Crucial Role of miR-433 in Regulating Cardiac Fibrosis

Abstract

Dysregulation of microRNAs has been implicated in many cardiovascular diseases including fibrosis. Here we report that miR-433 was consistently elevated in three models of heart disease with prominent cardiac fibrosis, and was enriched in fibroblasts compared to cardiomyocytes. Forced expression of miR-433 in neonatal rat cardiac fibroblasts increased proliferation and their differentiation into myofibroblasts as determined by EdU incorporation, α-SMA staining, and expression levels of fibrosis-associated genes. Conversely, inhibition of miR-433 exhibited opposite results. AZIN1 and JNK1 were identified as two target genes of miR-433. Decreased level of AZIN1 activated TGF-β1 while down-regulation of JNK1 resulted in activation of ERK and p38 kinase leading to Smad3 activation and ultimately cardiac fibrosis. Importantly, systemic neutralization of miR-433 or adeno-associated virus 9 (AAV9)-mediated cardiac transfer of a miR-433 sponge attenuated cardiac fibrosis and ventricular dysfunction following myocardial infarction. Thus, our work suggests that miR-433 is a potential target for amelioration of cardiac fibrosis.

Keywords: AZIN1; JNK1.; cardiac fibrosis; miR-433.

Figure 1

miR-433 is increased in cardiac fibrosis. A, dysregulated miRNAs in hearts from 21 days post-myocardial infarction (MI) versus sham control mice (n=4); B, upregulated miR-433 in ventricle samples from 21 days post-MI mice (n=4), a rodent model of doxorubicin (Dox)-induced cardiomyopathy (n=6), and human dilated cardiomyopathy (n=4); C-D, increased miR-433 in two in vitro cardiac fibrosis model induced either by TGF-β or Angiotensin II (n=6); E, expression of miR-433 in neonatal cardiac fibroblasts (NRCF) compared to cardiomyocytes (NRCM) (n=6); F, markers for pathological hypertrophy (ANP, BNP and Myh7) and extracellular matrix proteins (CTGF, TSP-1, Col1a1 and Col3a1) in cardiomyocytes with miR-433 overexpression (n=6). Scale bar: 50 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 2

Antagonizing miR-433 attenuates cardiac fibrosis and preserves ventricular function post-myocardial infarction. A, decreased miR-433 in hearts from mice treated with miR-433 antagomir (n=6); B, preserved left ventricular fractional shortening (FS) and ejection fraction (EF); C, reduced cardiac fibrosis; D, decreased collagen content in myocardial infarction (MI) with miR-433 inhibition, as evidenced by echocardiography (n=6), Masson's trichrome staining (n=4), and Sircol assay (n=4); E, no difference in the infarct size between mice treated with miR-433 antagomir or negative control 3 days post-MI (n=7). Scale bar: 100 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 3

Cardiac inhibition of miR-433 via AAV9 attenuates cardiac fibrosis and preserves ventricular function post-myocardial infarction. A, decreased miR-433 in hearts from mice treated with miR-433 sponge AAV9 (n=6); B, preserved left ventricular fractional shortening (FS) and ejection fraction (EF); C, reduced cardiac fibrosis; D, decreased collagen content in myocardial infarction (MI) interfered with miR-433 sponge AAV9, as evidenced by echocardiography (n=6), Masson's trichrome staining (n=4), and Sircol assay (n=4). Scale bar: 100 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 4

Antagonizing miR-433 attenuates cardiac fibroblasts proliferation and their differentiation into myofibroblasts in vivo. A-B, decreased cardiac fibroblasts proliferation; C, reduced differentiation into myofibroblasts in myocardial infarction (MI) with miR-433 inhibition, as determined by immunofluorescent staining for Vimentin and Ki-67 or pHH3 or α-SMA (n=4); D, decreased α-SMA, Col1a1, and Col3a1 in MI mice with miR-433 inhibition (n=4); E, Agilent gene arrays and KEGG pathway analysis identified extracellular matrix (ECM) receptor interaction as the most affected pathway in MI hearts with miR-433 inhibition (n=4); F, decreased TGF-β, CTGF, Col1a1, Col3a1 and α-SMA and increased MMP2 and MMP9 after treatment with miR-433 antagomir in MI mice (n=4). Scale bar: 20 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 5

miR-433 promotes cardiac fibroblasts proliferation and their differentiation into myofibroblasts in vitro.A, increased miR-433 in cardiac fibroblasts treated with miR-433 agomir (n=6); B, enhanced cardiac fibroblasts proliferation and their differentiation into myofibroblasts (n=4); C-D, upregulated fibrosis-related genes in cardiac fibroblasts with miR-433 overexpression in the absence of TGF-β stimulation, as evidenced by EdU/α-SMA staining (n=4), qRT-PCR (n=6), and Western blot analysis (n=4); E-F, upregulated fibrosis-related genes in cardiac fibroblasts with miR-433 overexpression in the absence of Angiotensin II stimulation. Scale bar: 50 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 6

Inhibition of miR-433 attenuates TGF-β/Ang II-induced cardiac fibroblasts proliferation and their differentiation into myofibroblasts in vitro. A, decreased miR-433 in cardiac fibroblasts treated with miR-433 antagomir (n=6); B, reduced cardiac fibroblasts proliferation and their differentiation into myofibroblasts (n=4); C-D, downregulated fibrosis-related genes in cardiac fibroblasts with miR-433 inhibition regardless of TGF-β stimulation, as evidenced by EdU/α-SMA staining (n=4), qRT-PCR (n=6), and Western blot analysis (n=4); E-F, downregulated fibrosis-related genes in cardiac fibroblasts with miR-433 inhibition regardless of Angiotensin II stimulation. Scale bar: 50 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 7

AZIN1 is a target gene of miR-433 in cardiac fibroblasts. A, Targetscan and Luciferase reporter assays identified AZIN1 as a direct target gene of miR-433 (n=6); B-C, AZIN1 was negatively regulated by miR-433 in cardiac fibroblasts (n=3); D-F, Overexpression of AZIN1 via pEGFP plasmid attenuated the pro-fibrotic effect of miR-433 agomir on cardiac fibroblasts proliferation and their differentiation into myofibroblasts. n=3 for Western blot, n=4 for EdU and α-SMA staining, n=6 for qRT-PCR. Scale bar: 50 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 8

AZIN1 inactivates TGF-β/Smad3 signaling in cardiac fibroblasts. A, Knockdown of AZIN1 could upregulate TGF-β expression and activate Smad3 phosphorylation (n=3); B, Overexpression of AZIN1 had opposite effects (n=3). *, P<0.05, **, P<0.01, ***, P<0.001 versus respective control.

Figure 9

JNK1 is a novel target gene of miR-433 in cardiac fibroblasts. A, Targetscan and Luciferase reporter assays identified JNK1 as a direct target gene of miR-433 (n=6); B-C, JNK1 was negatively regulated by miR-433 in cardiac fibroblasts (n=3); D-F, Overexpression of JNK1 via pEGFP plasmid attenuated the pro-fibrotic effect of miR-433 agomir on cardiac fibroblasts proliferation and their differentiation into myofibroblasts. n=3 for Western blot, n=4 for EdU and α-SMA staining, n=6 for qRT-PCR. Scale bar: 50 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 10

JNK1 knockdown significantly activates ERK and p38 kinase. A, JNK1 knockdown via siRNA increased ERK, p38, and Smad3 phosphorylation (n=3); B, Overexpression of JNK1 via pEGFP plasmid had opposite effects (n=3); C-D, reduced cardiac fibroblasts proliferation and their differentiation into myofibroblasts in cells treated with miR-433 agomir and inhibitor of p38, ERK, or Smad3. n=4 for EdU and α-SMA staining, n=6 for qRT-PCR. Scale bar: 50 μm. *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.

Figure 11

AZIN1 and JNK1 are downregulated by miR-433 antagomir in vivo. A-B, downregulated AZIN1 and JNK1 in ventricular samples from 21 day post-myocardial infarction (MI) mice, rodent model of doxorubicin (Dox)-induced cardiomyopathy, and human dilated cardiomyopathy (DCM). For Western blot, n=3. For qRT-PCR, n=3 for mice and n=4 for patients; C, Upregulated AZIN1 and JNK1, accompanied by an inactivation of ERK, p38, and Smad3 phosphorylation in MI hearts with miR-433 inhibition (n=4). *, P<0.05, **, P<0.01, ***, P<0.001 versus respective controls.