Systematic analysis of gene expression differences between left and right atria in different mouse strains and in human atrial tissue

Abstract

Background: Normal development of the atria requires left-right differentiation during embryonic development. Reduced expression of Pitx2c (paired-like homeodomain transcription factor 2, isoform c), a key regulator of left-right asymmetry, has recently been linked to atrial fibrillation. We therefore systematically studied the molecular composition of left and right atrial tissue in adult murine and human atria.

Methods: We compared left and right atrial gene expression in healthy, adult mice of different strains and ages by employing whole genome array analyses on freshly frozen atrial tissue. Selected genes with enriched expression in either atrium were validated by RT-qPCR and Western blot in further animals and in shock-frozen left and right atrial appendages of patients undergoing open heart surgery.

Results: We identified 77 genes with preferential expression in one atrium that were common in all strains and age groups analysed. Independent of strain and age, Pitx2c was the gene with the highest enrichment in left atrium, while Bmp10, a member of the TGFβ family, showed highest enrichment in right atrium. These differences were validated by RT-qPCR in murine and human tissue. Western blot showed a 2-fold left-right concentration gradient in PITX2 protein in adult human atria. Several of the genes and gene groups enriched in left atria have a known biological role for maintenance of healthy physiology, specifically the prevention of atrial pathologies involved in atrial fibrillation, including membrane electrophysiology, metabolic cellular function, and regulation of inflammatory processes. Comparison of the array datasets with published array analyses in heterozygous Pitx2c(+/-) atria suggested that approximately half of the genes with left-sided enrichment are regulated by Pitx2c.

Conclusions: Our study reveals systematic differences between left and right atrial gene expression and supports the hypothesis that Pitx2c has a functional role in maintaining "leftness" in the atrium in adult murine and human hearts.

Figure 1. Results of the mRNA expression array experiments.

A Venn diagram showing the overlap of transcripts (grey numbers) that are significantly different between LA and RA (Fold-Change>1.5, P<0.05) in 3-month-old MF1 mice (MF1_3), 12-month-old MF1 mice (MF1-12), and 12-month-old Swiss-Agouti mice (SA_12). The number of transcripts with increased level of expression in the left atrium (black) or right atrium (white) are given in brackets. The overlapping 83 transcripts in the centre of the diagram refer to 77 single genes (see Tables 1 and 2). B Two-dimensional hierarchical clustering of relative change in gene expression using the 83 transcripts common to mice of all strains and age groups.

Figure 2. Gene groups enriched in left atrium according to FatiGO gene ontology analysis.

220 genes significantly enriched in the left atrium in any of the three microarray datasets (MF1_3; MF1_12; SA_12) were used as input list against the genome. 9 biological process, 10 cellular component, and 8 molecular function enriched GO classes are highlighted in red (levels 3–9; P<0.01).

Figure 3. RT-qPCR validation of the differential expression of candidate genes in left and right atria from wild-type mice of three different strains.

A Relative Log2 expression ratios between LA and RA in MF1 mice (blue, n = 4), Swiss-Agouti mice (pink, n = 4), CD1 mice (yellow, n = 4), and in wild-type mice (grey, n = 12). B Corresponding average mRNA expression in left (black) and right (white) atrial tissues of MF1, SA, and CD1 mice (n = 4 each). Gapdh was used as control. Error bars indicate standard error of the mean (SEM). Statistically significant differences as assessed by unpaired Student's t-test are represented by asterisks (*P<0.05, **P<0.01).

Figure 4. A) RT-qPCR measurements of orthologous transcripts in left atrial (black) and right atrial (white) human appendages (n = 5).

Actb was used as control. Error bars indicate standard error of the mean (SEM). Statistically significant differences as assessed by paired Student's t-test are represented by asterisks (*P<0.05). B) and C) PITX2, IRX3, and BMP10 protein expression level measured by Western blot of total protein lysates from human left (black) and right (white) atrial appendages (n = 4). The data shown are averages of the results obtained from four separate samples. Error bars indicate standard deviation (SD). Statistically significant differences as assessed by unpaired Student's t-test are represented by asterisks (*P<0.05).