miR-23a participates in initiating hypertrophy. (A and B) Iso and Aldo induce hypertrophy. Cardiomyocytes were treated with different concentrations of isoproterenol (Iso) for 48 h (A) or aldosterone (Aldo) for 24 h (B), and then cell surface area was analyzed. *P < 0.05 vs. control. (C and D) Up-regulation of mature miR-23a, miR-27a, and miR-24 upon treatment with 10 μM Iso (C) or 1 μM Aldo (D). The expression of miR-23a, miR-27a, and miR-24 was analyzed by qRT-PCR, and the results were normalized to that of U6. (E–H) miR-23a antagomir inhibits hypertrophy induced by Iso. Cells were transfected with antagomir or antagomir-negative control (antagomir-NC). Twenty-four hours after transfection cells were treated with 10 μM Iso. Representative photos show sarcomere organization (E), (Scale bar, 20 μm.) Cell surface area measurement (F). Analysis of the transcripts for β-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) by qRT-PCR (G). The protein/DNA ratio analysis (H). *P < 0.05 vs. Iso alone. (I) miR-23a antagomir suppresses the expression of miR-23a. Cells were transfected with antagomir or antagomir-NC, and 24 h after transfection, treated with 10 μM Iso or 1 μM Aldo. *P < 0.05 vs. Aldo alone. ^#P < 0.05 vs. Iso alone.

NFATc3 regulates miR-23a cluster transcription. (A) Schematic representation of the 5′ upstream region of the rat miR-23a cluster. Sequences between –1,404 and + 75 bp of the rat miR-23a cluster are aligned with the corresponding sequences of human miR-23a cluster. The conservative NFAT-like site is shown in red. (B) ChIP analysis of in vivo NFATc3 binding to the promoter. ChIP assay was performed using cardiomyocytes treated with or without 10 μM Iso. The anti-MuRF1 antibody was used as a negative control. (C) NFATc3 activates miR-23a promoter activity. The promoter was synthesized and linked to luciferase (Luc) reporter gene. The mutations were introduced to the binding site (BS). Cardiomyocytes were infected with adenoviruses harboring β-gal or ΔNFATc3. Twenty-four hours after infection cells were transfected with the constructs of the empty vector (pGL-4.17), the constructs of wild type (WT) promoter, or the promoter with mutations in the binding site (m-BS), respectively. Firefly luciferase activities were normalized to Renilla luciferase activities. (D and E), Knockdown of endogenous NFATc3 leads to a reduction in miR-23a promoter activity. Cardiomyocytes were infected with adenovirus harboring NFATc3 RNAi or its scramble form, transfected with the wild type promoter construct, treated with 10 μM Iso (D) or 1 μM Aldo (E), and harvested at the indicated minutes (min). Firefly luciferase activities were normalized to Renilla luciferase activities.

MuRF1 is a target of miR-23a. (A) Sequence alignment of putative miR-23a targeting site in 3′UTR of MuRF1 shows a high level of complementarity and sequence conservation. (B) MuRF1 is down-regulated upon treatment with Iso or Aldo. Cardiomyocytes were treated with 10 μM Iso and 1 μM Aldo, respectively. MuRF1 protein levels were analyzed by immunoblot. (C) miR-23a inhibits the translation activity of MuRF1–3′UTR. HEK293 cells were transfected with the MuRF1–3′UTR luciferase construct, along with expression plasmids for miR-27a, miR-23a, and miR-23a angagomir. (D) miR-23a inhibits the expression of MuRF1. HEK293 cells were transfected with 0.8 μg pcDNA3.1-MuRF1–3′UTR, along with different amount of miR-23a. Forty-eight h after transfection, cells were collected for the analysis of MuRF1 by immunoblot. (E) Enforced expression of miR-23a reduces the protein levels of endogenous MuRF1. Cardiomyocytes were infected with adenoviruses harboring β-gal or miR-23a at 50 moi. MuRF1 mRNA levels were analyzed 24 h after infection by qRT-PCR (upper panel). MuRF1 protein levels were analyzed 48 h after infection by immunoblot (low panel). (F) MuRF1 target protector attenuates MuRF1 reduction and hypertrophy induced by miR-23a. Cells were infected with adenoviral miR-23a, and then transfected with the target protector (MuRF1-TP^miR23a) or the control (MuRF1-TP^control). Forty-eight h after transfection MuRF1 was analyzed by immunoblot (upper panel), and hypertrophy was assessed by measuring cell surface area (middle panel) and β-MHC (lower panel). *P < 0.05 vs. miR-23a alone. (G) miR-23a does not influence the expression of MuRF1 without 3′UTR. Cells were co-infected with adenoviruses harboring MuRF1 without 3′UTR, β-gal, or miR-23a. Forty-eight hours after infection cells were collected for immunoblot analysis. (H) Enforced expression of MuRF1 inhibits hypertrophy induced by miR-23a. Cardiomyocytes were treated as described for (G). Cell surface area (upper panel) and β-MHC (lower panel) were measured 48 h after infection. *P < 0.05 vs. miR-23a alone. (I) Enforced expression of MuRF1 inhibits hypertrophy induced by Iso or Aldo. Cardiomyocytes were infected with adenoviruses harboring MuRF1 or β-gal, and then treated with 10 μM Iso or 1 μM Aldo. *P < 0.05 vs. Iso alone. ^#P < 0.05 vs. Aldo alone.

miR-23a up-regulation depends on calcineurin and NFATc3. (A) The constitutively active form of calcineurin induces up-regulation of miR-23a. Cardiomyocytes were infected with adenovirus harboring the constitutively active form of calcineurin (caCN) or β-galactosidase (β-gal). miR-23a levels were analyzed by qRT-PCR. *P < 0.05 vs. control. (B) Inhibition of endogenous calcineurin attenuates miR-23a elevation induced by Iso. Cells were pretreated with 200 ng/mL cyclosporin, 25 ng/mL FK506, or adenovirus harboring cain or β-gal, and then treated with 10 μM Iso. miR-23a levels were analyzed by qRT-PCR. *P < 0.05 vs. Iso alone. (C) Knockdown of NFATc3 expression by RNAi. Cells were infected with adenovirus harboring NFATc3 RNAi or its scramble form (NFATc3-S-RNAi). Total NFATc3 and phosphorylated NFATc3 (P-NFATc3) levels were detected by immunoblot. (D and E) Knockdown of NFATc3 attenuates hypertrophy and miR-23a elevation induced by Iso. Cells were treated with RNAi as described for (C) and then exposed to 10 μM Iso. *P < 0.05 vs. Iso alone. (F and G) Knockdown of NFATc3 attenuates hypertrophy and miR-23a elevation induced by calcineurin. Cells were co-infected with adenoviruses harboring NFATc3 RNAi or caCN. *P < 0.05 vs. caCN alone.

miR-23a is able to convey the hypertrophic signal of calcineurin and NFATc3. Cardiomyocytes were infected with adenoviruses harboring the constitutively active NFATc3 (ΔNFATc3), calcineurin (caCN) or β-galactosidase (β-gal), and transfected with miR-23a antagomir or antagomir-NC. Representative photos show sarcomere organization, [Scale bar, 20 μm (A).] β-MHC mRNA levels (upper) and cell surface area measurement (lower) are shown in B. Protein/DNA ratio analysis is shown in (C). The expression levels of miR-23a are shown in (D). *P < 0.05 vs. ΔNFATc3 alone. ^#P < 0.05 vs. caCN alone.

miR-23a knockdown attenuates cardiac hypertrophy in animal model. (A) Iso infusion induces an elevation in miR-23a in hearts. The expression level of miR-23a was determined with qRT-PCR (n = 6). (B–D) Knockdown of miR-23a reduces hypertrophic responses induced by Iso infusion. Quantification of the miR-23a expression (B), *P < 0.05 vs. Iso alone, (n = 6). Heart weight/body weight ratio is shown in (C) (n = 7). Histological sections of hearts (left panels) and cross-sectional areas analyzed by staining with FITC-conjugated wheat germ agglutinin (WGA, right panels) are shown in (D). (Scale bar, 20 μm.) (E) Analysis of the transcripts for β-MHC, ANP, and BNP by qRT-PCR. *P < 0.05 vs. Iso alone. (F) miR-23a knockdown attenuates the reduction in MuRF1 levels induced by Iso infusion. A representative blot shows MuRF1 levels (upper panel). The results were densitometrically scanned. Quantitative analysis of MuRF1 levels is shown in the lower panel. *P < 0.05 vs. Iso alone. (n = 5).