Integrated microRNA and mRNA responses to acute human left ventricular ischemia

Abstract

MicroRNAs (miRNAs) play a significant role in ischemic heart disease. Animal models of left ventricular (LV) ischemia demonstrate a unique miRNA profile; however, these models have limitations in describing human disease. In this study, we performed next-generation miRNA and mRNA sequencing on LV tissue from nine patients undergoing cardiac surgery with cardiopulmonary bypass and cardioplegic arrest. Samples were obtained immediately after aortic cross clamping (baseline) and before aortic cross clamp removal (postischemic). Of 1,237 identified miRNAs, 21 were differentially expressed between baseline and postischemic LV samples including the upregulated miRNAs miR-339-5p and miR-483-3p and the downregulated miRNA miR-139-5p. Target prediction analysis of these miRNAs was integrated with mRNA expression from the same LV samples to identify anticorrelated miRNA-mRNA pairs. Gene enrichment studies of candidate mRNA targets demonstrated an association with cardiovascular disease, cell death, and metabolism. Therapeutics that intervene on these miRNAs and their downstream targets may lead to novel mechanisms of mitigating the damage caused by ischemic insults on the human heart.

Trial registration: ClinicalTrials.gov NCT00985049.

Keywords: mRNA; microRNA; myocardial infarction; sequencing.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Left ventricular microRNA (miRNA) expression profile. Scatter plot of mean normalized read counts (RPM) sequenced from pre- (baseline) and postischemic left ventricular heart miRNA pools. Only those miRNAs with mean normalized read count > 0.02 are charted. Dashed line represents 1:1 expression ratio between treatment groups.

Fig. 2.

Real-time quantitative PCR validation of differentially expressed miRNAs. Box plots of the relative expression of differentially expressed miRNAs assayed by real-time quantitative PCR; n = 5 paired baseline (black) and postischemic (gray) samples for all miRNAs. *Significance at P value < 0.05 by Wilcoxon signed-rank test.

Fig. 3.

Distribution of Pearson correlation coefficients of predicted miRNA-mRNA pairs. For each miRNA, we determined the correlation between the expression of that miRNA and the expression of a predicted mRNA target in all samples (n = 18) by calculating a Pearson correlation coefficient. This was repeated for all mRNA targets of that miRNA, and the distribution of these coefficients (blue line) is illustrated in a density plot. The control curve (red line) describes the distribution of an equal number of coefficients where each coefficient is derived from the correlation between the expression of that miRNA and the expression of a random (not a predicted target) mRNA. A left shift of the blue compared with the red curve indicates that predicted targets are preferentially more anticorrelated with that miRNA than random mRNAs. Only those miRNAs with a significant shift (by t-test P value < 0.05) are shown.

Fig. 4.

Network of putative mRNA targets of differentially expressed miRNAs. Ingenuity pathway analysis was used to generate associations between the top 14 significantly anticorrelated mRNA targets (shaded green) of differentially expressed miRNAs (shaded red). Target genes are linked in the network “lipid metabolism, small molecule biochemistry, and cell death and survival.” Master regulators that serve as central nodes in this network are shaded in magenta. Solid lines indicate direct relationships, while dotted lines represent indirect relationships.