Download Cardiac Hypertrophy-Related Pathways in Obesity

Chinese Journal of Physiology 57(3): 111-120, 2014
DOI: 10.4077/CJP.2014.BAB146
111
Cardiac Hypertrophy-Related Pathways
in Obesity
Wei-Kung Chen 1, * , Yu-Lan Yeh 2, 3, * , Yueh-Min Lin 2, 3, 4, Jing-Ying Lin 5, Bor-Show Tzang 6,
James A. Lin 7, Ai-Lun Yang 8, Fong-Li Wu 6, Fuu-Jen Tsai 9, Shiu-Min Cheng 10,
Chih-Yang Huang 7, 9, 11, #, and Shin-Da Lee 12, 13, #
1
Graduate Institute of Clinical Medical Science, China Medical University, Taichung 40402
Emergency Department, China Medical University Hospital, Taichung 40440
2
Department of Pathology, Changhua Christian Hospital, Changhua 50006
3
Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management,
Miaoli 35664
4
School of Medicine, Chung Shan Medical University, Taichung 40201
5
Department of Medical Imaging and Radiological Science, Central Taiwan University of Science
and Technology, Taichung 40601
6
Institutes of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 40201
7
Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402
8
Graduate Institute of Exercise Science, University of Taipei, Taipei 11153
9
Graduate Institute of Chinese Medicine, China Medical University and Hospital, Taichung 40440
10
Department of Psychology, Asia University, Taichung 41354
11
Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354
12
Department of Physical Therapy, Graduate Institute of Rehabilitation Science,
China Medical University, Taichung 40402
and
13
Department of Healthcare Administration, Asia University, Taichung 41354
Taiwan, Republic of China
Abstract
Obesity is often associated with the development of cardiac hypertrophy but the hypertrophyrelated pathways in obesity remain unknown. The purpose of this study was to evaluate cardiac
hypertrophy-related markers, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP),
tumor necrosis factor-alpha (TNFα) and hypertrophy-related pathways, interleukin (IL)-6-STAT3,
IL-6-MEK5-ERK5 and calcineurin-nuclear factor of activated T-cells (NFAT)3 in the excised hearts
from obese rats. Twelve obese Zucker rats were studied at 5-6 months of age and twelve age-matched
lean Zucker rats served as the control group. The cardiac characteristics, myocardial architecture,
ANP, BNP, TNFα levels, IL-6, STAT3, p-STAT3, MEK5, ERK5, p-ERK5, calcineurin and NFAT3 in
the left ventricle from the rats were measured by heart weight index, echocardiography, vertical cross
section, histological analysis, reverse transcription polymerase chain reaction and Western blotting.
Compared with the lean control, the whole heart weight, the left ventricule weight, the ratio of the
whole heart weight to tibia length, echocardiographic interventricular septum, left ventricular posterior
wall thickness, myocardial morphological changes and systolic blood pressure were found to increase in
the obese rats. The protein levels of ANP, BNP, TNFα, IL-6, STAT3, p-STAT3, MEK5, ERK5, p-ERK5,
calcineurin and NFAT3 were also significantly increased in the hearts of the obese rats. The results
showed that the hypertrophy-related markers, ANP, BNP and TNFα, the hypertrophy-related pathways IL-6-STAT3 and IL-6-MEK5-ERK5, and the calcineurin-NFAT3 hypertrophy-related pathways
Corresponding author: Shin-Da Lee, Ph.D., Department of Physical Therapy, China Medical University, 91 Hsueh-Shih Road, Taichung
40402, Taiwan, R.O.C. Tel: +886-4-22053366 ext. 7300, Fax: +886-4-22065051, E-mail: [email protected]
*Wei-Kung Chen and Yu-Lan Yeh share equal contribution.
#
Chih-Yang Huang and Shin-Da Lee share equal contribution.
Received: September 8, 2012; Revised: January 21, 2013; Accepted: August 8, 2013.
2014 by The Chinese Physiological Society and Airiti Press Inc. ISSN : 0304-4920. http://www.cps.org.tw
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Chen, Yeh, Lin, Lin, Tzang, Lin, Yang, Wu, Tsai, Cheng, Huang and Lee
were more active in obese Zucker rats, which may provide possible hypertrophic mechanisms for developing cardiac hypertrophy and pathological changes in obesity.
Key Words: calcineurin, ERK5, heart, hypertrophy, IL-6, MEK5, NFAT3, signaling
Introduction
The obese Zucker rat, a genetic model of morbid
obesity, presents many of the same cardiopulmonary
deficits noted in obese humans including respiratory
control dysfunction (24, 25, 27), chest wall limitations (12), upper airway narrowing (34), hypertension (2), myocardial hypertrophy (13), cardiac apoptosis
(29, 30) and poor exercise capacity (26, 28). Severe
obesity in human has long been recognized as causing
a form of cardiomyopathy characterized by increased
rates of hypertension, chronic volume overload, left
ventricular hypertrophy and the development of heart
failure (3, 8, 9, 31). However, the hypertrophy-related
pathways of cardiac hypertrophy in severe obesity remain uncertain.
Cardiac hypertrophy, a cardiac adaptive response
to stress, can exist in a state of compensation or progress to a decompensated state over time (10). It is wellrecognized that atrial natriuretic peptide (ANP) and
brain (B-type) natriuretic peptide (BNP) are markers
of cardiac hypertrophy and are elevated in conditions
of ventricular volume and pressure overload (16, 44).
Accumulating evidences indicate that the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα),
plays a pathogenic role in the myocardial remodeling
process, inducing cardiac hypertrophy (18, 32, 36).
Locally produced TNFα from the heart probably contributes to myocardial dysfunction via direct suppression of myocardial contractile function and the genesis
of cardiac hypertrophy (32). TNFα was found to provoke a hypertrophic growth response in adult mammalian cardiac myocytes (45). In addition, overexpression of TNFα in the heart of mice leads to
the transition from a hypertrophic to a dilated cardiac
phenotype and adverse cardiac remodeling (11). However, the roles of cardiac TNFα in obese animals and
humans are still unclear.
Cardiac hypertrophy, in which chamber volume
enlarges without a relative increase, or even with a
relative decrease, in wall thickness, is known to progress
to dilated cardiomyopathy, heart failure and sudden
death (37, 40). Interleukin (IL)-6, a ubiquitous cytokine, was found to have potent hypertrophic effects
on cardiomyocytes (21). IL-6 could be involved in an
autocrine and/or a paracrine network regulating myocardial hypertrophy (4). The IL-6 receptor (IL-6R)
system consists of an IL-6-specific binding molecule,
IL-6R, and a signal transducer, gp130. The multiple
intracellular signaling pathways are evoked by the IL-6
and MAPK extracellular signal regulated kinase (ERK)
pathways (18, 19, 38). The ERK5, also known as big
MAPK 1 (BMK1), plays a critical role in post-natal hypertrophy of the heart (6, 37). ERK5 and its upstream
MAPK-kinase 5 (MEK5) have a specific role in the
transduction of the cytokine signals that regulate
serial sarcomere assembly and in the induction of
cardiac hypertrophy that progresses to dilated cardiomyopathy and sudden death (37).
Calcineurin has been reported to be a critical
mediator for cardiac hypertrophy and cardiac myocyte
apoptosis (23, 33, 42). Transgenic mice that express
the activated forms of calcineurin in the heart develop
cardiac hypertrophy and heart failure that mimic human
heart diseases (33). It has been reported that the activation of Ca2+ induces the calcineurin-nuclear factor
activation transcription (NFAT) pathway and in turn
enhances hypertrophy (33, 46). Therefore, it is crucial to investigate the role of the cardiac hypertrophic markers, IL-6-STAT3, IL-6-MEK5-ERK5 and the
calcineurin-NFAT pathway in obesity.
The role of cardiac hypertrophy in obesity is not
well understood. In the current study, experimental
procedures were undertaken to investigate whether
cardiac hypertrophy in obesity was associated with
hypertrophy-related markers or pathways. We hypothesized that cardiac abnormality in obesity might
predispose the obese subjects to more activated ANP,
BNP and TNFα hypertrophy-related markers as well
as more activated IL-6-STAT3, IL-6-MEK5-ERK5 and
calcineurin-NFAT3 hypertrophy-related pathways.
Materials and Methods
Animal Model
The studies were performed on 12 lean (Fa/Fa
or Fa/fa) and 12 obese (fa/fa) age-matched 5-6-month
old male Zucker rats. Animals were supplied by
Zucker breeders purchased from Charles River Lab
in France. One lean and one obese rat, obtained from
the same breeder, were housed together. Ambient temperature was maintained at 25°C and the animals were
kept on an artificial 12-h light-dark cycle, with the
light period beginning at 7:00 am. Rats were provided
with standard laboratory chow (Lab Diet 5001; PMI
Nutrition International Inc., Brentwood, MO, USA)
and water ad libitum. All protocols were approved by
the Institutional Animal Care and Use Committee of
China Medical University, Taichung, Taiwan, ROC.
Cardiac Hypertrophy in Obesity
Blood Pressure Measurements and Echocardiography
The animals were loosely restrained. Systolic,
diastolic and mean arterial blood pressures were determined with an automated tail-cuff system (29SSP;
IITC/Life Science Instruments, Woodland Hills, CA,
USA) 16 h after the completion of all exposures to
hypoxia. Transthoracic echocardiographic images of
rats were performed using Philips M2424A ultrasound
systems (Andover, MA, USA) under anesthesia with
1% isoflurane via a nose cone. M-mode echocardiographic examination was performed using a 6-15
MHz linear transducer (15-6L) via parasternal long
axis approach. Left ventricular M-mode measurements
at the level of the papillary muscles included left ventricular internal end-diastolic dimensions (LVIDd), left
ventricular internal end-systolic dimensions (LVIDs),
interventricular septum (IVS) and left ventricular
posterior wall thicknesses (LVPW), and fractional
shortening (FS). FS% was calculated according to
the following equation:
FS% = [(LVIDd − LVIDs)/LVIDd] × 100.
Cardiac Characteristics
The animals were loosely restrained. Systolic,
diastolic and mean arterial blood pressures were determined with an automated tail-cuff system (29SSP;
IITC/Life Science Instruments). All rats were
weighed and decapitated. The hearts of eight lean and
eight obese animals were excised and cleaned with
ddH 2O. The left and right atrium and ventricle were
separated and weighed. The right tibias were also
separated and the tibia length was measured by an
electronic digital caliper (Mitutoyo Corporation,
Tokyo, Japan) to correct for the whole heart weight.
The ratios of the total heart weight to body weight,
the left ventricle weight to whole-heart weight, and the
whole-heart weight to tibial length were calculated.
Hematoxylin-Eosin Masson Trichrome Staining
After removal, the heart was soaked in 10% formalin and immersed in wax. Cross sections of the whole
hearts were sliced. Slides were prepared by deparaffinization and dehydration. The slides were passed
through a series of graded alcohols (100%, 95% and
75%) by immersion for 15 min in each solution. The
slides were then stained with mayer hematoxylin for
5-10 min followed by washing with tap water for 10-20
min. Each slide was then soaked in warm water until
it turned bright violet, followed by immersing in eosin
solution for 3-5 min. After gently rinsing with water,
each slide was soaked with 85% alcohol, 100% alcohol
I and II for 15 min each. Finally, the slides were soaked
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twice with xylene. Photomicrographs were obtained
using Zeiss Axiophot microscopes (Nikon, Japan).
Tissue Extraction
Cardiac tissue extracts were obtained by homogenizing left ventricle samples in a lysis buffer (20 mM
Tris, 2 mM EDTA, 50 mM 2-mercaptoethanol, 10%
glycerol, pH 7.4, proteinase inhibitor (Roche), phosphatase inhibitor cocktail (Sigma, St. Louis, MO USA)
at a ratio of 100 mg tissue/ml buffer for 1 min. The
homogenates were placed on ice for 10 min and then
centrifuged twice at 12,000 g for 40 min. The supernatant was collected and stored at -70°C for further
experiments.
Electrophoresis and Western Blots
The tissue extract samples were prepared as described by homogenizing with buffer. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was performed in 10% polyacrylamide gels. The samples
were electrophoresed at 140 V for 3.5 h and equilibrated for 15 min in 25 mM Tris-HCl, pH 8.3, containing 192 mM glycine and 20% (v/v) methanol.
Electrophoresed proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore,
Bedford, MA, 0.45 μm pore size) with a Bio-Rad
Scientific Instruments Transphor Unit at 100 V for 2 h.
PVDF membranes were incubated at room temperature for 1 h in a blocking buffer containing 100 mM
Tris-HCl, pH 7.5, 0.9% (w/v) NaCl. Antibodies directed
against ANP, BNP, TNFα, IL-6, STAT3, p-STAT3,
ERK5, p-ERK5, calcineurin, NFAT3 and α-tubulin
(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and
MEK-5 (BD, Franklin Lakes, NJ, USA) were diluted
to 1:500 in an antibody binding buffer containing 100
mM Tris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v)
Tween-20. Incubations were performed at 4°C overnight. The immunoblots were washed three times in
TBS buffer (Tris-Base, NaCl, Tween-20, pH 7.4) for
10 min and then immersed in the second antibody
solution containing goat anti-mouse IgG-HRP, goat
anti-rabbit IgG-HRP, or donkey anti-goat IgG-HRP
(Santa Cruz) for 1 h and diluted 500-fold in TBS buffer.
The immunoblots were then washed three times for
10 min in the blotting buffer. The immunoblotted
proteins were visualized by using an enhanced chemiluminescence ECL Western Blotting Luminal Reagent
(Santa Cruz) and quantified using a Fujifilm LAS-3000
chemiluminescence detection system (Tokyo, Japan).
RNA Extraction
Total RNA was extracted by the Ultraspec RNA
Isolation System (Biotecx Laboratories, Inc., Houston,
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Chen, Yeh, Lin, Lin, Tzang, Lin, Yang, Wu, Tsai, Cheng, Huang and Lee
Table 1. Characteristics of the lean and obese rats
Characteristics
Lean
Obese
Number
8
8
Body weight (BW), g
393 ± 32
555 ± 61*
Whole-heart weight (WHW), g
0.89 ± 0.06
1.29 ± 0.13*
Left ventricular weight (LVW), g
0.57 ± 0.07
0.87 ± 0.11*
-4
WHW/BW
22.7 ± 1.3 (× 10 )
23.3 ± 1.9 (× 10-4)
WHW/Tibia length, g/m
22.0 ± 1.2
34.1 ± 3.6*
LVW/BW
14.6 ± 1.5 (× 10-4)
15.6 ± 1.3 (× 10-4)
LVW/WHW
0.64 ± 0.05
0.67 ± 0.05
Blood Pressure
SBP, mmHg
117 ± 14
137 ± 12*
DBP, mmHg
83 ± 15
92 ± 14
MBP, mmHg
94 ± 16
107 ± 13
Blood Sampling
Fasting insulin, ng/ml
0.92 ± 0.06
4.93 ± 1.32*
Fasting glucose, mg/dl
102.4 ± 11.7
111.2 ± 19.5
Serum leptin, ng/l
3.4 ± 1.1
46.2 ± 4.2**
Values are means ± SD. *P < 0.05 and **P < 0.01 are significant differences between the lean and obese Zucker rats.
TX, USA) according to the manufacturer’s instructions. Each heart was thoroughly homogenized in 1
ml Ultraspec reagent/100 mg tissue with a Polytron
homogenizer. The homogenates were washed twice
with 70% ethanol by gentle vortexing. RNA precipitates were then collected by centrifugation at 12,000
g and dried under vacuum for 5-10 min before resuspending in 50 µl diethylpyrocarbonate-treated
water, and then incubated at 55-60°C for 10-15 min.
Reverse Transcription Polymerase Chain Reaction
(RT-PCR)
Total RNA was reverse transcribed and then amplified by the polymerase chain reaction using the Super
Script Preamplification System for first-strand cDNA
Synthesis kit and Taq DNA polymerase (Life Technologies, Rockville, MD, USA). RT-PCR products (45
μl) were separated on a 1.25% agarose gel (Life Technologies). Amplimers were synthesized based on
cDNA sequences from the GenBank. The rat GAPDH
was used as an internal standard. The primers used
were: Rat TNFα forward primer: 5’-TCGAGTGACAAGCCC GTAG-3’; Rat TNFα reverse primer: 5’CAGAGCAATGACTCCAAAGTAGAC-3’; Rat
GAPDH forward primer: 5’-GGGTGTGAACCACGAGAAAT-3’; Rat GAPDH reverse primer: 5’-CCACAGTCTTCTGAGTGGCA-3’ (MDBio Inc., Taipei,
Taiwan, ROC). Densitometric analysis of immunoblots
and PCR gels were performed using the AlphaImager
2200 digital imaging system (Digital Imaging System,
San Leandro, CA, USA).
Statistical Analysis
The data were compared between lean and obese
groups using Student’s t-test for two independent
samples. In all cases, a difference at P < 0.05 was
considered statistically significant.
Results
Body Weight and Cardiac Characteristics
Obese rats weighed about 41% more than agematched lean animals (555 ± 61 g versus 393 ± 32 g,
P < 0.01). The absolute whole-heart weight (WHW),
the left ventricular weight (LVW) and the ratio of
whole-heart weight to tibia length were significantly
increased in the obese group, compared with data of
the lean group (Table 1). From blood sampling, serum
leptin and fasting insulin levels in the obese rats were
significantly higher than those in lean rats (Table 1).
The averaged systolic blood pressure in the obese rats
was significantly higher than that in the lean rats
whereas DBP and MBP in the obese rats were not
significantly increased. From echocardiographic observations, the IVS, the LVPW and the LVIDd were
significantly increased. However, LVIDs in the obese
group was significantly decreased compared with that
of the lean group (Fig. 1). The ratio of average wall
thickness to diameter of left ventricles in the obese
group was significantly increased (P < 0.335 ± 0.032
versus 0.291 ± 0.012, n = 8) relative to the lean
group.
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Cardiac Hypertrophy in Obesity
(A) Representative Echocardiography
Lean
(A) H&E Staining (100)
Obese
Lean
(B) Echocardiographic Evaluation
Obese
(B) Masson Trichrome (100)
Lean
Obese
IVS, mm
1.70  0.31
LVPW, mm
1.79  0.28
3.08  0.25**
3.20  0.28*
LVIDd, mm
7.61  0.74
LVIDs, mm
3.35  0.65
4.84  0.51**
2.32  0.62*
FS, %
55.9  5.7
52.1  4.5
HR (beats/min)
325  13
313  12
Fig. 1. Echocardiography of lean and obese rats. (A) Representative echocardiography of lean and obese rats. Values
are means ± SD (n = number of animals). (B) Echocardiographic evaluation. IVS: interventricular septum;
LVPW: left ventricular posterior wall thickness; LVIDd:
internal dimension at diastole of left ventricle; LVIDs:
internal dimension at systole of left ventricle; FS: fractional shortening (LVIDd-LVIDs/LVIDd ×100). HR:
heart rates *P < 0.05 and **P < 0.01 are significant
differences between the lean and obese groups.
Fig. 2. Histopathological analysis of cardiac tissue sections
stained with hematoxylin and eosin (H&E) (A) and
Masson trichrome (B) in lean and obese Zucker rats.
The images of the myocardial architecture were magnified 100 times.
(A)
Lean
Obese
ANP
BNP
Cardiomyopathic Changes
ANP and BNP
The ANP and BNP protein levels showed significant increases (P < 0.01) in the cardiac tissues
excised from the obese rats as compared with the
age-matched lean rats (Fig. 3).
TNFα
The mRNA expression and protein levels of
TNFα were significantly increased (P < 0.01) in the
cardiac tissues excised from the obese rats compared
with age-matched lean rats (Fig. 4).
-Tubulin
(B)
ANP/-Tubulin
BNP/-Tubulin
The ventricular myocardium in the lean control
group showed normal architecture with normal interstitial space. In contrast, a disarray of myocardial
architecture, increased interstitial space and increased cardiac fibrosis were observed in the obese
group (Fig. 2).
**
1.2
ANP
BNP
**
0.8
0.4
0.0
Lean
Obese
Fig. 3. Analysis of ANP and BNP lean and obese rats. (A) Representative protein levels of ANP and BNP extracted from
the left ventricles of excised hearts in the lean and obese
age-matched Zucker rats, as measured by Western blotting analysis. (B) Relative quantification by densitometric analysis normalized to α-tubulin (n = 6 in each
group) indicating mean ± SD. **P < 0.01, significant
difference between the lean and obese groups.
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Chen, Yeh, Lin, Lin, Tzang, Lin, Yang, Wu, Tsai, Cheng, Huang and Lee
(A)
Obese
Lean
TNF
◄ 695 bp
◄ 295 bp
GADPH
(B)
Lean
Obese
TNF
◄ 19 kDa
-Tubulin
◄ 57 kDa
(C)
1.2
**
TNF/-Tubulin
TNF/GADPH
1.5
1.0
0.5
0.0
0.4
0.0
Obese
Lean
**
0.8
Lean
Obese
Fig. 4. Analysis of TNFα in lean and obese rats. The mRNA expression (A) and protein levels (B) of TNFα extracted from the left
ventricles of the hearts of lean and obese rats were measured by RT-PCR and Western blotting analysis. (C) Relative quantification (n = 6 in each group) normalized to GAPDH and α-tubulin, respectively. **P < 0.01, significant difference between
the lean and obese groups.
(A)
Obese
◄ 25 kDa
STAT3
◄ 96 kDa
p-STAT3
◄ 85 kDa
-Tubulin
◄ 57 kDa
0.8
**
0.6
0.4
0.2
0.0
Lean
Obese
0.3
**
0.2
0.1
0.0
Lean
Obese
p-STAT3/-Tubulin
IL-6
STAT3/-Tubulin
IL-6/-Tubulin
(B)
Lean
**
4
3
2
1
0
Lean
Obese
Fig. 5. Analysis of IL-6 and STAT3 in lean and obese rats. (A) The protein levels of IL-6, STAT3 and p-STAT3 extracted from the
left ventricles of the hearts in three lean and three obese rats, as measured by Western blotting analysis. (B) Relative quantification normalized to α-tubulin (n = 6 in each group). **P < 0.01, significant difference between the lean and obese groups.
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Cardiac Hypertrophy in Obesity
(A)
Lean
Obese
MEK5
◄ 50 kDa
ERK5
◄ 120 kDa
p-ERK5
◄ 125 kDa
-Tubulin
◄ 57 kDa
MEK5
ERK5
**
1.0
**
0.8
0.6
0.4
0.2
0.0
Lean
Obese
**
6
p-ERK5/-Tubulin
MEK5/-Tubulin
ERK5/-Tubulin
(B)
4
2
0
Lean
Obese
Fig. 6. Analysis of MEK-5 and ERK-5 in lean and obese rats. (A) The protein levels in MEK5, ERK5 and p-ERK5 extracted from
the left ventricles of excised hearts from three lean and three obese rats were measured by Western blotting analysis. (B)
Relative quantification normalized to α-tubulin (n = 6 in each group). **P < 0.01, significant difference between the lean
and obese groups.
IL-6-Related Pathway
The IL-6 protein levels showed significant
(P < 0.01) increases in the cardiac tissues excised
from the obese rats compared with the age-matched
lean rats (Fig. 5). STAT3 and p-STAT3 protein levels
were significantly increased (P < 0.05) in the obese
rats compared with the lean rats (Fig. 5). MEK5,
ERK5 and p-ERK5 protein levels were significantly
increased (P < 0.05) in the cardiac tissues excised
from the obese rats compared with the lean rats (Fig.
6). These findings suggest that the IL-6-STAT3 and
IL-6-MEK5-ERK5 pathways are activated in obese
rats compared with those of the lean group.
Calcineurin-NFAT3 Hypertrophy-Related Pathway
The protein levels of calcineurin and NFAT3 also
showed significant increases (P < 0.01) in the cardiac
tissues excised from the obese rats compared with the
lean rats (Fig. 7).
Discussion
Our main findings can be summarized as
follows: [1] Increased systolic blood pressure, increased heart wall thickness, increased whole heart
weight, increased ratio of whole-heart weight to tibia
length, abnormal myocardial architecture, increased
cardiac interstitial spaces and increased serum leptin
levels were observed in the obese group compared
with the lean group; [2] The ANP, BNP and TNFα
hypertrophy-related markers in the cardiac tissues of
the obese group were significantly increased; [3] Cardiac IL-6-STAT3 and IL-6-MEK5-ERK5 hypertrophyrelated signaling pathways were more active in the
hearts of the obese rats; [4] Cardiac calcineurin and
NFAT3 hypertrophy-related signaling pathways were
more active in the hearts of the obese rats.
Obesity is often associated with hemodynamic
overload, ventricular remodeling and higher cardiac
output due to an augmented stroke volume and an increase in the heart rate (1, 3). Obesity cardiomyopathy
typically occurs in persons with severe and longstanding obesity, which may progressively develop left
ventricular hypertrophy (3). In the current study, cardiac hypertrophy was found in 5-6-month old obese
Zucker rats based on evidences from echography,
heart weight index and myocardial architecture. We
speculate that obese rats progressively develop dele-
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Chen, Yeh, Lin, Lin, Tzang, Lin, Yang, Wu, Tsai, Cheng, Huang and Lee
(A)
Lean
Obese
Calcineurin
◄ 61 kDa
NFAT3
◄ 190 kDa
-Tubulin
◄ 57 kDa
(B)
Calcineurin
Calcineurin/-Tubulin
NFAT3/-Tubulin
1.2
NFAT3
**
0.8
**
0.4
0.0
Lean
Obese
Fig. 7. Analysis of calcineurin and NFAT3 in lean and obese rats. (A) The protein levels in calcineurin and NFAT3 extracted from
the left ventricles of the hearts in the three lean and three obese rats were measured by Western blotting analysis. (B) Relative
quantification normalized to α-tubulin (n = 6 in each group). **P < 0.01, significant difference between the lean and obese
groups.
terious cardiomyopathic changes through multiple
internal or external factors. Various potential factors
that enhance cardiac hypertrophy include hypertension, volume overload, hypoxia, oxidative stress and
the unbalance of some hormones (5, 17, 35). Therefore, we have to add a note of caution that any effect
on cardiomyopathic changes noted in the present investigation cannot be clearly attributed to any one specific factors. Furthermore, the ratio of whole-heart
weight to tibia length, used for normalizing rodent
skeletal sizes in the current study, was increased in
obese rats whereas the ratio of whole-heart weight
to whole-body weight, traditionally regarded as an
index of cardiac hypertrophy, was unchanged due to
proportionally increased body weight in obesity. In
obesity, cardiac hypertrophic effects are underestimated if only the index of the ratio of whole-heart
weight to the whole-body weight is used.
ANP and BNP are useful markers of preclinical
cardiac disease in obese humans (14). Increased left
ventricular ANP and a two-fold increase in cardiomyocyte volume were reported in diabetic Zucker
fatty rats (13). The development of obesity and metabolic disturbance is strongly related to increased levels
in pro-inflammatory cytokines IL-6 and TNFα, both of
which are secreted by adipocytes; concentrations of
these cytokines correlate with the percentage and distribution of fat tissue in the body (39). TNFα expression and peptide production are up-regulated in the
adult heart in response to pressure overload and in
response to myocardial infarction or ischemia (41).
TNFα provokes a hypertrophic growth response in a
dose-dependent manner and increases net actin and
myosin heavy chain synthesis in cardiac myocytes (45).
Over-expression of TNFα in the heart of mice leads
to adverse cardiac remodeling (11). No other studies
have directly observed TNFα in the hypertrophic
hearts of obese animals or obese humans. Our findings demonstrate that the more active TNFα was
found in the cardiac hypertrophy in the obese rats
compared with the age-matched lean rats. This may
imply that more active TNFα may partially enhance
adverse cardiac remodeling.
IL-6, a typical cytokine, was found to have a
potent hypertrophic effect on cardiomyocytes (21).
The overexpressed IL-6 in the myocardium under hypoxia or other stressors appears to play an important
role in the pathogenesis of cardiovascular diseases
and cardiac hypertrophy (21, 22). Over-expression of
IL-6 has also been confirmed in the injured myocar-
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Cardiac Hypertrophy in Obesity
dium in coexistence with hypertrophy in patients died
of acute myocardial infarction (22). In the current
study, the increased protein levels of IL-6 in the cardiac tissues were found in obese rat hearts, suggesting
that IL-6 may be responsible for pathophysiologic
challenges in obesity. No other study has directly observed IL-6 in the hypertrophic hearts in obese animals
or in obese human cardiac tissues. Further studies
clarifying the role of IL-6 in obese myocardial cells
or obese humans are needed.
IL-6 is also involved in multiple intracellular
signaling pathways including MEK5 and its downstream ERK5. The IL-6-MEK5-ERK5 pathway is believed to induce cardiac hypertrophy that progresses
to pathophysiologic cardiomyopathy and sudden death
(6, 37). However, the underlying mechanism of the
cardiac remodeling in hypertrophy has not yet been
well recognized. In addition, no other studies have
observed the MEK5-ERK5 pathway in the heart of
obese animals or obese humans. Therefore, no further
information is available to compare with our findings.
In the current study, more active IL-6, MEK5 and
ERK5 levels were found in the dilated heart excised
from obese group, compared with the lean group. We
suggest here that cardiac hypertrophy in obesity may
be partially mediated by the IL-6-associated MEK5ERK5 hypertrophy-related pathway.
Previous studies have shown that cardiac hypertrophy is induced by the calcium-dependent phosphatase calcineurin, which dephosphorylates the transcription factor NFAT3 enabling it to translocate to
the nucleus (46). Transgenic mice that express activated forms of calcineurin or NFAT3 in the heart
develop cardiac hypertrophy and heart failure that
mimic human heart disease (33). Our findings
showed that calcineurin and NFAT3 in obese heart
tissues were more active, which may be related to
one of the hypertrophy-related signal pathways to
cause cardiac hypertrophy and heart failure in obesity (33).
The underlying mechanisms determining which
pathways to be activated in cardiac remodeling of hypertrophy in obesity have not yet been recognized. Our
current findings that indicate more active cardiac ANP,
BNP, TNFα, IL-6-MEK5-ERK5 and calcineurin-NFAT3
pathway in obesity may provide possible mechanisms
to explain the development of cardiac hypertrophy and
heart failure in obesity. For therapeutic applications
based on this work, we further propose that TNFα,
IL-6-MEK5-ERK5 or the calcineurin-NFAT3 pathway
should be de-activated in obesity to prevent the development of obesity-related cardiac abnormality or
hypertrophy. In some previous studies, exercise has
been found to have beneficial effects on cardiovascular health (15), such as reducing hypertension (7,
43) and cardiac apoptosis (20). Further studies, ex-
ercise therapy and other therapeutic approaches are
required to clarify the possible mechanisms or treatments in obesity-related cardiac hypertrophy and abnormalities.
Acknowledgments
The work was partly supported by grant
NSC100-2314-B-039-016-MY from the National
Science Council, Taiwan. This study was partly
supported by CMU98-Asia-08 from Asia University
and China Medical University to Dr. Hsiu-Chung
Ou, and was supported in part by Taiwan Department of Health Clinical Trial and Research Center
of Excellence (DOH101-TD-B-111-004). We would
like to thank Dr. Timothy Williams and Professor
KB Choo for proofreading the manuscript.
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