Exposing H9c2 cells to H2O2 decreased cell viability, and this was attenuated by pretreating cells with leptin for 1 h, but not 24 h.. Further investigation of underlying mechanisms of l
Trang 1H2O2-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 research interest in recent years as an important mech-anistic link between obesity and various associated complications [6] The circulating adipokine profile is altered in obese individuals, and it is now clear that development of heart failure can be directly influenced
by adipokines [1] Leptin is the product of the obese (ob) gene, and its main function is to control appetite and energy expenditure by acting on the hypothalamus [7] There is a positive correlation between circulating leptin concentration and the body mass of an indiv-idual This suggests the existence of leptin resistance in
Keywords
apoptosis; caspase; heart failure; leptin;
mitochondria
Correspondence
G Sweeney, Department of Biology, York
University, Toronto, M3J 1P3 Canada
Fax: +1 416 736 5698
Tel: +1 416 736 2100 ext 66635
E-mail: gsweeney@yorku.ca
*These authors contributed equally to this
work
(Received 23 January 2008, revised 25
March 2008, accepted 14 April 2008)
doi:10.1111/j.1742-4658.2008.06465.x
Obesity is a known risk factor for induction of myocardial infarction, but, paradoxically, may also confer a protective effect against subsequent remodeling leading to heart failure In this study, we investigated the effect
of leptin, the product of the obese (ob) gene, on cardiomyocyte apoptosis,
a well-characterized component of cardiac remodeling after myocardial infarction Exposing H9c2 cells to H2O2 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 H2O2to increase phosphatidylserine exposure and annexin V binding Further investigation of underlying mechanisms of leptin’s protective effect demonstrated that the H2O2-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 H2O2-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
Abbreviations
JC-1, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazoyl carbocyanide iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PS, phosphatidylserine.
Trang 2the hypothalamus of such individuals; however,
whether the heart is leptin-resistant is still controversial
[8,9] Leptin’s action is mediated by six isoforms of
leptin receptors [10] These receptors can be classified
as secreted (Ob-Re), short (Ob-Ra, c, d and f) and
long (Ob-Rb) forms The adult heart has been shown
to express both long and short ObR isoforms, but
pre-dominantly short forms of the receptor [11], and it has
also been shown that the heart is a site of leptin
production [12] The local expression of leptin and
its receptors in the heart further suggests that leptin
can potentially affect cardiac function by directly
acting on the heart, and this has been confirmed by
several recent studies [13–15]
Exposure of cardiomyocytes to H2O2and other
reac-tive oxygen species is increased in the heart, especially
after short ischemia⁄ reperfusion, and excessive
oxida-tive stress contributes to the pathogenesis of heart
fail-ure [16] A high circulating leptin concentration is seen
in the majority of obese individuals In this study, we
have investigated the effects of leptin on H2O2-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 H2O2-induced decreases in cell
viability
The effect of H2O2 treatment on the cell viability of
H9c2 cells was measured by the uptake and reduction
of MTT to an insoluble formazan dye H2O2treatment
for 5 h significantly reduced the cell viability as
expected, and this effect was attenuated upon 1 h
pre-incubation with 6 nm leptin but not 24 h
pre-incuba-tion (Fig 1) The dose of leptin was chosen based on
preliminary experiments, previous work by ourselves
and others in vitro [17,18], and because it is relevant to
the circulating levels observed in obesity [19]
H2O2-induced phosphatidylserine exposure is
decreased by leptin treatment
Appearance of phosphatidylserine (PS) in the outer
leaflet of the phospholipid bilayer without disrupted
integrity of the membrane is one of the earliest char-acteristics of apoptotic cells In order to study the effect of leptin on H2O2-induced PS exposure, cells were incubated with appropriate treatments as indi-cated, and analyzed for the degree of annexin V binding to the surface of intact cells (Fig 2A,B) Cells were counterstained with propidium iodide to allow distinction between apoptosis and necrosis Leptin treatment alone did not affect PS exposure, but an increase in annexin V binding was observed after as little as 2 h H2O2 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 H2O2 Although apparently decreasing the effects
of H2O2, 24 h leptin pretreatment did not have a significant effect
Leptin pretreatment attenuates H2O2-induced loss of mitochondrial membrane potential The mitochondrial membrane potential (Y) is a critical factor in maintaining the integrity of mitochondria and subsequent regulation of apoptosis Loss of mito-chondrial membrane potential will lead to release of the cytochrome c from mitochondria, which in turn
Leptin (h)
0 0.2 0.4 0.6 0.8 1 1.2
*
Fig 1 Leptin pretreatment for 1 h but not 24 h attenuates the abil-ity of H 2 O 2 to decrease cell viability H9c2 cells were treated with
or without 6 n M leptin for 1 h or 24 h prior to exposure to H 2 O 2 (400 l M ) 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).
Trang 3activates 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 H2O2
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 H2O2
-induced changes (Fig 3)
Leptin pretreatment reduces cytochrome
c release from mitochondria
Release of cytochrome c from mitochondria is a
criti-cal step in progression of the intrinsic apoptotic
path-way [20,21] H2O2 treatment for 2 h increased the
release of cytochrome c from mitochondria, as can be
seen by the loss of co-localization of cytochrome c and mitochondria (Fig 4) The effect of H2O2 was again attenuated by preincubation with leptin for 1 h (Fig 4) Co-localization was unaffected in cells treated with leptin alone
The mechanism whereby leptin attenuates the intrinsic pathway of apoptosis involves reduced Bax integration in the mitochondrial membrane
To assess translocation of the apoptotic Bax pro-tein to the mitochondrial membrane, we utilized an approach exploiting the observation that the N-termi-nal domain is only exposed and recognized by a spe-cific antibody when this protein translocates and 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 H2O2, 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 H2O2-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 l M ) treatment with or without leptin pretreatment (6 n M , 1 h or 24 h) Cells were treated to allow detection of both ann-exin V (green) and propidium iodide (red), and images representative of those obtained for at least eight independent experiments are shown for each condition The results from all experiments (n > 3) were quanti-fied, and (C) shows the mean fluorescence (±SEM) The asterisk indicates a significant difference compared with H 2 O 2 alone (P < 0.05).
Trang 4H2O2-induced increases in caspase-3 cleavage
and activity are attenuated by leptin
pretreatment
Caspase-3 is an executioner of apoptosis, and is
involved in many important events that lead to the
completion of apoptosis [23] Cleavage of caspase-3 is
indicative of activation, and in cells treated with leptin
alone there was no change in the cleavage of caspase-3
compared to control H2O2 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 H2O2 treatment This effect of H2O2
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
H2O2 exposure When the number of living cells, as
determined by trypan blue exclusion, was counted
three days after exposure to H2O2, over 2.2-fold more
cells were viable when pretreated with leptin for 1 h as
opposed to exposure to H2O2alone (data not shown)
Discussion
There has been great interest in the relationship between circulating leptin levels and the development
of cardiovascular diseases, but the precise role of leptin
is still controversial [24] Hyperleptinemia, which is commonly seen in obese individuals, has been pro-posed to play a role in the development of various car-diovascular diseases [25,26] Heart failure is a common end-stage event resulting from various cardiovascular diseases, and it is now well established that cardiomyo-cyte apoptosis is an important component of cardiac remodeling, ultimately leading to heart failure An excellent recent study suggested that leptin can prevent the increased levels of apoptosis observed upon ageing
in ob⁄ ob mice [13] However, the direct effect of leptin
on cardiomyocyte apoptosis and the intracellular mechanisms involved are still unclear
H9c2 cells, together with use of H2O2to induce apop-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 H2O2-induced cell death Our results indicate that 1 h pretreatment with leptin is able
to significantly decrease the apoptotic effects of H2O2
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]
We have shown here that leptin’s cardioprotective effect against H2O2-induced apoptosis occurs through the prevention of activation of apoptotic markers at
an early stage, including PS exposure to the outer membrane – one of the first detectable signs of apopto-sis [35] The lack of significant propidium iodide stain-ing in our annexin V bindstain-ing studies suggests that 2 h treatment with H2O2 does not induce significant necro-sis in these cells Furthermore, upon investigation of the mechanisms underlying H2O2-induced apoptosis and their regulation by leptin, we observed changes in
Fig 3 Leptin attenuates H2O2-induced mitochondrial membrane
potential loss in H9c2 cells Quiescent H9c2 cells with or without
1 h leptin (6 n M ) pretreatment were exposed to 0.4 l M H2O2 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.
Trang 5major 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 H2O2-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 observations that, while the effects of leptin on satiety and energy metabolism were blunted, the sympatho-excitatory effects were maintained in obese individuals [9,37] Whether enhanced or suppressed myocardial leptin action, either direct or centrally mediated, exists pre- and post-myocardial infarction in obese
Merged
A
B
C
D
Fig 4 Leptin decreases H 2 O 2 -induced cyto-chrome c release from mitochondria Confo-cal analysis of H9c2 cells treated with or without leptin (6 n M , 1 h) prior to exposure
to H2O2(400 l M ) for 2 h shows immuno-staining of cytochrome c (green), Mitotrac-ker staining of mitochondria (red), and merged images of the two showing co-local-ization in yellow Upon release of cyto-chrome c from mitochondria, green 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 l M ) with or without leptin pretreatment (6 n M , 1 h).
Images are representative of three independent experiments.
Trang 6individuals is still a matter of some debate Our study
clearly indicates a direct, as opposed to systemic or
centrally mediated, role for leptin in mediating
cardio-myocyte apoptosis, and reinforces data from in vivo
studies suggesting a cardioprotective role for leptin via
mediation of anti-apoptotic effects As mentioned
above, leptin- or leptin receptor-deficient rodents
dis-play an increased rate of cardiac apoptosis The
increase in apoptotic rate and mortality was abolished
upon leptin injection in ob⁄ ob mice but not db ⁄ db
mice, indicating that leptin plays an important role in
cardioprotection [13] Furthermore, it has recently
been shown that perfusion of the heart with leptin
dur-ing a short reperfusion period (35 min) significantly
decreased mitochondrial membrane pore opening and
the infarct size induced by ischemia⁄ reperfusion [38]
In summary, our current in vitro study suggests that
leptin exerts a protective effect against H2O2-induced
apoptosis in H9c2 rat cardiomyocytes by preventing
activation of components of the
mitochondrial-depen-dent intrinsic pathway of apoptosis This is in keeping
with other recent data [13], butt the effect mediated by
leptin in vivo may depend on the development of leptin
resistance, the stage in progression of heart failure or
other variables Overall, the direct influence of leptin
on cardiac structure and function is still uncertain, but
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)1glucose supplemented with 10% (v⁄ v) fetal bovine
serum and 1% penicillin⁄ streptomycin (v ⁄ v) Cells were routinely grown to 80% confluence in 75 cm2 flasks at
37C with an atmosphere of 5% CO2prior 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 H2O2 (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 exclusion was used in some experiments, and the number of trypan blue-negative cells was counted using a hemocytom-eter 3 days after the end of H2O2treatment
Annexin V binding assay Annexin V Alexa Fluor 488 (Molecular Probes, Eugene,
OR, USA) was used to detect PS exposure to the outer sur-face of the cell membrane according to the manufacturer’s protocol Briefly, cells were grown in a 12-well plate with cover slips in each well They were treated with H2O2 follow-ing incubation with leptin Then the cells were washed with cold NaCl⁄ Piand 1· binding buffer (10 mm Hepes pH 7.4,
140 mm NaCl, 2.5 mm CaCl2) Cells were then incubated with annexin V Alexa Fluor 488 (1 : 20 dilution) and
1 lgÆmL)1propidium iodide diluted in 1· binding buffer for
15 min After incubation, cells were washed twice in 1· bind-ing buffer before mountbind-ing the cover slips on glass slides using DAKO fluorescent mounting medium (DakoCytoma-tion, Missisauga, Canada) Annexin V Alexa Fluor 488 was
H2O2 Leptin (24h) +
+ + + – –
– –
Cleaved caspase 3 Total caspase 3
Leptin (1h)
H2O2 + + + + – – – –
Cleaved caspase 3 Total caspase 3
0 0.5 1 1.5 2 2.5
H2O2
Leptin (h)
+ + + –
0 – –
0 1 24
24
1
Caspase 3 activity (f
A
B
Fig 6 H 2 O 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 l M , 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 a(mean-steri(mean-sk indicate(mean-s a
statistically significant difference from H 2 O 2
treatment alone (P < 0.05).
Trang 7excited 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
cover slips were used per condition and nine fields of view
from each cover slip were quantified
Immunofluorescent detection of conformational
changes in Bax (N-terminal exposure) by confocal
microscopy
For analysis of Bax immunofluorescence, cells grown on
cover slips were washed twice with NaCl⁄ Pi, fixed with 4%
paraformaldehyde in NaCl⁄ Pi for 15 min, permeabilized
with 0.2% Triton X-100 for 5 min and blocked using 3%
BSA in NaCl⁄ Pi for 1 h at room temperature Cells were
then incubated for 1 h at 37C with anti-Bax N-terminal
IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA;
1 : 150) in blocking buffer The unique feature of this assay
is that the N-terminal epitope is not detected when Bax is
retained in the cytosol, but is exposed and detected upon
Bax insertion into the mitochondrial membrane [22] The
cells were washed three times in NaCl⁄ Pi, and incubated
for 1 h at room temperature in anti-rabbit IgG Alexa
Fluor 488 serum (Molecular Probes; 1 : 2000) After
wash-ing, cells were mounted using DAKO mounting medium
and confocal images were analysed as above
Measurement of mitochondrial membrane
potential (Y) using JC-1
H9c2 cells were grown on cover-slips, treated as indicated
in Fig 3, and then washed twice with NaCl⁄ Pi The cells
were incubated with 5 lm JC-1 dye (Molecular Probes) in
serum-free medium for 15 min at 37C The medium was
then removed, and the cells were washed three times with
NaCl⁄ Pi 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⁄ Pi 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⁄ Pi 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 solution) for 1 h at room temperature, followed by staining with goat anti-mouse Alexa Fluor 488 (Molecular Probes;
1 : 1000) for 1 h at room temperature After washing, cells were mounted using DAKO mounting medium, and ana-lyzed by confocal microscopy
Caspase-3 activity assay Caspase-3 activity was measured using an Apo-ONE homo-geneous caspase-3 assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol Briefly, cells grown on 96-well plates were treated with H2O2 with or without leptin pretreatment After exposure to H2O2, Apo-ONE caspase-3⁄ 7 reagent was added, and the mixture incubated at room temperature for up to 18 h The level of fluorescence was measured using a Wallac 1420 Victor 3 apparatus (Perkin Elmer, Waltham, MA, USA) with excita-tion⁄ emission at 499 ⁄ 521 nm
Immunoblotting for total and cleaved forms of caspase-3
After appropriate treatment of cells, they were washed in NaCl⁄ Piand lysed using lysis buffer (0.5 m Tris⁄ HCl pH 6.8, 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 Na3VO4, 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 65C 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 4C, 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 4C 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)
Statistical analysis All data presented are expressed as means ± SEM Sta-tistical analysis was undertaken using Student’s t-test
Trang 8Differences between groups were considered significant at
P< 0.05
Acknowledgements
Funding for this work was provided by the Canadian
Institutes of Health Research (CIHR) via an operating
grant and a New Investigator award to GS
References
1 Abel ED, Litwin SE & Sweeney G (2008) Cardiac
remodeling in obesity Physiol Rev 88, 389–419
2 Hilfiker-Kleiner D, Landmesser U & Drexler H (2006)
Molecular mechanisms in heart failure focus on cardiac
hypertrophy, inflammation, angiogenesis, and apoptosis
J Am Coll Cardiol 48, A56–A66
3 Hall JA, French TK, Rasmusson KD, Vesty JC,
Roberts CA, Rimmasch HL, Kfoury AG & Renlund
DG (2005) The paradox of obesity in patients
with heart failure J Am Acad Nurse Pract 17, 542–546
4 Kunapuli S, Rosanio S & Schwarz ER (2006) ‘How do
cardiomyocytes die?’ Apoptosis and autophagic cell
death in cardiac myocytes J Card Fail 12, 381–391
5 Reeve JL, Duffy AM, O’Brien T & Samali A (2005)
Don’t lose heart – therapeutic value of apoptosis
prevention in the treatment of cardiovascular disease
J Cell Mol Med 9, 609–622
6 Kobayashi K (2005) Adipokines: therapeutic targets for
metabolic syndrome Curr Drug Targets 6, 525–529
7 Ahima RS & Flier JS (2000) Leptin Annu Rev Physiol
62, 413–437
8 Rahmouni K, Morgan DA, Morgan GM, Mark AL &
Haynes WG (2005) Role of selective leptin resistance in
diet-induced obesity hypertension Diabetes 54, 2012–
2018
9 Mark AL, Correia ML, Rahmouni K & Haynes WG
(2002) Selective leptin resistance: a new concept in
leptin physiology with cardiovascular implications
J Hypertens 20, 1245–1250
10 Sweeney G (2002) Leptin signaling Cell Signal 14, 655–
663
11 Matsui H, Motooka M, Koike H, Inoue M, Iwasaki
T, Suzuki T, Kurabayashi M & Yokoyama T (2007)
Ischemia⁄ reperfusion in rat heart induces leptin
and leptin receptor gene expression Life Sci 80, 672–
680
12 Purdham DM, Zou MX, Rajapurohitam V &
Karmazyn M (2004) Rat heart is a site of leptin
production and action Am J Physiol Heart Circ Physiol
287, H2877–H2884
13 Barouch LA, Gao D, Chen L, Miller KL, Xu W,
Phan AC, Kittleson MM, Minhas KM, Berkowitz
DE, Wei C et al (2006) Cardiac myocyte apoptosis is
associated with increased DNA damage and decreased survival in murine models of obesity Circ Res 98, 119–124
14 Madani S, De Girolamo S, Munoz DM, Li RK & Sweeney G (2006) Direct effects of leptin on size and extracellular matrix components of human pediatric ventricular myocytes Cardiovasc Res 69, 716–725
15 Ren J & Relling DP (2006) Leptin-induced suppression
of cardiomyocyte contraction is amplified by ceramide Peptides 27, 1415–1419
16 Sawyer DB, Siwik DA, Xiao L, Pimentel DR, Singh K
& Colucci WS (2002) Role of oxidative stress in myo-cardial hypertrophy and failure J Mol Cell Cardiol 34, 379–388
17 Tajmir P, Ceddia RB, Li RK, Coe IR & Sweeney G (2004) Leptin increases cardiomyocyte hyperplasia via extracellular signal-regulated kinase- and phosphatidyl-inositol 3-kinase-dependent signaling pathways Endocri-nology 145, 1550–1555
18 Palanivel R, Eguchi M, Shuralyova I, Coe I & Sweeney
G (2006) Distinct effects of short- and long-term leptin treatment on glucose and fatty acid uptake and metabo-lism in HL-1 cardiomyocytes Metabometabo-lism 55, 1067– 1075
19 Caro JF, Kolaczynski JW, Nyce MR, Ohannesian JP, Opentanova I, Goldman WH, Lynn RB, Zhang PL, Sinha MK & Considine RV (1996) Decreased cerebro-spinal-fluid⁄ serum leptin ratio in obesity: a possible mechanism for leptin resistance Lancet 348, 159–161
20 Suleiman MS, Halestrap AP & Griffiths EJ (2001) Mitochondria: a target for myocardial protection Phar-macol Ther 89, 29–46
21 Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death Biochem J 341, 233–249
22 Desbiens KM, Deschesnes RG, Labrie MM, Desfosses
Y, Lambert H, Landry J & Bellmann K (2003) c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade Biochem J 372, 631–641
23 Clerk A, Cole SM, Cullingford TE, Harrison JG, Jor-makka M & Valks DM (2003) Regulation of cardiac myocyte cell death Pharmacol Ther 97, 223–261
24 Ren J (2004) Leptin and hyperleptinemia – from friend to foe for cardiovascular function J Endocrinol 181, 1–10
25 Schulze PC & Kratzsch J (2005) Leptin as a new diag-nostic tool in chronic heart failure Clin Chim Acta 362, 1–11
26 Schulze PC, Kratzsch J, Linke A, Schoene N, Adams
V, Gielen S, Erbs S, Moebius-Winkler S & Schuler G (2003) Elevated serum levels of leptin and soluble leptin receptor in patients with advanced chronic heart failure Eur J Heart Fail 5, 33–40
Trang 927 Yasuoka C, Ihara Y, Ikeda S, Miyahara Y, Kondo T &
Kohno S (2004) Antiapoptotic activity of Akt is
down-regulated by Ca2+in myocardiac H9c2 cells Evidence
of Ca(2+)-dependent regulation of protein phosphatase
2Ac J Biol Chem 279, 51182–51192
28 Murata H, Ihara Y, Nakamura H, Yodoi J, Sumikawa
K & Kondo T (2003) Glutaredoxin exerts an
antiapop-totic effect by regulating the redox state of Akt J Biol
Chem 278, 50226–50233
29 Han H, Long H, Wang H, Wang J, Zhang Y &
Wang Z (2004) Progressive apoptotic cell death
trig-gered by transient oxidative insult in H9c2 rat
ven-tricular cells: a novel pattern of apoptosis and the
mechanisms Am J Physiol Heart Circ Physiol 286,
H2169–H2182
30 Tajmir P, Kwan JJ, Kessas M, Mozammel S & Sweeney
G (2003) Acute and chronic leptin treatment mediate
contrasting effects on signaling, glucose uptake, and
GLUT4 translocation in L6–GLUT4myc myotubes
J Cell Physiol 197, 122–130
31 Sweeney G, Keen J, Somwar R, Konrad D, Garg R &
Klip A (2001) High leptin levels acutely inhibit
insulin-stimulated glucose uptake without affecting glucose
transporter 4 translocation in L6 rat skeletal muscle
cells Endocrinology 142, 4806–4812
32 McGavock JM, Victor RG, Unger RH & Szczepaniak
LS (2006) Adiposity of the heart, revisited Ann Intern
Med 144, 517–524
33 Ghosh S & Rodrigues B (2006) Cardiac cell death in
early diabetes and its modulation by dietary fatty acids
Biochim Biophys Acta 1761, 1148–1162
34 Wagner R, Oberste-Berghaus C, Herpertz S, Blum WF, Pelz B, Hebebrand J, Senf W, Mann K & Albers N (2000) Time relationship between circadian variation of serum levels of leptin, insulin and cortisol in healthy subjects Horm Res 54, 174–180
35 Laimer M, Ebenbichler CF, Kaser S, Sandhofer A, Weiss
H, Nehoda H, Aigner F & Patsch JR (2002) Weight loss increases soluble leptin receptor levels and the soluble receptor bound fraction of leptin Obes Res 10, 597–601
36 Cook SA, Sugden PH & Clerk A (1999) Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: associ-ation with changes in mitochondrial membrane poten-tial Circ Res 85, 940–949
37 Correia ML, Haynes WG, Rahmouni K, Morgan DA, Sivitz WI & Mark AL (2002) The concept of selective leptin resistance: evidence from agouti yellow obese mice Diabetes 51, 439–442
38 Smith CC, Mocanu MM, Davidson SM, Wynne AM, Simpkin JC & Yellon DM (2006) Leptin, the obesity-associated hormone, exhibits direct cardioprotective effects Br J Pharmacol 149, 5–13
39 Wang L, Ma W, Markovich R, Lee WL & Wang PH (1998) Insulin-like growth factor I modulates induction
of apoptotic signaling in H9C2 cardiac muscle cells Endocrinology 139, 1354–1360
40 Eguchi M, Gillis LC, Liu Y, Lyakhovsky N, Du M, McDermott JC & Sweeney G (2007) Regulation of SOCS-3 expression by leptin and its co-localization with insulin receptor in rat skeletal muscle cells Mol Cell Endocrinol 267, 38–45