Exosomal miRNAs Differentiate Chronic Total Occlusion from Acute Myocardial Infarction

Abstract

Although coronary artery occlusion can have a negative effect on the myocardium, chronic total occlusion (CTO) exhibits different clinical features from those of acute myocardial infarction (AMI). In this study, we identify the differential associations of exosomal miRNAs with CTO and AMI. Exosomes were isolated from the plasma obtained from coronary arteries of patients undergoing percutaneous coronary intervention to treat CTO (n = 29) and AMI (n = 24), followed by small RNA sequencing, target gene predictions, and functional enrichment analyses. Promising miRNA markers were validated using real-time PCR in 35 CTO, 35 AMI, and 10 normal subjects. A total of 205 miRNAs were detected in all subjects, and 20 and 12 miRNAs were upregulated and downregulated in CTO compared to AMI patients, respectively (|fold change| > 4, FDR q < 0.05). The target genes of miRNAs that were higher in CTO patients were associated with "regulation of cell cycle phase transition", "cell growth", and "apoptosis". The target genes of miRNAs that were lower in CTO patients were enriched in terms such as "muscle cell differentiation", "response to oxygen levels", and "artery morphogenesis". On qRT-PCR analysis, the expression levels of miR-9-5p and miR-127-3p were significantly different between CTO and AMI patients. The miRNA expression levels accurately distinguished CTO from AMI patients with 79% specificity and 97% sensitivity. The miRNA contents of plasma exosomes were significantly different between CTO and AMI patients. The miRNAs may play important roles in CTO and AMI.

Keywords: acute myocardial infarction; biomarker; chronic total occlusion; exosome; microRNA.

Figure 1

Characterization of EVs from plasma. (A) Diameter and concentration distribution of EVs from plasma obtained from coronary artery. (B) Western blot analysis of the expression of the EV-markers CD9, CD63, and CD81, and a cytoplasmic marker beta-actin in EVs from plasma.

Figure 2

Differentially expressed miRNAs between patients with CTO and AMI. (A) Volcano plot shows the number and distribution of miRNAs. (B) Heatmap of the differentially expressed miRNAs.

Figure 3

Biological process of gene ontology (A) and KEGG pathway (B) enrichment of 247 genes targeted by 10 upregulated miRNAs in patients with CTO compared to AMI patients. The color intensity indicates Maximal Clique Centrality (MCC) rank.

Figure 4

Clusters (A–C) and key genes (D) of protein–protein interaction network of the 247 genes targeted by 10 upregulated miRNAs in patients with CTO.

Figure 5

Biological process of gene ontology (A) and KEGG pathway (B) enrichment of 219 genes targeted by three downregulated miRNAs in patients with CTO compared to AMI patients.

Figure 6

Clusters (A–C) and key genes (D) of protein–protein interaction network of the 219 genes targeted by 3 downregulated miRNAs in patients with CTO. The color intensity indicates Maximal Clique Centrality (MCC) rank.

Figure 7

The expression levels of miR-127-3p (A), miR-9-5p (B), and miR-21-5p (C) in plasma EVs analyzed using qRT-PCR and ROC curve (D) for miR-127-3p and miR-9-5p in discrimination between the CTO and AMI (AUC = 0.948 with 79% specificity and 97% sensitivity). Box plots represent median and quartile ranges.