Investigating gene-microRNA networks in atrial fibrillation patients with mitral valve regurgitation

Abstract

Background: Atrial fibrillation (AF) is predicted to affect around 17.9 million individuals in Europe by 2060. The disease is associated with severe electrical and structural remodelling of the heart, and increased the risk of stroke and heart failure. In order to improve treatment and find new drug targets, the field needs to better comprehend the exact molecular mechanisms in these remodelling processes.

Objectives: This study aims to identify gene and miRNA networks involved in the remodelling of AF hearts in AF patients with mitral valve regurgitation (MVR).

Methods: Total RNA was extracted from right atrial biopsies from patients undergoing surgery for mitral valve replacement or repair with AF and without history of AF to test for differentially expressed genes and miRNAs using RNA-sequencing and miRNA microarray. In silico predictions were used to construct a mRNA-miRNA network including differentially expressed mRNAs and miRNAs. Gene and chromosome enrichment analysis were used to identify molecular pathways and high-density AF loci.

Results: We found 644 genes and 43 miRNAs differentially expressed in AF patients compared to controls. From these lists, we identified 905 pairs of putative miRNA-mRNA interactions, including 37 miRNAs and 295 genes. Of particular note, AF-associated miR-130b-3p, miR-338-5p and miR-208a-3p were differentially expressed in our AF tissue samples. These miRNAs are predicted regulators of several differentially expressed genes associated with cardiac conduction and fibrosis. We identified two high-density AF loci in chromosomes 14q11.2 and 6p21.3.

Conclusions: AF in MVR patients is associated with down-regulation of ion channel genes and up-regulation of extracellular matrix genes. Other AF related genes are dysregulated and several are predicted to be targeted by miRNAs. Our novel miRNA-mRNA regulatory network provides new insights into the mechanisms of AF.

Fig 1. Transcriptome analysis of right atrial biopsies from AF patients compared to controls (SR).

A. Principal component analysis (PCA) showing the overall effect of variances between the transcriptome of samples analysed by RNA-sequencing. B. Volcano plot comparing expression of 44,852 genes in the right atrium of AF patients in relation to SR. Red dots represent genes with adjusted-p < 0.05 and log₂FC > 2 or < -2. C-D. Gene set enrichment analysis of differentially expressed genes. Top 10 enriched gene ontology terms and pathways with an enrichment p-value < 0.05 after Benjamini-Hochberg correction from up- (C) and down-regulated (D) genes were plotted. AF–atrial fibrillation; BP–biological processes; CC–cellular component; KEGG–Kyoto Encyclopedia of Genes and Genomes; MF–molecular function; NS–non-significant; FC—fold change.

Fig 2. Micro RNAs expression analysis of right atrial biopsies from AF patients compared to controls (SR).

Heatmap shows the 43 differentially expressed miRNAs in AF hearts. Columns represent samples and rows represent miRNAs. Red indicates increased expression, blue indicates decreased expression and yellow indicates low variation in relation to the mean expression.

Fig 3. miRNA-gene regulatory networks build using Cytoscape.

Network includes up-regulated miRNAs and candidate gene targets that were down-regulated in right atrial biopsies of AF patients compared to control subjects. The network helps to identify miRNAs targeting multiple DEGs and genes targeted by multiple miRNAs. Triangle nodes represent up-regulated miRNAs with red colour intensity according to FC. Rectangle nodes represent down-regulated genes with blue colouring according to FC. The darkness of the node to node edges correlates with the expression correlation value between a miRNA and its target gene. The darker the line, the closer to -1 is the correlation. All miRNA-gene pairs with correlation above -0.5 were excluded from the network. AF–atrial fibrillation; FC–fold-change.

Fig 4. Gene enrichment analysis of micro RNA target genes using GO terms and KEGG.

A. Analysis of up-regulated genes predicted to be targeted by down-regulated miRNAs. B. Analysis of down-regulated genes predicted to be targeted by up-regulated miRNAs. Enrichment cut-off was p <0.05 after Benjamini-Hochberg correction. BP–biological processes; CC–cellular component; GO–gene ontology; KEGG—Kyoto Encyclopedia of Genes and Genomes; MF–molecular function.

Fig 5. AF related miRNA-gene regulatory networks build using Cytoscape.

MiRNAs previously associated with AF were selected to create a subset of the regulatory networks. A. Up-regulated miRNAs and candidate gene targets down-regulated in right atrial biopsies of AF patients compared to control subjects. B. Down-regulated miRNAs and candidate gene targets up-regulated in AF patients. Triangle nodes represent miRNAs and rectangle nodes represent target genes. Red colour intensity varies according to expression increase in FC and blue colouring according to decrease in FC. Edges connecting miRNA and genes are coloured according to Spearman correlation of expression data. The darker the line, the closer to -1 is the correlation. AF–atrial fibrillation; FC–fold-change.

Fig 6. Chromosome enrichment of AF related genetic elements.

Enrichment includes AF related SNPs and differentially expresses genes and miRNAs in right atrium biopsies of AF patients compared to control subjects. A. Chromosome plots showing the genomic location of all 1008 elements included in the study. B. High-density cluster identified in the q arm of chromosome 14 including genes MYH6 and MYH7, miR-208a/b and two AF-associated SNPs. AF–atrial fibrillation; SNPs–single nucleotide polymorphisms; Mbp–million base pairs.