Myocardial microRNAs associated with reverse remodeling in human heart failure

Abstract

Background: In dilated cardiomyopathies (DCMs) changes in expression of protein-coding genes are associated with reverse remodeling, and these changes can be regulated by microRNAs (miRs). We tested the general hypothesis that dynamic changes in myocardial miR expression are predictive of β-blocker-associated reverse remodeling.

Methods: Forty-three idiopathic DCM patients (mean left ventricular ejection fraction 0.24 ± 0.09) were treated with β-blockers. Serial ventriculography and endomyocardial biopsies were performed at baseline, and after 3 and 12 months of treatment. Changes in RT-PCR (candidate miRs) or array-measured miRs were compared based on the presence (R) or absence (NR) of a reverse-remodeling response, and a miR-mRNA-function pathway analysis (PA) was performed.

Results: At 3 months, 2 candidate miRs were selectively changed in Rs, decreases in miR-208a-3p and miR-591. PA revealed changes in miR-mRNA interactions predictive of decreased apoptosis and myocardial cell death. At 12 months, 5 miRs exhibited selective changes in Rs (decreases in miR-208a-3p, -208b-3p, 21-5p, and 199a-5p; increase in miR-1-3p). PA predicted decreases in apoptosis, cardiac myocyte cell death, hypertrophy, and heart failure, with increases in contractile and overall cardiac functions.

Conclusions: In DCMs, myocardial miRs predict the time-dependent reverse-remodeling response to β-blocker treatment, and likely regulate the expression of remodeling-associated miRs.

Trial registration: ClinicalTrials.gov NCT01798992.

Funding: NIH 2R01 HL48013, 1R01 HL71118 (Bristow, PI); sponsored research agreements from Glaxo-SmithKline and AstraZeneca (Bristow, PI); NIH P20 HL101435 (Lowes, Port multi-PD/PI); sponsored research agreement from Miragen Therapeutics (Port, PI).

Conflict of interest statement

C.C. Sucharov, J.D. Port, and M.R. Bristow have stock (each less than 0.1% of total outstanding shares) in Miragen Therapeutics, and M.R. Bristow is member of the Miragen Scientific Advisory Board.

Figures

Figure 1. Box-and-whisker plots of RT-PCR RNA abundance data for a subset of candidate miRs as described in the text.

Boxes delineate 1st (lower border) and 3rd (upper border) quartiles from the median; the length of the whiskers is 1.5 times the interquartile distance (IQD), with values beyond the IQD plotted as outliers. miR expression was normalized to the combination of the small RNA U6 and miR-103, and is represented as fold difference from baseline. Comparisons were made to Non-Responder (NR) and Responder (R) as described in the Figure. (A) 3 months. (B) 12 months. Nonparametric Wilcoxon signed-rank test was performed plus Hochberg-corrected significance levels, as described in Methods. n values at 3 months: baseline = 41; NR = 9; R = 32. n values at 12 months: baseline= 35; NR = 11; R = 24.

Figure 2. miRs differentially regulated in response to β-blocker treatment at 3 and 12 months.

(A) Heat map of miRs differentially regulated in Responder (R) patients at 3 months. (B) Heat map of miRs differentially regulated in R patients at 12 months. All data were normalized to baseline levels. Red = upregulated. Green = downregulated. n values at 3 months: NR = 10; R = 35. n values at 12 months: NR = 11; R= 26.

Figure 3. Random forest (RF) analysis of miR arrays.

RF analysis of miR arrays based on outcomes: Responder and Non-Responder at 3 months (A) 12 months (B).

Figure 4. Multidimensional scaling of the estimated proximity matrix plots.

Classification performances to discriminate Responder from Non-Responder at 3 months (A) and 12 months (B) are displayed. The top 20 miRs are displayed.

Figure 5. Hierarchical clustering of Responder and Non-Responder patients based on the top 7 miRs as determined by random forest analysis in Figure 3.

(A) 3 months. (B) 12 months.

Figure 6. Receiver operating characteristic (ROC) curves.

ROC analysis was performed for top 7 miRs as determined by random forest analysis in Figure 3. Based on ROC the area under the curve (AUC) was calculated at 3 months (A) and 12 months (B).

Figure 7. Ingenuity pathway analysis (IPA) of mRNAs predicted to be targets of dysregulated miRs.

Selection of miRs is described in the text. Colors are indicative of activation or repression and are described in the Figure. P values and Z scores for predicted response are stated in the text. (A) 3 months (left panel, contraction of heart; middle panel, hypertrophy of heart; right panel, failure of heart). (B) 12 months (left to right: first panel, cell death of cardiomyocytes; second panel, function of heart; third panel, hypertrophy of heart; fourth panel, failure of heart).