Trimetazidine prevents pirarubicin-induced myocardial
damage: a possible antioxidant mechanism
Bao-Xin Ma1,4 , phD, Qun Li2, MD, Hua Li3, MD, Jie Li4 , phD, Sui-Sheng Wu4,
MD
1 Department of Cardiology, Affiliated Hospital of Binzhou Medical University,
Binzhou , Shandong Province, China
2 Department of Thyroid Surgery, First Hospital of Jilin University, Changchun , Jilin
Province, China
3 Department of Oncology, Affiliated Hospital of Binzhou Medical University,
Binzhou, Shandong Province, China
4 Department of Geriatrics, First Hospital of Jilin University, Changchun, Jilin
Province, China
Address correspondence and reprint request to: Dr. Jie Li, Department of Geriatrics,
First Hospital of Jilin University, Changchun, Shandong Province, China. E-mail:
[email protected]
Fax: +86(543)3258722
Telephone: +86(543)3258722
Mobilephone:+86-18454392981
Bao-xin Ma, E-mail:[email protected]
Qun Li, E-mail:[email protected]
Hua Li, E-mail:[email protected]
Jie Li, E-mail:[email protected]
Sui-sheng Wu,E-mail:[email protected]
1
Abstract
Objective: The present study aimed to investigate the effect and possible mechanism of
action of trimetazidine on myocardial damage induced by pirarubicin.
Methods: Thirty-six Wistar rats were randomly divided into a control group, model
group, and treatment group. Rats in the model group and treatment group were injected
with pirarubicin at 2.5 mg/kg through the vena caudalis once a week for six weeks. The
control group was injected in an equivalent manner with 0.9% NaCl for 6 weeks. Rats
in the treatment group received an intragastric infusion of trimetazidine 5.4 mg/kg/d for
8 weeks. The control and model groups were injected in an equivalent manner with
0.9% NaCl. At the end of the experiment, superoxide dismutase, cardiac enzymes, and
free radical mediators were measured. The myocardial tissue was examined by light
microscopy and electron microscopy.
Results: The levels of myoglobin, troponin, and alanine transaminase in the treatment
group were lower than those in the model group (p<0.05). After treatment with
trimetazidine, malondialdehyde (MDA) and nitric oxide (NO) levels were lower than
those in the model group (P < 0.05), and nonprotein sulfhydryl (NPSH) and superoxide
dismutase (SOD) levels were higher than those in the model group (P < 0.05). The
structure of myocardial cells arranged in a disorderly manner was severely damaged.
Myocardial myofilament dissolution and fracture were visible in the model group. In
the treatment group, the structure of the myocardial cells was orderly, and the structure
of the myocardium was basically preserved. Trimetazidine administration reduced
mitochondrial damage and relieved myocardial injury.
Conclusion: Trimetazidine has protective effects on cardiomyocytes damaged by
pirarubicin, and its mechanism may be related to antioxidative activities.
[Key words] trimetazidine; pirarubicin; myocardial damage; oxidative stress
Pirarubicin (THP) is a newer generation anthracycline antitumor antibiotic.1,2 THP
and THP-based combination chemotherapies have been shown to be effective against
various tumors.3,4 However, the toxic side effects of THP have seriously restricted its
clinical application. The development of a progressive dose-dependent
cardiomyopathy results in irreversible congestive heart failure5 that is mainly caused
by damage to the structure of mitochondria, which affects the metabolism of the heart
and ultimately has serious toxic effects on the heart.6
Trimetazidine (1-(2,3,4-trimethoxybenzyl) piperazine; TMZ), like ranolazine and
L-carnitine, is a cardioprotective drug.7,8 It blocks fatty acid oxidation and increases
glucose utilization, eventually leading to a reduction in intracellular acidosis.9
Numerous studies suggest that TMZ inhibits the production of free radicals and
protects myocyte structure and function.10,11 However, few studies have evaluated the
cardioprotective effect of TMZ after chemotherapy. The effect of clinical intravenous
chemotherapy and long-term trimetazidine intervention has not been reported.
Therefore, the present study examined the protective effect of TMZ and the
mechanism underlying this protection against THP-induced myocardial injury in rats.
2
Materials and methods. Animal treatment. A total of 36 healthy Wistar male rats
(280 ± 20 g, License: SCXK-(Ji) 2007-0003) were obtained from the Experimental
Animal Center of Jilin University (Changchun, China). This study was approved by
the ethics committee of the First Bethune Hospital of Jilin University. Rats were
divided into the following groups: control group (n=10), model group (n=13), and
treatment group (n=13). Rats were injected with pirarubicin (2.5 mg/kg, once a week)
through the vena caudalis for six weeks to establish a myocardial damage model.
Pirarubicin (Cat.1105c2) was purchased from Shenzhen Main Luck Pharmaceuticals
Inc (Shenzhen, China). TMZ (5.4 mg/kg/d, once a week for 8 weeks) was
administered by gavage one day before modeling. TMZ (Cat. 2000958) was
purchased from Servier.
Cardiac enzyme detection. Following the 8-week treatment period, oral feeding was
not provided for 12 hours before anesthesia administration. The eyeballs of rats were
removed under anesthesia to extract blood. After resting for 30 min at room
temperature, the serum was separated by centrifugation at 12,000 rpm at a low
temperature for 5 min. Troponin I and myoglobin were measured by
chemiluminescence (Johnson 5600 Biochemical Analyzer; kit: 20120110). Alanine
aminotransferase was measured by a continuous monitoring procedure (Japan Hitachi
Automatic Biochemical Analyzer1210; Kit: 20120218).
Estimation of the free radical mediator. Myocardial tissue was washed with cold
0.9% NaCl. Then, the water was removed completely. After the left ventricular
myocardium was cut and broken, 10% tissue homogenate was prepared by adding the
cold 0.9% NaCl. The supernatant was separated by centrifugation at 3500 rpm at 4C
for 15 min. After centrifugation, the supernatant was stored at -20C. The
dithio-bis-nitrobenzoic acid (DTNB) method was used with the nonprotein sulfhydryl
(NPSH) assay. The malondialdehyde (MDA) level was measured by the thiobarbituric
acid (TBA) method. Nitric oxide (NO) content was measured with the nitric reductase
method. The kits were provided by the Nanjing Jiancheng Bioengineering Institute.
Steps were carried out according to the kit instructions. The activity of superoxide
dismutase (SOD) was determined by the pyrogallol method (Johnson 5600
Biochemical Analyzer and Instrument; kit: 20120810).
Echocardiography detection. After 8 weeks of treatment, the rats were anesthetized
by intraperitoneal injection of 10% chloral hydrate. After shaving the chest, the rats
were fixed on the self-made fixed table in the left lateral decubitus position.
Ultrasound detection was carried out with the Japanese ALOKA-5500 ultrasonic
diagnostic instrument. All ultrasound measurements were performed by the same
experienced physician on the same ultrasound diagnostic apparatus.
Estimation of myocardial structure. Rats were anesthetized with diethyl ether. Two
pieces of myocardial tissue were taken from the left ventricular anterior wall. One was
fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin sections
were acquired and subjected to hematoxylin-eosin (HE) staining after routine
deparaffinization and hydration. Eight weeks following the commencement of the
experiment, the other sample was fixed in 4% glutaraldehyde at 4˚C. The heart pieces
3
(1x1x1 mm) were processed successively with 1% osmic acid fixations for 3 hours,
dehydrated in an ethanol series, embedded in Epon 812 epoxy resin, and sectioned.
Uranyl acetate and lead citrate double staining and transmission electron microscopy
(JEM-1200EX) were used to observe the sections, and photographic images were
captured.
Statistical analysis. Statistical analysis was performed using SPSS 11.5 software
(IBM–SPSS, Inc., Armonk, New York, USA). All data are presented as means ±
standard deviations. One-way analysis of variance (ANOVA) was applied for multiple
comparisons, and p values < 0.05 were considered to be statistically significant.
Results. Estimation of pirarubicin on animal. Symptoms occurred in the model group
and treatment group 1 week after the administration of pirarubicin and included
poorer mental function, diminished activity, and decreased diet. Some rats exhibited
depilation, diarrhea, and weight loss. The symptoms of the treatment group were not
as severe as those of the model group. When fed for 8 weeks, 3 rats died in the model
group and 1 rat died in the treatment group. The body weight of rats increased with
time. The increase in weight gain in the model group was lower than that in the
control group (379.23±47.81 vs. 499.11±59.98, P < 0.05). The increase in the
treatment group was greater than that in the model group (424.80±35.73 vs.
379.23±47.81; P < 0.05), as shown in Figure 1.
600
#
body weight(g)
500
*#
*&#
400
0 weeks
8 weeks
300
200
100
0
control
model
treatment
Figure 1 Weights of rats in various groups at 8 weeks.
*P < 0.05 compared with the control group; & P < 0.05 compared with the model group; # P <
0.05 compared with 0 weeks.
Estimation of cardiac enzymes. Compared with levels in the control group,
myoglobin, troponin, and alanine aminotransferase (ALT) levels were increased in the
treatment group. The levels in the rats that were administered trimetazidine were
lower than those in the model rats (myoglobin: 102.85±19.57 vs. 134.50±40.87,
troponi-I: 0.09±0.04 vs. 0.18±0.03, ALT: 39.51±5.26 vs. 51.19±9.81; P < 0.05).
4
200
180
160
140
120
100
80
60
40
20
0
*
#
myoglobin(ng/ml)
ALT(U/L)
*
#
control
model
treatment
Figure 2 Myocardial enzymes in rats in various groups at 8 weeks.
*P < 0.05 compared with the control group; #P < 0.05 compared with the model group.
Estimation of oxidative parameters. Compared with the control group, the levels of
MDA and NO increased and the levels of NPSH and SOD decreased in the model
group. MDA and NO were lower (MDA: 5.01±1.44 vs. 5.41±1.32, NO: 22.31±9.61 vs.
25.73±9.58; P < 0.05) and NPSH and SOD were higher (NPSH: 55.53±9.96 vs.
51.99±12.35, SOD: 17.58±0.97 vs. 13.34±3.21; P < 0.05) in the rats that were
administered trimetazidine than in the model group.
* #
* #
* #
control
model
treatment
SO
D(
U/
mg
pr
ot
)
* #
MD
A(
nm
ol
/g
pr
ot
)
NO
(u
mo
l/
gp
ro
t)
NP
SH
(u
mo
l/
gp
ro
t)
90
80
70
60
50
40
30
20
10
0
Figure 3 Levels of MDA, NO, and NPSH and activities of SOD in the myocardium of rats in the
three groups 8 weeks after administration.
*P < 0.05 compared with the control group; #P < 0.05 compared with the model group.
Estimation of echocardiography. Compared with the control group, the left
ventricular ejection fraction (EF) and fractional shortening (FS) decreased, but the left
ventricular internal diastolic diameter (LVIDd) and left ventricular internal dimension
systole (LVIDs) increased in the model group (EF: 76.98±1.52 vs. 83.04±3.55, FS:
40.46±1.40 vs. 47.24±4.55, LVIDd: 7.16±0.25 vs. 6.68±0.28, P < 0.01; LVIDs:
4.20±0.17 vs. 3.92±0.39, P < 0.05). EF and FS were greater than those in the model
group (EF: 79.47±1.54 vs. 76.98±1.52, FS: 43.09±0.68 vs. 40.46±1.40; P < 0.05) and
LVIDd and LVIDs were lower than those in the model group after treatment with
trimetazidine (LVIDd: 6.88±0.32 vs. 7.16±0.25, LVIDs: 3.91±0.25 vs. 4.20±0.17, P <
0.05).
5
100
90
80
70
60
50
40
30
20
10
0
* #&
control
model
treatment
* #&
* #&
EF(%)
FS(%)
LVIDd(mm)
# &
LVIDs(mm)
Figure 4 Echocardiography results in the three groups 8 weeks after administration.
*P < 0.01, #P < 0.05 compared with the control group; &P < 0.05 compared with the model group.
Estimation of myocardial structure by optical microscopy. At 8 weeks, myocardial
cells of the rats in the control group were arranged neatly, and the cellular structures
were intact (Figure 5A). In the model group, myocardial cells were arranged in a
disorderly manner, the structure was severely damaged. Myocardial myofilament
dissolution and fracture were visible (Figure 5B). In the treatment group, the structure
of the myocardial cells was orderly, the structure of the myocardium was basically
preserved, and partial dissolution and fracture were relatively absent (Figure 5C).
Figure 5 Observation of myocardial tissue in rats in various groups under an optical microscope (×200)
A: control group, B: model group, multiple visible myocardial cells (→), myofilament dissolved, fracture
(←), C: treatment group, partial dissolution, fracture (←)
Estimation of myocardial structure by electron microscopy. At 8 weeks, in the
control group, sarcomeres of cardiomyocytes were arranged in order, and the Z and M
lines were clear. There were many long, oval, and longitudinal arrangements of
mitochondria (Figure 6A). In the model group, myocardial myofilaments dissolved,
fractured, and disappeared, the number of mitochondria decreased, and cytoplasmic
matrices were cavitated (Figure 6B). Sarcomeres of cardiomyocytes were arranged
6
neatly after treatment with trimetazidine, the number of local myofilaments slightly
decreased, and the surrounding mitochondria were oval and arranged in parallel
between the myofilament bundles (Figure 6C). The application of trimetazidine
reduced mitochondrial damage and relieved myocardial injury.
Figure 6 Observation of myocardial tissue of rats in various groups under an electron microscope (×7500)
bar=500 nm
A: control group: the arrangement of cardiomyocyte sarcomeres were arranged in parellel, the Z and M lines
were clear, and the mitochondria were oval and arranged in order ().
B: model group: the myocardial muscle bundle had dissolved (↑), fractured, and disappeared, the number of
mitochondria decreased, and the cytoplasmic matrix was cavitated.
C: treatment group: the arrangement of cardiomyocyte sarcomeres was in line (), the number of local
myofilaments was slightly reduced, and the surrounding mitochondria were oval and arranged in parallel
between the muscle bundles.
Discussion. In the present study, we evaluated the effect and possible mechanism of
action of trimetazidine on myocardial damage induced by pirarubicin. The main
findings of the study were the following: 1. Injection of THP caused myocardial
damage, including myofilament dissolution and fracture, and 2. TMZ offered
protection against cardiotoxicity via the pathway of antioxidative activity, the
upregulation of NPSH and SOD, and the downregulation of MDA and NO.
THP is a derivative of anthracycline and exhibits strong antiproliferative activity.
However, its clinical use is severely limited by the development of cardiotoxicity.5,12
THP can intercalate into the mitochondrial membranes. These organelles are affected
and generate damaging reactive oxygen species (ROS). THP can increase the ROS
concentration beyond physiological levels.5 ROS directly or indirectly activates
several signaling pathways that lead to cardiomyocyte apoptosis and ultimately to
heart failure. Many studies suggest that antioxidative activities are the underlying
mechanism that protects against cardiotoxicity.13-16 Sun et al.14 showed that myricitrin
effectively reduced doxorubicin (DOX)-induced cell toxicity by counteracting
oxidative stress and increasing the activities of antioxidant enzymes. Das et al.
suggested that beet root juice could protect against DOX toxicity in cardiomyocytes
7
by reducing the DOX-induced generation of ROS.15
TMZ is an anti-ischemic agent that is widely used in the treatment of coronary
artery disease without affecting the hemodynamic determinants of myocardial oxygen
consumption.17,18 The anti-ischemic effects of TMZ have been experimentally
assessed in various models.19,20 It was reported that TMZ protected against
smoking-induced left ventricular remodeling. The underlying mechanism was the
attenuation of oxidative stress, apoptosis, and inflammation.10 It was also shown that
TMZ could shift fatty acid oxidation to glucose oxidation and attenuate myocardial
ischemia/reperfusion injury by activating AMPK and ERK signaling.8
In conclusion, the present study is the first to reveal that TMZ has protective
effects on cardiomyocytes damaged by pirarubicin, most likely related to its
antioxidant activity. The results of the present study may aid in the development of a
novel drug for the treatment of pirarubicin-induced cardiotoxicity.
8
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