Time Series Gene Expression Profiling and Temporal Regulatory Pathway Analysis of Angiotensin II Induced Atrial Fibrillation in Mice

Abstract

Background/Aim: Angiotensin II (Ang II) and hypertension play critical roles in the pathogenesis of the atrial remodeling that contributes to atrial fibrillation (AF). However, the gene expression profiles and signaling pathways in atria during the development of AF induced by Ang II remain unknown. Methods: Wild-type male mice (C57BL/6 background, 10 weeks old) were administered an infusion of Ang II (2000 ng/kg/min) using an osmotic pump for 1, 2, and 3 weeks. Blood pressure (BP) was measured by the tail-cuff method. AF was induced and recorded. Atrial enlargement and remodeling were examined by echocardiography and Masson's trichrome staining. Time-series microarray analyses were conducted to examine gene expression profiles and pathways. Results: Ang II infusion resulted in marked elevation of systolic BP, increased AF incidence and duration, atrial enlargement, fibrosis, and atrial infiltration of myofibroblasts and F4/80-positive macrophages in a time-dependent manner. Microarray results showed that 1,719 genes were differentially expressed in the atrium at weeks 1, 2, and 3 after Ang II infusion. Gene ontology showed that these genes participate mainly in immune system processes, and regulation of cell migration, cell adhesion, complement activation, and the inflammatory response. Significant pathways included lysosomal and phagosomal pathways, which are involved in antigen processing and presentation, as well as chemokine signaling, and extracellular matrix-receptor interaction, which are known to play important roles in Ang II-induced AF. Moreover, these differentially expressed genes were classified into 50 profiles by hierarchical cluster analysis. Of these, eight profiles were significant and contained a total of 1,157 genes. Gene co-expression network analysis identified that Pik3cg (also known as phosphoinositide-3-kinase regulatory subunit 3) was localized in the core of the gene network, and was the most highly expressed among the Pik3 isoforms at different time points. Conclusion: The present findings revealed that many genes are involved in Ang II-induced AF, and highlighted that Pik3cg may play a central role in this disease.

Keywords: Pik3cg; angiotensin II; atrial fibrillation; microarray; time series gene expression profiling.

Figure 1

Ang II infusion induces elevation of blood pressure (BP) and AF susceptibility in mice. (A) Wild-type mice were injected with Ang II (2000 ng/kg/min) for 1, 2, and 3 weeks. Systolic BP was measured by the tail cuff method (n = 10 per group). (B) Representative AF incidence and duration were detected at weeks 1, 2, and 3 after Ang II infusion (left). Quantification of AF incidence and AF duration (right, n = 10 per group). Data are expressed as mean ± SEM. ^∗P < 0.05; ^∗∗∗P < 0.001 vs. saline.

Figure 2

Ang II infusion promotes atrial remodeling. (A) Representative M-mode images of the left atrium at weeks 1, 2, and 3 after Ang II infusion. Quantification of atrial width (n = 10 per group). (B) Representative Masson trichrome staining (blue) of atrial sections, and quantification of fibrotic area (n = 8 per group). (C) Representative immunohistochemical staining of atrial sections with anti-α-SMA antibody (right), and quantification of α-SMA-positive fibroblasts (n = 8 per group). Representative immunohistochemical staining of atrial sections with anti-F4/80 antibody (right), and quantification of F4/80-positive macrophages (n = 8 per group). Scale bar: 50 μm. Data are expressed as mean ± SEM, and n represents the number of animals. ^∗∗P < 0.01; ^∗∗∗P < 0.001 vs. saline.

Figure 3

Analysis of gene ontology (GO) and KEGG pathways. (A) The heat map indicates the gene expression differences from the microarray between Ang II-infused atrium and saline control at weeks 1, 2, and 3 (n = 4 per group). The red color indicates up-regulation and green is for down-regulation. (B) GO analysis for differentially expressed genes. (C) Analysis of KEGG pathways for the differentially expressed genes in the atrium. LgP represents the logarithm of P-value. (D) The clustering analysis of differentially expressed genes in Ang II-infused atrium. Eight expression patterns (No. 47, 45, 49, 44, 29, 35, 0, and 12) of genes showed statistically significant difference (P < 0.00001) (colored boxes).

Figure 4

The time-series analysis of differentially expressed genes in each pattern (A–I): Nos. 47, 45, 49, 44, 29, 35, 0, 12, and 7 (n = 4 pr group). The horizontal axis shows the time points, and the vertical axis shows the time series of gene expression levels.

Figure 5

Validation of the results from microarray by qPCR analysis in Ang II-infused atrium at different time points. (A–I) The expression levels of selected 13 genes were verified by qPCR analysis (n = 4 per group), including Csf1r, Pik3r3, Plcb2, Cxcr4, Prckb, Cd8b1, Cx3cr1, Cacna1c, Cyp2s1, Aldh1a2, Pde6b, Pla2g10, and Gngt2. GAPDH as an internal control.

Figure 6

Analysis of gene co-expression network. (A) 37 genes selected from eight significant profiles (Nos. 47, 45, 49, 44, 29, 35, 0, and 12) were further analyzed by gene co-expression network with k-core algorithm. Cycle node shows genes, and edge between two nodes represents interaction between genes. (B) The expression of Pik3cg, Pik3cb, and Pik3r3 in the atrial tissues after Ang II infusion and saline control at weeks 1, 2, and 3 (n = 4 per group). (C) The mRNA level of Pik3cg was verified by qPCR analysis in Ang II-infused atrial tissues (n = 4 per group). Data are expressed as mean ± SEM, and n represents the number of animals. ^∗P < 0.05; ^∗∗P < 0.01 vs. control.