Using DNA microarray analysis we have obtained information about transcripts that are present in the human fetal heart at 18–22 weeks of gestation. The data indicate that approximately 50% of the 15,000 analyzed genes are expressed in human fetal heart. The percentage of Hsa21 genes expressed in fetal hearts was not very different.
Upregulation of Hsa21 genes
We found that Hsa21 genes are upregulated in all trisomic fetal hearts. This demonstrates that the concept of increased transcription of Hsa21 genes in Hsa21 trisomy applies to the human heart irrespective of CHD. The level of upregulation is modest. If we consider the mean for all Hsa21 genes analyzed, the fold increase in Hsa21 trisomy hearts versus controls is close to 1.5. This result is highly significant and confirms that microarrays can detect even small differences in gene expression levels.
This modest gene upregulation is in agreement with data obtained in developing human brain and heart [16, 17], as well as in the cerebellum [21, 22] and in different tissues [18–20] of adult Ts65Dn and Ts1Cje mice, two animal models of Hsa21 trisomy. Despite the different experimental conditions, an overall fold increase of ~1.5, which is consistent with a 3:2 ratio between trisomic and euploid fetuses, was reported in all these studies. However, the global 1.5-fold increase does not simply result from a 1.5-fold upregulation of each single gene. Indeed, whereas the fold increase for most genes is between 1.4 and 1.8, 8 genes are upregulated more than 2-fold in the DSH group and 25 genes are not upregulated in the DSH and NH groups. These results, which have been confirmed by qRT-PCR and are in agreement with another study , are indicative of either compensatory effects or heterogeneity in the regulatory mechanisms of Hsa21 genes. The gene expression level of individual Hsa21 genes in the heart of DS individuals is of central relevance in the effort to understand how Hsa21 trisomy causes CHD. Upregulation of a single gene or a combination of dysregulated genes might be at the base of CHD. Measurement of the levels of proteins corresponding to upregulated genes may shed light on the link between Hsa21 trisomy and CHD; a limited amount of information is available on this issue [34–36].
Downregulation of mitochondrial genes
Our finding that 441 extra-Hsa21 genes were dysregulated in all trisomic samples supports the hypothesis that genes outside of Hsa21 might play a role in determining DS features . Functional analysis of the genes differentially expressed between trisomic and control samples shows global downregulation of genes encoding mitochondrial proteins, especially enzymes involved in the oxidative phosphorylation pathway. Genes from all five complexes were downregulated suggesting that the corresponding proteins and enzymatic activities might be reduced, and that the mitochondrial function could be consequently impaired.
Protein levels of complex I, III and V were found to be decreased in cerebellar and brain regions [38, 39], and a reduction of mitochondrial enzyme activity has been reported in platelets from DS patients . Moreover, impaired mitochondrial function, indicated by reduced mitochondrial redox activity and membrane potential, has been observed in DS astrocytes and in primary cultures of DS fibroblasts, [26, 27]. More recently it has been reported that the brain of the DS mouse model Ts1Cje has decreased mitochondrial membrane potential and ATP production . These results are indicative of widespread mitochondrial dysfunction in DS. Our data suggest that mitochondrial dysfunction might also occur in DS hearts and that the reduced expression of mitochondrial genes might be the molecular basis of this dysfunction in the heart as well as in other DS tissues. This is probably not a primay effect of gene dosage because genes for mitochondrial function mapping on Hsa21 are either not expressed or not differentially expressed in human fetal hearts.
Upregulation of ECM genes
Functional analysis of the 473 differentially expressed genes also demonstrates global upregulation of ECM protein genes. This group includes genes encoding adhesion and ECM proteins that map on Hsa21 such as ADAMTS1, ADAMTS5, APP, JAM2, COL6A1, COL6A2 and COL18A1, which are dose-dependently upregulated in trisomic samples, and genes that do not map on Hsa21 such as fibronectin, fibulin, collagen type I, type III, type V, type XV, metalloproteases (MMPs) and several adhesion molecule genes. Overexpression of this gene family is likely to affect cell adhesion properties, possibly determining an increase in adhesiveness. Cells explanted from endocardial cushion derived structures of fetuses with Hsa21 trisomy are more adhesive in vitro than those from controls . A stochastic model has been proposed for septal defects in DS by which higher values of adhesiveness result in deficiencies of the atrio-ventricular canal development associated with clinical variability among individuals based on chance alone [42, 43].
Several of the ECM genes upregulated in trisomic samples have been investigated for their potential role in DS cardiopathy. These include collagen type VI and MMPs. Collagen type VI is expressed in the endocardial cushions during septation, persists during valve differentiation and is implicated in endocardial cushion differentiation. Its pronounced expression in Hsa21 trisomy might lead to AVSD . MMPs are involved in cardiac organogenesis by regulating cell proliferation, epithelial to mesenchymal transition, ECM remodeling and degradation. MMP2, in particular, coordinates prelooping stages, heart tube formation and selective ECM degradation. It has an important functional role in early cardiogenesis, neural crest cell and cardiac cushion migration and remodeling of the pharyngeal arches and cardiac heart tube [45, 46].
It is interesting that DNA microarray analysis from right ventricular biopsies of patients with tetralogy of Fallot demonstrated that genes encoding ECM proteins, such as collagen type I, III, IX, XV and fibronectin, were upregulated versus age-matched controls , suggesting that the increase of these ECM proteins has a potential role in CHD.
How might Hsa21 gene upregulation affect the expression of other genes?
A key issue of DS pathogenesis is to understand how upregulation of Hsa21 genes might dysregulate genes on different chromosomes. Recently, a link has been proposed between two Hsa21 genes, DYRK1A and DSCR1, and the NFATc family of genes. NFATc-null mice show phenotypic anomalies that resemble those observed in human DS and 65% of NFATc1–4-null mice have endocardial cushion defects . Even modest overexpression of DYRK1A decreases NFATc protein activity and levels and may induce vascular and cardiac defects . DSCR1 also encodes a regulatory protein that is expressed in heart tissue  and exerts an inhibitory effect on the calcineurin/NFAT signaling pathway , although different protein isoforms may have opposite effects . To verify whether these genes affect the heart of DS fetuses, we evaluated their differential expression both by microarray and by qRT-PCR. Our analyses show that DYRK1A is upregulated and that NFATc2 and NFATc3 are downregulated in Hsa21 trisomic samples. Moreover, an increase in DSCR1 expression in individual hearts correlated with a decrease in NFATc3 expression. Recent data show that the enzymatic activity of complex II and IV of the respiratory chain and mitochondrial oxidative activity are reduced in Nfatc3-/-; Nfatc4-/- cardiomyocytes  suggesting that the calcineurin/NFAT pathway affects mitochondrial activity during heart development. We propose that upregulation of DIRK1A and/or DSCR1 in DS might affect mitochondrial gene expression, and thereafter mitochondrial function, through the calcineurin/NFAT pathway. Mitochondrial abnormalities and a decrease of COX activity might also be induced by overproduction of beta APP , a gene mapping on Hsa21, although the TS1Cje mouse model, in which APP is not triplicated, also shows decreased mitochondrial function and ATP production . The transcription factor GABPalpha, which is encoded by a gene on Hsa21, is another regulator of the expression of genes involved in mitochondrial respiration . However, GABPalpha was not expressed in fetal hearts at 18–22 weeks of gestation, although it cannot be excluded that it is expressed at different embryonic or fetal stages.
Type VI collagen gene upregulation also sheds light on the potential interplay between Hsa21 genes and genes on different chromosomes. Type VI collagen, together with other collagens, is an activator of discoidin domain receptor tyrosine kinases (DDRs) [53, 54]. Activated DDRs, in turn, induce the expression of metalloproteases such as MMP1 and MMP2  and of other ECM proteins . Moreover, DDR1 and DDR2 are upregulated in trisomic heart samples (see Additional file 4) and DDR2 expression has been detected by confocal microscopy in developing heart, within the cardiac cushions and eventually within the septum .