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

Original article

NMR and cDNA array analysis prior to heart failure reveals an increase ofunsaturated 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, Francec 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 2006Available 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 isnot 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−/− SHHFrat 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 after16 months. We used a set of rat pangenomic high-density macroarrays to monitor left ventricle cardiac transcriptome regulation in 4- and 10-month-old, lean and obese animals. Comparative analysis of left ventricle of 4- and 10-month-old lean rat revealed 222 differentially expressedgenes while 4- and 10-month-old obese rats showed 293 differentially expressed genes. 1H NMR analysis of the metabolome of left ventricularextracts displayed a global decrease of metabolites, except for taurine, and lipid concentration. This may be attributed to gene expressionregulation and likely increased extracellular mass. The glutamine to glutamate ratio was significantly lower in the obese group. The relativeunsaturation 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 ischaracterized by a body mass index (BMI) over 30 kg/m2.Obesity prevalence is reaching around 10% to 30% of thepopulation in industrialized countries and this rate is doublingevery 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 Pharmacolo-gie, 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 metabolicsyndrome [3]. Obesity was shown to be an independent factorfor cardiovascular risk [4]. A positive correlation was noticedbetween overweight and cardiovascular mortality, even afteradjusting for other risk factors. Additionally, obesity is anindependent risk factor for heart failure (HF): a BMI over30 kg/m2 doubles the risk for HF in both genders [5].

Few works have studied specifically the cardiac abnorma-lities associated with obesity. Late stage obesity is characterizedby left ventricular hypertrophy (LVH), that leads to metabolicchange [6], and also by the alteration of the vegetative control ofcardiac frequency [7]. Severity and onset of these abnormalitiessuggest a specific role for obesity, independent from HTA, inthe development of cardiomyopathies.

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Adipose tissue is now considered as an endocrine organ thatsecretes a number of hormones and cytokines that areregrouped under the name “adipokines” [8,9]. The hypothesisof a specific role for adipose tissue has been supported by thefact that many adipose tissue secretions have been shown to beinvolved in cardiovascular pathologies [10]. We recently used adog nutritional model (hypercaloric high fat diet: HFD) ofobesity-related hypertension to show early-and late-specificcardiac 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 ofthe right auricle obtained from non-diabetic patients undergo-ing open heart surgery and heart bypass, we have showndifferent cardiac gene regulations that are specific to obesityand independent from obesity-associated hypertension [13].These observations are consistent with cardiac remodeling inresponse to obesity which could be mediated, at least in part, byadipokines.

In this work, our aim was to evaluate the molecularmechanisms involved in the development of a dilatedcardiomyopathy (DCM) during the onset of heart failure (HF)and the contribution of obesity in the early steps of thedevelopment of this disease. Therefore, we used the SHHF/Mcc-cp rat model of obesity, non-insulin dependent diabetesand congestive heart failure [14]. As obese rats are prone toheart failure several months before their lean counterpart [14],experiments were performed with 4 and 10 months old, lean andobese animals; before any sign of heart failure. We analyzed inparallel the cardiac transcriptome and metabolome using acDNA array and an NMR approach respectively.

2. Materials and methods

2.1. Animals and general procedures

All animal procedures were performed according to theguidelines of the French Ministry of Agriculture. Rats werehoused at the Toulouse IFR31 animal facility in a room lit 12 hper day (6 AM–6 PM) at an ambient temperature of 22±1 °C.Animals were allowed 1 week to adjust after arrival and hadaccess to regular rodent diet and tap water ad libitum during theexperiment.

Ten lean heterozygous (+/cp) and 10 obese homozygous(cp/cp) Spontaneously Hypertensive and Heart Failure-pronerats (SHHF), were provided by Charles River Laboratoriesand included in the study. This strain was obtained by back-crossing a Koletsky obese rat to a Spontaneously Hyperten-sive Rat (SHR/N). Obese males develop congestive heartfailure (CHF) at 10–14 months, lean males at 14–18 months[14,15]. From the Koletsky rat, SHHF rats harbor a nonsensemutation of the leptin receptor gene, designated fa, resultingin a premature stop codon in the leptin receptor. This mutationis a recessive autosomic trait. As a consequence, cp/cphomozygous animals for the fa mutation are obese. Lean ratsare heterozygous for this fa mutation or wild type for the cplocus.

After shaving of the chest, echocardiograms were performedby using the Vivid 7 pro 7 echocardiographic system (GEMedical System), equipped with a i13 L 14-MHz linear-arraytransducer. Images were obtained from rats lightly anesthetizedby 1–2% isoflurane (AErrane, Baxter) lying on their back sidewith transducer placed on the left hemithorax. Two-dimensionalparasternal long- and short-axis images of the left ventricle wereobtained, and two-dimensional targeted M-mode tracings wererecorded at a sweep speed of 200 mm/s. All measurements wereperformed according to the recommendations of the AmericanSociety for Echocardiography leading-edge method from threeconsecutive cardiac cycles (n) with the roundness of the leftventricular cavity (2D-image) as a criterion that the image wason axis, great effort was taken to achieve a good image quality tovisualize the endocardial and epicardial borders of the heart bygently moving and angulating the transducer. Measurements andcalculations used are as follows: percent LV fractionalshortening (FS), a measure of LV systolic function, was cal-culated as follows: FS=(EDD−ESD) /EDD×100, where EDDand ESD are end-diastolic and end-systolic diameters, respec-tively. All values were averaged over three consecutive cycles.

Twice a month, weight, systolic blood pressure (SBP) andheart 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, Austra-lia). Attention was given to the possible appearance of CHFsymptoms such as subcutaneous oedema, hydrothorax, ascites,dyspnea and cyanosis. At the end of the experiment, animalswere anesthetized, and surgery was quickly performed. Leftventricular and right auricle samples were taken from 5 lean and5 obese rats at the age of 4 months or 10 months. Samples wereimmediately washed in cold Phosphate Buffer Saline Buffer,snap frozen in liquid nitrogen and maintained at −80 °C untilanalysis.

2.2. RNA extraction and cDNA labeling

Total RNAwas isolated from left ventricular samples; qualitycheck, concentration control and labeling were performed aspreviously described [13].

2.3. cDNA array hybridization and expression analysis

cDNA array hybridization, washes, and expression analysiswere performed as previously described [13] using pan-genomic macroarrays membranes (RZPD, Berlin) containing26, 592 unique rat cDNA sequences spotted in duplicate. Thispangenomic array contained both known genes and non-annoted genes that required further computing analysis.Therefore, to achieve the maximum information for therespective probes on the membrane, the correspondingaccession numbers were used to identify both the respectiveNCBI-Unigene clusters [16] and the respective EST contigsprovided by the TIGR gene index [17].

To limit the number of false positive results and to increasespecificity of our data, we used the following selection criteriato determine whether the expression of a gene is up-regulated

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

or down-regulated in a statistically significant manner: (1)expression ratio of obese/lean was >1.5 or <0.67, (2) expressionlevel was required to be over 3 times above membranebackground after local background subtraction (membranebackground was calculated by averaging the signal intensityon empty spots i.e. generated by buffer deposition from DNAfree wells), (3) the difference between 2 experimental settingswas statistically significant by Student's t test, (4) attention wasgiven to avoid rejecting eventual highly induced genes withinitially low expression level (under 3 times the membranebackground). Statistical relevance of biological data and real-time PCR results was assessed with Student's t test or Mann–Whitney rank sum test when normality test failed, usingSigmaStat 3.0 software (SPSS).

Hierarchical clustering was performed on normalized to themean intensity of all spots X-dot reader dataset with Multi-Experiment Viewer 2.2 software (TIGR) [18]. The clusteringwas performed on the average linkage clustering method for thegenes and the arrays.

2.4. Real-time PCR control of differential expression

A set of genes was randomly chosen for real-time PCRvalidation of the observed differential expression [19]. Oligoswere synthesized by Proligo Company and designed withPrimer 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 quantificationwhich has been found to be a reliable internal control gene inour hands and others [20,21]. Real-time PCR data werestatistically analyzed with SigmaStat software (SPSS Science).

2.5. Heart left ventricular (LV) tissue dual phase metabolitesextraction

Frozen sections of LV (n=20, 90±39 mg) were powdered inliquid nitrogen with a mortar and pestle and then immediatelysubjected to a simultaneous extraction of lipids and water-soluble metabolites [22]. Briefly, frozen tissue fragments werehomogenized for 20 s in ice-cold purex-analytical-grademethanol and chloroform in a 2:1 ratio (1.5 ml) by using anUltra-Turrax homogenizer. After 15 min in contact with the firstsolvents at 4 °C, 0.5 ml chloroform and 0.5 ml distilled waterwere added and homogenized to form an emulsion. The sampleswere then centrifuged at 2000×g for 30 min, 4 °C. The aqueousphase was then separated from the organic phase. Both fractionswere dried in a Speed Vac concentrator. Extracts were main-tained at −80 °C until their preparation for NMR analysis. PriorNMR analysis, extracts were reconstituted in 600 μl of D2Owith10 μl of a 10 mM 3-(trimethylsilyl)-1-propanesulfonate sodiumsalt (TMPS) solution and 600 μl of CDCl3 /MeOD (2:1, v/v) foraqueous fractions and organic fractions, respectively.

2.6. NMR analysis

1H NMR spectra were recorded on a Bruker Avance 400NMR 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 resultingfrom 30° excitation pulses. Typical acquisition parameters werea SWof 9.8 kHz, 32 K data points and 2.5 s repetition time. Foranalysis of aqueous fractions, a presaturation pulse sequencewas used to suppress the residual intensity of the 1H waterresonance peak. Typical processing parameters were 65 K zero-filling 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 thebasis of 2D homonuclear correlation spectroscopy (COSY)experiments performed on the extracts (data not shown). Thesignal of TMPS (δ=0 ppm) for water-soluble metabolite andresidual CHCl3 (δ=7.36 ppm) for lipids served as referencesfor chemical shift and concentration. A 32 mM concentrationof residual CHCl3 was previously determined with trichlor-obenzene as standard for our batch of deuterated chloroform[29]. Only resonances giving a signal to noise ratio over 15were taken into account for statistical analysis. The number ofprotons giving rise to a signal was considered in thecalculations of relative and absolute concentrations. Signalsto quantify water-soluble metabolites (chemical shift value inppm, 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 chainswas determined using the area of the α-methylene resonancesat 2.31 ppm as 100% of fatty acyl chains (peak 8 in Fig. 4).Cholesterol was quantified using the area of the C-18 methylsinglet at 0.67 ppm (peak 1 in Fig. 4). n-3 fatty acyl chainswere quantified using the characteristic triplet at 0.96 ppm ofthe terminal-CH3 (peak 3 of Fig. 4). Total choline phospho-lipids were determined from the trimethyl group at 3.20 ppm(peak 10 in Fig. 4). Mean unsaturation was calculated as theratio of vinyl from fatty chain (–CHfCH–) determined fromthe signal at 5.35 ppm (peak 14 in Fig. 4) minus thecontribution of cholesterol proton, to the signal of total fattychains (peak 8 in Fig. 4). Mean poly-unsaturation wascalculated as the ratio of the signal of the allylic methyleneat 2.80 ppm (peak 9 in Fig. 4) to the signal of total fatty acylchains.

3. Results

3.1. Rat phenotype analysis

CHF symptoms such as subcutaneous oedema, hydrothorax,ascites, dyspnea and cyanosis were not observed in lean andobese 4- and 10-month-old animals. Four-month-old obese ratshad an average weight of 508±12 g versus 386±2 g for the leanrats: this 32% increase in weight is statistically significant(Student's t test, p=0.008) (Table 1). At 10 months thesedifferences in weight were even more evident as obese rats hadan average weight of 727±10 g and lean 477±5 g; p≤0.001).Clearly, obesity was due to increased fat mass as reported forthis rat model [14].

Table 1Body and heart weight, blood pressure, BNP levels and cardiac echographic analysis

SHHF 4 months SHHF 10 months

Lean (n=5) Obese (n=5) Lean (n=5) Obese (n=5)

Body weight (g) 386±2 508±12* 477±5† 727±10*,†

Heart weight (g) 1.27±0.04 1.35±0.02 1.7±0.04† 1.88±0.09†

SBP (mm Hg) 212±7 204±4 207±3 218±2*,†

BNP (ng/ml) 3.8±0.9 6.2±2.2 6.7±2.8 9.66±2.3Diastolic septal wall thickness (mm) 1.90±0.16 1.78±0.14 2.13±0.19* 2.18±0.13*Diastolic Posterior wall thickness (mm) 2.15±0.39 2.15±0.34 2.08±0.23 2.32±0.26Systolic septal wall thickness (mm) 3.21±0.18 3.18±0.20 3.35±0.25 3.53±0.26*Systolic Posterior wall thickness (mm) 3.41±0.52 3.16±0.37 3.2±0.31 3.32±0.27LV End diastolic diameter EDD (mm) 7.73±0.61 7.78±0.55 8.23±0.48 8.56±0.57*LV End systolic diameter ESD (mm) 4.30±0.65 4.54±0.49 4.87±0.57 5.20±0.57*LV Fraction shortening FS (%) 44.37±6.70 41.65±5.77 39.36±7.26 39.25±4.36

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). Dataare 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 versus10-month-old.

Table 2Comparison of DNA array analysis and real-time PCR

Gene name or GeneBank # Macroarray rt-PCR

Expr.level/bgd

Ratio p Ratio p

Lean analysis 10 m/4 m 10 m/4 mAA 858 801/NFKB1 12.2 2.59 0.0002 1.51 0.002AA963 792 17 2.41 0.002 1.43 0.090AA 998 657 10 2.57 0.002 2.47 0.001

Obese analysis 10 m/4 m 10 m/4 mAA 875 581/MRLCB 1.95 2.42 0.0014 2.26 0.017AI 028 924 8.9 2.65 0.017 2.46 0.003AI 054 986 9.2 2.87 0.01 2.16 0.018

Gene common to lean andobese analysis

10 m/4 m 10 m/4 m

AA 924 587 4.36 3.05 0.0005 1.63 0.038AA 819 584/ATP2A2 17.9 1.99 0.005 2.09 0.038

Differential expression validation by realtime PCR for a set of randomly chosengenes. Induction (if >1.5) and repression (if <0.6) ratios are represented. Meanvalues are indicated for 5 measurements. cDNA macroarrays analysis wereperformed on left ventricle (LV). m: months. Genes were randomly chosenamong those found to be statistically differentially regulated by Bioplot analysisand 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 Leangroup. Ratio 10m/4m: signal intensity in 10-month-old group/signal intensity in4-month-old group. NFKB1: nuclear factor kappa B p105 subunit; MRLCB:myosin regulatory light chain; ATP2A2: ATPase, Ca2+ transporting, cardiacmuscle, slow twitch 2.

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Macroscopic examination of the heart did not reveal anyabnormalities or any cardiac infarction lesions. No significantdifference in weight were observed between lean and obeseaverage 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 significantlyincreased in 10-month-old versus 4-month-old lean or obese rats.

Systolic blood pressure (SBP) was not statisticallysignificantly different: 212±7 mm Hg in 4-month-old leanrats versus 204±4 mm Hg in obese rats. In 10-month-oldanimals, SBP (207±3 mm Hg) was maintained in lean ratsand 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 clinicalsign of HF in 10-month-old animals (Table 1).

Echocardiographic analysis of obese SHHF and lean SHHFanimals demonstrated similar concentric LVH characterised byincreasedwall thicknesswith similar LV cavity dimensions (Table1). LV systolic function assessed by LV fractional shortening wassimilar in both groups (Table 1) at 4 and 10months. However, wenoted that in obese rats, there was an increase in diastolic andsystolic septal wall thickness and not in posterior wall thicknessbetween 4 and 10 months. End diastolic and systolic diameterswere also increased in obese rats. Moreover, echocardiographicanalysis did not reveal any left ventricular dysfunction.

3.2. Left ventricle transcriptome analysis

We first determined the number of detectable genes usingour cDNA macroarrays. Once the local background wassubtracted, we were able to detect an average of 9529 out of26,592 genes with a signal greater than 1-fold and up to 37-fold over the mean membrane background level (data notshown). 7 out of 8 differentially expressed genes harboringan expression level between 1.95 and 17.9 fold overbackground were confirmed by Realtime PCR (87%validation) (Table 2). According to this observation and pre-viously published works [30], we defined an expression level

that had to be over 3 fold over background to be the mini-mum expression level for which we could reliably monitordifferential gene expression. The following data take thisparameter into account.

3.2.1. 10-month-old versus 4-month-old lean and obese ratventricle transcriptome analysis

Comparison of gene expression between 10-month-oldanimals and 4-month-old animals revealed 222 differentially

Fig. 1. Differential expression analysis schematic diagram. Number ofdifferentially expressed genes is indicated. Arrows indicate the statisticalanalysis of the data performed using Student's t test. 102 genes were found to becommon to the differentially expressed genes list from the obese and the leananimals, 222 and 293 genes were found differentially expressed between 4- and10-month-old lean and obese animals respectively.

Table 3Differentially expressed genes selection, according to their well-knownfunction, resulting from lean and obese SHHF 4-month-old/10-month-oldcomparison

Gene name and Gene bankaccession #

Expr.level/bgd

Ratio p (Bioplot)

IL-6 R/AA 963 567 4.26 1.74 0.006CASP 1/AI 071 441 9.27 2.14 0.038GATA 4/AA 997 121 3.51 2.38 0.027TGFB/AI 548 079 3.26 1.64 0.033NPPA/AA 819 343 3.04 1.67 0.0061ACE/AI 556 575 5.68 1.66 0.010ECE 1/AA 817 947 3.21 2.11 0.039MYH 6/AA 819 464 4.87 0.58 0.042FN 1/AA 955 600 14.2 1.88 0.038SLC1A1/AA 996 752 5.31 2.03 0.044SLC25A10/AA 859 666 3.15 0.56 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-oldgroup. IL-6 R: InterLeukine 6 Receptor. CASP 1: Caspase 1. GATA 4: GATAbinding protein 4. TGFB: Transforming Growth Factor Beta. NPPA: NatriureticPeptide 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 carrierfamily 1, member 1. SLC25A10: solute carrier family 25, member 10.

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expressed genes (17 repressed and 205 over-expressed) and 293differentially expressed genes (30 repressed and 263 over-expressed) in lean and obese rats, respectively (Fig. 1).Homologies searches generated a list of 95 defined genesfrom the lean rat analysis and 132 defined genes from the obeserat analysis (Table available as a supplementary data on line).

3.2.2. 10 months versus 4 months differential expression of aset of cardiac-relevant genes

We identified a set of well-known function genes common tolean and obese analysis (Table 3). These genes differentiallyexpressed between 4- and 10-month-old animals are indicatorsof: 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 genesHierarchical clustering organization provided us with a

global view of the changes in cardiac gene expression inducedby the duration of hypertension in lean rats and the combinationof hypertension and obesity in obese rats. In addition,hierarchical clustering was efficient at grouping gene expres-sion profiles correctly without any intervention. Thus, geneexpression profiles were specific to the age of the lean or obeseSHHF rat hearts. Interpretation of the data is challenging forsome clusters due to a limited number of differentiallyexpressed genes, which limited cluster size. Nevertheless, onecould consider the set of 17 and 30 down-regulated genes in10 months, lean (Fig. 2A) and obese (Fig. 2B) rats, respectively.We first focused our attention on genes co-regulated in leananimals and down regulated in 10-month-old animals (cluster 1,Fig. 2A). We obtained gene identities or homologies for 11genes out of 17 in this cluster (Table 4A). For obese animal

clustering analysis, identity of 9 genes could be provided in themain cluster (Fig. 2B and Table 4B).

3.3. Left ventricle metabolome analysis

Characteristic signals arising from metabolites such ascreatine+phosphocreatine, taurine, glutamine, glutamate, ace-tate, alanine and lactate were detected in the 1H NMR spectrumof the aqueous fraction of LV heart extract (Fig. 3).Concentration of metabolites and statistical analysis of thedata showed a set of differences (Table 5). These differencesare related to the age and/or the obesity status. A significantdecrease in creatine and glutamate concentration was observedand exclusively correlated to the age. Taurine concentrationwas significantly decreased in the 10-month-old lean groupwhile its concentration was unchanged in other groups.Glutamine concentration was significantly increased in thelean group compared to that in the obese group. Moreover,glutamine concentration was correlated to the age of rats andsignificantly decreased in the 10-month-old lean groupcompared to that in the 4-month-old lean group. The glutamineto glutamate ratio was lower in obese animals, independent ofage.

A 1H NMR spectrum of total lipid extract of an obese SHHFheart rat is shown in Fig. 4. Characteristic signals of lipidmoieties allowed the determination of total fatty acyl chains,cholesterol, choline phospholipids (Table 6). Moreover, meanunsaturation which represents the number of vinyl moieties perfatty acyl chain and the mean poly-unsaturation whichrepresents the number of diallylic methylene per fatty acylchain were calculated. Choline phospholipids, cholesterol andfatty acyl chains contents were similar at the same ages inthe obese group and in the lean group. Surprisingly, we

Fig. 2. Hierarchical cluster analysis of differential expression. (A) Clustering performed with lean transcriptome analysis data. (B) Clustering performed with obesetranscriptome 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 bythe tree on the left side. Genes in cluster 1 from lean and obese analysis are detailed in Table 4.

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Fig. 2 (continued).

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Table 4Genes 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 homologyAA900596 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 No sequence homologyAI556930 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 No sequence homologyAA900578 ALAD 0.6 1.5 0.91 0.03495742 Aminolevulinate. delta-. dehydratase (Alad)

(Rattus norvegicus)AA925164 0.63 1.47 0.93 0.00010271 No sequence homologyAI547418 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 No sequence homologyAI575762 CABP1 0.61 1.55 0.95 0.00496229 Calcium binding protein 1 (Cabp1) (Rattus norvegicus)

highly similar to NP_839983.1sorting 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-bindingprotein. A cornified cell envelope precursor and anintermediate filament-associated (cross-linking) protein(Homo sapiens)

AI136136 CHMP1.5 0.62 1.55 0.95 0.00646823 CHMP1.5 protein (Homo sapiens) function: putativevesicle trafficking with VPS4 and putative chromatinstructure regulation in the nuclear matrix

AI555253 0.62 1.54 0.96 6.78e−05 Similar to KIAA1632 prot. proline–serine–threoninephosphatase interacting protein 2 (Homo sapiens)

AA925236 0.64 1.61 1.03 0.00126788 No sequence homologyAA956865 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 No sequence homologyAA900791 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 43AI501277 0.52 1.45 0.75 0.00210187 No sequence homologyAI136302 0.55 1.57 0.86 0.00989101 No sequence homologyAA926219 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 No sequence homologyAI043693 0.63 1.53 0.96 0.00859156 No sequence homologyAA925880 SPOCK2 0.58 2.16 1.25 0.00768181 Similar to testican-2 protein (LOC361840)AA858560 0.49 1.79 0.88 0.00540406 No sequence homologyAA859429 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 198AI145732 0.6 1.8 1.08 0.01230905 No sequence homologyAA858454 0.65 1.71 1.12 0.02969404 Transcribed locus. Moderately similar to XP_346694.1

hypothetical gene supported by NM_022857AI715210 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 No sequence homologyAI043993 0.62 1.45 0.9 0.00679676 No sequence homologyAI112905 0.65 2.22 1.45 0.00955169 No sequence homologyAI145673 0.65 1.97 1.28 0.00586599 Transcribed locus. Weakly similar to XP_346694.1

hypothetical gene supported byNM_022857

AI030972 0.48 1.58 0.75 0.00063431 No sequence homology

(continued on next page)

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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 No sequence homologyAA858808 0.63 2.09 1.32 0.0010659 No sequence homologyAA858866 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 No sequence homology

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

noticed a lower fatty acid chain concentration in 10-month-old animals when compared to the 4-month-old animals thatwas significant for the obese group. Obese animals displayeda higher level of n-3 fatty acid chains when compared totheir lean counterparts. In accordance with these observa-tions, 10-month-old obese animals displayed enhancedunsaturation and poly-unsaturation. Unsaturation levelsincreased with age only in obese animals. Therefore,increased unsaturation is related to the obesity state and itsduration.

4. Discussion

We combined cDNA macroarrays with NMR metabolicprofiling to characterize the cardiac transcriptome and metabo-lome of two groups of SHHF rats at 4 and 10 months of age

Fig. 3. 1H NMR spectrum of a water-soluble fraction of a heart extract from a leanresonances have been assigned to lactate (Lac), alanine (Ala), acetate (Acet), glutamSodium 3-(trimethyl-silyl)-1-propanesuffonate (TMPS); pH=7.4.

which differed in their state of obesity and the associated Xmetabolic syndrome [14,15]. Our main objective was to definethe molecular adaptation in heart at the onset of heart failuredevelopment and to evaluate the impact of obesity on themechanism of heart failure development. Prior to the molecularadaptations analysis, we examined the biological parameters ofthe subject animals. As previously described, the obese rats,homozygous for the fa mutation, displayed a significantincrease of weight (+32% at 4 months; +52% at 10 months;Table 1).

However, we noticed two major distinctions when analyzingthe biological parameters. First, both groups of animals werehypertensive and 10-month-old obese animals were significant-ly more hypertensive than their lean counterparts. This may be aconsequence of the obesity. Second, 10-month-old obeseanimals were expected to be at the onset of a DCM [14] but

SHHF rat. Within the aliphatic region (−0.2 to 4.5 ppm) of the NMR spectra,ate (Glu), glutamine (Gln), creatine+phosphocreatine (Cr+PCr), taurine (Taur),

Table 5Quantitation of water-soluble metabolites by 1H NMR

SHHF 4 months SHHF 10 months

Lean Obese Lean Obese

Taurine 33.70±3.55 a 34.71±5.98 28.48±3.32 33.01±3.52 b

Creatine+Phoshocreatine 16.55±1.20 a 15.24±2.72 a 12.19±1.37 10.98±1.27Glutamate (Glu) 5.72±0.56 a 5.75±0.93 a 4.43±0.76 4.53±0.83Glutamine (Gln) 7.82±0.71 a 6.56±1.30 b 6.71±0.78 5.47±0.89 b

Gln/Glu ratio 1.37±0.12 1.14±0.10 b 1.53±0.23 1.21±0.09 b

Acetate 1.70±0.44 1.62±0.55 1.71±0.65 1.14±0.37Alanine 2.20±0.12 a 2.07±0.05 b 1.75±0.32 2.09±0.44Lactate 14.49±5.09 21.45±9.06 a 11.03±1.72 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.

535J. Roncalli et al. / Journal of Molecular and Cellular Cardiology 42 (2007) 526–539

we did not observe any SBP normalization in this group ofanimals. This is evidence for the lack of development of a DCMin these 10-month-old animals. Nevertheless, our observationsare in accordance with previously published data [15]. Heartsweights at 4 and 10 months of age were not significantlydifferent in the obese and lean groups. Thus, 10-month-old heartweights were in accordance with published data [14] andcorrespond to LVH hearts when compared to normotensiveWistar–Kyoto or Sprague–Dawley rats of this age [14,31]. At10 months of age we did not notice a more pronouncedmacroscopic LVH in obese hearts when compared to leanhearts. This lack of macroscopic differences was also reflectedin the BNP levels that were not significantly increased by

Fig. 4. 1H NMR spectrum of a total lipid extract from an obese SHHF heart rat. Materminal of fatty acyl chain (FA) and Chol (C26, C27); 3, characteristic triplet of CH3

CH2–COO–; 7, –CH2–CHfCH–CH2–CHfCH–CH2–; 8, –CH2–COO–; 9, –CHfphosphatidylcholine; 12, mainly glycerol (C1 and C3) and –O–CH2–CH2N

+ of pho(C6); S, solvents (methanol+H2O).

obesity in 10-month-old rats (Table 1). However, echocardio-graphic analysis displayed an increased diastolic and systolicseptal wall thickness in obese 10-month-old animals (Table 1).This slight structural remodeling is likely a consequence ofblood volume increase observed in obese animals. Alternative-ly, it could be the result of altered kinetic gene regulations whichwere observed between lean and obese animals, as discussedbelow.

We confirmed differential expression of 2 fold abovebackground (Table 2 and data not shown), which is commonin microarray analysis. Therefore, to eliminate false positivesand focus on truly differentially expressed genes we set the limitfor reliable analysis of gene expression at 3-fold over

in peaks or regions are assigned as follows: 1, Cholesterol (Chol, C18); 2, CH3

terminal of (n-3) polyunsaturated FA; 4, Chol (C19); 5, (CH2)n of FA; 6, –CH2–CH–CH2–CHfCH–; 10, –N

+(CH3)3 of phosphatidylcholine; 11, –CH2N+ of

sphatidylcholine; 13, esterified glycerol (C2); 14, –CHfCH– of FA and Chol

Table 6Quantitation of lipids by NMR

SHHF 4 months SHHF 10 months

Lean Obese Lean Obese

Acyl chains (nmol/mg w.w.) 103.49±11.28 117.49±16.87 a 90.74±6.11 84.49±4.73(n-3) Acyl chains % 8.80±0.56 10.35±0.79 b 8.11±1.25 9.52±0.62 b

Cholesterol (nmol/mg w.w.) 5.09±0.33 5.33±0.95 4.61±0.62 5.11±0.78Choline phospholipids (nmol/mg w.w.) 14.54±1.00 15.72±2.06 a 13.57±0.89 12.72±082Mean unsaturation 1.70±0.09 1.78±0.12 1.78±0.10 1.90±0.02 b

Mean poly-unsaturation 1.17±0.06 1.23±0.08 a 1.23±0.07 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.

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

background. According to previously published work [30], thislowers the false positive rate to 0.7%. According to thesecriteria, we did not have any evidence of an effect of obesity in4- or 10-month-old animals. However, macroarrays analysis ona set of known cardiac genes recapitulated the differentialexpression of genes involved in hypertension and the develop-ment of heart failure (Table 3).

Hierarchical clustering analysis of gene expression from leananimals revealed in the same main cluster 3 genes out of 11encoding proteasome proteins COP9 subunit 4 (CSN4),aminolevulinate deltadehydratase (ALAD) and CHMP1.5(Table 4A) PKD1 is a cation channel regulator [32], a complexinvolved in many regulatory processes, including control ofdevelopment and regulationofmorphogenesis.Twogenes,UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase (B4GALT1) andtrichohyalin (THH), encode for proteins that have a structuralrole. UDP N-acetylglucosamine β-1,4 galactosyltransferase isa widely distributed enzyme which catalyzes the transfer ofgalactose to N-acetylglucosamine residues of glycoproteinsand glycolipids [33]. Elevated B4GALT1 expression wasalready shown to occur in the failing hearts of spontaneouslyhypertensive rats [34] and was found down regulated in ourexperiments that monitored rat heart gene expression prior toHF. In this cluster of down regulated genes, we also observed amember of a novel Ca2+-binding protein subfamily (CABP1),that is a component of Ca2+-mediated cellular signal transduc-tion in the heart [35]. We also noticed a down regulation ofgenes involved in cell proliferation and/or protein synthesis inmuscle and heart tissue such as the RNA binding protein(SEB4D) that was shown to be also down regulated in coloncancer [36]. FGFR1OP, which protects cells from apoptosis,was down regulated as well [37]. Cluster 1 in the obeseanalysis contained a set of genes encoding remodeling andstructural proteins and a second group of genes with designatedmiscellaneous functions (Table 4B). In the group of genesencoding remodeling and structural proteins, we noticed up-regulation of complement factor 1 (CF1). It is well known thatboth free radicals and complement activation can injure tissue.Local complement activation may represent a mechanism bywhich free radicals mediate tissue injury [38]. It has beenshown that complement activation is directly involved inchronically sustained myocardial damage [39]. Up-regulationof CF1 probably contributes to cardiac remodeling. The second

gene is B4GALT1, mentioned above and the third gene isSPOCK2. SPOCK2 encodes a protein called testican-2, whichis able to abrogate inhibition of membrane type metalloprotei-nases [40]. KS1 belongs to the largest family of zinc-fingertranscription factors containing the KRAB domain. Thefunctions proposed for members of the KRAB-containingprotein family are transcriptional repression of RNA polymer-ase and binding and splicing of RNA. KS1 counteractsneoplastic transformation induced by several oncogenes [41].Therefore KS1 could be involved in maintenance of thenucleolus, cell differentiation, cell proliferation, apoptosis andneoplastic transformation as found for other KRAB-containingproteins [42]. In addition, we found heart-type fatty acidbinding protein (FABP3) down-regulated in this cluster.FABP3 is involved in lipid metabolism and constitutes abiochemical marker of myocyte injury in HF [43] whoseexpression was found to be regulated in the heart in response tofatty acid levels [44]. Interestingly, troponin (TNNI3), a wellknown marker of HF [43], was also found down regulated inthis cluster. In the group of miscellaneous genes, we founddown regulation of 5′-nucleotidase (NT5). NT5 degrades theadenosine moiety of ATP. Ischemic preconditioning was foundto activate NT5. Moreover, it was found that plasma adenosinelevels are increased in patients with chronic HF. NT5 activityalso increased in the blood and the myocardium in patients withchronic HF, which may explain the increases in adenosinelevels in the plasma and the myocardium. In addition, it wasfound that further elevation of plasma adenosine levels due toeither dipyridamole or dilazep reduces the severity of chronicHF. Thus, endogenous adenosine was proposed for cardiopro-tection in chronic HF and against ischemia and reperfusioninjury [45]. The last gene in this cluster is KIR7.1, encoding foran inwardly rectifying potassium (Kir) channel. Kir channelsare ubiquitously expressed and serve functions as diverse asregulation 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 accor-dance with published data [47–49]. We noticed a general dropin metabolite concentrations linked to the age of the animals. Inaddition, the glutamine to glutamate ratio was significantlylowered in obese animals. Globally lowered metabolite levelscan be explained by an increase of non-cellular mass such as

537J. Roncalli et al. / Journal of Molecular and Cellular Cardiology 42 (2007) 526–539

excessive collagen type I and III that accumulate in themyocardium in hypertensive heart tissue [50]. A loweredglutamine to glutamate ratio could be a consequence ofincreased TGF-β stimulation since the TGF-β upregulates thephosphate dependant glutaminase and the Na+/H+ exchanger 3gene expression pathway [51,52] favoring intracellular gluta-mine conversion to glutamate. Moreover, plasmatic TGF-βcirculating levels are increased in obesity [53] and favorextracellular matrix expansion [54]. In addition, this loweredglutamine to glutamate ratio in obese may reflect the use ofglutamine to generate more ATP to fuel the cardiomyocytes andto respond to an increased energy need [52].

Fatty acyl chain concentrations were lower in 10-month-old animals when compared to 4-month-old obese animals. Inaccordance with this observation, FABP3, involved in lipiduptake, was down regulated in obese 10-month-old animalsversus 4-month-old (Table 4B). Besides, this apparentlowering of fatty acyl chain concentrations might also berelated to the increase of myocardial mass by extracellularmatrix accumulation as mentioned above [55]. In accordancewith this hypothesis, we observed a kinetic up-regulation offribronectin gene expression in the obese group (supplementalTable 2 on-line). Fibronectin is known to accumulate in theinterstitial space [55]. Since extracellular matrix accumulationis a known occurrence in the remodelling heart and globalmetabolite levels were lower in obese animals, (except fortaurine), this suggests that the obese heart is more fibroticthan its lean counterpart at the same age. This would explainearlier heart failure development in the obese compared to thelean SHHF rat [14]. Interestingly, n-3 fatty acyl chainspercentage was higher in obese animals. This observation wasconfirmed by an increased total fatty acyl unsaturation inobese animals. Hypertension and obesity are known to inducemyocardium remodelling [56,57]. Such omega-3 accumula-tion has already been recently observed during muscleregeneration [27] and may reflect mechanisms involved inthe increase of septal wall thickness as observed in the10 months obese rats (Table 1). The increase of unsaturatedlipids was accompanied by conservation of taurine levels(Table 5). Taurine was shown to preserve unsaturatedmembrane lipids from lipid peroxidation [58]. We proposethat cardiac remodelling may share some similarities withmuscle regeneration. This increase of omega-3 may contributeto the stimulation of membrane expansion from cardiac cellsas observed in PC12 cells [59].

In this present work we could identify a transcriptome kineticadaptation that was different in lean and obese animals (Figs. 1and 2) but the 10-month-old lean and obese animals had a verysimilar transcriptome. In our previous work performed onhuman right appendage biopsies, we had shown that obesehypertensive patients had clearly distinct cardiac gene expres-sion patterns when compared to hypertensive patients. Thus,several hypotheses are plausible. We could surmise that extremehypertension in the rat masks most obesity's contribution togene regulation. In addition, the duration of obesity is muchlonger in patients than in rats and this point may be important toallow for observation of the contribution of obesity to gene

regulations at the transcriptome level in hypertensive animals.Although, leptin is one element among a number of adipokinesthat are present in obese's bloodstream and that can regulateheart gene expression, the lack of leptin signaling in the SHHFobese rats may have some consequences on specific obesity-induced gene expression pattern. Indeed, it has been observedthat leptin added to cardiomyocytes in cell culture can inducehypertrophy [60] and hyperplasia [61]. In hypertensive, non-obese humans, plasmatic levels of leptin have been associatedwith myocardial wall thickness [62]. However, proteinsynthesis regulation is known to occur not only at the mRNAlevel and we did not perform a proteome analysis in this work toinvestigate for post-transcriptional or post-traductional generegulations but rather a 1H NMR metabolomic analysis whichmay be considered as a more downstream observation that takesinto account not only the protein level but also its enzymaticactivities. 1H NMR metabolomic analysis revealed significantdifferences in left ventricle metabolites concentration betweenobese and lean animals. Therefore, this work provides somenew insights both at the heart metabolome and transcriptomelevel during the early steps of heart failure development in theSHHF rat.

Acknowledgments

We thank Dr Sergueï Sokol (Toulouse Genomic CoreFacilities) respectively for array data WEB management. Weare indebted to Dr Peter J. Romanienko (Memorial Sloan-Kettering Cancer Center, New York, New York, USA) forcritical 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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