Biomedicine & Pharmacotherapy 109 (2019) 1372–1380

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Effects of nimodipine, vinpocetine and their combination on isoproterenol- T

induced myocardial infarction in rats
⁎
Mohd Asif Ansari, Ashif Iqubal, Rustam Ekbbal, Syed Ehtaishamul Haque
Department of Pharmacology, School of Pharmaceutical Education & Research (SPER), Jamia Hamdard, New Delhi, 110062, India

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Myocardial infarction (MI) remains a major cause of morbidity and mortality worldwide.
cGMP Nimodipine is a calcium (Ca2+) channel blocker as well as a PDE1 inhibitor and primarily used in subarachnoid
Nimodipine haemorrhage (SAH) due to its blood-brain barrier crossing property. Nimodipine and vinpocetine inhibit the
Subarachnoid haemorrhage degradation of phosphodiester bond which increases cGMP and cAMP levels causing vasodilation.
Troponin-T
Material and methods: We have divided rats randomly into Group I - Vehicle control; Group II - Toxic control (ISO
Vinpocetine
85 mg/kg, i.p.); Group III, IV and V - Nimodipine (5, 10 and 15 mg/kg, i.p. respectively) with ISO; Group VI-
Nimodipine (15 mg/kg) alone; Group VII – Nimodipine + Vinpocetine (10 mg/kg + 10 mg/kg) with ISO; Group
VIII - Nimodipine + Vinpocetine (10 mg/kg + 10 mg/kg) alone; Group IX- Diltiazem (25 mg/kg, p.o) with ISO;
Group X- Diltiazem (25 mg/kg) alone and Group XI- Vinpocetine (10 mg/kg, p.o.) with ISO for 7 days. After 24 h
of the last dose, haemodynamics were assessed then animals were sacrificed and biochemical, histopathological
and ultrastructural changes were measured.
Results: Treatment with ISO significantly deviated the haemodynamic parameters (HR, SAP, DAP and MAP),
biochemical parameters (CK-MB, LDH, SGOT, cGMP and Troponin-T) and antioxidant markers (TBARS, SOD,
CAT, GSH, GPx, GST and GR). Haemotoxylin and eosin staining of the cardiac tissue and ultrastructural study
also indicated significant myocardial damage. Pretreatment with nimodipine (10 and 15 mg/kg, i.p), vinpocetine
(10 mg/kg, p.o) and their combination significantly restored the antioxidant status, haemodynamic profile,
cellular architecture and ultrastructural changes in the heart.
Conclusion: Nimodipine and vinpocetine both showed cardioprotection when given alone. However, their
combination showed better restoration in terms of oxidative stress, cardiac membrane damage, haemodynamics,
histopathology and ultrastructural changes.

1. Introduction antioxidants become a valuable tool in preventing oxidative damage in

Heart failure is recognized by an irregularity of coronary blood
supply and demand, which prompts cardiac damage and harms cardi-
omyocytes [1]. Previous studies have revealed that oxidative stress
follows ischemic injury due to reactive oxygen species (ROS) generation
and this process plays an important role in the advancement of myo-
cardial infarction (MI) [2]. Prolonged MI causes ischemia and cardiac
cell death. MI remains a major reason for morbidity and mortality
worldwide and is a leading cause of mortality in India [3].
Cardiac injury in MI can be assessed through a change in the serum
levels of creatine kinase-muscle/brain (CK-MB), lactate dehydrogenase
(LDH), Troponin-T and serum glutamic oxaloacetic transaminase
(SGOT) [4,5]. These changes are accompanied by significant incre-
ments in ROS (superoxide anion and hydroxyl radicals) [6]. Thus
myocardial injury [7,8].
Isoproterenol (ISO) is a synthetic, non-selective β-agonist that exerts
positive chronotropic and inotropic effects at low dose and depletes the
energy reserve of cardiomyocytes at high dose. ISO-induced MI is a
widely-accepted murine model that can be used to evaluate the effects
of novel cardioprotective agents. This model exhibits numerous mor-
phological abnormalities that are similar to human MI [9–11].
Nimodipine is 1,4-dihydropyridine L-type calcium (Ca2+) channel
blocker [12–15]. It crosses the blood-brain barrier due to its lipophilic
nature and is useful in the treatment of migraine and dementia [16–19].
Nimodipine is also an antioxidant and has phosphodiesterase type 1
(PDE1) inhibitor activity. It works by regulating the binding of Ca2+
with calmodulin. It is clinically useful in patients with subarachnoid
haemorrhage (SAH) and reduces the severity of neurological deficits

⁎
Corresponding author.
E-mail address: haquepharm@gmail.com (S.E. Haque).

https://doi.org/10.1016/j.biopha.2018.10.199
Received 9 August 2018; Received in revised form 22 October 2018; Accepted 31 October 2018
0753-3322/ © 2018 Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M.A. Ansari et al. Biomedicine & Pharmacotherapy 109 (2019) 1372–1380

Table 1
Effect of nimodipine, vinpocetine and their combination on haemodynamic parameters during Isoproterenol-induced myocardial infarction in rats.
Groups HR (Beats/Min) SAP (mmHg) DAP (mmHg) MAP (mmHg)

Group I/ Vehicle control 336 ± 10.10 129 ± 2.98 96.6 ± 2.69 107 ± 2.68
Group II/ ISO 398 ± 12.30** 105.4 ± 3.40** 68.8 ± 2.20*** 82 ± 2.55***
Group III/ NIM-5 + ISO 361 ± 10.50 115 ± 2.88 72.2 ± 1.74 85.8 ± 1.93
Group IV/ NIM-10 + ISO 339 ± 15.70## 126 ± 5.02## 78.4 ± 2.86# 94 ± 3.51##
Group V/ NIM-15 + ISO 331 ± 13.50## 126.4 ± 3.39## 80.2 ± 2.71## 95.2 ± 2.82##
Group VI/ NIM per se 343 ± 9.52 123 ± 2.72 90.8 ± 2.15 101 ± 2.37
Group VII/ NIM + VIN + ISO 308 ± 12.90### 131 ± 5.66### 81.6 ± 1.75## 97.2 ± 2.62###
Group VIII/ NIM + VIN per se 327 ± 5.57 125 ± 2.70 92.6 ± 2.50 103 ± 0.89
Group IX/ DIL + ISO 320 ± 12.50### 128 ± 4.67## 81.4 ± 1.36## 96.4 ± 2.27##
Group X/ DIL per se 338 ± 11.50 127 ± 3.96 90.2 ± 2.15 103 ± 0.91
Group XI/ VIN + ISO 324 ± 13.80### 127 ± 5.15## 81 ± 2.55## 96.2 ± 2.60##

Data are expressed as mean ± S.E.M (n = 5 animals per group). Significance was determined by one-way ANOVA followed by Dunnett’s t-test.
Vehicle Control: Normal group, 1% gum acacia (1 ml orally) and normal saline solution (0.5 ml/kg, i.p.; ISO: Toxic control group (85 mg/kg, i.p.); NIM-5 + ISO: NIM
(5 mg/kg i.p.) + ISO (85 mg/kg, i.p.); NIM-10 + ISO: NIM (10 mg/kg, i.p.) + ISO (85 mg/kg i.p.); NIM-15 + ISO: NIM (15 mg/kg, i.p.) + ISO (85 mg/kg, i.p.); NIM
per se: NIM (15 mg/kg, i.p.); NIM + VIN + ISO: NIM (10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); NIM + VIN per se: NIM (10 mg/kg, i.p.) + VIN
(10 mg/kg, p.o.); DIL + ISO: DIL (25 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); DIL per se (25 mg/kg, p.o.); VIN + ISO: VIN (10 mg/kg, p.o.) + (85 mg/kg, i.p.).
** p < 0.01 as compared to the normal control group.
*** p < 0.001 as compared to the control group.
#
p < 0.05 as compared to the ISO group.
##
p < 0.01 as compared to the ISO group.
###
p < 0.001 as compared to the ISO group.

Table 2
Effect of nimodipine, vinpocetine and their combination on cGMP & Trop-T
parameters in serum during isoproterenol-induced myocardial infarction.
Group cGMP (pmol/ml) Troponin-T (ng/ml)

Group I/ Vehicle control 7.07 ± 0.58 0.16 ± 0.04

Group II/ ISO 3.10 ± 0.46*** 3.52 ± 0.22***
Group III/ NIM-5 + ISO 3.65 ± 0.32 3.44 ± 0.18
Group IV/ NIM-10 + ISO 5.08 ± 0.39# 2.85 ± 0.20#
Group V/ NIM-15 + ISO 5.60 ± 0.49## 2.73 ± 0.24##
Group VI/ NIM per se 7.24 ± 0.36 0.54 ± 0.05
Group VII/ NIM + VIN + ISO 9.95 ± 0.42### 1.54 ± 0.11###
Group VIII/ NIM + VIN per se 8.54 ± 0.63 0.45 ± 0.05
Group IX/ DIL + ISO 7.39 ± 0.34### 2.03 ± 0.17##
Group X/ DIL per se 7.55 ± 0.69 0.34 ± 0.05
Group XI/ VIN + ISO 8.00 ± 0.53### 2.14 ± 0.13##

Data are expressed as mean ± S.E.M (n = 5 animals per group). Significance

was determined by one-way ANOVA followed by Dunnett’s t-test.
Vehicle Control: Normal group, 1% gum acacia (1 ml orally) and normal saline
solution (0.5 ml/kg, i.p.; ISO: Toxic control group (85 mg/kg, i.p.); NIM-5 +
ISO: NIM (5 mg/kg i.p.) + ISO (85 mg/kg, i.p.); NIM-10 + ISO: NIM (10 mg/
Fig. 1. Data are expressed as mean ± S.E.M (n = 5 animals per group).
kg, i.p.) + ISO (85 mg/kg i.p.); NIM-15 + ISO: NIM (15 mg/kg, i.p.) + ISO
Significance was determined by one-way ANOVA followed by Dunnett’s t-test.
(85 mg/kg, i.p.); NIM per se: NIM (15 mg/kg, i.p.); NIM + VIN + ISO: NIM
Vehicle Control: Normal group, 1% gum acacia (1 ml orally) and normal saline
(10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); NIM + VIN per
solution (0.5 ml/kg, i.p.; ISO: Toxic control group (85 mg/kg, i.p.); NIM-5 +
se: NIM (10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.); DIL + ISO: DIL (25 mg/kg,
ISO: NIM (5 mg/kg i.p.) + ISO (85 mg/kg, i.p.); NIM-10 + ISO: NIM (10 mg/
p.o.) + ISO (85 mg/kg, i.p.); DIL per se (25 mg/kg, p.o.); VIN + ISO: VIN
kg, i.p.) + ISO (85 mg/kg i.p.); NIM-15 + ISO: NIM (15 mg/kg, i.p.) + ISO
(10 mg/kg, p.o.) + (85 mg/kg, i.p.).
(85 mg/kg, i.p.); NIM per se: NIM (15 mg/kg, i.p.); NIM + VIN + ISO: NIM
*** p < 0.001 as compared to the control group.
# (10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); NIM + VIN per
p < 0.05 as compared to the ISO group.
## se: NIM (10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.); DIL + ISO: DIL (25 mg/kg,
p < 0.01 as compared to the ISO group.
### p.o.) + ISO (85 mg/kg, i.p.); DIL per se (25 mg/kg, p.o.); VIN + ISO: VIN
p < 0.001 as compared to the ISO group.
(10 mg/kg, p.o.) + (85 mg/kg, i.p.).
*** p < 0.001 as compared to the control group.
caused by vasospasm. It has also been documented that nimodipine has #
p < 0.05 as compared to the ISO group.
blood pressure lowering effects [20–22]. ##
p < 0.01 as compared to the ISO group.
Vinpocetine is a synthetic drug produced from vincamine alkaloid. ###
p < 0.001 as compared to the ISO group.
It has antioxidant and neuroprotective property [23,24]. Vinpocetine is
also a PDE1 inhibitor. Phosphodiesterase-1 activation increases vaso-
atherosclerosis and lipid peroxidation [27–30].
constriction by decreasing the concentration of cyclic guanosine 3′, 5′-
Nimodipine and vinpocetine are promising agents in dementia,
monophosphate (cGMP) in arteries and is considerably reversed on
subarachnoid haemorrhage and memory loss [22,31] but they are least
treatment with PDE1 inhibitors like nimodipine and vinpocetine
explored on MI. Both of these drugs are PDE-1 inhibitors that restrict
[25,26]. Both cyclic adenosine monophosphate (cAMP) and cGMP play
the degradation of a phosphodiester bond in cAMP and cGMP and
a pivotal role in the inotropic regulation of human myocardium. Thus,
thereby lead to increase in the levels of cAMP and cGMP in cardiac
nimodipine and vinpocetine has a therapeutic action on heart, lung and
muscles causing vasodilation. Nimodipine and vinpocetine are both
vasculature. Vinpocetine is a potent antioxidant that checks
having antioxidant property. Nimodipine is an L-type calcium channel

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M.A. Ansari et al. Biomedicine & Pharmacotherapy 109 (2019) 1372–1380

Table 3
Effect of nimodipine, vinpocetine and their combination on antioxidant parameters in myocardial tissues during isoproterenol-induced myocardial infarction in rats.
Groups TBARS SOD CAT GSH (μmol GSH/ GST (nmol GPx (nmol CDNB/ GR (nmol NADPH/
(nmol MDA/ mg (unit/ mg of protien) (nmol H2O2/min/ mg protien) NADPH/min/mg min/mg protien) min/mg protien)
protien) mg protien) protien)

Group I/ Vehicle control 0.12 ± 0.01 5.122 ± 0.16 6.99 ± 0.23 1.93 ± 0.15 9.24 ± 0.46 7.07 ± 0.53 8.11 ± 0.61
Group II/ ISO 0.51 ± 0.03*** 1.88 ± 0.15*** 1.73 ± .14*** 0.62 ± 0.07*** 3.28 ± 0.28*** 2.15 ± 1.98 ±
0.39*** 0.25***
Group III/ NIM-5 0.48 ± 0.03 2.02 ± 0.19 1.78 ± 0.15 0.72 ± 0.09 3.44 ± 0.27 2.69 ± 0.32 2.43 ± 0.21
Group IV/ NIM-10 0.40 ± 0.03# 2.84 ± 0.24# 2.92 ± 0.22## 1.30 ± 0.13# 5.00 ± 0.43# 3.81 ± 0.32# 4.02 ± 0.28#
Group V/ NIM-15 0.38 ± 0.02## 3.14 ± 0.23## 3.26 ± .30### 1.37 ± 0.13## 5.08 ± 0.39# 4.04 ± 0.40# 4.28 ± 0.35#
Group VI/ NIM per se 0.15 ± 0.02 4.99 ± 0.20 6.72 ± 0.17 1.81 ± 0.14 8.51 ± 0.54 6.77 ± 0.39 8.06 ± 0.73
Group VII/ 0.26 ± 0.02### 4.17 ± 0.39### 4.82 ± .31### 1.94 ± 0.13### 6.29 ± 0.47### 5.48 ± .43### 6.00 ± 0.50###
NIM + VIN + ISO
Group VIII/ NIM + VIN 0.13 ± 0.01 5.31 ± 0.24 7.09 ± 0.24 1.96 ± 0.14 8.75 ± 0.44 6.9 ± 0.44 8.7 ± 0.75
per se
Group IX/ DIL + ISO 0.30 ± 0.02### 3.71 ± 0.28### 4.32 ± 0.29### 1.84 ± 0.17### 5.71 ± 0.42## 4.74 ± .44### 5.47 ± 0.33###
Group X/ DIL per se 0.13 ± 0.02 5.42 ± 0.21 7.06 ± 0.15 2.04 ± 0.18 8.96 ± 0.41 7.01 ± 0.43 8.43 ± 0.69
Group XI/ VIN + ISO 0.31 ± 0.02### 3.52 ± 0.22### 4.1 ± 0.26### 1.67 ± 0.14### 5.32 ± 0.45# 4.34 ± 0.40## 5.19 ±
0.30###

Data are expressed as mean ± S.E.M (n = 5 animals per group). Significance was determined by one-way ANOVA followed by Dunnett’s t-test.
Vehicle Control: Normal group, 1% gum acacia (1 ml orally) and normal saline solution (0.5 ml/kg, i.p.; ISO: Toxic control group (85 mg/kg, i.p.); NIM-5 + ISO: NIM
(5 mg/kg i.p.) + ISO (85 mg/kg, i.p.); NIM-10 + ISO: NIM (10 mg/kg, i.p.) + ISO (85 mg/kg i.p.); NIM-15 + ISO: NIM (15 mg/kg, i.p.) + ISO (85 mg/kg, i.p.); NIM
per se: NIM (15 mg/kg, i.p.); NIM + VIN + ISO: NIM (10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); NIM + VIN per se: NIM (10 mg/kg, i.p.) + VIN
(10 mg/kg, p.o.); DIL + ISO: DIL (25 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); DIL per se (25 mg/kg, p.o.); VIN + ISO: VIN (10 mg/kg, p.o.) + (85 mg/kg, i.p.).
*** p < 0.001 as compared to the control group.
#
p < 0.05 as compared to the ISO group.
##
p < 0.01 as compared to the ISO group.
###
p < 0.001 as compared to the ISO group.

blocker that can be used as a cardioprotective agent. day of treatment) to induce experimental myocardial infarction [32].
We, thus tried to evaluate the cardioprotective effect of nimodipine,
vinpocetine and their combination on ISO-induced myocardial infarc- 2.4. Experimental protocol
tion in Wistar rats, and compared with a standard drug diltiazem which
is a non-dihydropyridine calcium channel blocker. Our study also elu- A total of 55 Wistar albino rats were used for this study. After ac-
cidates the mechanism of therapeutic efficacy by substantiating the climatisation, they were randomly divided into eleven groups, con-
hemodynamic and biochemical changes with ultrastructural and his- sisting of five rats per group.
topathological studies.
2.4.1. Group I (Vehicle control)
2. Materials and methods Animals received 1% gum acacia (1 ml) orally for 7 days and normal
saline solution 0.5 ml/kg saline (i.p.) on 6th and 7th day.
2.1. Experimental animals
2.4.2. Group II (ISO)
The Jamia Hamdard animal ethics committee, New Delhi (India) Animals received 1% gum acacia (1 ml) orally for 7 days and ISO
approved the animal protocol. Wistar albino rats of male (180–200 (85 mg/kg, i.p.) on 6th and 7th day.
gms) were procured from Central Animal House Facility, Jamia
Hamdard, New Delhi (India). Animals were housed in polypropylene 2.4.3. Group III (NIM-5)
cages (5 in each cage) for one week to adjust to the normal conditions Animals received NIM 5 mg/kg/day (i.p) for 7 days and ISO (85 mg/
(temperature, 23 ± 2 °C; RH 60 ± 5% and a 12 h light: dark cycle). kg, i.p.) on 6th and 7th day.
They received commercial pellet diet (Nav Maharashtra Chakan Oil
Mills Ltd., Delhi, India) and water ad libitum. 2.4.4. Group IV (NIM-10)
Animals received NIM 10 mg/kg/day (i.p) for 7 days and ISO
2.2. Drugs and chemicals (85 mg/kg, i.p.) on 6th and 7th day.

Nimodipine was procured from USV Private Limited, Maharashtra, 2.4.5. Group V (NIM-15)
India; Diltiazem was procured from Torrent Pharma Gujrat, India; Animals received NIM 15 mg/kg/day (i.p) for 7 days and ISO
Vinpocetine was obtained as a gift sample from Micro Labs Limited, (85 mg/kg, i.p.) on 6th and 7th day.
Uttar Pradesh, India; Creatine kinase-MB (CK-MB) and lactate dehy-
drogenase (LDH) kits were purchased from Reckon Diagnostics Pvt.Ltd 2.4.6. Group VI (NIM- per se)
Gujrat, India; cGMP kit was obtained from Arbor assays, Mississippi, Animals received NIM 15 mg/kg/day (i.p.) for 7 days
USA. Troponin-T was procured from KinesisDx, California, USA. ISO
and other chemicals of analytical grade were purchased from Sigma 2.4.7. Group VII (NIM + VIN + ISO)
Chemicals, St. Louis, Missouri, USA. Animals received NIM 10 mg/kg/day (i.p.) and VIN 10 mg/kg/day
(p.o.) for 7 days and ISO (85 mg/kg, i.p.) on 6th and 7th day.
2.3. Induction of experimental MI
2.4.8. Group VIII (NIM + VIN- per se)
ISO (85 mg/kg, i.p.) in a normal saline solution injected to rats for Animals received NIM 10 mg/kg/day (i.p.) and VIN 10 mg/kg/day
the last 2 consecutive days at an interval of 24 h (i.e., on 6th and 7th (p.o.) for 7 days.

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(caption on next page)

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Fig. 2. A. Data are expressed as mean ± S.E.M (n = 5 animals per group). Significance was determined by one-way ANOVA followed by Dunnett’s t-test.
Vehicle Control: Normal group, 1% gum acacia (1 ml orally) and normal saline solution (0.5 ml/kg, i.p.; ISO: Toxic control group (85 mg/kg, i.p.); NIM-5 + ISO: NIM
(5 mg/kg i.p.) + ISO (85 mg/kg, i.p.); NIM-10 + ISO: NIM (10 mg/kg, i.p.) + ISO (85 mg/kg i.p.); NIM-15 + ISO: NIM (15 mg/kg, i.p.) + ISO (85 mg/kg, i.p.); NIM
per se: NIM (15 mg/kg, i.p.); NIM + VIN + ISO: NIM (10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); NIM + VIN per se: NIM (10 mg/kg, i.p.) + VIN
(10 mg/kg, p.o.); DIL + ISO: DIL (25 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); DIL per se (25 mg/kg, p.o.); VIN + ISO: VIN (10 mg/kg, p.o.) + (85 mg/kg, i.p.).
*** p < 0.001 as compared to the control group.
###
p < 0.001 as compared to the ISO group.
B. Photomicrographs of myocardial tissues of different groups (H&E, 10X) A - Group I/Vehicle Normal saline, B – Group II/ISO -Toxic control, C – Group III/NIM dose
5 mg/kg, D – Group IV/NIM Dose 10 mg/kg, E – Group V/NIM Dose 15 mg/kg, F – Group VI/NIM dose 15 mg/kg per se, G – Group VII/NIM + VIN + ISO, H – Group
VIII/NIM + VIN per se, I – Group IX/DIL 25 mg/kg, J - Group X/DIL per se, K- Group XI/ VIN 10 mg/kg.

Table 4 2.5. Biochemical estimations

Effect of nimodipine, vinpocetine and their combination on the histopatholo-
gical changes in the myocardial tissues during isoproterenol-induced myo- Activities of serum creatine kinase-MB (CK-MB), lactate dehy-
cardial infarction in rats. drogenase (LDH) (Reckon Diagnostics Pvt. Ltd., Baroda, India), Serum
Groups Edema Myocardial Inflammatory cells glutamic oxaloacetic transaminase (SGOT) (Span Diagnostics Ltd.,
necrosis Surat, India), cyclic Guanosine Monophosphate (cGMP) (Arbor assays,
Mississippi, USA) and Troponin-T (KinesisDx, California, USA) were
Group I/ Vehicle control – – –
estimated using commercially available kits as per the instructions. In
Group II/ ISO +++ +++ +++
Group III/ NIM-5 ++ +++ +++ myocardial tissue levels of malondialdehyde (MDA) [33], reduced
Group IV/ NIM-10 + + – glutathione (GSH) [34], superoxide dismutase (SOD) [35], catalase
Group V/ NIM-15 + – – (CAT) [36], glutathione peroxidase (GPx) [37], glutathione-S-trans-
Group VI/ NIM-15 per se – – –
ferase (GST) [38], and glutathione reductase (GR) [39] was also esti-
Group VII/ NIM + VIN + ISO – – –
Group VIII/ NIM + VIN per se – – –
mated
Group IX/ DIL + ISO + + –
Group X/ DIL per se – – –
2.6. Haemodynamic parameters
Group XI/ VIN + ISO + + –

The severity of changes was assessed semi-quantitatively and is denoted as
follows: (-): No changes; (+): Mild changes; (++): Moderate changes; (++
+): Severe changes.
Vehicle Control: Normal group, 1% gum acacia (1 ml orally) and normal saline
solution (0.5 ml/kg, i.p.; ISO: Toxic control group (85 mg/kg, i.p.); NIM-5 +
ISO: NIM (5 mg/kg i.p.) + ISO (85 mg/kg, i.p.); NIM-10 + ISO: NIM (10 mg/
kg, i.p.) + ISO (85 mg/kg i.p.); NIM-15 + ISO: NIM (15 mg/kg, i.p.) + ISO
(85 mg/kg, i.p.); NIM per se: NIM (15 mg/kg, i.p.); NIM + VIN + ISO: NIM
(10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.) + ISO (85 mg/kg, i.p.); NIM + VIN per
se: NIM (10 mg/kg, i.p.) + VIN (10 mg/kg, p.o.); DIL + ISO: DIL (25 mg/kg,
p.o.) + ISO (85 mg/kg, i.p.); DIL per se (25 mg/kg, p.o.); VIN + ISO: VIN
(10 mg/kg, p.o.) + (85 mg/kg, i.p.).
2.4.9. Group IX (DIL)
Animals received 25 mg/kg/day DIL (p.o.) for 7 days and ISO
(85 mg/kg, i.p.) on 6th and 7th day.
2.4.10. Group X (DIL- per se)
Animals received 25 mg/kg/day DIL (p.o.) for 7 days.
The hemodynamic parameters, viz., heart rate and blood pressure
were recorded on the 8th day for all the groups of albino rats, using a
non-invasive method of tail-cuff plethysmography, with a LE 5002
Storage pressure meter (LETICA Scientific Instruments, USA) [40].
2.7. Histopathological examination
Myocardial tissues were fixed in 10% formalin, which was then
processed and embedded in paraffin wax. It was then cut into 5 μm
thickness on glass slides, and after that, stained with hematoxylin (H)
and eosin (E). Slides were examined under a light microscope, by a
pathologist who was blinded to the study groups. Histological slides
were assessed by the semi-quantitative system of analysis [41,42]
considering 0–3 scores for the categories edema, myocardial necrosis,
and inflammatory cell infiltrates. The score 0–3 was assigned to 0 – no
damage, 1 - slight damage, 2 - moderate damage, and 3 – maximum
damage. The results obtained were then expressed as cumulative scores
from 0 to 9.
2.8. Transmission Electron Microscopy studies

2.4.11. Group XI (VIN + ISO)
Animals received VIN 10 mg/kg/day (p.o) for 7 days and ISO
(85 mg/kg, i.p.) on 6th and 7th day.
Where, i.p.-Intraperitoneal, NIM- Nimodipine, VIN- Vinpocetine and
DIL-Diltiazem.
24 h after the last dose, animals were weighed, recorded for the
heart rate and arterial blood pressure. After recording blood pressure,
blood was collected, centrifuged to separate serum, and stored at
−20 °C ± 5 °C for biochemical assays like Troponin-T, CK-MB, LDH,
cGMP and SGOT. Animals were then euthanized under mild anaesthesia
and hearts were removed, washed with normal saline, soaked and
weighed. A small piece of heart sample was preserved in formalin so-
lution (10%) for histopathology and in 2.5% glutaraldehyde and 2%
paraformaldehyde solution in 0.1 M sodium phosphate buffer (pH 7.2)
for transmission electron microscopy. Remaining heart tissues were
kept at −20 °C for biochemical estimations.
For ultrastructure study, 4–5 pieces of 1.0–1.5 mm dimension were
prepared from the left ventricle. These samples were rinsed several
times with a 0.1 M phosphate buffer (pH 7.2). Immediately, the samples
were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde solu-
tion in 0.1 M sodium phosphate buffer (pH 7.2) and stored at 4º C for
12 h. Ultra-thin sections from these samples were stained with 2% ur-
anyl acetate and 2% lead acetate, and observed under a Transmission
Electron Microscope (Tecnai) at All India Institute of Medical Sciences
and Research (AIIMS) New Delhi, India. [43].
2.9. Statistical analysis
Data were expressed as mean ± S.E.M (five rats per group). Groups
of data were compared by one-way analysis of variance (ANOVA) fol-
lowed by Dunnett’s t-test. Values p < 0.05 were considered statisti-
cally significant. Statistical analysis was carried out using Graph Pad
Prism 5.0 software (Graph Pad Software, San Diego, California, USA).

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M.A. Ansari et al. Biomedicine & Pharmacotherapy 109 (2019) 1372–1380

Fig. 3. Transmission electron microscopic study of the ventricle tissue of rat heart. Group I: (Normal control) group showing normal architecture of heart mi-
tochondria and myofilament. Groups II: (toxic group) treated with Isoproterenol (ISO; 85 mg/kg, i.p.) showing swollen heart mitochondria with the ruptured
mitochondrial cell membrane, Glycogen accumulation is seen, the band is totally damaged, protein got hampered and cell death was seen. Group III: group treated
with nimodipine (5 mg/kg, i.p) shows irregular Z-band, the arrangement is distorted and mitochondria are swollen. Group IV: group treated with nimodipine (10 mg/
kg, i.p) few lipid droplets seen with slight myofilament damage. Group V: group treated with nimodipine (15 mg/kg, i.p) showing an intact nucleus, two nucleoli are
seen and myofilament is normal. Group VI, VIII and X: (per se groups) with nimodipine, combination and diltiazem showing normal mitochondria along with good
appearance of myofilaments of the heart mitochondria and cristae is normal. Group VII: group treated with a combination of nimodipine (10 mg/kg, i.p) and
vinpocetine (10 mg/kg, p.o) showing properly arranged bands, myofilament is good and mitochondria is normal. Group IX: group is treated with diltiazem (25 mg/
kg, P.o) showing maintained architecture of myofilament and no vacuolation is seen. Group XI: treated with vinpocetine (10 mg/kg, P.o) normal mitochondria is seen
with normal cristae.

3. Results
3.1. Effect on haemodynamic parameters caused by isoproterenol
The effect of heart rate (HR), systolic arterial pressure (SAP), dia-
stolic arterial pressure (DAP) and mean arterial pressure (MAP) in the
treatment groups are represented in Table 1.
Rats treated with ISO, i.e. Group II, showed a significant
(P < 0.001) increase in the heart rate and decrease in SAP, DAP and
MAP as compared to the normal control i.e., Group I. Pre-treatment
with nimodipine (10 and 15 mg/kg) in Group IV and V, vinpocetine
(10 mg/kg) in Group XI, combination of nimodipine and vinpocetine
(10 mg/kg + 10 mg/kg) in Group VII and diltiazem (25 mg/kg) in
Group IX provided significant (p < 0.01 and p < 0.001) protection
from ISO-induced changes in Heart rate, SAP, DAP and MAP as com-
pared to ISO i.e., Group II. Nimodipine (5 mg/kg) in Group III did not
show any significant effect (p > 0.05) when compared with ISO Group
II.
3.2. Effect of nimodipine and vinpocetine on the level of cGMP and
Troponin-T
The effect of cGMP and Trop-T in the treatment groups are re-
presented in Table 2.
Rats treated with ISO (Group II) showed a significant (P < 0.001)
decrease in the level of serum cGMP and Trop-T as compared to the
normal control (Group I). Pre-treatment with nimodipine (10 and
15 mg/kg) in Group IV and V, vinpocetine (10 mg/kg) in Group XI,
combination of nimodipine and vinpocetine (10 mg/kg + 10 mg/kg) in
Group VII and diltiazem (25 mg/kg) in Group IX showed significant
(p < 0.01 and p < 0.001) reversal of these parameters when com-
pared to ISO (Group II). Nimodipine (5 mg/kg) in Group III did not
show any significant effect (p > 0.05) when compared with ISO
(Group II).
3.3. Effect of nimodipine and vinpocetine on CK-MB, LDH and SGOT level
The effect of CK-MB, LDH and SGOT in the treatment groups are
represented in Fig. 1.

1377
M.A. Ansari et al.
Rats treated with ISO (Group II) showed a significant (P < 0.001)
increase in the level of serum LDH, CK-MB and SGOT as compared to
the normal control (Group I). Pre-treatment with nimodipine (10 and
15 mg/kg) in Group IV and V, vinpocetine (10 mg/kg) in Group XI,
combination of nimodipine and vinpocetine (10 mg/kg + 10 mg/kg) in
Group VII and diltiazem (25 mg/kg) in Group IX showed significant
(p < 0.01 and p < 0.001) decrease in these parameters when com-
pared with ISO (Group II). Nimodipine (5 mg/kg) in Group III did not
show any significant effect (p > 0.05) when compared with ISO
(Group II).
3.4. Effect on the level of thiobarbituric acid-reactive substance (TBARS)
TBARS level was determined by evaluating myocardial mal-
ondialdehyde (MDA) content (Table 3). A significant (P < 0.001) rise
in the serum TBARS level was observed in the ISO (Group-II) when
compared with the control (Group-I). Pre-treatment with nimodipine
(10 and 15 mg/kg) in Group IV and V, vinpocetine (10 mg/kg) in Group
XI, combination of nimodipine and vinpocetine (10 mg/kg + 10 mg/
kg) in Group VII and diltiazem (25 mg/kg) in Group IX showed sig-
nificant (p < 0.05, p < 0.01 and p < 0.001) decrease in MDA level
as compared to the ISO group (Group-II). Nimodipine (5 mg/kg) in
Group III did not show any significant effect (p > 0.05) as compared to
ISO (Group II).
3.5. Effect of nimodipine and vinpocetine on the level of anti-oxidant
enzymes
The effect of superoxide dismutase (SOD), catalase (CAT),
Glutathione (GSH), Glutathione peroxidase (GPx), Glutathione re-
ductase (GR) and Glutathione S-transferases (GST) in the treatment
groups are represented in Table 3. Rats treated with ISO (Group II)
showed a significant (P < 0.001) decrease in the level of serum SOD,
CAT, GSH, GPx, GR and GST as compared to the normal control (Group
I). Pretreatment with nimodipine (10 and 15 mg/kg) in Group IV and V,
vinpocetine (10 mg/kg) in Group XI, combination of nimodipine and
vinpocetine (10 mg/kg + 10 mg/kg) in Group VII and diltiazem
(25 mg/kg) in Group IX showed a significant (p < 0.05, p < 0.01 and
p < 0.001) increase in their level when compared to ISO (Group II).
Nimodipine (5 mg/kg) in Group III did not show any significant effect
(p > 0.05) as compared to ISO (Group II).
3.6. Histopathological analysis
[Fig. 2A & B] and [Table 4] shows the histopathological changes in
various groups. Control and per se groups showed normal integrity of
myocardial tissue with striations, branched appearance and continuity
with adjacent myofibrils. The ISO-treated (Group-II) showed significant
myodegeneration, myocardial necrosis, edema, pyknotic nucleus, in-
filtration of macrophages and lymphocytes. Pretreatment with nimo-
dipine (10 and 15 mg/kg) in Group IV and V, vinpocetine (10 mg/kg) in
Group XI, combination of nimodipine and vinpocetine (10 mg/kg +
10 mg/kg) in Group VII and diltiazem (25 mg/kg) in Group IX sig-
nificantly reversed the changes to normal. Nimodipine (5 mg/kg) in
Group III did not show any significant effect (p > 0.05) as compared to
ISO (Group II).
3.7. Effect of nimodipine, vinpocetine and their combination on the
ultrastructure of cardiac tissue
Fig. 3 illustrates the ultrastructure of the left ventricle of the control,
toxic and experimental groups. In the present study, microscopic
images of the mitochondria in the ventricle of ISO-treated animals
(Group II) showed the extensive destruction of cristae and myofila-
ments with edema, and the presence of swollen and altered mi-
tochondrial membrane. Pretreatment with nimodipine and its
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Biomedicine & Pharmacotherapy 109 (2019) 1372–1380
combination with vinpocetine in the ISO-treated group showed a pre-
served architecture of the cell membrane and a maintained cristae
structure (Groups III, IV, V, VII, IX and XI). A combination of nimodi-
pine and vinpocetine per se, diltiazem per se and nimodipine per se
showed no pathological alterations (Groups VI, VIII and X). The normal
group showed a normal ultrastructure of the mitochondria of cardiac
tissue (Group I).
3.8. Per se effect of drugs
Per se treatment of nimodipine (15 mg/kg), diltiazem (25 mg/kg)
and combination of nimodipine and vinpocetine in group VI, VIII and X
for 7 days did not show any significant change (p > 0.05) with respect
to Hemodynamics, Biochemical Parameters, Histopathological studies
and Transmission Electron Microscopy as compared to Group I (Tables
1–4, Figs. 1, 2 and 3).
4. Discussion
The pathogenesis of cardiomyopathy is not clearly defined, but its
evaluation by ISO-induced cardiac damage has demonstrated the in-
volvement of oxidative stress. Our study, based on preclinical evalua-
tion in a murine model demonstrated that a combination of nimodipine
with vinpocetine exhibit strong cardioprotective effect. This combina-
tion improved haemodynamic parameters, reversed changes at the
histopathological and ultrastructural level, increased endogenous anti-
oxidants and maintained the serum levels of cardiac biomarkers CK-MB,
LDH, SGOT, Trop-T and cGMP.
Nimodipine is used for cerebral ischemia, cluster headache, de-
mentia, depression, head trauma, hypertension, migraine and majorly
in subarachnoid haemorrhage to reduce the severity of ischemic neu-
rological deficits [22]. Vinpocetine is reported to be an effective sca-
venger of free radicals. It has blood viscosity lowering properties. It is
used to increase cerebral blood flow and decrease platelet aggregation.
It is widely used for the improvement of memory and in geriatric,
neuropsychiatry, ophthalmology and otolaryngology [44].
Increase in the level of MDA is considered to be a clinical marker of
cardiac damage [45] and reflects decreased activity of antioxidant en-
zymes. Our study found an increase in the levels of MDA when the rats
were treated with ISO. Nimodipine at doses 10 mg/kg, 15 mg/kg and in
combination with vinpocetine, reversed the increased levels of MDA
thereby decreased lipid peroxidation
CAT, SOD, GPx, GR, GST and GSH are free radical-scavenging an-
tioxidant enzymes and are the first-line cellular defence against oxi-
dative stress. These enzymes eliminate oxygen radicals such as super-
oxide (O2-) and hydrogen peroxide (H2O2) and prevent the formation of
a more reactive hydroxyl radical (%OH) [46]. Pretreatment of animals
with nimodipine,vinpocetine and their combination increased the ac-
tivities of GSH, CAT, SOD, GPx, GR, and GST in ISO-subjected rats. The
results of our study demonstrated that free radicals produced by ISO
were significantly quenched by nimodipine, vinpocetine and their
combination. Thus this antioxidant effect accounts for the cardiopro-
tective property of these drugs.
Administration of ISO (85 mg/kg,i.p.) decreased the blood pressure
parameters (SAP, MAP, DAP) and raised HR to induce cardiac failure in
Wistar rats [47,48]. This effect was manifestedby intracellular Ca2+
overload and also due to disturbed sympathetic and parasympathetic
input to the heart. Nimodipine, a calcium channel blocker, can coun-
teract Ca2+ overload [14]. Pre-treatment with 10 mg/kg and 15 mg/kg
doses of nimodipine and its combination with vinpocetine prevented
cardiac injury with significant restoration of SAP, MAP and DAP
(P < 0.01, P < 0.001 and P < 0.01, respectively), and HR
(P < 0.01). This demonstrates improvement of myocardial oxygen
supply through the subendocardial region of ventricles andshows the
cardioprotective effects of nimodipine, vinpocetine and their combi-
nation.
M.A. Ansari et al.
We detected decreased levels of cGMP in ISO (85 mg/kg) treated
group. This was reversed significantly by nimodipine alone and by its
combination with vinpocetine. Cardiovascular disease impairs cGMP
signalling as cGMP–dependent protein kinase G pathway plays a pivotal
role in myocardial protection and preconditioning [49]. In the healthy
endothelium, vascular NO binds to the ferrous heme iron (Fe2+) and
activates a key signal transduction enzyme, soluble guanylate cyclase
(sGC), resulting in cGMP generation. This activation promotes various
actions such as vasodilation, inhibition of platelet aggregation, and
growth inhibition [50]. The increased level of cGMP in the serum
support the cardioprotective effects of nimodipine, vinpocetine and
theircombination.
Elevated serum CK-MB, LDH and SGOT levels are well-known
markers of myocardial infarction. Iso-induced MI causes cellular da-
mage due to oxygen and glucose deficit. This promotes rupture of
cardiac membrane and subsequent leakage of these enzymes into the
bloodstream. Elevated troponin levels predict the risk of both cardiac
death and subsequent infarction [51,52]. In our study, we also wit-
nessed increased levels of cardiac troponin-T in the serum of ISO-in-
duced rats. Pretreatment with nimodipine, vinpocetine and their com-
bination reduced the generation of oxygen radicals and helped in the
decrease of these enzymes showing preservation of the structural in-
tegrity of myocardium.
It has been reported that ISO administration could cause significant
damage to the myocardium. H and E staining of the myocardium is an
important technique to portrait the extent of injury qualitatively [53].
The toxic group (Group-II) showed the infiltration of macrophages and
myodegeneration. However, pre-treatment with nimodipine, vinpoce-
tine and theircombination efficiently prevented the damage done by
ISO.
The transmission electron micrographs of the mitochondria of the
heart in ISO-group showed swollen and ruptured mitochondrial cell
membrane, glycogen accumulation and damaged band protein leading
to cell death. This type of mitochondria is a characteristic of myocardial
ischemia. This swelling is due to an increase in the lipid peroxide by-
products formed due to GSH depletion [54,55]. Rats pre-treated with
nimodipine, vinpocetine and their combination in ISO-treated groups
showed normal mitochondrial cell membrane without glycogen accu-
mulation. Myofilament bands and protein were normal. In the per se
groups, no pathological changes were observed.
CK-MB and Troponin-T is a vital parameter which indicates myo-
cardial damage and their serum level increase due to the leakage from
the myocardium. In the case of SAH, it has been reported that these two
parameters also increase in the serum which means that SAH also
causes cardiac damage [56]. As our drug is restoring the cardiac da-
mage and decreasing the level of CK-MB and Troponin-T, we assume
that this is equally effective in both, i.e. MI and SAH. In summary, we
can say that ISO causes oxidative stress leading to cardiac membrane
damage, fall in cGMP and disturbance in haemodynamic parameters. As
we examined all these parameters using biochemical estimations, hae-
modynamic measurements, histopathological evaluations and Trans-
mission electron microscopy, we found significant restoration of all the
parameters to normal in presence of our test drugs.
5. Conclusion
Nimodipine and vinpocetine both showed cardioprotection when
given alone. However, their combination showed better restoration in
terms of oxidative stress, cardiac membrane damage, haemodynamics,
histopathology and ultrastructural changes. Thus, taking all the results
into consideration, we can conclude that nimodipine which is being
prescribed for SAH is potentially effective against ISO induced MI. The
current study is a good example of drug repurposing and provides
therapeutic value for the patients who develop MI in sub-arachnoid
haemorrhage.
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Biomedicine & Pharmacotherapy 109 (2019) 1372–1380
Financial support
We are thankful to University Grants Commission (UGC), Govt. of
India for providing a National fellowship of other backward classes
(NFOBC) to the first author.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
We express our gratitude to the Pharmacological Research Lab,
Department of Pharmacology, SPER, Jamia Hamdard for providing us
with various facilities and support during the study.
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