Left ventricular remodeling in swine after myocardial infarction: a transcriptional genomics approach

Abstract

Despite the apparent appropriateness of left ventricular (LV) remodeling following myocardial infarction (MI), it poses an independent risk factor for development of heart failure. There is a paucity of studies into the molecular mechanisms of LV remodeling in large animal species. We took an unbiased molecular approach to identify candidate transcription factors (TFs) mediating the genetic reprogramming involved in post-MI LV remodeling in swine. Left ventricular tissue was collected from remote, non-infarcted myocardium, 3 weeks after MI-induction or sham-surgery. Microarray analysis identified 285 upregulated and 278 downregulated genes (FDR < 0.05). Of these differentially expressed genes, the promoter regions of the human homologs were searched for common TF binding sites (TFBS). Eighteen TFBS were overrepresented >two-fold (p < 0.01) in upregulated and 13 in downregulated genes. Left ventricular nuclear protein extracts were assayed for DNA-binding activity by protein/DNA array. Out of 345 DNA probes, 30 showed signal intensity changes >two-fold. Five TFs were identified in both TFBS and protein/DNA array analyses, which showed matching changes for COUP-TFII and glucocorticoid receptor (GR) only. Treatment of swine with the GR antagonist mifepristone after MI reduced the post-MI increase in LV mass, but LV dilation remained unaffected. Thus, using an unbiased approach to study post-MI LV remodeling in a physiologically relevant large animal model, we identified COUP-TFII and GR as potential key mediators of post-MI remodeling.

Fig. 1

Workflow of our transcriptional genomics approach. RNA and nuclear proteins were isolated from LV tissue. The RNA was used in microarray analysis followed by TFBS analysis of differentially expressed genes. Nuclear protein extracts were used for protein/DNA array analysis to identify TFs that were increased or decreased in DNA-binding activity in post-MI hearts. Candidate TFs were identified by combining these two data sets. LV left ventricular, MI myocardial infarction, TFBS transcription factor binding site, DE differentially expressed, TF transcription factor

Fig. 2

Differential gene expression. a Principal component analysis of the data from the individual microarrays shows separation into two groups, with the sham (blue) and post-MI (red) samples forming separate clusters. Females (diamonds) and males (circles) do not form separate clusters. b Clustering of the differentially expressed genes into biological groups was performed with Ingenuity pathway analysis (p < 0.001). c, d The top two networks of differentially expressed genes. Rectangles in green and red represent genes downregulated and upregulated after MI compared with sham. Dual-colored rectangles depict a group of genes, some of which are upregulated and some are downregulated after MI. White rectangles are hub molecules, of which the expression is not altered, but that generally have a large number of connections with the genes. Uninterrupted and dashed lines indicate physical and indirect interactions between molecules, respectively

Fig. 3

Linking of TFs identified by protein/DNA array to differentially expressed genes. TFs that show less (green outline) or more (red outline) DNA-binding activity in post-MI LV tissue are depicted at the far left and right and are connected with upregulated (red) and downregulated (green) genes depicted in the center. TFs in yellow show matching changes in both protein/DNA array and TFBS analysis. A line was drawn between a TF and a gene if it was known from the literature that the TF can cause the expression of the gene. Uninterrupted and dashed lines indicate physical and indirect interactions between molecules

Fig. 4

Effect of GR antagonist mifepristone on cardiac remodeling and function following MI. Sham (n = 20), MI (n = 24) and MI + mifepristone (n = 7) animals were studied. a LVW/BW ratios. b LV-dilation as indicated by LV end-diastolic area. c LV 2D-Ejection fraction. d LV dP/dt_P40. *P < 0.05, ^† P < 0.01, ^‡ P < 0.001 vs. sham, ^§ P < 0.05 vs. MI. Measurements are mean ± SEM