Sex-specific pathways in early cardiac response to pressure overload in mice
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- Witt, H., Schubert, C., Jaekel, J. et al. J Mol Med (2008) 86: 1013. doi:10.1007/s00109-008-0385-4
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Pressure overload (PO) first causes cardiac hypertrophy and then heart failure (HF), which are associated with sex differences in cardiac morphology and function. We aimed to identify genes that may cause HF-related sex differences. We used a transverse aortic constriction (TAC) mouse model leading to hypertrophy without sex differences in cardiac function after 2 weeks, but with sex differences in hypertrophy 6 and 9 weeks after TAC. Cardiac gene expression was analyzed 2 weeks after surgery. Deregulated genes were classified into functional gene ontology (GO) categories and used for pathway analysis. Classical marker genes of hypertrophy were similarly upregulated in both sexes (α-actin, ANP, BNP, CTGF). Thirty-five genes controlling mitochondrial function (PGC-1, cytochrome oxidase, carnitine palmitoyl transferase, acyl-CoA dehydrogenase, pyruvate dehydrogenase kinase) had lower expression in males compared to females after TAC. Genes encoding ribosomal proteins and genes associated with extracellular matrix remodeling exhibited relative higher expression in males (collagen 3, matrix metalloproteinase 2, TIMP2, and TGFβ2, all about twofold) after TAC. We confirmed 87% of the gene expression by real-time polymerase chain reaction. By GO classification, female-specific genes were related to mitochondria and metabolism and males to matrix and biosynthesis. Promoter studies confirmed the upregulation of PGC-1 by E2. Less downregulation of metabolic genes in female hearts and increased protein synthesis capacity and deregulation of matrix remodeling in male hearts characterize the sex-specific early response to PO. These differences could contribute to subsequent sex differences in cardiac function and HF.
KeywordsPressure overload Hypertrophy Gene expression Sex Heart
Pressure overload (PO), as in hypertension or aortic stenosis, initially leads to left ventricular hypertrophy (LVH) and later to heart failure (HF). In the population, women with aortic stenosis develop less HF at similar mechanical load than men. HF affects about 10% of persons aged 70 years and increases thereafter [1, 2, 3, 4]. Women more commonly develop HF with preserved ejection fraction, whereas men more often have HF with reduced ejection fraction [5, 6, 7]. In addition, women with HF have a better outcome at similarly reduced systolic function [8, 9]. Estrogens improve myocardial adaptation to PO in hypertensive women [10, 11] and in mouse models [12, 13]. Several transgenic LVH models have shown that female animals develop HF and death at a later time point than males [14, 15, 16]. PO in rodent models leads to LVH with normal systolic function at an early stage and impaired systolic function at later stages with significant sex differences [17, 18, 19]. The exact time point when sex differences appear depends on the model, the degree of PO, animal age, and strain. In a rat transverse aortic constriction (TAC) model with moderate PO, males showed more LVH and depressed contractile reserve than females [18, 20]. With severe PO, male mice had worse LVH than females by 2 weeks . Gene expression has identified hypertrophy-related genes mainly in males or in mixed populations [17, 21, 22, 23]. The profiles suggested pathways that included functionally related pro- and antihypertrophy genes or estrogen receptor (ERα and ERβ)-driven genes [22, 24]. However, only a single microarray study on sex differences in cardiac hypertrophy has been published so far in mice . Sex differences that might underlie the functional differences at later stages were not identified. We aimed to study this issue and used a novel bioinformatics approach to define networks of functionally related genes [25, 26].
Materials and methods
All animal experiments followed the “Principles of laboratory animal care” (NIH publication No. 86-23, revised 1985) as well as the current version of the German Law on the Protection of Animals.
In the first cohort, we randomized 12–14-week-old male and female C57BL/6J mice to either sham operation or banding of the thoracic aorta . Four animals per group (sham-operated males, females, TAC males, and females) underwent echocardiography 2 weeks after surgery and were killed 2 days later. A two-dimensional short-axis view and M-mode tracings of the left ventricle were obtained with a 45-MHz transducer (Vevo 770™, VisualSonics, Canada). Wall thickness and left ventricular diameters (LVEDD/LVESD) were measured according to the American Society for Echocardiography. Left ventricles were immediately frozen in liquid nitrogen and used for microarray analysis and real-time polymerase chain reaction (PCR). Hemodynamic parameters were evaluated in a second cohort of 4 × 4 male and female mice that underwent TAC or sham surgery, parallel to the main experiment. A pressure transducer catheter (Millar Instruments) was introduced into the right carotid artery and advanced into the left ventricle to assess ventricular function. Data were analyzed using a computerized data acquisition system (Power Lab, ADI Instruments, Melbourne, Australia) . In a third cohort, echocardiography was done at weeks 2, 6, and 9 after TAC or sham surgery to determine LV mass (n = 62, males/females/sham/TAC).
Four individual hearts per group from cohort 1 (sham male, sham female, TAC male, TAC female) were analyzed on 16 single arrays. The amplification and labeling of the RNA samples were carried out according to standard protocols (Affymetrix, Santa Clara, CA, USA). Fifteen micrograms of each cRNA sample was hybridized to Affymetrix RAE 430A GeneChip arrays. Arrays were scanned at 3 mm resolution using the Affymetrix GeneChip System confocal scanner 2500 (Electronic supplementary material), and raw signal intensities were normalized globally with a scaling factor using Affymetrix GeneChip® Operating Software (GCOS). The gene expression data were deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/projects/geo/) as GEO accession GSE6970.
Quantification of mRNA by real-time PCR
Total RNA preparation (RNABee, BIOZOL, Germany), desoxyribonuclease (DNase) digestion (DNAse I, Invitrogen, Germany), and reverse transcription (SuperScript, Invitrogen, Germany) were performed according to the manufacturer’s recommendations. A “hot start” real-time PCR procedure with SYBR Green was performed in duplicates with the ABI Prism 7700 Sequence Detection System. The mRNA content of the target genes was normalized to the expression of GAPDH in the same sample. Primers for all target genes were designed using Primer3 Software (Table e1, Electronic supplementary material) . To correct differences in PCR efficiencies, a calibration curve containing 25, 12.5, 6.25, 3.13, and 1.56 ng cDNA template pooled from all samples analyzed was run with each primer pair.
Morphological and functional data were analyzed using the Mann–Whitney test and one-way analysis of variance (ANOVA) followed by Tukey’s test. Differences were considered significant at p < 0.05. For the identification of differentially expressed genes in the comparison of two conditions with microarrays (male vs. female, TAC vs. sham), we used the significance analysis of microarrays (SAM) . The SAM statistic identifies significant changes in gene expression by performing a set of gene-specific t tests. For each gene, a score is calculated on the basis of expression change relative to the standard deviation of repeated measurements for that gene. Genes with scores greater than a threshold delta were defined as significantly deregulated. Manual adjustment of this threshold delta allows the identification of smaller or larger gene cohorts. In addition, based on random permutations of all measurements, a false discovery rate was estimated. For the identification of differentially expressed genes in the comparison of four conditions (sham female, sham male, TAC female, and TAC male) we used a two-way ANOVA to test for the interaction between hypertrophy (TAC/sham) and sex (male/female) without correction for multiple testing. A p value < 0.05 indicates that the gene is deregulated differentially after PO in males and females. For functional annotation and pathway analysis, we classified the genes as relatively upregulated in females or males, respectively. For example, a relative upregulation in females would correspond to: first, a relative stronger induction (female TAC/female sham > male TAC/male sham > 1); second, a weaker repression (1 > female TAC/female sham > male TAC/male sham); or third, an opposite regulation (female TAC/female sham > 1 > male TAC/male sham) of gene expression.
Functional annotation and pathway analysis
For the detection of gene ontology (GO, www.geneontology.org) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG, www.kegg.com) pathways with a significant overrepresentation of genes in a given group compared to the whole genome, the web-based Database for Annotation Visualization and Integrated Discovery tool (DAVID, National Institute of Allergy and Infectious Disease) was used [25, 26]. Fisher’s exact test was applied to determine whether or not the proportion of those genes falling into each GO category or KEGG pathway differed significantly between the input data set and the whole genome. Networks of biologically related genes were created with the help of the Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com). The sex-specific regulated genes obtained from the microarray analysis were used to build literature-based sex-specific networks. In the resulting networks, genes or gene products are represented as nodes, and the biological relationship between two nodes is shown as a line. All lines are supported by at least one reference from literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base. For each network, a score is calculated based on the p value of a right-tailed Fisher’s exact test. This score calculates the approximate fit between each network and the focus genes from the input data set and indicates whether or not a network contains more genes than expected by chance.
Cell culture and transient transfection reporter assays
Human genomic DNA isolated from peripheral blood samples of healthy volunteers was used as template to generate the reporter construct containing the 5′-flanking region of the PGC-1α gene by PCR with PGC-1α-1.7 kb-Fw: GATCGGATCCGTGCTGGTGAACTGTATTCAGC, PGC-1α-0.6 kb-Fw: GATCGGATCCCGCTTTCAAACACTCCCTCAATG, respectively, and PGC-1α-RV: GATCGGATCCTCCTGAATGACGCCAGTCAAGC primers. PGC-1α promoter fragments (−1,655 bp/+114 bp and −515 bp/+114 bp, relative to the transcriptional start site +1) were cloned into pJET1.2 vector (Fermentas) and confirmed by sequencing. The individual promoter fragments were subcloned into the luciferase reporter vector pGL3-basic (Promega) using BglII restriction sites in sense and antisense orientation. AC16 cells (human cardiomyocyte cell line, Davidson et al. ) were cultured in phenol red-free DMEM/F12, supplemented with 12.5% charcoal-stripped fetal calf serum (FCS; Biochrom AG), penicillin/streptomycin (100 U/ml/100 μg/ml, PAA), and amphotericin B (0.25 μg/ml) at 37°C under 5% CO2. For transient transfection experiments, 500 ng of the PGC-1α promoter-luciferase reporter construct and 100 ng of the internal reference Renilla luciferase reporter plasmid phRL-TK vector (Promega) were co-transfected into AC16 cells using FuGENE® HD reagent (Roche Diagnostics). In some experiments, ERα-pSG5 plasmid (HEGO-vector) or its empty vector pSG5 were co-transfected by using a similar transfection procedure. Twenty-four hours after transfection, cells were treated with or without E2 (10−8 M, Sigma) in phenol red-free DMEM containing 2.5% charcoal-stripped FCS for additional 24 h. Firefly and Renilla luciferase activities were measured using the Dual-Glo™ Luciferase Assay System (Promega). Transfections were carried out in triplicates for at least three times. The authors had full access to the data and take responsibility for its integrity. All authors have read and agreed to the manuscript as written.
Sex differences in LVH were present at 6 weeks but not at 2 weeks
PO-induced gene regulation
Sex-specific gene expression
A small group of genes showed differential expression between males and females (Fig. 4b, c) independent from TAC (all males vs. all females). As in previous studies for somatic tissues , the overall number was relatively low (20 genes, false discovery rate <10%, SAM). Six genes showed higher expression in females including several X-linked genes. Fourteen genes showed higher expression in males including members of the X-degenerated region of the Y chromosome (Table e2, Electronic supplementary material).
Sex-specific gene regulation in hypertrophy by microarray and confirmatory real-time PCR
Functional classification of sex-specific regulated genes
Genes with sex differences in their expression pattern were GO-annotated at level 4 of the categories “biological process” and “cellular localization” and were sorted in KEGG pathways
No. of genes
Ratio >1 (relatively upregulated in females)
Cellular macromolecule metabolism
Intracellular membrane-bound organelle
Mitochondrial inner membrane
Propanoate metabolism (MMU00640)
Pyruvate metabolism (MMU00620)
Insulin signaling (MMU04910)
Ratio <1 (relatively upregulated in males)
Cytosolic ribosome (sensu Eukaryota)
Intracellular membrane-bound organelle
Focal adhesion (MMU04510)
Ribosomal genes with relative upregulation in males
Probe set IDa
Ratio female vs. male
Ratio male vs. female
Mitochondrial ribosomal protein L10
Mitochondrial ribosomal protein L27
Mitochondrial ribosomal protein L32
Mitochondrial ribosomal protein S18A
Ribosomal protein L10
Ribosomal protein L13a
Ribosomal protein L18
Ribosomal protein L21
Ribosomal protein L23
Ribosomal protein L23a
Ribosomal protein L29
ribosomal protein S11
Ribosomal protein S15
Ribosomal protein S16
Ribosomal protein S24
Ribosomal protein S3a
Ribosomal protein S5
Ribosomal protein S7
Ribosomal protein S8
Ribosome binding protein 1
The most prominent male network included 32 genes and three external genes (Fig. 6b, Table e7, Electronic supplementary material). In the male-specific network, genes related to extracellular matrix synthesis were overrepresented. These genes included procollagen type III alpha 1 (Col3a1) and procollagen type V alpha 2 (Col5a2), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 4 (TIMP4), transcription factors from the sons of mothers against decapentaplegic (SMAD) family, and transforming growth factor beta2 (TGFβ2). The second best male network included the prohypertrophic transcription factor myocyte enhancer factor 2C (MEF2; Fig. e1, Table e7, Electronic supplementary material). MEF2c was downregulated in females, whereas the gene was almost unchanged in males (ratio female/maleArray = 0.54; ratio female/maleRT-PCR = 0.58).
Regulation of the PGC-1α promoter activity by E2
We found that many genes were differentially regulated in male and female mice before sex-specific morphological or functional changes occurred. We consigned these genes to networks that feature the predominant early response in the male and female hearts to PO. Less downregulation of genes encoding mitochondrial function and fatty acid oxidation characterized the network of the female hearts and relative upregulation of matrix remodeling and ribosomal genes the network of the male hearts. The data support the concept that better adaptation of energy metabolism in the female hearts, and greater induction of matrix turnover and ribosomal protein synthesis in the male hearts, are important mediators of sex-dependent remodeling during early stages of PO. These processes may represent the molecular basis for the sex-specific functional changes observed at late stages of PO and LVH.
In transgenic LVH models, HF and death occur earlier in male than in female animals [14, 15, 16, 31]. TAC-induced PO represents a global stimulus for LVH that is initially concentric and leads at later stages to dilatation and HF. Rats and mice exhibit sex differences in the LVH response to PO and the transition to HF, which are accompanied by changes in gene expression [17, 18, 19, 20, 32]. However, sex differences in gene expression that occur after the manifestation of sex differences in cardiac function may represent a response to differences in hemodynamic load rather than its cause. We therefore searched to detect the early sex differences in gene expression following TAC that may determine the sex differences in cardiac function at later stages. We euthanized mice 2 weeks after TAC to measure gene expression at a time point when hypertrophy was already present, yet without sex differences. LVH-related sex differences developed only at later stages after TAC in our model, similar to other reports [17, 18, 19, 33]. To obtain evidence that our microarray analysis had sufficient sensitivity and specificity to detect biologically relevant gene expression changes, we analyzed gene expression profiles of classical markers of hypertrophy and sex chromosome-linked genes. Indeed, we observed the upregulation of a comprehensive hypertrophy marker set induced by TAC as described earlier, as well as Y chromosomal genes in males [21, 22, 34].
A significant overrepresentation of male-upregulated genes was found in the gene ontology categories of biosynthesis and cell organization, which agrees with published data analyzing the effects of TAC on gene expression and showing strong activation of matrix remodeling [21, 22, 34]. A group of ribosomal genes linked to protein biosynthesis also showed relative upregulation in male hearts, which may be explained by the greater need of protein synthesis for increased matrix turnover. Accordingly, the most prominent male network assembles matrix regulating genes, namely collagens, matrix metalloproteinases (MMP), and tissue inhibitors of MMPs (TIMPs). An increase of MMP14 after PO in males was also reported earlier in a similar setting . Our data correspond well to the analyses of PO-induced gene expression in a similar TAC model by Mirotsou et al. . First, a large number of hypertrophy-related genes that they found to be regulated were also detected in our study, including ANP, collagens, and CTGF. Second, genes with functions related to cell growth, morphology, differentiation, and ECM activity were directly correlated with LVH, which is also in agreement with our study showing upregulation of matrix-related and ribosomal genes, predominantly and most pronounced in males . Based on our findings, male TAC mice have a higher collagen turnover than females, which may lead to an altered collagen composition with a different amount of cross linking and collagen subtypes in both sexes. Indeed, the modulation of extracellular matrix synthesis has been linked to sex differences in cardiac stiffness underlying sex differences in diastolic function in clinical syndromes [6, 17, 20].
Female hearts were characterized by the relatively better maintenance of metabolic capacity. An unbiased analysis placed the PPARγ-coactivator 1α (PGC-1) in a central position in the most relevant female network . PGC-1 stimulates mitochondrial biogenesis and fatty acid oxidation and reduces glucose oxidation in the myocardium [36, 37]. Accordingly, we found less downregulation of PGC-1-controlled genes involved in mitochondrial fatty acid metabolism and the respiratory chain in females. Examples included acyl-CoA dehydrogenase, carnitine-palmitoyl transferase, cytochrome oxidase, and pyruvate dehydrogenase kinase 4, which downregulates glucose metabolism. Mirotsou et al. also found an inverse correlation of energy metabolism-related genes and mitochondrial function with left ventricular hypertrophy after TAC in a cohort of females and males, which was confirmed by our data with the additional finding of a stronger downregulation in males than in females . Recent work by Wagner et al. in male hearts also showed that mitochondrial energy metabolism is decreased after TAC . O’Lone et al. demonstrated that a large number of energy metabolism-related and mitochondrial function-related genes are controlled by estrogen via ERα or ERβ in the vasculature . This finding corresponds well to the group of genes that were upregulated in our female network, namely genes associated with energy metabolism, mitochondrial genes, and genes involved in electron transport and fatty acid oxidation. Interestingly, Haddad et al. found in a gene expression profiling on idiopathic dilated cardiomyopathy in humans that metabolic genes are mainly deregulated in females and genes related to muscular contraction and extracellular matrix in males . Altogether these studies indicate that our findings are not restricted to our model, but represent general mechanisms in the sex-specific adaptation processes of the heart.
Limitations of the study
Our findings on sex differences in hypertrophy-related gene expression in mice are consistent with the concepts discussed in previous reports [17, 19]. Nevertheless, larger assay numbers are needed to analyze age dependency and mouse strain specificity. To describe the pathways involved and to prove their mediating role, more experimental approaches are required that alter the function of one or more of the genes identified.
Species differences between humans and mice have to be analyzed in further studies as well as the effect of hormonal status of the females. We found that employing synchronized females did not reduce inter-animal variability in gene expression . Finally, to work out the whole complexity of sex-specific responses in hypertrophy, systems biology, proteomic, and metabolomic approaches must be developed to complement the expression studies.
In the early response to pressure overload, a more efficient metabolic regulation in female hearts, and greater induction of matrix remodeling in male hearts, may set the stage for the relative protection of the female hearts. Translation of these findings to human PO-induced hypertrophy and HF could benefit both women and men.
We gratefully acknowledge the critical comments of Noel Bairey Merz, Medical Director and Endowed Chair, Women’s Health Program, Cedars-Sinai Medical Center and Friedrich Luft, Director of the Experimental and Clinical Research Center in Berlin-Buch. We thank Pierre Chambon (Institute of Genetics, Molecular and Cellular Biology, CNRS/INSERM, College de France, Illkirch Cedex, France) for his of ERα-pSG5 plasmid (HEGO vector). We thank Stefanie Roehner for excellent secretarial work and Jenny Thomas for excellent technical assistance. The EU FP6 grant LSHM-CT-2005-018833, EUGeneHeart, the Deutsche Forschungsgemeinschaft Graduate Studies Grant 754 on myocardial hypertrophy, DFG Re 662/6-1, and Grohe 729/12-1 supported this work. We thank Joerg Isensee for helping with the graphical illustrations. Parts of the doctoral thesis of Juliane Jaekel have been incorporated into this article.