Novel Antiarrhythmic and Cardioprotective Effects of Brilliant Blue G

OMAR M.E. ABDEL-SALAM

Department of Toxicology and Narcotics

Medical Research and Clinical Studies Institute, National Research Centre

Tahrir Street, Dokki, Cairo

EGYPT

MARAWAN ABD EL BASET

Department of Pharmacology

Medical Research and Clinical Studies Institute, National Research Centre

Tahrir Street, Dokki, Cairo

EGYPT

FATMA A. MORSY

Department of Pathology

Medical Research and Clinical Studies Institute, National Research Centre

Tahrir Street, Dokki, Cairo

EGYPT

AMANY A. SLEEM

Department of Pharmacology

Medical Research and Clinical Studies Institute, National Research Centre

Tahrir Street, Dokki, Cairo

EGYPT

Abstract: - In this study, we investigated the effects of the purinergic P2X7 receptor antagonist brilliant blue G

(BBG) on cardiac arrhythmia and myocardial injury induced by intravenously (i.v.) administered epinephrine in

anesthetized rats. We also examined the possible involvement of beta-adrenergic receptors or cholinergic

mechanisms in the effects of BBG. Sprague-Dawley rats were treated with epinephrine (10 μg/kg, i.v.).

Brilliant blue G (100 mg/kg) was intraperitoneally (i.p.) administered thirty minutes prior to i.v. epinephrine.

The effects of pretreatment with propranolol (2 mg/kg, i.p.) or atropine (2 mg/kg, i.v.) given prior to BBG and

epinephrine were examined. The control group received saline. Moreover, the effects of only BBG on

electrocardiogram (ECG) parameters were investigated. Results showed that compared with the saline control,

BBG caused significant bradycardia (from 405.8 ± 1.18 to 239.4 ± 6.69 beats/min), increased RR interval (from

0.149 ± 0.002 to 0.254± 0.007 sec) and PR interval (from 0.051 ± 0.0008 to 0.059 ± 0.0004 sec), increased R

wave amplitude (from 0.238 ± 0.019 to 0.548 ± 0.009 mv), and shortened QTc interval (from 0.169 ± 0.006 to

0.141 ± 0.003 sec) over 15 minutes after of BBG administration. BBG did not cause cardiac arrhythmia.

Meanwhile, epinephrine produced significant bradycardia (209.8 ± 28.78 vs. 405.8 ± 1.18 beats/min), increased

PR interval, prolonged the QRS complex, shortened QTc interval, decreased R wave amplitude and induced

ventricular tachycardia. Brilliant blue G given prior to epinephrine increased heart rate and completely

suppressed the epinephrine-induced ventricular arrhythmia. The inhibitory effect of BBG on the arrhythmia

caused by epinephrine was prevented by atropine. In contrast the epinephrine induced arrhythmia was

completely suppressed with propranolol and BBG. The histopathological study showed that epinephrine caused

necrosis and apoptosis of cardiac muscle cells, degeneration of cardiac muscle fibers, and interstitial

haemorrhages. These changes were markedly prevented by BBG alone, propranolol/BBG and to a less extent

by atropine/BBG pretreatment. The study provided the first evidence for a cardioprotective and anti-

arrhythmogenic actions for BBG against epinephrine-induced arrhythmia and myocardial damage, and

suggested that cholinergic mechanisms are involved in its anti-arrhythmogenic action.

Key-Words: - cardiac arrhythmia; epinephrine; brilliant blue G; epinephrine; myocardial injury; beta adrenergic

receptors; atropine; cardioprotection

Received: February 15, 2023. Revised: February 15, 2024. Accepted: May 13, 2024. Published: June 5, 2024.

MOLECULAR SCIENCES AND APPLICATIONS

DOI: 10.37394/232023.2024.4.2

Omar M. E. Abdel-Salam, Marawan Abd El Baset,

Fatma A. Morsy, Amany A. Sleem

E-ISSN: 2732-9992

Volume 4, 2024

1 Introduction

The vasopressor agent epinephrine is a mainstay in

the treatment of life threatening conditions

characterized by circulatory collapse such as

cardiogenic shock, septic shock, and anaphylaxis

[1], [2], [3]. Epinephrine has potent stimulatory

action on β1- and β2- adrenoceptors in cardiac tissue

with powerful inotropic and chronotropic effects on

the heart, increasing myocardial contractility and

heart rate, while its action on α1 adrenoceptors on

arterial smooth muscle cells causes vasoconstriction

[1]. The intravenous use of epinephrine also has the

risk of provoking serious ventricular arrhythmia [4],

[5]. A high dose of epinephrine can cause direct

cardiac muscle damage, focal areas of necrosis ]6],

[7], and apoptosis of cardiomyocytes [6], [8],

attributable to coronary vasoconstriction and to the

oxidation products of catecholamines aminochromes

and reactive oxygen species, leading to the

formation of quinoproteins and depletion of

intracellular reduced glutathione levels [9], [10],

[11], [12]. Intravenously administered epinephrine

is thus widely used as a model of ventricular

arrhythmia for studying the pathogenetic

mechanisms involved, and testing potential anti-

arrhythmic drugs [13], [14].

Purinergic P2X7 receptors are members of the P2X

family of ionotropic ATP-gated receptors, activated

primarily by extracellular adenosine 5′-triphosphate

(ATP). P2X7 receptors are expressed in immune

cells such as macrophages, microglia, neurons,

cardiac smooth muscle cells, epithelial cells, and

endothelial cells. When stimulated by high

concentrations of extracellular ATP, the P2X7

receptor acts as a nonselective cation channel,

leading to K+ efflux and the intracellular influx of

Na+ and Ca2+. Excessive stimulation of P2X7

receptors can also open large transmembrane pores

permeable to large molecular weight molecules and

ions. The result is the activation of a number of

downstream signaling events, including activation

of inflammasome, release of proinflammatory

cytokines such as interleukin-1beta (IL-1β) and

tumour necrosis factor-alpha (TNF-α), increased

generation of oxygen free radicals, and ultimately

cell death [15], [16]. There is accumulating

evidence for the involvement of purinergic P2X7

receptors in several cardiovascular disorders such as

atherosclerosis, arrhythmia post-myocardial

infarction, and cardiac fibrosis [17], [18], [19], [20].

Antagonists of P2X7 receptor are thus an intriguing

approach to elucidate the role of these receptors in

cardiovascular function under physiologic and

pathophysiologic conditions.

Brilliant blue G (BBG), also known as Coomassie

brilliant blue, is used to stain proteins in biomedical

applications [21] and as an ophthalmic solution in

vitreoretinal surgery [22]. The dye is a

noncompetitive antagonist of P2X7 receptors with

nanomolar affinity [23]. Blockade of P2X7

receptors with BBG has been shown to attenuate

systemic inflammation [24], [25]. Whether P2X7

antagonism by BBG would be protective against

catecholamine-induced cardiac arrhythmia is not

known.

The aims of this study were therefore to: (i)

investigate the effects of P2X7 receptor antagonist

BBG in the epinephrine model of cardiac

arrhythmia and myocardial injury; (ii) examine the

role of beta-adrenoreceptors or cholinergic

mechanisms in the effects of BBG.

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

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

Brilliant blue G (Sigma Chemical Co., St. Louis,

MO, U.S.A), epinephrine (Nile Co., Egypt), and

propranolol (AstraZeneca-Egypt) were used in the

study and freshly dissolved in saline before the

experiments to obtain the necessary doses.

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Omar M. E. Abdel-Salam, Marawan Abd El Baset,

Fatma A. Morsy, Amany A. Sleem

E-ISSN: 2732-9992

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2.3 Experimental Groups

Rats were randomly divided into equal treatment

groups (6 rats per group). The following groups

were studied:

Group 1 received i.p. saline and served as normal

control.

Group 2 received i.p. saline thirty minutes before

i.v. epinephrine (10 µg/kg), and served as

epinephrine control.

Group 3 received i.p. brilliant blue G (100 mg/kg).

Group 4 received i.p. brilliant blue G (100 mg/kg)

thirty minutes before i.v. epinephrine injection.

Group 5 received i.v. atropine (2 mg/kg) followed

thirty minutes later by i.p. brilliant blue G (100

mg/kg), and thirty minutes thereafter by i.v.

epinephrine.

Group 6 received i.p. propranolol (2 mg/kg)

followed thirty min later by i.p. brilliant blue G (100

mg/kg), and thirty minutes thereafter by i.v.

epinephrine.

2.4 Electrocardiography

After 30 minutes of drug or saline administration,

rats were anesthetized with 10% chloral hydrate

(300 mg/kg, i.p.). 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 was

recorded thereafter for 15 min in each group. The

average heart rate, RR interval, PR interval, QRS

interval, QTc interval (corrected QT interval), R

wave amplitude, ST segment height, number of

ventricular premature beats, ventricular arrhythmia,

and duration of ventricular arrhythmia after different

treatments were determined over a period of 15

minutes [26], [27]. The mean value of three

successive 5 min of ECG recordings for each group

obtained in the first 15 min after saline, BBG or in

the first 15 min after the i.v. injection of epinephrine

was used for the calculations. Arrhythmia was

assessed by counting the number of premature

ventricular beats, missed beats, and runs of

ventricular tachycardia during the first 15 min after

different agents or after i.v. epinephrine.

Arrhythmias were defined according to the Lambeth

conventions [28]. Ventricular extrasystole is defined

as a single premature ventricular complex, and

ventricular tachycardia is defined as 4 or more

consecutive ventricular premature beats.

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 over a period of 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 Changes in electrocardiographic

parameters

The representative ECG changes in the different

treated groups are shown in Figs. 1-7. Results of the

ECG parameters are presented in tables 1 & 2 plus

Figs. 8 & 9.

3.1.1 Effects of brilliant blue G

Compared with the saline control, BBG

administration resulted in significant bradycardia,

increased RR and PR intervals, increased R wave

amplitude, shortened QTc interval but did not cause

arrhythmia. Heart rate decreased significantly by

41% (from 405.8 ± 1.18 to 239.4 ± 6.69 beats/min)

over 15 min after BBG administration. This

reduction in heart rate by BBG was consistent

during the successive 5 min of recordings that

followed its i.p. administration. Values were 218.5 ±

6.72, 227.2 ± 8.36, 272.4 ± 2.70 compared with the

corresponding saline control values of 410.1 ± 2.95,

404.0 ± 0.18, 403.4 ± 0.05 beats/min, respectively.

Meanwhile, the RR interval significantly increased

by 69.4% from 0.149 ± 0.002 to 0.254 ± 0.007 sec,

and the PR interval increased by 16.6% from 0.051

± 0.0008 to 0.059 ± 0.0004 sec). The QRS complex

was not changed compared with the saline control

but the R wave amplitude increased by 130.2% from

0.238 ± 0.019 to 0.548 ± 0.009 mv. The QTc

interval fell from 0.169 ± 0.006 to 0.141 ± 0.003 sec

over 15 min of ECG recording. BBG significantly

increased the ST amplitude relative to the saline

control (Table 1, Figs. 8 & 9). The representative

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Omar M. E. Abdel-Salam, Marawan Abd El Baset,

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ECG tracings of the saline and BBG only groups are

shown in Figs. 1 & 2.

Fig. 1 Representative ECG tracing in saline control

group.

Fig. 2 Representative ECG tracings in brilliant blue

G only group showing sinus bradycardia. These are

ECG recordings from 5, 10 and 15 min time periods

after i.p. BBG.

3.1.2 Effects of epinephrine

Compared with the saline control, i.v. epinephrine

caused significant bradycardia, increased RR and

PR intervals, decreased R wave amplitude,

increased QRS duration, and shortened QTc

interval. The heart rate significantly decreased by

88.1% from 410.1 ± 2.95 to 48.69 ± 1.78 beats/min

in the first 5 minutes that followed epinephrine

injection. It decreased by 48.3% from 405.8 ± 1.18

to 209.8 ± 28.78 beats/min in the 15 minutes after

i.v. epinephrine injection.

The RR and PR intervals significantly increased

from 0.149 ± 0.002 to 0.554 ± 0.115 sec and from

0.051 ± 0.0008 to 0.056 ± 0.002 sec. In contrast,

QTc interval significantly decreased from 0.169 ±

0.006 to 0.099 ± 0.015 sec over 15 min of

epinephrine injection. Epinephrine significantly

decreased R wave amplitude from 0.238 ± 0.019 to

0.183 ± 0.013 mv, and increased QRS duration from

0.0187 ± 0.0007 to 0.0418 ± 0.005 sec. Epinephrine

injection resulted in significantly depressed ST

segment compared to the saline control (Table 1,

Figs. 8 & 9). Multiple premature ventricular

contractions and runs of ventricular tachycardia

occurred after epinephrine administration (Table 2,

Figs. 3).

Fig. 3 Representative ECG tracings of the changes

induced by intravenous epinephrine (10 µg/kg)

showing bardycardia (upper two tracings) and

polymorphic ventricular tachycardia (lowest

tracing). These are ECG recordings from 5, 10 and

15 min time periods after epinephrine injection.

3.1.3 Effects of brilliant blue G and

epinephrine

Brilliant blue G given prior to i.v. epinephrine

resulted in increased heart rate, decreased RR

interval, prolonged PR interval, increased R wave

amplitude and normalized QRS duration and QTc

interval compared to epinephrine-treated control

rats. The reflex bradycardic effect observed early

after i.v. epinephrine was largely prevented by

BBG. The heart rate significantly increased from

48.69 ± 1.78 to 196.4 ± 10.54 beats/min in the first 5

minutes and from 209.8 ± 28.78 to 267.4 ± 12.75

beats/min in the 15 minutes that followed

epinephrine injection. The RR interval significantly

decreased from 0.554± 0.115 to 0.236± 0.014 sec.

The PR interval did not change significantly (0.061

± 0.0004 vs. 0.056 ± 0.002 sec). Brilliant blue G in

addition, increased R wave amplitude from 0.183 ±

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DOI: 10.37394/232023.2024.4.2

Omar M. E. Abdel-Salam, Marawan Abd El Baset,

Fatma A. Morsy, Amany A. Sleem

E-ISSN: 2732-9992

Volume 4, 2024

0.013 to 0.612 ± 0.012 mv and caused significantly

raised ST segment compared to epinephrine control

group (Table 1, Figs. 8 & 9). Brilliant blue G

completely inhibited the development of

epinephrine-induced arrhythmia (Table 2, Fig. 4).

Fig. 4 Representative ECG tracings of the changes

induced by i.p. BBG administration prior to i.v.

epinephrine (10 µg/kg). These are ECG recordings

from 5, 10 and 15 min time periods after

epinephrine injection.

3.1.4 Effects of atropine, BBG and

epinephrine

No significant difference in heart rate was observed

in the 15 minutes that followed epinephrine

injection between rats that received

atropine/BBG/epinephrine and the BBG/epinephrine

group. Values were 275.0 ± 16.18 and 267.4 ± 12.76

beats/min, respectively.

The PR interval normalized, but the QRS duration

increased from 0.0181 ± 0.0006 to 0.0251 ± 0.001

sec, the QTc interval decreased from 0.152 ± 0.006

to 0.093 ± 0.008 sec, the R wave decreased in

amplitude from 0.612 ± 0.012 to 0.288 ± 0.076 mv,

and the ST segment was significantly depressed

compared with the BBG/epinephrine group (Table

1, Figs. 8 & 9). The inhibitory effect of BBG on

arrhythmia caused by epinephrine was almost

prevented by atropine. The duration of arrhythmia

increased in atropine/BBG/epinephrine group with

respect to the BBG/epinephrine group (Table 2).

The ECG showed marked bradycardia, ventricular

premature beats, and sinus arrest (Fig. 5 & 6).

Fig. 5 Representative ECG tracings of the changes

induced by i.p. atropine and BBG given prior to i.v.

epinephrine (10 µg/kg) showing bradycardia and ST

segment elevation (upper and middle tracings), and

sinus pause (lowest tracing). These are ECG

recordings from 5, 10 and 15 min time periods after

epinephrine injection.

Fig. 6 Representative ECG tracings of the changes

induced by i.p. atropine and BBG given prior to i.v.

epinephrine (10 µg/kg) showing ventricular

premature beat and sinus pause (upper and middle

tracings), monomorphic ventricular tachycardia

(lower tracing). These are ECG recordings from 5,

MOLECULAR SCIENCES AND APPLICATIONS

DOI: 10.37394/232023.2024.4.2

Omar M. E. Abdel-Salam, Marawan Abd El Baset,

Fatma A. Morsy, Amany A. Sleem

E-ISSN: 2732-9992

Volume 4, 2024