Cardiac myocyte miR-29 promotes pathological remodeling of the heart by activating Wnt signaling

Abstract

Chronic cardiac stress induces pathologic hypertrophy and fibrosis of the myocardium. The microRNA-29 (miR-29) family has been found to prevent excess collagen expression in various organs, particularly through its function in fibroblasts. Here, we show that miR-29 promotes pathologic hypertrophy of cardiac myocytes and overall cardiac dysfunction. In a mouse model of cardiac pressure overload, global genetic deletion of miR-29 or antimiR-29 infusion prevents cardiac hypertrophy and fibrosis and improves cardiac function. Targeted deletion of miR-29 in cardiac myocytes in vivo also prevents cardiac hypertrophy and fibrosis, indicating that the function of miR-29 in cardiac myocytes dominates over that in non-myocyte cell types. Mechanistically, we found cardiac myocyte miR-29 to de-repress Wnt signaling by directly targeting four pathway factors. Our data suggests that, cell- or tissue-specific antimiR-29 delivery may have therapeutic value for pathological cardiac remodeling and fibrosis.

Fig. 1

Genetic deletion of miR-29 in a mouse model for left ventricular pressure overload. a Expression of miR-29 family members in left myocardium from wildtype (WT), miR-29 ab1 ^−/− b2c ^+/− and miR-29 ab1 ^+/+ b2c ^−/− mice; n = 4–6 mice/group. b Echocardiographic analysis of fractional shortening as a measure of left ventricular function; n = 4–10 mice/group. A Student’s t-test was used to calculate the P values. c (Left) Representative stainings of myocardial tissue from WT or knockout mice (tissue fixation 21 days after sham surgery or transverse aortic constriction, TAC) by hematoxylin/eosin and Sirius Red/Fast Green stainings. Scale bar: 2 mm. (Right) Ratio between heart weight and tibia length (HW/TL) as a measure of cardiac hypertrophy; n = 6–14 mice/group. P values were determined by two-way ANOVA followed by Bonferroni’s post hoc test. d (Left) Representative wheat germ agglutinin (WGA)-staining of midventricular sections to assess hypertrophy of cardiac myocytes. Scale bar: 50 µm. (Right) Quantitative analysis; n = 5–8 mice/group. P values were calculated using two-way ANOVA followed by Bonferroni’s post hoc test. e (Left) Representative image sections from Sirius Red/Fast Green-stained myocardium of the indicated groups and (Right) quantitative analysis of fibrosis; n = 3–11 mice/group. P values were determined by two-way ANOVA followed by Bonferroni’s post hoc test. f Real-time PCR quantification of markers for cardiac remodeling in left ventricular tissue from WT, miR-29 ab1 ^−/− b2c ^+/− and miR-29 ab1 ^+/+ b2c ^−/− mice. Collagen mRNAs and the following markers of cardiac myocyte hypertrophy were assessed: Nppa, atrial natriuretic peptide; Myh7/Myh6, ratio of mRNAs encoding β- and α-myosin heavy chain. Tissues were collected 21 days after TAC surgery; data are from 4 to 9 independent experiments, with 2 replicates each. WT TAC means were compared to that of miR-29 ab1 ^−/− b2c ^+/− and miR-29 ab1 ^+/+ b2c ^−/− mice by a one-way ANOVA followed by a Bonferroni’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 for all panels. All quantitative data are reported as means ± SEM

Fig. 2

Pharmacological inhibition of miR-29 prevents cardiac remodeling and dysfunction. a (Left) Design of the miR-29 family inhibitor (antimiR-29). Sequences of miR-29a, b1, b2 and c display a high degree of sequence similarity with identical seed regions (depicted in blue), thus permitting the design of a single antimiR molecule. (Right) Design of the study. b Cardiac expression of miR-29 family members in mice, determined weeks after the first injection of antimiR-29, a control molecule (antimiR-Ctrl) or PBS. c Echocardiographic analysis of left ventricular fractional shortening in sham-/TAC-operated mice 3 weeks after injection with antimiR-29/-ctrl or PBS (determined by echocardiography), indicating reduced TAC effects in the antimiR-29-treated group. d Heart weight-to-tibia length ratio and e WGA staining of left ventricular tissue from mice described in c for determination of cardiac and CM hypertrophy. f (Left) Representative left ventricular sections stained with Sirius Red/Fast Green of the indicated treatment groups and (right) quantification of interstitial fibrosis. g Quantitative real-time PCR analysis of molecular markers for cardiac myocyte hypertrophy (Nppa, Myh7/Myh6) and of fibrosis-associated collagens. All scale bars: 50 µm. All quantifications derive from n = 5–14 mice/group, PCR performed with 2 replicates each. All quantitative data in panels c–g are reported as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 determined by two-way ANOVA followed by Bonferroni’s post hoc test

Fig. 3

Expression of miR-29 members in cardiac cells and their deregulation in disease. a–d Individual quantifications (by qPCR) of miR-29 variants a, b and c in primary cardiac cells or in left ventricular myocardium. a Age-dependent cardiac expression of miR-29 variants in mice (w: week); n = 4–5 mice per group. b Endogenous levels of miR-29 family members in cardiac myocytes from mice 21 days after sham treatment or, for the TAC group, at denoted time points; n = 3–6 mice per group. c Endogenous levels of miR-29 family members in CM and CF freshly isolated from adult mouse myocardium; n = 12–13 independent cell isolations. d Quantification of miR-29 variants in human left ventricular myocardium from 23 healthy individuals or from 24 patients with aortic valve stenosis. All quantitative data are reported as means ± SEM. ***P < 0.001 calculated using Student’s t-test

Fig. 4

Deletion of miR-29 in cardiac myocytes in vivo protects from cardiac remodeling. Tropism of adeno-associated virus 9 for cardiac myocytes in vivo was employed to deliver improved Cre recombinase (AAV9-iCre) to miR-29 b2c ^fl/fl mice for the deletion of this cluster (with miR-29 b2c ^+/+ littermates serving as controls). a Design of the study. 5 × 10¹¹ viral particles (AAV9-iCre) were delivered to 5 day-old mice via intrapericardial injection. Seven weeks later, mice were subjected to TAC or sham surgery and sacrificed another 3 weeks later (after echocardiographic analysis). b Expression of miR-29b and miR-29c in cardiac tissue from mice treated as in a; n = 4–6 mice per group. c Left ventricular fractional shortening as determined by echocardiographic analysis; n = 4–8 mice per group. d (Left) Representative hematoxylin eosin stainings of myocardial sections; scale bar = 2 mm. (Right) Heart weight-to-tibia length ratio; n = 4–11 mice per group. e (Left) Representative myocardial sections stained for fibrosis with Sirius Red/Fast Green and (right) quantitative analysis of the results; n = 4–9 mice per group; scale bar: 2 mm. All quantitative data are reported as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s t-test b or two-way ANOVA followed by Bonferroni’s post hoc test c–e

Fig. 5

miR-29 targets key components of the Wnt signaling pathway. a Design of the study. b Volcano-plot of fold changes of individual proteins from NRCM transfected with antimiR-29 or antimiR-ctrl. Dark gray symbols highlight significantly deregulated proteins (P < 0.05) and orange symbols those with known or predicted profibrotic function. LUM Lumican, CTSL Cathepsin L, LGALS3BP Galectin 3 Binding Protein, CST3 Cystatin C, CDH2 Cadherin 2, ECM1 Extracellular Matrix Protein 1, APP Amyloid Beta Precursor Protein. c Significant GO enrichment of transcription factor binding sites in the deregulated secreted factors. d (Left) Wnt activity using a TCF/LEF reporter assay in NRCM 48 h after transfection with miR-29a or miR-ctrl. (Right) NFAT activity in NRCM 48 h after transfection with miR-29a miR-ctrl; 4–5 independent experiments in triplicate. e miR-29 directly regulates the Gsk3β, Ctnnbip1, Hbp1 and Glis2 3´-UTRs. HEK293 cells were transfected with miR-29a or miR-ctrl. Ratiometric analysis of fluorescent emissions from a dual fluorescent reporter carrying the Gsk3β, Ctnnbip1, Hbp1 and Glis2 3´-UTRs or seed mutants. Data are from 8 independent experiments performed in triplicate. f The Wnt-inhibitor IWR-1 (10 μM for 96 h) prevented miR-29-induced cardiac myocyte hypertrophy. (Up) Representative segmentation images of NRCM transfected with either miR-29 or miR-ctrl in the presence or absence of IWR-1 scale bar: 100 μm. NRCM were identified based on α-actinin detection (green) and are assigned green nuclei, whereas non-myocytes were assigned red nuclei. (Down) Quantitative analysis of the results. Data are from four independent experiments performed in triplicate. g Proposed mechanism how miR-29 promotes Wnt signaling in CM and signals to fibroblasts. In cardiac myocytes, miR-29 (by targeting Gsk3b Ctnnbip1, Hbp1 and Glis2) activates the Wnt signaling pathway, as well as NFAT activity. Activation of Wnt and NFAT signaling pathways promotes cardiac myocyte hypertrophy and secretion of profibrotic factors, which act in cardiac fibroblasts. Pharmacological inhibition or genetic deletion of miR-29 in cardiac myocytes prevents cardiac hypertrophy and fibrosis. All quantitative data are reported as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s t-test d, e or two-way ANOVA followed by Bonferroni’s post hoc test f