Exploring Cardiac Exosomal RNAs of Acute Myocardial Infarction

Abstract

Background: Myocardial infarction (MI), often a frequent symptom of coronary artery disease (CAD), is a leading cause of death and disability worldwide. Acute myocardial infarction (AMI), a major form of cardiovascular disease, necessitates a deep understanding of its complex pathophysiology to develop innovative therapeutic strategies. Exosomal RNAs (exoRNA), particularly microRNAs (miRNAs) within cardiac tissues, play a critical role in intercellular communication and pathophysiological processes of AMI.

Methods: This study aimed to delineate the exoRNA landscape, focusing especially on miRNAs in animal models using high-throughput sequencing. The approach included sequencing analysis to identify significant miRNAs in AMI, followed by validation of the functions of selected miRNAs through in vitro studies involving primary cardiomyocytes and fibroblasts.

Results: Numerous differentially expressed miRNAs in AMI were identified using five mice per group. The functions of 20 selected miRNAs were validated through in vitro studies with primary cardiomyocytes and fibroblasts.

Conclusions: This research enhances understanding of post-AMI molecular changes in cardiac tissues and investigates the potential of exoRNAs as biomarkers or therapeutic targets. These findings offer new insights into the molecular mechanisms of AMIs, paving the way for RNA-based diagnostics and therapeutics and therapies and contributing to the advancement of cardiovascular medicine.

Keywords: acute myocardial infarction; cardiac tissue; exosomal RNA sequencing; exosome; microRNA.

Figure 1

Experimental overview and exosome characterization of extracted from heart tissues. (A) Schematic representation of the experimental procedure employed in the current study. (B) Group-specific heart images from the myocardial infarction (MI) mouse models, both with and without triphenyltetrazolium chloride (TTC) staining, illustrating the morphological differences among the groups. (C) Transmission electron microscopy (TEM) images of isolated exosome stained with phosphotungstic acid, showcasing their structural features. (D) Immunoblotting analysis of the isolated exosomes using exosomal markers, including CD9, CD63, and CD81, alongside internal controls. Group designations: sham, control group; MI-1day, MI induced for 1 day; MI-3day, MI induced for 3 days.

Figure 2

Comprehensive analysis of exosomal RNA (exoRNA) sequencing data. (A) Depicts the count distribution of various read types for each group, including trimmed reads, nonadapter reads, short reads and low-quality reads. (B) Shows the remaining reads (blue) after the removal of ribosomal RNA (rRNA; red). (C) Illustrates the distribution of read lengths across each sample. (D) Provides a breakdown of the smRNA composition within each sample, categorizing them into various types: miRNA (microRNA), piRNA (PIWI-interacting RNA), snoRNA (small nucleolar RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), siRNA (small interfering RNA), Y RNA, scRNA (single-cell RNA).

Figure 3

Differential Expression (DE) miRNA analysis from exosomal RNA (exoRNA) sequencing. (A) Correlation matrix: This panel presents the correlation matrix of all samples, calculated using Pearson’s coefficient based on normalized values. The correlation coefficient (r) ranges from −1 to 1, where values closer to 1 indicate a higher similarity between samples. (B) Hierarchical clustering: The left part of this panel shows the hierarchical clustering of samples based on their normalized expression normalized value, where samples with higher expression similarities are grouped together (distance metric = Euclidean distance, linkage method = complete linkage) (left). The right part features a heat map of the two-way hierarchical clustering, utilizing Z-scores of Log2-transformed normalized values for visualization. (C) Quantitative analysis of microRNAs (miRNAs): This section displays the number of mature miRNAs that are either up-regulated or down-regulated based on a fold change (|FC| > 2) and p-value (p < 0.05) for each comparison pair. (D) Smear plots: This panel illustrates smear plots representing the expression level of miRNAs. The plots are designed to visually represent the distribution and variance of miRNA expression across samples.

Figure 4

Comprehensive functional enrichment analysis of differentially expressed miRNAs. (A) Depicts the top seven gene ontology (GO) terms, providing insights into the biological processes, cellular components, and molecular functions most affected by the dysregulated miRNAs. (B) Illustrates the top seven pathways identified in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, highlighting critical pathways impacted by the altered expression of mRNAs across different groups.

Figure 5

Identification and selection of key candidate microRNAs (miRNAs). (A) A diagrammatic representation of the number of miRNAs exhibiting increased (up) or decreased (down) expression upon comparative analysis between groups. This visualization aids in understanding the overall distribution and directional trends of miRNA expression changes. (B) A broken line graph displaying the expression profiles of the 20 selectively chosen miRNAs across the groups, enabling a comparative and detailed view of their expression dynamics.

Figure 6

Analysis of cytotoxicity using selected miRNA mimics in primary cardiomyocytes and fibroblasts. On the left, immunofluorescence analysis confirms the presence of specific markers in isolated cells: troponin-T in primary cardiomyocytes and vimentin in primary fibroblasts. The right side of the figure investigates the impact of miRNA mimic treatment on cell survival under hypoxic conditions. In the accompanying bar graph, white and black bars represent the control (miRNA mimic negative control-treated) group, while gray bars denote groups treated with miRNA mimics. Statistical significance between the control and treated groups was assessed using ANOVA. p value annotations indicate levels of significance: * p < 0.05 and ** p < 0.01, highlight key differences in cytotoxicity responses. The error bar represents the standard deviation between the five wells of the 96-well plates used in the cytotoxicity assay. N, normoxic condition; H, hypoxic condition; FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; miR, microRNA.