Leptin protects H9c2 rat cardiomyocytes from
H
2
O
2
-induced apoptosis
Megumi Eguchi*, Yuantao Liu*, Eyun-Jung Shin and Gary Sweeney
Department of Biology, York University, Toronto, Canada
The rapid increase in the prevalence of obesity to epi-
demic proportions is a serious concern, as obesity is
associated with the development of many complications
including type 2 diabetes, hypertension and heart failure
[1]. Heart failure is a leading cause of mortality in indus-
trialized countries, and is accompanied by progressive
left ventricular remodeling characterized by hypertro-
phy of the myocytes, impaired vascularization in the
heart, abnormal extracellular matrix composition (fibro-
sis) and elevated cardiomyocyte cell death [2]. However,
in addition to increasing the risk for initial myocardial
infarction, obesity may confer protective effects that
limit cardiac remodeling post-infarction, the so-called
obesity paradox [3]. Necrosis was initially viewed as the
major pathway by which cardiomyocytes are lost during
remodeling; however, research in the past 10–15 years
has indicated that apoptosis has important pathophysio-
logical consequences in the development and progres-
sion of heart failure [4,5]. Indeed, the apoptotic rate is
significantly increased (from 0.001% to 0.08%) in the
failing heart [2].
Adipokines, collectively referring to factors derived
from adipose tissue, have attracted tremendous

infarction. Exposing H9c2 cells to H
2
O
2
decreased cell viability, and this
was attenuated by pretreating cells with leptin for 1 h, but not 24 h. Leptin
also attenuated the ability of H
2
O
2
to increase phosphatidylserine exposure
and annexin V binding. Further investigation of underlying mechanisms of
leptin’s protective effect demonstrated that the H
2
O
2
-induced decrease in
mitochondrial membrane potential (Y) leading to cytochrome c release was
attenuated by leptin pretreatment, and this was associated with reduced
translocation of the pro-apoptotic Bax protein to the mitochondrial mem-
brane. Finally, leptin prevented H
2
O
2
-induced increases in caspase-3 cleav-
age and activity, although again 24 h leptin pretreatment did not confer
significant protection. In summary, we have demonstrated that acute leptin
pretreatment mediates anti-apoptotic effects in H9c2 rat cardiomyocytes,
which may be of significance in clarifying the direct impact of leptin on the
heart.

O
2
-induced
cell death in H9c2 cells, the most appropriate in vitro
model of cardiomyocytes currently available. This
was accomplished by analyses of apoptotis [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT) assay and annexin V binding], together with
investigation of the mechanistic role played by the
intrinsic pathway of apoptosis (change in mitochon-
drial membrane potential (Y), cytochrome c release
and caspase-3 activity).
Results
Leptin treatment for 1 h but not 24 h protects
H9c2 cells from H
2
O
2
-induced decreases in cell
viability
The effect of H
2
O
2
treatment on the cell viability of
H9c2 cells was measured by the uptake and reduction
of MTT to an insoluble formazan dye. H
2
O
2

after as little as 2 h H
2
O
2
treatment. No increase in
propidium iodide staining was apparent under these
conditions, but was seen in positive control experi-
ments (data not shown), indicating that the cell
death was predominantly due to apoptosis. Quanti-
tative assessment of fluorescence (Fig. 2C) showed
that 1 h leptin pretreatment significantly attenuated
the level of annexin V binding detected in response
to H
2
O
2
. Although apparently decreasing the effects
of H
2
O
2
, 24 h leptin pretreatment did not have a
significant effect.
Leptin pretreatment attenuates H
2
O
2
-induced
loss of mitochondrial membrane potential
The mitochondrial membrane potential (Y) is a critical

O
2
(400 lM) for 5 h, and cell viability was measured using the MTT
assay. Data represent mean ± SEM (n = 4). The asterisk indicates
a statistically significant difference from H
2
O
2
treatment alone
(P < 0.05).
M. Eguchi et al. Regulation of cardiomyocyte apoptosis by leptin
FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS 3137
activates downstream caspases to cause apoptosis
[20,21]. 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimi-
dazoyl carbocyanide iodide (JC-1) accumulates as
aggregates in the normal hyperpolarized mitochondria,
resulting in red fluorescence, but JC-1 exists in the
monomeric form in apoptotic cells and stains cells
green. Here we observed that untreated control cells
exhibit numerous brightly stained mitochondria that
emit red fluorescence (Fig. 3). Cells treated with H
2
O
2
exhibited fewer red JC-1 aggregates, and more green
fluorescence of monomers appeared in the cytoplasm,
indicating dissipation of the mitochondrial membrane
potential. Leptin pretreatment attenuated these H
2
O

integrates into the membrane [22]. In viable control
cells, or those treated with leptin, little or no Bax
immunofluorescence was observed (Fig. 5). However,
when cells were exposed to H
2
O
2
, we observed pro-
nounced staining for Bax, with a maximal effect after
4 h, and this was clearly attenuated in cells pretreated
with leptin for 1 h (Fig. 5).
A
B
C
Fig. 2. H
2
O
2
-induced annexin V binding to
the cell surface decreases with leptin
pretreatment. Phosphatidylserine externali-
zation was assessed via annexin V binding
in the absence (A) or presence (B) of 2 h
H
2
O
2
(400 lM) treatment with or without
leptin pretreatment (6 n
M, 1 h or 24 h). Cells

O
2
treatment increased gener-
ation of the cleaved form of caspase-3, and this was
attenuated by 1 h leptin pretreatment (Fig. 6A). The
levels of cleaved caspase-3 correlated well with enzy-
matic activity, which was increased 1.8-fold compared
to control upon H
2
O
2
treatment. This effect of H
2
O
2
was again significantly reduced by leptin 1 h pretreat-
ment, but not significantly by 24 h pretreatment
(Fig. 6B). In order to determine the functional conse-
quences of the above findings, we examined whether
the protective effect of leptin on cell viability was
observed after a prolonged time period subsequent to
H
2
O
2
exposure. When the number of living cells, as
determined by trypan blue exclusion, was counted
three days after exposure to H
2
O

tosis, have been used on many occasions as a model
system to study regulation of cardiomyocyte cell death
[27–29]. Here we used this model system to show the
effects of short-term (1 h) and long-term (24 h) exposure
of H9c2 cells to leptin on H
2
O
2
-induced cell death. Our
results indicate that 1 h pretreatment with leptin is able
to significantly decrease the apoptotic effects of H
2
O
2
on H9c2 cells and thus protect them from death. How-
ever, when 24 h preincubation was used, a protective
effect was not observed. This is not entirely without
precedent, as we have previously shown that acute and
chronic leptin treatments have distinct effects on insulin
signaling and subsequent regulation of glucose uptake
in skeletal muscle cells [30,31]. These results suggest that
transient intracellular effects stimulated by acute leptin
treatment play an important role in the cardioprotective
role of leptin, and that the enhanced lipid accumulation
found after 24 h treatment with leptin [18] may convey
deleterious effects [32,33]. The effects observed after a
short period of leptin exposure may be of physiological
relevance given the fact that circulating leptin levels
fluctuate with diurnal rhythm and are not consistently
high for 24 h [34].

M) pretreatment were exposed to 0.4 lM H
2
O
2
for
30 min. JC-1 fluorescence was measured by confocal microscopy,
assessing the emission shift from green (530 nm) to red (590 nm)
using 488 nm excitation. Composite red and green fluorescence is
shown. Results are representative of those from three separate
experiments.
M. Eguchi et al. Regulation of cardiomyocyte apoptosis by leptin
FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS 3139
major components of the intrinsic pathway of apopto-
sis. Notably, the mechanism whereby leptin prevents
activation of the intrinsic pathway of apoptosis
appears to involve prevention of the H
2
O
2
-induced
change in the cellular localization and activity level of
the pro-apoptotic Bax protein [36] detected by immu-
nofluorescence microscopy using a conformation-sensi-
tive antibody [22]. Accordingly, attenuation of a
decrease in mitochondrial membrane potential, and of
the subsequently increased cytochrome c release and
caspase-3 activation was also observed in cells pre-
treated with leptin.
The theory of selective leptin resistance occurring in
obese individuals has been suggested based upon

fluorescence can be seen independently. (A)
Control, (B) leptin treatment for 1 h, (C)
H
2
O
2
treatment, (D) H
2
O
2
treatment with
1 h leptin pretreatment. Images shown
are representative of four independent
experiments.
Fig. 5. Leptin attenuates H
2
O
2
-induced exposure of the Bax N-ter-
minus. Immunofluorescence staining (green) of Bax using Bax
N-terminal (N20) antibody, which only detects Bax localized in mito-
chondrial membrane. The results are for cells after 4 h exposure to
H
2
O
2
(400 lM) with or without leptin pretreatment (6 nM, 1 h).
Images are representative of three independent experiments.
Regulation of cardiomyocyte apoptosis by leptin M. Eguchi et al.
3140 FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS

appears to be of growing importance.
Experimental procedures
Culture of H9c2 rat cardiomyocytes
The rat embryonic ventricular myocardial cell line H9c2
was maintained as described previously [39] in DMEM with
4.5 gÆL
)1
glucose supplemented with 10% (v ⁄ v) fetal bovine
serum and 1% penicillin ⁄ streptomycin (v ⁄ v). Cells were
routinely grown to 80% confluence in 75 cm
2
flasks at
37 °C with an atmosphere of 5% CO
2
prior to passage and
seeding for experiments. All cell-culture materials were pur-
chased from Wisent (Quebec, Canada). For the induction
of cell death, cells were exposed to H
2
O
2
(400 lm, Sigma-
Aldrich, St Louis, MO, USA) for various time periods as
indicated following treatment with leptin (6 nm). We
analyzed ObR expression in these cells by PCR, and found
expression of both long (ObRb) and short (ObRa) receptor
isoforms (data not shown).
Determination of cell viability
The MTT assay was performed as described previously [40]
as a measure of cell viability. In addition, trypan blue

using DAKO fluorescent mounting medium (DakoCytoma-
tion, Missisauga, Canada). Annexin V Alexa Fluor 488 was
H
2
O
2
Leptin (24h)
+
+
+
+
–
–
– –
Cleaved
caspase 3
To ta l
caspase 3
Leptin (1h)
H
2
O
2
+
+
+
+
–
–
– –

2
-induced cleavage and activa-
tion of caspase-3 are reduced in leptin pre-
treated cells. (A) Representative western
blots of cell lysates prepared after H
2
O
2
treatment (400 lM, 4 h) with or without lep-
tin pretreatment (6 n
M, 1 h or 24 h). Levels
of the cleaved form of caspase-3
(17 ⁄ 19 kDa) as well as changes in total cas-
pase-3 levels (35 kDa) were analysed by
western blotting. (B) Quantitative analysis of
the activity of caspase-3 measured using a
specific caspase-3 activity assay kit (mean-
s ± SEM, n = 3). The asterisk indicates a
statistically significant difference from H
2
O
2
treatment alone (P < 0.05).
M. Eguchi et al. Regulation of cardiomyocyte apoptosis by leptin
FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS 3141
excited at 495 nm, and the fluorescence of cells was deter-
mined using a confocal microscope (Olympus Fluoview
Center Valley, PA, USA). Quantification was performed by
analyzing the fluorescence intensity per cell, and the data
shown are means ± SEM of all experiments, in which two

potential (Y) using JC-1
H9c2 cells were grown on cover-slips, treated as indicated
in Fig. 3, and then washed twice with NaCl ⁄ P
i
. The cells
were incubated with 5 lm JC-1 dye (Molecular Probes) in
serum-free medium for 15 min at 37 °C. The medium was
then removed, and the cells were washed three times with
NaCl ⁄ P
i
. The cells were examined immediately under a con-
focal microscope. JC-1 fluorescence was measured to assess
the emission shift from green (530 nm) to red (590 nm) in
polarized mitochondria at 488 nm excitation.
Immunofluorescent detection of intracellular
cytochrome c localization by confocal microscopy
To detect cytochrome c release from the mitochondria, cells
grown on cover slips were first treated to stain mitochondria
by incubation for 10 min at room temperature with 10 nm
MitoTracker CMTMRos dye (Molecular Probes) in
NaCl ⁄ P
i
. Cells were fixed with 4% paraformaldehyde for
15 min, permeabilized with 0.2% Triton X-100 for 5 min,
and blocked using 3% serum dissolved in NaCl ⁄ P
i
for
30 min at room temperature. Cells were then probed
with monoclonal anti-cytochrome c IgG (BD Biosciences
Pharmingen, Oakville, Canada; 1 : 250 dilution in blocking

2% v ⁄ v SDS, 15% v ⁄ v glycerol 10% v ⁄ v 2-mercaptoethanol,
0.2 mm phenylmethanesulfonyl fluoride, 10 lgÆmL
)1
leupep-
tin, 1 mm pepstatin A, 0.5 mm Na
3
VO
4
, 0.2 mm E64, 2 mm
okadoic acid, a few grains of bromophenol blue). Centri-
fugation at 1500 g was used to precipitate floating cells,
which were collected and lysed with the cells growing in
culture dish. Each lysate was collected and transferred to
Eppendorf tubes, which were heated to 65 °C for 15 min,
and the cells were further lysed by passing five times through
a 25-gauge needle ⁄ syringe. After centrifuging each sample at
12 000 g for 2 min at 4 °C, 35 lL aliquots were loaded onto
a 10% SDS–PAGE gel. After protein transfer to poly(vinyli-
dene difluoride) membrane, the membrane was incubated
with the primary caspase-3 antibody solution (1 : 1000, Cell
Signaling Technology, Beverly, MA, USA) at 4 °C overnight.
The antibody detects both total (35 kDa) and cleaved
(17 ⁄ 19 kDa) forms of caspase-3. Then the membrane was
incubated in horseradish peroxidase-linked secondary anti-
body solution (1 : 10 000) for 1 h and analyzed by enhanced
chemilunenescence. The b-actin content was routinely
checked to confirm the accuracy of protein loading on gels
(data not shown). Quantification of band intensity upon wes-
tern blotting was conducted using nih image software
(National Institutes of Health, Bethesda, MD, USA).

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