Possible involvement of downregulation of the apelin

Am J Physiol Heart Circ Physiol 308: H931–H941, 2015.
First published February 13, 2015; doi:10.1152/ajpheart.00703.2013.
Possible involvement of downregulation of the apelin-APJ system in
doxorubicin-induced cardiotoxicity
Juri Hamada,1,2 Altansarnai Baasanjav,1 Natsumi Ono,1 Kazuya Murata,2 Koichiro Kako,1 Junji Ishida,1,2
and Akiyoshi Fukamizu1,2
1
Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan; and 2Life Science Center,
Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan
Submitted 12 September 2013; accepted in final form 9 February 2015
apelin; APJ; doxorubicin; cardiotoxicity
(Dox) is an anthracycline antitumor drug that has
long been used in cancer chemotherapy. The antitumor action
of Dox is based on its intercalation into DNA double helix,
which, in turn, leads to inhibition of DNA replication, repair,
and transcription. This mechanism inhibits cell growth and
exerts a cell-killing effect against diverse malignant tumors.
Despite the powerful antitumor effect of Dox, its clinical use is
limited by dose-dependent cardiotoxicity, including arrhythmia, acute left ventricular (LV) dysfunction, and degenerative
cardiomyopathy (29). Once it occurs, Dox-induced cardiomyopathy is irreversible and often progresses to congestive heart
failure. Moreover, this disease has an extremely poor prognosis, as a cohort study (10) has reported that the 3-yr survival
DOXORUBICIN
Address for reprint requests and other correspondence: A. Fukamizu, Life
Science Center, Graduate School of Life and Environmental Sciences, Tsukuba Advanced Research Alliance, Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan (e-mail: [email protected]).
http://www.ajpheart.org
rate in patients with heart failure secondary to Dox-induced
cardiomyopathy was ⬍50%. Prevention of cardiotoxicity is
therefore important in Dox chemotherapy, as is monitoring the
onset and progression of cardiomyopathy.
APJ [a putative receptor protein related to ANG II type 1
(AT1) receptor] is a member of a G protein-coupled receptor
superfamily with seven transmembrane domains. APJ was first
identified as a gene possessing homology with the AT1 receptor but was found not to bind ANG II (23). Thereafter,
following a period of orphan designation, APJ was finally
deorphanized when it was discovered to be an apelin receptor
(31). Apelin peptides exist in multiple short forms of different
sizes produced by COOH-terminal cleavage of the 77-amino
acid preproapelin. Peptides with amino acid lengths of 12, 13,
17, and 36 have been found in plasma and cardiovascular
tissues in rodents and humans and are known to be biologically
active (31, 39). In addition, both apelin and APJ mRNAs are
highly expressed in the heart, and, therefore, the cardiac
apelin-APJ system has been implicated in cardiovascular physiology and pathophysiology (14, 20).
Since the discovery of apelin, APJ has emerged as having a
potential role in vasodilation, cardiac contraction, and cardioprotection in rodent models. Apelin stimulates nitric oxide
(NO) release from the vascular endothelium through endothelial NO synthase activation, leading to vessel relaxation (12,
32). Apelin exerts a direct positive inotropic action on the heart
both ex vivo and in vivo (2, 28). In addition, apelin treatment
confers significant cardioprotection in rodent models of cardiac
ischemia-reperfusion injury as well as ANG II- or isoproterenol-induced cardiac remodeling (15, 24, 25, 30). More importantly, it has been reported that apelin knockout mice showed
impaired cardiac contractility with aging and developed progressive heart failure induced by pressure overload (19). Another study (4) found that both apelin knockout mice and APJ
knockout mice showed not only modest declines in cardiac
function under basal conditions but also clear decrements in
exercise capacity under physiological stress conditions. These
studies support the view that the endogenous apelin-APJ system contributes to the maintenance of cardiac function.
Recently, clinical studies have demonstrated that several
human cardiovascular diseases are accompanied by changes in
the expression of cardiac apelin and APJ. A large cohort study
(7) reported that blood apelin levels were decreased in patients
with chronic heart failure after LV systolic dysfunction. Implantation of a LV assist device in the patients with severe
heart failure resulted in an elevation of APJ mRNA and apelin
concentrations in the LV (5). These studies seem to support the
notion that the endogenous apelin-APJ system has a cardioprotective role and that APJ is a potential therapeutic target for
0363-6135/15 Copyright © 2015 the American Physiological Society
H931
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.1 on October 18, 2016
Hamada J, Baasanjav A, Ono N, Murata K, Kako K, Ishida J,
Fukamizu A. Possible involvement of downregulation of the apelinAPJ system in doxorubicin-induced cardiotoxicity. Am J Physiol
Heart Circ Physiol 308: H931–H941, 2015. First published February
13, 2015; doi:10.1152/ajpheart.00703.2013.—Apelin peptide is an
endogenous ligand of APJ (a putative receptor protein related to the
angiotensin II type 1 receptor), which is a member of a G proteincoupled receptor superfamily with seven transmembrane domains.
Recent findings have suggested that the apelin-APJ system plays a
potential role in cardiac contraction and cardioprotection. In the
present study, we show that the apelin-APJ system is disrupted in
doxorubicin (Dox)-induced cardiotoxicity. We found downregulation
of apelin and APJ mRNA expression in C57Bl/6J mouse hearts on
days 1 and 5 after Dox administration (20 mg/kg ip). Plasma apelin
levels and cardiac APJ protein expression were significantly decreased on day 5 after Dox injection. Cardiac apelin contents were
reduced on day 1 but increased to basal levels on day 5 after Dox
injection. We also examined the effects of APJ gene deletion on
Dox-induced cardiotoxicity. Compared with wild-type mice, APJ
knockout mice showed a significant depression in cardiac contractility
on day 5 after Dox (15 mg/kg ip) treatment followed by a decrease in
14-day survival rates. Moreover, Dox-induced myocardial damage,
cardiac protein carbonylation, and autophagic dysfunction were accelerated in APJ knockout mice. Rat cardiac H9c2 cells showed
Dox-induced decreases in viability, which were prevented by APJ
overexpression and the combination with apelin treatment. These
results suggest that the suppression of APJ expression after Dox
administration can exacerbate Dox-induced cardiotoxicity, which may
be responsible for depressed protective function of the endogenous
apelin-APJ system. Modulation of the apelin-APJ system may hold
promise for the treatment of Dox-induced cardiotoxicity.
H932
EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
cardiovascular diseases. Taken together, we hypothesized that
the apelin-APJ system may play a role in the pathogenesis of
Dox-induced cardiotoxicity and the development of subsequent heart failure.
To test our hypothesis, we created a mouse model of Doxinduced acute cardiotoxicity and analyzed gene expression. We
also assessed protein expressions of apelin and APJ and evaluated the effects of APJ gene deletion on this model. Finally,
we examined the effect of apelin on Dox-induced cell toxicity
in vitro.
MATERIALS AND METHODS
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Experimental animals. Male C57Bl/6J and Balb/cA mice were
purchased from CLEA Japan, (Tokyo, Japan). APJ knockout mice
were generated as previously described (12). In the present study, we
used APJ knockout mice from the F9 generation (backcrossed to
C57Bl/6J) to equalize the effect of genetic background. Cross-fertilization between APJ heterozgous mice (APJ⫹/⫺ ⫹ APJ⫹/⫺ mice) did
not produce the expected Mendelian genotype distribution, and few
APJ homozygotes were obtained (data not shown). Cross-fertilization
between APJ-deficient mice (APJ⫺/⫺ ⫹ APJ⫺/⫺ mice) produced few
offspring due to intrauterine and postnatal deaths. Nurturing offspring
by genetic maternal and Balb/cA mice together partially improved the
breeding rate. With both types of cross-fertilization, surviving APJ⫺/⫺
mice grew into adults after weaning and exhibited a normal physical
appearance. Although we did not examine the cause of low APJ⫺/⫺
breeding rates, recent reports (4, 17) have suggested that embryonic
deaths of APJ knockout mice are caused by impaired cardiovascular
development.
Animal experiments were carried out in a humane manner with
approval by the Institutional Animal Experiment Committee of the
University of Tsukuba. Experiments were performed in accordance
with the Regulation of Animal Experiments of the University of
Tsukuba and Fundamental Guidelines for Proper Conduct of Animal
Experiments and Related Activities in Academic Research Institutions
under jurisdiction of the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
Genotyping of APJ knockout mice was performed by PCR using
genomic DNA from tail biopsies of 2 mm. The tail genome was
extracted with KAPA Express Extract, and multiplex allele-specific
PCR was carried out with KAPA2G Robust HotStart ReadyMix with
dye (Kapa Biosystems, Woburn, MA) according to the manufacturer’s
protocol using the following primers: a common forward primer
(5=-untranslated region of the APJ gene, 5=-TGGAGGGTGCAAGGAAGTGTCTTTAGCTGAGC-3=), two reverse primers specific for the wild-type (WT) allele (APJ coding DNA sequences,
5=-CGTAGTCGCATTCAGACTGGTTGTCAGCCC-3=), and the
knockout allele (nuclear localization signal-lacZ cassette, 5=CTCAGGAAGATCGCACTCCAGCCAGCTTTC-3=). The genotype
was determined by the PCR product size as follows: 317 bp for the
WT allele and 592 bp for the knockout allele (data not shown).
Intraperitoneal injection of Dox. Experiments were performed
using 8- to 12-wk-old male mice. Dox hydrochloride (D1515, SigmaAldrich, St. Louis, MO) was resolved in saline (Otsuka Pharmaceutical, Tokushima, Japan) at 3 or 4 mg/ml concentrations and then
administered by a single intraperitoneal injection of 5 ml/kg mouse
body wt (Dox dose: 15 or 20 mg/kg body wt). In experiments of gene
expression analysis and apelin measurements using C57Bl/6J mice,
Dox was given to mice at a dose of 20 mg/kg body wt. In experiments
using APJ knockout mice, we reduced the dose to 15 mg/kg body wt,
because most animals died between days 5 and 7 after Dox injection
at the dose of 20 mg/kg body wt. This dose allowed us enough time
to analyze survival.
Echocardiographic assessment and survival analysis. On day 5
after the administration of either Dox or saline as vehicle, mice were
anesthetized with 1% isoflurane in O2, and chest hair was removed
with a depilatory cream. Using a digital ultrasound system Vevo 2100
equipped with a MS550D Microscan transducer with a center frequency of 40 MHz (VisualSonics, Toronto, ON, Canada), M-mode
images of the LV short axis at the papillary muscle level were
captured with monitoring heart rate. LV fractional shortening (FS)
was calculated as follows: FS ⫽ [(LV interior diameter at diastole ⫺
LV interior diameter at systole)/LV interior diameter at diastole] ⫻
100. For survival analysis of the mice given Dox, we recorded 14-day
survival times and performed necropsies on dead mice.
Plasma and tissue preparation. At times of interest (24 h, day 5,
and day 14) after Dox injection, blood was collected from the inferior
vena cava into 0.5 M EDTA (pH 8.0, final concentration: 7.5–10 mM)
or heparin (final concentration: 15–20 U/ml) under inhalational anesthesia with isoflurane. Blood samples were rapidly centrifuged at 800
g for 15 min at 4°C, and the obtained plasma samples were frozen in
liquid nitrogen and stored at ⫺80°C. Hearts were then excised,
immersed in PBS, and allowed to keep beating for a few seconds to
remove remaining blood. After heart weight was measured, the ratio
of heart weight to body weight was determined for each animal. For
RNA and protein analysis, hearts were immediately transferred into
liquid nitrogen and then stored at ⫺80°C. For immunofluorescent
analysis, hearts were embedded in OCT compound (Tissue-Tek,
Torance, CA) and then frozen in 2-methylbutane prechilled with
liquid nitrogen. For histomorphological analysis, beating hearts were
arrested with 50 mM KCl and PBS, fixed in 4% paraformaldehyde for
⬃24 h at 4°C, and then processed and embedded in paraffin.
RNA isolation and quantitative real-time RT-PCR. Frozen hearts
were ground into a fine powder using a Multi-Beads Shocker (Yasui
Kikai, Osaka, Japan). A subset of frozen tissue powder was homogenized in Isogen (Nippongene, Tokyo, Japan), and total RNA was
isolated according to the manufacturer’s protocol. After treatment
with DNase I and ethanol precipitation with ethachinmate (Nippongene), total RNA was heated at 65°C for 10 min and immediately
chilled on ice to denature its secondary structure. Total RNA was then
reverse transcribed using ReverTra Ace (TRT-101, Toyobo, Osaka,
Japan) and random hexamer (Takara Bio, Shiga, Japan). The reaction
was carried out as follows: 30°C for 10 min, 42°C for 90 min, and
99°C for 5 min. The obtained sample containing an equivalent of 100
ng cDNA was used in a 20-␮l volume for RT-PCR containing 10 ␮l
of 2⫻ SYBR Premix Ex Taq (Perfect Real Time, RR081A, Takara
Bio) and 0.2 ␮M forward and reverse primers. The primers used for
amplification of target cDNAs are shown in Table 1. Two-step
amplification and calculation of the threshold cycle (Ct) value by the
second derivative maximum method were performed in duplicate
using the Thermal Cycler Dice Real Time System (Takara Bio).
Expression levels of target genes were corrected for GAPDH expression levels using the ⌬⌬Ct method. The amplification efficiency of
primers for each target gene was confirmed to be equal using serial
dilutions of cDNA.
Apelin-12 enzyme immunoassay. A subset of frozen tissue powder
was homogenized in 10 eye-measured volumes of 0.1 M acetate on
ice. Next, the homogenate was boiled at 100°C for 10 min and rapidly
cooled on ice. After centrifugation at 17,800 g for 10 min at 4°C, the
clear supernatant was obtained, and its protein concentration was
determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories,
Hercules, CA) according to the manufacturer’s protocol. The supernatant was evaporated to dryness and dissolved with assay buffer from
the apelin-12 enzyme immunoassay (EIA) kit (EK-057-23, Phoenix
Pharmaceuticals, Burlingame, CA). The obtained crude peptide extract (150 ␮g equivalent of protein) was applied onto the apelin-12
EIA plate in duplicate. To measure plasma apelin levels, 8- or 16-fold
diluted plasma/EDTA samples with assay buffer were directly applied
onto the EIA plates in duplicate. The assay was performed according
to an EIA protocol recommended by the manufacturer with a slight
modification as follows: the first incubation step for the competitive
immunoreaction between the sample and biotinylated apelin-12 was
EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
H933
Table 1. Primers used for quantitative real-time relative PCR analysis
Accession Number
Forward Primer
Reverse Primer
Product Size, bp
GAPDH
APJ
Apelin
angiotensinogen
ANG II type 1a receptor
ANG II type 1b receptor
Endothelin type A receptor
Endothelin type B receptor
Preproendothelin-1
␤1-Adrenergic receptor
␤2-Adrenergic receptor
␤-Myosin heavy chain
Brain natriuretic peptide
Atrial natriuretic peptide
Light chain 3B
p62
Beclin-1
NM_008084.2
NM_011784.3
NM_013912.3
NM_007428.3
NM_177322
NM_175086
NM_010332.2
NM_007904
NM_010104
NM_007419.2
NM_007420.3
NM_080728.2
NM_008726
NM_008725
NM_026160.4
NM_011018.2
NM_019584.3
5=-TCACTGGCATGGCCTTCC-3=
5=-CGCCAGGCCATTCTCAAAG-3=
5=-CAGGCCTATTCCCAGGCTCA-3=
5=-AAGCTGCCCACCCTTTTG-3=
5=-ACTCACAGCAACCCTCCAAG-3=
5=-CAGTTTCAACCTCTACGCCAGT-3=
5=-GCTGGTTCCCTCTTCACTTAAGC-3=
5=-TCAGAAAACAGCCTTCATGC-3=
5=-CCTGGACATCATCTGGGTC-3=
5=-GCTGATCTGGTCATGGGATT-3=
5=-GCCCCTGGTGGTGATGGTCTTTG-3=
5=-AACCAGACGGCACTGAAGAG-3=
5=-GGGCTGTAACGCACTGAAG-3=
5=-GGTAGGATTGACAGGATTGGAG-3=
5=-AAGTGATCGTCGCCGGAGT-3=
5=-AGACCCCTCACAGGAAGGAC-3=
5=-CCGGGAAGTAGCTGAAGACC-3=
5=-CAGGCGGCACGTCAGATC-3=
5=-AGCTGCCTCTGCTGCTATCTGTC-3=
5=-CAAGATCAAGGGCGCAGTCA-3=
5=-TGTTCCTCCTCTCCTGCTTTG-3=
5=-TCAGACACTGTTCAAAATGCAC-3=
5=-GGGTGGACAATGGCTAGGTA-3=
5=-TCATGGTTGCCAGGTTAATGC-3=
5=-GCGGCAAGCAGAAGTAGAA-3=
5=-TGTGGCCTTATTGGGAAG-3=
5=-AAGTCCAGAGCTCGCAGAAG-3=
5=-TCCGCCCATCCTGCTCCACC-3=
5=-TGCCCACTTTGACTCTAGGATG-3=
5=-ACTTCAAAGGTGGTCCCAGAG-3=
5=-GCAGAATCGACTGCCTTTTC-3=
5=-CCGGACATCTTCCACTCTTTGT-3=
5=-CATCTGGGAGAGGGACTCAA-3=
5=-GACGCCTTAGACCCCTCCAT-3=
65
99
116
119
61
75
129
77
109
100
131
102
109
97
113
103
141
carried out at 4°C overnight. This process improved the accuracy and
reproducibility of the apelin-12 EIA in our laboratory.
Histology. Paraffin sections (5 ␮m thickness) of the short axis of
mouse hearts at the papillary muscle level were prepared using a
Microtome HM 340 E (Microm, Walldorf, Germany). Deparaffinized
sections were stained with hematoxylin and eosin, and images were
obtained using a SZ61 microscope, a BX53 microscope, and DP21
digital camera (Olympus, Tokyo, Japan).
Creatine kinase-MB assay. Creatine kinase (CK)-MB activities in
heparinized plasma samples were measured with a Fuji Dri-Chem
7000Vz (Fujifilm, Tokyo, Japan) according to the manufacturer’s
protocol.
Protein carbonylation assay and Western blot analysis. A portion
of frozen heart powder was homogenized in RIPA buffer containing
20 mM Tris (pH 7.8), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, 1 mM sodium
vanadate, 10 mM sodium fluoride, 20 mM ␤-glycerophosphate, 1 mM
PMSF, 1 ␮g/ml leupeptin, and 2.1 ␮g/ml aprotinin. After centrifugation at 17,800 g for 15 min at 4°C, the supernatant was obtained, and
its protein concentration was determined as described above. Protein
samples were mixed with Laemmuli sample buffer containing 100
mM DTT and treated at 100°C for 3 min. Fifty or one hundred
micrograms of the protein sample were subjected to SDS-PAGE and
transferred to an Immobilon-P Transfer Membrane (Millipore, Bedford, MA). Protein carbonylation was then detected using the Protein
Carbonyls Western Blot Detection Kit (Shima Laboratories, Tokyo,
Japan) according to the manufacturer’s protocol. For Western blot
analysis, membranes were blocked with skim milk and then blotted
with the following primary antibodies: anti-light chain (LC)3 antibody
(1:1,000, rabbit polyclonal, MBL, Woburn, MA), anti-p62 (1:1,000,
rabbit monoclonal, EPITOMICS, Burlingame, CA), anti-beclin-1 (1:
1,000, rabbit polyclonal, H-300, Santa Cruz Biotechnology, Santa
Cruz, CA), and anti-␣-tubulin (1:5,000, mouse monoclonal, clone
B-5-1-2, Sigma-Aldrich). After incubation, blots were incubated with
the corresponding secondary antibodies conjugated to horseradish
peroxidase. Visualization via chemiluminescent detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL). Data images were
captured by LAS-3000 (Fujifilm), and densitometric analysis was
performed using Multi Gauge (version) 3.0 software (Fujifilm).
Immunofluorescent staining. Fresh frozen tissue sections (10 ␮m
thickness) of mouse hearts were fixed with 4% paraformaldehyde for
10 min followed by a wash with PBS. Sections were then blocked
with 5% BSA and PBS for 30 min. After endogenous biotin blockade
using an endogenous avidin-biotin blocking kit (Nichirei, Tokyo,
Japan) according to the manufacturer’s protocol, sections were incu-
bated overnight with anti-APJ antibody that we had previously generated (1:10, rabbit polyclonal) (11). For doublestaining, sections
were blocked with MOM Blocking Reagent (Vector laboratories,
Burlingame, CA) according to the manufacturer’s protocol and incubated for 1 h with anti-dystrophin (1:100, mouse monoclonal, clone
1808, Abcam, Cambridge, UK). Primary antibody binding was detected with biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), Alexa fluor 488-conjugated streptavidin (Jackson ImmunoResearch Laboratories), and Alexa fluor 568-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad,
CA). Deparaffinized sections (5 ␮m thickness) of mouse hearts were
heated to boiling in a microwave oven for 10 min in antigen retrieval
buffer (10 mM sodium citrate buffer, pH 6.0). Sections were then
blocked with 0.25% gelatin, 0.5% Triton X-100, and 50 mM Tris·HCl
(pH 7.6) for 30 min. After endogenous biotin blockade using an
endogenous avidin-biotin blocking kit (Nichirei) according to the
manufacturer’s protocol, sections were incubated overnight with the
following primary antibodies: anti-p62 (1:200, rabbit monoclonal,
EPITOMICS) and anti-ubiquitin (1:200, rabbit polyclonal, Dako,
Carpinteria, CA). Primary antibody binding was detected with biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories) and Alexa fluor 488-conjugated streptavidin (Jackson ImmunoResearch Laboratories). For membrane and nuclear staining, CF
640R wheat germ agglutinin (Biotium, Hayward, CA) and Hoechst
33258 were used, respectively. Fluorescence was visualized using a
Fluoview FV10i confocal laser-scanning microscope (Olympus).
TUNEL assay. A TUNEL assay was performed on paraffin sections
(5 ␮m thickness) of mouse hearts using an in situ Cell Death
Detection Kit (Roche Diagnostics, Mannheim, Germany) according to
the manufacturer’s protocol. For nuclear counterstaining, sections
were subsequently stained with Hoechst 33258. Fluorescence was
visualized, and the percentage of TUNEL-positive cells in five randomly selected LV wall areas was determined using a BIOREVO
BZ-9000 (Keyence, Osaka, Japan).
Cell culture and generation of stable cell lines. H9c2 (2-1) rat
cardiomyoblasts were obtained from DS Pharma Biomedical (Osaka,
Japan). Cells were grown in DMEM supplemented with 10% heatinactivated FBS, 100 U/ml penicillin, and 100 ␮g/ml streptomycin at
37°C (5% CO2 and 95% humidity). To establish transformants stably
expressing human APJ, cells were transfected with the coding region
of the human APJ gene with pcDNA3.1/Zeo (⫹) (11) using Genejuice
(Novagen, Madison, WI). Stable transformants (“H9c2/hAPJ”) were
selected and propagated with 200 ng/ml zeocin (Invitrogen). H9c2
cells stably transfected with pcDNA3.1/Zeo (⫹) (“H9c2/mock”) were
used as a control.
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Gene Name
H934
EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
RESULTS
Dox-induced downregulation of cardiac apelin and APJ
mRNA expression in mice. Because Dox has been known to
acutely alter the expression of multiple genes, we first
performed a quantitative real-time relative PCR analysis of
hormones, receptors, and cardiac stress markers in mouse
hearts on days 1 and 5 after the administration of Dox (20
mg/kg body wt). Figure 1, A and B, shows that both apelin
and APJ mRNA drastically declined on days 1 and 5 after
Dox injection. In contrast, mRNAs of angiotensinogen and
␤-myosin heavy chain (known as a cardiac stress marker)
were increased on day 1 and further strengthened on day 5
(Fig. 1, C and D). mRNA of brain natriuretic peptide (also
Fig. 1. Gene expressions in mouse hearts after doxorubicin (Dox) injection. C57Bl/6J mice were treated with saline or Dox (20 mg/kg, single intraperitoneal
injection), and hearts were then subjected to quantitative real-time relative PCR analysis. The graphs (A–L) show relative expression levels of each gene in salineand Dox-treated groups. Note that apelin (A) and APJ (B) mRNA showed significant decreases in the Dox-treated group compared with the saline-treated group
on days 1 and 5. This observation was different from changes in other genes (C–L). C: angiotensinogen (Agt); D: ␤-myosin heavy chain (␤-MHC); E: brain
natriuretic peptide (BNP); F: endothelin (ET) type A (ETA) receptor; G: ANG II type 1a (AT1a) receptor; H: ET type B (ETB) receptor; I: AT1b receptor; J:
preproET-1 (ppET-1); K: ␤1-adrenergic receptor (␤-AR1); L: ␤2-adrenergic receptor (␤-AR2). Each bar represents mean ⫾ SE for 5 mice/group in all cases. *P ⬍
0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. the corresponding saline-treated group.
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Cell viability assay. Cells were plated at 1,000 cells/well into
poly-D-lysine-coated 96-multiwell plates and allowed to adhere overnight in growth medium at 37°C (5% CO2 and 95% humidity). Cells
were starved for 12 h in DMEM supplemented with 0.5% FBS
followed by drug treatment. Cells were incubated with pE-apelin-13
for 1 h before exposure to Dox. After Dox treatment at 1 ␮M for 24 h,
cell viability was determined by measurement of ATP levels using a
CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison,
WI). Sample luminescence was measured on a Wallac ARVOSX
Multilabel Counter (Perkin-Elmer, Tokyo, Japan).
Statistics. All data are presented as means ⫾ SE. Statistical analysis
was performed using Prism 5 (version 5.0a, GraphPAD Software, San
Diego, CA). Unpaired data were compared by Student’s t-test,
Welch’s t-test, nonparametric Mann-Whitney’s U-test, or one-way
ANOVA with a Bonferroni test. Survival rates were compared by a
log rank (Mantel-Cox) test.
EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
Fig. 2. Plasma apelin contents and protein expressions of apelin and APJ in the
heart after Dox injection. C57Bl/6J mice were treated with saline or Dox (20
mg/kg, single intraperitoneal injection), and plasma and hearts were then
subjected to apelin measurements. The graphs (A and B) show apelin peptide
contents in saline- and Dox-treated groups. Plasma apelin levels were reduced
on day 5 after Dox injection (A). Apelin contents of hearts significantly
declined on day 1 but increased to basal levels on day 5 after Dox injection (B).
Each bar represents means ⫾ SE for 5– 8 mice/group. *P ⬍ 0.05 and ***P ⬍
0.001 vs. the corresponding saline-treated group. C: representative immunofluorescence images of APJ. Top, APJ distribution. Middle, merged images
(APJ: green; cardiac membrane: red; and nuclear: cyan). Scale bar ⫽ 20 ␮m.
Bottom, magnified views of corresponding squares from the top and middle
images. A patchy distribution of APJ on the cardiac membrane was observed
in saline-treated APJ⫹/⫹ mice (arrowheads) but not in Dox-treated APJ⫹/⫹ and
APJ⫺/⫺ [knockout (KO)] mice.
the Dox-treated group of APJ⫺/⫺ mice was even lower than
that in the Dox-treated group of APJ⫹/⫹ mice, but these groups
showed a similar decreasing ratio in FS (average percent
decrease: 33.74 ⫾ 3.50%, mean ⫾ SE).
To determine whether the Dox-induced FS reduction in
APJ⫺/⫺ mice was caused at least in part by lack of the APJ
gene or not, we also measured FS in APJ⫹/⫺ mice. The
saline-treated group of APJ⫹/⫺ mice showed normal FS, similar to the saline-treated group of APJ⫹/⫹ mice, but the Dox-
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known as a cardiac stress marker) was significantly reduced
on day 1 but was increased on day 5 (Fig. 1E). mRNA of the
endothelin (ET) type A receptor showed small increases on
days 1 and 5 after Dox injection (Fig. 1F). mRNA of the
AT1a receptor was slightly decreased on day 1 but was
increased to the same level as the control group on day 5
(Fig. 1G). mRNA of the ET type B receptor was not altered
on day 1 but fractionally increased on day 5 after Dox
injection (Fig. 1H). Besides the above, mRNA levels of the
AT1b receptor, preproET-1, ␤1-adrenergic receptor, and ␤2adrenergic receptor were unaffected at the observed time
points (Fig. 1, I–L). Taken together, these results show that
Dox administration causes changes in the expression of
cardiac genes and, in particular, downregulates mRNA expression of apelin and APJ.
Changes in plasma and cardiac tissue levels of apelin and
suppression of APJ expression in Dox-treated mice. We examined plasma and cardiac tissue apelin contents and found that
plasma apelin levels were clearly diminished on day 5 after
Dox injection (Fig. 2A). Cardiac apelin peptide levels were
significantly reduced on day 1 but were increased to the same
levels as the control group on day 5 after Dox injection (Fig.
2B). Next, we performed an immunofluorescent staining of
heart tissues on day 5 after Dox injection (Fig. 2C). In the
saline-treated group, immunoreactivity against anti-APJ antibody was observed in association with cardiomyocytes (dystrophin positive) in a patchy fashion. In contrast, in Doxtreated and knockout groups, most of immunoreactivity against
anti-APJ antibody disappeared. These data suggest that Dox
administration impacts on contents of systemic and cardiac
apelin and suppresses APJ expression in mouse hearts.
Effects of APJ deletion on Dox-induced cardiac contractile
dysfunction in mice. To evaluate the potential involvement of
the apelin-APJ system in Dox-induced cardiotoxicity, we examined the effects of APJ gene deletion on Dox-induced
cardiac dysfunction in mice. It has been well established that
Dox administration impairs cardiac function in rodent models,
and we therefore measured factional shortening by echocardiography on day 5 after Dox injection (15 mg/kg body wt).
First, we confirmed that lowering of the Dox dose to 15 mg/kg
body wt had a similar effect as high-dose Dox (20 mg/kg body
wt) on mRNA levels of apelin and APJ in hearts. The administration of Dox (15 mg/kg body wt) resulted in persistent
downregulation of mRNA expression of apelin and APJ in
APJ⫹/⫹ mouse hearts at all observed time points (Fig. 3, A and
B). In contrast, in APJ⫹/⫺ mouse hearts, the apelin mRNA
level was significantly reduced on day 1 but showed an
increasing tendency to the same level as the saline-treated
group through day 14 from day 5 after Dox administration
(Fig. 3A). In addition, the APJ mRNA level in APJ⫹/⫺ mouse
hearts was significantly reduced on days 1 and 5 but showed an
increasing tendency to the same level as the saline-treated
group on day 14 after Dox administration (Fig. 3B). We also
observed a significant decrease in the apelin mRNA level in
APJ⫺/⫺ mouse hearts on day 5 after Dox injection (Fig. 3A).
The Dox-treated group of APJ⫹/⫹ mice showed a significant
decrease in FS compared with the saline-treated group of
APJ⫹/⫹ mice (average percent decrease: 36.38 ⫾ 4.45%, mean ⫾
SE; Fig. 3, C and D). On the other hand, FS in the salinetreated group of APJ⫺/⫺ mice was substantially lower than that
in the saline-treated group of APJ⫹/⫹ mice. Furthermore, FS in
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EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
treated group of APJ⫹/⫺ mice suffered from a marked decline
in FS to almost the same level as the Dox-treated group of
APJ⫺/⫺ mice (average percent decrease: 51.95 ⫾ 2.65%, mean ⫾
SE; Fig. 3, C and D). It was therefore hypothesized that FS
in the Dox-treated groups of APJ⫺/⫺ and APJ⫹/⫺ mice had
bottomed out. All these changes in FS were caused by
increases in LV internal diameter at systole, not diastole
(Table 2), indicating systolic dysfunction. Heart rate, body
weight, and heart weight were reduced to similar levels among
all Dox-treated groups, but heart weight-to-body weight ratios
were not affected in either saline- or Dox-treated groups (Table
2). These findings indicate that a deficiency in the apelin-APJ
system can underlie Dox-induced cardiac contractile dysfunction in mice.
Effects of APJ deletion on survival of Dox-treated mice. To
investigate the effect of APJ gene deletion on the prognosis
after Dox treatment, we analyzed survival rates of APJ⫹/⫹,
APJ⫹/⫺, and APJ⫺/⫺ mice. Fourteen-day survival rates did not
differ between APJ⫹/⫹ and APJ⫹/⫺ mice but were markedly
decreased in APJ⫺/⫺ mice (Fig. 3E and Table 3). In most dead
mice, there were visible signs related to heart failure at necropsy, such as pleural effusion and ascite retention. These
findings led us to consider that Dox-administered mice died
mainly from acute heart failure and that APJ⫺/⫺ mice were
susceptible to Dox-induced heart failure. Therefore, it is suggested that a defect of the apelin-APJ system can facilitate the
development of Dox-induced heart failure in mice associated
with its cardiotoxicity. Hence, we used APJ⫺/⫺ mice to inves-
Table 2. Echocardiographic assessment of LV function and other parameters
Saline
LV internal diameter at diastole, mm
LV internal diameter at systole, mm
Fractional shortening, %
Heart rate, beats/min
Heart weight, mg
Body weight, g
Heart weight/body weight, mg/g
Doxorubicin
APJ⫹/⫹
APJ⫹/⫺
APJ⫺/⫺
APJ⫹/⫹
APJ⫹/⫺
APJ⫺/⫺
3.558 ⫾ 0.2699
1.908 ⫾ 0.1888
46.7 ⫾ 3.5
452.1 ⫾ 63.55
115.0 ⫾ 6.481
24.48 ⫾ 1.092
4.698 ⫾ 0.2343
3.629 ⫾ 0.1951
1.917 ⫾ 0.2151
47.3 ⫾ 3.9
436.6 ⫾ 61.14
115.5 ⫾ 6.704
24.71 ⫾ 1.066
4.683 ⫾ 0.3502
4.012 ⫾ 0.3617c
2.640 ⫾ 0.3098d
34.2 ⫾ 3.9d
425.4 ⫾ 65.50
117.1 ⫾ 10.22
23.90 ⫾ 1.590
4.914 ⫾ 0.5216
3.566 ⫾ 0.3966
2.522 ⫾ 0.4910b
29.7 ⫾ 7.2b
277.1 ⫾ 66.98b
93.46 ⫾ 7.734b
20.52 ⫾ 1.561b
4.576 ⫾ 0.5046
3.738 ⫾ 0.2934
2.895 ⫾ 0.3415b,e
22.7 ⫾ 4.3b,f
229.9 ⫾ 36.09b
91.14 ⫾ 6.661b
20.75 ⫾ 1.037b
4.396 ⫾ 0.3154
3.458 ⫾ 0.3720a
2.677 ⫾ 0.3455
22.7 ⫾ 3.4b,e
256.7 ⫾ 43.16b
93.36 ⫾ 8.530b
20.50 ⫾ 1.044b
4.558 ⫾ 0.3945
Values are expressed as means ⫾ SE for 8 –12 mice/group in each genotype. LV, left ventricular. aP ⬍ 0.05 and bP ⬍ 0.001 vs. the corresponding saline-treated
group; cP ⬍ 0.05 and dP ⬍ 0.001 vs. the saline-treated group of APJ⫹/⫹ mice; eP ⬍ 0.05 and fP ⬍ 0.01 vs. the doxorubicin-treated group of APJ⫹/⫹ mice.
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Fig. 3. Expression analysis of apelin and APJ mRNA, echocardiographic assessment of left ventricular (LV) function, and survival rate in APJ KO mice. APJ
KO mice were treated with saline or Dox (15 mg/kg, single intraperitoneal injection), and hearts were then subjected to quantitative real-time relative PCR
analysis. The graphs show relative expression levels of apelin (A) and APJ (B) mRNAs in saline- and Dox-treated groups. Each bar represents means ⫾ SE for
5– 6 mice/group in all cases. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. the corresponding saline-treated group. LV function was assessed by
echocardiography on day 5, and 14-day survival rates after Dox administration were analyzed. Representative echocardiographic images (C) and quantified data
of fractional shortening (FS; D) are shown. The saline-treated group of APJ⫺/⫺ mice exhibited a substantial decline in FS compared with saline-treated groups
of APJ⫹/⫹ and APJ⫹/⫺ mice. Dox-treated groups of APJ⫹/⫺ and APJ⫺/⫺ mice showed a severe deterioration in FS compared with APJ⫹/⫹ mice. Values are
expressed as means ⫾ SE for 8 –10 mice/group in each genotype. ***P ⬍ 0.001 vs. the corresponding saline-treated group; ###P ⬍ 0.001 vs. the saline-treated
group of APJ⫹/⫹ mice; †P ⬍ 0.05 and ††P ⬍ 0.01 vs. the Dox-treated group of APJ⫹/⫹ mice. E: comparison of survival curves showed the significant
susceptibility of APJ⫺/⫺ mice () to Dox exposure (n ⫽ 12). ***P ⬍ 0.001 vs. APJ⫹/⫹ mice (, n ⫽ 41) and APJ⫹/⫺ mice (Œ, n ⫽ 41).
EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
Table 3. Survival rate analysis and necropsy findings of
doxorubicin-treated mice
⫹/⫹
APJ
APJ⫹/⫺
APJ⫺/⫺
Survival (Alive/Total)
Pleural Effusions
Ascites
26/41
29/41
3/12
12/15
11/12
8/9
8/15
6/12
5/9
treated group of APJ⫹/⫹ mice and was even higher in the
Dox-treated group of APJ⫺/⫺ mice (Fig. 4C). Dox-induced
cardiotoxicity is well characterized by oxidative damage, including lipid peroxidation and protein carbonylation (6). We
therefore examined carbonyl formation in heart proteins of
APJ⫹/⫹ and APJ⫺/⫺ mice on day 5 after Dox administration.
Protein carbonylation levels were similar among saline- and
Dox-treated groups of APJ⫹/⫹ mice but were significantly
elevated in the Dox-treated group of APJ⫺/⫺ mice (Fig. 4, D
and E). These results suggest that a compromised apelin-APJ
system can aggravate Dox-induced cardiac damage in mice.
Effects of APJ gene deletion on autophagy in mouse hearts.
Recent reports (18, 21, 26) have suggested that Dox triggers
cardiac autophagy and apoptosis. To assess the effect of APJ
gene deletion on Dox-induced myocardial autophagy, we performed Western blot analysis for autophagy-related proteins in
mouse hearts on day 5 after treatment (Fig. 5A). The LC3-IIto-LC3-I ratio, an established autophagy index, was significantly increased in the Dox-treated group of APJ⫺/⫺ mice. In
addition, p62 protein, an adaptor protein targeting ubiquitinated proteins to autophagosomes and also a selective au-
Fig. 4. Aggravation of Dox-induced cardiac damages in
APJ⫺/⫺ mouse hearts. Tissue damage of hearts from
APJ KO mice on day 5 after Dox injection (15 mg/kg)
was analyzed. A and B: representative photomicrographs of hematoxylin and eosin-stained transverse
heart sections. A: saline-treated groups of APJ⫹/⫹ and
APJ⫺/⫺ mice showed normal cardiac morphology, but
Dox-treated groups of APJ⫹/⫹ and APJ⫺/⫺ mice appeared have cardiac atrophy. Scale bar ⫽ 2 mm. B: at
the LV myocardial area, Dox-treated groups of APJ⫹/⫹
and APJ⫺/⫺ mice showed myofibrillar loss (arrowheads). Scale bar ⫽ 50 ␮m. Sections were analyzed for
3– 4 mice/group. C: creatine kinase (CK)-MB activity in
plasma of APJ KO mice was measured. APJ gene
deletion promoted the Dox-induced elevation of plasma
CK-MB activity. Values are expressed as means ⫾ SE
for 10 –12 mice/group in each genotype. ***P ⬍ 0.001
vs. the corresponding saline-treated group; †P ⬍ 0.05
vs. the Dox-treated group of APJ⫹/⫹ mice. D: protein
carbonyl formation was detected by Western blot analysis (top), and loaded protein samples on membranes
were confirmed to be equal by CBB stain (bottom).
Carbonylated BSA was loaded as a positive control
(PC). Molecular weight (MW) markers are indicated in
kilodaltons on the left. E: quantification analysis of pixel
number of the respective lanes showed that APJ gene
deletion significantly increased the Dox-induced carbonylation of cardiac proteins. AU, arbitrary units. Values are expressed as means ⫾ SE for 3 mice/group in all
cases. ***P ⬍ 0.001 vs. the saline-treated group of
APJ⫹/⫹ mice; ###P ⬍ 0.001 vs. the corresponding
saline-treated group; ††P ⬍ 0.01 vs. the Dox-treated
group of APJ⫹/⫹ mice.
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tigate the effects of loss of the apelin-APJ system on Doxinduced cardiotoxicity.
Effects of APJ deletion on Dox-induced cardiac damage in
mice. We observed histological sections stained with hematoxylin and eosin from hearts of APJ knockout mice on day 5 after
Dox administration. Saline-treated groups of APJ⫹/⫹ and
APJ⫺/⫺ mice showed normal cardiac morphology and myofibril architecture, but Dox-treated groups of APJ⫹/⫹ and
APJ⫺/⫺ mice underwent atrophy with local deterioration of
myofibrils (Fig. 4, A and B). To assess Dox-induced cardiac
damage quantitatively, we measured plasma CK-MB activity,
the most specific biomarker for myocardial necrosis. CK-MB
activity in plasma was significantly increased in the Dox-
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EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
tophagy substrate, was also significantly increased in the Doxtreated group of APJ⫺/⫺ mice. Protein expression levels of
beclin-1, required for autophagosome formation, were not
changed in any group. To determine whether p62 protein and
ubiquitinated proteins had accumulated in cardiomocytes, we
carried out immunofluorescent staining. We found that p62positive and ubiquitin-positive aggresome-like structures appeared in cardiomyocytes, in much higher numbers in the
Dox-treated group of APJ⫺/⫺ mice compared with the Doxtreated group of APJ⫹/⫹ mice, whereas saline-treated groups
showed no such structures (Fig. 5B). In Dox-treated groups,
mRNA levels of LC3B and p62 were significantly increased to
a similar extent compared with the saline-treated group of
APJ⫹/⫹ mice, suggesting that Dox administration showed the
same upregulatory effect on expression levels of LC3B and
p62 in these mice (Fig. 5C). Levels of beclin-1 mRNA did not
change significantly in any group. As reported in a previous
study (18), Dox-induced myocardial accumulation of LC3 and
p62 proteins indicates a possible impairment of autophagic
degradation. Thus, our results indicate that Dox-induced cardiotoxicity can be accompanied by the activation of myocardial
autophagy in the presence or absence of the APJ gene, but that
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Fig. 5. Excessive activation of autophagy in Dox-treated APJ⫺/⫺ mouse hearts. Myocardial autophagy in APJ KO mice on day 5 after Dox injection (15 mg/kg)
was analyzed. A: Western blot analysis of whole heart homogenates and quantification analysis of respective bands were performed. The graphs showed that
levels of light chain (LC)3 and p62 were significantly increased in the Dox-treated group of APJ⫺/⫺ mice, but levels of beclin-1 were not changed significantly
in any group. Values are normalized with levels of ␣-tubulin and expressed as means ⫾ SE for 3 mice/group in all cases. *P ⬍ 0.05 vs. the saline-treated group
of APJ⫹/⫹ mice; #P ⬍ 0.05 vs. the corresponding saline-treated group; †P ⬍ 0.05 and ††P ⬍ 0.01 vs. the Dox-treated group of APJ⫹/⫹ mice. B: representative
immunofluorescence images of myocardial p62 and ubiquitin (membrane: red, nuclear: cyan). Scale bar ⫽ 20 ␮m. Marked accumulation of p62 (top) and
ubiquitin (bottom)-positive aggregates (green) in cardiomyocytes was observed in APJ⫺/⫺ mice. Quantitative relative PCR analysis showed that mRNA levels
of LC3 and p62 were significantly increased in Dox-treated groups compared with saline-treated APJ⫹/⫹ mice. WGA, wheat germ agglutinin. C: beclin-1 mRNA
levels were not affected in any group. Values are expressed as means ⫾ SE for 5– 6 mice/group in all cases. *P ⬍ 0.05 vs. the saline-treated group of APJ⫹/⫹
mice. D: representative TUNEL-stained images (TUNEL-positive nuclei: red, arrowheads; nuclei: blue; left) and quantified data of percentages of TUNELpositive cells (right). Scale bar ⫽ 100 ␮m. TUNEL-positive nuclei were significantly increased in Dox-treated groups, but there was no significant difference
in each genotype. Values are expressed as means ⫾ SE for 4 mice/group in each genotype. ***P ⬍ 0.001 vs. the corresponding saline-treated group.
EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
DISCUSSION
In the present study, we demonstrated that Dox administration decreased the expression of apelin and APJ mRNA in
mouse hearts followed by a significant reduction of APJ at the
protein level. Genetic deletion of APJ in mice exacerbated
Dox-induced cardiotoxicity, including LV contractile dysfunction, myocardial necrosis, oxidative damage, and impaired
autophagy. Furthermore, genetic deletion of APJ in mice increased the mortality from heart failure after Dox administra-
Fig. 6. Attenuation of Dox-induced cell toxicity by activation of the apelin-APJ
system in H9c2 cells. The graph shows percent cell viability of H9c2/mock and
H9c2/hAPJ cells treated with 1 ␮M Dox for 24 h in the presence or absence
of pE-apelin-13. After serum starvation, cells were treated with Dox at 1 ␮M
for 24 h. Cell viability was determined by measurement of ATP levels.
Pretreatment with pE-apelin-13 for 1 h before exposure of Dox did not affect
the Dox-induced decrease in the viability of H9c2/mock cells but attenuated
the Dox-induced decrease in the viability of H9c2/hAPJ cells in a dosedependent manner. Each bar represents means ⫾ SE of four independent
experiments.
tion. We also found that apelin treatment attenuated the Doxinduced cell toxicity in vitro. These results suggest that the
apelin-APJ system is one of potential targets of Dox and that its
dysfunction is associated with the onset or progression of heart
failure in mice.
Dox has potentially serious cardiotoxic side effects that limit
its use in cancer chemotherapy, and the mechanisms underlying this toxicity have been studied for a number of years.
Genetic approaches and pharmacological blockade have provided evidence that several humoral factors, including ANG II
and ET-1, are involved in Dox-induced cardiotoxicity. Genetic
deletion of AT1a receptor and administration of an ANG II
receptor blocker attenuated Dox-induced cardiac dysfunction
independent of blood pressure control, suggesting that ANG II
could act as a positive mediator of Dox-induced cardiotoxicity
(33). Recently, counterregulatory actions of the apelin-APJ
system against the ANG II-AT1 receptor system have been
examined. Downregulation of apelin and APJ mRNA expression was observed in failing hearts of Dahl salt-sensitive and
ANG II-infused rats, and this was reversed by administration
of an ANG II receptor blocker (13). Apelin antagonized ANG
II-mediated vasoconstriction and vascular remodeling in an
NO-dependent fashion and also inhibited ANG II-induced
intracellular signal transduction (8, 27). These actions may
result from a possible heterodimer formation between APJ and
AT1 receptor. In the present study, we observed upregulation
of angiotensinogen mRNA expression in mouse hearts after
Dox administration (Fig. 1C), and thus we consider that Dox
may exert potential adverse cardiac effects via disruption of
possible links between the apelin-APJ system and Ang II-AT1
receptor system. On the other hand, it has also been reported
that preproET-1 mRNA and ET-1 were increased in mouse
hearts after Dox administration and that the ET receptor antagonist bosentan suppressed Dox-induced cardiotoxicity (3).
However, we observed no effect on preproET-1 mRNA expression in mouse hearts after mice received the same dose of
Dox (Fig. 1J). This discrepancy might be due to differences in
the environment and mouse strains. Our study collectively
shows that Dox treatment induces the downregulation of apelin
and APJ mRNA expression in mouse hearts.
During the last decade, there has been growing evidence that
the apelin-APJ system is involved in modulating cardiac function in animal models. Consistent with a previous study (4), we
also observed that APJ knockout mice showed a significant
decline in basal cardiac contractility regardless of age (data not
shown), suggesting that the apelin-APJ system is an important
component in cardiac contraction. Furthermore, the apelin-APJ
system has been shown to protect hearts from oxidative stress
and cardiac cell death under conditions of ischemia-reperfusion
(37). With regard to Dox-induced cardiac oxidative stress,
there are several possible sources of Dox-related ROS generation. It has been suggested that a redox cycle of Dox in
cardiac mitochondria could generate superoxide anion as a
byproduct (9) and that an iron-Dox complex could produce
highly reactive hydroxyl free radical (36). Moreover, a recent
report (38) has shown that myocardial activation of the ANG
II-AT1 receptor-NADPH oxidase 2 pathway was involved in
Dox-induced cardiac oxidative stress. Therefore, it has been
speculated that Dox-induced downregulation of the apelin-APJ
system may lead to susceptibility to cardiac oxidative stress. In
fact, we have provided genetic evidence that loss of APJ in
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APJ gene deletion can accelerate dysfunctional changes in the
autophagic degradation pathway after Dox treatment. Furthermore, we performed a TUNEL assay on heart sections. On day
5 after Dox administration, TUNEL-positive cells were increased in mouse hearts of each genotype, but no significant
difference was found among Dox-treated groups (Fig. 5D),
indicating that APJ gene deletion did not affect Dox-induced
cardiac apoptotic cell death.
Effects of APJ overexpression and apelin treatment on
Dox-induced cell death in vitro. We next evaluated a potential
role of the apelin-APJ system in cardioprotection from Doxinduced cardiotoxicity in vitro. We verified the abundant APJ
mRNA expression in neonatal mouse hearts, but it was suppressed in vitro primary cultured neonatal mouse cardiac myocytes when prepared (data not shown). Therefore, we used
immortalized cardiac myoblasts from embryonic rat H9c2 cells
as a commonly used model for the study on Dox toxicity. We
also detected little expression of APJ in H9c2 cells and thus
established the transformants stably expressing human APJ.
The cell viability of control H9c2/mock cells with 1 ␮M Dox
for 24 h was remarkably reduced in the presence or absence of
pE-apelin-13. In contrast, the Dox-induced decrease in cell
viability of H9c2/hAPJ cells was attenuated with pE-apelin-13
in a dose-dependent manner (Fig. 6). Interestingly, overexpression of APJ showed a tolerance to Dox-induced cardiotoxicity
in the absence of pE-apelin-13. Taken together, these results
indicate that the apelin-APJ system has preventive effect
against Dox-induced cell death.
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EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
when cardiac APJ is expressed above certain thresholds. Furthermore, our data using cultured cell lines indicated that
apelin did mitigate Dox-induced cytotoxicity.
Because of the broad tissue distribution of Dox, it can affect
a variety of tissues, and its toxicity is not confined to the heart
(1, 34), which may be responsible in part for the increased
mortality of Dox-treated APJ⫺/⫺ mice. In addition to susceptibility to Dox-induced cardiotoxicity, the apelin-APJ system
may have some further aspects of sensitivity against Doxinduced systemic toxicity, including nephrotoxicity and hepatotoxicity. At this time, it still remains to be unsettled whether
the basal FS reduction in APJ⫺/⫺ mice can affect on Doxinduced cardiotoxicity. On this point, the use of APJ⫹/⫺ and/or
APJ-overexpressing mice treated with apelin-13 will have a
great advantage for further study, which is required to elucidate
the pathophysiological relevance of the apelin-APJ system to
Dox-induced cardiotoxicity.
Based on our observations in vivo and in vitro, we concluded that Dox-induced cardiotoxicity is associated with a risk
of impairment of the apelin-APJ system in the heart, which is
responsible, at least in part, for the aggravation of Dox-induced
cardiotoxicity. Although the acute model used in this study
differed from the actual clinical case in Dox chemotherapy, our
work generates important findings in the field of Dox-induced
cardiotoxicity. Modulating of apelin-APJ signaling and the
expression of apelin and APJ without a simultaneous reduction
in Dox’s antitumor efficacy may improve the outcomes of
cancer patients receiving Dox chemotherapy.
ACKNOWLEDGMENTS
The authors thank members of the Fukamizu Laboratory for the helpful
discussions and encouragement. The authors also thank Dr. H. Kidoya and
Prof. N. Takakura for general support. This article is dedicated to the memory
of Prof. Alejandro M. Bertorello, who kindly discussed this APJ project with
the authors, in Karolinska Institutet, Stockholm, Sweden.
GRANTS
This work was supported by a Grant-In-Aid for Young Scientists (B) (to J.
Hamada) and a Grant-In-Aid for Scientific Research (B) (C) (to J. Ishida) from
the Ministry of Education, Culture, Sports, Science and Technology of Japan.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: J.H., J.I., and A.F. conception and design of research;
J.H., A.B., and N.O. performed experiments; J.H., A.B., N.O., and K.M.
analyzed data; J.H., A.B., N.O., K.M., K.K., J.I., and A.F. interpreted results
of experiments; J.H. prepared figures; J.H., J.I., and A.F. drafted manuscript;
J.H., J.I., and A.F. edited and revised manuscript; J.H., A.B., N.O., K.M., K.K.,
J.I., and A.F. approved final version of manuscript.
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mice led to marked enhancement of Dox-induced carbonyl
formation in cardiac proteins, cardiac necrosis, cardiac dysfunction, and mortality. Taken together, these observations
suggest a possible protective role of the endogenous apelinAPJ system in cardiac oxidative stress and the onset or progression of heart failure secondary to Dox-induced cardiotoxicity in mice.
Cardiac autophagy serves an essential role in not only
maintenance of cardiac homeostasis but also in the clearance of
damaged organelles and aggregated proteins under pathological conditions such as cardiac ischemia-reperfusion injury and
heart hypertrophy (22, 35). Recently, several animal studies
have reported the possible involvement of autophagy in Doxinduced cardiotoxicity. It has been suggested that Dox can
promote cardiac autophagy to protect the heart from Doxrelated toxicity (26). However, another study (21) has suggested that Dox-induced excessive autophagy causes cardiomyocyte cell death. In addition, it has also been reported that
Dox impairs cardiac autophagosome formation and its degradation, resulting in cardiac dysfunction (18). Although it has
not yet been completely elucidated whether autophagy has
beneficial or causal effects on Dox-induced cardiotoxicity, our
study suggests that downregulation of the apelin-APJ system
by Dox treatment may progress Dox-induced cardiotoxicity
through a functional disturbance of the autophagic degradation
pathway. More recently, it has been reported that apelin treatment can attenuate glucose deprivation-induced excessive autophagy in rat cardiomyocytes (16). Excess ROS activate
autophagy by producing damages to mitochondria and can
cause necrosis due to ATP depletion (22). Therefore, the
endogenous apelin-APJ system may play a suppressive role in
cardiac oxidative stress, and Dox-induced downregulation of
the apelin-APJ system may consequently accelerate cardiac
autophagic cell death and necrosis.
Since there is no effective means of preventing or treating
Dox-induced cardiotoxicity, we figured out a part of the mechanisms underlying this toxicity, which involves the breakdown
of the apelin-APJ system. In the present study, despite the
reduced apelin mRNA levels, apelin contents in the heart were
increased to basal levels on day 5 after Dox injection. The
discrepancy between apelin expression and its peptide contents
may be attributed to the suppression of the number of APJ
molecules available for apelin binding, resulting an apparent
recovery in apelin contents. At this moment, neither clear
preventive effects nor improvement have been observed in our
experimental model involving systemic coadministration of
apelin-13 (data not shown), which can be explained by concomitant downregulation of APJ expression. Correspondingly,
we observed notable adverse effects in APJ knockout mice,
suggesting that the degree of APJ loss can impact the pathophysiological changes in mouse hearts after Dox administration.
An important issue raised by our findings is why APJ⫺/⫺
mice showed higher Dox-induced mortality rates regardless of
similar levels of cardiac dysfunction compared with APJ⫹/⫺
mice. In this regard, we found that mRNA levels of apelin and
APJ in APJ⫹/⫺ mouse hearts were restored to basal levels on
day 14 after Dox injection (Fig. 3, A and B), suggesting a
functional recovery of the apelin-APJ system. Accordingly, we
consider that the apelin-APJ system has the potential to delay
the progression of heart failure arising from Dox treatment
EFFECTS OF APJ GENE DELETION ON DOX-INDUCED CARDITOXICITY
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