Published online before print February 24, 2003, doi: 10.1161/01.HYP.0000057573.32425.95
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(Hypertension. 2003;41:414.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
Cardiac Transcriptome Analysis in Obesity-Related Hypertension
From INSERM U586, Faculté de Médecine, Laboratoire de Pharmacologie Médicale et Clinique, Toulouse Cedex, France.
Correspondence to Philippe Rouet, INSERM U586, Faculté de Médecine, Laboratoire de Pharmacologie Médicale et Clinique, 37 Allées Jules Guesde. 31073 Toulouse Cedex. France. E-mail Philippe.rouet{at}toulouse.inserm.fr
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Abstract |
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Obesity is associated with volumetric arterial hypertension and with early increase in heart rate and decreased heart rate variability. The consequences of obesity-related hypertension on heart gene regulation are poorly known and were investigated in a model of obesity-related hypertension induced by high fat diet in dogs. When compared with control animals (n=6), a 9-week high fat diet (n=6) provoked significant weight gain and increased blood pressure load and heart rate but failed to significantly change left ventricular mass assessed by echocardiography. Subtractive hybridization of dog heart cDNA libraries were used to generate sublibraries containing differentially expressed cDNAs that were in turn spotted onto membranes to create custom microarrays. Hybridizations of these microarrays with complex probes representing mRNAs expressed in right atria and left ventricles from obese hypertensive and control dogs were performed. Thirty-eight differentially expressed genes were identified; altered expression was confirmed by Northern blot analysis in 15. In addition, real-time quantitative polymerase chain reaction confirmed differential expression for 80% of the randomly chosen tested genes. Once identified, transcripts were categorized into groups involved in metabolism, cell signaling, ionic regulation, cell proliferation, protein synthesis, and tissue remodeling. In addition, we found a set of 11 cDNAs encoding proteins with unknown functions. This study clearly shows that obesity-related hypertension, lasting for only 9 weeks, causes marked changes in gene expression in right atrium as well as the left ventricle that may contribute to early functional changes in heart function and to long-term structural changes such as left ventricular hypertrophy and remodeling.
Key Words: obesity • hypertension, arterial • heart rate • heart • dogs
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Introduction |
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Obesity is the most prevalent nutritional disorder in developed countries1 and plays an important role in cardiovascular morbidity through multiple mechanisms,2 including well-known risk factors such as hypertension,3 diabetes,4,5 and dyslipidemia.6–8 Moreover, as suggested for leptine and arterial hypertension,9 a direct interaction of adipose tissue, through its multiple secretions, with the cardiovascular system could also be involved in the pathophysiology of cardiovascular disorders associated with obesity. Previous studies have shown that overweight is associated with increased cardiac output10 and resting heart rate and with decreased heart rate variability,11 which is considered to be a prognostic indicator of cardiovascular mortality in humans.12,13 Obesity is also associated with structural changes in the heart, including eccentric and concentric hypertrophy,14 thus explaining the increased risk of cardiac failure.15 However, the molecular mechanisms underlying these changes in cardiac function and structure are poorly understood. We and others have developed a dietary model of obesity-related hypertension that closely mimics the cardiovascular, hormonal, and metabolic abnormalities found in human obesity.11,16,17 This model is produced by feeding dogs ad libitum a hypercaloric, high fat diet (HFD) that induces abdominal obesity, hyperinsulinemia and insulin resistance, and arterial hypertension within a few weeks. Left ventricular hypertrophy (LVH) also develops but only after 20 weeks of HFD.11,18 In this model, parasympathetic drive to the heart is blunted and atrial but not ventricular M2-muscarinic receptors are downregulated shortly after starting HFD.19 In addition, we found an upregulation of endothelial nitric oxide synthase specifically in the atrium (Pelat et al, unpublished results). These observations suggest that HFD induces a set of molecular alterations that could be distinct and earlier in the atrium than in ventricle.
Currently, very little is known about early cardiac molecular mechanisms induced by obesity-related hypertension, especially shortly after weight gain acquisition and before major structural changes in the heart have occurred. Therefore, we investigated, at the transcriptome level, the early changes in cardiac gene expression in the dog model of obesity-related hypertension before observable LVH. We prepared dog heart cDNA libraries and used subtractive hybridization to generate sublibraries and microarrays containing differentially expressed cDNAs20–22 and compared the patterns of regulation in the right atrium and the left ventricle.
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Methods |
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Animals and General Procedures
Twelve adult Beagle-Harrier dogs (Harlan SA, France), 22±3 months of age, were included in the study. An implantable telemetry measurement system (Data Science International) allowing long-term recording of blood pressure (BP) and heart rate (HR) was surgically installed in the femoral artery under general anesthesia (1 mg/kg acepromazine plus 10 mg/kg tiletamine-zolazepam) at least 15 days before the beginning of the experiment. After recovery, animals were randomized into 2 groups. A resting period of 4 weeks was given before the initiation of HFD to determine the normal canine diet intake (Royal Canin M25, Royal Canin) required to maintain constant body weight. The control group was fed a normal canine diet. The HFD group was fed ad libitum a hypercaloric hyperlipidic diet (normal canine diet mixed with uncooked beef fat). Body weight, BP, and HR were measured twice per week during the whole study.
After 9 weeks of HFD, cardiac echocardiography was performed as previously described11 to detect LVH or cardiac failure. Telesystolic and telediastolic diameters and wall thickness of left ventricle were measured from time-movement recordings by Devereux’s method.23 Telesystolic and telediastolic volumes (cm3) of the left ventricle were calculated according to Cube’s method. Left ventricular mass (g) was evaluated by Devereux’s formula.23 Septum and posterior wall thickness ratio were calculated as an index of ventricular asymmetry. Left ventricular function was evaluated by means of left ventricular ejection fraction (%), defined as the ratio between the difference of telediastolic and telesystolic volumes and telediastolic volume.
At the end of the experiment, animals were anesthetized, and surgery was quickly performed. Right atrium and left ventricle tissue samples were frozen in liquid nitrogen and maintained at -80°C until RNA preparation.
All animal procedures were performed according to the guidelines of the French Ministry of Agriculture.
Blood Pressure and Heart Rate Measurement
A receiver unit (RL 200) was used to monitor and amplify signal from each implantable transmitter and to convert it into a series of digital pulses to be decoded and evaluated by computer.24 Systolic and diastolic BP and HR were obtained from the femoral artery pressure waveform. BP and HR signal were digitalized at 500 Hz. Systolic and diastolic BP were computed for each cycle and extracted at 2 Hz, then stored in a compatible IBM-PC for analysis. BP and HR measurements were performed between 9 and 10 AM on quiet, unrestrained animals in their individual cages.
RNA Extraction and Generation of Differentially Expressed cDNA
RNA was extracted by disrupting tissues in Trizol reagent (Invitrogen) and prepared according to recommended procedures (Clontech). We used 500 ng of total RNA to produce cDNA that was polymerase chain reaction (PCR)-amplified with a SMART PCR cDNA synthesis kit (Clontech). Amplification linearity was checked by fluorescent monitoring of the PCR products using PicoGreen (Molecular Probes) and a plate fluorometer (Fluoroscan, Labsystems). To generate enriched cDNA libraries for differentially expressed genes, the PCR-select cDNA subtraction kit reagents and protocol (Clontech) were used. Two separate subtractions, obese-control to enrich for genes upregulated in obese, and the reciprocal control-obese to enrich for genes upregulated in control (ie, downregulated in obese), were performed as follows: In a first subtraction, cDNAs from obese dogs were used as "tester" and control cDNA as "driver" to generate a library containing upregulated genes in the obese phenotype. In the reciprocal reaction, we used cDNA from obese dogs as "driver" for subtraction, enriched the resulting library in downregulated genes. Subtraction reaction efficiency was checked by agarose gel electrophoresis. Subtracted products were ligated to pGEM-T vector DNA (Promega) and electroporated in DH5-
Electromax-competent cells (Invitrogen). This established a library of potentially upregulated and downregulated cDNAs. The complexity of these libraries was determined to be 
1000, so 1200 cDNA of each library were PCR-amplified from bacterial clones and spotted in duplicates onto nylon membranes at Toulouse Genomic Core Facilities with a BioRobotics apparatus.
DNA Array Hybridization and Expression Analysis
Labeling with -[32P]-dATP (NEN) of unsubtracted cDNA from obese hypertensive or control dogs, hybridization to the membranes, and washes were performed as described in the PCR-select differential screening kit manual (Clontech). Membranes were exposed for 1 to 3 hours in a cassette and revealed with a Storm 860 PhosphorImager (Molecular Dynamics). Signal intensity was analyzed with X-dot reader software (COSE) and normalized to the mean intensity from all the measured values. Since cDNA subtraction generally enriches up to 9% of differential expressed cDNA in the library,25 and that subtraction was done to enrich in upregulated and reciprocally to enrich in downregulated sequences, differentially expressed cDNA will not affect dramatically the overall signal of a membrane. Considering the large number of clones screened and that the percentage of differentially expressed genes was expected to be <10%, global normalization has been considered as appropriate26,27 and preferred to normalization with housekeeping genes, which is sometimes used successfully with lower numbers of clones.28 In addition, because housekeeping gene expression can also vary significantly,29 the use of housekeeping genes to normalize expression data could have led to erroneous conclusions.30 Results are shown as ratio of obese/control of the mean from 10 normalized measurements. Expression was considered as induced (ratio>1.2; 
) and repressed (ratio<0.8; 
).
DNA Sequencing and Sequence Analysis
PCR products (200 to 1500 bp) were directly sequenced according to the Applied Biosystems AmpliTaq FS dye terminator kit and loaded on an ABI 373 automated sequencer that provided an average of 400 bases of good quality sequence. Sequence homologies were searched with an on-line BLAST server (NCBI) set with default parameters. Gene identity was given if BLAST probability values were <10-30. The probability value is the smallest sum probability that by chance one would observe a matched score as high as the observed high score when searching the database with a random sequence of the same size as the query sequence. This value allowed identification sequences of at least 83% homology to known human or rodent sequences.
Verification of mRNA Levels by Northern Blot Analysis
Twenty micrograms of total RNA was loaded per lane, and Northern blot analysis was performed with the use of standard procedures. PCR products from differentially expressed genes were used as probes and labeled with -[32P]-dATP according to the NEB blot kit manual (New England Biolabs). Quickhyb solution (Stratagene) was used for overnight hybridization, and washes were done as recommended. Membranes were exposed 6 to 24 hours in a PhosphorImager cassette, and exposure was analyzed in a SI445 PhosphorImager (Molecular Dynamics). Band intensity was determined with the use of ImageQuant software (Molecular Dynamics).
Real-Time Quantitative PCR
Primers were designed with the use of Primer Express 2.0 software (Applied Biosystems). Oligos sequences were as follows: Cytochrome oxidase subunit 1: AGGAATAGATGTAGACACAC-GAGCAT and CTCCCGTTGGAATAGCGATAAT; Matrix metalloproteinase-9: CCCTTGAACACGCATGACAT and AAGCGGTCCTGGCAGAAGTA: Lectomedin-3: ATTGCCAGCGGCGAATAC and GGGCGGTCTCGTTATGGTTA; NRF2: GCAGGGTGATTTTCTTCCTTATGA and CTGTGGACCGTGTGTTGACAA; ß-actin: GACCCAGATCATGTTCGAGACTT and AGCCTGGATGGCCACGTA; ANP: TGGACCATTTGGAAGAAAAGATG and CGGCTCACTGAGCACT-TGTG; tensin: GGTATCTGCCGTCAGGTCATC and CAGCTTGGCCTGGCATTT; VDAC 2: ATTTGGTTTTGGGTTGGTGAAA and TTAGATGAACCGGACGTTGAAA; ALS 2: TTCCACAAAAACAAAGGGTGAA and TCTACACTCTATTGACCTCAGCACAA; NADH dehydrogenase: GTGAAGTCCCCTCCCAATACC and GCGAGGCTTGATATTGCTAGTATG; hsp70: TGGCACACTGGACCCTGTAG and CCCACCAAGACAATATCATGGA; SERCA2: TCGAACCTTCCCACAAGTCAA and TCACACCATCCCCAGTCATG; 18 S: CGCCGCTAGAGGTTGAAATTC and TCCGACTTTCGTTCTTGATTAATG.
One microgram of DNAse I-treated (DNAfree, Ambion) total RNA was retrotranscribed in the presence of random primers and Thermoscript enzyme (Invitrogen) according to the manufacturer’s protocol. Real-time PCR reactions were carried out with Sybergreen PCR Master Mix (Applied Biosystems) in a GenAmp 5700 apparatus (Applied Biosystems). The standard curve method was used for relative quantification of the PCR products, and gene expression was normalized to 18 S RNA quantification.
Statistical Analysis
All results are depicted as mean±SEM. Multiple comparisons (Table 1) were analyzed by ANOVA, followed, when appropriate, by the Dunnett post hoc test with Statview 4.5 software (Abacus Concepts). Single comparisons (Table 2 and echocardiographic parameters on Table 1) were performed by means of unpaired Student t test, with a value of P
0.05 considered significant. Preliminary microarray experiments independent from the present study but using the same methods allowed us to calculate that 8 hybridizations were necessary to reach enough statistical power to detect a significant variation of expression of at least 20% with a risk of error of 5%. Therefore, we performed 10 experimental hybridizations on our custom microarrays to ensure sufficient statistical power.
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Results |
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Body Weight, Blood Pressure, and Heart Rate
Body weight, BP, and HR were similar in the 2 groups before starting the diet. After 9 weeks of HFD, significant increases in BP, HR, and body weight were observed in the HFD group compared with the control group (Table 1). Echocardiography indicated absence of difference in wall thickness, ejection fraction (data not shown), and in left ventricular mass (Table 1) between the 2 groups at the end of the experiment.
Identification of Differentially Expressed Genes in Atrium
RNA from 6 atrial tissue samples from each group were pooled to provide average complex probes to screen our subtracted libraries. According to our statistical analysis and criteria, we found 200 genes that were differentially regulated with at least 20% of variation in expression in atria from obese dogs compared with control animals. BLAST homology searches of sequenced clones identified 32 single known genes with identifiable function and 13 new genes that were potentially altered in atria of obese compared with lean dogs (Table 2). A Student t test with microarray data revealed statistically relevant significance for differential expression of 29 known genes and 11 new genes. Four of these 40 genes (ß-actin, Myoferlin, ß-hemoglobin, and Tensin) were found to be not differentially expressed in Northern blot experiments. Lectomedin-3 and VDAC2, which were not found to be statistically differentially expressed by microarray analysis, were found to be differentially expressed by Northern blot analysis. Therefore a total of 38 genes can be considered as differentially expressed in atria.
To assemble a molecular physiological view of the adaptation processes, we organized genes into groups representing cellular functions (Table 2). These functions included extracellular matrix remodeling processes, cytoskeletal structure, nuclear and sarcolemma structure, energy metabolism, ionic flux, cell proliferation, stress response, signal transduction, hormones, and proteins associated with translation. We also found 11 new genes of unknown function that will require further investigation.
Northern analysis confirmed differential expression for 15 genes of 20 tested, and loading was controlled for by means of 18 S rRNA hybridization. A difference of expression of at least 20% was detectable in the 15 genes from atria of dogs subjected to HFD (Table 2). Two genes, sarcoplasmic reticulum Ca2+ ATPase (SERCA2) and ß-hemoglobin, which were found to be downregulated in the microarrays, were found to be upregulated by Northern blot analysis in 2 separate trials (Table 2 and data not shown).
Quantitative PCR performed on cDNA obtained from each animal confirmed expression of 12 genes of 15 randomly chosen in the known gene list (Table 2). Expression of 3 genes (Tensin, ALS 2, and NRF2) displayed high variability, and differential expression was not statistically relevant. Interestingly, real-time PCR confirmed downregulation of SERCA-2.
Identification of Differentially Expressed Genes in Ventricle
Differentially expressed genes identified in the atrium were also tested in left ventricle samples by the use of our cDNA microarrays. This experiment revealed differential expression for 14 genes of 38. Of the 24 genes that showed no differential expression in ventricles of obese compared with lean dogs, 6 genes were randomly selected for Northern blot analysis. Results obtained with microarrays were confirmed by Northern blot for 6 of 6 genes (Table 2).
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Discussion |
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Although obesity is recognized as a cause of major cardiovascular diseases such as heart failure,15 the molecular mechanisms involved in left ventricular hypertrophy and remodeling are poorly understood. In the present study, we applied gene expression profiling as a technique to identify potential candidate genes that may play a role in the pathophysiology of changes in cardiac structure and function in an experimental model of obesity and arterial hypertension induced by a 9-week HFD regimen in dogs. We identified 38 genes that were differentially expressed in the atria of obese dogs compared with lean dogs, and 15 of these were confirmed by Northern blot analysis. Many of these differentially expressed genes are known to be involved in multiple cell functions, including extracellular matrix remodeling processes, cytoskeletal structure, nuclear and sarcolemma structure, energy metabolism, ionic flux, cell proliferation, stress response, signal transduction, hormones, and proteins associated with translation. Moreover, as further discussed below, we found in several instances that expression of genes that were differentially expressed in atria was not significantly modified in ventricles of obese dogs. This different pattern of regulation between right atrium and left ventricle could be explained by several factors. The first one is the precocity of the analysis. We deliberately chose a short period of exposure to HFD. This choice was made to get information in a rather stable condition, that is, at the end of the dynamic phase of weight gain and of endocrine and metabolic changes induced by fat diet11 but before the occurrence of significant changes in left ventricular mass reflecting LVH, which needs longer exposure to HFD to develop.18 The second factor explaining the differences in atrium and ventricle responses to obesity-related hypertension is the volumetric character of the arterial hypertension in this experimental model. As previously reported by our group, obesity-related hypertension induced by short-term HFD is associated with an increase in right auricular pressures without changes in peripheral arterial resistances.11 Finally, the present findings indicating more prominent changes in atrium genes pattern of expression could be relevant to explain early changes in atrial function such as tachycardia and decreased HR variability11 or modification in M2-muscarinic receptors.18
Previous studies have provided considerable evidence that in dogs, HFD closely mimics cardiovascular and metabolic changes observed in obese humans.31 Although this model has provided important information about some of the physiological events that underlie the mechanisms of arterial hypertension and of other cardiovascular adaptations to obesity, a molecular understanding of these processes has been limited by the lack of tools for functional genomic studies in dogs. To our knowledge, there have been no previous reports of cardiac gene expression profiling in obese dogs. This contrasts to the large amount of data available concerning gene regulation in other experimental models of ventricular overload or of cardiac failure in rodents. Therefore, we prepared our own dog cDNA arrays with a focus on differentially expressed genes using a subtractive-hybridization approach (SSH).20 The use of cDNA microarrays to further screen such libraries has been shown to be efficient,32 and these new tools will be of great importance in future studies. SSH cannot be considered as exhaustive but enriches rare, differentially expressed transcripts by >1000-fold.20 One major advantage of the combined approach of SSH and cDNA microarrays is the identification of differentially expressed genes without the availability of already-cloned dog cDNA. The use of custom-made cDNA arrays allowed us to perform enough hybridizations to gain accuracy for monitoring gene expression and to assess variations as small as 20% in transcript level. This low threshold for detection of changes in gene expression, which required a high degree of repeatability in 10 microarray hybridizations, for each experiment, is important because many physiological variables change by 20% or less in the early phases of obesity and structural changes in the heart and blood vessels are modest.33 Therefore, one might also expect modest changes in expression of many genes involved in the early stages of cardiovascular adaptation to an HFD, thus requiring sensitive and repeatable methods of detection. Using 10 microarray hybridizations that were highly repeatable for each experiment, we found 38 genes that were upregulated or downregulated in atria from dogs made obese and hypertensive by HFD. Confirmation was obtained in almost half of them with Northern analyses (15 successful confirmations out of 20 Northern blots) and with a high success rate by quantitative PCR (12 confirmations out of 15 randomly chosen genes), thus indicating, as previously reported, that microarrays can be reliable indicators of altered gene expression. In addition, quantitative PCR evaluated individual variability as low for expression of 80% of the tested genes and further confirmed differential expression for 2 genes (VDAC and Lectomedin-3) that were not statistically differentially expressed with microarray analysis. Despite this measure of reliability, microarrays can also generate some artifacts, as shown in our comparison of expression profiles between microarrays and Northern blots for SERCA2 and ß-hemoglobin. Even taking into account limitations, our data globally show that in a dietary model of obesity produced by feeding dogs ad libitum an HFD, gene-regulated reprogramming occurs. The importance of these early mRNA level modifications for the pathophysiology of cardiovascular morbidity related to obesity-related hypertension remains to be elucidated. In the same way, the respective roles of increased fat mass and of hypertension in theses genic adaptations will need further studies, which could, for instance, investigate the pattern of gene expression in hearts from transgenic obese mice without arterial hypertension.
The qualitative aspects of our findings and their relevance for the pathophysiology of heart changes related to HFD deserves some comments in view of the differences observed between atrium and ventricle changes in pattern of gene expression. A first set of genes was found to exhibit similar regulation in both right atrium and left ventricle, suggesting that weight gain and arterial hypertension induced by HFD are associated with early and diffuse cardiac reprogramming. Among the genes that belong to this group, we found induction of MMP-9. Overexpression of MMP-9, a gelatinase B whose substrates include gelatin, collagen IV, V, and XIV, aggrecan, elastin, entactin, and vibronectin,34 as previously reported in LVH in humans.35 These genes could be involved in the pathophysiological mechanisms leading to the genesis and progression of heart failure. We also found upregulation, in both chambers, of mRNA levels encoding proteins of the mitochondria respiratory chain such as cytochrome C oxidase subunit I and VIIb, NADH dehydrogenase, and ATP synthase F0 subunit. These changes probably reflect increased energy and ATP consumption secondary to the increased venous return and cardiac preload as well as to arterial hypertension. Similar positive regulations of ATP synthesis have also been described in left ventricular mitochondria from spontaneously hypertensive rats.36,37 These findings showing that even if more marked in atrium, gene expression pattern changes also concern left ventricle, contrasts with the absence of major ventricular macroscopic alterations and of increased left ventricular mass measured with echocardiography after 9 weeks of an HFD. However, one can speculate that we detected initial molecular changes in the extracellular matrix that will participate in tissue remodeling that is usually seen after 20 weeks of an HFD.18
A second group of genes was differentially regulated in the atria from obese dogs but not in ventricle. This feature could be explained by the specific function and structure of the atria, which acts as a volume sensor38 that is prone to distension by volume overload, a key feature of obesity-related hypertension.17 Interestingly, mRNA from genes involved in cardiac excitation-contraction coupling such as phospholamban (PLB) and SERCA-2 were found to be differentially regulated in the atria from obese dogs. This regulation may contribute to the marked increase in heart rate and may be a consequence of decreased baroreflex efficiency observed in obese dogs.11 Thyroid hormones, which are also increased during caloric overfeeding,39 have also been shown to increase PLB mRNA levels.40 However, the relevance of these changes to explain changes in atrial gene expression in obesity-related hypertension remains to be elucidated. In obese dogs, we also observed a specific 9-fold reduction of N-acetyl galactosaminyl transferase mRNA levels in atrium by Northern blot analysis, whereas its expression was not significantly modified in left ventricle. This enzyme, which mediates acquisition of carbohydrate side-chains in O-glycosidic linkage to either threonine or serine, modifies the structure of polypeptide backbone and heavily O-glycosylated proteins involved in cell interactions such as mucin glycoproteins.41 In addition, O-glycans function as ligands for receptors mediating tumor cell adhesion.42 The levels of mRNA encoding lectomedin-3 were also found to be upregulated. This protein harbors a galactose binding domain and is a G-protein-coupled receptor that also regulates cell adhesion by means of an inside-out effector signaling pathway. Overexpression of this protein can promote adhesion to ligands present in the extracellular matrix or on opposing cells resulting in chemotaxis and extravasation.43 Such proteins have recently been identified and may be triggered by cell-cell or cell-matrix interactions that implicate them in planar polarization during organogenesis.43 The last gene from this group is atrial natriuretic peptide, whose induction has been considered an index for cardiac remodeling toward a fetal pattern.44
Reorganization of tissue structure is often concomitant with cell proliferation.45 We observed the expression of a set of genes that have not yet been studied in heart. First of all, nucleophosmin (NPM), which is known to accumulate in nuclei of exponential growing HeLa cells46 and is strongly upregulated during estrogen-induced cell proliferation in MCF-7 breast cancer cells,47 was upregulated in atria of obese dogs. NPM has several potentially important roles in regulating cell function and signaling. Specifically, NPM is a chaperone for nuclear import of proteins.48,49 This feature lends itself to chromatin dynamics50 and plays an important role in the regulation of cellular mitogenesis. In this same group of genes, we found upregulation of mRNA levels for ERK-3, a mitogen-activated protein kinase present in the nucleus.51 ERK3, also called p97, is activated by protein kinase C52 and is responsive to different growth factors, implying a mechanism for specificity in cellular signaling53 in response to activation of the p38 pathway.54 As expected in a situation of tissue growth, we observed upregulation of RNA levels encoding ribosomal proteins and elongation factor-1
, a protein involved in mRNA translation. Taken together, these changes in gene expression are in accordance with a situation of cell proliferation in response to extracellular stimuli associated with an HFD. These findings point toward several potential molecular pathways that may offer a better understanding of the mechanisms involved in atrial and ventricular adaptations to a long-term HFD.
We also found 11 cDNA sequences representative of mRNA coding for new proteins of unknown functions to be differentially expressed in atria and in ventricle after 9 weeks of an HFD. Analysis of the encoded proteins and their functions will be undertaken in future works.
Perspectives
Our work shows that HFD-induced obesity not only is associated with arterial hypertension and other important cardiovascular changes but also causes early profound and specific modifications of the cardiac transcriptome both at the atrium and at the ventricular level. However, because obesity is associated in our model with arterial hypertension, it is not possible to conclude that changes observed are specific to obesity-related hypertension. Further studies in experimental models of obesity without changes in blood pressure are therefore necessary.
Since we focused on modifications that occur early after the installation of obesity and hypertension, the observed changes constitute one basis for the study of the mechanisms linking the increase in fat mass and myocardium. In fact, the search for the potential mechanisms that initiate alterations in cardiac gene expression secondary to HFD represents an important area for further studies that could be fruitful for a better understanding of the pathophysiology of obesity-related cardiovascular morbidity.
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Acknowledgments |
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The authors thank Veronique Leberre, Cécile Tonon, and Serguei Sokol (Toulouse Genomic Core Facilities), Jean-José Maoret (INSERM IFR 31), and Nathalie Laplace (Service de Pharmacologie, Paul Sabatier University), respectively, for their excellent technical assistance in microarray spotting, data analysis, DNA sequencing, and laboratory logistics; and Peter J. Romanienko (National Institutes of Health, Bethesda, Md) and John. E. Hall (University of Mississippi Medical Center, Jackson) for critical reading of the manuscript.
Received September 25, 2002; first decision October 16, 2002; accepted January 10, 2003.
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