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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 Jul 2;106(29):12103–12108. doi: 10.1073/pnas.0811371106

miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy

Zhiqiang Lin a,1, Iram Murtaza a,1, Kun Wang a, Jianqin Jiao a, Jie Gao a, Pei-Feng Li a,b,2
PMCID: PMC2715539  PMID: 19574461

Abstract

Cardiac hypertrophy is accompanied by maladaptive cardiac remodeling, which leads to heart failure or sudden death. MicroRNAs (miRNAs) are a class of small, noncoding RNAs that mediate posttranscriptional gene silencing. Recent studies show that miRNAs are involved in the pathogenesis of hypertrophy, but their signaling regulations remain to be understood. Here, we report that miR-23a is a pro-hypertrophic miRNA, and its expression is regulated by the transcription factor, nuclear factor of activated T cells (NFATc3). The results showed that miR-23a expression was up-regulated upon treatment with the hypertrophic stimuli including isoproterenol and aldosterone. Knockdown of miR-23a could attenuate hypertrophy, suggesting that miR-23a is able to convey the hypertrophic signal. In exploring the molecular mechanism by which miR-23a is up-regulated, we identified that NFATc3 could directly activate miR-23a expression through the transcriptional machinery. The muscle specific ring finger protein 1, an anti-hypertrophic protein, was identified to be a target of miR-23a. Its translation could be suppressed by miR-23a. Our data provide a model in which the miRNA expression is regulated by the hypertrophic transcriptional factor.

Keywords: calcineurin, microRNA, MuRF1

Cardiac hypertrophy is an early milestone of many heart diseases (1, 2), which is associated with changes in gene expression (3). MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate pathophysiological processes such as cell differentiation, apoptosis, cell proliferation, and organ development (4, 5). Recently, the work on miRNAs renovates our understanding about the regulation of cardiac hypertrophy (6, 7). Functional studies reveal that different miRNAs have distinct effects on cardiac hypertrophy. For example, inhibition of miR-133 causes significant cardiac hypertrophy (8). In contrast, miR-208 is required for cardiomyocyte hypertrophy in response to stress and hypothyroidism (9). Overexpression of miR-195 in mice hearts results in severe cardiac hypertrophy (10). Overexpression of miR-214, miR-24, or miR-23a in the cardiomyocytes also causes significant hypertrophy (10). Thus, it appears that miRNAs play multiple and essential roles in the regulation of cardiac hypertrophy.

The levels of many miRNAs have been demonstrated to be altered in cardiac hypertrophy by a series of high-throughput miRNA microarray analysis (10–12). Nevertheless, the signaling pathways that regulate the expression of miRNAs during cardiac hypertrophy remain largely unknown. It is reported that serum response factor (SRF) can directly bind to the promoter of miR-1–1 and miR-1–2 genes and activate their expression (13). Nuclear factor of activated T cells (NFAT) is a transcription factor. Currently, 5 isoforms of NFAT have been identified (14) of which NFATc3 is well-documented to play a key role in mediating the hypertrophic signal of calcineurin as well as other stimuli (15). It is not yet clear whether NFATc3 can regulate cardiac hypertrophy through targeting miRNAs.

The muscle specific ring finger protein 1 (MuRF1) is an anti-hypertrophic factor. MuRF1 knockout mice demonstrate exaggerated cardiac hypertrophy in response to pressure overload (16). It remains unknown whether MuRF1 is a target of miRNAs in the hypertrophic cascades.

The present work aimed at elucidating whether miRNAs are involved in the hypertrophic pathway regulated by NAFTc3. Our results show that miR-23a is a downstream mediator of NFATc3 in induction of hypertrophy. NFATc3 can directly bind to the promoter region of miR-23a and activate its expression. Consequently, miR-23a may convey the hypertrophic signal by suppressing the translation of MuRF1. These results reveal a model to understanding the molecular regulation of miRNAs in cardiac hypertrophy.

Results

miR-23a But Not miR-27a and miR-24 Participate in Initiating Hypertrophy Induced by Iso and Aldo.

Both Iso and Aldo are able to induce cardiac hypertrophy (17–20). We attempted to understand whether miRNAs participate in conveying their hypertrophic signals. As shown in Fig. 1 A and B, Iso- or Aldo-treated cells displayed an increase in cell surface area. We analyzed the expression levels of miR-23a, miR-27a, and miR-24, which are located in the miR-23a≈27a≈24–2 cluster. Their expression levels were significantly up-regulated upon treatment with Iso (Fig. 1C) or Aldo (Fig. 1D).

Fig. 1.

Fig. 1.

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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.

The elevations of these miRNAs led us to consider whether they all are essential for mediating the hypertrophic effect of Iso or Aldo. To this end, the antagomirs of miR-23a, miR-24, and miR-27a were used to knockdown their expression, respectively. miR-23a knockdown could attenuate Iso-induced hypertrophic responses assessed by the visualization of sarcomere organization (Fig. 1E), cell surface area measurement (Fig. 1F), assessment of the hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) (Fig. 1G), and the protein/DNA ratio analysis (Fig. 1H). A similar result was obtained from the hypertrophic model of Aldo (Fig. S1 A, B, C). Surprisingly, knockdown of miR-24 or miR-27a resulted in no significant alterations in the hypertrophic responses induced by Iso or Aldo (Fig. S1D). We detected whether the administration of the antagomirs indeed could influence the levels of miR-23a, miR-27a, and miR-24. The levels of miR-23a were decreased by the administration of miR-23a antagomirs (Fig. 1I), which was also the case for miR-27a and miR-24 (Fig. S1E). These results indicate that miR-23a is involved in mediating the hypertrophic effects of Iso and Aldo.

miR-23a Up-regulation Is Dependent on Calcineurin-NFATc3 Pathway.

Calcineurin is a mediator of Iso- or Aldo-induced hypertrophy (18, 21). To understand the relationship between calcineurin and miR-23a in the hypertrophic cascades of Iso and Aldo, we tested whether calcineurin is able to influence miR-23a levels. Enforced expression of the constitutively active calcineurin could significantly increase the levels of miR-23a (Fig. 2A). Calcineurin inhibitors includingcyclosporin and FK506 were able to attenuate miR-23a elevation induced by Iso. Furthermore, cain, a specific endogenous suppressor of calcineurin (22), could suppress the expression of miR-23a (Fig. 2B). Cain also inhibited Aldo-induced up-regulation of miR-23a (Fig. S2A). These data suggest that Iso- and Aldo-induced miR-23a up-regulation depends on calcineurin.

Fig. 2.

Fig. 2.

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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.

NFATc3 Is a Downstream Mediator of Calcineurin in Hypertrophy (23, 24).

Calcineurin-dependent miR-23a up-regulation encouraged us to explore the relationship between NFATc3 and miR-23a in the hypertrophic cascades of Iso and Aldo. To this end, NFATc3 RNAi was produced, and it could decrease the expression levels of total NFATc3 and the phosphorylated NFATc3 (Fig. 2C). Knockdown of NFATc3 inhibited the hypertrophic responses (Fig. 2D) and the expression of miR-23a (Fig. 2E) upon treatment with Iso. Knockdown of NFATc3 also could lead to a reduction in the hypertrophic response and the levels of miR-23a upon treatment with Aldo (Fig. S2 B and C). These data demonstrate that NFATc3 controls the expression of miR-23a during cardiomyocyte hypertrophy induced by Iso or Aldo.

To further confirm whether miR-23a is a downstream target of calcineurin-NFATc3 pathway, we tested whether calcineurin regulates miR-23a through NFATc3. The constitutively active calcineurin (caCN) could lead to hypertrophy (Fig. 2F) as well as an elevation of miR-23a (Fig. 2G) in the absence but not presence of NFATc3-RNAi. Taken together, it appears that NFATc3 is required for calcineurin to regulate miR-23a levels.

miR-23a Is a Direct Target of NFATc3.

NFATc3 is a transcription factor (14, 25), whose consensus-binding site is [A/T]GGAAA[A/N][A/T/C]N (26). The involvement of NFATc3 in regulating miR-23a levels led us to consider whether miR-23a is a transcriptional target of NFATc3. To test this hypothesis, we first compared the promoter sequence of the miR-23a≈27a≈24–2 cluster between human and rat using rvista (http://rvista.dcode.org). We observed that there is 1 optimal and conservative NFAT binding site in the promoter region of the miR-23a≈27a≈24–2 cluster (Fig. 3A). Second, we tested whether NFATc3 could directly bind to the promoter region. An association of NFATc3 with the promoter of miR-23a≈27a≈24–2 could be observed (Fig. 3B). Third, we tested whether NFATc3 can influence the promoter activity of miR-23a≈27a≈24–2. A fragment from [minsu]799 to −1 of the rat premiR-23a was produced and fused to a luciferase reporter gene. The transcriptional activity of the miR-23a≈27a≈24–2 promoter was significantly increased in the presence of the constitutively active NFATc3 (ΔNFATc3). Mutations in the NFAT binding site of the promoter abolished its response to ΔNFATc3 (Fig. 3C). Finally, we tested whether endogenous NFATc3 is able to influence the promoter activity of miR-23a≈27a≈24–2. The promoter of miR-23a≈27a≈24–2 was activated upon Iso (Fig. 3D) or Aldo (Fig. 3E) treatment, but its activation was abolished when NFATc3 was knocked down by RNAi. Taken together, these data suggest that miR-23a is a direct transcriptional target of NFATc3.

Fig. 3.

Fig. 3.

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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.

miR-23a Is Able to Convey the Hypertrophic Signal of Calcineurin and NAFT.

We tested whether miR-23a participates in mediating the hypertrophic signal of calcineurin and NFATc3. miR-23a antagomir could abolish the constitutively active calcineurin- and NFATc3-induced cardiomyocyte hypertrophy as revealed by sarcomere organization (Fig. 4A), cells surface and the hypertrophic marker measurement (Fig. 4B), and protein/DNA ratio analysis (Fig. 4C). Administration of miR-23a antagomir led to a reduction in miR-23a levels (Fig. 4D). These results demonstrate that miR-23a is a component of calcineurin-NFATc3 hypertrophic pathway.

Fig. 4.

Fig. 4.

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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.

MuRF1 Is a Target of miR-23a.

To find out the molecular target of miR-23a, we searched the potential targets of miR-23a using the program of target scan (http://www.targetscan.org/). MuRF1 has a conservative miR-23a ‘seed’ sequence in its 3′UTR (Fig. 5A). MuRF1 is an anti-hypertrophic molecule (16, 27), and MuRF1 knockout mice demonstrate exaggerated cardiac hypertrophy in response to pressure overload (16). These lines of evidence encouraged us to test whether MuRF1 is a downstream target of miR-23a in the hypertrophic pathway. We detected the expression levels of MuRF1 in the hypertrophic model of Iso and Aldo. MuRF1 protein levels were decreased upon treatment with Iso or Aldo (Fig. 5B). Subsequently, we analyzed the effect of miR-23a on MuRF1 translation. miR-23a could significantly decrease the luciferase activity of MuRF1–3′UTR. miR-23a antagomir was able to rescue MuRF1–3′UTR luciferase activity (Fig. 5C). To understand whether miR-23a indeed can influence the protein translation of MuRF1, we analyzed the protein levels of MuRF1 in cells co-transfected with the constructs of miR-23a and MuRF1–3′UTR. MuRF1 expression could be suppressed by miR-23a in a dose-dependent manner (Fig. 5D). We then overexpressed miR-23a in the cardiomyocytes to test if endogenous MuRF1 is regulated by miR-23a. MuRF1 protein but not mRNA levels were significantly decreased in the presence of miR-23a (Fig. 5E), suggesting that miR-23a predominantly suppresses MuRF1 translation. To definitively test whether miR-23a targets MuRF1, we used target protector technology (28). Administration of MuRF1 target protector against miR-23a could attenuate the reduction in MuRF1 levels as well as hypertrophic responses induced by miR-23a (Fig. 5F). These data suggest that MuRF1 is a target of miR-23a.

Fig. 5.

Fig. 5.

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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-TPmiR23a) or the control (MuRF1-TPcontrol). 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.

To understand whether MuRF1 plays a functional role in hypertrophic cascades of Iso or Aldo, we tested whether enforced expression of MuRF1 can influence hypertrophy induced by these stimuli. We constructed adenovirus harboring MuRF1 which only contains the protein coding sequence. As shown in Fig. 5G, miR-23a was unable to influence the expression of MuRF1 without 3′UTR. Enforced expression of miR-23a resulted in cardiomyocyte hypertrophy that was attenuated by MuRF1 (Fig. 5H). MuRF1 also was able to inhibit hypertrophy induced by Iso or Aldo (Fig. 5I). These data suggest that miR-23a and MuRF1 are both involved in the regulation of hypertrophy.

To further confirm the involvement of MuRF1 in hypertrophy, we analyzed the protein and ubiquitination levels of troponin I, which has been shown to be degraded by ubiquitin system through MuRF1-dependent manner (29). As shown in Fig. S3, Iso treatment resulted in an elevation in troponin I protein levels (upper panel), and a reduction in troponin I ubiquitination levels (lower panel), that could be attenuated by enforced expression of MuRF1. Thus, it appears that troponin I, a downstream target of MuRF1, is altered in the hypertrophic model of Iso.

miR-23a Is Necessary for the Induction of Hypertrophy In Vivo.

Finally, we tested whether miR-23a is necessary for Iso to induce hypertrophy in the animal model. The expression levels of miR-23a were up-regulated in hearts upon Iso treatment (Fig. 6A). To understand the functional role of miR-23a in vivo, we used its antagomir to test if knockdown of miR-23a can influence the effect of Iso on cardiac hypertrophy. miRNA silence through infusion with minipumps requires the amount of antagomir at the milligram level (8). We tried antagomir at 5 (Fig. S4A), 12.5 (Fig. S4B), or 25 mg/kg/d, and observed that 25 mg/kg/d could effectively reduce miR-23a levels (Fig. 6B). The antagomir could be delivered to the heart as revealed by fluorescent analysis (Fig. S4C). To further understand the delivery of the antagomir, we detected its distribution and effect in the liver because the liver expresses miR-23a under physiological conditions (30). The antagomir could be observed in the liver (Fig. S5A). Concomitantly, the miR-23a levels were reduced in the liver (Fig. S5B). However, the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were not significantly altered (Fig. S5C). miR-23a antagomir inhibited Iso-induced hypertrophy revealed by heart weight/body weight ratio (Fig. 6C), hypertrophic phenotype and cross-sectional areas (Fig. 6D), the expression levels of hypertrophic markers including ANP, BNP, and β-MHC (Fig. 6E), as well as cardiac size and function (Table S1). Finally, we observed that suppression of miR-23a by its antagomir restored the expression of MuRF1 (Fig. 6F). These data suggest that miR-23a is required for Iso to induce cardiac hypertrophy in vivo.

Fig. 6.

Fig. 6.

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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).

Role of miR-23a in Alternative Hypertrophic Pathway of Iso.

Our above results reveal that miR-23a can be a downstream mediator of calcineurin and NFATc3 in conveying the hypertrophic signal, and MuRF1 is a target of miR-23a. We asked whether miR-23a plays a role in other hypertrophic pathways in the Iso model. Protein kinase A (PKA) transgenic mice demonstrate cardiac hypertrophy (31). However, inhibition of PKA does not attenuate hypertrophic responses upon treatment with Iso at a low dose (32). These studies suggest that PKA activation is conditional in the hypertrophic model of Iso. To understand the involvement of PKA in our experimental conditions, we tested whether inhibition of PKA can influence hypertrophy. Administration of H89, a PKA inhibitor, slightly attenuated hypertrophy induced by Iso at 10 μM, but significantly inhibited hypertrophy induced by Iso at 30 μM (Fig. S6A). These results suggest that PKA pathway is significantly activated when Iso is at a high dose. Knockdown of miR-23a (Fig. S6B) or NFATc3 (Fig. S6C), inhibition of calcineurin by cain (Fig. S6D), or enforced expression of MuRF1 (Fig. S6E) could partially attenuate hypertrophic responses upon treatment with 30 μM Iso. These data suggest that miR-23a is not the predominant mediator of PKA pathway.

Discussion

miRNAs remain at a constant level under physiological conditions. However, their levels are altered in response to the pathological insults, thereby leading to the pathogenesis of heart diseases. A key question as to how the expression of miRNAs is regulated in cardiac hypertrophy remains to be answered. The calcineurin-NFATc3 pathway plays an important role in conveying the signals for a variety of hypertrophic stimuli (14, 33, 34). Our present study reveals that miR-23a can mediate the hypertrophic signals. Furthermore, miR-23a is transcriptionally regulated by NFATc3. In addition, MuRF1 is a target of miR-23a in inducing hypertrophy. Taken together, our data provide evidence indicating that the miRNA expression is regulated by the hypertrophic transcription factor, NFATc3.

miRNAs levels are altered in response to hypertrophic stimulation (10, 12). Our present work shows that miR-23a is up-regulated by hypertrophic stimuli. Knockdown of miR-23a is crucial for the formation of cardiomyocyte hypertrophy upon treatment with Iso and Aldo. miR-23a has been found to be up-regulated during cardiac hypertrophy induced by pressure overload, and overexpression of miR-23a is able to initiate hypertrophic responses (10). Thus, it appears that miR-23a indeed is a pro-hypertrophic miRNA, and it widely participates in the regulation of cardiac hypertrophy.

miR-23a, miR-24, and miR-27a are in the same cluster, but the up-regulation of miR-27a and miR-24 occurred later than that of miR-23a (Fig. 1). One explanation for such diversity is that miR-23a is closest to the binding site of NFATc3. Another is that because there are more than 70 nucleotides between miR-23a and miR-24 as well as miR-27a in the rat genome, we cannot exclude the possibility that miR-24 and/or miR-27a are also under the control of other known or unknown transcriptional factors. Surprisingly, miR-23a but not miR-27a and miR-24 is necessary for cardiomyocyte hypertrophy upon treatment with Iso and Aldo. Thus, it is apparent that miR-23a already initiates the hypertrophic program, whereas miR-27a and miR-24 cannot exert a synergistic effect. A dozen of miRNAs including miR-27a and miR-24 have been shown to be up-regulated in heart upon pressure overload (10). Future studies are required to specifically elucidate the role of miR-27a and miR-24 in initiating a hypertrophic program.

To further investigate the function of miR-23a, we searched its targets and found that MuRF1 is its downstream target. MuRF1 is a crucial anti-hypertrophic factor (16, 29, 35). It appears that different miRNAs have distinct mechanisms in regulating hypertrophy. For example, miR-133 inhibits hypertrophy through targeting RhoA and Cdc42 (8). miR-208 initiates cardiomyocyte hypertrophy by regulating triiodothyronine-dependent repression of β-MHC (9). A miRNA may have multiple targets. To understand if miR-23a can influence the mRNA levels of other genes, we performed the transcriptome microarray and found that the expression levels of a variety of genes are altered upon stimulation with miR-23a (Fig. S7). For example, miR-23a can stimulate early growth response 1 (Egr-1) expression, whereas the latter has been shown to be up-regulated in cardiac hypertrophy (36, 37). Pituitary tumor-transforming gene-1 (Pttg1), a molecule in the hypertrophic cascades (38), can be up-regulated by miR-23a. It would be interesting to study how miR-23a directly and indirectly targets these molecules. In addition, we analyzed the potential targets of miR-23a using http://www.targetscan.org. Those factors that may be potentially related to cardiac hypertrophy are listed in Table S2.

Enforced expression of MuRF1 alone does not significantly induce cardiomyocyte atrophy (35). Our present study obtained a similar result. It could be possible that MuRF1 alone is unable to induce cardiomyocyte atrophy, or it may hold true under different conditions.

To understand the expression levels of other microRNAs in response to the treatment with Iso, we performed miRNA microarray analysis. miR-23a levels were up-regulated. However, miR-24 and miR-27a elevation could not be observed, although our data obtained from qRT-PCR in Fig. 1C shows their elevation. One explanation for such a discrepancy can be the sensitivity of qRT-PCR is higher than that of microarray. A variety of other miRNAs was up- or down-regulated (Fig. S8). Their roles in the hypertrophic program of Iso will be investigated in future studies.

Cardiac hypertrophy induced by Iso and Aldo can be controlled by complex molecular mechanisms or signaling pathways. Although the present work shows that miR-23a can be regulated by calcineurin and NFATc3, our results do not exclude the involvements of any other molecules and/or pathways that can regulate miR-23a directly or indirectly dependent on calcineurin and NFATc3. Other calcineurin- and NFATc3-independent pathways in the hypertrophic model of Iso and Aldo as well as their roles in regulating miR-23a remain to be further identified.

In conclusion, our present study demonstrates that miR-23a is transcriptionally regulated by NFATc3. miR-23a can mediate the hypertrophic signal of NFAFc3 as well as its upstream regulator, calcineurin. Furthermore, miR-23a conveys the hypertrophic signal by targeting the anti-hypertrophic protein, MuRF1. Our results may provide important information for further studies to explore the beneficial effect of targeting miRNAs as a biological means for the treatment of maladaptive hypertrophy as well as heart failure.

Materials and Methods

Cardiomyocyte Culture, Cell Surface Area Measurement, and Protein/DNA Ratio Analysis.

Neonatal rat cardiomyocytes were isolated from 1- to 2-day-old Wistar rats. Details are in SI Materials and Methods.

Quantitative Reverse Transcription-PCR (qRT-PCR).

Stem-loop qRT-PCR for mature miRNAs was performed on an Applied Biosystems AB 7000 Real Time PCR system. Total RNA was extracted using TRIzol reagent. RNA was reverse transcribed with reverse transcriptase (ReverTra Ace, Toyobo). Details are in SI Materials and Methods.

Chromatin Immunoprecipitation (ChIP).

The purified DNA was used as template and amplified with the following primer sets: 5′-TTGAGCCACCAGGTGCAACTGT-3′; 5′-GAAAGCCCGAGTAAGCCGAGT-3′. Details are in SI Materials and Methods.

miR-23a Silencing.

Chemically modified antagomir complementary to miR-23a were used to inhibit its expression. The antagomir sequence is 5′-GGAAAUCCCUGGCAAUGUGAU-3′. Chemically modified oligonucleotides 5′-CAGUACUUUUGUGUAGUACAA-3′ were used as a negative control (antagomir-NC). All of the bases were 2′-OMe modified. Antagomir oligonucleotides were synthesized and purified with high-performance liquid chromatography by GenePharma Co. Ltd. Cells were transfected with antagomir or antagomir-NC using Lipofectamin 2000 (Invitrogen).

Plasmids and Adenovirus Constructions, Target Protector Preparations, Promoter Constructions and Luciferase Assay, Immunoprecipitation and Immunoblot Analysis, Microarray Analysis, and Animal Experiments.

Details are in SI Materials and Methods.

Supplementary Material

Supporting Information
supp_106_29_12103__index.html (784B, html)

Acknowledgments.

We thank Qing Xu for her excellent technical assistance in echocardiography. This work was supported by National Natural Science Foundation of China Grants 30730045 and 30871243), and National Basic Research Program of China 973 Program Grant 2007CB512000.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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