C dna array analysis prior to heart failure reveals an incr

Journal of Molecular and Cellular Cardiology 42 (2007) 526 – 539
www.elsevier.com/locate/yjmcc

Original article

NMR and cDNA array analysis prior to heart failure reveals an increase of
unsaturated lipids, a glutamine/glutamate ratio decrease and a specific
transcriptome adaptation in obese rat heart
J. Roncalli a , F. Smih a , F. Desmoulin b , N. Dumonteil a , R. Harmancey a , S. Hennig c , L. Perez a ,
A. Pathak a , M. Galinier a , P. Massabuau a , M. Malet-Martino b , J.M. Senard a , P. Rouet a,⁎
a

Unite de recherches sur les obesités, INSERM UPS U586, Institut Louis-Bugnard, Université Paul-Sabatier, CHU Rangueil,
BP 84225, 31432 Toulouse cedex 4, France
b
Groupe de RMN Biomédicale, Laboratoire SPCMIB (UMR CNRS 5068), Université Paul Sabatier, Toulouse, France
c
Deutsches Ressourcenzentrum für Genomforschung GmbH, Heubnerweg 6, D-14059 Berlin, Germany
Received 12 June 2006; received in revised form 13 October 2006; accepted 8 November 2006
Available online 11 January 2007

Abstract
Obesity is a risk factor for heart failure through a set of hemodynamic and hormonal adaptations, but its contribution at the molecular level is
not clearly known. Therefore, we investigated the kinetic cardiac transcriptome and metabolome in the Spontaneous Hypertensive Heart Failure
(SHHF) rat. The SHHF rat is devoid of leptin signaling when homozygous for a mutation of the leptin receptor (ObR) gene. The ObR−/− SHHF
rat is obese at 4 months of age and prone to heart failure after 14 months whereas its lean counterpart ObR−/+ is prone to heart failure after
16 months. We used a set of rat pangenomic high-density macroarrays to monitor left ventricle cardiac transcriptome regulation in 4- and 10month-old, lean and obese animals. Comparative analysis of left ventricle of 4- and 10-month-old lean rat revealed 222 differentially expressed
genes while 4- and 10-month-old obese rats showed 293 differentially expressed genes. 1H NMR analysis of the metabolome of left ventricular
extracts displayed a global decrease of metabolites, except for taurine, and lipid concentration. This may be attributed to gene expression
regulation and likely increased extracellular mass. The glutamine to glutamate ratio was significantly lower in the obese group. The relative
unsaturation of lipids increased in the obese heart; in particular, omega-3 lipid concentration was higher in the 10-month-old obese heart. Overall,
several specific kinetic molecular patterns act as a prelude to heart failure in the leptin signaling deficient SHHF obese rat.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Obesity; Heart failure; NMR; Transcriptome; Metabolome; Arrays

1. Introduction
Obesity is defined by excessive adipose mass, which is
characterized by a body mass index (BMI) over 30 kg/m2.
Obesity prevalence is reaching around 10% to 30% of the
population in industrialized countries and this rate is doubling
every 10 years [1]. Obesity is leading to enhanced mortality [2]
and cardiovascular morbidity by means of arterial hypertension
(HTA) and endocrino-metabolic abnormalities like type II
⁎ Corresponding author. Faculté de Médecine, Laboratoire de Pharmacologie, INSERM U586, 37 allées Jules Guesde, 31073 Toulouse cedex 7, France.
Tel.: +33 5 61 14 59 98; fax: +33 5 61 25 51 16.
E-mail address: Philippe.Rouet@toulouse.inserm.fr (P. Rouet).
0022-2828/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.yjmcc.2006.11.007

diabetes or dyslipidemia, that are associated in the X metabolic
syndrome [3]. Obesity was shown to be an independent factor
for cardiovascular risk [4]. A positive correlation was noticed
between overweight and cardiovascular mortality, even after
adjusting for other risk factors. Additionally, obesity is an
independent risk factor for heart failure (HF): a BMI over
30 kg/m2 doubles the risk for HF in both genders [5].
Few works have studied specifically the cardiac abnormalities associated with obesity. Late stage obesity is characterized
by left ventricular hypertrophy (LVH), that leads to metabolic
change [6], and also by the alteration of the vegetative control of
cardiac frequency [7]. Severity and onset of these abnormalities
suggest a specific role for obesity, independent from HTA, in
the development of cardiomyopathies.

J. Roncalli et al. / Journal of Molecular and Cellular Cardiology 42 (2007) 526–539

Adipose tissue is now considered as an endocrine organ that
secretes a number of hormones and cytokines that are
regrouped under the name “adipokines” [8,9]. The hypothesis
of a specific role for adipose tissue has been supported by the
fact that many adipose tissue secretions have been shown to be
involved in cardiovascular pathologies [10]. We recently used a
dog nutritional model (hypercaloric high fat diet: HFD) of
obesity-related hypertension to show early-and late-specific
cardiac genes regulated at the transcriptome level [11,12].
These transcriptome regulations are responsible, at least in part,
for the cardiac remodeling that leads to LVH. More recently,
using human heart samples (right appendage) representative of
the right auricle obtained from non-diabetic patients undergoing open heart surgery and heart bypass, we have shown
different cardiac gene regulations that are specific to obesity
and independent from obesity-associated hypertension [13].
These observations are consistent with cardiac remodeling in
response to obesity which could be mediated, at least in part, by
adipokines.
In this work, our aim was to evaluate the molecular
mechanisms involved in the development of a dilated
cardiomyopathy (DCM) during the onset of heart failure (HF)
and the contribution of obesity in the early steps of the
development of this disease. Therefore, we used the SHHF/
Mcc-cp rat model of obesity, non-insulin dependent diabetes
and congestive heart failure [14]. As obese rats are prone to
heart failure several months before their lean counterpart [14],
experiments were performed with 4 and 10 months old, lean and
obese animals; before any sign of heart failure. We analyzed in
parallel the cardiac transcriptome and metabolome using a
cDNA array and an NMR approach respectively.
2. Materials and methods

527

After shaving of the chest, echocardiograms were performed
by using the Vivid 7 pro 7 echocardiographic system (GE
Medical System), equipped with a i13 L 14-MHz linear-array
transducer. Images were obtained from rats lightly anesthetized
by 1–2% isoflurane (AErrane, Baxter) lying on their back side
with transducer placed on the left hemithorax. Two-dimensional
parasternal long- and short-axis images of the left ventricle were
obtained, and two-dimensional targeted M-mode tracings were
recorded at a sweep speed of 200 mm/s. All measurements were
performed according to the recommendations of the American
Society for Echocardiography leading-edge method from three
consecutive cardiac cycles (n) with the roundness of the left
ventricular cavity (2D-image) as a criterion that the image was
on axis, great effort was taken to achieve a good image quality to
visualize the endocardial and epicardial borders of the heart by
gently moving and angulating the transducer. Measurements and
calculations used are as follows: percent LV fractional
shortening (FS), a measure of LV systolic function, was calculated as follows: FS = (EDD − ESD) / EDD × 100, where EDD
and ESD are end-diastolic and end-systolic diameters, respectively. All values were averaged over three consecutive cycles.
Twice a month, weight, systolic blood pressure (SBP) and
heart rate (HR) were measured in both lean and obese groups.
SBP and HR were recorded on vigil animals, after 15 min rest,
using a PowerLab System apparatus (ADInstruments, Australia). Attention was given to the possible appearance of CHF
symptoms such as subcutaneous oedema, hydrothorax, ascites,
dyspnea and cyanosis. At the end of the experiment, animals
were anesthetized, and surgery was quickly performed. Left
ventricular and right auricle samples were taken from 5 lean and
5 obese rats at the age of 4 months or 10 months. Samples were
immediately washed in cold Phosphate Buffer Saline Buffer,
snap frozen in liquid nitrogen and maintained at − 80 °C until
analysis.

2.1. Animals and general procedures
2.2. RNA extraction and cDNA labeling
All animal procedures were performed according to the
guidelines of the French Ministry of Agriculture. Rats were
housed at the Toulouse IFR31 animal facility in a room lit 12 h
per day (6 AM–6 PM) at an ambient temperature of 22 ± 1 °C.
Animals were allowed 1 week to adjust after arrival and had
access to regular rodent diet and tap water ad libitum during the
experiment.
Ten lean heterozygous (+/cp) and 10 obese homozygous
(cp/cp) Spontaneously Hypertensive and Heart Failure-prone
rats (SHHF), were provided by Charles River Laboratories
and included in the study. This strain was obtained by backcrossing a Koletsky obese rat to a Spontaneously Hypertensive Rat (SHR/N). Obese males develop congestive heart
failure (CHF) at 10–14 months, lean males at 14–18 months
[14,15]. From the Koletsky rat, SHHF rats harbor a nonsense
mutation of the leptin receptor gene, designated fa, resulting
in a premature stop codon in the leptin receptor. This mutation
is a recessive autosomic trait. As a consequence, cp/cp
homozygous animals for the fa mutation are obese. Lean rats
are heterozygous for this fa mutation or wild type for the cp
locus.

Total RNA was isolated from left ventricular samples; quality
check, concentration control and labeling were performed as
previously described [13].
2.3. cDNA array hybridization and expression analysis
cDNA array hybridization, washes, and expression analysis
were performed as previously described [13] using pangenomic macroarrays membranes (RZPD, Berlin) containing
26, 592 unique rat cDNA sequences spotted in duplicate. This
pangenomic array contained both known genes and nonannoted genes that required further computing analysis.
Therefore, to achieve the maximum information for the
respective probes on the membrane, the corresponding
accession numbers were used to identify both the respective
NCBI-Unigene clusters [16] and the respective EST contigs
provided by the TIGR gene index [17].
To limit the number of false positive results and to increase
specificity of our data, we used the following selection criteria
to determine whether the expression of a gene is up-regulated

528


or down-regulated in a statistically significant manner: (1)
expression ratio of obese/lean was > 1.5 or <0.67, (2) expression
level was required to be over 3 times above membrane
background after local background subtraction (membrane
background was calculated by averaging the signal intensity
on empty spots i.e. generated by buffer deposition from DNA
free wells), (3) the difference between 2 experimental settings
was statistically significant by Student's t test, (4) attention was
given to avoid rejecting eventual highly induced genes with
initially low expression level (under 3 times the membrane
background). Statistical relevance of biological data and realtime PCR results was assessed with Student's t test or Mann–
Whitney rank sum test when normality test failed, using
SigmaStat 3.0 software (SPSS).
Hierarchical clustering was performed on normalized to the
mean intensity of all spots X-dot reader dataset with MultiExperiment Viewer 2.2 software (TIGR) [18]. The clustering
was performed on the average linkage clustering method for the
genes and the arrays.
2.4. Real-time PCR control of differential expression
A set of genes was randomly chosen for real-time PCR
validation of the observed differential expression [19]. Oligos
were synthesized by Proligo Company and designed with
Primer Express 2.0 software (supplemental Table 1 on-line).
Real time PCR was performed as previously described [11–13];
gene expressions were normalized to 18S RNA quantification
which has been found to be a reliable internal control gene in
our hands and others [20,21]. Real-time PCR data were
statistically analyzed with SigmaStat software (SPSS Science).
2.5. Heart left ventricular (LV) tissue dual phase metabolites
extraction
Frozen sections of LV (n = 20, 90 ± 39 mg) were powdered in
liquid nitrogen with a mortar and pestle and then immediately
subjected to a simultaneous extraction of lipids and watersoluble metabolites [22]. Briefly, frozen tissue fragments were
homogenized for 20 s in ice-cold purex-analytical-grade
methanol and chloroform in a 2:1 ratio (1.5 ml) by using an
Ultra-Turrax homogenizer. After 15 min in contact with the first
solvents at 4 °C, 0.5 ml chloroform and 0.5 ml distilled water
were added and homogenized to form an emulsion. The samples
were then centrifuged at 2000 × g for 30 min, 4 °C. The aqueous
phase was then separated from the organic phase. Both fractions
were dried in a Speed Vac concentrator. Extracts were maintained at − 80 °C until their preparation for NMR analysis. Prior
NMR analysis, extracts were reconstituted in 600 μl of D2O with
10 μl of a 10 mM 3-(trimethylsilyl)-1-propanesulfonate sodium
salt (TMPS) solution and 600 μl of CDCl3 / MeOD (2:1, v/v) for
aqueous fractions and organic fractions, respectively.
2.6. NMR analysis
1

H NMR spectra were recorded on a Bruker Avance 400
NMR spectrometer, equipped with a z-gradient 5 mm TBO

probe. Fully relaxed 1H spectra (without saturation effects)
were obtained in 12 min by accumulating 128 FID resulting
from 30° excitation pulses. Typical acquisition parameters were
a SW of 9.8 kHz, 32 K data points and 2.5 s repetition time. For
analysis of aqueous fractions, a presaturation pulse sequence
was used to suppress the residual intensity of the 1H water
resonance peak. Typical processing parameters were 65 K zerofilling and a LB of 1 Hz applied prior to Fourier transform.
Characteristic metabolites [23–25] and lipid signals [26–28]
have been assigned by reference to literature data and on the
basis of 2D homonuclear correlation spectroscopy (COSY)
experiments performed on the extracts (data not shown). The
signal of TMPS (δ = 0 ppm) for water-soluble metabolite and
residual CHCl3 (δ = 7.36 ppm) for lipids served as references
for chemical shift and concentration. A 32 mM concentration
of residual CHCl3 was previously determined with trichlorobenzene as standard for our batch of deuterated chloroform
[29]. Only resonances giving a signal to noise ratio over 15
were taken into account for statistical analysis. The number of
protons giving rise to a signal was considered in the
calculations of relative and absolute concentrations. Signals
to quantify water-soluble metabolites (chemical shift value in
ppm, and relative number of protons) were as fellows: lactate
(1.33, 3); alanine (1.48, 3) acetate (1.92, 3); glutamate (2.35,2);
glutamine (2.44, 2) creatine + phosphocreatine (3.04, 3), taurine
(3.41, 2; this resonance was chosen according to its specificity);
TMPS (0, 9); see also Fig. 3. Concentration of fatty acyl chains
was determined using the area of the α-methylene resonances
at 2.31 ppm as 100% of fatty acyl chains (peak 8 in Fig. 4).
Cholesterol was quantified using the area of the C-18 methyl
singlet at 0.67 ppm (peak 1 in Fig. 4). n-3 fatty acyl chains
were quantified using the characteristic triplet at 0.96 ppm of
the terminal-CH3 (peak 3 of Fig. 4). Total choline phospholipids were determined from the trimethyl group at 3.20 ppm
(peak 10 in Fig. 4). Mean unsaturation was calculated as the
ratio of vinyl from fatty chain (–CHfCH–) determined from
the signal at 5.35 ppm (peak 14 in Fig. 4) minus the
contribution of cholesterol proton, to the signal of total fatty
chains (peak 8 in Fig. 4). Mean poly-unsaturation was
calculated as the ratio of the signal of the allylic methylene
at 2.80 ppm (peak 9 in Fig. 4) to the signal of total fatty acyl
chains.
3. Results
3.1. Rat phenotype analysis
CHF symptoms such as subcutaneous oedema, hydrothorax,
ascites, dyspnea and cyanosis were not observed in lean and
obese 4- and 10-month-old animals. Four-month-old obese rats
had an average weight of 508 ± 12 g versus 386 ± 2 g for the lean
rats: this 32% increase in weight is statistically significant
(Student's t test, p = 0.008) (Table 1). At 10 months these
differences in weight were even more evident as obese rats had
an average weight of 727 ± 10 g and lean 477 ± 5 g; p ≤ 0.001).
Clearly, obesity was due to increased fat mass as reported for
this rat model [14].


529

Table 1
Body and heart weight, blood pressure, BNP levels and cardiac echographic analysis
SHHF 4 months
Lean (n = 5)
Body weight (g)
Heart weight (g)
SBP (mm Hg)
BNP (ng/ml)
Diastolic septal wall thickness (mm)
Diastolic Posterior wall thickness (mm)
Systolic septal wall thickness (mm)
Systolic Posterior wall thickness (mm)
LV End diastolic diameter EDD (mm)
LV End systolic diameter ESD (mm)
LV Fraction shortening FS (%)

386 ± 2
1.27 ± 0.04
212 ± 7
3.8 ± 0.9
1.90 ± 0.16
2.15 ± 0.39
3.21 ± 0.18
3.41 ± 0.52
7.73 ± 0.61
4.30 ± 0.65
44.37 ± 6.70

SHHF 10 months
Obese (n = 5)

Lean (n = 5)

Obese (n = 5)

†

508 ± 12*
1.35 ± 0.02
204 ± 4
6.2 ± 2.2
1.78 ± 0.14
2.15 ± 0.34
3.18 ± 0.20
3.16 ± 0.37
7.78 ± 0.55
4.54 ± 0.49
41.65 ± 5.77

727 ± 10*,†
1.88 ± 0.09†
218 ± 2*,†
9.66 ± 2.3
2.18 ± 0.13*
2.32 ± 0.26
3.53 ± 0.26*
3.32 ± 0.27
8.56 ± 0.57*
5.20 ± 0.57*
39.25 ± 4.36

477 ± 5
1.7 ± 0.04†
207 ± 3
6.7 ± 2.8
2.13 ± 0.19*
2.08 ± 0.23
3.35 ± 0.25
3.2 ± 0.31
8.23 ± 0.48
4.87 ± 0.57
39.36 ± 7.26

Values statistically relevant are indicated by * for p < 0.05 (obese versus lean) and by † for 10-month-old versus 4-month-old statistical analysis (Student's t test). Data
are mean of five measurements per group ± S.E.M. Left ventricle (LV) percent left ventricular fractional shortening (FS) was calculated as follows: FS = (EDD − ESD) /
EDD × 100, where EDD and ESD are end-diastolic and end-systolic diameters, respectively. *p < 0.05 with Student's t test for obese or lean rats at 4-month-old versus
10-month-old.

Macroscopic examination of the heart did not reveal any
abnormalities or any cardiac infarction lesions. No significant
difference in weight were observed between lean and obese
average weight of the hearts in 4- or 10-month-old rats
(respectively 1.27 ± 0.04 g versus 1.35 ± 0.02 g; and 1.7 ±
0.04 g versus 1.88 ± 0.09 g). Nevertheless, heart mass significantly
increased in 10-month-old versus 4-month-old lean or obese rats.
Systolic blood pressure (SBP) was not statistically
significantly different: 212 ± 7 mm Hg in 4-month-old lean
rats versus 204 ± 4 mm Hg in obese rats. In 10-month-old
animals, SBP (207 ± 3 mm Hg) was maintained in lean rats
and was significantly increased in obese (218 ± 2 mm Hg).
Cardiac frequencies were similar in each group of animals
(data not shown). Moreover, we did not observe any clinical
sign of HF in 10-month-old animals (Table 1).
Echocardiographic analysis of obese SHHF and lean SHHF
animals demonstrated similar concentric LVH characterised by
increased wall thickness with similar LV cavity dimensions (Table
1). LV systolic function assessed by LV fractional shortening was
similar in both groups (Table 1) at 4 and 10 months. However, we
noted that in obese rats, there was an increase in diastolic and
systolic septal wall thickness and not in posterior wall thickness
between 4 and 10 months. End diastolic and systolic diameters
were also increased in obese rats. Moreover, echocardiographic
analysis did not reveal any left ventricular dysfunction.
3.2. Left ventricle transcriptome analysis
We first determined the number of detectable genes using
our cDNA macroarrays. Once the local background was
subtracted, we were able to detect an average of 9529 out of
26,592 genes with a signal greater than 1-fold and up to 37fold over the mean membrane background level (data not
shown). 7 out of 8 differentially expressed genes harboring
an expression level between 1.95 and 17.9 fold over
background were confirmed by Realtime PCR (87%
validation) (Table 2). According to this observation and previously published works [30], we defined an expression level

that had to be over 3 fold over background to be the minimum expression level for which we could reliably monitor
differential gene expression. The following data take this
parameter into account.
3.2.1. 10-month-old versus 4-month-old lean and obese rat
ventricle transcriptome analysis
Comparison of gene expression between 10-month-old
animals and 4-month-old animals revealed 222 differentially
Table 2
Comparison of DNA array analysis and real-time PCR
Gene name or GeneBank # Macroarray
Expr.
Ratio
level/bgd
Lean analysis
AA 858 801/NFKB1
AA963 792
AA 998 657
Obese analysis
AA 875 581/MRLCB
AI 028 924
AI 054 986
Gene common to lean and
obese analysis
AA 924 587
AA 819 584/ATP2A2

12.2
17
10

1.95
8.9
9.2

p

Ratio

p

10 m/4 m
10 m/4 m
2.59
0.0002 1.51
0.002
2.41
0.002 1.43
0.090
2.57
0.002 2.47
0.001
10 m/4 m
10 m/4 m
2.42
0.0014 2.26
0.017
2.65
0.017 2.46
0.003
2.87
0.01
2.16
0.018
10 m/4 m

4.36
17.9

rt-PCR

3.05
1.99

10 m/4 m
0.0005 1.63
0.005 2.09

0.038
0.038

Differential expression validation by realtime PCR for a set of randomly chosen
genes. Induction (if > 1.5) and repression (if < 0.6) ratios are represented. Mean
values are indicated for 5 measurements. cDNA macroarrays analysis were
performed on left ventricle (LV). m: months. Genes were randomly chosen
among those found to be statistically differentially regulated by Bioplot analysis
and harboring at least a 2 fold over the background expression intensity value.
Expr. level/bgd: ratio of signal intensity for a gene/mean background intensity.
Ratio Obese/Lean: signal intensity in Obese group/signal intensity in Lean
group. Ratio 10m/4m: signal intensity in 10-month-old group/signal intensity in
4-month-old group. NFKB1: nuclear factor kappa B p105 subunit; MRLCB:
myosin regulatory light chain; ATP2A2: ATPase, Ca2+ transporting, cardiac
muscle, slow twitch 2.

530


clustering analysis, identity of 9 genes could be provided in the
main cluster (Fig. 2B and Table 4B).
3.3. Left ventricle metabolome analysis

Fig. 1. Differential expression analysis schematic diagram. Number of
differentially expressed genes is indicated. Arrows indicate the statistical
analysis of the data performed using Student's t test. 102 genes were found to be
common to the differentially expressed genes list from the obese and the lean
animals, 222 and 293 genes were found differentially expressed between 4- and
10-month-old lean and obese animals respectively.

expressed genes (17 repressed and 205 over-expressed) and 293
differentially expressed genes (30 repressed and 263 overexpressed) in lean and obese rats, respectively (Fig. 1).
Homologies searches generated a list of 95 defined genes
from the lean rat analysis and 132 defined genes from the obese
rat analysis (Table available as a supplementary data on line).
3.2.2. 10 months versus 4 months differential expression of a
set of cardiac-relevant genes
We identified a set of well-known function genes common to
lean and obese analysis (Table 3). These genes differentially
expressed between 4- and 10-month-old animals are indicators
of: neurohormonal activation (NPPA, ACE, ECE 1); apoptosis
(CASP1); inflammation (Il-6 receptor), fibrosis (TGF-β);
energetic metabolism (SLCA1, SLC25A10); hypertrophy
(GATA4); structure (MYH 6).
3.2.3. Hierarchical clustering of differentially expressed genes
Hierarchical clustering organization provided us with a
global view of the changes in cardiac gene expression induced
by the duration of hypertension in lean rats and the combination
of hypertension and obesity in obese rats. In addition,
hierarchical clustering was efficient at grouping gene expression profiles correctly without any intervention. Thus, gene
expression profiles were specific to the age of the lean or obese
SHHF rat hearts. Interpretation of the data is challenging for
some clusters due to a limited number of differentially
expressed genes, which limited cluster size. Nevertheless, one
could consider the set of 17 and 30 down-regulated genes in
10 months, lean (Fig. 2A) and obese (Fig. 2B) rats, respectively.
We first focused our attention on genes co-regulated in lean
animals and down regulated in 10-month-old animals (cluster 1,
Fig. 2A). We obtained gene identities or homologies for 11
genes out of 17 in this cluster (Table 4A). For obese animal

Characteristic signals arising from metabolites such as
creatine + phosphocreatine, taurine, glutamine, glutamate, acetate, alanine and lactate were detected in the 1H NMR spectrum
of the aqueous fraction of LV heart extract (Fig. 3).
Concentration of metabolites and statistical analysis of the
data showed a set of differences (Table 5). These differences
are related to the age and/or the obesity status. A significant
decrease in creatine and glutamate concentration was observed
and exclusively correlated to the age. Taurine concentration
was significantly decreased in the 10-month-old lean group
while its concentration was unchanged in other groups.
Glutamine concentration was significantly increased in the
lean group compared to that in the obese group. Moreover,
glutamine concentration was correlated to the age of rats and
significantly decreased in the 10-month-old lean group
compared to that in the 4-month-old lean group. The glutamine
to glutamate ratio was lower in obese animals, independent of
age.
A 1H NMR spectrum of total lipid extract of an obese SHHF
heart rat is shown in Fig. 4. Characteristic signals of lipid
moieties allowed the determination of total fatty acyl chains,
cholesterol, choline phospholipids (Table 6). Moreover, mean
unsaturation which represents the number of vinyl moieties per
fatty acyl chain and the mean poly-unsaturation which
represents the number of diallylic methylene per fatty acyl
chain were calculated. Choline phospholipids, cholesterol and
fatty acyl chains contents were similar at the same ages in
the obese group and in the lean group. Surprisingly, we

Table 3
Differentially expressed genes selection, according to their well-known
function, resulting from lean and obese SHHF 4-month-old/10-month-old
comparison
Gene name and Gene bank
accession #

Expr.
level/bgd

Ratio

p (Bioplot)

IL-6 R/AA 963 567
CASP 1/AI 071 441
GATA 4/AA 997 121
TGFB/AI 548 079
NPPA/AA 819 343
ACE/AI 556 575
ECE 1/AA 817 947
MYH 6/AA 819 464
FN 1/AA 955 600
SLC1A1/AA 996 752
SLC25A10/AA 859 666

4.26
9.27
3.51
3.26
3.04
5.68
3.21
4.87
14.2
5.31
3.15

1.74
2.14
2.38
1.64
1.67
1.66
2.11
0.58
1.88
2.03
0.56

0.006
0.038
0.027
0.033
0.0061
0.010
0.039
0.042
0.038
0.044
0.040

Expr. level/bgd: ratio of signal intensity for a gene/mean background intensity.
Ratio: signal intensity in 10-month-old group/signal intensity in 4-month-old
group. IL-6 R: InterLeukine 6 Receptor. CASP 1: Caspase 1. GATA 4: GATA
binding protein 4. TGFB: Transforming Growth Factor Beta. NPPA: Natriuretic
Peptide Precursor type A. ACE: Angiotensin Converting Enzyme. ECE 1:
Endothelin Converting Enzyme 1. MYH 6: Myosin Heavy chain polypeptide 6
(Myosin Heavy Chain alpha). FN 1: FibroNectine 1. SLC1A1: solute carrier
family 1, member 1. SLC25A10: solute carrier family 25, member 10.


531

Fig. 2. Hierarchical cluster analysis of differential expression. (A) Clustering performed with lean transcriptome analysis data. (B) Clustering performed with obese
transcriptome analysis data. 1 to 5: 4-month-old animals; 6 to 10: 10-month-old animals. Genes close to each other harboring correlated expressions are illustrated by
the tree on the left side. Genes in cluster 1 from lean and obese analysis are detailed in Table 4.

532


Fig. 2 (continued).


533

Table 4
Genes identities for cluster 1 from Figs. 2A and B
#Gene ID

Gene name

Ratio

4 months

10 months

p-value

Identities and homologies

(A) Gene co-regulated in lean animals and down regulated in 10-month-old animals (cluster 1, Fig. 2A)
AA926219
B4GALT1
0.54
1.42
0.77
0.00025917
UDP-Gal:betaGlcNAc beta 1.4-galactosyltransferase.
Polypeptide 1 (Rattus norvegicus)
AI454214
0.59
1.54
0.91
0.04882843
No sequence homology
AA900596
CSN4
0.27
1.43
0.38
0.04986411
Similar to COP9 complex subunit 4 (Homo sapiens)
AA957598
0.27
1.68
0.46
0.03521052
AI556930
PKD1
1.52
1.93
2.93
0.01708796
Polycystic kidney disease 1 (Pkd 1) and tuberous
sclerosis 2 (Tsc2) genes (Homo sapiens)
AI556150
0.66
1.49
0.98
0.04582136
AA900578
ALAD
0.6
1.5
0.91
0.03495742
Aminolevulinate. delta-. dehydratase (Alad)
(Rattus norvegicus)
AA925164
0.63
1.47
0.93
0.00010271
AI547418
0.64
1.85
1.19
8.90e − 05
Similar to XP_141317.3 RIKEN cDNA A430105I19
gene (Mus musculus)
AA925172
FGFR1OP
0.65
1.39
0.9
0.00086072
FGFR1 oncogene partner (Homo sapiens)
AA957860
0.66
1.49
0.98
0.00555646
AI575762
CABP1
0.61
1.55
0.95
0.00496229
Calcium binding protein 1 (Cabp1) (Rattus norvegicus)
highly similar to NP_839983.1
sorting nexin 26 [Mus musculus]. Human trichohyalin.
Potential multiple
AI454874
THH
0.63
1.44
0.9
0.00654817
Roles as a functional EF-hand-like calcium-binding
protein. A cornified cell envelope precursor and an
intermediate filament-associated (cross-linking) protein
(Homo sapiens)
AI136136
CHMP1.5
0.62
1.55
0.95
0.00646823
CHMP1.5 protein (Homo sapiens) function: putative
vesicle trafficking with VPS4 and putative chromatin
structure regulation in the nuclear matrix
AI555253
0.62
1.54
0.96
6.78e − 05
Similar to KIAA1632 prot. proline–serine–threonine
phosphatase interacting protein 2 (Homo sapiens)
AA925236
0.64
1.61
1.03
0.00126788
AA956865
SEB4D
0.66
1.6
1.06
0.0010949
Similar to dJ259A10.1 (ssDNA binding protein (SEB4D).
RNA-binding region (RNP1. RRM) (Homo sapiens)
(B) Genes co-regulated in obese animals and down regulated in 10-month-old animals (cluster 1, Fig. 2B)
AA859561
0.08
1.51
0.11
0.04220966
AA900791
CFI
1.54
1.7
2.62
0.04129383
Complement factor I. (Rattus norvegicus)
AA925981
0.09
1.48
0.14
0.00609678
Similar to 2410001C21Rik protein (Homo sapiens)
chromosome 20 open reading frame 43
AI501277
0.52
1.45
0.75
0.00210187
AI136302
0.55
1.57
0.86
0.00989101
AA926219
B4GALT1
0.56
1.55
0.87
0.00531375
UDP-Gal:betaGlcNAc beta 1.4-galactosyltransferase.
polypeptide 1 (Rattus norvegicus)
AA925093
0.58
1.45
0.84
0.00311088
AI043693
0.63
1.53
0.96
0.00859156
AA925880
SPOCK2
0.58
2.16
1.25
0.00768181
Similar to testican-2 protein (LOC361840)
AA858560
0.49
1.79
0.88
0.00540406
AA859429
0.59
1.63
0.96
0.01117883
Similar to hypothetical protein MGC31967(function:
translation initiation factor activity) (Rattus norvegicus)
AI145122
Centa2
0.62
1.65
1.03
0.03227733
Centaurin-alpha2 protein (Centa2) (Rattus norvegicus)
AI136080
0.63
1.73
1.09
0.037447
Similar to zinc finger protein 198
AI145732
0.6
1.8
1.08
0.01230905
AA858454
0.65
1.71
1.12
0.02969404
Transcribed locus. Moderately similar to XP_346694.1
hypothetical gene supported by NM_022857
AI715210
KS1
0.64
1.5
0.96
0.0019953
KRAB/zinc finger suppressor protein 1
(KS1)(LOC246264)(Rattus norvegicus)
AI113019
0.57
2.21
1.25
0.00374868
AI043993
0.62
1.45
0.9
0.00679676
AI112905
0.65
2.22
1.45
0.00955169
AI145673
0.65
1.97
1.28
0.00586599
Transcribed locus. Weakly similar to XP_346694.1
hypothetical gene supported by
NM_022857
AI030972
0.48
1.58
0.75
0.00063431
(continued on next page)

534


Table 4 (continued)
#Gene ID

Gene name

Ratio

4 months

10 months

p-value

Identities and homologies

(B) Genes co-regulated in obese animals and down regulated in 10-month-old animals (cluster 1, Fig. 2B)
AA900732
0.61
1.45
0.88
0.00367707
Transcribed locus. Similar to NP_080656.1
RIKEN cDNA 6530413N01 gene (Mus musculus)
AI060068
FABP3
0.62
1.44
0.9
0.00212252
Fatty acid binding protein 3 (Fabp3)
(Rattus norvegicus)
AA926237
TNNI3
0.66
2.06
1.36
0.01630635
Troponin 1. Type 3 (Tnni3) (Rattus norvegicus)
AI029660
0.52
1.56
0.82
0.00171603
AA858808
0.63
2.09
1.32
0.0010659
AA858866
NT5
0.6
1.81
1.09
0.00320482
5 Nucleotidase (Nt5) (Rattus norvegicus)
AA925124
KIR7.1
0.63
1.98
1.24
0.00392794
Inward rectifier potassium channel Kir7.1
[(Rattus norvegicus)
AI137849
0.66
1.57
1.04
0.01128975

noticed a lower fatty acid chain concentration in 10-monthold animals when compared to the 4-month-old animals that
was significant for the obese group. Obese animals displayed
a higher level of n-3 fatty acid chains when compared to
their lean counterparts. In accordance with these observations, 10-month-old obese animals displayed enhanced
unsaturation and poly-unsaturation. Unsaturation levels
increased with age only in obese animals. Therefore,
increased unsaturation is related to the obesity state and its
duration.
4. Discussion
We combined cDNA macroarrays with NMR metabolic
profiling to characterize the cardiac transcriptome and metabolome of two groups of SHHF rats at 4 and 10 months of age

which differed in their state of obesity and the associated X
metabolic syndrome [14,15]. Our main objective was to define
the molecular adaptation in heart at the onset of heart failure
development and to evaluate the impact of obesity on the
mechanism of heart failure development. Prior to the molecular
adaptations analysis, we examined the biological parameters of
the subject animals. As previously described, the obese rats,
homozygous for the fa mutation, displayed a significant
increase of weight (+ 32% at 4 months; + 52% at 10 months;
Table 1).
However, we noticed two major distinctions when analyzing
the biological parameters. First, both groups of animals were
hypertensive and 10-month-old obese animals were significantly more hypertensive than their lean counterparts. This may be a
consequence of the obesity. Second, 10-month-old obese
animals were expected to be at the onset of a DCM [14] but

Fig. 3. 1H NMR spectrum of a water-soluble fraction of a heart extract from a lean SHHF rat. Within the aliphatic region (−0.2 to 4.5 ppm) of the NMR spectra,
resonances have been assigned to lactate (Lac), alanine (Ala), acetate (Acet), glutamate (Glu), glutamine (Gln), creatine + phosphocreatine (Cr + PCr), taurine (Taur),
Sodium 3-(trimethyl-silyl)-1-propanesuffonate (TMPS); pH = 7.4.


535

Table 5
Quantitation of water-soluble metabolites by 1H NMR
SHHF 4 months
Lean
Taurine
Creatine + Phoshocreatine
Glutamate (Glu)
Glutamine (Gln)
Gln/Glu ratio
Acetate
Alanine
Lactate

SHHF 10 months
Obese

a

33.70 ± 3.55
16.55 ± 1.20 a
5.72 ± 0.56 a
7.82 ± 0.71 a
1.37 ± 0.12
1.70 ± 0.44
2.20 ± 0.12 a
14.49 ± 5.09

Lean

Obese

34.71 ± 5.98
15.24 ± 2.72 a
5.75 ± 0.93 a
6.56 ± 1.30 b
1.14 ± 0.10 b
1.62 ± 0.55
2.07 ± 0.05 b
21.45 ± 9.06 a

28.48 ± 3.32
12.19 ± 1.37
4.43 ± 0.76
6.71 ± 0.78
1.53 ± 0.23
1.71 ± 0.65
1.75 ± 0.32
11.03 ± 1.72

33.01 ± 3.52 b
10.98 ± 1.27
4.53 ± 0.83
5.47 ± 0.89 b
1.21 ± 0.09 b
1.14 ± 0.37
2.09 ± 0.44
10.26 ± 1.39

Data are means ± S.E. (in nmol/mg wet wt) from 5 hearts in each group.
a
Obese 4-month-old is significantly different from obese 10-month-old, or lean 4-month-old is significantly different from lean 10-month-old, Student's t test
p < 0.05.
b
Obese is significantly different from lean of the same age, Student's t test p < 0.05.

we did not observe any SBP normalization in this group of
animals. This is evidence for the lack of development of a DCM
in these 10-month-old animals. Nevertheless, our observations
are in accordance with previously published data [15]. Hearts
weights at 4 and 10 months of age were not significantly
different in the obese and lean groups. Thus, 10-month-old heart
weights were in accordance with published data [14] and
correspond to LVH hearts when compared to normotensive
Wistar–Kyoto or Sprague–Dawley rats of this age [14,31]. At
10 months of age we did not notice a more pronounced
macroscopic LVH in obese hearts when compared to lean
hearts. This lack of macroscopic differences was also reflected
in the BNP levels that were not significantly increased by

obesity in 10-month-old rats (Table 1). However, echocardiographic analysis displayed an increased diastolic and systolic
septal wall thickness in obese 10-month-old animals (Table 1).
This slight structural remodeling is likely a consequence of
blood volume increase observed in obese animals. Alternatively, it could be the result of altered kinetic gene regulations which
were observed between lean and obese animals, as discussed
below.
We confirmed differential expression of 2 fold above
background (Table 2 and data not shown), which is common
in microarray analysis. Therefore, to eliminate false positives
and focus on truly differentially expressed genes we set the limit
for reliable analysis of gene expression at 3-fold over

Fig. 4. 1H NMR spectrum of a total lipid extract from an obese SHHF heart rat. Main peaks or regions are assigned as follows: 1, Cholesterol (Chol, C18); 2, CH3
terminal of fatty acyl chain (FA) and Chol (C26, C27); 3, characteristic triplet of CH3 terminal of (n-3) polyunsaturated FA; 4, Chol (C19); 5, (CH2)n of FA; 6, –CH2–
CH2–COO–; 7, –CH2–CHfCH–CH2–CHfCH–CH2–; 8, –CH2–COO–; 9, –CHfCH–CH2–CHfCH–; 10, –N+(CH3)3 of phosphatidylcholine; 11, –CH2N+ of
phosphatidylcholine; 12, mainly glycerol (C1 and C3) and –O–CH2–CH2N+ of phosphatidylcholine; 13, esterified glycerol (C2); 14, –CHfCH– of FA and Chol
(C6); S, solvents (methanol + H2O).

536


Table 6
Quantitation of lipids by NMR
SHHF 4 months
Lean
Acyl chains (nmol/mg w.w.)
(n-3) Acyl chains %
Cholesterol (nmol/mg w.w.)
Choline phospholipids (nmol/mg w.w.)
Mean unsaturation
Mean poly-unsaturation

103.49 ± 11.28
8.80 ± 0.56
5.09 ± 0.33
14.54 ± 1.00
1.70 ± 0.09
1.17 ± 0.06

SHHF 10 months
Obese
117.49 ± 16.87
10.35 ± 0.79 b
5.33 ± 0.95
15.72 ± 2.06 a
1.78 ± 0.12
1.23 ± 0.08 a

Lean
a

Obese

90.74 ± 6.11
8.11 ± 1.25
4.61 ± 0.62
13.57 ± 0.89
1.78 ± 0.10
1.23 ± 0.07

84.49 ± 4.73
9.52 ± 0.62 b
5.11 ± 0.78
12.72 ± 082
1.90 ± 0.02 b
1.33 ± 0.02 b

Data are means ± S.E. from 5 hearts in each group.
a
Obese 4 months is significantly different from obese 10 months, or lean 4-month-old is significantly different from lean 10-month-old, Student's t test p < 0.05.
b
Obese is significantly different from lean of the same age, Student's t test p < 0.05.

background. According to previously published work [30], this
lowers the false positive rate to 0.7%. According to these
criteria, we did not have any evidence of an effect of obesity in
4- or 10-month-old animals. However, macroarrays analysis on
a set of known cardiac genes recapitulated the differential
expression of genes involved in hypertension and the development of heart failure (Table 3).
Hierarchical clustering analysis of gene expression from lean
animals revealed in the same main cluster 3 genes out of 11
encoding proteasome proteins COP9 subunit 4 (CSN4),
aminolevulinate deltadehydratase (ALAD) and CHMP1.5
(Table 4A) PKD1 is a cation channel regulator [32], a complex
involved in many regulatory processes, including control of
development and regulation of morphogenesis. Two genes, UDPGal:betaGlcNAc beta 1,4-galactosyltransferase (B4GALT1) and
trichohyalin (THH), encode for proteins that have a structural
role. UDP N-acetylglucosamine β-1,4 galactosyltransferase is
a widely distributed enzyme which catalyzes the transfer of
galactose to N-acetylglucosamine residues of glycoproteins
and glycolipids [33]. Elevated B4GALT1 expression was
already shown to occur in the failing hearts of spontaneously
hypertensive rats [34] and was found down regulated in our
experiments that monitored rat heart gene expression prior to
HF. In this cluster of down regulated genes, we also observed a
member of a novel Ca2+-binding protein subfamily (CABP1),
that is a component of Ca2+-mediated cellular signal transduction in the heart [35]. We also noticed a down regulation of
genes involved in cell proliferation and/or protein synthesis in
muscle and heart tissue such as the RNA binding protein
(SEB4D) that was shown to be also down regulated in colon
cancer [36]. FGFR1OP, which protects cells from apoptosis,
was down regulated as well [37]. Cluster 1 in the obese
analysis contained a set of genes encoding remodeling and
structural proteins and a second group of genes with designated
miscellaneous functions (Table 4B). In the group of genes
encoding remodeling and structural proteins, we noticed upregulation of complement factor 1 (CF1). It is well known that
both free radicals and complement activation can injure tissue.
Local complement activation may represent a mechanism by
which free radicals mediate tissue injury [38]. It has been
shown that complement activation is directly involved in
chronically sustained myocardial damage [39]. Up-regulation
of CF1 probably contributes to cardiac remodeling. The second

gene is B4GALT1, mentioned above and the third gene is
SPOCK2. SPOCK2 encodes a protein called testican-2, which
is able to abrogate inhibition of membrane type metalloproteinases [40]. KS1 belongs to the largest family of zinc-finger
transcription factors containing the KRAB domain. The
functions proposed for members of the KRAB-containing
protein family are transcriptional repression of RNA polymerase and binding and splicing of RNA. KS1 counteracts
neoplastic transformation induced by several oncogenes [41].
Therefore KS1 could be involved in maintenance of the
nucleolus, cell differentiation, cell proliferation, apoptosis and
neoplastic transformation as found for other KRAB-containing
proteins [42]. In addition, we found heart-type fatty acid
binding protein (FABP3) down-regulated in this cluster.
FABP3 is involved in lipid metabolism and constitutes a
biochemical marker of myocyte injury in HF [43] whose
expression was found to be regulated in the heart in response to
fatty acid levels [44]. Interestingly, troponin (TNNI3), a well
known marker of HF [43], was also found down regulated in
this cluster. In the group of miscellaneous genes, we found
down regulation of 5′-nucleotidase (NT5). NT5 degrades the
adenosine moiety of ATP. Ischemic preconditioning was found
to activate NT5. Moreover, it was found that plasma adenosine
levels are increased in patients with chronic HF. NT5 activity
also increased in the blood and the myocardium in patients with
chronic HF, which may explain the increases in adenosine
levels in the plasma and the myocardium. In addition, it was
found that further elevation of plasma adenosine levels due to
either dipyridamole or dilazep reduces the severity of chronic
HF. Thus, endogenous adenosine was proposed for cardioprotection in chronic HF and against ischemia and reperfusion
injury [45]. The last gene in this cluster is KIR7.1, encoding for
an inwardly rectifying potassium (Kir) channel. Kir channels
are ubiquitously expressed and serve functions as diverse as
regulation of resting membrane potential, maintenance of K+
homeostasis, control of heart rate and hormone secretion [46].
NMR analysis allowed the quantitation of a number of
metabolites. Metabolite contents determined were in accordance with published data [47–49]. We noticed a general drop
in metabolite concentrations linked to the age of the animals. In
addition, the glutamine to glutamate ratio was significantly
lowered in obese animals. Globally lowered metabolite levels
can be explained by an increase of non-cellular mass such as


excessive collagen type I and III that accumulate in the
myocardium in hypertensive heart tissue [50]. A lowered
glutamine to glutamate ratio could be a consequence of
increased TGF-β stimulation since the TGF-β upregulates the
phosphate dependant glutaminase and the Na+/H+ exchanger 3
gene expression pathway [51,52] favoring intracellular glutamine conversion to glutamate. Moreover, plasmatic TGF-β
circulating levels are increased in obesity [53] and favor
extracellular matrix expansion [54]. In addition, this lowered
glutamine to glutamate ratio in obese may reflect the use of
glutamine to generate more ATP to fuel the cardiomyocytes and
to respond to an increased energy need [52].
Fatty acyl chain concentrations were lower in 10-monthold animals when compared to 4-month-old obese animals. In
accordance with this observation, FABP3, involved in lipid
uptake, was down regulated in obese 10-month-old animals
versus 4-month-old (Table 4B). Besides, this apparent
lowering of fatty acyl chain concentrations might also be
related to the increase of myocardial mass by extracellular
matrix accumulation as mentioned above [55]. In accordance
with this hypothesis, we observed a kinetic up-regulation of
fribronectin gene expression in the obese group (supplemental
Table 2 on-line). Fibronectin is known to accumulate in the
interstitial space [55]. Since extracellular matrix accumulation
is a known occurrence in the remodelling heart and global
metabolite levels were lower in obese animals, (except for
taurine), this suggests that the obese heart is more fibrotic
than its lean counterpart at the same age. This would explain
earlier heart failure development in the obese compared to the
lean SHHF rat [14]. Interestingly, n-3 fatty acyl chains
percentage was higher in obese animals. This observation was
confirmed by an increased total fatty acyl unsaturation in
obese animals. Hypertension and obesity are known to induce
myocardium remodelling [56,57]. Such omega-3 accumulation has already been recently observed during muscle
regeneration [27] and may reflect mechanisms involved in
the increase of septal wall thickness as observed in the
10 months obese rats (Table 1). The increase of unsaturated
lipids was accompanied by conservation of taurine levels
(Table 5). Taurine was shown to preserve unsaturated
membrane lipids from lipid peroxidation [58]. We propose
that cardiac remodelling may share some similarities with
muscle regeneration. This increase of omega-3 may contribute
to the stimulation of membrane expansion from cardiac cells
as observed in PC12 cells [59].
In this present work we could identify a transcriptome kinetic
adaptation that was different in lean and obese animals (Figs. 1
and 2) but the 10-month-old lean and obese animals had a very
similar transcriptome. In our previous work performed on
human right appendage biopsies, we had shown that obese
hypertensive patients had clearly distinct cardiac gene expression patterns when compared to hypertensive patients. Thus,
several hypotheses are plausible. We could surmise that extreme
hypertension in the rat masks most obesity's contribution to
gene regulation. In addition, the duration of obesity is much
longer in patients than in rats and this point may be important to
allow for observation of the contribution of obesity to gene

537

regulations at the transcriptome level in hypertensive animals.
Although, leptin is one element among a number of adipokines
that are present in obese's bloodstream and that can regulate
heart gene expression, the lack of leptin signaling in the SHHF
obese rats may have some consequences on specific obesityinduced gene expression pattern. Indeed, it has been observed
that leptin added to cardiomyocytes in cell culture can induce
hypertrophy [60] and hyperplasia [61]. In hypertensive, nonobese humans, plasmatic levels of leptin have been associated
with myocardial wall thickness [62]. However, protein
synthesis regulation is known to occur not only at the mRNA
level and we did not perform a proteome analysis in this work to
investigate for post-transcriptional or post-traductional gene
regulations but rather a 1H NMR metabolomic analysis which
may be considered as a more downstream observation that takes
into account not only the protein level but also its enzymatic
activities. 1H NMR metabolomic analysis revealed significant
differences in left ventricle metabolites concentration between
obese and lean animals. Therefore, this work provides some
new insights both at the heart metabolome and transcriptome
level during the early steps of heart failure development in the
SHHF rat.
Acknowledgments
We thank Dr Sergueï Sokol (Toulouse Genomic Core
Facilities) respectively for array data WEB management. We
are indebted to Dr Peter J. Romanienko (Memorial SloanKettering Cancer Center, New York, New York, USA) for
critical reading of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.yjmcc.2006.11.007.
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