Protective role of CoQ10 or L-carnitine on the integrity of the myocardium in doxorubicin induced

Tissue and Cell 49 (2017) 410–426

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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 Clinical Biochemistry Department, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia Medical Biochemistry Department, National Research Centre, Cairo, Egypt d Pharmacology Department, National Research Center, Cairo, Egypt b c

a r t i c l e

i n f o

Article history: Received 16 January 2017 Received in revised form 15 March 2017 Accepted 31 March 2017 Available online 2 April 2017 Keywords: Dox CoQ10 and L-carnitine Cardiotoxicity eNOS Vimentin

a b s t r a c t Doxorubicin (DOX) is a chemotherapeutic agent used for treatment of different cancers and its clinical usage is hindered by the oxidative injury-related cardiotoxicity. This work aims to declare if the harmful effects of DOX on heart can be alleviated with the use of Coenzyme Q10 (CoQ10) or L-carnitine. The study was performed on seventy two female Wistar albino rats divided into six groups, 12 animals each: Control 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 of 10 mg/kg DOX that injected intraperitoneally (IP) then the treatment continued for 10 days. At the end of the study, serum biochemical parameters of cardiac damage, oxidative stress indices, and histopathological changes were investigated. CoQ10 or L-carnitine showed a noticeable effects in improving cardiac functions evidenced reducing serum enzymes as serum interleukin-1 beta (IL-1 ␤), tumor necrosis factor 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 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 transplantation 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). http://dx.doi.org/10.1016/j.tice.2017.03.007 0040-8166/© 2017 Elsevier Ltd. All rights reserved.

(Potemski et al., 2006). Cardiomyocytes suffer from relative deficiency of antioxidant enzymes so they are more vulnerable for the deleterious effects produced by free radical result from DOX administration (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 cardiotoxicity (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 acetylcoenzyme 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).

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 regenerating other antioxidants such as ascorbate and tocopherol. Also, CoQ10 causes scavenging of free radicals, inhibition of lipid peroxidation (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 myofibroblastlike 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 function, 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 failure (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 controlled (24 ± 1 ◦ C) with a 12 h light/dark cycle and 60 ± 5% humidity and were provided with standard laboratory diet and water ad libitum. 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 single 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 Powerlab/8sp and Animal Bio-Amplifier, Australia, in addition to Lab Chart 7 software with ECG analyzer (Hajrasouliha et al., 2004).

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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 centrifuge. 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 glutathione (GSH) levels using colorimetric assay kits (Catalogues No. MD 25 29, GR 25 11 respectively) in accordance with the manufacturer’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 metabolites, nitrite and nitrate. Determination of nitrite and nitrate (NOx) in body fluid and tissues is widely used as a marker of NO production 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, stable 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 manufacturer’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 carried 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 thirdgeneration 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), purchased from LifeSpan BioSciences international Inc., USA.

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Table 1 Effect of CoQ10 and L-carnitine on body weight, heart weight, heart/body weight% and mortality No. DOX N = 9

Groups

Control N = 12

CoQ10 N = 12

L-carnitine N = 12

Body weight (g)

152.3 ± 2.31

153.01 ± 3.41 NS

151.33 ± 5.61 NS

Heart weight (g)

0.810 ± 0.019

0.79 ± 0.021 NS

0.83 ± 0.017 NS

Heart weight/Body weight% Mortality No.

0.532 ± 0.002

0.516 ± 0.006 P ≤ 0.001

0

0.548 ± 0.008 P ≤ 0.001

135 ± 6.51 P ≤ 0.001

1

0.482 ± 0.040 P1 ≤ 0.001 0.357 ± 0.007 P ≤ 0.001

1

1

1

0

0

3

DOX + CoQ10 N = 11

DOX + Lcarnitine N = 11

146.07 ± 3.80 P ≤ 0.05 2 P ≤ 0.001 0.603 ± 0.011 1 P ≤ 0.001 2 P ≤ 0.001 0.413 ± 0.003 1 P ≤ 0.001 2 P ≤ 0.001 1

144.4 ± 5.03 1 P ≤ 0.01 2 P ≤ 0.001 0.594 ± 0.026 1 P ≤ 0.001 2 P ≤ 0.001 0.411 ± 0.001 1 P ≤ 0.001 2 P ≤ 0.001 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 (bpm)

P-R (s)

QRS Interval (s)

QTc duration (s)

P amplitude (mV)

T amplitude (mV)

S-T Height (mV)

Control (n = 12) DOX Significance (n = 9) CoQ10 Significance (n = 12) L-carnitine Significance (n = 12) DOX + CoQ10 Significance (n = 11) DOX + Lcarnitine Significance (n = 11)

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

281.930 ± 22.591 1 P = 0.013

0.214 ± 0.019 1 P = 0.0001

0.016 ± 0.004 1 P = 0.624

0.134 ± 0.077 1 P = 0.024

0.057 ± 0.057 1 P = 0.743

0.145 ± 0.105 1 P = 0.141

0.090 ± 0.066 1 P = 0.006

0.017 ± 0.002 P = 0.275; 2 P = 0.443 0.015 ± 0.001 1 P = 0.698; 2 P = 0.447 0.017 ± 0.002 1 P = 0.140; 2 P = 0.276 0.014 ± 0.003

0.101 ± 0.005 1 P = 0.811; 2 P = 0.082 0.103 ± 0.014 1 P = 0.773; 2 P = 0.148 0.096 ± 0.011 1 P = 0.975; 2 P = 0.009 0.105 ± 0.036

0.066 ± 0.007 1 P = 0.796; 2 P = 0.625 0.066 ± 0.009 1 P = 0.774; 2 P = 0.604 0.047 ± 0.031 1 P = 0.260; 2 P = 0.422 0.068 ± 0.29

0.174 ± 0.021 1 P = 0.750; 2 P = 0.409 0.209 ± 0.058 1 P = 0.482; 2 P = 0.071 0.127 ± 0.063 1 P = 0.022; 2 P = 0.486 0.170 ± 0.075

0.059 ± 0.030 1 P = 0.405; 2 P = 0.116 0.023 ± 0.019 1 P = 0.335; 2 P = 0.002 0.065 ± 0.008 1 P = 0.216; 2 P = 0.154 0.037 ± 0.035

1

1

1

1

1

286.167 ± 10.913 1 P = 0.061; 2 P = 0.815 307.483 ± 44.887 1 P = 0.520; 2 P = 0.161 340.989 ± 8.550 1 P = 0.130; 2 P = 0.0001 311.536 ± 36.491 1 2

P = 0.560; P = 0.030

0.187 ± 0.024 P = 0.059; 2 P = 0.016 0.182 ± 0.031 1 P = 0.111; 2 P = 0.003 0.179 ± 0.007 1 P = 0.123; 2 P = 0.0001 0.188 ± 0.022 1

1 2

P = 0.015; P = 0.002

1

2

P = 0.336; P = 0.119

2

P = 0.603; P = 0.069

2

P = 0.588; P = 0.386

2

P = 0.050; P = 0.036

2

P = 0.755; 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 collagen 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 quantitated from none (0) to severe (4) based on the degree of necrosis, cytoplasmic vacuolations, myocardial disorganization, degeneration 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 pretreated with 3% H2 O2 (hydrogen peroxide) to block endogenous peroxidase activity. Microwave-assisted antigen retrieval was performed 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 peroxidase) 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 counterstained 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 technique. 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).

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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 (nM/g)

Reduced glutathione (␮M/g)

Nitric oxide (␮M/g)

Control (n = 12) DOX Significance (n = 9) CoQ10 Significance (n = 12) L-carnitine Significance (n = 12) DOX + CoQ10 Significance (n = 11) DOX + Lcarnitine Significance (n = 11)

11.79 ± 2.89

1.06 ± 0.19

10.00 ± 0.65

0.82 ± 0.17 P = 0.005

27.71 ± 8.96 1 P = 0.0001

17.18 ± 1.66 1 P = 0.202; 2 P = 0.0001 19.10 ± 2.24 1 P = 0.101; 2 P = 0.0001 40.77 ± 7.44 1 P = 0.0001; 2 P = 0.0001 41.03 ± 10.73

1.26 ± 0.11 1 P = 0.022; 2 P = 0.0001 1.31 ± 0.06 1 P = 0.004; 2 P = 0.0001 1.15 ± 0.10 1 P = 0.266; 2 P = 0.0001 1.08 ± 0.08

10.49 ± 0.52 1 P = 0.840; 2 P = 0.0001 11.34 ± 0.59 1 P = 0.579; 2 P = 0.0001 18.77 ± 1.38 3 P = 0.001; 2 P = 0.001 16.29 ± 1.51

1

1

1

65.60 ± 9.46 1 P = 0.0001

2

P = 0.0001; P = 0.0001

1

2

P = 0.840; P = 0.003;

2

P = 0.015; P = 0.0001

Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.

P: compared to control. 2 P: compared to DOX. nM/g: nanomolar/gram. ␮M/g: micromolar/gram.

1

2.6.3. Morphometric study Ten non-overlapping fields for each animal were selected randomly 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 percentage 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

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Fig. 2. A: Comparison of electrocardiographic changes in different studied groups [Heart Rate (bpm)]. B: Comparison of electrocardiographic changes in different studied 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

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

Groups

Interleukin-1␤ (pg/ml)

Tumor necrosis factor-␣ (pg/ml)

Leptin (pg/ml)

Lactate dehydrogenase (U/ml)

Control (n = 12) DOX Significance (n = 9) CoQ10 Significance (n = 12) L-carnitine Significance (n = 12) DOX + CoQ10 Significance (n = 11) DOX + Lcarnitine Significance (n = 11)

40.35 ± 7.47

40.25 ± 4.48

27.12 ± 3.45

100.80 ± 12.24

286.01 ± 24.55 1 P = 0.0001

198.00 ± 12.43 1 P = 0.0001

120.14 ± 10.51 1 P = 0.0001

403.40 ± 37.83 1 P = 0.0001

45.43 ± 8.82 1 P = 0.597; 2 P = 0.0001 42.28 ± 5.27 1 P = 0.840; 2 P = 0.0001 84.44 ± 8.82 1 P = 0.0001; 2 P = 0.0001 143.51 ± 21.67 1 2

P = 0.0001; P = 0.0001

53.86 ± 22.80 P = 0.077; 2 P = 0.0001 37.19 ± 3.93 1 P = 0.682; 2 P = 0.0001 72.24 ± 6.52 1 P = 0.0001; 2 P = 0.0001 113.61 ± 7.90 1

1 2

P = 0.0001; P = 0.0001

27.54 ± 6.86 P = 0.932; 2 P = 0.0001 27.36 ± 7.19 1 P = 0.961; 2 P = 0.0001 48.66 ± 6.35 1 P = 0.0001; 2 P = 0.0001 84.72 ± 9.80 1

1 2

P = 0.0001; P = 0.0001

96.20 ± 18.10 P = 0.740; 2 P = 0.0001 105.00 ± 17.92 1 P = 0.761; 2 P = 0.0001 131.60 ± 18.37 1 P = 0.034; 2 P = 0.0001 182.80 ± 15.51 1

1 2

P = 0.0001; P = 0.0001

Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.

P: compared to control. 2 P: compared to DOX. pg/ml: Picograms per Millilitre. U/ml: Units per Millilitre.

1

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

Groups

Cardiotrophin1 (pg/ml)

Cardiac specificcreatine kinase (ng/ml)

Troponin-I (ng/ml)

Troponin-T (ng/ml)

Control (n = 12) DOX Significance (n = 9) CoQ10 Significance (n = 12) L-carnitine Significance (n = 12) DOX + COQ10 Significance (n = 11) DOX + Lcarnitine Significance (n = 11)

68.20 ± 8.47

100.80 ± 12.24

0.72 ± 0.06

0.39 ± 0.08

237.36 ± 18.01 1 P = 0.0001

403.40 ± 37.83 1 P = 0.0001

5.80 ± 0.74 1 P = 0.0001

1.81 ± 0.55 1 P = 0.0001

62.45 ± 6.19 1 P = 0. 0.375; 2 P = 0.0001 64.08 ± 6.22 1 P = 0. 0.523; 2 P = 0.0001 109.78 ± 9.10 1 P = 0.0001; 2 P = 0.0001 167.08 ± 7.05

96.20 ± 18.10 1 P = 0.740; 2 P = 0.0001 105.00 ± 17.92 1 P = 0.761; 2 P = 0.0001 131.60 ± 18.37 1 P = 0.034; 2 P = 0.0001 182.80 ± 15.51

0.70 ± 0.09 1 P = 0.952; 2 P = 0.0001 0.68 ± 0.15 1 P = 0.886; 2 P = 0.0001 1.44 ± 0.38 1 P = 0.011; 2 P = 0.0001 2.37 ± 0.55

0.43 ± 0.07 1 P = 0.850; 2 P = 0.0001 0.44 ± 0.13 1 P = 0.792; 2 P = 0.0001 0.98 ± 0.33 1 P = 0.004; 2 P = 0.0001 1.39 ± 08

1

1

1

1

2

P = 0.0001; P = 0.001

2

P = 0.0001; P = 0.0001

2

P = 0.0001; P = 0.0001

2

P = 0.0001; P = 0.23

Values are means ± SD. ANOVA followed by Bonferroni’s post hoc test.

P: compared to control. 2 P: compared to DOX. pg/ml: picograms per Millilitre. ng/ml: Nanograms per Millilitre.

1

were cut by ultramicrotome (NOVA, LKB 2188, Bromma, Sweden); and stained with uranyl acetate and lead citrate. Then tissues were examined with Philips 201 transmission electron microscope (Philips Industries, Eindhoven, Netherlands) at 60–80 kv at the Transmission Electron Microscope Unit (Mustafa and Hussein, 2015).

tistical Package for the Social Sciences (SPSS), version 23. The values were considered significant when P < 0.05 (Mustafa, 2015).

3. Results 2.7. Statistical analysis

3.1. General assessment

Statistical Analysis. Quantitative data were expressed as the mean and standard deviations. Data were analyzed using a oneway analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. All statistical analyses were implemented using the Sta-

The results revealed a significant decrease in the heart/body weight ratio in DOX group. The administration of CoQ10 or L-carnitine significant increase in the heart/body weight ratio (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.

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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 significantly 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, Lcarnitine 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 + Lcarnitine 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 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 (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 Cardiotrophin1, 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 control as regard H&E and Masson’s trichrome stains.

DOX group. H&E showed necrosis and swollen of the cardiomyocytes 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 statistical 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 (Masson’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 + Lcarnitine 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 significant 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 minimal 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 + Lcarnitine 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 architecture 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 mitochondria 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).

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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.

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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.

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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 The cardiotoxicity of doxorubicin (DOX) limits its use in cancer chemotherapy; the cells that are most affected by DOX are those

with a large number of mitochondria, which include cardiac and liver cells. New approaches are therefore needed to decrease the oxidative side effects of doxorubicin (Chao et al., 2011). DOX possesses cardiotoxic properties that affect both the conductivity and

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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).

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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 Degeneration and vacuolations Edema Inflammatory cell infiltrate

0* 0 0 0**

+4 +4 +3 +3

+1 +1 +1 +1

+1 +1 +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 N = 12

DOX N = 9

Cardiomyocyte diameter (␮m)

14.24 ± 2.71

15.35 ± 3.07

13.84 ± 2.3

10.01 ± 0.97 1 P ≤ 0.001

Area percentage of collagen (␮m2 ) Area percentage of ␣-SMA Area percentage of vimentin Area percentage of eNOS

4.57 ± 1.52

3.82 ± 1.27

5.32 ± 1.77

23.17 ± 3.61 1 P ≤ 0.001

0.16 ± 0.05

0.91 ± 0.3

0.66 ± 0.22

12.67 ± 1.97 1 P ≤ 0.001

0.37 ± 0.12

1.12 ± 0.37

0.87 ± 0.29

31.21 ± 4.45 1 P ≤ 0.001

1.23 ± 0.36

1.48 ± 0.49

0.98 ± 0.33

6.45 ± 2.15 1 P ≤ 0.001

DOX + CoQ10 N = 11 16.92 ± 1.082 P ≤ 0.01 2 P ≤ 0.001 9.01 ± 0.70 1 P ≤ 0.001 2 P ≤ 0.001 5.12 ± 1.36 1 P ≤ 0.001 2 P ≤ 0.001 10.02 ± 2.13 1 P ≤ 0.001 2 P ≤ 0.001 1.97 ± 0.32 1 P = NS 2 P ≤ 0.001 1

DOX + LCarnitine N = 11 16.25 ± 1.071 P ≤ 0.05 2 P ≤ 0.001 11.72 ± 1.41 1 P ≤ 0.001 2 P ≤ 0.001 6.32 ± 1.14 1 P ≤ 0.001 2 P ≤ 0.001 9.97 ± 1.08 1 P ≤ 0.001 2 P ≤ 0.001 1.64 ± 0.54 1 P = NS 2 P ≤ 0.001 1

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 generate ROS, which results in lipid peroxidation of both the cellular and mitochondrial membrane, ending in the injury of myocardio-

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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 structure 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 efficiency 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 elimination of toxic compounds; maintaining the integrity of the

mitochondrial membrane’s permeability; and promoting the elimination 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 preservation 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

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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 oxidative 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 peroxidation 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 significant 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, confirming 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 congestion 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 components 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 cardiac myocytes (Rashikh et al., 2011). With CoQ10 or L-carnitine,

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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 disorganization, 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 peroxidation, 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, ascribing 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 extracellular matrix accumulation in pathological disorders (Ma et al., 2014). In cardiac disease, cardiomyocytes are wasted due to necrosis, 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 tissue 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 intracellular 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).

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The interaction between DOX and NOS is a complex. DOX converts eNOS from a nitric oxide donor to a superoxide generator (Octavia et al., 2012). DOX-induced hydrogen peroxide creation (H2 O2 ) is responsible for apoptotic cell death and DOXtoxicity (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.

References İzgüt-Uysal, V.N., Ağaç, A., Derin, N., 2003. Effect of L-Carnitine on carrageenan-induced inflammation in aged rats. Gerontology 49, 287–292.

Šimůnek, T., Štěrba, M., Popelová, O., Adamcová, M., Hrdina, R., Geršl, V., 2009.

Anthracycline-induced cardiotoxicity: overview of studies examining the roles of oxidative stress and free cellular iron. Pharmacol. Rep. 61, 154–171.

Agarwal, A., Said, T.M., 2004. Carnitines and male infertility. Reprod Biomed Online 8, 376–384.

Ahmed, H.H., Mannaa, F., Elmegeed, G.A., Doss, S.H., 2005. Cardioprotective activity of melatonin and its novel synthesized derivatives on doxorubicin-induced cardiotoxicity. Bioorg. Med. Chem. 13, 1847–1857.

Aliev, G., Liu, J., Shenk, J.C., Fischbach, K., Pacheco, G.J., Chen, S.G., Obrenovich, M.E., Ward, W.F., Richardson, A.G., Smith, M.A., 2009. Neuronal mitochondrial amelioration by feeding acetyl-L-carnitine and lipoic acid to aged rats. J. Cell.

Mol. Med. 13, 320–333.

Alpsoy, S., Aktas, C., Uygur, R., Topcu, B., Kanter, M., Erboga, M., Karakaya, O., Gedikbasi, A., 2013. Antioxidant and anti-apoptotic effects of onion (Allium cepa) extract on doxorubicin-induced cardiotoxicity in rats. J. Appl. Toxicol. 33, 202–208.

Arcamone, F., Cassinelli, G., Fantini, G., Grein, A., Orezzi, P., Pol, C., Spalla, C., 2000.

Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius. reprinted from biotechnology and bioengineering, vol.

XI, issue 6, pages 1101–1110 (1969). Biotechnol. Bioeng. 67, 704–713.

Atas, E., Kismet, E., Kesik, V., Karaoglu, B., Aydemir, G., Korkmazer, N., Demirkaya, E., Karslioglu, Y., Yurttutan, N., Unay, B., Koseoglu, V., Gokcay, E., 2015. Cardiac troponin-I, brain natriuretic peptide and endothelin-1 levels in a rat model of doxorubicin-induced cardiac injury. J. Cancer Res. Ther. 11, 882–886.

Balli, E., Mete, U.O., Tuli, A., Tap, O., Kaya, M., 2004. Effect of melatonin on the cardiotoxicity of doxorubicin. Histol. Histopathol. 19, 1101–1108.

Bertinchant, J.P., Polge, A., Juan, J.M., Oliva-Lauraire, M.C., Giuliani, I., Marty-Double, C., Burdy, J.Y., Fabbro-Peray, P., Laprade, M., Bali, J.P., Granier, C., de la Coussaye, J.E., Dauzat, M., 2003. Evaluation of cardiac troponin I and T levels as markers of myocardial damage in doxorubicin-induced cardiomyopathy rats, and their relationship with echocardiographic and histological findings. Clin. Chim. Acta 329, 39–51.

Bhagavan, H.N., Chopra, R.K., 2006. Coenzyme Q10: absorption, tissue uptake, metabolism and pharmacokinetics. Free Radic. Res. 40, 445–453.

Chao, H.-H., Liu, J.-C., Hong, H.-J., Lin J.-w. Chen, C.-H., Cheng, T.-H., 2011.

L-carnitine reduces doxorubicin-induced apoptosis through a

425

prostacyclin-mediated pathway in neonatal rat cardiomyocytes. Int. J. Cardiol.

146, 145–152.

Chatterjee, K., Zhang, J., Honbo, N., Karliner, J.S., 2010. Doxorubicin cardiomyopathy. Cardiology 115, 155–162.

Chen, P.-Y., Hou, C.-W., Shibu, M.A., Day, C.H., Pai, P., Liu, Z.-R., Lin, T.-Y., Viswanadha, V.P., Kuo, C.-H., Huang, C.-Y., 2017. Protective effect of Co-enzyme Q10 On doxorubicin-induced cardiomyopathy of rat hearts. Environ. Toxicol.

32, 679–689.

Crespo, M.J., Zalacain, J., Dunbar, D.C., Cruz, N., Arocho, L., 2008. Cardiac oxidative stress is elevated at the onset of dilated cardiomyopathy in streptozotocin-diabetic rats. J. Cardiovasc. Pharmacol. Ther. 13, 64–71.

Di, S., Marotta, M., Salvatore, G., Cudemo, G., Cuda, G., De Vivo, F., Di, B., Ciaramella, F., Caputo, G., de Divitiis, O., 2000. Changes in myocardial cytoskeletal intermediate filaments and myocyte contractile dysfunction in dilated cardiomyopathy: an in vivo study in humans. Heart 84, 659–667.

Dong, Q., Chen, L., Lu, Q., Sharma, S., Li, L., Morimoto, S., Wang, G., 2014. Quercetin attenuates doxorubicin cardiotoxicity by modulating Bmi-1 expression. Br. J. Pharmacol. 171, 4440–4454.

Dudka, J., Gieroba, R., Korga, A., Burdan, F., Matysiak, W., Jodlowska-Jedrych, B., Mandziuk, S., Korobowicz, E., Murias, M., 2012. Different effects of resveratrol on dose-related Doxorubicin-induced heart and liver toxicity. Evid. Based Complement Alternat. Med. 2012, 606183.

El-Agamy, D.S., Abo-Haded, H.M., Elkablawy, M.A., 2016. Cardioprotective effects of sitagliptin against doxorubicin-induced cardiotoxicity in rats. Exp. Biol. Med. (Maywood) 241, 1577–1587.

Elbaky, N.A., Ali, A.A., Ahmed, R.A., 2010. Cardioprotective effect of simvastatin on doxorubicininduced oxidative cardiotoxicity in rats. J. Basic Appl. Sci. 6, 29–38.

Elsherbiny, N.M., Salama, M.F., Said, E., El-Sherbiny, M., Al-Gayyar, M.M., 2016.

Crocin protects against doxorubicin-induced myocardial toxicity in rats through down-regulation of inflammatory and apoptic pathways. Chem. Biol.

Interact. 247, 39–48.

Gala, A.A.A., 2013. Protective effect of Zingiber officinale (ginger) on doxorubicin induced oxidative cardiotoxicity in rats. Life Sci. J. 10, 2924–2934.

Garrido-Maraver, J., Cordero, M.D., Oropesa-Ávila, M., Fernández Vega, A., De La Mata, M., Delgado Pavón, A., De Miguel, M., Pérez Calero, C., Villanueva Paz, M., Cotán, D., 2014. Coenzyme q10 therapy. Mol. Syndromol. 5, 187–197.

Gianni, L., Salvatorelli, E., Minotti, G., 2007. Anthracycline cardiotoxicity in breast cancer patients: synergism with trastuzumab and taxanes. Cardiovasc. Toxicol.

7, 67–71.

Goyal, S.N., Mahajan, U.B., Chandrayan, G., Kumawat, V.S., Kamble, S., Patil, P., Agrawal, Y.O., Patil, C.R., Ojha, S., 2016. Protective effect of oleanolic acid on oxidative injury and cellular abnormalities in doxorubicin induced cardiac toxicity in rats. Am. J. Transl. Res. 8, 60–69.

Guan, Y., Zheng, X., Yang, Z., Li, S., 2009. [Protective effects of L-carnitine upon testicular ischemia-reperfusion damage in rats]. Zhonghua yi xue za zhi 89, 1858–1861.

Hadi, N., Yousif, N.G., Al-Amran, F.G., Huntei, N.K., Mohammad, B.I., Ali, S.J., 2012.

Vitamin E and telmisartan attenuates doxorubicin induced cardiac injury in rat through down regulation of inflammatory response. BMC Cardiovasc. Disord.

12, 1.

Hajrasouliha, A.R., Tavakoli, S., Jabehdar-Maralani, P., Shafaroodi, H., Borhani, A.A., Houshmand, G., Sadeghipour, H., Dehghani, M., Dehpour, A.R., 2004. Resistance of cholestatic rats against epinephrine-induced arrhythmia: the role of nitric oxide and endogenous opioids. Eur. J. Pharmacol. 499, 307–313.

Heling, A., Zimmermann, R., Kostin, S., Maeno, Y., Hein, S., Devaux, B., Bauer, E., Klovekorn, W.P., Schlepper, M., Schaper, W., Schaper, J., 2000. Increased expression of cytoskeletal, linkage, and extracellular proteins in failing human myocardium. Circ. Res. 86, 846–853.

Ikeda, Y., Aihara, K., Akaike, M., Sato, T., Ishikawa, K., Ise, T., Yagi, S., Iwase, T., Ueda, Y., Yoshida, S., Azuma, H., Walsh, K., Tamaki, T., Kato, S., Matsumoto, T., 2010.

Androgen receptor counteracts Doxorubicin-induced cardiotoxicity in male mice. Mol. Endocrinol. 24, 1338–1348.

Jagetia, G., Venkatesh, P., 2015. An indigenous plant bael (Aegle marmelos (L.) correa) extract protects against the doxorubicin-Induced cardiotoxicity in mice. Biochem. Physiol. 4, 2.

Jones, S.P., Greer, J.J., Kakkar, A.K., Ware, P.D., Turnage, R.H., Hicks, M., van Haperen, R., de Crom, R., Kawashima, S., Yokoyama, M., 2004. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury. Am. J. Physiol.-Heart Circulatory Physiol. 286, H276–H282.

Kalyanaraman, B., Joseph, J., Kalivendi, S., Wang, S., Konorev, E., Kotamraju, S., 2002. Doxorubicin-induced apoptosis: implications in cardiotoxicity. Mol. Cell.

Biochem. 234–235, 119–124.

Kwong, L.K., Kamzalov, S., Rebrin, I., Bayne, A.C., Jana, C.K., Morris, P., Forster, M.J., Sohal, R.S., 2002. Effects of coenzyme Q(10) administration on its tissue concentrations, mitochondrial oxidant generation, and oxidative stress in the rat. Free Radic. Biol. Med. 33, 627–638.

Ma, Y., de Castro Brás, L.E., Toba, H., Iyer, R.P., Hall, M.E., Winniford, M.D., Lange, R.A., Tyagi, S.C., Lindsey, M.L., 2014. Myofibroblasts and the extracellular matrix network in post-myocardial infarction cardiac remodeling. Pflugers Arch. 466, 1113–1127.

Mandziuk, S., Gieroba, R., Korga, A., Matysiak, W., Jodlowska-Jedrych, B., Burdan, F., Poleszak, E., Kowalczyk, M., Grzycka-Kowalczyk, L., Korobowicz, E., Jozefczyk, A., Dudka, J., 2015. The differential effects of green tea on dose-dependent doxorubicin toxicity. Food Nutr. Res. 59, 29754.

426

H.N. Mustafa et al. / Tissue and Cell 49 (2017) 410–426

Mantawy, E.M., El-Bakly, W.M., Esmat, A., Badr, A.M., El-Demerdash, E., 2014.

Chrysin alleviates acute doxorubicin cardiotoxicity in rats via suppression of oxidative stress, inflammation and apoptosis. Eur. J. Pharmacol. 728, 107–118.

Mescka, C.P., Rosa, A.P., Schirmbeck, G., da Rosa, T.H., Catarino, F., de Souza, L.O., Guerreiro, G., Sitta, A., Vargas, C.R., Dutra-Filho, C.S., 2016. L-carnitine prevents oxidative stress in the brains of rats subjected to a chemically induced chronic model of MSUD. Mol. Neurobiol. 53, 6007–6017.

Miranda, K.M., Espey, M.G., Yamada, K., Krishna, M., Ludwick, N., Kim, S., Jourd’heuil, D., Grisham, M.B., Feelisch, M., Fukuto, J.M., 2001. Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion. J. Biol.

Chem. 276, 1720–1727.

Mitry, M.A., Edwards, J.G., 2016. Doxorubicin induced heart failure: phenotype and molecular mechanisms. Int. J. Cardiol. Heart Vasc. 10, 17–24.

Moshage, H., Kok, B., Huizenga, J.R., Jansen, P.L., 1995. Nitrite and nitrate determinations in plasma: a critical evaluation. Clin. Chem. 41, 892–896.

Mustafa, H.N., Hussein, A.M., 2016. Does allicin combined with vitamin B-complex have superior potentials than alpha-tocopherol alone in ameliorating lead acetate-induced Purkinje cell alterations in rats? An immunohistochemical and ultrastructural study. Folia Morphol (Warsz) 75 (1), 76–86.

Mustafa, H.N., El Awdan, S.A., Hegazy, G.A., Jaleel, G.A.A., 2015. Prophylactic role of coenzyme Q10 and Cynara scolymus L on doxorubicin-induced toxicity in rats: biochemical and immunohistochemical study. Indian J. Pharmacol. 47, 649–656.

Mustafa, H.N., 2015. Potential alleviation of chlorella vulgaris and zingiber officinale on lead-Induced testicular toxicity: an ultrastructural study. Folia Biol. (Praha) 63, 269–278.

Mustafa, H.N., 2016. The role of curcumin in streptozotocin-induced hepatic damage and the trans-differentiation of hepatic stellate cells. Tissue Cell 48, 81–88.

Nascimento, A.R., Machado, M.V., Gomes, F., Vieira, A.B., Gonçalves-de-Albuquerque, C.F., Lessa, M.A., Bousquet, P., Tibiriçá, E., 2016.

Central sympathetic modulation reverses microvascular alterations in a rat model of high-Fat diet-Induced metabolic syndrome. Microcirculation 23, 320–329.

Naugle, J.E., Olson, E.R., Zhang, X., Mase, S.E., Pilati, C.F., Maron, M.B., Folkesson, H.G., Horne, W.I., Doane, K.J., Meszaros, J.G., 2006. Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling. Am. J. Physiol.-Heart Circulatory Physiol. 290, H323–H330.

Neilan, T.G., Blake, S.L., Ichinose, F., Raher, M.J., Buys, E.S., Jassal, D.S., Furutani, E., Perez-Sanz, T.M., Graveline, A., Janssens, S.P., 2007. Disruption of nitric oxide synthase 3 protects against the cardiac injury, dysfunction, and mortality induced by doxorubicin. Circulation 116, 506–514.

Octavia, Y., Tocchetti, C.G., Gabrielson, K.L., Janssens, S., Crijns, H.J., Moens, A.L., 2012. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J. Mol. Cell. Cardiol. 52, 1213–1225.

Oktem, G., Uysal, A., Oral, O., Sezer, E.D., Olukman, M., Erol, A., Akgur, S.A., Bilir, A., 2012. Resveratrol attenuates doxorubicin-induced cellular damage by modulating nitric oxide and apoptosis. Exp. Toxicol. Pathol. 64, 471–479.

Olukman, M., Can, C., Erol, A., Öktem, G., Oral, O., 2009. Reversal of doxorubicin-induced vascular dysfunction by resveratrol in rat thoracic aorta: is there a possible role of nitric oxide synthase inhibition? Anatol. J. Cardiol. 9, 260–266.

Potemski, P., Polakowski P. a. Wiktorowska-Owczarek, A.K., Owczarek, J., Pluzanska, A., Orszulak-Michalak, D., 2006. Amifostine improves hemodynamic parameters in doxorubicin-pretreated rabbits. Pharmacol. Rep. 58, 966.

Pott, C., Steinritz, D., Bolck, B., Mehlhorn, U., Brixius, K., Schwinger, R.H., Bloch, W., 2006. eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium. Am. J. Physiol. Cell Physiol.

290, C1437–1445.

Pradegan, N., Vida, V.L., Geva, T., Stellin, G., White, M.T., Sanders, S.P., Padera, R.F., 2016. Myocardial histopathology in late-repaired and unrepaired adults with tetralogy of Fallot. Cardiovasc. Pathol. 25, 225–231.

Rashikh, A., Abul Kalam, N., Akhtar, M., Mahmood, D., Pillai, K.K., Ahmad, S.J., 2011.

Protective effects of aliskiren in doxorubicin-induced acute cardiomyopathy in rats. Hum. Exp. Toxicol. 30, 102–109.

Rastogi, S., Mishra, S., Zaca, V., Mika, Y., Rousso, B., Sabbah, H.N., 2008. Effects of chronic therapy with cardiac contractility modulation electrical signals on cytoskeletal proteins and matrix metalloproteinases in dogs with heart failure.

Cardiology 110, 230–237.

Reagan, W.J., York, M., Berridge, B., Schultze, E., Walker, D., Pettit, S., 2013.

Comparison of cardiac troponin I and T, including the evaluation of an ultrasensitive assay, as indicators of doxorubicin-induced cardiotoxicity.

Toxicol. Pathol. 41, 1146–1158.

Sahu, B.D., Kumar, J.M., Kuncha, M., Borkar, R.M., Srinivas, R., Sistla, R., 2016.

Baicalein alleviates doxorubicin-induced cardiotoxicity via suppression of myocardial oxidative stress and apoptosis in mice. Life Sci. 144, 8–18.

Schaper, J., Froede, R., Hein, S., Buck, A., Hashizume, H., Speiser, B., Friedl, A., Bleese, N., 1991. Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation 83, 504–514.

Sharov, V.G., Kostin, S., Todor, A., Schaper, J., Sabbah, H.N., 2005. Expression of cytoskeletal, linkage and extracellular proteins in failing dog myocardium.

Heart Fail. Rev. 10, 297–303.

Sun, Z., Yan, B., Yu, W.Y., Yao, X., Ma, X., Sheng, G., Ma, Q., 2016. Vitexin attenuates acute doxorubicin cardiotoxicity in rats via the suppression of oxidative stress, inflammation and apoptosis and the activation of FOXO3a. Exp. Ther. Med. 12, 1879–1884.

Surai, P.F., 2015. Antioxidant action of carnitine: molecular mechanisms and practical applications. EC Veterinary Sci. 2, 66–84.

Viswanatha Swamy, A.H., Wangikar, U., Koti, B.C., Thippeswamy, A.H., Ronad, P.M., Manjula, D.V., 2011. Cardioprotective effect of ascorbic acid on doxorubicin-induced myocardial toxicity in rats. Indian J. Pharmacol. 43, 507–511.

Weber, K.T., Sun, Y., Bhattacharya, S.K., Ahokas, R.A., Gerling, I.C., 2013.

Myofibroblast-mediated mechanisms of pathological remodelling of the heart.

Nat. Rev. Cardiol. 10, 15–26.

Yin, W., Zhang, P., Huang, J.H., Zhang, Q.Y., Fan, R., Li, J., Zhou, J.J., Hu, Y.Z., Guo, H.T., Zhang, S.M., Wang, Y.M., Kaye, A.D., Gu, C.H., Liu, J.C., Cheng, L., Cui, Q., Yi, D.H., Pei, J.M., 2009. Stimulation of kappa-opioid receptor reduces isoprenaline-induced cardiac hypertrophy and fibrosis. Eur. J. Pharmacol. 607, 135–142.

Zhang, S., Liu, X., Bawa-Khalfe, T., Lu, L.S., Lyu, Y.L., Liu, L.F., Yeh, E.T., 2012.

Identification of the molecular basis of doxorubicin-induced cardiotoxicity.

Nat. Med. 18, 1639–1642.

Zhang, Y.P., Song, C.Y., Yuan, Y., Eber, A., Rodriguez, Y., Levitt, R.C., Takacs, P., Yang, Z., Goldberg, R., Candiotti, K.A., 2013. Diabetic neuropathic pain development in type 2 diabetic mouse model and the prophylactic and therapeutic effects of coenzyme Q10. Neurobiol. Dis. 58, 169–178. de Salvi Guimaraes, F., de Moraes, W.M., Bozi, L.H., Souza, P.R., Antonio, E.L., Bocalini, D.S., Tucci, P.J., Ribeiro, D.A., Brum, P.C., Medeiros, A., 2017.

Dexamethasone-induced cardiac deterioration is associated with both calcium handling abnormalities and calcineurin signaling pathway activation. Mol. Cell.

Biochem. 424, 87–98.