miR-21 is associated with fibrosis and right ventricular failure - PubMed (original) (raw)

miR-21 is associated with fibrosis and right ventricular failure

Sushma Reddy et al. JCI Insight. 2017.

Abstract

Combined pulmonary insufficiency (PI) and stenosis (PS) is a common long-term sequela after repair of many forms of congenital heart disease, causing progressive right ventricular (RV) dilation and failure. Little is known of the mechanisms underlying this combination of preload and afterload stressors. We developed a murine model of PI and PS (PI+PS) to identify clinically relevant pathways and biomarkers of disease progression. Diastolic dysfunction was induced (restrictive RV filling, elevated RV end-diastolic pressures) at 1 month after generation of PI+PS and progressed to systolic dysfunction (decreased RV shortening) by 3 months. RV fibrosis progressed from 1 month (4.4% ± 0.4%) to 3 months (9.2% ± 1%), along with TGF-β signaling and tissue expression of profibrotic miR-21. Although plasma miR-21 was upregulated with diastolic dysfunction, it was downregulated with the onset of systolic dysfunction), correlating with RV fibrosis. Plasma miR-21 in children with PI+PS followed a similar pattern. A model of combined RV volume and pressure overload recapitulates the evolution of RV failure unique to patients with prior RV outflow tract surgery. This progression was characterized by enhanced TGF-β and miR-21 signaling. miR-21 may serve as a plasma biomarker of RV failure, with decreased expression heralding the need for valve replacement.

Keywords: Cardiology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1

Figure 1. Clinical and histologic characteristics recapitulate the clinical progression of PI+PS.

Sham-operated mice and mice with PI+PS were assessed using echocardiogram, cardiac catheterization, and exercise testing at 1, 2, and 3 months. Early RV diastolic dysfunction manifested as an increase in (A and B) tricuspid inflow E/A ratio by echocardiogram and (C) RV end diastolic pressures (RVEDp) by cardiac catheterization. Late RV systolic dysfunction characterized by (D) progressive decline in exercise duration by modified treadmill testing, (E) decrease in RV outflow tract (RVOT) shortening fraction (SF) by echocardiogram at 3 months, and (F) decrease in dP/dt by RV catheterization after an early increase. (G and H) Development of overt heart failure compared with generalized edema. (I and J) Histologic progression_:_ A progressive increase in myocyte cell area was seen by wheat germ agglutinin staining of the cell wall in green, during the stage of diastolic dysfunction (1 month and 2 months) and plateaued with systolic dysfunction (3 months). Scale bar: 50 μm. (K and L) Progressive increase in subendocardial fibrosis was seen with trichrome staining. n = 6–9 sham for each time point at 1, 2, and 3 months; n = 4–10 PI+PS for each time point at 1, 2, and 3 months. Scale bar: 100 μm. PI+PS, pulmonary insufficiency and pulmonary stenosis; RV, right ventricle. Data represent mean ± SEM and were analyzed using ANOVA with multiple testing correction. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Figure 2. TGF-β pathway is upregulated with PI+PS.

(A) Transcriptional profiling of the failing right ventricle. Agilent whole-genome oligonucleotide microarrays were used to evaluate gene expression at 1 and 2 months (diastolic dysfunction) as well as 3 months (systolic dysfunction) following surgery and compared with sham-operated controls. Metabolic, transport, and ion channel pathways were downregulated over time, while structural, inflammation, and fibrosis-related pathways were upregulated (fold change ≥ 2, Benjamini-Hochberg multiple testing correction with corrected P < 0.05). n = 4–6/sham and PI+PS/time point. (B) Schematic of TGF-β signaling and its regulation of profibrotic miRs. White box, unchanged protein expression; green box, upregulated protein and miR expression; orange box, downregulated protein expression. (C) Phosphorylated/total SMAD2/3 protein expression is upregulated in PI+PS during the stages of diastolic (1 month and 2 months) and systolic dysfunction (3 months). (D and E) miR-21 downstream target proteins involved in ECM regulation, Smad 7 and Sprouty 1, were downregulated as miR-21 expression increased, while (F) PTEN expression was not changed. n = 4–6/sham and PI+PS. PI+PS, pulmonary insufficiency and pulmonary stenosis; ECM, extracellular matrix. Data are presented as mean ± SEM. Microarray data was analyzed using GeneSpring GX 11.5 software. An unpaired, 2-tailed Student’s t test was utilized for 2-group comparisons and ANOVA with multiple testing correction for 3 or more group comparisons. *P < 0.05, **P < 0.01.

Figure 3

Figure 3. miR-21 is a biomarker of RV dysfunction in mice.

(A) RV expression of profibrotic miR-21 was upregulated during the stages of diastolic (1 month and 2 months) and systolic (3 months) dysfunction compared with sham-operated controls. n = 6–8/sham and PI+PS/time point. (B) The expression of miR-21 positively correlated with the degree of RV fibrosis as diastolic dysfunction progressed, but that correlation was lost once systolic dysfunction developed. (C) miR-21 expression is upregulated in the hearts in PI+PS mice and not in the livers, spleens, or lungs. n = 5/sham; n = 8/PI+PS. (D) miR-21 is expressed predominantly in nonmyocytes. n = 4/sham; n = 6–8/PI+PS. (E) Plasma miR-21 was upregulated with diastolic dysfunction but switched to being downregulated with the development of systolic dysfunction, despite persistent tissue-level upregulation (n = 9–10/sham and PI+PS/time point); miR-21 is expressed in plasma exosomes. (F) TEM demonstrating exosomes (arrows) 50–100 nm in size, with characteristic cup shape (arrowhead) and surrounding halo. Scale bar: 500 nm (top); 200 nm (bottom). (G) Exosome marker expression was similar at all time points. n = 2/group. (H) miR-21 is enriched in exosomes in the plasma compared with the nonexosome fraction. n = 3/sham; n = 4/PI+PS. (I) Plasma miR-21 correlated with RV end-diastolic area (RVED) as diastolic dysfunction progressed and (J) switched to a negative correlation with the onset of systolic dysfunction. (K and L) A similar correlation was seen with fibrosis. RV, right ventricle; PI+PS, pulmonary insufficiency and pulmonary stenosis; TEM, transmission electron microscopy. Data are presented as mean ± SEM. An unpaired, 2-tailed Student’s t test was utilized for 2-group comparisons and ANOVA with multiple testing correction for 3 or more group comparisons. Correlation was assessed using Pearson’s correlation. *P < 0.02, ** P < 0.001.

Figure 4

Figure 4. RV miR-21 is upregulated in children with PI+PS and is associated with increasing fibrosis.

(A) RV miR-21 expression is upregulated in children with PI+PS (n = 4) and end-stage RV failure (n = 2) compared with those with mild RVOTO (n = 3) and infants with unrepaired TOF (n = 7). (B and C) Increasing fibrosis is seen in children with PI+PS (n = 4) and end-stage RV failure (n = 2). Scale bar: 100 μm. RV, right ventricle; RVOTO, RV outflow tract obstruction; TOF, tetralogy of Fallot; PI+PS, pulmonary insufficiency and pulmonary stenosis. Data are presented as mean ± SEM. ANOVA with multiple testing correction was used. Correlation was assessed using Pearson’s correlation. *P < 0.01, **P < 0.001.

Figure 5

Figure 5. miR-21 is a potential biomarker for RV dysfunction in patients with tetralogy of Fallot.

(A) Plasma miR-21 expression is increased in patients with PI+PS without RV systolic dysfunction (n = 7) but switches to being decreased in patients with systolic dysfunction (n = 9) compared with controls (n = 12). (B) miR-21 expression correlates negatively with RV end-diastolic volume and (C) positively with RV ejection fraction. (D) TEM demonstrating exosomes (arrow) with characteristic cup shape (arrowhead). Scale bar: 500 nm (top); 200 nm (bottom). (E) Exosome marker expression was similar between control and PI+PS. n = 2/group. (F) Electropherogram of exosomes showing small RNAs in the 10- to 40-nucleotide region indicative of miRs and no contamination from larger RNAs. (G) miR-21 expression is enriched in exosomes compared with the nonexosome fraction. n = 4/control; n = 9/PI+PS. RV, right ventricle; PI+PS,; TEM, transmission electron microscopy. Data are presented as mean ± SEM. An unpaired, 2-tailed Student’s t test was utilized for 2-group comparisons and ANOVA with multiple testing correction for 3 or more group comparisons. Correlation was assessed using Pearson’s correlation. *P < 0.01, ***P < 0.001.

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