Tissue and Cell 49 (2017) 410–426

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Tissue and Cell

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

Protective role of CoQ10 or L-carnitine on the integrity of the

myocardium in doxorubicin induced toxicity
Hesham N. Mustafa a,∗ , Gehan A. Hegazy b,c , Sally A. El Awdan d , Marawan AbdelBaset d
a
Anatomy Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
b
Clinical Biochemistry Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
c
Medical Biochemistry Department, National Research Centre, Cairo, Egypt
d
Pharmacology Department, National Research Center, Cairo, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Doxorubicin (DOX) is a chemotherapeutic agent used for treatment of different cancers and its clinical
Received 16 January 2017 usage is hindered by the oxidative injury-related cardiotoxicity. This work aims to declare if the harmful
Received in revised form 15 March 2017 effects of DOX on heart can be alleviated with the use of Coenzyme Q10 (CoQ10) or L-carnitine. The study
Accepted 31 March 2017
was performed on seventy two female Wistar albino rats divided into six groups, 12 animals each: Control
Available online 2 April 2017
group; DOX group (10 mg/kg); CoQ10 group (200 mg/kg); L-carnitine group (100 mg/kg); DOX + CoQ10
group; DOX + L-carnitine group. CoQ10 and L-carnitine treatment orally started 5 days before a single dose
Keywords:
of 10 mg/kg DOX that injected intraperitoneally (IP) then the treatment continued for 10 days. At the end
Dox
CoQ10 and L-carnitine of the study, serum biochemical parameters of cardiac damage, oxidative stress indices, and histopatho-
Cardiotoxicity logical changes were investigated. CoQ10 or L-carnitine showed a noticeable effects in improving cardiac
eNOS functions evidenced reducing serum enzymes as serum interleukin-1 beta (IL-1 ␤), tumor necrosis factor
Vimentin alpha (TNF-␣), leptin, lactate dehydrogenase (LDH), Cardiotrophin-1, Troponin-I and Troponin-T. Also,
alleviate oxidative stress, decrease of cardiac Malondialdehyde (MDA), Nitric oxide (NO) and restoring
cardiac reduced glutathione levels to normal levels. Both corrected the cardiac alterations histologically
and ultrastructurally. With a visible improvements in ␣-SMA, vimentin and eNOS immunohistochemical
markers. CoQ10 or L-carnitine supplementation improves the functional and structural integrity of the
myocardium.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction (Potemski et al., 2006). Cardiomyocytes suffer from relative defi-

Chemotherapy is not a risk-free experience due to the unwanted
effects of these medications on the healthy cells. DOX (Adriamycin)
is one of the chemotherapeutic medications that is widely used
and occurs within anthracycline list of antibiotics. It is produced by
Streptomyces peucetius var caesius algae (Arcamone et al., 2000). It
is used in the management of lymphomas, leukemia and different
solid tumors like carcinoma of ovaries, breast, lung and thyroid
gland (Gianni et al., 2007).
DOX has a dose dependent cardiotoxicity (Elbaky et al., 2010).
DOX toxicity is responsible for 2–3% of all cases of heart trans-
plantation patients (Mitry and Edwards, 2016). DOX-induced
cardiotoxicity is mainly mediated via oxidative stress and apoptosis
∗ Corresponding author at: King Abdulaziz University, Faculty of Medicine,
Anatomy Department, Building No. 8 − P.O. Box: 80205, Jeddah, 21589, Saudi Arabia.
E-mail address: hesham977@hotmail.com (H.N. Mustafa).
ciency of antioxidant enzymes so they are more vulnerable for the
deleterious effects produced by free radical result from DOX admin-
istration (Dong et al., 2014). DOX-induced cardiotoxicity may result
in evident cardiac damage before clinical signs become apparent,
hence the importance of detecting biochemical markers possess
high sensitivity and specificity that indicates early damage to be
used for screening and early diagnoses of DOX-induced cardiotox-
icity (Atas et al., 2015).
L-carnitine is a naturally occurring compound biosynthesized
from the amino acids methionine and lysine in the kidneys and
liver. It is found at a high concentration in skeletal and cardiac
muscles (Aliev et al., 2009). L-carnitine exerts an antioxidant action
that protect against lipid peroxidation of membrane phospholipid
(Guan et al., 2009). L-carnitine eliminates extracellular toxic acetyl-
coenzyme A that is responsible for mitochondrial ROS (reactive
oxygen species) (Agarwal and Said, 2004). L-carnitine revealed a
varied range of biological actions comprising anti-inflammatory
and anti-apoptotic properties (İzgüt-Uysal et al., 2003).

http://dx.doi.org/10.1016/j.tice.2017.03.007
0040-8166/© 2017 Elsevier Ltd. All rights reserved.
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426
CoQ10 is a compound which is synthesized endogenously that
is a potent lipophilic antioxidant capable of recycling and regen-
erating other antioxidants such as ascorbate and tocopherol. Also,
CoQ10 causes scavenging of free radicals, inhibition of lipid perox-
idation (Zhang et al., 2013). CoQ10 is a cofactor that plays a crucial
role in the mitochondria respiratory chain and ATP production
(Bhagavan and Chopra, 2006).
␣-Smooth muscle actin (␣-SMA) is a marker for myofibroblast-
like cells and hepatic stellate cells (HSC) (Mustafa, 2016). Vimentin
is an intermediate filament (IF) protein that is expressed frequently
in the cells of mesodermal origin as endothelial cells, that forms
an irregular network in endomysium and perimysium sheaths of
the myocardium (Heling et al., 2000). Vimentin expression occurs
during myocardial stress as heart failure (Sharov et al., 2005).
Endothelial nitric oxide synthase (eNOS) is shown in the
endothelium, within the heart, in cardiac conduction tissue and
in cardiac myocytes (Jones et al., 2004). In cardiac muscle, eNOS
arranges NO physiological action, as organizing endothelial func-
tion, platelet aggregation, vascular tone and cardiac contractility
(Pott et al., 2006). The expression of eNOS in the myocardium is
modulated in dilated cardiomyopathy with evidence of heart fail-
ure (Crespo et al., 2008). Thus, the aim of this work is to declare the
possible ameliorative potentials of CoQ10 or L-carnitine on DOX
induced cardiotoxicity.
2. Material and methods
2.1. Ethical approval
The study was conducted after approval by the Medical Research
Ethics Committee of the National Research Centre, Cairo, Egypt and
followed the recommendations of the National Institutes of Health
Guide for Care and Use of laboratory Animals (NIH Publications No.
8023, revised 1978).
2.2. Animals
The study was performed on female Wistar albino rats (n = 72),
8–10 weeks of age and weighed ranging 150–200 g that were bred
and obtained from Animal House Colony, National Research Centre,
Cairo, Egypt. All animals were housed in cages in a temperature con-
trolled (24 ± 1 ◦ C) with a 12 h light/dark cycle and 60 ± 5% humidity
and were provided with standard laboratory diet and water ad libi-
tum. DOX was provided by Sigma, which was dissolved in sterile
saline. CoQ10 and L-carnitine were obtained from Mepaco, Egypt.
2.3. Experiment
Rats were divided into six groups, including 12 animals: Control
group; DOX group (10 mg/kg) (Mustafa et al., 2015); CoQ10 group
(200 mg/kg)(Mustafa et al., 2015); L-carnitine group (100 mg/kg)
(Mescka et al., 2016); DOX + CoQ10 group; DOX + L-carnitine group.
CoQ10 and L-carnitine treatment orally started 5 days before a sin-
gle dose of 10 mg/kg DOX that was injected intraperitoneally (IP)
then the treatment was continued for 10 days. At the beginning and
at the end of the study the animals body weights were measured.
2.4. Echocardiographic study
ECG was recorded at the beginning of the experiment to ensure
the normal ECG pattern of the rats. At the last day of the experiment,
rats were anesthetized by dimethyl ether and ECG was recorded for
1 min. Heart rate, P duration, QRS Interval, QTc, and ST Height were
monitored using ECG Powerlab module which consists of Power-
lab/8sp and Animal Bio-Amplifier, Australia, in addition to Lab Chart
7 software with ECG analyzer (Hajrasouliha et al., 2004).
411
At the end of treatment, the animals were kept for an overnight
fasting and the blood samples were collected from retroorbital
plexus and allowed to clot for 30 min at room temperature. After
blood collection, all animals were rapidly sacrificed and the hearts
were dissected and immediately homogenized in 50 mM ice-cold
phosphate buffer (pH 7.4) to give 10% homogenate (w/v). The
homogenate was centrifuged at 3200 rpm for 20 min in cooling cen-
trifuge. The supernatant was used for the determination of different
parameters.
2.5. Biochemical measurements
2.5.1. Measurement of malondialdehyde (MDA) and reduced
glutathione (GSH)
Measurement of malondialdehyde (MDA) and reduced glu-
tathione (GSH) levels using colorimetric assay kits (Catalogues No.
MD 25 29, GR 25 11 respectively) in accordance with the manufac-
turer’s instructions (Bio Diagnostic, Cairo, Egypt).
2.5.2. Measurement of Nitric oxide (NO)
Nitric oxide metabolites (NO) were determined according to
the method described by Miranda et al. (Miranda et al., 2001)
and expressed as ␮M/g wet tissue using colorimetric assay kits
(Catalogue No. NO 25 33) in accordance with the manufacturer’s
instructions (Bio Diagnostic, Cairo, Egypt). Nitric oxide has a short
biological half-life and is rapidly converted into its stable metabo-
lites, nitrite and nitrate. Determination of nitrite and nitrate (NOx)
in body fluid and tissues is widely used as a marker of NO produc-
tion Miranda et al. (Miranda et al., 2001). Nitric oxide measured
as nitrite was determined using Griess reagent, according to the
method of Moshage et al. (Moshage et al., 1995), where nitrite, sta-
ble end product of nitric oxide radical, is mostly used as an indicator
for the production of nitric oxide.
2.5.3. Assessment of inflammatory cytokines
Serum interleukin-1beta (IL-1 ␤), tumor necrosis factor (TNF-
␣) and Leptin levels were measured using an enzyme-linked
immunosorbent assay (ELISA) kit (Catalogues No. RAB0277 Sigma,
RAB0479 Sigma and RAB0335 Sigma respectively) according to the
manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, United
States). All samples were tested in duplicate and averaged.
2.5.4. Assessment of cardiac markers
Lactate dehydrogenase (Catalog No. MA5-17242) and cardiac
specific creatinine kinase levels were measured using commercial
kits (Catalog No. LF-MA0233) purchased from Invitrogen (Thermo
Fisher Scientific, Inc., Waltham, MA, USA) according to the manu-
facturer’s protocol. All measurements were performed in duplicate.
2.5.5. Quantitative estimation of serum troponin I
Quantitative estimation of serum troponin I levels were car-
ried out by ELISA technique-using kit (Catalog No. LS-F127394)
purchased from Lifespan BioSciences international Inc., USA
2.5.6. Measurement of serum Troponin-T (cTnT) levels
Troponin-T (cTnT) levels were measured cTnT with a third-
generation cardio-specific assay (Catalog No. 04660307190)
(ElecsysR Troponin T STATimmunoassay manufactured by Roche
Diagnostics, France)
2.5.7. Measurement of Cardiotrophin-1
Cardiotrophin-1 is measured based on the sandwich ELISA
principle following the manufacturer’s instructions using Rat
CTF1/Cardiotrophin-1 ELISA Kit (Catalog No. LS-F127394), pur-
chased from LifeSpan BioSciences international Inc., USA.
412 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426

Table 1
Effect of CoQ10 and L-carnitine on body weight, heart weight, heart/body weight% and mortality No.

Groups Control N = 12 CoQ10 N = 12 L-carnitine DOX N = 9 DOX + CoQ10 DOX + L-

N = 12 N = 11 carnitine
N = 11

Body weight 152.3 ± 2.31 153.01 ± 3.41 151.33 ± 5.61 135 ± 6.51 146.07 ± 3.80 144.4 ± 5.03
(g) NS NS 1
P ≤ 0.001 1
P ≤ 0.05 1
P ≤ 0.01
2
P ≤ 0.001 2
P ≤ 0.001
Heart weight 0.810 ± 0.019 0.79 ± 0.021 0.83 ± 0.017 0.482 ± 0.040 0.603 ± 0.011 0.594 ± 0.026
(g) NS NS P1 ≤ 0.001 1
P ≤ 0.001 1
P ≤ 0.001
2
P ≤ 0.001 2
P ≤ 0.001
Heart 0.532 ± 0.002 0.516 ± 0.006 0.548 ± 0.008 0.357 ± 0.007 0.413 ± 0.003 0.411 ± 0.001
weight/Body 1
P ≤ 0.001 1
P ≤ 0.001 1
P ≤ 0.001 1
P ≤ 0.001 1
P ≤ 0.001
weight% 2
P ≤ 0.001 2
P ≤ 0.001
Mortality No. 0 0 0 3 1 1

Values are means ± SD (Control n = 12& DOX = 9 & treated = 11). ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX.

Table 2
Comparison of electrocardiographic changes in different studied groups.

Groups Heart Rate P-R (s) QRS Interval (s) QTc duration P amplitude T amplitude S-T Height
(bpm) (s) (mV) (mV) (mV)

Control 318.386 ± 47.582 0.164 ± 0.017 0.015 ± 0.002 0.096 ± 0.005 0.061 ± 0.018 0.185 ± 0.043 0.035 ± 0.033
(n = 12)
DOX 281.930 ± 22.591 0.214 ± 0.019 0.016 ± 0.004 0.134 ± 0.077 0.057 ± 0.057 0.145 ± 0.105 0.090 ± 0.066
1 1 1 1 1 1 1
Significance P = 0.013 P = 0.0001 P = 0.624 P = 0.024 P = 0.743 P = 0.141 P = 0.006
(n = 9)
CoQ10 286.167 ± 10.913 0.187 ± 0.024 0.017 ± 0.002 0.101 ± 0.005 0.066 ± 0.007 0.174 ± 0.021 0.059 ± 0.030
1 1 1 1 1 1 1
Significance P = 0.061; P = 0.059; P = 0.275; P = 0.811; P = 0.796; P = 0.750; P = 0.405;
2 2 2 2 2 2 2
(n = 12) P = 0.815 P = 0.016 P = 0.443 P = 0.082 P = 0.625 P = 0.409 P = 0.116
L-carnitine 307.483 ± 44.887 0.182 ± 0.031 0.015 ± 0.001 0.103 ± 0.014 0.066 ± 0.009 0.209 ± 0.058 0.023 ± 0.019
1 1 1 1 1 1 1
Significance P = 0.520; P = 0.111; P = 0.698; P = 0.773; P = 0.774; P = 0.482; P = 0.335;
2 2 2 2 2 2 2
(n = 12) P = 0.161 P = 0.003 P = 0.447 P = 0.148 P = 0.604 P = 0.071 P = 0.002
DOX + CoQ10 340.989 ± 8.550 0.179 ± 0.007 0.017 ± 0.002 0.096 ± 0.011 0.047 ± 0.031 0.127 ± 0.063 0.065 ± 0.008
1 1 1 1 1 1 1
Significance P = 0.130; P = 0.123; P = 0.140; P = 0.975; P = 0.260; P = 0.022; P = 0.216;
2 2 2 2 2 2 2
(n = 11) P = 0.0001 P = 0.0001 P = 0.276 P = 0.009 P = 0.422 P = 0.486 P = 0.154
DOX + L- 311.536 ± 36.491 0.188 ± 0.022 0.014 ± 0.003 0.105 ± 0.036 0.068 ± 0.29 0.170 ± 0.075 0.037 ± 0.035
carnitine
1 1 1 1 1 1 1
Significance P = 0.560; P = 0.015; P = 0.336; P = 0.603; P = 0.588; P = 0.050; P = 0.755;
2 2 2 2 2 2 2
(n = 11) P = 0.030 P = 0.002 P = 0.119 P = 0.069 P = 0.386 P = 0.036 P = 0.004

Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX.
Bpm: beat per minute. S: seconds. mV: millivolts.

2.6. Histological studies
2.6.1. Light microscopic study
Tissues were fixed in 10% neutral buffered formalin and 5 ␮m in
thickness sections were prepared. For each specimen, at least three
to five slides were stained with H&E (hematoxylin and eosin) for
general examination, Masson’s trichrome stain to demonstrate col-
lagen fibers. Slides were observed with Olympus BX53 microscope
equipped with DP73 camera (Olympus, Tokyo, Japan) (Mustafa,
2015). The scoring system for the severity of changes was quan-
titated from none (0) to severe (4) based on the degree of necrosis,
cytoplasmic vacuolations, myocardial disorganization, degenera-
tion edema and inflammatory cell infiltrate (Alpsoy et al., 2013;
Dudka et al., 2012; Gala, 2013; Mandziuk et al., 2015).
2.6.2. Immunohistochemical study
Streptavidin–biotin peroxidase technique was applied to
paraffin-embedded tissue. 5 ␮ sections were de-waxed and pre-
treated with 3% H2 O2 (hydrogen peroxide) to block endogenous
peroxidase activity. Microwave-assisted antigen retrieval was per-
formed for 10 min in 0.01 M sodium citrate buffer (pH 6.0) at
95 ◦ C, and then, the slides were cooled at room temperature for
20 min. Blocking non-specific binding by incubating in 3% BSA/PBS
(Bovine Serum Albumin/Phosphate buffered saline) for 10 min.
Then, slides were incubated overnight at 4 ◦ C with the primary
antibody against ␣-SMA (a mouse monoclonal antibody [Dako,
Carpinteria, California, USA] with a dilution of 1:1000; cellular
site was cytoplasmic) to evaluate the fibrosis. They were similarly
incubated with vimentin (a Mouse monoclonal antibody [Dako,
Carpinteria, California, USA] with a dilution of 1:400; cellular site
was cytoplasmic) as a cytoskeleton marker for cardiac fibroblasts
and endothelial cells and pericytes. They were incubated with
eNOS (a rabbit polyclonal antibody [Santa Cruz Biotechnology, CA,
USA] with a dilution of 1:50; cellular site was cytoplasmic) is
involved in the modulation of cardiac myocyte function. Sections
were incubated at room temperature with HRP (horseradish per-
oxidase) conjugate as a secondary antibody (Invitrogen, Zymed,
Burlington, ON, Canada). Sections were then incubated with DAB
(3,3 -diaminobenzidine tetrachloride; Vector Laboratories, Orton
Southgate, Peterborough, United Kingdom) substrate chromogen
solution (1 drop of DAB chromogen/1 mL of substrate buffer)
for 5 min to detect immunoreactivity. All sections were counter-
stained with Mayer’s hematoxylin and negative control sections
were prepared by omitting the primary antibody. While positive
control standard slides were used to prove the success of the tech-
nique. All slides were examined and the presence of labeled cells
was documented. Absence of staining was recognized as a negative
result (−), while the presence of brown staining was recognized as
positive result (+) (Mustafa, 2016).
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426 413

Fig. 1. Effect of CoQ10 and L-carnitine on body weight, heart weight and heart/body weight%.
Values are means ± SD (Control n = 12& DOX = 9 & treated = 11). ANOVA followed by Bonferroni’s post hoc test.
1P: compared to control. 2P: compared to DOX.

Table 3
Comparison of measured oxidative stress parameters in heart tissue homogenate in different studied groups.

Groups Malondialdehyde Reduced Nitric oxide

(nM/g) glutathione (␮M/g)
(␮M/g)

Control 11.79 ± 2.89 1.06 ± 0.19 10.00 ± 0.65

(n = 12)
DOX 65.60 ± 9.46 0.82 ± 0.17 27.71 ± 8.96
1 1 1
Significance P = 0.0001 P = 0.005 P = 0.0001
(n = 9)
CoQ10 17.18 ± 1.66 1.26 ± 0.11 10.49 ± 0.52
1 1 1
Significance P = 0.202; P = 0.022; P = 0.840;
2 2 2
(n = 12) P = 0.0001 P = 0.0001 P = 0.0001
L-carnitine 19.10 ± 2.24 1.31 ± 0.06 11.34 ± 0.59
1 1 1
Significance P = 0.101; P = 0.004; P = 0.579;
2 2 2
(n = 12) P = 0.0001 P = 0.0001 P = 0.0001
DOX + CoQ10 40.77 ± 7.44 1.15 ± 0.10 18.77 ± 1.38
1 1 3
Significance P = 0.0001; P = 0.266; P = 0.001;
2 2 2
(n = 11) P = 0.0001 P = 0.0001 P = 0.001
DOX + L- 41.03 ± 10.73 1.08 ± 0.08 16.29 ± 1.51
carnitine
1 1 1
Significance P = 0.0001; P = 0.840; P = 0.015;
2 2 2
(n = 11) P = 0.0001 P = 0.003; P = 0.0001

Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX. nM/g: nanomolar/gram. ␮M/g: micromolar/gram.

2.6.3. Morphometric study
Ten non-overlapping fields for each animal were selected ran-
domly and analyzed to determine cardiomyocytes’ diameter of
H&E stained sections. Cardiomyocytes with centrally located visible
nuclei intact cell membrane were selected and the measurements
were done along their short axis (de Salvi Guimaraes et al., 2017;
Nascimento et al., 2016; Pradegan et al., 2016). The area percent-
age of collagen fibers in Masson’s trichrome, ␣-SMA, vimentin and
eNOS-stained sections. Quantitative measurements were analyzed
with the use of Image-Pro Plus v6 (Media Cybernetics Inc., Bethesda,
Maryland, USA) and ImageJ (NIH, 1.51; Melville, NY, USA), which
was calibrated for distance, color and area before its use (Mustafa
and Hussein, 2015).
2.6.4. Ultrastructure study
One mm3 samples were immersed in 2.5% glutaraldehyde in
0.1 M phosphate buffer at 4 ◦ C for 3 hs and post-fixed in 1% OsO4
(osmium tetraoxide). Then, tissues were embedded in Epon 812 and
semithin sections were prepared, stained with toluidine blue and
observed with a microscope. Ultrathin sections of 50–60 nm thick
414 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426

Fig. 2. A: Comparison of electrocardiographic changes in different studied groups [Heart Rate (bpm)]. B: Comparison of electrocardiographic changes in different stud-
ied groups [P-R duration (seconds)]. C: Comparison of electrocardiographic changes in different studied groups [QRS Interval (seconds)]. D: Comparison of electrocardiographic
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426 415

Table 4
Comparison of measured inflammatory parameters in different studied groups.

Groups Interleukin-1␤ Tumor necrosis Leptin (pg/ml) Lactate

(pg/ml) factor-␣ dehydrogenase
(pg/ml) (U/ml)

Control 40.35 ± 7.47 40.25 ± 4.48 27.12 ± 3.45 100.80 ± 12.24

(n = 12)
DOX 286.01 ± 24.55 198.00 ± 12.43 120.14 ± 10.51 403.40 ± 37.83
1 1 1 1
Significance P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
(n = 9)
CoQ10 45.43 ± 8.82 53.86 ± 22.80 27.54 ± 6.86 96.20 ± 18.10
1 1 1 1
Significance P = 0.597; P = 0.077; P = 0.932; P = 0.740;
2 2 2 2
(n = 12) P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
L-carnitine 42.28 ± 5.27 37.19 ± 3.93 27.36 ± 7.19 105.00 ± 17.92
1 1 1 1
Significance P = 0.840; P = 0.682; P = 0.961; P = 0.761;
2 2 2 2
(n = 12) P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
DOX + CoQ10 84.44 ± 8.82 72.24 ± 6.52 48.66 ± 6.35 131.60 ± 18.37
1 1 1 1
Significance P = 0.0001; P = 0.0001; P = 0.0001; P = 0.034;
2 2 2 2
(n = 11) P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
DOX + L- 143.51 ± 21.67 113.61 ± 7.90 84.72 ± 9.80 182.80 ± 15.51
carnitine
1 1 1 1
Significance P = 0.0001; P = 0.0001; P = 0.0001; P = 0.0001;
2 2 2 2
(n = 11) P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001

Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX.
pg/ml: Picograms per Millilitre. U/ml: Units per Millilitre.

Table 5
Comparison of measured heart parameters in different studied groups.

Groups Cardiotrophin- Cardiac Troponin-I Troponin-T

1 specific- (ng/ml) (ng/ml)
(pg/ml) creatine kinase
(ng/ml)

Control 68.20 ± 8.47 100.80 ± 12.24 0.72 ± 0.06 0.39 ± 0.08

(n = 12)
DOX 237.36 ± 18.01 403.40 ± 37.83 5.80 ± 0.74 1.81 ± 0.55
1 1 1 1
Significance P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
(n = 9)
CoQ10 62.45 ± 6.19 96.20 ± 18.10 0.70 ± 0.09 0.43 ± 0.07
1 1 1 1
Significance P = 0. 0.375; P = 0.740; P = 0.952; P = 0.850;
2 2 2 2
(n = 12) P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
L-carnitine 64.08 ± 6.22 105.00 ± 17.92 0.68 ± 0.15 0.44 ± 0.13
1 1 1 1
Significance P = 0. 0.523; P = 0.761; P = 0.886; P = 0.792;
2 2 2 2
(n = 12) P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
DOX + COQ10 109.78 ± 9.10 131.60 ± 18.37 1.44 ± 0.38 0.98 ± 0.33
1 1 1 1
Significance P = 0.0001; P = 0.034; P = 0.011; P = 0.004;
2 2 2 2
(n = 11) P = 0.0001 P = 0.0001 P = 0.0001 P = 0.0001
DOX + L- 167.08 ± 7.05 182.80 ± 15.51 2.37 ± 0.55 1.39 ± 08
carnitine
1 1 1 1
Significance P = 0.0001; P = 0.0001; P = 0.0001; P = 0.0001;
2 2 2 2
(n = 11) P = 0.001 P = 0.0001 P = 0.0001 P = 0.23

Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX. pg/ml: picograms per Millilitre. ng/ml: Nanograms per Millilitre.

were cut by ultramicrotome (NOVA, LKB 2188, Bromma, Sweden); tistical Package for the Social Sciences (SPSS), version 23. The values
and stained with uranyl acetate and lead citrate. Then tissues were considered significant when P < 0.05 (Mustafa, 2015).
were examined with Philips 201 transmission electron micro-
scope (Philips Industries, Eindhoven, Netherlands) at 60–80 kv at
the Transmission Electron Microscope Unit (Mustafa and Hussein,
2015).
3. Results

2.7. Statistical analysis 3.1. General assessment

Statistical Analysis. Quantitative data were expressed as the The results revealed a significant decrease in the heart/body
mean and standard deviations. Data were analyzed using a one- weight ratio in DOX group. The administration of CoQ10 or
way analysis of variance (ANOVA) followed by Bonferroni’s post L-carnitine significant increase in the heart/body weight ratio
hoc test. All statistical analyses were implemented using the Sta- (Table 1, Fig. 1).

changes in different studied groups [T amplitude (mV)]. E: Comparison of electrocardiographic changes in different studied groups [S-T Height (mV)]. (F&G): ECG of control
group. ECG of DOX group. Rats were anesthetized and ECG was recorded for 1 min. PVC: premature ventricular complex. AV block: Atrio-ventricular block.
416 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426
3.2. ECG findings
Heart rate was significantly lower in DOX group than control,
DOX + CoQ10 and DOX + L-carnitine groups. P-R duration was sig-
nificantly higher in DOX + L-carnitine group versus control and was
significantly higher in DOX group than control, CoQ10, L-carnitine,
DOX + CoQ10 and DOX + L-carnitine groups. QTc was significantly
higher in DOX group than control and DOX + CoQ10. T amplitude
was significantly lower in DOX group than DOX + L-carnitine. S-T
height was significantly lower in DOX group than control, L-
carnitine and DOX + L-carnitine (Table 2, Fig. 2A–I).
3.3. Heart tissue homogenate levels of oxidative stress markers
Heart tissue homogenate levels of MDA was significantly higher
in DOX group than control, CoQ10, L-carnitine, DOX + CoQ10
and DOX + L-carnitine groups and in DOX + CoQ10 and DOX + L-
carnitine groups versus control. Heart tissue homogenate levels
of NO was significantly higher in DOX group than control, CoQ10,
L-carnitine, DOX + CoQ10 and DOX + L-carnitine groups and in
DOX + CoQ10 and DOX + L-carnitine groups versus control. Heart
tissue homogenate levels of reduced glutathione was significantly
lower in DOX group than control, CoQ10, L-carnitine, DOX + CoQ10
and DOX + L-carnitine groups but was significantly higher in CoQ10
and L-carnitine groups versus control (Table 3, Fig. 3A–C).
3.4. Serum levels of inflammatory cytokines
Serum levels of IL-1 beta, TNF-␣ and leptin were significantly
higher in DOX group than control, CoQ10, L-carnitine, DOX + CoQ10
and DOX + L-carnitine groups and in DOX + CoQ10 and DOX and
L-carnitine groups versus control. Serum level of LDH was sig-
nificantly higher in DOX group than control, CoQ10, L-carnitine,
DOX + CoQ10 and DOX + L-carnitine groups and in DOX + CoQ10 and
DOX + L-carnitine groups versus control (Table 4, Fig. 4A–D).
3.5. Serum levels of cardiac parameters
Serum levels of Cardiotrophin-1, Cardiac specific-creatine
kinase, and Troponin-I were significantly lower in control, CoQ10,
L-carnitine, DOX +CoQ10 and DOX + L-carnitine than DOX group
while regarding Troponin-T no significant difference between DOX
group and DOX + L-carnitine group. Serum levels of Cardiotrophin-
1, Cardiac specific-creatine kinase, Troponin-I and Troponin-T in
DOX + CoQ10 and DOX + L-carnitine groups versus control showed
a significant increase (Table 5, Fig. 5A–D).
3.6. Histological studies
Control group. H&E stained sections of control heart tissues
showed normal cardiac myocytes with their centrally placed nuclei
(Fig. 6A). Sections stained with Masson’s trichrome stain showed
scanty green stained connective tissue surrounding the muscle
fibers (Fig. 7A). Groups treated with only L-carnitine and CoQ10
revealed no significant differences between these groups and con-
trol as regard H&E and Masson’s trichrome stains.
DOX group. H&E showed necrosis and swollen of the car-
diomyocytes with an increase in the diameter. Pyknotic nuclei,
mononuclear cellular infiltration and dilated blood vessels were
observed (Fig. 6B). Masson’s trichrome stained sections showed
intense increase in collagen fibers of the surrounding endomysium
(Fig. 7B). These results were confirmed by morphometric and sta-
tistical study. Cardiomyocytes diameter of DOX group showed a
significant decrease in the mean cardiomyocyte diameter (P < 0.01)
when compared with the control. Area percentage of collagen (Mas-
son’s trichrome stain) of DOX group showed a significant increase
in the area percentage of collagen (P < 0.001) when compared with
the control (Tables 6, 7).
DOX + CoQ10 group: H&E showed nearly normal microscopic
architecture of cardiomyocytes with minimal changes in nuclei
were observed (Fig. 6C). Masson’s trichrome stained sections
showed mild reaction (Fig. 7C). These results were confirmed
by morphometric and statistical study (Tables 6, 7). DOX + L-
carnitine group: H&E showed apparently normal microscopic
histo-architecture of cardiomyocytes with mild changes in nuclei
were observed (Fig. 6D). Masson’s trichrome stained sections
showed mild reaction (Fig. 7D). These results were confirmed by
morphometric and statistical study (Tables 6, 7). Cardiomyocytes
diameter of groups treated with CoQ10 and L-carnitine showed a
significant improvement as compared with DOX group (Table 6,
Fig. 11). Area percentage of collagen (Masson’s trichrome stain) of
groups treated with CoQ10 and L-carnitine showed a significant
improvement as compared with the DOX group (Tables 6, 7 Fig. 11).
3.6.1. Immunohistochemical results for ˛-SMA
Immunohistochemical Results for ␣-SMA of the control group
revealed minimal immune expression (Fig. 8A). DOX group
showed extensive immune expression (Fig. 8B). DOX + CoQ10 group
revealed mild immune expression (Fig. 8C). DOX + L-carnitine
group showed moderate immune expression (Fig. 8D). Mean area%
of ␣–SMA immunopositive cells of DOX group showed a signifi-
cant increase in the mean area% of ␣–SMA immunoreactivity when
compared with the control. Also, groups treated with CoQ10 and
L-carnitine showed a significant improvement as compared with
the DOX group (Table 7, Fig. 11).
3.6.2. Immunohistochemical results for vimentin
Immunohistochemical Results for vimentin of the control
group revealed faint immune expression in the myocardium
(Fig. 9A). DOX group showed wide positive immunoreactivity in
the myofibroblasts (Fig. 9B). DOX + CoQ10 group revealed mini-
mal immune expression (Fig. 9C). DOX + L-carnitine group showed
slight immune expression (Fig. 9D). Mean area% of vimentin of DOX
group showed a significant increase in the mean area% of vimentin
immunoreactivity when compared with the control. In addition,
groups treated with CoQ10 and L-carnitine showed a significant
improvement as compared with the DOX group (Table 7, Fig. 11).
3.6.3. Immunohistochemical Results for vimentin
Immunohistochemical Results for eNOS of the control group
revealed faint or no immune expression (Fig. 10A). DOX group
showed strong immune expression (Fig. 10B, C). DOX + CoQ10
group revealed minimal immune expression (Fig. 10D). DOX + L-
carnitine group showed moderate immune expression (Fig. 10E).
Mean area% of eNOS of DOX group showed a significant increase
in the mean area% of eNOS immunoreactivity when compared
with the control. Also, groups treated with CoQ10 and L-carnitine
showed a significant improvement as compared with the DOX
group (Table 7, Fig. 11).
3.6.4. Immunohistochemical Results for eNOS
Ultrastructural results. Control group showed normal architec-
ture of the cardiomyocytes (Figs. 12 and 13A). DOX group revealed
degeneration and fragmentation of myofibrils and loss of light
bands with broadening and interruption of Z lines. The mitochon-
dria appeared electron dense with a moth eaten appearance among
the muscle fibers (Figs. 12 and 13B). DOX + CoQ10 group revealed
well-organized myofibrils and the mitochondria looked normal
with tightly packed cristae (Figs. 12 and 13C). DOX + L-carnitine
showed an improvement of the myofibrils organization (Figs. 12
and 13D).
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426 417

Fig. 3. A: Comparison of measured oxidative stress parameters in heart tissue homogenate in different studied groups for Malondialdehyde (nM/g). B: Comparison of
measured oxidative stress parameters in heart tissue homogenate in different studied groups for reduced glutathione (␮M/g). C: Comparison of measured oxidative stress
parameters in heart tissue homogenate in different studied groups for Nitric oxide (␮M/g).
Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX. nM/g: nanomolar/gram.
418 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426

Fig. 4. A: Comparison of measured inflammatory parameters in different studied groups [Interleukin-1␤ (pg/ml)]. B: Comparison of measured inflammatory parameters in
different studied groups [Tumor necrosis factor-␣ (pg/ml)]. C: Comparison of measured inflammatory parameters in different studied groups [Leptin (pg/ml)]. D: Comparison
of measured inflammatory parameters in different studied groups [Lactate dehydrogenase (U/ml)].
Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX.
pg/ml: Picograms per Millilitre.

Fig. 5. A: Comparison of measured heart parameters in different studied groups [Cardiotrophin-1 (pg/ml)]. B: Comparison of measured heart parameters in different studied
groups [Cardiac specific-creatine kinase (ng/ml)]. C: Comparison of measured heart parameters in different studied groups [Troponin-I (ng/ml)]. D: Comparison of measured
heart parameters in different studied groups [Troponin-T (ng/ml)].
Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.
1
P: compared to control. 2 P: compared to DOX. pg/ml: picograms per Millilitre.
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426 419

Fig. 6. (A). Photomicrograph of control showed cardiac myocytes with centrally placed nuclei (arrow). (B). DOX treated group showed cardiac myocytes showing massive
necrosis with focal marked fragmentation and nuclear changes in the form of pyknosis (p), karyolysis (k) and chromatin margination (c). (C). CoQ10 and DOX showed nearly
normal architecture of the cardiac myocytes with focal necrosis. (D): L-carnitine and DOX showed apparently regular architecture of the cardiac myocytes with focal necrosis
(H&E, Scale bar 20 ␮m).

Fig. 7. (A). Photomicrograph of control showed scanty green colored collagen fibers (arrow) between the cardiomyocytes. (B). DOX treated group showed an intense of
greenish colored collagen fibers (arrow) between swollen cardiomyocytes. (C). CoQ10 and DOX showed mild reaction. (D): L-carnitine and DOX showed mild reaction
(Masson’s trichrome, Scale bar 20 ␮m). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

4. Discussion with a large number of mitochondria, which include cardiac and

liver cells. New approaches are therefore needed to decrease the
The cardiotoxicity of doxorubicin (DOX) limits its use in cancer oxidative side effects of doxorubicin (Chao et al., 2011). DOX pos-
chemotherapy; the cells that are most affected by DOX are those sesses cardiotoxic properties that affect both the conductivity and
420 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426

Fig. 8. (A). Photomicrograph of control showed faint immunoreactivity in the myocardium (arrow). (B). DOX treated group showed wide positive immunoreactivity in the
myofibroblasts, which are attached together by their processes (arrow). (C). CoQ10 and DOX showed minimal immunoreactivity. (D). L-carnitine and DOX showed slight
immunoreactivity (arrow) (arrow) (␣-SMA. Scale bar 20 ␮m).

Fig. 9. (A) Photomicrograph of control showed minimal immune reaction in the blood capillaries wall (curved arrows) and interstitial cells (arrow). With an immune negative
cardiac muscle fibers (arrowhead). (B). DOX treated group showed strong immune reaction in endomysium and perimysium connective tissues (star), in the blood capillaries
wall (curved arrows), and interstitial cells (arrow). (C). CoQ10 and DOX showed mild immune reaction in the endomysium and perimysium (star), in the blood capillaries wall
(curved arrows) and interstitial cells (arrow). With an immune negative reaction in cardiac muscle fibers (arrowhead). (D): L-carnitine and DOX showed moderate immune
reaction (Vimentin. Scale bar 20 ␮m).
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426 421

Fig. 10. (A). Photomicrograph of control showed faint or no immune reaction cardiac muscle fibers. (B, C). DOX treated group showed strong immune reaction (arrowhead)
in cardiac muscle fibers and endothelial cells of blood capillaries. (C). CoQ10 and DOX showed minimal immune reaction (arrowhead). (E): L-carnitine and DOX showed
moderate immune reaction (arrowhead) (eNOS. Scale bar 5 ␮m).

Table 6
Effect of CoQ10and L-carnitine on the heart tissues treated with DOX.

Groups Control DOX DOX + CoQ10 DOX + L-Carnitine

Necrosis 0* +4 +1 +1
Degeneration and vacuolations 0 +4 +1 +1
Edema 0 +3 +1 +1
Inflammatory cell infiltrate 0** +3 +1 +1

A single animal may be represented more than once in the listing of individual histological changes. *Massive necrosis/changes limited to single cardiomyocytes. **Massive
inflammatory infiltration/disseminate mononuclear cells between cadiomyocytes.

Table 7
Cardiomyocyte diameter, area percentage of collagen, vimentin, ␣-SMA and eNOS Immunohistochemistry of the different groups.

Groups Control N = 12 CoQ10 N = 12 L-Carnitine DOX N = 9 DOX + CoQ10 DOX + L-

N = 12 N = 11 Carnitine
N = 11

Cardiomyocyte 14.24 ± 2.71 15.35 ± 3.07 13.84 ± 2.3 10.01 ± 0.97 16.92 ± 1.082 16.25 ± 1.071
diameter (␮m) 1
P ≤ 0.001 1
P ≤ 0.01 1
P ≤ 0.05
2
P ≤ 0.001 2
P ≤ 0.001
Area 4.57 ± 1.52 3.82 ± 1.27 5.32 ± 1.77 23.17 ± 3.61 9.01 ± 0.70 11.72 ± 1.41
percentage of 1
P ≤ 0.001 1
P ≤ 0.001 1
P ≤ 0.001
collagen (␮m2 ) 2
P ≤ 0.001 2
P ≤ 0.001
Area 0.16 ± 0.05 0.91 ± 0.3 0.66 ± 0.22 12.67 ± 1.97 5.12 ± 1.36 6.32 ± 1.14
percentage of 1
P ≤ 0.001 1
P ≤ 0.001 1
P ≤ 0.001
␣-SMA 2
P ≤ 0.001 2
P ≤ 0.001
Area 0.37 ± 0.12 1.12 ± 0.37 0.87 ± 0.29 31.21 ± 4.45 10.02 ± 2.13 9.97 ± 1.08
percentage of 1
P ≤ 0.001 1
P ≤ 0.001 1
P ≤ 0.001
vimentin 2
P ≤ 0.001 2
P ≤ 0.001
Area 1.23 ± 0.36 1.48 ± 0.49 0.98 ± 0.33 6.45 ± 2.15 1.97 ± 0.32 1.64 ± 0.54
percentage of 1
P ≤ 0.001 1
P = NS 1
P = NS
eNOS 2
P ≤ 0.001 2
P ≤ 0.001

Values are means ± SD (Control n = 12& DOX = 9 & treated = 11). ANOVA followed by Bonferroni’s post hoc test.
1P: compared to control. 2P: compared to DOX.

rhythmicity of cardiac muscle, as shown by its effect on heart rate
in addition to the associated elongation of the corrected QT interval
(QTc), ST elevation, and shortening of the T amplitude (Mantawy
et al., 2014).
The results of this study showed significant abnormalities that
affected ECG in the DOX group in agreement with previous studies
(Goyal et al., 2016; Jagetia and Venkatesh, 2015). These changes
include reflected arrhythmias, conduction abnormalities, and the
attenuation of left ventricular function (Mantawy et al., 2014).
This study illustrated that DOX induces oxidative damage and
nitrosative stress in the cardiac muscle. These results align with
those from other studies (Goyal et al., 2016; Jagetia and Venkatesh,
2015). These results could be explained by the ability of DOX to gen-
erate ROS, which results in lipid peroxidation of both the cellular
and mitochondrial membrane, ending in the injury of myocardio-
422 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426

Fig. 11. Cardiomyocyte diameter and Area% of collagen,␣-SMA, vimentin and eNOS. The mean is given in columns, and error bars represent the standard deviation (SD).

cytes (Sahu et al., 2016). Moreover, DOX creates free radicals that
cause destruction in DNA and proteins and interfere with the struc-
ture of the cytoskeleton (Ikeda et al., 2010). Oxidative stress could
injure mitochondrial cell membranes, increasing the membrane’s
permeability and making it vulnerable to rupture (Viswanatha
Swamy et al., 2011).
L-carnitine produces its antioxidant effects through different
mechanisms, including the scavenging of free radical activity either
directly or by inhibition of its production, maintaining the effi-
ciency of the mitochondrial electron transport chain, stimulating
the activation of antioxidant enzymes, and synthesis of antioxidant
molecules like reduced glutathione (Surai, 2015).
L-carnitine protects myocardial integrity by controlling the
intra-mitochondrial percentage of acyl-CoA/CoA, resulting in elim-
ination of toxic compounds; maintaining the integrity of the
mitochondrial membrane’s permeability; and promoting the elim-
ination of free radicals (Chao et al., 2011).
CoQ10 plays an important role in energy metabolism and is
part of the electron transport chain that is responsible for ATP
synthesis. Moreover, it is one of the most efficient endogenous
antioxidants and protects cellular DNA, lipids, and protein from
oxidative damage (Garrido-Maraver et al., 2014). CoQ10 protects
myocardial integrity through many mechanisms, including preser-
vation of myocardial ATP levels and powerful antioxidant effects.
CoQ10 may exert its effects directly by acting as a scavenger of free
radicals or through the regeneration of tocopherol and ascorbic acid
from their oxidized state (Chen et al., 2017).
The results of this study confirm that DOX toxicity has specific
inflammatory effects, as evidenced by the significant increase in
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426 423

Fig. 12. (A). Electron micrograph of control showed a cardiomyocyte with an elongated nucleus (N) with an evenly dispersed chromatin pattern and regular nuclear membrane
(↑). Numerous mitochondria (M) appear with apparent cristae between the longitudinally arranged myofibrils. That exhibit a normal cross-striated pattern Z lines (Z). (B).
DOX treated group showed disorganized, fragmented, degenerated myofibrils with loss of cross striations (↑). Distorted mitochondria (M) with dense matrix, unapparent
cristea, with different shapes and sizes irregularly arranged between the myofibrils and wide intercellular spaces (star) in the sarcoplasm of the cardiac myocytes. (C). CoQ10
and DOX showed regularly arranged myofilaments between successive Z lines (Z) in the sarcomeres. Mitochondria (M) arranged in rows between the myofibrils. The nucleus
(N) of a cardiac muscle fiber with slightly irregular nuclear membrane (↑). (D): L-carnitine and DOX showed mitochondria (M) appear distorted, with different shapes and
sizes around the nucleus (N) and between the myofibrils. Nuclear membrane indentations is observed (↑). Note the wide intercellular space (*) between adjacent muscle
fibers. (Scale bar 2 ␮m).

inflammatory cytokines. These results are in agreement with other
studies (Elsherbiny et al., 2016; Sun et al., 2016).
These results might be explained by the fact that ROS produced
by DOX can initiate inflammatory responses, mainly via NF-␬B,
which results in the release of cytokines such as tumor necrosis
factor-alpha [TNF-␣] and interleukin 1 beta [IL-1 ␤] (Sun et al.,
2016).
Leptin is considered one of acute response markers in oxida-
tive stress; it is involved in the prediction of coronary heart disease
due to the known relation between C-reactive protein and leptin
(Ahmed et al., 2005). In addition, this study showed that DOX led
to significant myocardial damage, as evidenced by increased serum
levels of both CK-MB and LDH. These results, in accordance with
those of other studies (El-Agamy et al., 2016; Sun et al., 2016), can
be explained by the increase in oxidative stress leading to lipid per-
oxidation and disruption of the cell membranes of myocardiocytes,
along with the release of biochemical markers in the serum and
plasma. CK-MB is one of the most important biochemical diagnostic
markers for myocardial damage (El-Agamy et al., 2016).
Treatment with CoQ10 and L-carnitine resulted in a sig-
nificant decrease of these enzymes that is attributable to a
decrease in oxidative stress and stabilization of cardiomyocyte
cell membranes. Furthermore, specific cardiac markers for acute
cardiotoxicity have been measured, including cardiac troponin
I (cTnL), T (cTnT), and cardiotrophin-1. All of these parameters
showed significant elevation in the group treated with DOX (Atas
et al., 2015). These results are concordant with those from other
studies (Atas et al., 2015; Bertinchant et al., 2003; Reagan et al.,
2013) showing an increase in the levels of cTnI and cTnT, confirm-
ing that these are sensitive and specific markers for cardiac injury
that may be elevated in the blood of patients treated with DOX
before cardiac damage is evident. Therefore, these markers can
be used for the prediction of future left ventricular dysfunction.
They are used in early detection of necrosis, before CK-MB levels
significantly increase in the heart (Atas et al., 2015).
In the current study, the DOX group showed visible conges-
tion in between cardiomyocytes, a finding that coincided with
other findings that noted the presence of marked blood cells
in the peri-capillary space (Hadi et al., 2012). The vacuoles are
ascribed to the expansion of cytoplasmic membranous compo-
nents due to redistribution of the intra-cellular electrolytes and
water (Balli et al., 2004). The diameter of cardiomyocytes was
increased, in agreement with other reports in which myocytic
diameter increased, with the presence of hyperchromatic nuclei,
disorganization of myofibrils, and loss of cross-striation of car-
diac myocytes (Rashikh et al., 2011). With CoQ10 or L-carnitine,
424 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426

Fig. 13. (A). Electron micrograph of control showed a cardiomyocyte contains strands of myofibrils formed of light bands (I), Z lines (Z), dark bands (A), H zone (H), sarcomere
(S) and mitochondrial rows (M). (B). DOX treated group showed destruction, fragmentation and lysis of myofibrils (arrows) with absence of light bands and broadening of Z
lines (Z). Moth-eaten appearance of degenerated mitochondria (ME) with variable sizes were seen among the myofibrils. Note lipofuscin pigment (star). (C). CoQ10 and DOX
showed myofibrils with preserved cross-banding pattern, intercalated disc (IC) and euchromatic nucleus (N). The mitochondria (m) looks normal with tightly packed cristae
and relative increase in number. (D): L-carnitine and DOX showed well-organized myofibrils with few interrupted Z lines (arrow. Preserved healthy mitochondria (M) and
Dilated SER (SER). (Scale bar 500 nm).

the histopathological findings were improved such that they were
consistent with other studies that found decreases in myofibril dis-
organization, exudation, and inflammatory cell infiltration in the
myocardium (Kwong et al., 2002). Analysis of the ultrastructure
morphology images showed peripheral chromatin condensation,
deformity and fragmentation of the nuclei, and apoptosis (Zhang
et al., 2012), supporting the hypothesis that apoptosis is one of
the mechanisms of DOX-cardiotoxicity, DOX-induced lipid per-
oxidation, reactive oxygen species (ROS) production, disturbed
mitochondrial metabolism, and direct cardiotoxicity (Oktem et al.,
2012).
The results were in agreement with those of previous
researchers who observed that myocardial stress increases the
mean number of ␣-SMA positive myofibroblasts. This was
attributed to myofibroblasts, which are considered the key cells
responsible for extracellular matrix and collagen deposition in
myocardial fibrosis (Naugle et al., 2006). Other researchers have
observed a rise in fibronectin and in collagen types I and III, ascrib-
ing this to collagen synthesis related to ␤-adrenergic receptor
activation in fibroblasts (Yin et al., 2009). The contractile fibers of
myofibroblasts contain ␣-SMA and are linked to exaggerated extra-
cellular matrix accumulation in pathological disorders (Ma et al.,
2014). In cardiac disease, cardiomyocytes are wasted due to necro-
sis, and myofibroblasts are stimulated to launch restorative fibrosis.
Myofibroblasts also generate angiotensin II and fibrogenic growth
factors, which play a crucial role in fibrosis and collagen type I
synthesis (Weber et al., 2013). With CoQ10 or L-carnitine, there
is a decrease in the transformation of fibroblasts to myofibroblasts,
which are a source of collagen, thus restraining cardiac fibrosis.
In the current work, an increase in vimentin area percentage
expressed in the arterial walls was observed in the DOX group;
similar results were revealed in dilated cardiomyopathy, where
vimentin immunoreaction was increased in the interstitial tis-
sue cells (Di et al., 2000). The increased vimentin was linked
to an increase of collagen and fibrosis (Schaper et al., 1991).
Scientists revealed an adverse connection between myocardial
vimentin overexpression and the sliding rate of actin myosin. It
was proposed that disarrangement of cytoskeleton proteins occurs
with participation of vimentin in the modulations of coupling of
myocytes to the extracellular matrix, myocyte functions, and intra-
cellular signaling during cardiac failure and hypertrophy (Rastogi
et al., 2008). Moreover, investigators noticed the proliferation of
T-tubules linked to vimentin overexpression in cardiomyopathy.
This can lead to recovery of an inappropriate cardiac function by
substituting for the contractile elements (Di et al., 2000). Fibrosis
is responsible for an increased stiffness and decrease of ventricular
compliance (Heling et al., 2000).
 H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426
The interaction between DOX and NOS is a complex. DOX
converts eNOS from a nitric oxide donor to a superoxide gen-
erator (Octavia et al., 2012). DOX-induced hydrogen peroxide
creation (H2 O2 ) is responsible for apoptotic cell death and DOX-
toxicity (Octavia et al., 2012). In turn, H2 O2 promotes endothelial
nitric oxide synthase (eNOS) transcription in endothelial cells and
cardiomyocytes (Kalyanaraman et al., 2002). Up-regulated eNOS
expression can play a key role in DOX-cardiac dysfunction by
affecting ROS-mediated apoptosis of endothelial cells (Neilan et al.,
2007). Genetic disruption of eNOS transcription protects against
DOX-induced cardiotoxicity and mortality, while overexpression
exaggerates the toxic effects of DOX (Šimůnek et al., 2009). Studies
about endothelial dysfunction have demonstrated a considerable
attenuation of endothelial vasodilation after DOX administration,
suggesting dysfunctional eNOS activity (Olukman et al., 2009).
The current results provide proof that CoQ10 and L-carnitine
attenuates DOX-induced generation of free radicals. Also, prevent
eNOS uncoupling by reducing superoxide formation, increasing
NO bioavailability, and inhibiting upregulation of the activity and
expression of the vascular NAD (P) H oxidase (Chatterjee et al.,
2010).
5. Conclusion
Supplementation with CoQ10 or L-carnitine defends the
myocardium through their antioxidant activity, as was proven by
the improvement of different biochemical markers and oxidative
status and the restoration of the myocardium’s structural integrity
and function.
Disclosure of interest
The authors declare that they have no conflicts of interest.
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