Transcriptional changes associated with advancing stages of heart failure underlie atrial and ventricular arrhythmogenesis

Figures

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  Fig 1. Principal component and clustering show a clear separation between controls and heart failure. (A) Three-dimensional principal component analysis (PCA) plot of multi-array average–normalized data for early and late stages of HF in RA (red) and RV (blue). (B) Unsupervised hierarchical clustering of the global gene expression data. (C) Unsupervised hierarchical clustering of the 534 identified differentially expressed genes (DEGs) for the averaged data. (ANOVA, p<0.01, n = 3). Data are log transformed and normalized. Upregulated genes are displayed in red and downregulated genes in blue. Dendrogram provides a measure of the relatedness of gene expression in each sample (at left) and for each gene (at top).

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  Fig 2. Comparison of differentially expressed genes (DEGs) between early and late stages of heart failure (HF). The Venn diagrams show the number of DEGs in each comparison and the number of upregulated (red) and downregulated (blue) overlapping genes between the early and late stages of HF in a canine model for (A) RA and (B) RV. All samples are compared to their respective control. (C) The Volcano plots show the total gene expression with positive (red) and negative (blue) log2 fold difference ≥2 (x-axis) against adjusted P-value ≤0.05 (y-axis). All other genes with adjusted P-value >0.05 are indicated in black.

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  Fig 3. Enriched gene ontology (GO) terms belonging to biological processes. Enriched GO terms upregulated differentially expressed genes for comparisons between early and late stages of HF. Significantly enriched GO terms (Fisher's exact test; adjusted P-value ≤0.01) are shown in dark blue according to the color key.

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  Fig 4. Several genes participate in more than one biological process. Heat map of top scored signaling pathways enriched in heart failure microarray analysis. Every row represents a gene, and every column represents a pathway. Top significant signaling pathways for commonly enriched genes are presented on the heat map in black. Pathway enrichment analysis was done using Advaita iPathway Guide, and full list of pathways can be found in S1–S3 Tables. Only genes that have fold change above two were presented in the heat map.

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  Fig 5. RT-qPCR validates the differentially expressed genes (DEGs) identified in microarray analysis. (A) Heat map of genes differentially expressed in heart failure vs. control dogs (ANOVA, p<0.05, fold change >2 or <−2, n = 3). Upregulated gene expression is displayed in red and downregulated gene expression in blue. Genes are presented in alphabetical order. (B) Relative expression of DEGs associated with fibrosis/wound healing and immune response. Mean fold change with cut-off filter criteria of at least a 2-fold change and an adjusted P-value <0.05 was used to identify upregulation. (C) Correlation between microarray expression and qPCR for selected genes. Expression levels normalized to GAPDH (n = 5). Data presented as fold change relative to control levels. One-way ANOVA with Bonferroni post hoc analysis.