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Obesity is a major risk factor for the development of cardiovascular diseases such as hypertension, atherosclerosis, and
heart failure[1,2]. Leptin is the product of the ob gene, and its concentration is increased in obese individuals. Animal studies
indicate that long-term administration of leptin results in a sustained increase in artery pressure. Clinical evidence indicates
that leptin is a potential independent risk factor for coronary heart disease. Recent studies have supplied evidence that leptin
also induces hypertrophy in rat neonatal cardiomyocytes
in vitro[3,4]. Clinical evidence also indicates that increased plasma
leptin levels are correlated with cardiac hypertrophy and congestive heart
failure[5,6]. These results suggest that leptin is an
important factor in the cause of obesity-related cardiovascular diseases.
Statins are inhibitors of 3-hydroxy-3-methylglutaryl
coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme of cholesterol synthesis. Emerging evidence from
both animal and human studies indicates that mechanisms independent of cholesterol lowering effects contribute to the
observed clinical benefits of
statins[7,8]. Recent studies (including our own) have demonstrated that statins prevent cardiac
hypertrophy and heart failure in animals with aortic stenosis and angiotensin
II-infusion[9,10]. However, it is unknown
whether statins can inhibit obesity-related cardiac hypertrophy.
In the present study, we hypothesized that simvastatin inhibited leptin-induced cardiomyocyte hypertrophy. Our results
suggest that leptin stimulates hypertrophy via reactive oxygen species (ROS) generation in cultured neonatal rat
cardiomyocytes. We found that treatment with simvastatin or the antioxidants butylated hydroxyanisole (BHA) and catalase
inhibited leptin-induced ROS production and hypertrophy in cultured neonatal rat cardiomyo-cytes.
Materials and methods
Cell culture Primary cultures of cardiomyocytes were prepared according to previously published
methods[11,12]. Briefly, hearts from 1-day-old Sprague-Dawley rats that were obtained from the Medical Laboratory Animal Center (Guangdong,
China) were dissected, minced and placed in a Petri dish. The tissue was trypsinized at 37 ĄăC in D-HanksĄŻ balanced salt
solution (in g/L: NaCl 8.00, KCl 0.4,
KH2PO4
0.06, NaHCO3 0.35,
Na2HPO4.7H2
O 0.09 and 0.125% trypsin). After centrifugation (200
g, 15 min), cells were collected and suspended in M199 medium (containing 10% fetal calf serum, insulin 5 µg/mL, transferrin 5 µg/mL, penicillin 100 U/mL, and
streptomycin 100 µg/mL (Sigma, St Louis, USA). Cells were then preplated at 37 ĄăC for 1 h. The cells were diluted to
5ĄÁ106 cells/mL, plated in 96-well, 24-well or 6-well plates, and were cultured for 48-64 h in medium containing 0.1 mmol/L
bromodeoxyuridine to prevent proliferation of nonmyocytes. On the fifth day, after being cultured in a non-serum medium for
72 h, cells were washed twice with HanksĄŻ balanced salt solution at 37 ĄăC. The washed cells were incubated at 37 ĄăC
for 4 h and in a non-serum medium containing leptin (100
ng/mL, R&D Systems, Minneapolis, MN, USA) for 72 h, leptin (100 ng/mL)
plus simvastatin (1-100 nmol/L, Merck, Sharp & Dohme, NJ, USA), leptin (100 ng/mL) plus the antioxidant butylated
hydroxyanisole (BHA; 10 µmol/L;
Sigma)[13] or leptin (100 ng/mL) plus catalase (200 U/mL, Sigma), an enzyme that specifically
decomposes hydrogen peroxide
(H2O2) to water and molecular oxygen.
ROS determination Intracellular ROS was assessed by using the ROS-specific probe 2ĄŻ,7ĄŻ-dichlorofluorescein
diacetate (DCF-DA, Molecular Probes, Eugene, OR,
USA)[11,14]. On culture d 4, the cultured cardiomyocytes were washed with HanksĄŻ
solution, and then incubated with DCF-DA (5 mmol/L) in HanksĄŻ solution at 37 ĄăC. After incubation for 1 h, cardiomyocytes
were washed with HanksĄŻ solution. Fluorescence signals were obtained with a fluorescence microscope (TE300-ECI; Nikon,
Tokyo, Japan) and assayed by using its image processing and analysis system. In each case, 5 randomly selected fields in
each well were selected for examination.
RNA content measurement The RNA content was determined by using the RNA-sensitive fluorescence probe propidium
iodide (PI; Molecular Probes) after DNase
treatment[11,15]. Cardiomyocytes were treated with diluent (control), leptin (100
ng/mL), leptin (100 ng/mL) plus simvastatin
(1-100 nmol/L), or leptin (100 ng/mL) plus catalase (200 U/mL) from culture d 4 to d 7. On culture d 7, the cells were washed
with HanksĄŻ solution and fixed with 75% ethanol for 10 min. Ethanol-fixed cells were rinsed in HanksĄŻ solution and incubated
at 36 ĄăC for 40 min in a solution containing DNase (1 mg/mL), sucrose (0.25 mol/L),
MgCl2 (5 mmol/L), and Tris-HCl (20
mmol/L; pH 6.5). After incubation, 1 mL of HanksĄŻ solution containing PI (0.05 mg/mL) was added to each well and left for 30 min.
Then the fluorescence signal was analyzed with a fluorescence microscope. In each case, five randomly selected fields in
each well were selected for examination.
Cardiomyocyte surface area assay Cardiomyocyte surface area was measured according to a previously described
method[16]. Cell images were captured with a digital camera
(Nikon) fixed to a microscope (Nikon). Cardiomyocyte surface area
was analyzed using the Leica Image Processing and Analysis System. One hundred cells from randomly selected fields in 3
wells were examined for each condition.
Cardiomyocyte surface area was determined after 3-d treatment with leptin, simvastatin,
or the antioxidant catalase in comparison with control cells treated
with diluent.
Protein content evaluation Cultured cardiomyocytes were treated with leptin, simvastatin, catalase, or diluent (control)
from culture d 4 to d 7. The cells were washed with PBS and then treated with 5% trichloroacetic acid at
4 ĄăC for 1 h to precipitate the
protein[11,16]. The precipitates were dissolved
in NaOH (0.1 mol/L). The protein content was measured using
the Bio-Rad (Hercules, CA , USA) DC protein
assay.
Statistical analysis All results are expressed as meanĄŔSD. One-way ANOVA was used for multiple comparisons. A value
of P<0.05 was considered significant.
Results
After 72 h treatment, leptin (100 ng/mL) significantly increased RNA levels in cultured cardiomyocytes. Treatment with
simvastatin, BHA, or catalase significantly inhibited the effect of leptin on RNA levels (Figure 1).
Protein content in cultured cardiomyocytes treated with leptin (100 ng/mL) for 72 h was significantly increased.
Treatment with simvastatin, BHA, or catalase markedly inhibited the effect of leptin on protein content (Figure 2).
Cell surface area in cultured cardiomyocytes treated with leptin for 72 h was significantly increased. Treatment with
simvastatin, BHA, or catalase markedly inhibited the effect of leptin on cell surface area (Figure 3).
After treatment for 4 h, leptin markedly increased the ROS levels of cultured cardiomyocytes, an effect that was blunted
by treatment with simvastatin, BHA, or catalase (Figure 4).
Discussion
The present study demonstrated that leptin induced hypertrophy through a ROS-dependent pathway and that simvastatin
inhibited leptin-induced hypertrophy in cultured neonatal rat cardiomyocytes.
Clinical evidence has shown that plasma leptin levels are increased in obese individuals, and that enhanced plasma leptin
levels are correlated with cardiac hypertrophy and other cardiovascular
diseases[6,17]. This observation is supported by
recent studies in vitro[3]. The results of these studies indicate that leptin induces hypertrophy in cultured rat neonatal
cardiomyocytes in a concentration-dependent manner. These results provide a link between leptin and cardiac hypertrophy
in obese individuals. However, the mechanisms by which leptin induces cardiac hypertrophy are still unknown. Therefore,
in the present study, we examined how leptin induced cardiac hypertrophy. Our results indicate that leptin induces
ROS generation and hypertrophy, which were significantly inhibited by simvastatin and catalase (an enzyme that specifically
decomposes hydrogen peroxide to water and molecular oxygen) in cultured neonatal rat cardiomyocytes. Collectively, these
data suggest that oxidative stress is involved in leptin-induced hypertrophy in cultured neonatal rat
cardiomyocytes.
It is also well known that oxidative stress contributes to cardiac hypertrophy, because hypertrophic substances such as
endothelin-1, norepinephrine, angiotensin II, and cytokines such as tumor necrosis factor
(TNF)-a induce cardiomyocyte hypertrophy via ROS
generation[11,16]. Furthermore, clinical and animal studies have demonstrated that the failing heart is
subjected to increased oxidative stress, and antioxidant therapy has been shown to preserve left ventricular function during
the development of chronic heart
failure[19,20]. Recent evidence has also indicated that obesity was a state of chronic oxidative
stress and inflammation[2,18].
However, it is unknown whether oxidative stress contributes to cardiac hypertrophy in the
obese state. Our present results show that leptin induces ROS generation and hypertrophy, which can be reversed by
treatment with the antioxidants catalase and simvastatin, in cultured neonatal rat cardiomyocytes. Thus, these results
suggest that elevated leptin levels in obese subjects may increase cardiac oxidative stress, which results in cardiac hypertrophy.
Therefore, antioxidant therapy aimed at reducing oxidative stress induced by leptin may provide an effective means to
improve cardiac dysfunction in obese humans.
Animal studies have also shown that statins induce the regression of cardiac hypertrophy
in vivo[9,10] and inhibit the hypertrophy of cardiomyocytes induced by norepinephrine and angiotensin II
in vitro[11,16]. Inhibition of the Rac1-ROS
pathway represents an important mechanism by which statins inhibit cardiac
hypertrophy[21,22]. Our present results indicate
that simvastatin inhibits leptin-induced ROS-mediated hypertrophy in cultured neonatal rat cardiomyocytes. The results
further show that statins are effective against cardiac hypertrophy in different animal models, and highlight the need for
clinical trials to determine the effects of statins on cardiac hypertrophy and fibrosis in humans.
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