Beneficial role of tamoxifen in experimentally induced

Pharmacological Reports 66 (2014) 264–272
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Pharmacological Reports
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Original research article
Beneﬁcial role of tamoxifen in experimentally induced cardiac hypertrophy
Bhoomika M. Patel *, Vishal J. Desai
Institute of Pharmacy, Nirma University, Ahmedabad, India
A R T I C L E I N F O
Article history:
Received 23 March 2013
Received in revised form 22 August 2013
Accepted 16 September 2013
Available online 2 March 2014
Keywords:
Tamoxifen
Cardiac hypertrophy
Isoproterenol (ISO)
Partial abdominal aortic constriction
(PAAC)
A B S T R A C T
Background: Protein kinase C (PKC) activation is associated with cardiac hypertrophy (CH), ﬁbrosis,
inﬂammation and cardiac dysfunction. Tamoxifen is a PKC inhibitor. Despite these, reports on effect of
tamoxifen on cardiac hypertrophy are not available. Hence, we have investigated effect of tamoxifen
(2 mg/kg/day, po) on CH.
Methods: In isoproterenol (ISO) induced CH, ISO (5 mg/kg/day, ip) was administered for 10 days in
Wistar rats. For partial abdominal aortic constriction (PAAC), abdominal aorta was ligated by 4-0 silk
thread around 7.0 mm diameter blunt needle. Then the needle was removed to leave the aorta partially
constricted for 30 days. Tamoxifen was given for 10 days and 30 days, respectively, in ISO and PAAC
models and at end of each studies, animals were sacriﬁced and biochemical and cardiac parameters were
evaluated.
Results: ISO and PAAC produced signiﬁcant dyslipidemia, hypertension, bradycardia, oxidative stress
and increase in serum lactate dehydrogenase and creatine kinase-MB, C-reactive protein. Treatment
with tamoxifen signiﬁcantly controlled dyslipidemia, hypertension, bradycardia, oxidative stress and
reduced serum cardiac markers. ISO control and PAAC control rats exhibited signiﬁcantly increased
cardiac and left ventricular (LV) hypertrophic index, LV thickness, cardiomyocyte diameter. Treatment
with tamoxifen signiﬁcantly reduced these hypertrophic indices. There was a signiﬁcant increase in LV
collagen level, decrease in Na+K+ATPase activity, and reduction in the rate of pressure development and
decay. Tamoxifen signiﬁcantly reduced LV collagen, increased Na+K+ATPase activity and improved
hemodynamic function. This was further supported by histopathological studies, in which tamoxifen
showed marked decrease in ﬁbrosis and increase in extracellular spaces in the treated animals.
Conclusions: Our data suggest that tamoxifen produces beneﬁcial effects on cardiac hypertrophy and
hence may be considered as a preventive measure for cardiac hypertrophy.
ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.
z o.o. All rights reserved.
Introduction
Cardiac hypertrophy (CH) is an important predictor of
cardiovascular morbidity and mortality, associated with diastolic
dysfunction [17]. The heart adapts in response to an array of
stimuli by increasing myocardial mass through the induction of a
hypertrophic response [27]. Studies in humans and animal models
have demonstrated that cardiac hypertrophy signiﬁcantly affects
myocardial electronic cell-to-cell coupling, leading to disturbance
in action potential duration and sudden cardiac death [29].
Currently, cardiac hypertrophy is associated with several disorders
including diabetes mellitus [45] and there is no treatment for
* Corresponding author.
E-mail address: [email protected] (B.M. Patel).
reversal of cardiac hypertrophy and hence, preventive measures
are strictly required [48].
Protein kinase C (PKC) is a group of closely related serinethreonine protein kinases associated with cardiac hypertrophy,
ﬁbrosis, and cardiac dysfunction [40]. Additionally, it has been
reported that inhibition of PKC bII prevents cardiac hypertrophy
and enhances cardiac contractility [24]. In addition to PKC,
estrogen also plays an important role on cardiovascular system.
Estrogen effects are mediated by ERa and ERb, receptors, both of
which are expressed in cardiac myocytes, ﬁbroblasts, and vascular
cells in human and rodent heart [2]. ERa and ERb agonist 17bestradiol attenuates cardiac hypertrophy [46]. It has been reported
that pre-menopausal women have a lower prevalence of left
ventricular hypertrophy (LVH) than age-matched men [1], and that
hormone replacement therapy (HRT) with estradiol reverses left
ventricular hypertrophy in postmenopausal women [36]. Similar
results have been obtained from animal studies [30,59]. From this
http://dx.doi.org/10.1016/j.pharep.2014.02.004
1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
B.M. Patel, V.J. Desai / Pharmacological Reports 66 (2014) 264–272
perspective, it is clear that PKC and estrogen play important role in
the prevention of heart disease.
Tamoxifen is a well-known drug from selective estrogen
receptor modulator class; basically, used in treatment of breast
cancer. In addition to its effect on estrogen receptor, it is also
reported to inhibit PKC [23]. Tamoxifen shows inhibitory effect on
L-type Ca2+ channels in vascular smooth muscle cells and reduced
smooth muscle contractility [53]. McDonald et al. [34] have
reported that tamoxifen produced a reduction in risk of myocardial
infraction. Further, Rutqvist and Mattsson [50] also suggested that
tamoxifen decreases cardiac morbidity with long-term treatment.
Despite above-mentioned facts, direct reports of effect of tamoxifen on cardiac hypertrophy are not available. Hence, objective of
the present study was to evaluate the effect of tamoxifen on
experimentally induced cardiac hypertrophy.
265
PAAC induced cardiac hypertrophy respectively. The dose of
tamoxifen was selected on the basis of the clinical dose converted
into rat dose based on body surface area and weights by previously
described methods [18,21].
Blood sample collection and serum analysis
At the end of experimental period, blood samples were collected
from the retro orbital plexuses under light ether anesthesia, serum
was separated and analyzed for total cholesterol, HDL-cholesterol,
Triglycerides, LDL-cholesterol. C-reactive protein (CRP), lactate
dehydrogenase (LDH), creatinine kinase (CK) spectrophotometrically (Shimadzu UV-1601, Japan) using biochemical diagnostic kits
(Labcare Diagnostics Pvt. Ltd., India) [19,43].
Measurement of hemodynamic parameters
Materials and methods
The protocol of the experiment was approved by our
institutional animal ethical committee as per the guidance of
the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), Ministry of Social Justice and
Empowerment, Government of India (IPS/PCOL/MPH10-11/009, 17
August 2011 and IPS/PCOL/MPH 10-11/2002, 14 January 2011).
At the end of the respective treatment, the carotid artery behind
the trachea was exposed and cannulated for the measurement of
hemodynamic parameters using a transducer (BP 100) and
Labscrib Systems (IWORX, New Hampshire, USA) under anesthetic
conditions. The hemodynamic parameters observed were mean
arterial blood pressure, heart rate, rate of pressure development
(dp/dtmax) and rate of pressure decay (dp/dtmin). All the data
were analyzed using Labscrib Software (Version 118) [41].
Animals
Measurement of cardiovascular parameters
Adult female Wistar rats of 6- to 8-week of age were chosen for
the study and maintained under well-controlled conditions of
temperature (22 2 8C), humidity (55 5%) and 12 h/12 h light–
dark cycle. Standard laboratory rat chew and UV-ﬁltered water were
provided ad libitum.
Isoproterenol (ISO) induced cardiac hypertrophy
Adult female Wistar rats were used for the procedure. The rats
were randomly divided into four groups: CON – control animals,
TAM – control animals treated with tamoxifen, DIS – hypertrophic
control animals treated with isoproterenol, DIS + TAM – hypertrophic animals treated with treated with isoproterenol and tamoxifen. The rats were injected intraperitoneally (ip) with 5 mg/kg
isoproterenol in 0.9% sodium chloride solution daily for 10 days.
Control rats received equivalent amount of isotonic saline alone.
Partial abdominal aortic constriction (PAAC) induced cardiac
hypertrophy
At the end of the study, animals were sacriﬁced, hearts were
excised, extraneous tissues were separated and wet weight of the
entire heart, left ventricle (LV) and right ventricle (RV), femur
length and LV wall thickness was noted down using screw gauge
micrometer. Cardiac hypertrophic index was calculated as wet
heart weight to femur length ratio and left ventricular hypertrophic index was calculated as wet left ventricle weight to wet heart
weight ratio [20,42]. Na+K+ATPase activity was performed
according to method described by Tsimaratos et al. [58].
Quantiﬁcation of LV myocardial hydroxyproline concentrations
[16], malondialdehyde (MDA) [39], reduced glutathione (GSH) [4]
and superoxide dismutase (SOD) levels [35] were measured. The
LV was subjected for histopathological studies for hematoxylin and
eosin (HE) staining. The sections were observed and desired areas
were photographed in an Olympus photomicroscope under 40
and 100 magniﬁcation and cell diameter measurements were
done using Image J analyzer 1.45.
Statistical analysis
Adult female Wistar rats were used for procedure. The rats were
randomly divided into four groups: CON – sham control, TAM –
sham control animals treated with tamoxifen, DIS – hypertrophic
control animals, DIS + TAM – hypertrophic animals treated with
tamoxifen. Treatment of tamoxifen was started from 0th day in
sham treated and PAAC treated animals. Surgical procedure was
done on 3rd day in PAAC control and PAAC treated animals under
anesthesia produced by ketamine (20 mg/kg) and xylazine (10 mg/
kg). Incision was made in abdominal wall to expose abdominal
aorta. Abdominal aorta was ligated suprarenally with 4.0 silk
suture along with 7-0 mm blunt needle. Thereafter needle was
removed to leave abdominal aorta partially constricted. Sham
control and sham treated animal underwent the same procedure
except constriction of abdominal aorta.
Treatment protocol
Tamoxifen was dissolved in saline and was administered orally
(po) at a dose of 2 mg/kg/day, po for 10 days and 4 weeks in ISO and
Results are presented as mean SEM. Statistical differences
between the mean of the various groups were evaluated using oneway analysis of variance (ANOVA) followed by Tukey’s test. Data were
considered statistically signiﬁcant at p value < 0.05.
Results
Serum lipid proﬁle
ISO control and PAAC control rats exhibited a signiﬁcantly
(p < 0.05) increased level of serum total cholesterol, LDL and
triglyceride and signiﬁcantly (p < 0.05) decreased levels of serum
HDL as compared to normal control rats. Treatment with
tamoxifen (2 mg/kg/day, po) showed a signiﬁcant (p < 0.05)
reduction in serum cholesterol and serum LDL levels but did not
produce any signiﬁcant (p < 0.05) effect on serum triglyceride and
HDL levels in both the models of cardiac hypertrophy, i.e. ISOinduced and PAAC-induced hypertrophy (Table 1).
B.M. Patel, V.J. Desai / Pharmacological Reports 66 (2014) 264–272
266
Table 1
Effect of tamoxifen on serum lipid proﬁle and serum cardiac markers.
Parameters
Serum cholesterol (mg/dl)
Serum LDL (mg/dl)
Serum triglyceride (mg/dl)
Serum HDL (mg/dl)
LDH (U/L)
CK-MB (U/L)
CRP (mg/L)
Model of ISO induced cardiac hypertrophy
Model of PAAC induced cardiac hypertrophy
CON
TAM
DIS
DIS + TAM
CON
TAM
DIS
DIS + TAM
74.34 2.98
44.86 2.32
56.4 1.70
34.7 1.69
967.90 62.72
628.50 41.27
3.83 0.93
68.53 3.70
45.52 2.35
56.95 2.32
37.72 3.92
1082.2 41.12
657.10 46.43
2.46 0.42
112.17* 2.66
65.27* 5.14
82.21* 3.51
25.74* 2.74
1491.77* 58.17
838.32* 38.84
22.43* 0.58
97.36# 3.05
35.40# 3.78
74.28 3.57
28.58 2.75
1183.81# 55.30
689.51# 20.24
14.55# 0.24
81.19 2.73
81.19 2.73
63.71 3.82
45.4 3.34
539.72 30.30
342.46 22.43
4.01 2.7
78.65 3.06
78.65 3.06
53.47 2.79
44.95 2.94
485.17 50.16
445.83 18.93
4.56 1.04
128.27* 6.86
128.27* 6.86
75.22* 3.97
36.53* 2.43
976.93* 56.73
673.04* 51.85
20.82* 1.18
93.07# 3.55
93.07# 3.55
68.49 2.08
38.77 1.73
673.70# 39.38
385.20# 46.01
12.17# 2.27
Values are expressed as mean SEM of 6 rats.
ISO – isoproterenol, PAAC – partial abdominal aortic constriction, CON – control animals (model of ISO induced cardiac hypertrophy)/sham control (model of PAAC induced cardiac
hypertrophy), TAM – control animals treated with tamoxifen (model of ISO induced cardiac hypertrophy)/sham control animals treated with tamoxifen (model of PAAC induced
cardiac hypertrophy), DIS – hypertrophic control animals, DIS + TAM – hypertrophic animals treated with tamoxifen.
*
Signiﬁcantly different from control (p < 0.05).
#
Signiﬁcantly different from disease control (p < 0.05).
Table 2
Effect of tamoxifen on hypertrophic parameters.
Parameters
Model of ISO induced cardiac hypertrophy
CON
TAM
DIS
Model of PAAC induced cardiac hypertrophy
DIS + TAM
CON
TAM
DIS
DIS + TAM
Cardiac hypertrophy index (mg/mm)
22.52 0.24 21.5 0.45 33.04* 1.08 28.20# 1.10 23.28 0.61 22.89 0.47 30.36* 0.75 26.52 # 0.52
Left ventricular hypertrophy index (mg/mg) 0.72 0.0077 0.72 0.0082 0.79* 0.0059 0.74# 0.0095 0.71 0.0076 0.71 0.0109 0.76* 0.0081 0.73# 0.0089
LVW/RVW (mg/mg)
4.63 0.22 4.77 0.09
6.54* 0.19
5.48# 0.07
4.29 0.10
4.58 0.24
5.50* 0.28
4.69# 0.18
Values are expressed as mean SEM of 6 rats.
ISO – isoproterenol, PAAC – partial abdominal aortic constriction, CON – control animals (model of ISO induced cardiac hypertrophy)/sham control (model of PAAC induced cardiac
hypertrophy), TAM – control animals treated with tamoxifen (model of ISO induced cardiac hypertrophy)/sham control animals treated with tamoxifen (model of PAAC induced
cardiac hypertrophy), DIS – hypertrophic control animals, DIS + TAM – hypertrophic animals treated with tamoxifen.
*
Signiﬁcantly different from control (p < 0.05).
#
Signiﬁcantly different from disease control (p < 0.05).
There was a signiﬁcant (p < 0.05) increase in serum CK-MB,
LDH and CRP levels in ISO control and PAAC control rats as
compared to control rats. Treatment with tamoxifen (2 mg/kg/day,
po) showed a signiﬁcant (p < 0.05) reduction in serum CK-MB, LDH
and CRP levels in both the models of cardiac hypertrophy, i.e. ISOinduced and PAAC-induced hypertrophy (Table 1).
in both ISO induced and PAAC induced models of cardiac
hypertrophy. Further, isoproterenol control and PAAC control rats
showed a signiﬁcant (p < 0.05) increase in the left ventricular
collagen level as compared to control rats. Treatment with
tamoxifen (2 mg/kg/day, po) showed a signiﬁcant (p < 0.05)
reduction in collagen levels in both the models of cardiac
hypertrophy, i.e. ISO induced and PAAC induced hypertrophy
(Fig. 2b).
Hypertrophic parameters
Hemodynamic parameters
ISO control and PAAC control rats exhibited a signiﬁcantly
(p < 0.05) increased cardiac hypertrophy index, left ventricular
hypertrophy index and LV weight to RV weight ratio (LVW/RVW)
as compared to control group (Table 2). Treatment with tamoxifen
(2 mg/kg/day, po) signiﬁcantly (p < 0.05) reduced cardiac hypertrophy index, left ventricular hypertrophy index and LVW/RVW
(Table 2) in both the models of cardiac hypertrophy, i.e. ISOinduced and PAAC-induced hypertrophy. Further, ISO control and
PAAC control rats showed a signiﬁcantly (p < 0.05) increased LV
wall thickness and cardiomyocyte diameter as compared to
control rats. Treatment with tamoxifen (2 mg/kg/day, po) in
hypertrophic treated rats showed a signiﬁcant (p < 0.05) reduction in LV wall thickness (Fig. 1a) and cardiomyocyte diameter
(Fig. 1b) in both ISO-induced and PAAC-induced models of cardiac
hypertrophy.
ISO control and PAAC control rats showed signiﬁcant (p < 0.05)
increased in blood pressure and signiﬁcant (p < 0.05) reduction in
heart rate as compare to control rats (Table 3). Treatment with
tamoxifen (2 mg/kg/day, po) in hypertrophic treated group did not
produce signiﬁcant (p < 0.05) change in blood pressure and heart
rate in both ISO induced and PAAC induced models of cardiac
hypertrophy (Table 3). Further, hypertrophic control rats showed a
signiﬁcant (p < 0.05) reduction in rate of pressure development
and decay as compare to control rats. Treatment with tamoxifen
(2 mg/kg/day, po) in hypertrophic treated group produced
signiﬁcantly (p < 0.05) increased in rate of pressure development
(Fig. 3a) and decay (Fig. 3b) in both ISO induced and PAAC induced
models of cardiac hypertrophy.
LV tissue quantiﬁcation (LV collagen and Na+K+ATPase activity)
There was a signiﬁcant (p < 0.05) reduction in LV SOD,
glutathione levels in ISO control and PAAC control rats as compare
to normal control rats (Table 3). Treatment with tamoxifen (2 mg/
kg/day, po) in hypertrophic treated rats in both ISO induced and
PAAC induced models of cardiac hypertrophy, showed signiﬁcant
(p < 0.05) increase in LV SOD, glutathione levels (Table 3).
Serum cardiac markers
ISO control and PAAC control rats exhibited a signiﬁcantly
(p < 0.05) decreased Na+K+ATPase activity as compared to control
rats. Treatment with tamoxifen (2 mg/kg/day, po) signiﬁcantly
(p < 0.05) increased Na+K+ATPase activity, in treated rats (Fig. 2a),
Oxidative stress parameters
[(Fig._1)TD$IG]
B.M. Patel, V.J. Desai / Pharmacological Reports 66 (2014) 264–272
267
Fig. 1. Effect of tamoxifen on change in (a) LV wall thickness and (b) LV cardiomyocyte diameter. Each bar represents mean SEM of 6 experiments. * Signiﬁcantly different
from control (p < 0.05), # Signiﬁcantly different from disease control (p < 0.05). ISO, isoproterenol, PAAC, partial abdominal aortic constriction; CON – control animals (model of ISO
induced cardiac hypertrophy)/sham control (model of PAAC induced cardiac hypertrophy), TAM – control animals treated with tamoxifen (model of ISO induced cardiac
hypertrophy)/sham control animals treated with tamoxifen (model of PAAC induced cardiac hypertrophy), DIS – hypertrophic control animals, DIS + TAM – hypertrophic animals
treated with tamoxifen.
Hypertrophic control rats showed signiﬁcantly (p < 0.05) increased LV MDA levels as compare to control rats. Treatment
with tamoxifen (2 mg/kg/day, po) in hypertrophic treated group
showed signiﬁcant (p < 0.05) reduction in LV MDA levels in both
the models of cardiac hypertrophy, i.e. ISO induced and PAAC
induced hypertrophy (Table 3).
Histopathological studies
Histologically, control rats showed no pathological changes in
the LV cardiomyocyte (Fig. 4a and b) or in LV myocardial ﬁbers
(Fig. 5a and b). Histopathological examination of sections of LV
cardiomyocyte (Fig. 4e and f) and LV ﬁbers (Fig. 5e and f) from
untreated hypertrophic rats (DIS) showed marked microscopic
changes like reduction in extracellular space, intense ﬁbrosis and
ﬁber disarray. Tamoxifen treated section (DIS + TAM) showed
increase in extracellular space (Fig. 4g and h) and reduced ﬁbrosis
and ﬁber disarray (Fig. 5g and h) as compared to the hypertrophic
control rats (DIS) in both the models of cardiac hypertrophy, i.e. ISO
induced and PAAC induced hypertrophy. The control treated LV
cardiomyocyte (Fig. 4c and d) and LV ﬁbers (Fig. 5c and d) did not
show any histological alterations.
[(Fig._2)TD$IG]
Fig. 2. Effect of tamoxifen on change in (a) Na+ K+ ATPase activity and (b) LV collagen levels. Each bar represents mean SEM of 6 experiments. * Signiﬁcantly different from
control (p < 0.05), # Signiﬁcantly different from disease control (p < 0.05). For other explanations: see Fig. 1.
268
B.M. Patel, V.J. Desai / Pharmacological Reports 66 (2014) 264–272
Table 3
Effect of tamoxifen on hemodynamic and oxidative stress parameters.
Parameters
Blood pressure (mmHg)
Heart rate (beats/min)
LV SOD (units/min/mg protein)
LV glutathione (mg/mg protein)
LV MDA (nmol/mg protein)
Model of ISO induced cardiac hypertrophy
Model of PAAC induced cardiac hypertrophy
CON
TAM
DIS
DIS + TAM
CON
TAM
DIS
DIS + TAM
115 3
360 11
2.85 0.12
3.47 0.19
4.24 0.18
114 5
360 17
3.05 0.17
3.24 0.25
4.34 0.28
162* 4
266* 18
1.26* 0.05
2.25* 0.12
8.09* 0.26
157 3
294 12
2.01# 0.08
2.80# 0.10
6.92# 0.25
123 3
346 12
3.41 0.09
4.78 0.46
4.40 0.36
120 2
345 10
3.21 0.14
4.22 0.22
4.49 0.16
154* 4
238* 9
1.12* 0.11
2.49* 0.15
10.31* 0.15
152 4
251 9
2.23# 0.17
3.63# 0.28
6.86# 0.18
Values are expressed as mean SEM of 6 rats.
ISO – isoproterenol, PAAC – partial abdominal aortic constriction, CON – control animals (model of ISO induced cardiac hypertrophy)/sham control (model of PAAC induced cardiac
hypertrophy), TAM – control animals treated with tamoxifen (model of ISO induced cardiac hypertrophy)/sham control animals treated with tamoxifen (model of PAAC induced
cardiac hypertrophy), DIS – hypertrophic control animals, DIS + TAM – hypertrophic animals treated with tamoxifen.
*
Signiﬁcantly different from control (p < 0.05).
#
Signiﬁcantly different from disease control (p < 0.05).
[(Fig._3)TD$IG]
Fig. 3. Effect of tamoxifen on change in (a) rate of pressure development and (b) rate of pressure decay. Each bar represents mean SEM of 6 experiments. * Signiﬁcantly
different from control (p < 0.05), # Signiﬁcantly different from disease control (p < 0.05). For other explanations: see Fig. 1.
Discussion
The present investigation was carried out using 2 models of
cardiac hypertrophy viz. chemical induced hypertrophy using ISO
and pressure overload induced hypertrophy produced by PAAC.
The effects of ISO on heart are mediated through b-adrenoceptors,
producing relative ischemia or hypoxia due to myocardial
hyperactivity and coronary hypotension. Additionally, ISO causes
myocardial damage and enhances protein synthesis [27]. Pressure
overload hypertrophy results in thickening of ventricular walls and
cardiac myocytes and increased sarcomeric protein content with
expression of fetal cardiac genes [51].
Dyslipidemia is characterized by increased levels and altered
composition of very low density lipoprotein cholesterol (VLDL-C)
and reduced levels and altered composition of high density
lipoprotein cholesterol (HDL-C). PAAC is a model of pathological
hypertrophy and is associated with decreased fatty acid oxidation
[12,13]. In the present study, increase in serum total cholesterol,
low-density lipoprotein cholesterol (LDL-C), triglyceride level and
decrease in serum HDL level was found in hypertrophic control rat.
Treatment with tamoxifen signiﬁcantly reduced elevated serum
total cholesterol and LDL levels. However, tamoxifen did not
produce any effect on serum triglyceride and HDL levels. It has
been reported that tamoxifen produces inhibition of sterol-D8,7-
isomerase, which prevents the conversion of zymosterol into
cholesterol and affects cholesterol esteriﬁcation by inhibiting
acetyl-coenzyme A acetyltransferase [22]. Moreover, tamoxifen
stimulates the expression and activity of the LDL receptor [54].
Thus, these could be possible mechanism of tamoxifen in reduction
in LDL and triglyceride levels. Our reports are consistent with other
reports. Vinitha et al. [60] have reported that tamoxifen decreases
cholesterol level in atherosclerosis suffering animals. Hozumi et al.
[26] also reported tamoxifen decreases the concentration of total
and low-density lipoprotein cholesterol in patients with breast
cancer of postmenopausal onset. Thus, above ﬁnding provides
evidence that tamoxifen could be beneﬁcial in coronary heart
disease.
Concentration of CK-MB is higher in ventricular myocardial
tissue in animal models of hypertrophy or ischemia and in humans
with several cardiac conditions [37,61]. In the present study, there
was signiﬁcant increase in serum CK-MB level in hypertrophic rats,
and treatment with tamoxifen signiﬁcantly reduced CK-MB level
indicating decrease in myocardial damage.
A signiﬁcant increased level of LDH is found in case of
myocardial damage due to hypertrophy [47]. In the present study,
we found signiﬁcant increase in serum LDH level in both animal
models. Treatment with tamoxifen signiﬁcantly reduced serum
LDH level indicative of reduction in myocardial damage. It has been
[(Fig._4)TD$IG]
B.M. Patel, V.J. Desai / Pharmacological Reports 66 (2014) 264–272
269
Fig. 4. Representative ﬁgure of left ventricular cardiomyocyte from (a) normal control, (b) sham control, (c) control treated, (d) sham treated, (e) hypertrophic control from
isoproterenol group, (f) hypertrophic control from PAAC group, (g) hypertrophic treated from isoproterenol group, and (h) hypertrophic treated control from PAAC group.
Magniﬁcation 10.
reported that oxalate-induced PKC activation in LLC-PK1 cells is
responsible for LDH release [55]. Thus, tamoxifen being PKC
inhibitor, the decrease in LDH levels is justiﬁed.
Elevated serum CRP levels are associated with traditional
cardiovascular risk factors and obesity [7]. In the present study, we
found elevated CRP level in hypertrophic rat while treatment with
tamoxifen signiﬁcantly lowered level of CRP in hypertrophic rats.
Bonanni et al. [5] have shown that tamoxifen reduced CRP level in
healthy hysterectomized women. Thus, our reports are in
consistence with previous reports.
Hypertension and bradycardia in ISO and PAAC models has been
previously described [16,28]. ISO and PAAC induced increase in
mean arterial blood pressure and decrease in heart rate have been
noted in the present study which were not reduced by tamoxifen
treatment. Various studies have demonstrated that increase in left
ventricular collagen content may produce cardiac stiffness and
ﬁbrosis disrupting coordination of myocardial excitation–contraction coupling in both diastole and systole [9,44]. In the present study,
the left ventricular dysfunction assessed in terms of dp/dtmax and
dp/dtmin, was decreased in hypertrophic rats. Treatment with
tamoxifen signiﬁcantly increased dp/dtmax and dp/dtmin. Chintalgattu et al. [9] reported PKC-d association in angiotensin II induced
ERK activation in ﬁbroblasts. Activation of PKC by phorbol ester in
the heart leads to signiﬁcant loss of contractile function [8].
Tamoxifen is a PKC inhibitor and this may be the possible reason
behind improvement in cardiovascular functioning.
The Na+K+ATPase transporter plays a major role in electrical–
mechanical activity of cardiac muscle for maintaining cell
[(Fig._5)TD$IG]
270
B.M. Patel, V.J. Desai / Pharmacological Reports 66 (2014) 264–272
Fig. 5. Representative ﬁgure of left ventricular myocardial ﬁbers from (a) normal control, (b) sham control, (c) control treated, (d) sham treated, (e) hypertrophic control from
isoproterenol group, (f) hypertrophic control from PAAC group, (g) hypertrophic treated from isoproterenol group, (h) hypertrophic treated control from PAAC group.
Magniﬁcation 45.
homeostasis and in regulation of intracellular calcium in cardiomyocyte [57]. In the present study, hypertrophic rats showed
signiﬁcantly reduction in Na+K+ATPase activity. Treatment with
tamoxifen in hypertrophic treated rats signiﬁcantly restored
Na+K+ATPase activity. Activity of the Na+K+ATPase during injury
and disease is regulated by PKC, which produces phosphorylation
of serine and threonine residues on the Na+K+ATPase, reducing ion
transport function [14]. Thus, restoration of Na+K+ATPase activity
by tamoxifen suggests its beneﬁcial effect in maintaining cardiac
cells homeostasis.
Excessive accumulation of collagen leads to tissue stiffness, and
adverse effects on the elasticity of the myocardium producing
ventricular systolic and diastolic dysfunction [33]. Collagen levels
are reported to increase in ISO and PAAC induced cardiac
hypertrophy [15,31,32]. In the present study, we also found
signiﬁcant increase in LV collagen level in hypertrophic control rats
which was reduced by tamoxifen treatment. Neugarten et al. [38]
has reported inhibition of type I and IV collagen by tamoxifen
which is mediated through inhibition of COL4A1 gene transcription and type IV collagen protein synthesis inhibition. It has also
been reported that hemodynamic overload may stimulate cardiac
hypertrophy and induce cardiac injury ﬁbrosis through PKC
activation [31]. Tamoxifen being an inhibitor of PKC, this might
be possible mechanism of inhibition of collagen deposition.
Excess accumulation of extracellular matrix leads to decrease in
tissue compliance and function [52]. It has been reported that PKC
[(Fig._6)TD$IG]
B.M. Patel, V.J. Desai / Pharmacological Reports 66 (2014) 264–272
271
Fig. 6. Summarized effects of tamoxifen mainly mediated through protein kinase C. # – decrease, " – increase, PKC – protein kinase C, LDL – low density lipoprotein, LDH –
lactate dehydrogenase.
activation is one of the regulator of isoproterenol induced cardiac
hypertrophy [10]. Further, Bayer et al. [3] and Braun et al. [6] have
reported PKC upregulation in pressure overload cardiac hypertrophy. The ISO induced cardiac hypertrophy and PAAC induced
cardiac hypertrophy models employed in the present study have
depicted cardiac hypertrophy index, LV hypertrophic index, LV
wall thickness, LV protein content suggesting the development of
cardiac hypertrophy [49]. Treatment with tamoxifen signiﬁcantly
reduced cardiac hypertrophy. PKC is responsible for the hypertrophic gene regulation in cardiac hypertrophy, thus prevention of
cardiac hypertrophy by tamoxifen is justiﬁed. Improvement in
cardiac hypertrophy was further evident by histopathological
study of the transversection of left ventricle tissue. There was
reduction in extracellular space and increase in cardiomyocyte
diameter in hypertrophic control rats as compare to control rats.
Treatment with tamoxifen in hypertrophic treated rats showed
less reduction in extracellular space and less increase in
cardiomyocyte diameter as compared to hypertrophic control
rats.
ISO causes myocardial ischemia due to excessive production of
free radicals resulting from oxidative metabolism of catecholamine
[16]. ROS aggravate cardiac hypertrophy in pressure overload
induced cardiac hypertrophy [25]. In the present study, in both
models of cardiac hypertrophy, we found oxidative stress, which
was evident by increase in LV MDA and decrease in reduced
glutathione and SOD levels. Treatment with tamoxifen signiﬁcantly prevented this oxidative stress. The study reported by
Thangaraju et al. [56] showed a signiﬁcantly decreased concentration of malondialdehyde, an end product of lipid peroxidation and
remarkably increased levels of enzymic and non-enzymic antioxidants in tamoxifen-treated patients. Also, inhibition of lipid
peroxidation and antioxidant effect of tamoxifen and its metabolites have been reported [62]. Tamoxifen enhanced MnSOD
expression is responsible for its cardioprotective action against
ADR-induced cardiomyocytes injury [11]. Thus tamoxifen prevents
oxidative stress and thereby preserves cardiovascular function.
Conclusions
In conclusion (Fig. 6), our data suggests that tamoxifen
produces beneﬁcial effects on cardiac hypertrophy as evident
from reduction in hypertrophic parameters including collagen
levels and thereby preserves LV systolic and diastolic function.
Thus, this may be considered as preventive measure for cardiac
hypertrophy.
Conﬂict of interest
The authors declare no conﬂict of interest.
Funding
The work was not funded by any external agency, governmental
body or any other sponsor.
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