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Basic Science for Clinicians

MicroRNAs Add a New Dimension toCardiovascular Disease

Eric M. Small, PhD*; Robert J.A. Frost, MD, PhD*; Eric N. Olson, PhD

Cardiovascular disease is the predominant cause of humanmorbidity and mortality in developed countries. Thus,

extraordinary effort has been devoted to determining themolecular and pathophysiological characteristics of the dis-eased heart and vasculature with the goal of developing noveldiagnostic and therapeutic strategies to combat cardiovascu-lar disease. The collective work of multiple research groupshas uncovered a complex transcriptional and posttranscrip-tional regulatory circuit, the integrity of which is essential forthe maintenance of cardiac homeostasis. Mutations in oraberrant expression of various transcriptional and posttran-scriptional regulators have now been correlated with humancardiac disease, and pharmacological modulation of theactivity of these target genes is a major focus of ongoingresearch. Recently, a novel class of small noncoding RNAs,called microRNAs (miRNAs), was identified as importanttranscriptional and posttranscriptional inhibitors of gene ex-pression thought to “fine tune” the translational output oftarget messenger RNAs (mRNAs).1,2 miRNAs are implicatedin the pathogenesis of various cardiovascular diseases andhave become an intriguing target for therapeutic intervention.This review focuses on the basic biology and mechanism ofaction of miRNAs specifically pertaining to cardiovasculardisorders and addresses the potential for miRNAs to betargeted therapeutically in the treatment of cardiovasculardisease.

miRNA Processing and FunctionmiRNAs originate from longer precursor RNAs called pri-mary miRNAs that are regulated by conventional transcrip-tion factors and transcribed by RNA polymerase II. PrimarymiRNAs are hundreds to thousands of nucleotides long andare processed in the nucleus into an �70- to 100-nucleotidehairpin-shaped precursor miRNA by the RNase III enzymeDrosha and the double-stranded RNA binding proteinDGCR8. The precursor miRNA is then transported into thecytoplasm by the nuclear export factor exportin 5 and furtherprocessed into an �19- to 25-nucleotide double-strandedRNA by the RNaseIII enzyme Dicer. This duplex miRNA isthen incorporated into the RNA-induced silencing complex.One strand remains in the RNA-induced silencing complex

and becomes the “mature” miRNA, whereas the other strandis often rapidly degraded and is called the “star” strand(miRNA*). On being loaded into the RNA-induced silencingcomplex, the mature miRNA associates with target mRNAsand acts as a negative regulator of gene expression bypromoting mRNA degradation or inhibiting translation.3

Translational inhibition seems to be the predominant mech-anism in mammals; however, target genes that are stronglydownregulated on the protein level often show a reducedmRNA level,4 suggesting that mRNA destabilization is amajor contributor to gene silencing.

A mature miRNA typically regulates gene expression viaan association with the 3� untranslated region (UTR) of anmRNA with complementary sequence, although emergingevidence suggests that miRNAs may also target 5� UTRs orexons and may potentially even undergo base pairing withregulatory DNA sequences to regulate transcription. OnmiRNA binding to a 3� UTR, the degree of mRNA degrada-tion and/or translational repression is affected by multiplemechanisms, including the overall complementarity betweenthe miRNA and target mRNA, the secondary structure of theadjacent sequences, the distance of the miRNA binding site tothe coding sequence of the mRNA, and the number of targetsites within the 3� UTR.5 Complementarity between nucleo-tides 2 through 8 of the miRNA, called the “seed” region,appears to be essential for 3� UTR identification. Therefore,miRNAs with high sequence homology and identical seedregion are commonly grouped into miRNA families that arelikely to target similar sets of mRNAs.6

Up to 1000 miRNAs are predicted to exist in the humangenome, each of which could potentially target hundreds ofmRNAs. Most 3� UTRs contain potential binding sites for alarge number of individual miRNAs, allowing redundancy orcooperative interactions between various seemingly unrelatedmiRNAs. Furthermore, the targets of many miRNAs canmodulate the expression of additional miRNAs or groups ofmiRNAs, generating positive or negative feedback loops.Finally, miRNA maturation seems to be posttranscriptionallyregulated in a sequence-specific manner,7 potentially explain-ing why genetically clustered and cotranscribed miRNAs areoften expressed at different levels.

From the Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/121/8/1022/DC1.*Drs Small and Frost contributed equally to this work.Correspondence to Dr Eric N. Olson, UT Southwestern Medical Center, 5323 Harry Hines Blvd, NA8.602, Dallas, TX 75390-9148. E-mail

[email protected](Circulation. 2010;121:1022-1032.)© 2010 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.109.889048

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Multiple miRNA target prediction tools are now available(summarized in the Table in the online-only Data Supple-ment). Generally, in silico target prediction algorithms use astandard scheme to identify and rank potential targets.8

Briefly, potential targets are ranked on the basis of thecomplementarity between miRNA and 3� UTR and thedegree of conservation of the miRNA and the 3� UTR targetsequence across species. A particular miRNA target is con-sidered to be more meaningful if the sequence is evolution-arily conserved.

Identification and validation of miRNA targets remain amajor hurdle in the study of miRNA function because manyputative targets display little or no detectable regulation whentested in vitro. This is likely due, at least partially, to therelatively modest effect of any single miRNA on the trans-lational output of the target mRNA. Therefore, many pre-dicted miRNA binding sites are probably not true targets, andexperimental validation is essential for confirming targetgenes.

A surprising number of published miRNA targets do notconform to the traditional rules of target prediction outlinedabove. For example, in several instances, cross-species con-servation is not observed in the target sequence, even betweenrodents and humans. Of course, for a miRNA target to betherapeutically viable, the miRNA target sequence must beconserved in humans, not simply present in the modelorganism studied. Therefore, this review focuses primarily onpublished miRNAs that target 3� UTR binding sites that areconserved in humans.

A key insight into the mechanism of miRNA action hasbeen that a large number of miRNAs apparently targetmultiple functionally related mRNAs. The coordinated regu-lation of multiple steps in a complex physiological process by1 miRNA or a group of similarly expressed miRNAs is animportant characteristic of miRNA biology that lends itselfto therapeutic applications. In contrast to therapeuticallymodulating a single target with a conventional drug,miRNA biology can, in principle, modulate multiple levelsof a pathological process by targeting a single nucleic acidmolecule.

Expression of miRNAs Implicated inCardiovascular Disease

As summarized in Figure 1, miRNA expression profiling hasidentified a subset of miRNAs expressed in the normal heartand modulated during cardiovascular disease. The 18 moststrongly expressed miRNAs and miRNA families account for�90% of all miRNA expressed in the adult mouse heart9

(Figure 1), although the specific degree of expression likelyvaries between species and depends on the precise location ofcardiac tissue harvested. In this respect, it is interesting thatseveral of the most abundant miRNAs in the heart belong tofamilies, with the let-7 family accounting for �14%, themiR-30 family for �5%, and the miR-29 family for �4% ofall miRNAs expressed in the murine heart9 (Figure 1).Surprisingly, nearly all of the 18 most enriched miRNAswithin the heart display altered expression during cardiacdisease, indicating an extremely dynamic regulation of miR-NAs in the adult heart and pointing toward the importance of

miRNAs as modifiers of gene expression programs in car-diovascular disease.

Re-expression of a fetal cardiac gene program is a hallmarkof various models of cardiovascular disease and human heart

Figure 1. Regulated miRNAs in cardiovascular disease. The dis-ease state is listed in the top row; the particular study for each col-umn is stated below. Heat map illustrates level of upregulation ordownregulation based on color scheme in legend. All miRNAs thatwere similarly regulated in at least 2 independent studies are listedon the left of the heat map. The last column lists the most abun-dant cardiac miRNAs in order of expression level in mice, whichmight be slightly different from the expression in humans. miRNAswith the same seed region (nucleotides 2 to 8) were combined intofamilies based on putatively similar functions and on technical diffi-culties in distinguishing very similar miRNAs with microarrays,Northern blotting. or real-time polymerase chain reaction. The fam-ily was labeled as regulated if at least 1 member was changed.The let-7 family includes let-7b, c, d, e, f, g, h, i, j; miR-15 family,miR-15a,b/16/195/424/497; miR-29 family, miR-29a, b, c; miR-30family, miR-30a, b, c, d, e; and miR-17 family, miR-17-5p/20a,b/93/106a, b. TAC indicates thoracic aortic constriction; BZ, borderzone; RM, remote myocardium; HF, heart failure; DCM, dilated car-diomyopathy; ICM, ischemic cardiomyopathy; AS, aortic stenosis;and CAA, carotid artery angioplasty.

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failure. Upregulated genes include the fetal isoform of themyosin heavy chain gene (�MHC) and the atrial and brainnatriuretic factor genes, among others. Recently, it has beensuggested that a fetal miRNA program also exists that isreactivated on cardiac stress and may contribute to thereactivation of the fetal gene mRNA program during cardiacdisease.10

Although some of the miRNAs regulated during cardio-vascular disease are not highly expressed outside the heart, alarge number of such miRNAs are broadly expressedthroughout the body. For example, miR-21, which typicallydisplays dramatic alterations in expression after variouscardiac or vascular stresses, is detectable in nearly everytissue analyzed. Inhibition of miRNAs that are specificallyexpressed, or highly enriched, in the cardiovascular systemmay circumvent side effects resulting from miRNA activityin additional organs.

miRNAs Experimentally Implicated inVarious Cardiovascular Disease Settings

and RegenerationMyocardial RemodelingMyocardial remodeling is typically characterized by cardiomyo-cyte hypertrophy, cardiomyocyte apoptosis, interstitial fibrosis,and aberrant cardiac conduction, which ultimately impair theelectromechanical performance of the myocardium. The fol-lowing sections review the potential roles of miRNAs in eachof these processes (see Figure 2) and suggest potentialtherapeutic implications.

Cardiomyocyte HypertrophyPathological hypertrophy is a maladaptive process that ulti-mately leads to reduced cardiac output and is an independent

risk factor in heart failure.11 Pathological cardiac hypertrophyoccurs primarily on pressure overload resulting from arterialhypertension or stenosis of the aortic valve, as well asinherited mutations in sarcomeric and cytoskeletal proteins.Myocardial infarction (MI) also frequently leads to hypertro-phic growth of the remote myocardium as a means ofcompensating for lost contractile function.

The general importance of miRNAs for the homeostasis ofcardiomyocytes was demonstrated by cardiomyocyte-specificdeletions of Dicer12 and Dgcr8,9 2 essential components ofthe machinery required to generate miRNAs. Embryonicdeletion of Dicer in cardiomyocytes resulted in embryonic orearly postnatal death, depending on the time point of Dicerdeletion.13,14 Early postnatal deletion of Dicer induced fatalarrhythmias, whereas deletion of Dicer in adult mice led tothe development of severe heart failure.15 Perinatal dele-tion of Dgcr8 also resulted in rapid development of severeheart failure and premature death.9 It is interesting tospeculate that the downregulation of Dicer in patients withheart failure might contribute to the disease pathogenesisand progression.13

Several individual miRNAs are transcriptionally regulatedduring cardiac hypertrophy and heart failure (Figure 1). Someof them have been experimentally verified to play importantroles in cardiac development and disease and are reviewedbelow.

miR-1miR-1 is encoded by 2 genes (miR-1-1 and miR-1-2), each ofwhich is coexpressed bicistronically with 1 copy of the 2miR-133a genes. miR-1 expression is restricted to heart andskeletal muscle and is regulated by the transcription factorsserum response factor and myocyte enhancer factor-2

Figure 2. Published target mRNAs and functional role of disease-related miRNAs in cardiomyocytes and fibroblasts. Targets that couldnot be validated in later publications are labeled with a question mark. The arrow adjacent to miRNA illustrates whether the miRNA isupregulated or downregulated in cardiac disease. Green- and purple-shaded regions indicate stimulation or repression of a process incardiac disease, respectively. AP-1 indicates activator protein 1; CDC42, cell division cycle 42; CTGF, connective tissue growth factor;Cx43, connexin 43; Hand2, heart and neural crest derivatives expressed 2; HCN2, hyperpolarization-activated cyclic nucleotide-gatedpotassium channel 2; HIF1�, hypoxia-inducible factor; Hop, homeodomain-only protein; Hsp, heat shock protein; Irx5, Iroquoishomeobox protein 5; Kcnd2, potassium voltage-gated channel subfamily D member 2; Kcnj2, potassium inwardly rectifying channel,subfamily J, member 2; MuRF 1, muscle RING-finger protein 1; PDCD4, programmed cell death 4; PHD2, prolyl hydroxylase 2; PP2A,B56a regulatory subunit of protein phosphatase 2a; PTEN, phosphatase and tensin homolog; RhoA, Ras homolog gene family, memberA; Sirt1, sirtuin; SRF, serum response factor; and Thrap1, thyroid hormone receptor associated protein 1.

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(MEF2)/MyoD, respectively.16 During cardiogenesis, miR-1is believed to control the balance between differentiation andproliferation of cardiac precursor cells by targeting Hand2, atranscription factor that promotes expansion of ventricularcardiomyocytes.16 Overexpression of miR-1 in embryoniccardiomyocytes under the control of the �MHC promoterresulted in thin-walled ventricles and developmental arrest atembryonic day 13.5.16 Homozygous deletion of just 1 miR-1gene (miR-1-2) in mice resulted in late embryonic/postnatalmortality of �50% of the offspring caused mainly byventricular septal defects.14 Some of the survivors diedpostnatally as a result of heart failure, but the majoritysuccumbed to sudden cardiac death caused by arrhythmiaswith no obvious alterations in cardiac morphology. Thissevere phenotype seen by deletion of only 1 miR-1 gene issurprising given that most miRNA knockout mice show onlymild phenotypes under basal conditions. One possible expla-nation might be that miR-1 is by far the strongest expressedmiRNA in the murine heart9,14 (Figure 1).

Besides the crucial role of miR-1 during cardiac develop-ment, it is currently unclear whether miR-1 contributes toadverse remodeling in human heart failure. Some studiessuggest upregulation of miR-1 expression in heart failure,10,17

whereas others report downregulation.18 Ikeda et al19 alsofound miR-1 downregulated in hearts of calcineurin trans-genic mice, a well-established murine heart failure model.Adenoviral overexpression of miR-1 in the heart attenuatedcardiomyocyte hypertrophy most likely via downregulationof calmodulin and MEF2a.19

miR-21miR-21 is the most upregulated miRNA in cardiac disease,although under basal conditions it is only weakly expressed(Figure 1). miR-21 is also highly upregulated in severalcancers and is believed to function as an oncogene byinhibiting apoptosis.20 The role of miR-21 in cardiac diseaseis controversial at present. Some studies find an induction ofcardiomyocyte hypertrophy by miR-21 in vitro21 and indi-rectly in vivo via fibroblasts.22 In contrast, other studiesreport an antihypertrophic effect of miR-21 in isolated car-diomyocytes,23 finding a promotion of cellular outgrowths ofcardiomyocytes,24 a reduction in infarct size by miR-21,25 oran inhibition of H2O2-induced apoptosis of isolated cardio-myocytes.26 The reasons for the discrepancy between thesestudies are unclear; however, miR-21 is expressed predomi-nantly in cardiac fibroblasts, not cardiomyocytes.22 It ispossible that much of the miR-21 enrichment seen duringcardiac disease might be due primarily to an increase incardiac fibroblast cell number, implying a rather limitedphysiological relevance of miR-21, at least in cardiomyo-cytes. This is supported by the fact that cardiomyocyte-specific overexpression of miR-21 in vivo does not evoke anobvious cardiac phenotype.22 miR-21 is broadly expressed inmultiple tissues, including the vasculature, which hampersthe discrimination of primary and secondary cardiac effects inmodels that use ubiquitous knockdown of miR-21 such asintravenous injection of antagomiRs (antisense oligonucleo-tide inhibitors of miRNAs).

miR-23There are 2 miR-23 genes that differ by only 1 nucleotide inthe mature miRNA sequence. Each miR-23 gene is closelyclustered with a miR-24 and a miR-27 gene, suggesting thatthey are transcribed as a common transcript. Accordingly,several groups found that miR-23, miR-24, and miR-27 areall upregulated in heart failure and murine cardiac hypertro-phy (Figure 1). van Rooij et al27 showed that adenoviraloverexpression of miR-23a induces hypertrophy of isolatedcardiomyocytes. Lin et al28 showed recently that miR-23aexpression in cardiomyocytes is regulated by nuclear factorof activated T cells (NFATc3) and that miR-23a promotescardiomyocyte hypertrophy by downregulation of themuscle-specific RING-finger protein 1, an antihypertrophicprotein. Knockdown of miR-23a by injection of a specificantagomiR attenuated isoproterenol-induced cardiachypertrophy.28

miR-133The miR-133 family contains 3 miRNA genes, miR-133a-1,miR-133a-2, and miR-133b, which are each transcribed asbicistronic transcripts together with miR-1-2, miR-1-1, andmiR-206, respectively. The expression of the 2 miR-1/133aclusters is regulated by MEF2 and serum response factor andis restricted to cardiac and skeletal muscle.29 The miR-206/133b cluster is expressed only in skeletal muscle.

Partial knockdown of miR-133 in mice with specificantagomiRs was shown to induce cardiac hypertrophy, sug-gesting that pharmacological elevation of miR-133 expres-sion might prevent cardiac hypertrophy during cardiac dis-ease.30 In contrast, Liu et al31 reported that mice with agenetic deletion of either miR-133a gene were phenotyp-ically normal and showed a normal hypertrophic responseto pressure overload of the left ventricle despite an �50%decrease in miR-133 expression in the heart. Deletion ofboth miR-133a genes resulted in late embryonic or neona-tal lethality because of ventricular-septal defects, accom-panied by abnormalities in cardiomyocyte proliferation,apoptosis, and aberrant expression of smooth muscle genesin the heart.31 About a quarter of the double-knockout micesurvived to adulthood, developed extensive myocardial fibro-sis without evidence of cardiomyocyte hypertrophy, andultimately died of heart failure or sudden death. Cardiac-restricted overexpression of miR-133a under the control ofthe �MHC promoter resulted in embryonic lethality causedby inhibition of cardiomyocyte proliferation.31

Many of the phenotypic abnormalities observed in miR-133a knockout mice such as ectopic expression of smoothmuscle genes and aberrant cardiomyocyte proliferation couldbe ascribed at least partially to inappropriate expression of themiR-133 target genes serum response factor and cyclin D2.31

It is not clear why genetic deletion of miR-133 and knock-down by antagomiRs provoke different effects; however, apartial and transient knockdown with antagomiRs might havedifferent consequences than a complete genetic deletion inwhich the gene is eliminated throughout the whole life.Perhaps long-term genetic deletion allows compensatorymechanisms that do not occur in response to transient miRNAknockdown. Further studies are required to determine



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whether therapeutic modulation of miR-133 expression mightrepresent an interesting target for the treatment of cardiacdisease.

miR-208aThe cardiac-specific miR-208a is encoded by an intron of the�MHC gene. Thyroid hormone (T3) stimulates the expres-sion of �MHC and miR-208a after birth while repressing theexpression of the embryonically predominant �MHC iso-form. Cardiac disease is associated with a reactivation of thefetal gene program with increased expression of �MHC anddecreased expression of �MHC, although a correspondingdecrease in miR-208a levels is not observed in short-termstudies because miR-208a is very stable. The results of vanRooij et al32 demonstrated that miR-208a regulates theinduction of �MHC expression on cardiac stress in adultmice. Callis et al33 further demonstrated that this results in theconcomitant upregulation of miR-208b, which is encoded byan intron of the �MHC gene. Genetic deletion of miR-208a inmice resulted in blunted hypertrophy and reduced cardiacfibrosis in response to thoracic aortic constriction, whereascardiomyocyte-specific overexpression of miR-208a inducedcardiomyocyte hypertrophy.32,33 The regulation of cardio-myocyte growth by miR-208a might be due at least partiallyto the repression of thyroid hormone receptor–associatedprotein 1 and myostatin, 2 negative regulators of musclegrowth and hypertrophy.32,33 Although initially protectiveagainst acute cardiac stress–induced remodeling, long-termdeletion of miR-208a led to a decrease in cardiac contractil-ity,32 possibly resulting from perturbations in the cardiacconduction system causing atrial fibrillation of miR-208aknockout mice.33 The latter seems to be caused by misregu-lation of GATA4, homeodomain-only protein, and connexin40.33 It would be interesting to determine whether partialknockdown of miR-208 might prevent hypertrophy whilecircumventing the side effects of complete miR-208a knock-out, thereby enhancing the potential of miR-208 as a thera-peutic target.

Cardiomyocyte Apoptosis and RegenerationBecause the adult heart has only limited regenerative capac-ities, an excessive loss of cardiomyocytes after myocardialischemia or infarction can significantly decrease cardiacperformance. Some miRNAs seem to play important roles inthe regulation of cardiomyocyte apoptosis in vivo and arediscussed below. MiRNAs are certainly crucial regulators ofcell fate determination and differentiation of stem cells34;however, it is currently unclear whether they play a role in theregeneration of the adult heart.

miR-195miR-195 belongs to a family including miR-15a, miR-15b, miR-16-1, miR-16-2, miR-424, and miR-497 (the miR-15 family).The miR-15 family was consistently found to be stronglyupregulated in cardiac ischemia and heart failure (Figure 1).Cardiomyocyte-specific overexpression of miR-195 resulted incardiac hypertrophy and rapid progression to fatal dilatedcardiomyopathy.27 The exact function of the miR-15 familyin the heart is not clear, but studies in other cell types suggestthat the miR-15 family might induce apoptosis by downregu-

lation of the antiapoptotic factor Bcl-2.20 In this respect,antagomiR-induced knockdown of the miR-15 family mightbe a means to prevent ischemia-induced cardiomyocyteapoptosis.

miR-199aThe miR-199 family contains 3 miRs, miR-199a-1, miR-199a-2,and miR-199b, that are all encoded by the antisense strand of anintron of a dynamin gene (Dnm2, Dnm3, and Dnm1, respec-tively). Furthermore, miR-199a-2 is cotranscribed with miR-214. The transcriptional regulation of miR-199 in the heart isunknown, but Rane et al35 reported recently that miR-199a wasrapidly downregulated in cardiomyocytes on hypoxic condi-tions, most likely via a posttranscriptional mechanism becausethe expression level of the miR-199a precursor was unaf-fected. Downregulation of miR-199a derepressed the expres-sion of hypoxia-inducible factor-1�, the most importanttranscription factor for the induction of gene expression onhypoxia.35 miR-199a downregulation also resulted in thederepression of Sirtuin 1, which was responsible for down-regulation of prolyl hydroxylase 2, the enzyme that hydroxy-lates hypoxia-inducible factor-1� to induce its degradation.35

The results of this study demonstrated that knockdown ofmiR-199a during hypoxia induced apoptosis, whereas knock-down of miR-199a before hypoxia surprisingly mirroredpreconditioning and protected cardiomyocytes against hy-poxic damage.35

miR-320In contrast to the upregulation of the miR-15 family, miR-320expression was decreased on ischemia/reperfusion injury.36

Transgenic mice with cardiac-specific overexpression ofmiR-320 exhibited increased apoptosis and infarct size afterischemia/reperfusion injury. Conversely, administration ofmiR-320 antagomiRs reduced infarct size, probably at leastpartially by derepression of the cardioprotective heat-shockprotein 20.36 It is early to speculate, but targeting miR-320with antagomiRs after MI could be a new treatment option todecrease cardiomyocyte loss.

Cardiac ConductionMembrane excitability, a special characteristic of cardiomyo-cytes, is regulated via ion channels. Specifically, Na�, Ca2�,and K� channels and gap junction proteins such as connexin43 are important regulators of cardiomyocyte polarizationand depolarization during contraction and relaxation, respec-tively. Several miRNAs, including miR-1 and miR-133, arepredicted to target ion channels and might therefore playimportant roles in cardiac conduction and the onset ofarrhythmias during cardiac disease.

Zhao et al14 reported that adult miR-1-2 knockout miceshowed several ECG alterations such as reduced heart rate,shortened PR interval, and widened QRS complexes and diedas a result of cardiac arrhythmia. The ECG alterations werepresumably due at least partially to elevated expression of themiR-1 target Irx5, a transcription factor that regulates theexpression of Kcnd2, a potassium channel important fornormal cardiac repolarization.14 Further, Yang et al37 showedthat miR-1 is upregulated in coronary artery disease. Over-expression of miR-1 exacerbated and antagomiR-induced



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knockdown relieved arrhythmogenesis on MI, most likely viaregulation of connexin 43 and the K� channel Kir2.1 sub-unit.37 Additionally, miR-1 regulated Ca2� signaling viatargeting of the B56a regulatory subunit of the proteinphosphatase PP2A.38 Downregulation of B56a by miR-1resulted in hyperphosphorylation of L-type calcium channelsand ryanodine receptor 2 by disrupting localization of PP2Aactivity to these channels.38 These studies suggest thatmiRNA-1 and potentially other miRNAs might be promisingtargets to treat cardiac arrhythmia.

FibrosisFibroblast activation and proliferation during cardiac diseaselead to inappropriate secretion of extracellular matrix proteinsand concomitant interstitial fibrosis. Fibrosis results in im-paired cardiac contractility and alters the electromechanicalcharacteristics of the myocardium, often leading to arrhyth-mias, an important cause of mortality in heart disease. Theexpression of several miRNAs is altered after MI or otherfibrotic pathologies, some of which have been shown to playboth direct and indirect roles in the regulation of cardiacfibrosis.

As mentioned above, adult miR-133a double-knockoutmice develop severe fibrosis and heart failure.31 In line withthose data, Duisters et al39 showed that miR-133 could targetconnective tissue growth factor. Downregulation of miR-133during cardiac disease might therefore result in increasedexpression and secretion of connective tissue growth factorfrom cardiomyocytes, which consecutively stimulates extra-cellular matrix synthesis in fibroblasts.

The fibroblast-enriched miR-21 is upregulated in failingand hypertrophic myocardium, possibly as a consequence offibroblast proliferation. Thum et al22 demonstrated thatmiR-21 increases fibroblast survival and fibrosis possibly viainhibition of sprouty homolog 1 and consecutive extracellularreceptor kinase–mitogen-activated protein kinase activation.Knockdown of miR-21 with antagomiRs attenuated intersti-tial fibrosis and cardiac remodeling after aortic banding.22

miR-21 is also upregulated in the border zone, a fibroblast-rich region adjacent to the infarct, after MI and was shown topromote MMP-2 expression via repression of phosphataseand tensin homolog.40

van Rooij et al41 found that all members of the miR-29-family downregulated after MI, particularly in the borderzone. miR-29 is predicted to target myriad genes that areinvolved in fibrosis such as collagens, fibrillins, and elastinand is a prime example of the ability to modulate a largeportion of a particular pathology by pharmacologically tar-geting 1 miRNA. Knockdown of miR-29 by intravenousinjection of an antagomiR resulted in the upregulation ofcollagens in the heart, suggesting that miR-29 indeed acts asan inhibitor of cardiac fibrosis.41 In the future, it will beinteresting to determine the effect of miR-29 replacementstrategies on the outcome of fibrotic pathologies such as MI.

miRNAs in the Vascular System andVascular Disease

Vessel injury is characterized by profound phenotypicchanges in molecular and physiological identity; in particular,

vascular smooth muscle cells (VSMCs) undergo a program ofdedifferentiation and become more proliferative and migra-tory after injury. These changes contribute to neointimalthickening during proliferative vascular diseases such asatherosclerosis, hypertension, and restenosis. Expression pro-filing has identified a signature pattern of miRNA expressionin VSMCs and endothelial cells of the major vessels that arespecifically altered in vessel injury (Figure 1).

Restenosis

miR-21Several groups have demonstrated roles for various miRNAsin SMC phenotypic modulation and the response of thevasculature to injury. Antisense-mediated knockdown ofmiR-21, which is normally increased after vessel injury(Figure 1), blunted the formation of a neointimal lesion inresponse to balloon angioplasty of the carotid artery.42 Theresults of Ji et al42 demonstrated that miR-21 promoted SMCproliferation after vessel injury via inhibition of phosphataseand tissue homolog and the subsequent activation of thePI3K/Akt signaling pathway, which is partially blocked onknockdown of miR-21 (Figure 3).

miR-145Recently, the SMC-enriched miR-143/miR-145 gene cluster,which is downregulated in the carotid artery after mechanicalinjury, has been implicated in the regulation of SMC contrac-tility and the stress response to vessel injury. Mice harboringdeletions of miR-143, miR-145, or both reveal a role for thesemiRNAs in the acquisition of a contractile SMC phenotype;vessels of knockout mice are thin and distended, and nullVSMCs appear to have acquired a “synthetic” phenotypebased on reduced actin stress fiber formation and increasedrough endoplasmic reticulum production.43,44 Furthermore,miR-143/miR-145–null mice display decreased vascular toneand a reduction in systolic blood pressure. However, mutantmice were viable and appeared to have functional SMCs,albeit shifted slightly toward the synthetic phenotype. This isin contrast to in vitro analyses that demonstrated a criticalrole for miR-145 in the differentiation of cultured SMCs andembryonic stem cells,43,45 likely via repression of Klf4 andKlf5, transcriptional repressors that have previously beenshown to inhibit a differentiated state.

Adenovirus-mediated overexpression of miR-145 at theonset of carotid artery injury blunted the phenotypic modu-lation of VSMCs and reduced neointima formation in re-sponse to angioplasty.46 Surprisingly, Xin et al43 described adiminution of neointima formation in miR-145–null miceafter ligation of the carotid artery. This disparity mightsuggest that a fully differentiated state at baseline may beessential for appropriate phenotypic modulation of the VSMCstate. It is also possible that the reduced vascular tone inmiR-145–mutant mice impinges on the responsiveness of thevessel wall to vascular injury and that the phenotype reflectsan inability to “sense” the injury as opposed to an inability to“respond” to injury. Finally, miR-143/miR-145 targets adisproportionate number of genes involved in actin cytoskel-etal rearrangements and SMC migration (Figure 3), and it ispossible that deficiencies in stress-induced migration account



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for the protection from neointima formation in mutant mice.43

Additional studies should be undertaken to determine theviability of pharmacological modulation of miR-145 as atherapy for restenosis.

miR-221miR-221, although not VSMC specific, is induced in VSMCson platelet-derived growth factor stimulation.47 Activation ofthe platelet-derived growth factor signaling pathway resultsin the switch of VSMCs from a fully differentiated, contrac-tile state to a less differentiated, synthetic state typified byincreases in proliferation and cell migration, contributing tothe formation of a neointimal lesion after arterial injury.Indeed, miR-221 expression is moderately elevated aftercarotid artery angioplasty (Figure 1).42 In vitro data suggestthat miR-221 contributes to SMC phenotypic switching byaffecting multiple cellular responses: miR-221 targets cKitfor repression, resulting in a decrease in SMC differentiation,and inhibits p27Kip1, thereby increasing SMC prolifera-tion.47 Therefore, knockdown of miR-221 after vessel injurymay block neointima formation. Further examination inanimal models should be forthcoming to ascertain its thera-peutic potential.

AngiogenesisNeoangiogenesis plays an essential role in the process ofcardiac repair after ischemic injuries such as MI by promot-ing vascularization of the infarcted tissue through growth ofcollateral vessels that bypass the infarcted artery. Variousgrowth factors such as vascular endothelial growth factor andfibroblast growth factor are required for proper generation ofblood vessels after MI.

Recently, a role for miRNAs has been suggested in theregulation of angiogenesis. Genetic deletion of Dicer in miceresulted in abnormal blood vessel formation and embryoniclethality between embryonic days 12.5 and 14.5.48 si-RNA–

mediated knockdown of Dicer in endothelial cells resulted ina failure of capillary sprouting and tube formation.49,50

Individual miRNAs have since been shown to play positiveand negative regulatory roles in angiogenesis, as presented inFigure 3 and discussed below.

miR-221 and miR-222Studies using human umbilical vein endothelial cells demon-strated that miR-221 and miR-222 regulate angiogenesis inresponse to stem cell factor, presumably by directly repress-ing the levels of cKit and attenuating cell survival, migration,and endothelial tube formation.51 Although it was not clearfrom this study whether the expression of miR-221/miR-222is altered during the physiological process of capillary for-mation in response to stem cell factor, it is clear fromoverexpression studies that modulation of miR-221/miR-222affects endothelial tube formation and represents a potentialavenue for therapeutic modulation of angiogenesis.

miR-210miR-210 expression is elevated during hypoxic conditionsand has been shown to possess proangiogenic properties invitro.52,53 Overexpression of miR-210 in endothelial cellsresulted in accelerated tube formation under normoxic con-ditions and enhanced vascular endothelial growth factor–dependent migration. Conversely, knockdown of miR-210blocked capillary formation in response to hypoxia. Thisstudy demonstrated ephrin-A3 as an important target ofmiR-210, the inhibition of which is a major contributor tomiR-210–mediated cell survival, migration, and tube forma-tion in response to hypoxia (Figure 3). A recent studysuggests that cytoprotection afforded by cardiac ischemia/reperfusion may be partially mediated by the induction ofmiR-210, which promoted mesenchymal stem cell survivalby blocking caspase-8–associated protein-2.54 The results ofthese studies suggest that miR-210 should be further exam-

Figure 3. Published target mRNAs and functional role of disease-related miRNAs in vascular remodeling and angiogenesis. Spindle-shaped yellow cells represent VSMCs, and cuboidal gray cells represent endothelial cells (ECs). Purple- and green-shaded regions indi-cate miRNAs with “proangiogenic” and “antiangiogenic” characteristics, respectively. ACE indicates angiotensin-converting enzyme;SuFu, suppressor of fused; Shh, sonic hedgehog; HGS, hepatocyte growth factor–regulated tyrosine kinase substrate; IGF, insulin-likegrowth factor; ITGA5, integrin-�5; PIK3R2, phosphoinositol-3 kinase regulatory subunit 2 (p85�); PTEN, phosphatase and tensinhomolog; Spred-1, sprouty-related EVH domain-containing protein-1; and VCAM-1, vascular cell adhesion molecule.



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ined for a potential cardioprotective role after ischemicinjury.

miR-126miR-126 has been implicated in the maintenance of vascularintegrity and promotion of vessel growth as a proangiogenicfactor both in vitro and in vivo.55,56 Genetic deletion ofmiR-126 resulted in profound vascular defects, phenotypesthat were previously ascribed to the host gene Egfl7, becauseof inadvertent deletion of miR-126 in the Egfl7-null mouse.57

Although a significant fraction of miR-126–null mice diedembryonically because of vessel leakage, those mice thatescaped neonatal lethality were particularly susceptible tovascular rupture after MI as a result of a deficit in neovas-cularization of the infarcted tissue. The proangiogenic effectof miR-126 was attributed, at least in part, to the repression ofSpred-1, an intracellular inhibitor of vascular endothelialgrowth factor– and fibroblast growth factor–mediated angio-genesis and phosphatidylinositol-3-kinase regulatory subunitPIK3R2 (p85�) (Figure 3). Interestingly, miR-126 is alsoexpressed in hematopoietic progenitor cells, a stem cellpopulation that might contribute to post-MI cardiac regener-ation. It is tempting to speculate that miR-126 may alsoinfluence cardiac repair by affecting the homing ability ofcirculating hematopoietic progenitor cells. In addition to adirect role in angiogenesis, miR-126 may play a role in theprocess of leukocyte infiltration and vascular inflammation.Vascular cell adhesion molecule expression is modulated bymiR-126, thus affecting tumor necrosis factor-�–stimulatedleukocyte adherence to endothelial cells and vessel inflam-mation.58 Taken together, these studies suggest that pharma-cological elevation of miR-126 levels may be a viabletherapeutic strategy to enhance neoangiogenesis and cardiacrepair in ischemic myocardium.

miR-17-92 ClusterMembers of the miR-17-92 cluster were demonstrated toeither positively or negatively influence angiogenesis, de-pending on the cellular context. The endothelial cell–enriched miR-92a, which is upregulated after ischemia, is anegative regulator of vessel growth.59 In vivo knockdown ofmiR-92a with antisense oligonucleotides resulted in im-proved recovery from ischemic damage because of acceler-ated vessel growth. The mechanism for the antiangiogenicrole of miR-92a was attributed to repression of the proangio-genic factor integrin �5 (ITGA5; Figure 3). Conversely, othermembers of the miR-17-92 cluster (particularly miR-18 andmiR-19) suppressed antiangiogenic factors in tumor cells andpromoted angiogenesis and the vascularization of tumors invivo.60,61 These studies demonstrated the importance of miR-NAs in the modulation of angiogenesis in a pathologicalsetting and highlighted the potential for pharmacologicalintervention by inhibition of an miRNA with an antisenseapproach.

miRNAs as Therapeutic Targets inCardiovascular Disease

miRNAs are rapidly becoming an intriguing pharmacologicaltarget in the treatment of cardiovascular disease. The devel-opment of antisense oligonucleotide-mediated (antimiR)

knockdown and miRNA-mimic–mediated overexpressiontechniques might soon allow the regulation of any givenmiRNA in cardiovascular disease.

Antisense-Mediated miRNA KnockdownAntimiRs are antisense oligonucleotides with the reversecomplementary sequence of the target miRNA. They can beconjugated to a cholesterol moiety to increase cellular uptakeand often contain a modified sugar backbone that increasesstability. On cellular uptake, antimiRs bind to the maturemiRNA, thereby inhibiting its activity.62 Because miRNAstypically act as repressors of gene expression, inhibition of anmiRNA should result in the derepression of mRNAs that aredirectly targeted by the miRNA. Thus, the primary effect ofan miRNA inhibitor is activation of gene expression.Krutzfeldt et al62 reported the first mammalian antimiRknockdown using cholesterol-conjugated, 2�-O-methyl–modified antimiRs, called antagomiRs, that resulted inderepression of putative target mRNAs. In a later study,they showed that systemic antagomiR delivery via intra-venous injection efficiently reduced the level of a givenmiRNA in multiple tissues over an extended period oftime.63 Several animal model studies have now demon-strated the value of antagomiRs in downregulating theexpression of specific miRNAs in the heart and therebyinfluencing myocardial remodeling.22,28,30,36,41,59

Besides antagomiRs, other antimiRs with different modi-fications are now being developed that display distinctpharmacokinetics and possibly mechanisms of action. Forexample, antimiRs with 2�-O-methoxyethyl phosphorothioatesubstitutions have proven useful for the inhibition of miR-122in the liver.64 A very promising new approach might be theuse of locked nucleic acid chemistry, which has already beendemonstrated in the downregulation of miRNAs in nonhumanprimates65 and is currently being tested in the first humanclinical trial of miRNA inhibition.

miRNA MimicsmiRNA mimics are synthetic RNA duplexes in which 1strand is identical to the mature miRNA sequence (guidestrand) and is designed to “mimic” the function of theendogenous miRNA. The other strand (passenger strand) isoften only partially complementary to the guide strand andtypically linked to cholesterol to enhance cellular uptake. Thedouble-stranded structure is required for efficient recognitionand loading of the guide strand into the RNA-inducedsilencing complex.66 The use of miRNA mimics would beparticularly useful for enhancing the expression of miRNAsthat are downregulated in cardiovascular disease; however,their in vivo efficacy has not yet been experimentally vali-dated in a pathological setting. It is important to note that theuse of miRNA mimics also results in their uptake in tissuesthat do not normally express the miRNA and may result inunanticipated side effects, which could be overcome bytissue-specific targeting strategies.

Intravenously injected antagomiRs and miRNA mimicshave been the primary method of systemic delivery to date;however, they are preferentially targeted to the liver, kidney,and spleen. Therefore, a major challenge will be the devel-



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opment of strategies for the enrichment of antimiRs or miRmimics in the cardiovascular system. One possible approachwould be a conjugation strategy with the nucleic acid linkedto targeting molecules such as peptides, antibodies, or otherbioactive molecules, which may promote homing of theantimiR/miRNA mimic to the heart. Alternatively, the anti-miR/miRNA mimic could be encapsulated into a lipid-basedformulation that enhances cardiac uptake. Finally, the spe-cific application of the antimiR/miR mimic to the heart, eg,into the coronary vessels during cardiac catheterization onMI, might improve cardiac uptake.

There are several difficulties to overcome to promotemiRNAs as a viable therapeutic target. As stated above,results obtained from pharmacological knockdown oroverexpression by administration of antimiRs and miRmimics often differ from those observed with geneticmouse models. Several possibilities might explain thesefindings. First, miRNAs often have hundreds or eventhousands of predicted mRNA targets, but at physiologicalexpression levels, a miRNA most likely targets only a smallfraction of them. Forced overexpression of a miRNA, how-ever, might result in the regulation of physiologically irrele-vant targets. Second, antimiRs similarly may potentially haveconsiderable off-target effects that in some cases might bemore responsible for the observed effects than the actualknockdown of the miRNA. Third, because systemic deliveryof antagomiRs and miRNA mimics affects miRNA expres-sion globally, effects observed in the heart could also besecondary to effects in extracardiac tissues, eg, as a result ofaltered blood pressure or a change in the level of circulatinghormones. Finally, a short-term and partial knockdown withantagomiRs might have different consequences than a com-plete genetic deletion in which the gene is eliminated fromembryogenesis to adulthood; genetic deletion may result inembryonic phenotypes that may complicate the analysis ofadult disease models. Furthermore, compensatory mecha-nisms could be activated in genetic knockout mice thataccount for mild phenotypes compared with transient knock-down. Nevertheless, the most valuable model to study thefunction of a miRNA is genetic deletion, which should specifi-cally derepress only those mRNAs that are physiologicallyrepressed by the miRNA. In summary, it is apparent that miRNAbiology must be examined through the use of a combination ofgenetic models and pharmacological manipulation.

Concluding RemarksThe demonstration that miRNAs play a crucial role incardiovascular disease and can be easily regulated in vitroand in vivo by antimiRs and miR mimics has tremendouslyaccelerated miRNA research and has nourished hopes that thedrugs used and verified in animal models could be used inhumans. Currently, therapeutic applications of miRNA biol-ogy focus only on affecting translational regulation. Besidesthe use of miRNAs as therapeutic targets, they might serve avaluable diagnostic and prognostic function for various car-diovascular pathologies because cardiac damage results indetectable levels of cardiac miRNAs in the blood.67 miRNAsrepresent a relatively young field of basic biological andtranslational research into new and innovative therapeutic

applications. However, the rapid advancement toward viablemiRNA-driven therapeutic options increases the odds that thenext “small RNA step” will be a “giant leap” for the treatmentof cardiovascular disease.

Sources of FundingThis work was supported by grants from the National Institutes ofHealth, the Donald W. Reynolds Clinical Cardiovascular ResearchCenter, the Sandler Foundation for Asthma Research, and the RobertA. Welch Foundation (Dr Olson).

DisclosuresDr Olson is cofounder of Miragen Therapeutics. The other authorsreport no conflicts.

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KEY WORDS: antagomiR � cardiovascular diseases � microRNAsoligonucleotides, antisense � remodeling � RNA



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Eric M. Small, Robert J.A. Frost and Eric N. OlsonMicroRNAs Add a New Dimension to Cardiovascular Disease

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Supplemental Table 1. MicroRNA target prediction resources

Algorithm Web link Target criteria Reference TargetScan http://www.targetscan.org/ Short seed;

conservation 1

DIANA-microT http://diana.cslab.ece.ntua.gr/microT/ Conserved and non-conserved 3’

complimentarity; Links to potential

functional pathway

2, 3

miRanda http://www.microrna.org/ Thermodynamic stability;

conservation

4

PicTar http://www.pictar.org/ Conservation strongly weighted

5

MicroInspector http://bioinfo.uni-plovdiv.bg/microinspector/

Allows for user inputs of UTR sequence and

thermodynamic cutoffs

6

Rna22 http://cbcsrv.watson.ibm.com/rna22.html No conservation analysis

7

miTarget http://cbit.snu.ac.kr/~miTarget/ Weights position 4, 5, and 6 in seed. SVM classifier

8

PITA http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html

Scans user defined miRNA or 3’UTR

for target sites

9

miRTarget2 http://mirdb.org/miRDB/index.html Target prediction by SVM learning

machine

10, 11

miRecords http://mirecords.umn.edu/miRecords/ Convenient integration of major

target prediction programs. Includes

validated targets.

12

Supplemental References

1. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by

adenosines, indicates that thousands of human genes are microRNA targets. Cell.

2005;120:15-20.

2. Maragkakis M, Alexiou P, Papadopoulos GL, Reczko M, Dalamagas T,

Giannopoulos G, Goumas G, Koukis E, Kourtis K, Simossis VA, Sethupathy P,

Vergoulis T, Koziris N, Sellis T, Tsanakas P, Hatzigeorgiou AG. Accurate

microRNA target prediction correlates with protein repression levels. BMC

Bioinformatics. 2009;10:295.

3. Maragkakis M, Reczko M, Simossis VA, Alexiou P, Papadopoulos GL,

Dalamagas T, Giannopoulos G, Goumas G, Koukis E, Kourtis K, Vergoulis T,

Koziris N, Sellis T, Tsanakas P, Hatzigeorgiou AG. DIANA-microT web server:

elucidating microRNA functions through target prediction. Nucleic Acids Res.

2009;37:W273-276.

4. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human

MicroRNA targets. PLoS Biol. 2004;2:e363.

5. Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da

Piedade I, Gunsalus KC, Stoffel M, Rajewsky N. Combinatorial microRNA target

predictions. Nat Genet. 2005;37:495-500.

6. Rusinov V, Baev V, Minkov IN, Tabler M. MicroInspector: a web tool for

detection of miRNA binding sites in an RNA sequence. Nucleic Acids Res.

2005;33:W696-700.

7. Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, Lim B,

Rigoutsos I. A pattern-based method for the identification of MicroRNA binding

sites and their corresponding heteroduplexes. Cell. 2006;126:1203-1217.

8. Kim SK, Nam JW, Rhee JK, Lee WJ, Zhang BT. miTarget: microRNA target

gene prediction using a support vector machine. BMC Bioinformatics.

2006;7:411.

9. Kertesz M, Iovino N, Unnerstall U, Gaul U, Segal E. The role of site accessibility

in microRNA target recognition. Nat Genet. 2007;39:1278-1284.

10. Wang X, El Naqa IM. Prediction of both conserved and nonconserved microRNA

targets in animals. Bioinformatics. 2008;24:325-332.

11. Wang X. miRDB: a microRNA target prediction and functional annotation

database with a wiki interface. RNA. 2008;14:1012-1017.

12. Xiao F, Zuo Z, Cai G, Kang S, Gao X, Li T. miRecords: an integrated resource

for microRNA-target interactions. Nucleic Acids Res. 2009;37:D105-110.

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