miR-3113-5p, miR-223-3p, miR-133a-3p, and miR-499a-5p are sensitive biomarkers to diagnose sudden cardiac death

Abstract

Background: Sudden cardiac death (SCD) remains a great health threat and diagnostic challenge, especially those cases without positive autopsy findings. Molecular biomarkers have been urgently needed for the diagnosis of SCD displaying negative autopsy results. Due to their nature of stability, microRNAs (miRNAs) have emerged as promising diagnostic biomarkers for cardiovascular diseases.

Methods: This study investigated whether specific cardio-miRNAs (miR-3113-5p, miR-223-3p, miR-499a-5p, and miR-133a-3p) could serve as potential biomarkers for the diagnosis of SCD. Thirty-four SCD cases were selected, 18 categorized as SCD with negative autopsy (SCD-negative autopsy) findings and 16 as SCD with positive autopsy (SCD-positive autopsy) findings such as coronary atherosclerosis and gross myocardial scar. Carbon monoxide (CO) intoxication (n = 14) and fatal injury death (n = 14) that displayed no pathological changes of myocardium were selected as control group, respectively. Histological analyses were performed to reveal the pathological changes and real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the expression of those miRNAs.

Results: It showed that heart samples from the SCD-negative autopsy group displayed no remarkable difference with regard to the expression of cleaved-caspase3, CD31, and CD68 and the extent of fibrotic tissue accumulation when compared with control samples. The four cardio-miRNAs were significantly up-regulated in the SCD samples as compared with control. When discriminating SCD from controls, receiver operating characteristic (ROC) curve analysis revealed that the areas under the curve (AUC) of these 4 miRNAs were from 0.7839 to 0.9043 with sensitivity of 64.71-97.06% and specificity of 70-100%. Moreover, when discriminating the specific causes of SCD, the four miRNA expressions increased in the heart from the SCD-negative autopsy group as relative to that from the SCD-positive autopsy group, and a combination of two miRNAs presented higher diagnostic value (AUC = 0.7407-0.8667).

Conclusion: miR-3113-5p, miR-223-3p, miR-499a-5p, and miR-133a-3p may serve as independent diagnostic biomarkers for SCD, and a combination of two of these miRNAs could further discriminate detailed causes of SCD.

Keywords: Biomarkers; Molecular pathology; Sudden cardiac death; miRNA.

Fig. 1

Representative photographs of the morphological changes in the heart tissues of fatal injury, carbon monoxide (CO) intoxication, sudden cardiac death with positive autopsy (SCD-positive autopsy), and sudden cardiac death with negative autopsy (SCD-negative autopsy) by H&E staining and PicroSirius Red (PSR) staining. The SCD-positive autopsy group presented with severe coronary stenosis, calcification, and fibrotic tissue accumulation, whereas the fatal injury and CO intoxication groups did not show notable pathological changes. SCD-negative autopsy group showed slight histological changes such as interstitial edema, fiber rupture, and patchy hypereosinophilia of cardiomyocytes. Scale bar = 200 μm

Fig. 2

Representative photographs of cleaved-Caspase3 (cl-Casp3), CD31, and CD68 expression in heart tissues stained by immunohistochemistry. SCD-positive autopsy group showed increases in the expression of cl-Casp3 (cytoplasm positivity in apoptotic cells), CD31 (lumen positivity), and CD68 (a marker of macrophage), whereas fatal injury and CO intoxication cases were absent from strong positivity of the above markers. SCD-negative autopsy cases showed patchy positivity of cl-Casp3 and dim CD31 signal, and were absent of CD68 positivity. Scale bar = 200 μm

Fig. 3

The relative expression level of the four miRNAs among fatal injury (n = 14), CO intoxication (n = 14) and SCD groups (n = 34). The expression of miR-3113-5p (A), miR-223-3p (B), miR-499a-5p (C) and miR-133a-3p (D) were detected by RT-qPCR. Results were normalized with U6 gene, *p < 0.05, **p < 0.01

Fig. 4

Diagnostic performance of the four miRNAs were analyzed by ROC curve. (A, B) ROC curve of miR-3113-5p. (C, D) ROC curve of miR-223-3p. (E, F) ROC curve of miR-499a-5p. (G, H) ROC curve of miR-133a-3p

Fig. 5

The relative expression level of the four miRNAs between SCD-positive autopsy (n = 18) and SCD-negative autopsy groups (n = 16). miR-3113-5p (A), miR-223-3p (B), miR-499a-5p (C), and miR-133a-3p (D) were upregulated in SCD-negative autopsy group as compared to the SCD-positive autopsy. Results were normalized to U6 gene, *p < 0.05 versus SCD-positive autopsy

Fig. 6

Diagnostic performance of miRNAs was analyzed by ROC curve. The ROC analysis for detection of SCD-positive autopsy and SCD-negative autopsy using a combination of miR-3113-5p and miR-223-3p (A), a combination of miR-3113-5p and miR-499a-5p (B), a combination of miR-3113-5p and miR-133a-3p (C), a combination of miR-499a-5p and miR-133a-3p (D), a combination of miR-223-3p and miR-499a-5p (E), a combination of miR-223-3p and miR-133a-3p (F) were shown