Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers

Abstract

Heart failure (HF) is associated with high morbidity and mortality and its incidence is increasing worldwide. MicroRNAs (miRNAs) are potential markers and targets for diagnostic and therapeutic applications, respectively. We determined myocardial and circulating miRNA abundance and its changes in patients with stable and end-stage HF before and at different time points after mechanical unloading by a left ventricular assist device (LVAD) by small RNA sequencing. miRNA changes in failing heart tissues partially resembled that of fetal myocardium. Consistent with prototypical miRNA-target-mRNA interactions, target mRNA levels were negatively correlated with changes in abundance for highly expressed miRNAs in HF and fetal hearts. The circulating small RNA profile was dominated by miRNAs, and fragments of tRNAs and small cytoplasmic RNAs. Heart- and muscle-specific circulating miRNAs (myomirs) increased up to 140-fold in advanced HF, which coincided with a similar increase in cardiac troponin I (cTnI) protein, the established marker for heart injury. These extracellular changes nearly completely reversed 3 mo following initiation of LVAD support. In stable HF, circulating miRNAs showed less than fivefold differences compared with normal, and myomir and cTnI levels were only captured near the detection limit. These findings provide the underpinning for miRNA-based therapies and emphasize the usefulness of circulating miRNAs as biomarkers for heart injury performing similar to established diagnostic protein biomarkers.

Keywords: body fluids; cardiovascular disease; development; exRNA; miRNA-mRNA regulation.

Fig. 1.

Number of individuals in each group and tissue with the number of samples shown in parentheses. Advanced HF group at LVAD implantation 3 mo (3M LVAD) or 6 mo (6M LVAD) after LVAD implantation and at LVAD explantation.

Fig. 2.

miRNA cistron abundance changes and target gene regulation in failing and fetal myocardium. (A and B) Relative changes in miRNA cistron abundance compared with nonfailing postnatal hearts. Cistrons with a false discovery rate (FDR) of <10% are colored red if up- regulated and blue if down-regulated. Cistrons contributing to the top 85% sequencing reads are to the right and residual cistrons to the left of the dotted vertical line. Labeled in both A and B for comparison are (i) cistrons consistently changed in DCM and ICM HF, (ii) myomirs, and (iii) mir-216a(3) and mir-29a(4) due to their large differences. (C) Unsupervised hierarchical clustering of miRNA cistrons representing the cumulative top 85% miRNA sequence reads with residual cistrons represented as “all–other” at the bottom of the heatmap. The row dendrogram has been omitted. The column labels at the bottom represent the unique IDs for each subject; demographic details on the individual subjects and for each sequencing sample can be found in Dataset S1 (Tables 34 and 35). (D and E) Cumulative distribution function (CDF) showing the changes in mRNA abundance (x axis) for transcripts with different target sites (colored) for family miR-1-1(3) in the 3′UTR compared with transcripts without a site in the 3′UTR (black line). Colored points at bottom of the graph indicate the median of the CDF; P values are from a one-sided Kolmogorov–Smirnov test.

Fig. 3.

miRNA composition in circulation. (A) Unsupervised hierarchical clustering of miRNA cistrons contributing to the cumulative top 85% sequence reads in plasma samples compared with red blood cells (RBCs), peripheral blood mononuclear cells (PBMCs), and HUVECs. (B) Unsupervised clustering of the same samples as in A but restricted to myomir cistrons with other miRNAs summarized in “all–other” at the bottom of the heatmap. The order of the annotation tracks above the heatmap and the legend are the same for A and B. The column labels at the bottom of the heatmaps are the unique IDs for each subject; demographic details on the individual subjects and for each sequencing sample can be found in Dataset S1 (Tables 34 and 35). For corresponding heatmaps with all serum and plasma samples, see Fig. S5 G and H.

Fig. 4.

Circulating miRNA dynamics in HF. (A) Changes of muscle-specific and other selected miRNA cistrons in circulation comparing the conditions as indicated in the column headings. Red bars represent higher levels and blue bars lower levels in each comparison. The gray shading marks changes that have an FDR < 10%. (B and C) Correlation of cistron mir-208b(1) with cTnI (B) and BNP (C) levels showing the fit (blue line) and its 95% confidence interval (CI) (gray shading). (D) Receiver operating characteristic curve comparing the performances for cTnI and mir-208b(1) in distinguishing patients with advanced HF at the time of LVAD implantation from healthy control individuals. Areas around the blue and red lines represent 95% CI. The cross marks represent the best threshold value, which was 0.044 ng/mL (95% CI: 0.01, 0.05) for cTnI and a relative frequency of 0.0018% (95% CI: 0.0015, 0.0019) for mir-208b(1).