ± 0.002
0.048 ±
0.005*
0.024 ± 0.003+#
R wave amplitude (mv)
0.49 ± 0.004
0.30 ± 0.003*
0.53 ± 0.005*+#
ST segment height
(mv)
-0.005± 0.003
-0.07 ± 0.005*
-0.02 ± 0.004+#
Duration of bradycardia
(s)
0.0 ± 0.0
831.7 ± 16.9*
66.25 ± 15.0+
Number of
extrasystoles
0.0 ± 0.0
7.92 ± 0.56*
1.5 ± 0.19+
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MB: methylene blue. Data were expressed as mean
± SE (n = 12). Data were analyzed by one-way
ANOVA followed by Tukey’s multiple comparison
test. *p<0.05: significantly different from normal
control group. +p<0.05: significantly different from
epinephrine control group. #p<0.05: significantly
different from the MB 50 mg/kg group.
N or m al
E p i n ep h r in e
M B (5 0 m g /k g)
M B (1 00 m g /k g)
0.0
0.2
0.4
0.6
0.8
R R in te rva l (s)
*
+#
**+
N or m al
E p i n ep h r in e
M B (5 0 m g /kg)
M B (1 00 m g /k g)
0.0 0
0.0 2
0.0 4
0.0 6
0.0 8
P R in ter va l (s)
**
N or m al
E p i n ep h r in e
M B (5 0 m g /kg)
M B (1 00 m g /k g)
0.0 0
0.0 2
0.0 4
0.0 6
0.0 8
Q T (s)
N or m al
E p i n ep h r in e
M B (5 0 m g /kg)
M B (1 00 m g /k g)
0
100
200
300
400
H eart ra te (b pm )
*
**
++#
N or m al
E p i n ep h r in e
M B (5 0 m g /kg)
M B (1 00 m g /k g)
0.0 0
0.0 2
0.0 4
0.0 6
Q R S d ur ation (s )
*
*
+ #
N or m al
E p i n ep h r in e
M B (5 0 m g /kg)
M B (1 00 m g /k g)
0.0 0
0.0 5
0.1 0
0.1 5
Q T c (s )
#
*
*+
+
Figure 5. Effects of treatment with methylene blue
(MB) on the epinephrine-induced changes in heart
rate, RR interval, PR interval, QT, QTc, and QRS
duration. *p<0.05: significantly different from
normal control group. +p<0.05: significantly
different from epinephrine control group. #p<0.05:
significantly different from MB 50 mg/kg group.
N or m al
E p i n ep h r in e
M B (5 0 m g /kg)
M B (1 00 m g /k g)
0
200
400
600
800
1000
D ura tio n of h ea rt b lo ck (s )
*
*
+
+
*#
N orm a l
E p i n ep h r in e
M B (5 0 m g /k g)
M B (1 00 m g /k g)
0
5
10
15
N um b er o f ex tr a sy stole
*
*++ #
N orm a l
E p i n ep h r in e
M B (5 0 m g /k g)
M B (1 00 m g /k g)
0.0
0.2
0.4
0.6
0.8
1.0
R am p litu de (m V )
+
+
*
*
*#
N or m al
E p i n ep h r in e
M B (5 0 m g /kg)
M B (1 00 m g /k g)
-0 .1 5
-0 .1 0
-0 .0 5
0.0 0
ST heigh t (m V )
*
#+
+
*
Fig. 6: Effects of treatment with methylene blue
(MB) on the epinephrine-induced changes in R
wave amplitude, ST wave height, duration of
bradycardia and number of ventricular extrasystoles.
*p<0.05: significantly different from normal control
group. +p<0.05: significantly different from
epinephrine control group. #p<0.05: significantly
different from MB 50 mg/kg treatment group.
3.2 Histopathological Results
In the saline control group, the myocardium was
formed of longitudinally striated cardiac myocytes
with central oval pale nuclei. They were joined
together by intercalated discs and appeared
branching and anastomosing forming muscle sheets.
In-between the cardiac myocytes, there was a
delicate layer of connective tissue with well-
demonstrated blood vessels (Fig.7A). In the
adrenaline only group, the myocardium
demonstrated structural disorganization with
features of inflammation and cellular damage,
necrosis, widening of the intercellular spaces with
active fibroblasts and thickened delicate connective
tissue. There were many dilated, congested blood
vessels, haemorrhage between muscle fibres, and
pyknotic nuclei (Fig. 7B).
Sections of the adrenaline and methylene blue at 50
mg/kg group presented mild to moderate features of
myocardial lesion with dilated congested blood
vessels, inflammatory cells infiltrations, mild deeply
stained (pyknotic) nuclei and mild thickened
delicate connective tissue (Fig. 7C). Whereas,
sections of the adrenaline and methylene blue at 100
mg/kg group showed nearly normal histological
architecture, except for the presence of dilated
congested blood vessels, mild thickened delicate
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connective tissue, and very few shrunken darkly
stained nuclei (Fig. 7D).
Fig. 7: Representative photomicrographs of (A):
Saline control showing 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)
Adrenaline control showing disorganization cardiac
structure, with features of inflammation (arrow),
necrosis (arrowhead) with widening of the
intercellular spaces with active fibroblasts (F),
thickened delicate connective tissue (Ct), dilated,
congested blood vessels (Star), haemorrhage (H)
between muscle fibres, and pyknotic nuclei (P). (C)
Adrenaline and methylene blue 50 mg/kg showing
moderate features of myocardial lesion with mild
cellular inflammation (arrow), mild necrosis
(arrowhead), pyknotic nuclei (P), slight congested
blood vessels (star). (D) Adrenaline and methylene
blue 100 mg/kg showing most of cardiac muscle
fibres appearing more or less normal. Few areas still
show cellular inflammation (arrow), mild necrosis
(arrowhead), pyknotic nuclei (P), and slight
congested blood vessels (star).
4 Discussion
The results of the present study showed that
epinephrine-induced arrhythmia and ECG changes
were reduced by prior with methylene blue. The i.v.
administration of 10 µg/kg epinephrine caused
severe bradycardia and first degree heart block as
well as polymorphic ventricular premature beats.
The ECG showed increased RR, PR and QTc
intervals, QRS widening, a decrease in R wave
height and ST segment. Methylene blue prevented
these epinephrine-induced changes accompanied by
marked decrease in the duration of heat block and
number of ventricular extrasystoles. The study
supports and extends previous work in which
methylene blue protected against arrhythmias and
restored contractility in mouse cardiac myocytes
after intoxication with hydrogen sulfide [16].
The mechanism of epinephrine-induced arrhythmia
and cardiac muscle injury is thought to involve
direct stimulation of β-adrenoceptors in
cardiomyocytes resulting in increased cyclic AMP
and intracellular Ca2+, while stimulation of α-
adrenergic receptors in coronary arteries induces
coronary spasm and myocardial ischemia. Added to
this are the effects evoked by the oxidation products
adrenochromes and oxyradicals [17]. In the perfused
rat heart preparation, the leakage of lactate
dehydrogenase induced by epinephrine is prevented
by the β1-adrenoceptor antagonist atenolol [18]. It
has also been suggested that epinephrine induces
cardiac arrhythmia partly by the local release of
acetylcholine i.e. a cholinergic mechanism is
involved [19]. Moreover, intracerebroventricular
(i.c.v.) injection of dynorphin caused an increase in
the threshold for epinephrine-induced ventricular
arrhythmias which is opposed by i.c.v. or i.v.
atropine sulfate, suggesting mediation by central
cholinergic mechanisms [20]. Other studies showed
that the antiarrhythmic action of bradykinin [15] or
ATP-sensitive potassium channel opener nicorandil
[21] against epinephrine arrhythmia is mediated by
nitric oxide.
Methylene blue has been used in treatment of
hypotension and reduced systemic vascular
resistance i.e., vasoplegic shock, that occurs during
cardiopulmonary bypass despite intravenous
norepinephrine and fluids [10]. This condition is
thought to be caused in part by initiation of a
systemic inflammatory response resulting in
increased production of reactive oxygen
metabolites, cytokines, and increased production of
nitric oxide by the enzyme nitric oxide syntheses
(NOS) [6,7]. The administration of ethylene blue in
the event of cardiopulmonary bypass results in
improved hemodynamic, reduced the requirement of
vasopressin agents and decreased serum lactate
levels [22]. Methylene blue was also reported to
increase mean arterial pressure and improve cardiac
function in septic shock [23]. The beneficial effect
of methylene blue is largely attributed to
counteracting the vasorelaxant action of the
excessively released nitric oxide [10, 24].
Methylene blue inhibits nitric oxide production
through inhibition of nitric oxide synthases [9].
Moreover, methylene blue binds to the heme moiety
of the enzyme guanylate cyclase. In this way, the
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dye prevents the activation of guanylyl cyclase, the
increase in cyclic guanosine 3’5’ -monophosphate
and the subsequent vascular relaxation [9, 24]. It is
not clear, however, that this action of methylene
blue in inhibiting nitric oxide production have
accounted for its antiarrhythmic effect reported
herein.
Methyblue is highly lipophilic and readily crosses
the blood brain barrier [25] and exert
neuroprotective effects [26,27]. The dye and its
metabolites are also a reversible and competitive
inhibitor of both acetylcholinesterase and
butyrylcholinesterase [28-30]. In view of data
suggestive of a cholinergic mechanism being
involved in epinephrine arrhythmia [19,20], an
intriguing possibility is that the above action of
methylene blue may underlie at least in a part its
antiarrhythmic properties.
Oxidative stress has been involved in the genesis of
cardiac arrhythmias [31,32]. In rats with epinephrine
arrhythmia, the glutathione precursor N-acetyl-L-
cysteine or vitamin E were reported to decrease the
duration and increase the time of onset of
arrhythmias, possibly by reducing the level of the
oxidation products of catecholamines aminochromes
[33]. These products increase intracellular Ca2+,
induce coronary spasm, depletion of high energy
stores, subcellular alterations and cause ventricular
arrhythmias and myocardial cell damage [17].
Methylene blue has antioxidants effects. It was
shown to inhibit the enzyme xanthine oxidase and
the subsequent generation of superoxide radicals
[34]. The dye is a redox-cycling agent, a blue cation
which is reduced by nicotinamide adenine
dinucleotide phosphate (NADPH) or thioredoxin to
give the uncharged and colorless leucoMB to be re-
oxidized by O2 [24] Methylene blue reduces the
formation of brain mitochondrial reactive oxygen
radicals and thus protects and enhances
mitochondrial function and affords neuroprotection
[35]. It also enhances the electron transport chain,
thereby promoting oxygen consumption [36]. In
cardiac mitochondria isolated from diabetic and
normal rat hearts, methylene blue improved
mitochondrial respiratory function [37]. The dye
was also shown to restore ATP levels in
cardiomyocytes exposed to toxic concentrations of
hydrogen sulfide [16]. The above mentioned effects
of methylene blue may explain its antiarrhythmic
and cardioprotective effects reported in the present
study.
5 Conclusion
We conclude that the epinephrine-induced
bradyarrhythmia, ventricular premature beats and
myocardial injury is prevented by prior
administration of methylene blue. The mechanism is
not clear but may involve antioxidant action and
improved mitochondrial function. Further work is
needed in order to elucidate the exact mechanism by
which methylene blue exerts its antiarrhythmic and
cardioprotective effects.
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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 Sayed
conducted the experiments. Enayat Omara
performed the histopathology and its 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
This work was not supported by research grants.
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
_US
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.7
Omar M. E. Abdel-Salam,
Marawan Abd El Baset Mohamed Sayed,
Enayat A Omara, Amany A. Sleem
E-ISSN: 2224-2902
72
Volume 20, 2023