MicroRNA-133 controls cardiac hypertrophy

© 2007 Nature Publishing Group http://www.nature.com/naturemedicine

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MicroRNA-133 controls cardiac hypertrophy Alessandra Care`1,11, Daniele Catalucci2,3,11, Federica Felicetti1, De´sire´e Bonci1, Antonio Addario1, Paolo Gallo3,4, Marie-Louise Bang2,3, Patrizia Segnalini1, Yusu Gu2, Nancy D Dalton2, Leonardo Elia2, Michael V G Latronico3,4, Morten Høydal5, Camillo Autore6, Matteo A Russo7, Gerald W Dorn II8, Øyvind Ellingsen5, Pilar Ruiz-Lozano9, Kirk L Peterson2, Carlo M Croce10, Cesare Peschle1,11 & Gianluigi Condorelli2,3,11 Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by ‘decoy’ sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease. MicroRNAs (miRNAs) are small conserved RNA molecules of B22 nucleotides1 which negatively modulate gene expression in animals and plants, primarily through base paring to the 3¢ untranslated region (UTR) of target mRNAs; this leads to mRNA cleavage and/or translation repression1. MiRNAs are involved in a variety of basic biological processes, for example, cell proliferation and apoptosis2–4 and stress responses5. In fact, bioinformatic analysis predicts that each miRNA may regulate hundreds of targets, suggesting that miRNAs may play a role in almost every biological pathway6. Furthermore, miRNAs are implicated in cancer, where they can act as tumor suppressors or oncogenes7. Our studies focused on the possible functional role of miRNAs in cardiac hypertrophy. Cardiac myocytes respond to stress by under-

going hypertrophy, which is mediated by extracellular stimuli, including cytokines or pressure overload that activate diverse signal transduction pathways8. These in turn induce a reprogramming of cardiac gene expression and the activation of ‘fetal’ cardiac genes8. A few miRNAs, particularly miR-133 and miR-1, which are included in the same bicistronic unit, are specifically expressed in skeletal muscle and cardiac myocytes (refs. 9,10). Notably, miR-133 and miR-1 play key roles in skeletal myoblast proliferation and differentiation, respectively9. Here we investigated the expression and functional role of miR-133 in cardiac myocyte hypertrophy. Some expression and functional studies also examined miR-1. We initially explored the expression profile of miR-133 and miR-1 in different tissues (Supplementary Fig. 1 online). Microarray and northern blot analyses revealed that both miR-133 and miR-1 are expressed only in the heart and skeletal muscle of human embryos and adults. We found a similar expression pattern in mice. Furthermore, microarray analysis revealed increased miR-133 expression in developing mouse embryos from embryonic day (E) 12 through at least E18. In situ hybridization analysis confirmed that miR-133 is selectively expressed in embryonic heart and skeletal muscle, whereas it is virtually absent from other tissues. We then assessed miR-133 and miR-1 expression levels in three murine models of cardiac hypertrophy: transverse aortic arch– constricted (TAC) mice, transgenic (Tg) mice with selective cardiac overexpression of a constitutively active mutant of the Akt kinase11 (hemodynamic data in Supplementary Table 1 online), and exercised rats. In the first model, multiple signal transduction pathways are induced simultaneously by pressure overload, leading to cardiac myocyte hypertrophy12. In the second, hypertrophy is mediated by the downstream effects of Akt on mRNA translation and gene expression11. In the third, exercised rats are analyzed as a model of adaptive cardiac hypertrophy, in which the signaling cascade, activated by insulin growth factor (IGF)-1 and/or insulin and including phosphatidyl-inositol 3-kinase (PI-3K) and Akt, plays a key role in

1Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore Sanita`, 00161 Rome, Italy. 2Department of Medicine, Division of Cardiology, University of California San Diego, La Jolla, California 92093, USA. 3Istituto di Ricovero e Cura a Carattere Scientifico Multimedica, 20099 Milan, Italy. 4San Raffaele Biomedical Science Park, 00128 Rome, Italy. 5The Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway. 6II Faculty of Medicine, University ‘La Sapienza’, 00161 Rome, Italy. 7Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele Pisana, 00163 Rome, Italy. 8Division of Cardiology, University of Cincinnati, Cincinnati, Ohio 45267, USA. 9The Burnham Institute for Medical Research, La Jolla, California 92037, USA. 10Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA. 11These authors contributed equally to this work. Correspondence should be addressed to C.P. ([email protected]) or G.C. ([email protected]).

Received 26 January; accepted 27 March; published online 22 April 2007; doi:10.1038/nm1582

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Figure 1 miR-133 expression in cardiac hypertrophy. (a) Northern blot analysis and relative expression values obtained by densitometric analysis of miR-133 expression in sham-operated, transverse aortic arch–constricted (TAC) and Akt transgenic mice (representative results, mean ± s.d. of a minimum of n ¼ 5 mice per group). (b) As in a, but for sedentary versus exercised rats. n ¼ 5 rats per group. (c) As in a, but for humans with myocardial diseases (hypertrophic cardiomyopathy or atrial dilatation). Expression in ventricles and in atria was measured in patients with hypertrophic cardiomyopathy (n ¼ 4 for both disease and control samples) and atrial dilatation (n ¼ 3 for both disease and control samples), respectively. N, control samples; D, disease samples. *P o 0.05, **P o 0.01. (In a, P-values indicate comparisons to sham.)

cardiac remodeling13. As shown by northern blot analysis, cardiac hypertrophy in all three models resulted in reduced expression levels of both miR-133 and miR-1 (Fig. 1a,b and Supplementary Fig. 2 online). The decrease was particularly prominent in the left ventricle and atria. In the right ventricle, both miR-133 and miR-1 expression were significantly (P o 0.05) reduced in Akt Tg mice, whereas there was no decrease in the TAC model, which is not associated with right ventricular hypertrophy (Supplementary Table 1). To determine whether miR-133 and miR-1 expression is also modulated in human heart disease, we conducted two northern blot analyses: in atria from patients with mitral stenosis and dilated atria versus those from control patients with normal atria; and in myectomies of the interventricular septum from young patients undergoing curative surgery for hypertrophic cardiomyopathy versus those from control patients with valvular defects but with normal wall thickness. We found that miR-133 expression in diseased ventricles and atria was reduced by 50% compared to that in controls (Fig. 1c). We observed a similar expression pattern for miR-1 (Supplementary Fig. 2). Taken together, the murine and human data indicate an inverse correlation between miR-133 and miR-1 expression and myocardial hypertrophy. We then performed a series of functional studies both in vitro and in vivo to determine the roles of miR-133 and miR-1 in cardiac hypertrophy. We first examined miR-133 overexpression in vitro by infecting neonatal mouse cardiac myocytes with an adenoviral vector containing an expression cassette of 683 base pairs (bp) from the mouse miR-133a-2 precursor sequence (Ad133). This precursor is processed into the mature miRNA (data not shown). To determine the effect of miR-133 overexpression on cardiac myocyte hypertrophy, we induced hypertrophy by phenylephrine or endothelin-1 treatment (ref. 8 and Fig. 2a,b). Infection with Ad133 inhibited the hallmark parameters of in vitro agonist–induced hypertrophy: these parameters include increased cell size; enhanced protein synthesis, as measured by 3[H]leucine incorporation; upregulation of fetal genes, including those encoding atrial natriuretic factor (Nppa), skeletal muscle and cardiac a-actin (Acta1 and Actc1, respectively) and a- and b-myosin heavy chain (Myh6 and Myh7, respectively); acto-myosin chain rearrangement and subsequent cytoskeletal reorganization; and perinuclear localization of atrial natriuretic factor protein (Fig. 2c,d, Supplementary Fig. 3 online and data not shown). We obtained similar results in

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adult as in neonatal mouse cardiac myocytes infected with Ad133 (Supplementary Fig. 3). After 48 h of phenylephrine treatment (that is, when the increase in cell size reached a plateau), we performed northern blot and qRT-PCR analyses, and found decreased expression of miR-133 in cardiac myocytes (Fig. 2a and data not shown), thus confirming an inverse correlation between miR-133 levels and hypertrophy. To assess the functional consequences of silencing endogenous miR-133 in vitro, we used neonatal mouse cardiac myocytes infected with an adenoviral vector in which a 3¢ UTR with tandem sequences complementary to mouse miR-133 is linked to the enhanced green fluorescent protein (EGFP) reporter gene. The complementary sequences act as a decoy, sequestering endogenous miR-133. In cells infected with this decoy adenovirus (AdDecoy), phenylephrineinduced hypertrophy was associated with a marked increase in EGFP expression, compared to that in unstimulated AdDecoy-infected cells (data not shown). This result indicates that hypertrophic stimuli promote a reduction in miR-133 expression, thus reducing its binding to decoy sequences in the 3’ UTR and enabling EGFP mRNA translation. In functional assays, AdDecoy infection of neonatal murine cardiac myocytes caused marked hypertrophy, while suppressing miR-133 expression (Fig. 2). Notably, the infection induced an increase of 3[H]leucine incorporation significantly higher than that induced by phenylephrine (Fig. 2c). Fetal gene expression, perinuclear localization of atrial natriuretic factor and cell size were also markedly increased by AdDecoy infection (Fig. 2d and data not shown). We obtained similar results in adult as in neonatal mouse cardiac myocytes infected with AdDecoy (Supplementary Fig. 3). To analyze the effects of miR-1 overexpression in vitro, we used cardiac myocytes infected with an adenovirus vector expressing a miR-1 transgene (Ad1). Notably, miR-1 overexpression negatively affected protein synthesis, as measured by 3[H]leucine incorporation, as well as the expression of selected fetal genes. In a representative experiment, mean 3[H]leucine incorporation, relative to that in the control, was 46% with Ad1 and 39% with Ad133. Expression of a- and b-myosin heavy chain was reduced by Ad1 treatment, to 62% and 54%, respectively, of that in the control. These findings raise the possibility that miR-1 and miR-133 cooperate in the development of cardiac hypertrophy.

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Figure 2 Infection of neonatal cardiac myocytes with Ad133 and AdDecoy. (a) Hypertrophy, evaluated Control Ad AdDecoy as [3H]leucine incorporation in neonatal cardiac myocytes infected with Ad133 or control adenovirus 2 * * (control Ad; multiplicity of infection, 100), and with or without 100 mM phenylephrine (PE) or 100 nM * endothelin-1 (ET1). *P o 0.01 versus control Ad; 1P o 0.01 versus control Ad + PE or control Ad + 1 ET1. Also shown are northern blot analyses of miR-133 in cardiac myocytes after PE stimulation, and after infection with Ad133 or control Ad. Northern blots are representative of at least three experiments. 0 (b) Dot blot analysis performed with total RNA extracted from neonatal cardiac myocytes. The expression Acta1 Actc1 Myh6 Myh7 Nppa level of fetal cardiac genes (Acta1, encoding skeletal a-actin; Actc1, encoding cardiac a-actin; Myh6, encoding myosin heavy chain-a; Myh7, encoding myosin heavy chain-b; Nppa, encoding atrial natriuretic factor), normalized for Gapdh expression, is evaluated as fold induction over that in control Ad cells. *P o 0.01 versus control Ad; DP o 0.05 and 1P o 0.01 versus control Ad + PE. (c) [3H]leucine incorporation in cardiac myocytes. Northern blot analysis of miR-133 is also shown. (d) Dot blot analysis of expression of fetal cardiac genes (see legend to panel b) in AdDecoy-infected cardiac myocytes, evaluated as fold induction over that in control Ad-infected controls. *P o 0.05 and **P o 0.01 versus control Ad; 1P o 0.01 versus control + PE. In each panel, data represent mean ± s.d., with a minimum of 3 experiments.

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histological analysis (Fig. 3a). Furthermore, we noted that cardiac hypertrophy in antagomir-treated mice was associated with a reinduction of fetal gene expression (Fig. 3b). Northern blot analysis revealed that the miR-133 level in antagomir-treated mice was 70% lower than that in controls (Fig. 3c). We confirmed these results in short-term experiments using transcoronary gene delivery15. In a miR-133 gain-of-function model, we tested the effect of Ad133 infection on cardiac hypertrophy in Akt Tg mice (Supplementary Fig. 4 online). At 14 d after Ad133 infection, overexpression of miR-133 resulted in a significant reduction in the size of left ventricular cardiac myocytes and a significant decrease in the expression of fetal genes, except for that encoding skeletal muscle

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To investigate the role of miR-133 in cardiac remodeling in vivo, we implanted mice subcutaneously with osmotic minipumps for a continuous delivery of a chemically modified (ref. 14) antisense RNA oligonucleotide (termed an antagomir) targeted to miR-133 (antagomir-133). Antagomir oligonucleotides can efficiently and stably knockdown specific miRNAs in living cells14. At 1 month after minipump implantation, echocardiographic analysis showed a marked increase in key hypertrophic parameters, such as diastolic left ventricular posterior wall and diastolic interventricular septum thickness, left ventricular mass index and the ratio of left ventricle weight to body weight, in antagomir-treated compared to saline-treated mice (Fig. 3a and Supplementary Table 1). We confirmed this effect by

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Figure 3 In vivo effects of antagomir-133 administration on cardiac hypertrophy. (a) Histological evaluation of hearts from saline- versus antagomir-treated mice. Echocardiographic parameters of cardiac hypertrophy were evaluated (IVSd, diastolic interventricular septum thickness (mm); LVPWd, diastolic left ventricular posterior wall thickness (mm); LVM, left ventricular mass (mg); LVW/BW, ratio of left ventricle weight to body weight). (b) Dot blot analysis of fetal cardiac genes (see legend to Fig. 2b for abbreviations). (c) Northern blot and densitometric analysis of miR-133. n ¼ 8 per group (in all panels). *P o 0.05 versus saline control.

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to Wolf-Hirschhorn syndrome, which is characterized by cardiac dysgenesis as well as several other abnormalities18. Rhoa RhoA Several lines of evidence indicate that Rhoa, Tubulin Gapdh 100 Cdc42 and NELFA/Whsc2 are regulated by * miR-133 in cardiac hypertrophy. In TAC125 125 ** 50 treated and Akt Tg mice, the expression 100 100 level of miR-133 was inversely related to the 75 75 amount of these proteins (Supplementary 0 50 50 Wt Mut Wt Mut Figs. 5 and 6; see also Fig. 1). Furthermore, ** 25 25 Control miRNA + – + – + – + – in both neonatal and adult cardiac myocytes, miR-133 – + – + – + – + 0 0 miR-133 suppression with AdDecoy infection, Site 1 - 3′ UTR Site 2 - 3′ UTR or overexpression following Ad133 infection, caused an increase or a decrease in the level of these three proteins, respectively; mRNA c d levels, however, were not affected (Fig. 4 150 and Supplementary Fig. 6). These data sugCdc42 Cdc42 gest that Rhoa, Cdc42 and Whsc2 mRNAs are Gapdh Actin 100 targeted by miR-133 in cardiac myocytes. To test this possibility, we performed luciferase 125 300 ** reporter assays in HeLa cells, which do not 50 250 100 ** ** express miR-133. We identified at least 200 75 two ‘high score’ seed sequences in the 3¢ 150 0 50 UTR of each of the three candidate target 100 Wt Mut Wt Mut 25 50 genes. Cotransfection of miR-133 with the Control miRNA + – + – + – + – ** 0 0 luciferase reporter gene linked to the miR-133 – + – + – + – + wild-type 3’ UTR of Rhoa, Cdc42 or Whsc2 Site 1 - 3′ UTR Site 2 - 3′ UTR resulted in a significant (P o 0.01) decrease in luciferase activity (Fig. 4 and SupplemenFigure 4 Analysis of the miR-133 target genes Rhoa and Cdc42. (a) Top, representative northern (left) tary Fig. 6). In contrast, cotransfection of and western (right) blot analyses for RhoA after infection of cardiac myocytes with control adenovirus a control miRNA (not complementary to the (control Ad), Ad133 or AdDecoy. Bottom, relative densitometric analyses (mean ± s.d. of a minimum 3¢ UTRs of these three genes) with the wildof n ¼ 5 mice per group). (b) Luciferase reporter assays (mean ± s.d., minimum of 4 experiments type 3¢ UTR constructs did not result in a per group) performed by cotransfection of miR-133 oligonucleotide with a luciferase reporter gene decrease in luciferase activity; similarly, linked to the Rhoa 3¢ UTR, containing either wild-type (Wt) or mutated or deleted (Mut) miR-133 complementary sites; a control nontargeting oligonucleotide (control miRNA) was also included. cotransfection of miR-133 with constructs Mutated 3’ UTR sequences are listed in the Supplementary Methods. (c) As in a, but measuring containing mutated or deleted 3¢ UTR expression of Cdc42. (d) As in b, but using the Cdc42 3¢ UTR. *P o 0.05 and **P o 0.01 versus sequences also did not result in a decrease control Ad (a and c) or control miRNA (b and d). in luciferase activity (Fig. 4 and Supplementary Fig. 6). To test the effects of Whsc2 overexpression on cardiac hypertrophy, actin. In a miR-133 loss-of-function model, we tested the effect of AdDecoy infection on cardiac hypertrophy in wild-type mice. At 14 d we infected cardiac myocytes in vitro with an adenovirus vector after gene transfer, we observed a significant increase in the size of left expressing a Whsc2 transgene (AdWhsc2). Although Whsc2 overventricular cardiac myocytes and upregulation of cardiac hypertrophy expression resulted in reduced cardiomyocyte protein synthesis (data markers, as compared to that in controls infected with the EGFP virus not shown), reactivation of fetal gene expression clearly showed (Supplementary Fig. 4). Notably, in the control group, only B40% of induction of the hypertrophic gene program (Supplementary left ventricle cardiac myocytes were infected (on the basis of EGFP Fig. 6). Whsc2 overexpression also upregulated RhoA protein expresexpression, data not shown), and the miR-133 level was only mildly sion (Supplementary Fig. 6). In addition, we tested the effect of reduced (mean ± s.e.m., 0.69 ± 0.04% of control values). In view of AdWhsc2 infection on cardiac hypertrophy in wild-type mice. At 14 d these findings, the effects seen on hypertrophy may underestimate the after infection, overexpression of Whsc2 resulted in a notable increase consequences of complete suppression of miR-133. Notably, miR-1 in fetal gene expression (Supplementary Fig. 6). Taken together, our studies indicate a key role for miR-133 in the expression was also reduced (0.81 ± 0.07% of control values), regulation of cardiac hypertrophy. First, miR-133 expression was suggesting a link between the two miRNAs. Using a bioinformatic approach, we then searched for candidate inversely related to cardiac hypertrophy in three different murine miR-133 target genes that have been reported to be involved in cardiac models. Second, functional studies performed on neonatal and adult hypertrophy. This analysis led to the identification of Rhoa, Cdc42 and cardiac myocytes in vitro showed that overexpression of miR-133 Whsc2 (human gene: NELFA), whose mRNA 3¢ UTR regions comprise inhibited the increase in cardiac myocyte size and other hallmarks of ‘seed’ sequences and flanking nucleotides matching miR-133 (Fig. 4, hypertrophy; suppression of endogenous miR-133 using a decoy and Supplementary Figs. 5 and 6 online). RhoA and Cdc42 are sequence induced a marked cardiac myocyte hypertrophy in the members of the Rho subfamily (RhoA, Rac1 and Cdc42) of small absence of any hypertrophic stimulus. Third, a single infusion GTP-binding proteins and are involved in cardiac hypertrophy16,17. in vivo of an antagomir oligonucleotide suppressing miR-133 induced NELF-A/Whsc2, a negative regulator of RNA polymerase II, is linked a marked and sustained cardiac hypertrophy. Last, myocardial

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LETTERS miR-133 expression was downregulated in the hearts of patients with hypertrophic cardiomyopathy or atrial dilatation. We identified three targets of miR-133—Rhoa, Cdc42 and NELFA/Whsc2—relevant to cardiac hypertrophy development. RhoA and Cdc42 are associated with cytoskeletal and myofibrillar rearrangements during hypertrophy16,17, whereas NELF-A/Whsc2 is not known to be involved in hypertrophy18. In gene transfer studies, we showed that Whsc2 overexpression upregulates myocardial fetal gene expression but not protein synthesis (Supplementary Fig. 6), suggesting that NELF-A/Whsc2 plays a role in only some aspects of the hypertrophy gene program. Recent studies have suggested that miRNAs may function according to a ‘combinatorial circuitry model’, whereby a single miRNA targets multiple mRNAs and several coexpressed miRNAs may target a single mRNA (refs. 19,20). In line with this model, we identified multiple targets of miR-133 implicated in heart hypertrophy. Although other miRNAs may also be involved13, our results indicate that miR-133 functions as a critical gene for establishing and sustaining the hypertrophy gene program. The expression of miR-133 and miR-1 in the same bicistronic unit raises the possibility that they may cooperate in cardiac hypertrophy. In skeletal myoblast culture, miR-133 and miR-1 promote differentiation and proliferation, respectively9. In cardiac hypertrophy, we found that miR-133 and miR-1 were downregulated in an identical pattern, suggesting that these two miRNAs may functionally interact to regulate the translation of two complementary sets of target mRNAs involved in hypertrophy development. This model is in line with in vitro functional studies on miR-1 reported by others21 and by us in this study. The regulation of miR-133 and miR-1 transcription is also of interest. In mouse skeletal muscle, MyoD and myogenin modulate the transcription of miR-1 and miR-133 according to a regulatory loop22, as suggested for miR-223 and its target, the NFI-A transcription factor23. However, MyoD and myogenin are not expressed in the heart (ref. 24 and data not shown), whereas it has been suggested that serum response factor (SRF) regulates miR-1 expression in transgenic mice25 and Drosophila26. Our studies may have important clinical implications. Notably, in vivo miR-133 levels are downmodulated not only in murine models of cardiac hypertrophy, but also in human disease states associated with myocardial hypertrophy. More importantly, the effects of antagomir-133 on hypertrophy imply that modulation of miR-133 expression by oligonucleotide administration may have future therapeutic application in the clinical setting. METHODS Human tissues and animal experiments. Human embryos and fetuses were obtained by legal abortions at 5–10 weeks after fertilization, according to institutional guidelines (Istituto Superiore di Sanita`); written, informed consent was obtained in advance from the mothers. The age of the embryo was carefully established by morphologic staging according to standard multiple criteria. Different organs were dissected under an inverted microscope and stored under liquid nitrogen. Samples from human hearts were obtained according to institutional regulations (University ‘‘La Sapienza’’); written, informed consent was obtained in advance from the patients. Experiments on mice were performed according to institutional guidelines of the University of California, San Diego, Animal Subjects Committee. Tissues were harvested, frozen, and stored at –80 1C for RNA and protein extraction. Pressure overload cardiac hypertrophy. We used 10- to 12-week old C57BL/6 female mice (Harlan). The pressure overload model was obtained through transverse aortic arch constriction (TAC) under anesthesia, as described27. Akt transgenic mice. Transgenic mice with cardiac-specific overexpression of constitutively active Akt have previously been described11.

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Endurance training. We analyzed a model of adaptive hypertrophy in Sprague Dawley rats. The apparatus and method have been previously described and validated28. Briefly, rats ran uphill on a treadmill for 1.5 h, alternating between 8 min at an exercise intensity corresponding to 85–90% of maximal oxygen uptake (VO2max) and 2 min recovery at 50–60% VO2max. Rats performed this exercise 2 d per week over 8 weeks; controls were age-matched rats that remained sedentary. Isolation, culture and treatment of mouse cardiac myocytes. We isolated and cultured neonatal and adult cardiac myocytes using standard techniques29. We performed adenoviral infection in serum-free medium; 5 h after infection, the medium was replaced and cells further incubated for 48 h (Supplementary Methods online). miR-133 silencing by antagomir treatment. Chemically modified antisense oligonucleotides (antagomir) have been used to inhibit miR-133 expression14. The antagomir sequence complementary to miR-133 is 5¢-P-ACAGCUGGUUG AAGGGGACAA-3¢. The 3¢ end of the oligonucleotides was conjugated to cholesterol; all the bases were 2¢-OMe modified. Antagomir oligonucleotides were deprotected, desalted and purified by high-performance liquid chromatography (HPLC; Dharmacon). C57BL/6female mice (8 weeks old) received antagomirs at doses of 80 mg/kg body weight through Alzet osmotic minipumps (model 1003D, Alza). Minipumps were prepared and placed in a petri dish filled with sterile 0.9% saline at 37 1C, for at least 4 h before implantation, in order to prime the pumps for continuous delivery of the drug. Controls received a saline minipump. Transcoronary gene delivery. Details are in Supplementary Methods. Luciferase assays. We performed luciferase reporter experiments in the HeLa cell line. 3¢ UTR segments of Rhoa, Cdc42 and Whsc2 predicted to interact specifically with miR-133 were subcloned by standard procedures into the pGL3 promoter vector (Promega) immediately downstream of the stop codon of the luciferase gene. Mutagenesis was performed as described by the manufacturer (Invitrogen). We made short constructs (80–100 bases), encompassing wild-type or mutated (six point mutations) seed sequences, in order to separately analyze the functional role of each seed (Supplementary Methods). The seed sequences are indicated in Supplementary Figures 5 and 6. Using Lipofectamine 2000 (Invitrogen), cells were transfected with 0.8 mg of pGL3-3¢ UTR plasmid, 20 pmol of either a stability-enhanced 2¢-O-methyl nontargeting RNA control or miR-133a oligonucleotides (Dharmacon), and an emerald GFP–expressing plasmid (to evaluate the percentage of transfected cells). At 48 h after transfection, cells were lysed and luciferase activity was measured (FemtomasterFB 12, Zylux). Plasmids and vectors. See Supplementary Methods. Accession numbers. GenBank: Rhoa, BC068115; Cdc42, NM_009861; Whsc2, NM_011914. Note: Supplementary information is available on the Nature Medicine website. ACKNOWLEDGMENTS We thank M. Blasi, M. Fontana and V. Michetti for editorial assistance, and G. Loreto for graphics. This work was supported by grants from the US National Institutes of Health (HL078797-01A1 to G.C., HLO65484 to P.R.-L. and 1R01HL63168 to C.P.), the Marie Curie Outgoing Fellowship 6th European Framework Programme (D.C.), EUGeneHeart (LSHM-CT-2005-018833 to G.C.), the Italian Ministry of Scientific Research (G.C. and C.P.), the Italian Ministry of Health (C.P. and G.C.) and the Italy-USA miR Oncology Program, Istituto Superiore di Sanita`, Rome (C.P.). AUTHOR CONTRIBUTIONS D.C., A.C., D.B., F.F., A.A., M.V.G.L., P.S., M.-L.B. and L.E. conducted the in vitro and in vivo experiments. D.C. and P.R-L. performed the assessment of miRNAs in mouse cardiac development. P.G., Y.G., N.D.D., G.W.D., Ø.E. and K.L.P. performed the in vivo models of cardiac hypertrophy. C.A. and M.A.R. collected human samples. C.M.C. conducted the miRNA microarray analysis. A.C., D.C., C.P. and G.C. planned the experiments. D.C., A.C., M.-L.B., P.R.-L., C.P. and G.C.

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LETTERS wrote the manuscript. C.P. and G.C. were responsible for research coordination and strategy. COMPETING INTERESTS STATEMENT The authors declare no competing financial interests.


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