miRNA-mRNA crosstalk in myocardial ischemia induced by calcified aortic valve stenosis

Abstract

Aortic valve stenosis is the most common cause of morbidity and mortality in valvular heart disease in aged people. Both microRNA (miRNA) and mRNA are potential targets for the diagnosis and therapeutic intervention of myocardial ischemia induced by calcified aortic valve stenosis (CAVS), with unclear mechanisms. Here, 3 gene expression profiles of 47 male participants were applied to generate shared differentially expressed genes (DEGs) with significant major biological functions. Moreover, 20 hub genes were generated by a Weighted Genes Co-Expression Network Analysis (WGCNA) and were cross-linked to miRNA based on miRanda/miRwalk2 databases. Integrated miRNA/mRNA analysis identified several novel miRNAs and targeted genes as diagnostic/prognostic biomarkers or therapeutic targets in CAVS patients. In addition, the clinical data suggested that myocardial hypertrophy and myocardial ischemia in CAVS patients are likely associated with hub genes and the upstream regulatory miRNAs. Together, our data provide evidence that miRNAs and their targeted genes play an important role in the pathogenesis of myocardial hypertrophy and ischemia in patients with CAVS.

Keywords: calcified aortic valve stenosis (CAVS); hub genes; miRNA-mRNA crosstalk; mitochondrial dysfunction; myocardial ischemia.

Figure 1

Overview of DEGs between calcified aortic valve stenosis and normal aortic valve tissues. (A) Volcano plot of DEGs and boxplot of normalized gene expression profiles. In the volcano plots, each color dot represents a downregulated or upregulated gene; The red color indicated high expressed genes and the blue color indicated low expressed genes, above and below the median, respectively. (B) Gene modules’ dendrogram plots of DEGs, and bar plots of eigengenes’ module significance and correlation. In the dendrogram plots, each leaf (short vertical lines) in the dendrogram corresponds to a gene and the branches are expression modules of highly interconnected groups of genes with a color to indicate its module assignment.

Figure 2

Microarray analysis for DEGs of integrated multiple datasets. (A) Fisher’s method tests the integrated multiple profiles of the shared DEGs’ expression; (B) Integrated heat map of significantly expressed DEGs with each row representing a probe and each column representing a sample. Expression levels are depicted according to the color scale, shown at the bottom. The red color indicated high expressed genes and the blue color indicated low expressed genes, above and below the median, respectively. The magnitude of deviation from the median is represented by the color saturation.

Figure 3

Functional enrichment related to mitochondria. (A) GSEA enrichment results related to mitochondrial functions; (B) ROS production after H9C2 hypoxic simulated myocardial ischemia, bar=100μm; (C) mitochondrial transmembrane potential after H9C2 hypoxic simulated myocardial ischemia, bar=50μm; (D) mPTP opening after H9C2 hypoxic simulated myocardial ischemia, bar=100μm; (E) TUNEL staining after H9C2 hypoxic simulated myocardial ischemia, bar=100μm. *P<0.05, **P<0.01.

Figure 4

Mitochondrial dysfunctions after myocardial ischemia in CAVS patients. (A) The chest CT of the CAVS patient. The yellow arrow refers to the calcified aortic valve; (B) The cardiac multifunctional color Doppler ultrasound image of the CAVS patient; (C) The electronic microscopy observation of mitochondria in CAVS myocardial tissues; (D) Confocal microscopy observation of mitochondrial morphology in H9C2 hypoxic simulated myocardial ischemia, bar=10μm. Quantitation is done in triplicate and scored into three categories: foreshortened, middle and elongated mitochondria. *P<0.05, **P<0.01.

Figure 5

Biological processes and hub genes related to mitochondrial functions in CAVS. (A) Co-expression network approaches to reveal hub genes associated with mitochondrial functions in CAVS; (B) a. overview of hub genes and connected miRNAs. b. subnetwork of the integrated miRNAs and hub genes. The triangle and dot represent the hub genes and miRNA, respectively. Red color and up-direction represent up-regulated hub genes, blue color and down-direction represent down-regulated hub genes. The size color saturation of the nodes was weighted by the connectedness of the hub gene interacted with other miRNAs. c. two tail T-test of top-ten hub genes expression based on data profiles; C. Real-time PCR results of mRNA expression of these top-ten hub genes based on clinical samples; D. Western Blot results of protein expression of these top-ten hub genes based on clinical samples; *P<0.05, **P<0.01.

Figure 6

Molecular and subcellular events leading to apoptosis and calcification. ETC: electron transport chain; ROS: reactive oxygen species; ER: endoplasmic reticulum; mPTP: mitochondrial permeability transition pore; LRRK2: leucine-rich repeat kinase 2; DRP1: dynamin-related protein-1. Hypoxia and ER stress had been known as the key factors in calcium overflow. ETC dysfunction and ROS reaction induced the opening of mPTP pores to release of Cyt-C which was thought to be important in the apoptotic process. DRP1 and LRRK2 were two major dysregulated genes in CAVS patients and may be used as potential therapeutic targets for microRNAs. The main of our findings have revealed the critical roles of miRNAs in the regulation of target genes that influence the mitochondrial function and muscle cell death. Concomitant with the overflow of calcium ions, the calcification of biological process was induced to activate. Therefore, hypoxia and calcification formed a vicious-loop in cardiovascular disease, exacerbating stenosis and calcification of heart valves.