Reciprocal Repression Between MicroRNA-133 and Calcineurin Regulates Cardiac Hypertrophy
A Novel Mechanism for Progressive Cardiac Hypertrophy
Cardiac hypertrophy involves a remodeling process of the heart in response to diverse pathological stimuli. Both calcineurin/nuclear factor of activated T cells pathway and microRNA-133 (miR-133) have been shown to play a critical role in cardiac hypertrophy. It has been recognized that the expression and activity of calcineurin increases and miR-133 expression decreases in the hypertrophic heart, and inhibition of calcineurin or increase of miR-133 expression protects against cardiac hypertrophy. Here we tested the interaction between miR-133 and calcineurin in cardiac hypertrophy. Cardiac hypertrophy in vivo and in vitro was induced by transverse aortic constriction and phenylephrine treatment. mRNA levels were measured by using real-time PCR methods. Luciferase assays showed that transfection of miR-133 in HEK293 cells downregulated calcineurin expression, which was reversed by cotransfection with the miR-133–specific 2′-O-methyl antisense inhibitory oligoribonucleotides. These results were confirmed in cultured primary cardiomyocytes. miR-133 expression was downregulated, and calcineurin activity was enhanced in both in vivo and in vitro cardiac hypertrophy models. Treatment of cells and animals with cyclosporin A, an inhibitor of calcineurin, prevented miR-133 downregulation. Moreover, the antisense oligodeoxynucleotides against the catalytic subunits of calcineurin Aβ and the decoy oligodeoxynucleotides targeting nuclear factor of activated T cells transcription factor, a calcineurin downstream effector, increased miR-133 expression in cultured primary cardiomyocytes. Our data show that reciprocal repression between miR-133 and calcineurin regulates cardiac hypertrophy.
MicroRNAs (miRNAs) are small noncoding RNA molecules that silence their cognate target genes by specifically cleaving messenger RNAs or inhibiting their translations.1 MicroRNAs have emerged as important regulators of cardiac hypertrophy, and several miRNAs involved in heart hypertrophy have been identified, that is, miR-133, miR-195, miR-1, miR-208, miR-21, and so forth.2–6 Carè et al2 reported that the expression of microRNA-133 (miR-133) decreased in mouse and human models of cardiac hypertrophy. Overexpression of miR-133 in vitro inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by “decoy” sequences induced hypertrophy. Therefore, changes of miR-133 were considered as the requisites for determining cardiac hypertrophy. Calcineurin is a calcium/calmodulin-activated serine-threonine phosphatase that dephosphorylates the transcription factor, nuclear factor of activated T cells (NFAT), which translocates into the nucleus to bind to DNA and activate hypertrophic response genes. Cardiac-specific activation of calcineurin or its downstream effector NFAT is sufficient to induce cardiac hypertrophy,7,8 and calcineurin protein levels and phosphatase activities increase in hypertrophic hearts.9,10 In light of the antithetical functions of calcineurin and miR-133, we hypothesized that calcineurin is the target of miR-133, and the expression of miR-133 is regulated by the calcineurin/NFAT signaling pathway.
Construction of Luciferase-miRNA Target-Site Fusion Plasmids
To construct reporter vectors bearing miRNA target sites, we synthesized fragments (by Invitrogen) containing the target sites for miR-133 and inserted these fragments into the multiple cloning sites downstream of the luciferase gene (HindIII and SacI sites) in the pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, Inc), as reported previously.11
Synthesis of miRNAs and Anti-miRNA Antisense Inhibitors
Detailed methods are described in the online Data Supplement (please see http://hyper.ahajournals.org).
Luciferase Activity Assay
Detailed methods are described in the online Data Supplement.
Real-Time PCR Analysis
Unless otherwise stated, real-time PCR experiments were repeated 5 times. Detailed methods are described in the online Data Supplement.
Western Blot for Calcineurin and Calcineurin Phosphatase Assay
Detailed methods are described in the online Data Supplement.
Pressure-Overload Cardiac Hypertrophy
Detailed methods are described in the online Data Supplement.
Preparation of Primary Rat Cardiomyocytes
Cardiomyocyte cultures were prepared by dissociation of 1-day–old neonatal rat (Wistar) hearts and were differentially plated to remove fibroblasts.7 To induce the hypertrophic response, phenylephrine was added to cardiomyocyte cultures at 50 μmol/L, the concentration which has been proven to induce stable cardiomyocyte hypertrophy in our preliminary tests and another previous study.12,13 The culture medium containing phenylephrine was changed every 12 hours for a period of 72 hours. Cyclosporin A (CsA) was present at 0.5 μg/mL to inhibit cardiomyocyte hypertrophy.7 Cardiomyocytes were prepared for immunocytochemistry, as described previously.7 Monoclonal antibody against sarcomeric α-actinin (Sigma) was added at dilutions of 1:200. Nuclear staining was performed with 1.3 μmol/L of bisbenzimide (Sigma). The relative surface area of the cell was calculated from the number of pixels by using Image-Pro Plus (version 5.0.1).
Synthesis of NFAT Decoy Oligodeoxynucleotide and Calcineurin Aβ Antisense Oligodeoxynucleotide
NFAT decoy oligodeoxynucleotides (ODNs) are 5′-CGCCCAAAGAGGAAAATTTGTTTC-3′ and 3′-GAAACAAATTTTCCTCTTTGGGCG-5′. Scrambled decoy ODNs are 5′-TAGTTATGCATCACGACCTGAT-3′ and 3′-ATCAGGTCGTGATGCATAACTA-5′. Phosphorothioate-modified ODNs (20 bases in length) were used as antisense ODNs against the catalytic subunits of calcineurin Aβ and scrambled ODNs. Sequence (5′ to 3′) of antisense-Aβ is GCCATGCTGGGCCCGGGGCT; sequence (5′ to 3′) of scrambled-Aβ is GCGCAGTGCGGCGGTCCGTC. These antisense ODNs have been described and evidenced previously.14,15 All of the ODNs were synthesized by Shanghai Sangon Biological Engineering and Technology and Service Co, Ltd.
Transfections of microRNA, 2′-O-methyl antisense oligonucleotides (AMO), the decoy ODN, or antisense ODN were performed by using Lipofectamine 2000 (Invitrogen, Life Technologies), according to the procedure specification.
Data are presented as mean±SEM. Significance was determined by using the Student t test for comparison of 2 groups and 1-way ANOVA for comparison of 2 more groups in SigmaStat Analysis Software. For t test, if normality failed, Mann–Whitney rank-sum test was used. P<0.05 was considered significant.
miR-133 Targets Calcineurin
We compared miR-133 nucleotide sequences with the sequences in the 3′-untranslated region (3′UTR) of calcineurin mRNA in rats and mice and found that the 3′UTR of calcineurin mRNA in the 2 types of species contains sequences that were imprecisely complementary to miR-133. There are ≥6 nucleotides matching the 2 to 10 nucleotides from the 5′ end of miR-133 in mice and ≥6 matching nucleotides in 1 site (site 1) in rats (Figure 1A). Because there are 2 possible binding sites in the 3′UTR of calcineurin mRNA in rats, we synthesized the fragments containing both site 1 and site 2, respectively. We performed luciferase reporter assays in HEK293 cells that do not express miR-133 (miRNA Research Guide, http://www.ambion.com/techlib/guides/Ambion_miRNA_res.pdf) to test whether miR-133 targets calcineurin in rats and mice. Compared with the negative control (NC) using chimeric constructs, transfection of miR-133 with the luciferase reporter gene linked to the wild-type 3′-UTR of calcineurin resulted in a significant decrease (P<0.01) of luciferase activity, and coapplication of miR-133 with its inhibitor, miR-133-specific 2′-O-methyl antisense inhibitory oligoribonucleotide (AMO-133), alleviated (P<0.05) the decrease of luciferase activity, whereas AMO-133 alone or mutant miR-133 had no effect (Figure 1B). These data suggest that calcineurin is the target of miR-133. To identify the importance of site 1 and site 2 in the interaction between miR-133 and calcineurin in rats, we further applied the luciferase reporter gene linked to the wild-type 3′-UTR of calcineurin with site 1 and site 2, respectively. Results showed that the effect of site 1 was equivalent to that of both sites, but site 2 had less effect than both sites and site 1 alone (see Figure S2 in the online Data Supplement), indicating that site 1 was predominant in the 2 sites.
Next, we tested the regulation of calcineurin by miR-133 in cultured primary neonatal rat cardiomyocytes. We examined the reliability of transfection with lipofectamine 2000 reagent. miR-133 level in cultured primary cardiomyocytes transfected with miR-133 increased ≈10-fold compared with the nontransfection group (Figure 2A, left). Mir-1, another skeletal specific microRNA, was the control and showed no difference between miR-133 transfection and nontransfection groups (P>0.05; Figure 2A, left). Because the cultured primary cardiomyocytes express endogenous miR-133, we checked the effects of AMO-133 and NC on the miR-133 level. Results showed that the NC had no effect on the miR-133 level, but AMO-133 decreased the miR-133 level significantly (Figure 2A, right). These data indicated that the experimental conditions were reliable.
In the cultured primary cardiomyocytes without phenylephrine treatment, transfection with miR-133 or cotransfection with miR-133 and AMO-133 did not affect the protein and mRNA level of calcineurin, but transfection of AMO-133 alone significantly increased the protein (Figure 2B) and mRNA level (Figure 2C) of calcineurin and induced cardiomyocyte hypertrophy (Figure 2D; P<0.01). In cultured primary cardiomyocytes treated with phenylephrine (50 μmol/L), transfection of miR-133 significantly decreased the elevated protein and mRNA level of calcineurin induced by phenylephrine (P<0.01), and coapplication of AMO-133 with miR-133 antagonized the effects of miR-133 (P<0.05; Figure 2E and 2⇑F). The changes of cell area were consistent with those of calcineurin protein and mRNA levels (Figure 2G). In addition, we also examined the protein level of calcineurin-B1 (CnB) in cultured primary cardiomyocytes to identify the specificity of miR-133 for calcineurin. In general, the protein phosphatase holoenzyme is composed of a catalytic subunit and regulatory subunit. CnB is a regulatory subunit. There were no statistical differences in the CnB protein levels among the NC, miR-133, miR-133+AMO-133, and AMO-133 groups (Figure S3A). However, compared with NC, phenylephrine treatment significantly increased the CnB protein level, and the increased CnB protein level was not affected by transfection of miR-133, miR-133+AMO-133, or AMO-133 (Figure S3B). These data indicate that miR-133 specifically targets calcineurin.
Calcineurin/NFAT Signaling Regulates miR-133 Expression
Conversely, it is not clear whether calcineurin regulates miR-133 expression. We established a cardiac hypertrophy model in vivo by transverse aortic constriction (TAC) in mice. The left heart ventricular tissues were used for examination of calcineurin protein level, calcineurin activity, and microRNA expression. As shown in Figure 3A and 3B, TAC induced cardiac hypertrophy, which was inhibited by treatment of the calcineurin inhibitor CsA. The protein level of calcineurin increased in the hypertrophic heart, and the increased calcineurin protein level was inhibited by CsA (Figure 3C). P-release represented the calcineurin activity, and the hypertrophic heart showed increased P-release, which was inhibited by CsA (Figure 3D). The expression of modulatory calcineurin interacting protein 1 (MCIP1, also known as regulator of calcineurin 1) was upregulated in response to calcineurin activation16; therefore, we further measured MCIP1 mRNA as a reflection of endogenous calcineurin activity. As shown in Figure 3E, MCIP1 mRNA expression was significantly increased in hypertrophic hearts, and the increased MCIP1 mRNA expression was inhibited by CsA treatment. These results were similar to the previous report.9 As shown in Figure 3F, the cardiac hypertrophy resulted in reduced miR-133 expression, as was reported by Carè et al.2 However, the decreased miR-133 expression was recovered by inhibiting calcineurin with CsA (Figure 3F). At the same time, we examined miR-195, which is another important miRNA involved in cardiac hypertrophy.3 As shown in Figure 3G, cardiac hypertrophy led to the increase in miR-195 expression, which was not affected by calcineurin inhibition, suggesting that there exists a specific correlation between calcineurin and miR-133. We also examined the protein level of CnB and fibrosis in sham and hypertrophic hearts treated with or without CsA. CnB protein level increased in TAC-induced hypertrophic hearts, and the increased CnB protein level was inhibited by CsA treatment (Figure S4). CsA also prevented cardiac fibrosis induced by pressure overload (Figure S5). Next, we confirmed the relationship between calcineurin and miR-133 in cultured primary cardiomyocytes that were treated with phenylephrine to induce hypertrophy in vitro. Phenylephrine induced cultured primary cardiomyocytes hypertrophy, which was inhibited by CsA treatment (Figure 4A and 4B). The protein level (Figure 4C) and activity (Figure 4D and 4⇓E) of calcineurin increased and miR-133 expression (Figure 4F) decreased in phenylephrine-treated cardiomyocytes. CsA inhibited cardiac hypertrophy (Figure 4A and 4⇓B), decreased calcineurin protein level (Figure 4C) and activity (Figure 4D and 4⇓E), and increased miR-133 expression (Figure 4F) in phenylephrine-treated cardiomyocytes.
Although CsA is a specific inhibitor of calcineurin, we further used antisense Aβ ODNs (antisense ODNs against the catalytic subunits of calcineurin Aβ) to test the effect of calcineurin on miR-133 expression in cultured primary cardiomyocytes. Results showed that antisense Aβ increased the expression of miR-133, and scrambled Aβ showed no effects (Figure 5A). NFAT is the immediate downstream effector of calcineurin. We inhibited NFAT by using NFAT-specific decoy ODNs (cis-element double-stranded oligodeoxynucleotides) to confirm that NFAT signaling regulated miR-133 expression. Results showed that NFAT decoy ODNs increased the expression of miR-133, and scrambled ODNs showed no effects (Figure 5B). These data suggest that calcineurin/NFAT signaling regulates miR-133 expression.
A clue to the mechanism of interaction between miR-133 and calcineurin in cardiac hypertrophy comes from the fact that the increased activity and expression of calcineurin were accompanied by the decreased miR-133 expression in the hypertrophic heart, which implies that there existed a specific relationship between calcineurin and miR-133. We found that miR-133 regulated calcineurin expression, whereas calcineurin/NFAT signaling regulated miR-133 expression. On the basis of our results, a model between miR-133 and calcineurin in cardiac hypertrophy was established. As shown in Figure 6, miR-133 or calcineurin regulates its own expression via a positive feedback, and the reciprocal repression between miR-133 and calcineurin regulates cardiac hypertrophy. Cardiac hypertrophy is a progressive process, and the mechanism remains obscure; the present study provides a novel understanding.
MicroRNAs are a class of endogenously expressed, noncoding tiny RNAs of 20 to 23 nucleotides, which are largely conserved in sequences and structures. miR-133 is divided into miR-133a and miR-133b, which are only different in 1 base in the terminal 3′ position. This differential base had no influence on complementary matching to mRNA. miR-133a is also divided into miR-133a1 and miR-133a2, for which the sequences are the same. miR-133a was used in the present study. Empirically, ≥6 nucleotides matching the 2 to 10 nucleotides from the 5′ end of miRNAs indicate that there would be existence of the target regulation by miRNAs.11 We identified target sites for miR-133 in calcineurin on the basis of complementarity, with ≥6 nucleotides matching the 2 to 10 nucleotides from 5′-end of miR-133 in mice and ≥6 matching nucleotides in 1 site (site 1) in rats (Figure 1A). Although there are 2 possible binding sites in the 3′UTR of calcineurin mRNA in rats, site 1 is predominant.
We found that transfection of AMO-133 alone significantly increased the protein and mRNA level of calcineurin and induced hypertrophy in the cultured primary cardiomyocytes without phenylephrine treatment, suggesting that endogenous miR-133 has physiological significance. On the other hand, in the cultured primary cardiomyocytes without phenylephrine treatment, transfection with miR-133 or cotransfection with miR-133 and AMO-133 did not affect the protein and mRNA level of calcineurin, suggesting that the amount of endogenous miR-133 is sufficient to inhibit calcineurin in the normal state; even if the miR-133 level is increased by exogenous transfection, calcineurin cannot be inhibited further.
Most animal miRNAs are imprecisely complementary to their mRNA targets, and they inhibit protein synthesis without interfering with the stability of the mRNA target. miR-133 is imprecisely complementary to calcineurin mRNA (Figure 1A); thus, calcineurin mRNA should not be cleaved and destructed directly. We found that the change of calcineurin mRNA was paralleled with that of calcineurin protein level, which might be attributed to the positive feedback mechanism of calcineurin itself. It has been reported that the calcineurin promoter is specifically activated through a positive feedback mechanism involving calcineurin-induced NFAT in the heart17; therefore, when calcineurin protein level decreases, the mRNA level will decrease correspondingly. For this reason, we speculate that the decrease of calcineurin mRNA level is not as a result of direct destruction induced by miR-133.
Although CsA is a specific inhibitor of calcineurin and has been used to specifically inhibit calcineurin in our and other previous works,7–9 we further used antisense Aβ ODNs and NFAT decoy ODNs to test the effect of the calcineurin/NFAT pathway on miR-133 expression. These 3 approaches, CsA, antisense-Aβ, and NFAT decoy ODNs, confirm that calcineurin/NFAT signaling regulates miR-133 expression. NFAT is a transcriptional factor, and the putative binding site for NFAT is 5′-A/TGGAAANA/T/C-3′.18 We found that the genes of miR-133 in rats and mice are Mib1 and EG622283 (from the genome database at the National Center for Biotechnology Information), respectively; however, the putative binding sites for NFAT could not be found from 700 bp upstream to 300 bp downstream of the transcription start site of these genes (the genome database at the National Center for Biotechnology Information). On the basis of the present knowledge, the ideal explanation for the regulation of transcriptional factors on miRNA expression should be the existence of binding sites for transcriptional factors, but the regulation of miRNA expression is so complicated that multiple factors may be involved in the procession from gene expression to miRNA maturity. Mature miRNAs are initially transcribed as primary miRNAs that require the RNase III enzyme Drosha in the nucleus to be trimmed into precursor miRNAs. The latter are exported by the nuclear export factor exportin 5 to the cytoplasm, where they are cropped to become mature miRNAs by another RNase III enzyme, Dicer.19 From this, it can be seen that it is unreliable to explain the regulation of transcriptional factors on miRNA expression simply by matching the binding sites. The possibility is not excluded that there exits an indirect regulation of miR-133 by calcineurin/NFAT pathway. Presently, the regulation of miRNAs is not understood in depth. We could not elucidate the exact molecular mechanism of NFAT regulating miR-133, which is the limitation of the present study.
Calcineurin/NFAT pathway or miR-133 plays a critical role in cardiac hypertrophy. Here we report that the reciprocal repression between miR-133 and calcineurin regulates cardiac hypertrophy. miR-133 regulates the expression of calcineurin through posttranscriptional repression. Conversely, calcineurin/NFAT signaling also regulates miR-133 expression. Our results indicate that, once calcineurin/NFAT signaling is activated, miR-133 expression will decrease with a loss of repression on calcineurin, and the heart will be progressively hypertrophic. We provide a novel understanding of progressive cardiac hypertrophy.
We gratefully acknowledge Xiao-Bin Luo (Research Center, Montreal Heart Institute, Montreal, Quebec, Canada) for providing sequences of NFAT Decoy ODNs.
Sources of Funding
This work was supported by National Natural Science Foundation of China (30873064), A Foundation for the Author of National Excellent Doctoral Dissertation of PR China (2007B72), and Program for New Century Excellent Talents in University of China.
- Received July 19, 2009.
- Revision received August 7, 2009.
- Accepted January 22, 2010.
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