Sildenafil augments early protective transcriptional changes after ischemia in mouse myocardium

Abstract

Recently, targeting cyclic-GMP specific phosphodiesterase-5 (PDE5) has attracted much interest in several cardiopulmonary diseases, in particular myocardial ischemia (MI). Although multiple mechanisms were postulated for these beneficial effects at cellular level, early transcriptional changes were unknown. The aim of present study was to examine gene expression profiles in response to MI after 24 h of ischemia in murine model and compare transcriptional modulation by sildenafil, a popular phosphodiesterase 5 (PDE5) inhibitor. Mice were divided into four groups: Control sham (C), Sildenafil sham (S), Control MI (CMI) and Sildenafil MI (SMI). Sildenafil was given at a dose of 0.7 mg/kg intraperitoneally 30 min before LAD occlusion. cDNA microarray analysis of peri-infarct tissue was done using a custom cloneset and employing a looped dye swap design. Replicate signals were median averaged and normalized using LOWESS algorithm. R/MAANOVA analysis was used and false discovery rate corrected permutation p-values <0.005 were employed as significance thresholds. 156 genes were identified as significantly regulated demonstrating fold difference >1.5 in at least one of the four groups. 52 genes were significantly upregulated in SMI compared to CMI. For a randomly chosen subset of genes (9), microarray data were confirmed through real time RT-PCR. The differentially expressed genes could be classified into following groups based on their function: phosphorylation/dephosphorylation, apoptosis, differentiation, ATP binding. Our results suggest that sildenafil treatment might regulate early genetic reprogramming strategy for preservation of the ischemic myocardium.

Fig 1

A. Experimental protocol: Mice were randomly divided into 4 groups: (1) Control (C) (2) Control Myocardial infarction (CMI) (3) Sildenafil (S) and (4) Sildenafil MI (SMI). They were given either saline or sildenafil intraperitoneally at a dose of 0.7 mg/kg in treatment group 30 minutes before permanent LAD occlusion. The left ventricular tissues were collected for microarray analysis after 24 hours of MI (n = 6 in each group) B. Experimental design: Loop design for gene expression analysis. Samples: C (Control or Sham), CMI (Control Myocardial Infaction), S (Sildenafil treated) and SMI (Sildenafil Myocardial Infarction). The red and green dots in each sample represent the “dye-flip” concept. Color-coded lines indicate the pairs of differentially labeled samples (red or green) co-hybridized on the different arrays.

Fig 2. Chilibot text mining analysis

Chilibot text mining analysis of real time RT-PCR validated genes of known relationships between angiogenesis and statistically significant differentially expressed genes within enriched biological process terms of the GO analysis. Circles represent color coded interactive relationships: gray is neutral, green is stimulatory, red is inhibitory and yellow is both stimulatory and inhibitory. The numerical values within each icon present the number of PubMed abstracts used to validate the relationship (default is 50). Icons containing “_” represent parallel relationships, whereas gray rhomboidal icons indicate co-occurrence.

Fig 3. Confirmation of cDNA microarray data by real time RT-PCR

The relative mRNA expression in all four groups is shown for NFKBIA, GADD45B, ADAM15, NOTCH4, PYCARD, TGTP, BCL2L1, UBE2D2, MAP3K6P. The obtained data confirmed the cDNA microarray data in the transcriptional direction. (n = 6 animals for each group)