Sexually dimorphic gene expression in the heart of mice and men

Abstract

The prevalence and clinical manifestation of several cardiovascular diseases vary considerably with sex and age. Thus, a better understanding of the molecular basis of these differences may represent a starting point for an improved gender-specific medicine. Despite the fact that sex-specific differences have been observed in the cardiovascular system of humans and animal models, systematic analyses of sexual dimorphisms at the transcriptional level in the healthy heart are missing. Therefore we performed gene expression profiling on mouse and human cardiac samples of both sexes and young as well as aged individuals and verified our results for a subset of genes using real-time polymerase chain reaction in independent left ventricular samples. To tackle the question whether sex differences are evolutionarily conserved, we also compared sexually dimorphic genes between both species. We found that genes located on sex chromosomes were the most abundant ones among the sexually dimorphic genes. Male-specific expression of Y-linked genes was observed in mouse hearts as well as in the human myocardium (e.g. Ddx3y, Eif2s3y and Jarid1d). Higher expression levels of X-linked genes were detected in female mice for Xist, Timp1 and Car5b and XIST, EIF2S3X and GPM6B in women. Furthermore, genes on autosomal chromosomes encoding cytochromes of the monoxygenase family (e.g. Cyp2b10), carbonic anhydrases (e.g. Car2 and Car3) and natriuretic peptides (e.g. Nppb) were identified with sex- and/or age-specific expression levels. This study underlines the relevance of sex and age as modifiers of cardiac gene expression.

Fig. 1

Experimental design to screen for genes with sex-biased expression in the heart of mice and human donors. Total RNA was isolated from mouse whole heart samples and from myocardial tissue of human donors. Cyanine dye labelled cDNAs generated from pooled RNAs were hybridised on Agilent cDNA microarrays. In addition, human myocardial expression datasets were downloaded from CardioGenomics and filtered for sex-biased genes. Candidate gene lists were compared to identify sexual dimorphisms in young as well as aged individuals and both species and platforms. Expression levels of several candidate genes were quantified individually by real-time PCR (QPCR) in the same samples used for microarray analysis and in left ventricular samples from female mice at different stages of the oestrous as well as from males (n = 8 per group). Numbers of individuals analysed by QPCR are shown in brackets. FDR, false discovery rate; y, years; mon, months

Fig. 2

Candidate genes identified by microarray approaches. a False colour representation of the 13 overlapping genes (see also intersection in the Venn diagram) identified with sex-biased expression in 2 as well as in 8 months old mice. Fold-changes were calculated from four technical replicates using pooled total RNA isolated from male and female whole mouse hearts of both age groups (n = 6 per group). Numbers of genes identified with sex-biased expression in each group (90 and 33, respectively) are shown in the Venn diagram. b False colour representation of the 14 overlapping genes (see also intersection in the Venn diagram) identified with sex-biased expression in human left ventricular samples of both age groups. Fold-changes were calculated from four technical replicates using pooled total RNA isolated from three to five samples per group. Numbers of genes identified with sex-biased expression in each age group (93 and 125, respectively) are shown in the Venn diagram. c False colour representation of the 16 genes identified in the Cardiogenomics dataset with sex-biased expression in myocardial tissue of male (mean age = 53.6 years) and female (mean age = 49.3 years) human donors (n = 7 per group). Note that for some genes sex-biased expression was detected in both species (e.g. Ddx3y and Jarid1d) as well as in both platforms (USP9Y and RPS4Y1). Shades of red represent female-biased expression and blue, male-biased expression in individual samples (FDR <5%, fold-change >2)

Fig. 3

Analysis of sex-biased gene expression of carbonic anhydrase 3 (Car3), Car2 and cytochrome P450 2b10 (Cyp2b10) in individual samples. The expression levels of these genes were quantified using real-time PCR (QPCR) in whole hearts (a, d, g; n = 6 per group) as well as in isolated left ventricles of mice (b, e, h; n = 8; 3 months of age) and human donors (c, f, i; n = 3 for <40-year-old women, n = 6 for <40-year-old men, n = 7 for 50–65-year-old women and men). Data represent mean values of relative expression levels normalised to the geometric mean of reference gene expression levels (HPRT, GAPDH and 18S) ±SEM. Asterisks indicate levels of significance determined by two-tailed t tests assuming unequal variance of the biological replicates. (Asterisks, p < 0.05; double asterisk, p < 0.01)

Fig. 4

Analysis of sex-biased gene expression of natriuretic peptide precursor A (Nppa), Nppb and nuclear receptor 4a1 (Nr4a1) in individual samples. The expression levels of these genes were quantified using real-time PCR (QPCR) in whole hearts (a, d, g; n = 6 per group) as well as in isolated left ventricles of mice (b, e, h; n = 8; 3 months of age) and human donors (c, f, i; n = 3 for <40-year-old women, n = 6 for <40-year-old men, n = 7 for 50–65-year-old women and men). Data represent mean values of relative expression levels normalised to the geometric mean of reference gene expression levels (HPRT, GAPDH and 18S) ±SEM. Asterisks indicate levels of significance determined by two-tailed t tests assuming unequal variance of the biological replicates. (Asterisks, p < 0.05; double asterisk, p < 0.01)