Inhibition of Gene Expression of Carnitine Palmitoyltransferase I and Heart Fatty Acid Binding...

Inhibition of Gene Expression of Carnitine Palmitoyltransferase Iand Heart Fatty Acid Binding Protein in Cyclophosphamideand Ifosfamide-Induced Acute Cardiotoxic Rat Models

Mohamed M. Sayed-Ahmed • Meshan L. Aldelemy •

Othman A. Al-Shabanah • Mohamed M. Hafez • Khaled A. Al-Hosaini •

Naif O. Al-Harbi • Shakir D. Al-Sharary • Mohamed M. Al-Harbi

� Springer Science+Business Media New York 2014

Abstract This study investigated whether cyclophos-

phamide (CP) and ifosfamide (IFO) therapy alters the

expression of the key genes engaged in long-chain fatty

acid (LCFA) oxidation outside rat heart mitochondria, and

if so, whether these alterations should be viewed as a

mechanism during CP- and IFO-induced cardiotoxicity.

Adult male Wistar albino rats were assigned to one of the

six treatment groups: Rats in group 1 (control) and group 2

(L-carnitine) were injected intraperitoneal (i.p.) with nor-

mal saline and L-carnitine (200 mg/kg/day), respectively,

for 10 successive days. Animals in group 3 (CP group)

were injected i.p. with normal saline for 5 days before and

5 days after a single dose of CP (200 mg/kg, i.p.). Rats in

group 4 (IFO group) received normal saline for 5 successive

days followed by IFO (50 mg/kg/day, i.p.) for 5 successive

days. Rats in group 5 (CP-carnitine supplemented) were

given the same doses of L-carnitine as group 2 for 5 days

before and 5 days after a single dose of CP as group 3. Rats

in group 6 (IFO-carnitine supplemented) were given the

same doses of L-carnitine as group 2 for 5 days before and

5 days concomitant with IFO as group 4. Immediately, after

the last dose of the treatment protocol, blood samples were

withdrawn and animals were killed for biochemical, histo-

pathological and gene expression studies. Treatment with

CP and IFO significantly decreased expression of heart fatty

acid binding protein (H-FABP) and carnitine palmitoyl-

transferase I (CPT I) genes in cardiac tissues. Moreover, CP

but not IFO significantly increased acetyl-CoA carboxyl-

ase2 mRNA expression. Conversely, IFO but not CP sig-

nificantly decreased mRNA expression of malonyl-CoA

decarboxylase. Both CP and IFO significantly increased

serum lactate dehydrogenase, creatine kinase isoenzyme

MB and malonyl-CoA content and histopathological lesions

in cardiac tissues. Interestingly, carnitine supplementation

completely reversed all the biochemical, histopathological

and gene expression changes induced by CP and IFO to the

control values, except CPT I mRNA, and protein expression

remained inhibited by IFO. Data from the current study

suggest, for the first time, that (1) CP and IFO therapy is

associated with the inhibition of the expression of H-FABP

and CPT I genes in cardiac tissues with the consequent

inhibition of mitochondrial transport and oxidation of

LCFA. (2) The progressive increase in cardiotoxicity

enzymatic indices and the decrease in H-FABP and CPT I

expression may point to the possible contribution of these

genes to CP- and IFO-induced cardiotoxicity.

Keywords Cyclophosphamide � Ifosfamide � CPT I �H-FABP � Cardiotoxicity � L-Carnitine

Introduction

Cyclophosphamide (CP) and ifosfamide (IFO) are nitrogen

mustard alkylating prodrugs and commonly used in most

cancer chemotherapy and immunosuppressive protocols [1,

2]. Experimental and clinical studies reported that high ther-

apeutic doses of CP and IFO are associated with acute and

lethal cardiotoxicity [3–7]. The incidence of CP-induced

M. M. Sayed-Ahmed (&) � O. A. Al-Shabanah �M. M. Hafez � K. A. Al-Hosaini � N. O. Al-Harbi �S. D. Al-Sharary � M. M. Al-Harbi

Department of Pharmacology and Toxicology, College of

Pharmacy, King Saud University,

P. O. Box 2457, Riyadh 11451, Kingdom of Saudi Arabia

e-mail: [email protected]

M. L. Aldelemy

King Abdullah International Medical Research Center,

P. O. Box 2457, Riyadh 11451, Kingdom of Saudi Arabia

123

Cardiovasc Toxicol

DOI 10.1007/s12012-014-9247-1

cardiotoxicity was reported to be 22 % with 11 % showing

fatal cardiotoxicity [4, 8]. In patients who never had prior

anthracyclines and radiotherapy, the incidence of symptom-

atic CP-induced cardiotoxicity was 25 % with 12 % mortality

[9]. Also, acute heart failure has been reported 1 week after

CP administration [8, 9]. Cardiac effects reported with IFO

therapy include supraventricular arrhythmias with a 30 %

incidence of congestive heart failure [5, 10]. The pathogenesis

of CP- and IFO-induced acute cardiotoxicity was attributed to

an increase in free oxygen radicals and decrease in the anti-

oxidant defense mechanism in the myocardium [11, 12].

Hypercholesterolemia, hypertriglyceridemia and impaired

secretion of heart lipoprotein lipase have been reported in CP-

treated rabbits [11, 13, 14]. Increasing inner mitochondrial

membrane permeability to calcium in cardiac tissues by CP

has been reported [15].

It has been demonstrated that carnitine deficiency and car-

nitine insufficiency are risk factors and provoke CP- and IFO-

induced acute cardiotoxicity and nephropathy [16–18]. Recent

study in our laboratory demonstrated that CP- and IFO-induced

cardiotoxicity was primarily to the decrease in myocardial

carnitine content following the inhibition of organic cation/

carnitine transporter (OCTN2) mRNA and protein expression

[19]. Since L-carnitine is the major player in the mitochondrial

transport of long-chain fatty acids (LCFA), inhibition of

OCTN2 could inhibit LCFA oxidations [20, 21]. The pathway

of LCFA oxidation includes several events including its

transport to the outer mitochondrial membrane via heart fatty

acid binding protein (H-FABP), its activation at the outer

mitochondrial membrane by acyl-CoA synthetase, its transport

across the mitochondrial membranes via the carnitine palmi-

toyltransferases (CPT I, carnitine-acylcarnitinetranslocase

(CACT) and CPT II) and finally its oxidation in the mito-

chondrial matrix through b-oxidation [22, 23]. Earlier studies

reported that chloroacetaldehyde (CAA) and thiodiaglycolic

acid (TDGA), the two major toxic metabolites of CP and IFO,

inhibit the oxidation of LCFA (carnitine dependent) but not

medium-chain fatty acids (carnitine independent) [24, 25].

Therefore, it seems that CP and IFO inhibit LCFA oxidation

possibly at site(s) outside mitochondria, including H-FABP and

CPT I and its related genes. Accordingly, this study investigated

whether CP and IFO therapy alters the expression of the key

genes engaged in LCFA oxidation outside rat heart mitochon-

dria, and if so, whether these alterations should be viewed as a

mechanism during CP- and IFO-induced cardiotoxicity.

Materials and Methods

Materials

Endoxan and Holoxan vials (Baxter oncology GmbH,

Germany) were gifted from King Khalid University

Hospital drug store, King Saud University, Kingdom of

Saudi Arabia. Each Endoxan vial contains 500 mg CP,

whereas each Holoxan vial contains 1 g IFO in a dry

lyophilized powder form. The content of each vial was

freshly dissolved in sterile water for injection immediately

before injection. L-carnitine was kindly supplied by Dr.

ZavenOrfalian, Sigma-Tau Pharmaceuticals, Pomezia,

Italy. It has been supplied as white powder in non-com-

mercial plastic bottles containing 100 g, and it was freshly

dissolved in normal saline prior to injection. Primers and

probes were designed using Primer Express 3.0 (Applied

biosystem, life technology, USA) and purchased from

Metabion International AG (Germany). CPT I and GAPDH

monoclonal antibodies have been purchased from Santa

Cruz biotechnology, Inc. (Heidelberg, Germany). All other

chemicals used were of the highest analytical grade.

Animals

Adult male Wistar albino rats, weighing 180–200 g, were

obtained from the Animal Care Center, College of Phar-

macy, King Saud University, Riyadh, Kingdom of Saudi

Arabia, and were housed in metabolic cages under con-

trolled environmental conditions (25 �C and a 12 h light/

dark cycle). Animals had free access to pulverized standard

rat pellet food and tap water. The protocol of this study has

been approved by Research Ethics Committee of College

of Pharmacy, King Saud University, Riyadh, Kingdom of

Saudi Arabia.

Experimental Design

To achieve the ultimate goals of this study, a total of 60

adult male Wistar albino rats were used and divided at

random into 6 groups of 10 animals each. Rats of group 1

(control group) received i.p. injection of normal saline

(2.5 ml/kg/day) for 10 successive days. Animals in group 2

(carnitine-supplemented group) were given L-carnitine

(200 mg/kg/day, i.p.) for 10 successive days as previously

described [17]. Animals in group 3 (CP group) received

normal saline for 5 days before and 5 days after a single

dose of CP (200 mg/kg, i.p.) according to previously

published studies [12, 16]. Rats of group 4 (IFO group)

received normal saline for 5 successive days followed by

IFO (50 mg/kg/day, i.p.) for 5 successive days [26].

Animals in group 5 (CP-carnitine supplemented) received

L-carnitine (200 mg/kg/day, i.p.) for 5 days before and

5 days after a single dose of CP (200 mg/kg, i.p.) [16]. Rats

in group 6 (IFO-carnitine supplemented) received L-carni-

tine (200 mg/kg/day, i.p.) for 5 successive days before and

5 days concomitant with IFO (50 mg/kg/day, i.p.) as pre-

viously described [17]. Immediately after the last dose of

the treatment protocol, animals were then killed by

Cardiovasc Toxicol

123

decapitation after exposure to ether in a desiccator kept in a

well-functioning hood and blood samples were obtained.

Serum was separated for measurement of serum lactate

dehydrogenase (LDH) and creatine phosphokinase isoen-

zyme (CK-MB). Hearts were quickly excised for mea-

surement of malonyl-CoA content, mRNA and protein

expression of CPT I, and mRNA expression of H-FABP,

ACC2, and MCD genes. Moreover, heart specimens from

each group were removed to be examined histopathologi-

cally; they were fixed in 10 % neutral buffered formalin,

sectioned at 3 lm and stained with Hematoxylin and Eosin

(H & E) stain for light microscopic examination.

Methods

Quantification of mRNA Expression by Real-Time

Polymerase Chain Reaction

Total RNA Extraction Total RNA was extracted from

heart tissues by Trizol method according to the standard

protocol as previously described [27]. Briefly, RNA was

extracted by homogenization of cardiac tissues (Polytron;

Kinematica, Lucerne, Switzerland) in TRIzol reagent

(GibcoBRL) at maximum speed for 90–120 s. The

homogenate was incubated for 5 min at room temperature.

A 1:5 volume of chloroform was added, and the tube was

vortexed and subjected to centrifugation at 12,000g for

15 min. The aqueous phase was isolated, and the total

RNA was precipitated by cold absolute ethanol. After

centrifugation and washing, the total RNAwas finally

eluted in 20 ll of diethyl pyrocarbonate-treated water. The

quantity was characterized using a UV spectrophotometer

(NanoDrop 8000, Thermo Scientific, USA). The isolated

RNA has a 260/280 ratio of 1.9–2.1.

First-Strand cDNA Synthesis Using SuperScript II RT

First-strand cDNA was synthesized from 1 lg of total

RNA by reverse transcription with a SuperScriptTM first-

strand synthesis system kit (Invitrogen, CA, USA),

according to the manufacturer’s instructions.

Real-Time PCR Real-time reaction was performed using

the KAPA PROBE FAST qPCR kit master mix (KAPA

Biosysems, USA) and the 2-DDCt method. GAPDH gene

was used as the endogenous control. PCR assay was opti-

mized by varying the PCR conditions such as the con-

centration of cDNA, primers and probes, amplification

cycle number and annealing temperature. Briefly, a stan-

dard 25 ll reaction mixture contained in final concentra-

tion of 19 KAPA PROBE FAST qPCR master mix buffer,

0.4 lM of each forward and reverse primers, 0.2 lM probe

for H-FABP, CPT I, ACC2, MCD and GAPDH (Table 1),

100 ng of cDNA and RNase, and DNase-free water. The

reaction was done in an ABI 96-well optical reaction plate

placed on ice before cDNA template was added. The

standard thermal cycling conditions of initial 50 �C for

2 min and 95 �C for 10 min followed by 40 cycles at 95 �C

for 15 s and 60 �C for 1 min were used. All reactions were

performed using an ABI 7500 System (Applied Biosystem,

USA). Experiments were performed in triplets for all data

points. Each qPCR included no-template controls.

Western Blot Analysis of CPT I Protein

For Western blot analysis, heart tissues were washed with ice-

cold PBS and the protein extracts were prepared using ice-

cold cell lysis buffer supplemented with protease inhibitor

cocktail (IBI SCIENTIFIC, Peosta, USA). Protein concen-

trations were measured using the Bradford assay (Bio-Rad,

CA, USA) according to manufacturer’s protocol. Proteins

were separated on 10 % sodium dodecyl sulfate–polyacryl-

amide (SDS-PAGE) gels and transferred to a nitrocellulose

membrane. The membrane was blocked with 5 % skimmed

milk in TBS-T (10 mM Tris–HCl, 150 mM NaCl, 0.25 %

Tween 20, pH 7.5) at room temperature for 2 h followed by

incubation with 2 lg/ml of primary antibody for CPT I and

the endogenous control, GAPDH, diluted in TBS and 5 %

skimmed milk overnight at 4 �C. After washing with TBS-T

buffer, the membrane was incubated with 1 lg/ml of horse-

radish peroxidase (HRP)-labeled secondary antibody diluted

in TBS-T buffer for 2 h at room temperature, followed by

three washes with TBS-T buffer. The detection was per-

formed using chemiluminescence assay (Amersham, GE

Healthcare, life science, UK). Membranes were exposed to

X-ray film to observe the bands (Kodak, Rochester, NY).

Protein bands in treated and untreated (control) groups were

quantified using the Kodak Scientific ID software.

Determination of Malonyl-CoAlevelin Rat Cardiac Tissue

Malonyl-CoA was determined in heart tissues using HPLC

system (Jasco Corporation, Ishikawa-Cho, Hachioji,

Tokyo, Japan) [28]. In brief, heart tissue was homogenized

in ice-cold 6 % perchloric acid, centrifuged at 1,000 rpm

for 15 min at 0.5 �C and the supernatant fluid was injected

into HPLC after neutralization to pH 6–7. Chromato-

graphic separation was performed using ODS-Hypersil,

150 9 4.6 mm I.D., 5 lm column (Supelco SA, Gland,

Switzerland). The UV detector was operated at 254 nm and

set at 0.005. A mobile phase of 220 mM potassium phos-

phate containing 0.05 % dithioglycol (A) and 98 % meth-

anol, 2 % chloroform (B) was used. The flow rate was

0.6 ml/min, and the gradient was as follows: at zero time,

94 % A and 6 % B; at 8 min, 92 % A and 8 % B; at

14 min, 87 % A and 13 % B; at 25 min, 80 % A and 20 %

Cardiovasc Toxicol

123

B; at 40 min, 55 % A and 45 % B; at 45 min, 55 % A and

45 % B; and at 60 min, 94 % A and 6 % B.

Assessment of Serum Creatine Kinase (CK-MB)

and Lactate Dehydrogenase (LDH) Activity

Serum activities of LDH and CK-MB were determined

according to the methods of Buhl and Jackson [29] and Wu

and Bowers [30], respectively.

Statistical Analysis

Differences between obtained values (mean ± SEM,

n = 10) were carried out by one way analysis of variance

(ANOVA) followed by the Tukey–Kramer multiple com-

parison test using GraphPad Prism 5 software. P B 0.05

was taken as a criterion for a statistically significant

difference.

Results

To investigate the effects of CP and IFO on the transport of

LCFA and their intermediates to the sites of their metabolic

conversion, H-FABP mRNA expression was measured in

cardiac tissues using RT-PCR (Fig. 1). Treatment with

either CP or IFO significantly decreased the expression of

H-FABP mRNA in cardiac tissues. A significant 45 and

47 % decrease was obtained after CP and IFO, respec-

tively, as compared to the control group. Daily adminis-

tration of L-carnitine alone for 10 successive days showed

nonsignificant change, as compared to the control. Fasci-

natingly, administration of L-carnitine in combination with

CP or IFO resulted in a complete reversal of CP- and IFO-

induced decrease in H-FABP mRNA expression to the

control values.

To investigate the effects of CP and IFO on the mito-

chondrial transport of LCFA, CPT I mRNA and protein

expression were investigated in cardiac tissues using

RT-PCR and Western blot analysis, respectively (Fig. 2).

Treatment with either CP or IFO significantly decreased

the expression of CPT I on the mRNA (Fig. 2a) and the

protein (Fig. 2b) levels in cardiac tissues. A significant 46

and 33 % decrease in mRNA expression was obtained after

CP and IFO, respectively, as compared to the control

group. Similarly, CP and IFO significantly decreased CPT I

protein expression by 82 and 83 %, respectively, as com-

pared to the control group. Daily administration of

L-carnitine alone for 10 successive days resulted in a sig-

nificant 41 % increase in CPT I mRNA expression as

compared to the control. Interestingly, administration of

L-carnitine for 5 days before and 5 days after a single dose

of CP resulted in a complete reversal of CP-inducedTa

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Cardiovasc Toxicol

123

decrease in CPT I mRNA expression to the control values.

However, CP-induced decrease in CPT I protein expression

was partially recovered by L-carnitine. Conversely,

administration of L-carnitine to IFO-treated rats did not

affect IFO-induced decrease in CPT I mRNA and protein

expression.

To investigate the indirect effects of CP and IFO on the

activity of CPT I in cardiac tissues, the level of malonyl-

CoA, the well-known physiological and potent inhibitor of

CPT I enzyme, was measured using HPLC (Fig. 3).

Administration of a single dose of CP and 5 doses of IFO

resulted in a significant 32 and 29 % increase in malonyl-

CoA level in heart tissues, respectively, as compared to the

control group. On the other hand, daily administration of

L-carnitine alone for 10 successive days resulted in a sig-

nificant 30 % decrease as compared to the control group.

Interestingly, administration of L-carnitine in combination

with CP and IFO resulted in a complete reversal of the

increase in myocardial malonyl-CoA content, induced by

CP and IFO, to the control values.

Acetyl-CoA carboxylase is an enzyme responsible for the

synthesis of malonyl-CoA via carboxylation of acetyl-CoA.

The effects of CP and IFO, L-carnitine and their combination

on ACC2 mRNA expression in heart tissues are shown in

Fig. 4. Treatment with single dose of CP (200 mg/kg)

resulted in a significant 10-fold increase in ACC2 mRNA

expression, as compared to the control group. On the other

hand, administration of IFO (50 mg/kg) for 5 successive days

showed nonsignificant changes in ACC2 mRNA expression

compared to control group. Similarly, carnitine supplemen-

tation alone for 10 successive days showed nonsignificant

changes in ACC2 mRNA expression. Interestingly,

administration of L-carnitine to CP-treated rats resulted in a

complete reversal of CP-induced increase in ACC2 mRNA

expression to the control values.

Malonyl-CoA decarboxylase (MCD) is an enzyme

responsible for the degradation of malonyl-CoA to acetyl-

CoA and carbon dioxide. Figure 5 shows the effects of CP

and IFO, L-carnitine and their combination on the expression

of MCD mRNA expression in rat heart tissues. Treatment

with IFO (50 mg/kg) for 5 successive days resulted in a

significant 60 % decrease in the expression of MCD mRNA,

as compared to the control group. On the other hand,

administration of a single dose of CP (200 mg/kg) showed

nonsignificant changes in MCD mRNA expression as com-

pared to the control group. Similarly, treatment with

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Fig. 1 Effects of cyclophosphamide (CP), ifosfamide (IFO),

L-carnitine and their combination on H-FABP mRNA expression in

cardiac tissues. Data are presented as mean ± SEM (n = 10). *, # and$ indicate significant change from control, CP and IFO, respectively,

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L-carnitine and their combination on CPT I mRNA (2A) and

protein (2B) expression in cardiac tissues. Data are presented as

mean ± SEM (n = 10). * and # indicate significant change from

control and CP, respectively, at p \ 0.05 using ANOVA followed

by Tukey–Kramer as a post-ANOVA test

Cardiovasc Toxicol

123

L-carnitine (200 mg/kg/day) for 10 successive days alone

showed nonsignificant increase in MCD m RNA expression

as compared to control group. Fascinatingly, administration

of L-carnitine to IFO-treated rats resulted in a complete

reversal of IFO-induced decrease in MCD mRNA expression

to the control values.

Figure 6 shows the effects of CP and IFO, L-carnitine

and their combination on cardiotoxicity enzymatic indices,

CK-MB (A) and LDH (B), in rats. Administration of a

single dose of CP (200 mg/kg) resulted in a significant 77

and 92 % increase in serum CK-MB and LDH, respec-

tively, as compared to the control group. Treatment with

L-carnitine (200 mg/kg/day) for 10 successive days showed

nonsignificant changes. Interestingly, administration of

L-carnitine for 5 days before and 5 days after a single dose

of CP resulted in a complete reversal of CP-induced

increase in serum CK-MB and LDH to the control values.

On the other hand, administration of IFO (50 mg/kg/day)

for 5 successive days resulted in 52 and 67 % increase in

serum CK-MB and LDH, respectively, as compared to the

control group. Carnitine supplementation by daily admin-

istration of L-carnitine for 5 days before and 5 days con-

comitant with IFO resulted in a complete reversal of IFO-

induced increase in serum CK-MB and LDH to the control

values.

Figure 7 shows the histopathological changes in cardiac

tissues induced by CP and IFO in carnitine-supplemented

rats. Cardiac tissues of control rats showed normal-looking

cardiac muscle with no degenerative changes (Fig. 7a). On

the other hand, animals treated with CP alone showed pro-

gressive subendocardial and interstitial fibrosis (Fig. 7b).

Also, treatment with IFO alone showed focal subendocardial

fibrosis, myocarditis with lymphohistiocytic infiltrate and

mild perivascular fibrosis (Fig. 7c). Heart specimens from rats

treated with CP plus L-carnitine showed minimal interstitial

fibrosis (Fig. 7d). Similarly, rats treated with IFO plus L-car-

nitine showed minimal myocarditis and fibrosis (Fig. 7e).

Discussion

The pathway of LCFA oxidation in the heart is unique from

the other tissues in the following. First, fatty acids are the

major substrates for energy production in the adult working

heart [31]. Under normal physiological conditions, fatty acid

oxidation supplies the heart with approximately 60–90 % of

ATP and the remaining 40–10 % of ATP is supplied via

glucose, lactate and ketone bodies utilization [20, 21].

Second, the existence of specific heart-type fatty acid

binding protein (H-FABP) in the cytosol of cardiac myo-

cytes plays a crucial role in eliminating the toxicity of free

fatty acids and their intermediates in the cytosol by trans-

porting these compounds to their sites of metabolic con-

version [32, 33]. Third, carnitine is an obligatory cofactor

for the oxidation of LCFA [22, 23]. Finally, inhibition of

this vital pathway in the heart is associated with cardio-

myopathy [34–37]. Earlier in vivo and in vitro studies

reported that CAA and TDGA, the two major toxic

metabolites of CP and IFO, inhibit the oxidation of LCFA

(carnitine dependent) but not medium-chain fatty acids

(carnitine independent) [24, 25]. Authors found that CAA

and TDGA inhibited the oxidation of (1-14C) palmitic acid

but not (1-14C) octanoic acid, (1-14C) succinic acid and

(1-14C) palmitoyl-L-carnitine. Normal oxidation of octanoic

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ssio

n


L-carnitine and their combination on acetyl-CoA carboxylase2

(ACC2) mRNA expression in cardiac tissues. Data are presented as

mean ± SEM (n = 10). *, # and $ indicate significant change from

control, CP and IFO, respectively, at p \ 0.05 using ANOVA

followed by Tukey–Kramer as a post-ANOVA test

Cardiovasc Toxicol

123

acid, succinic acid and palmitoyl-L-carnitine in the presence

of TDGA and CAA demonstrates intact function of CACT,

CPT II, b-oxidation enzymes and Krebs’s cycle [24, 25].

Accordingly, one can anticipate that CP and IFO inhibit

LCFA oxidation possibly at site(s) outside mitochondria.

Therefore, this study has been initiated to investigate whether

CP and IFO therapy alters the expression of H-FABP and

CPT 1 and its related genes in cardiac tissues, and if so,

whether these alterations should be viewed as a mechanism

during development of CP- and IFO-induced cardiotoxicity.

Data presented here clearly demonstrate that both CP and

IFO induced inhibition of H-FABP mRNA expression in

cardiac tissues. The inhibition of H-FABP expression by CP

and IFO in the heart could increase the accumulation of free

fatty acids and their toxic intermediates secondary to the

inhibition of their delivery by H-FABP to the CPT I which is

responsible for their translocation into mitochondria [38, 39].

Inhibition of H-FABP mRNA expression in doxorubicin-

related cardiotoxicity has been previously reported [40]. In

doxorubicin-induced chronic cardiomyopathic rat mode,

earlier studies reported that carnitine supplementation com-

pletely restored doxorubicin-induced inhibition of H-FABP m

RNA expression [40, 41]. L-carnitine is known to prevent the

accumulation of toxic fatty acid intermediates in the myo-

cardium [37, 42]. Moreover, L-carnitine through its reported

antioxidant defense against generation of ROS could protect

against CP- and IFO-induced inhibition of H-FABP mRNA

expression in cardiac tissues [16–19, 26, 74].

Data presented here demonstrated that CP and IFO

significantly inhibited mRNA and protein expression of

CPT I. Accordingly, one can anticipate that both CP and

IFO could inhibit the translocation of LCFA from cyto-

plasmic compartment into mitochondrial compartment.

This observed decrease in CPT I expression by CP and IFO

was parallel to the increase in cardiotoxicity enzymatic

indices and the histopathological lesions in cardiac tissues

which may point to the possible consideration of the

inhibition of CPT I as a mechanism in CP- and IFO-

induced acute cardiotoxicity. Inhibition of CPT I and its

contribution to cancer chemotherapy-dependent [42–44]

and chemotherapy-independent [45–47] cardiomyopathies

have been previously reported. The observed stronger

inhibitory effects of both CP and IFO on CPT1 at protein

level than mRNA level could be due to post-transcription

modification, protein half-life and/or protein damage fol-

lowing oxidative stress induced by CP and IFO treatment.

It is well documented that malonyl-CoA is a potent

inhibitor of CPT I and a key regulator of LCFA oxidation

in the heart [48–51]. Accordingly, increased cardiac mal-

onyl-CoA levels are associated with a decrease in mito-

chondrial LCFA uptake and oxidation, whereas its decrease

in cardiac tissues results in an increase in fatty acid

Contro

l

L-Car

nitin

e CP

IFO

CP+L-C

arni

tine

IFO+L

-Car

nitin

e 0.0

0.5

1.0

1.5

2.0

*#

$$

MC

D m

RN

A F

old

Exp

ress

ion


L-carnitine and their combination on malonyl-CoA decarboxylase

(MCD) mRNA expression in cardiac tissues. Data are presented as




Control

L-Car

nitine

CPIF

O

CP+L-Car

nitine

IFO+L-C

arniti

ne0

250

500

750

**

#$

A

CK

-MB

(U

/L)

Control

L-Car

nitine

CPIF

O

CP+L-C

arniti

ne

IFO+L

-Car

nitine

0

250

500

750B*

*

#$

LD

H (

U/L

)


L-carnitine and their combination on serum cardiotoxicity enzymatic

indices, CK-MB (a) and LDH (b), in rats. Data are presented as




Cardiovasc Toxicol

123

oxidation [52–55]. Data presented in the current study

showed that CP and IFO increased malonyl-CoA produc-

tion in cardiac tissues, suggesting that both CP and IFO

could inhibit CPT I activity with the consequent decrease

in mitochondrial transport and oxidation of LCFA. The

question now arises, how CP and IFO increased malonyl-

CoA level in cardiac tissues under our experimental con-

dition? It is well known that the level of malonyl-CoA in

the heart depends on the rate of its synthesis by the car-

boxylation of acetyl-CoA by ACC [20, 54, 56] and its

degradation to acetyl-CoA by MCD [54, 55, 57].

Acetyl-CoA carboxylase enzyme is present in two iso-

forms: ACCa or ACC1 which is found predominantly in

lipogenic tissues and ACCb or ACC2 which is the major

isoform expressed in skeletal muscle and heart [58–63].

Because of its predominant location in skeletal and cardiac

muscle, it has been proposed that ACC2 is involved in the

regulation of fatty acid oxidation rather than fatty acid

biosynthesis [61, 64, 65]. On the other hand, MCD cata-

lyzes the decarboxylation of malonyl-CoA [52–55] and it is

found in many compartments, including mitochondria,

peroxisomes and cytosol [66]. Our results showed that CP

but not IFO increased ACC2 mRNA expression. Con-

versely, IFO but not CP decreased mRNA expression of

MCD. Accordingly, it seems from our results that both CP

and IFO increased malonyl-CoA level in cardiac tissues by

different mechanisms. CP increased malonyl-CoA level by

increasing its synthesis by ACC2, whereas, IFO increased

malonyl-CoA level via decreasing its degradation by MCD.

ACC2 activity is dependent on the supply of its substrate,

acetyl-CoA and its phosphorylation by AMP-activated

protein kinase [67–69]. It is worth mentioning that results

of mRNA expression of H-FABP, MCD and ACC2 pre-

sented in our study warrant detailed mechanistic studies to

monitor the activity and the protein expression codified by

these genes as well as mRNA and protein expression of

AMPK. This might explain the observed differential effects

of CP and IFO on MCD and ACC2 mRNA expression.

Data presented in the current study showed that carnitine

supplementation significantly increased mRNA and protein

expression of CPT I. Yoon et al. [43] reported that daily

administration of carnitine (200 mg/kg) for 2 weeks

increased activity of CPT I. This increase in CPT I

expression and activity could be explained on the data


L-carnitine and their combination on histopathological changes in

rat cardiac tissues. a Heart from control rat showing normal cardiac

histology (940). b Heart of rat treated with CP alone showing

progressive subendocardial and interstitial fibrosis (940). c Heart of

rat treated with IFO alone showing focal subendocardial fibrosis,

myocarditis with lymphohistiocytic infiltrate and mild perivascular

fibrosis (940). d Heart of rat treated with CP plus L-carnitine showed

minimal interstitial fibrosis (940). e Heart of rat treated with IFO plus

L-carnitine showed minimal myocarditis and fibrosis (940)

Cardiovasc Toxicol

123

presented in this study which demonstrated that carnitine

decreased malonyl-CoA production, the well-known

inhibitor of CPT I. Normal expression of both ACC2 and

MCD in the presence of carnitine suggests that carnitine

regulates malonyl-CoA production by other mechanism.

Since acetyl-CoA is the precursor of malonyl-CoA, this

observed decrease in malonyl-CoA level by carnitine could

be explained on the basis that carnitine may decrease the

cytosolic acetyl-CoA. It is well documented that carnitine

decreases acetyl-CoA accumulation by stimulating its

efflux in the form of acetylcarnitine in a reaction mediated

by CAT [28, 70, 71]. Results from this study demonstrated

that carnitine supplementation completely reversed CP-

and IFO-induced alteration in the expression of ACC2 and

MCD genes to the normal values. In CP plus carnitine

group, carnitine supplementation completely reversed CP-

induced decrease in CPT1 mRNA but not protein expres-

sion. This discrepancy could be due to post-transcription

modification, protein half-life and/or protein damage fol-

lowing oxidative stress induced by CP treatment.

Although L-carnitine completely reversed CP- and IFO-

induced decrease in malonyl-CoA, CPT I mRNA and protein

expression remained inhibited by IFO but not CP. It seems

that IFO induced irreversible inhibition of CPT I. This

hypothesis is consistent with data presented in our recent

study which has reported that IFO but not CP induced irre-

versible inhibition of carnitine transporter gene, organic cat-

ion/carnitine transporter, on mRNA and protein expression

levels in both kidney and heart tissues [19]. Moreover, earlier

studies have demonstrated that the inhibition of CPT I is

associated with the accumulation of ceramide, a sphingolipid

that has been implicated in apoptotic response of cells to

death inducers, such as Fas/Fas ligand, TNF-a, growth factor

withdrawal, hypoxia and DNA damage [72, 73]. In IFO-

induced Fanconi syndrome rat model, carnitine supplemen-

tation normalized the increase in the expression of apoptotic

genes and the decrease in the expression of antiapoptotic ones

induced by IFO [19, 74]. It has been recognized that L-car-

nitine is an important factor in the regulation of apoptotic

processes both at the mitochondrial level and at the level of

ceramide synthesis and signaling [75]. It has been reported

that accumulation of ceramide occurs after cytotoxic stress

including chemotherapeutic drugs and radiation. It has been

reported that apoptosis induced by doxorubicin is associated

with the activation of sphingomyelinase, leading to sphing-

omyeline hydrolysis and concomitant ceramide accumulation

[76]. Authors concluded that L-carnitine treatment attenuated

ceramide accumulation by interacting with the sphingomye-

linase, leading to the inhibition of doxorubicin-induced car-

diac apoptosis. [76]. Earlier study indicated that L-carnitine is

able to inhibit CD95-induced apoptosis, by inhibiting the

acidic sphingomyelinase, the key enzyme in ceramide syn-

thesis [77–79].

Conclusions

Data obtained from the current study suggest that CP and

IFO therapy is associated with the inhibition of the

expression of H-FABP and CPT I genes in cardiac tissues.

The progressive increase in cardiotoxicity enzymatic

indices and the decrease in H-FABP and CPT I expression

may point to the possible contribution of H-FABP and CPT

I as a mechanism during development of CP- and IFO-

induced cardiotoxicity.

Acknowledgments Authors thank the Deanship of Scientific

Research at KSU for funding this work through the research group

project no. RGP-VPP-142.

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Inhibition of Gene Expression of Carnitine Palmitoyltransferase I and Heart Fatty Acid Binding...

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