Metabolic gene profile in early human fetal heart development

Abstract

The primitive cardiac tube starts beating 6-8 weeks post fertilization in the developing embryo. In order to describe normal cardiac development during late first and early second trimester in human fetuses this study used microarray and pathways analysis and created a corresponding 'normal' database. Fourteen fetal hearts from human fetuses between 10 and 18 weeks of gestational age (GA) were prospectively collected at the time of elective termination of pregnancy. RNA from recovered tissues was used for transcriptome analysis with Affymetrix 1.0 ST microarray chip. From the amassed data we investigated differences in cardiac development within the 10-18 GA period dividing the sample by GA in three groups: 10-12 (H1), 13-15 (H2) and 16-18 (H3) weeks. A fold change of 2 or above adjusted for a false discovery rate of 5% was used as initial cutoff to determine differential gene expression for individual genes. Test for enrichment to identify functional groups was carried out using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Array analysis correctly identified the cardiac specific genes, and transcripts reported to be differentially expressed were confirmed by qRT-PCR. Single transcript and Ontology analysis showed first trimester heart expression of myosin-related genes to be up-regulated >5-fold compared with second trimester heart. In contrast the second trimester hearts showed further gestation-related increases in many genes involved in energy production and cardiac remodeling. In conclusion, fetal heart development during the first trimester was dominated by heart-specific genes coding for myocardial development and differentiation. During the second trimester, transcripts related to energy generation and cardiomyocyte communication for contractile coordination/proliferation were more dominant. Transcripts related to fatty acid metabolism can be seen as early as 10 weeks and clearly increase as the heart matures. Retinol receptor and gamma-aminobutyric acid (GABA) receptor transcripts were detected, and have not been described previously in human fetal heart during this period. For the first time global gene expression of heart has been described in human samples to create a database of normal development to understand and compare with known abnormal fetal heart development.

Keywords: cardiac development; fetal; human; microarray; renin-angiotensin.

Figure 1

(A) Genes with the highest differential expression in the heart. This figure is based on pooled tissue samples from 14 fetal hearts from 10 to 18 weeks of gestational age for genes with 20-fold change or greater expression level. Human Gene 1.0 ST array (Affymetrix) was used. Genes were chosen based on fold change compared with mRNA, after confirming a differential expression >95% with an false discover rate (FDR) of 5%. * = genes verified by qRT–PCR. (B) Genes with the highest differential expression in the brain. This figure is based on pooled tissue samples from 10 fetal brains from 10 to 18 weeks of gestational age for genes with 20-fold change or greater expression level. Human Gene 1.0 ST array (Affymetrix) was used. Genes were chosen based on fold change compared with mRNA, after confirming a differential expression >95% with an FDR of 5%. * = genes verified by qRT–PCR. (C) Differentially expressed genes measured by fold change in mRNA levels between the hearts at different gestational ages. The late first trimester hearts (H1) showed an increased number of differentially expressed genes (green bars) when compared with the early second trimester hearts (H3). Tissue samples correspond to hearts from 10–12 weeks of gestational age (H1) and 16–18 weeks of gestational age (H3). Differences are measured by fold change (>2), after adjusting for an FDR of 5% with a differential expression >95% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. (D) Genes with the highest differential expression in the late first trimester hearts (H1). Tissue samples correspond to hearts from 10–12 weeks of gestational age (H1) and 16–18 weeks of gestational age (H3) pooled on two separate Human Gene 1.0 ST array, according to gestational age. Differences are measured by fold change (>2), after adjusting for an FDR of 5% with a differential expression >95% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. * = gene verified by qRT–PCR.

Figure 2

(A) mRNA gene expression of regulators of cardiomyocyte differentiation. All transcripts are increased in the late first trimester (H1) versus the early second trimester (H3). Differences are measured by mRNA level on genes with a differential expression >95%, after adjusting for an FDR of 5% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. SLN, Sarcolipin; MYL1, Myosin light chain 1; MYH3, Myosin heavy chain 3; ACTG2, Actin gamma. * = gene verified by qRT–PCR. (B) Bar chart showing terms from Gene Ontology in the heart, classified according to biological process, cellular component and molecular function using test for enrichment of common functions among sets of differentially expressed genes (Gene Ontology Annotations and the KEGG). Gene families involved in common functions for muscular development like myosin filament and fatty acid metabolism including chylomicron, cholesterol, lipoprotein binding proteins and triglyceride lipoprotein are the highest ranked in the cardiomyocytes from 10 to 18 weeks of gestational age (pooled data). (C) Genes with the highest differential expression in the early second trimester hearts (H3). Tissue samples correspond to hearts from 10–12 weeks of gestational age (H1) and 16–18 weeks of gestational age (H3) pooled on two separate Human Gene 1.0 ST array, according to gestational age. Differences are measured by fold change (>2), after adjusting for an FDR of 5% with a differential expression >95% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. *= gene verified by qRT–PCR.

Figure 3

(A) mRNA gene expression of regulators of the cardiac fatty acid metabolism and remodeling. All transcripts are increased in the early second trimester as the heart matures, compared with the late first trimester. Differences are measured by mRNA level on genes with a differential expression >95%, after adjusting for an FDR of 5% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), to identify differentially expressed genes in any two conditions. *= genes verified by qRT–PCR. (B) mRNA expression of regulators of glucose energy generation by the cardiomyocytes. All transcripts are high in both groups showing no significant differential expression (<0.005), after adjusting for an FDR of 5%. (C) mRNA gene expression of insulin growth factor and angiotensin. Specific transcripts were examined due to the their suggested importance in previous studies (see introduction). mRNA levels detected in each group are shown. Differential expression >95%, after adjusting for an FDR of 5% using EBarrays (Newton et al., 2001; Kendziorski et al., 2003), was not achieved suggesting that these genes are not differentially controlled across early gestation. (D) Reference genes in the heart. The graph shows mRNA expression of two candidate genes without significant change in expression, regardless of the experimental group (H1, H2 or H3). Reference genes were validated by the method of Stern-Straeter et al. (2009). All H1, H2 and H3 groups of hearts were analyzed under exact conditions. Differential expression was <0.005 for both genes under the two conditions. RPLP0: Ribosomal protein, large; TBS: TATA box binding protein.