micrornas in cardiac development and regeneration...early cardiac progenitors at e8.5 (embryonic day...

Clinical Science (2013) 125, 151–166 (Printed in Great Britain) doi: 10.1042/CS20130011

microRNAs in cardiac developmentand regenerationEnzo R. PORRELLO

School of Biomedical Sciences, The University of Queensland, St Lucia, Queensland 4072, Australia

AbstractHeart development involves the precise orchestration of gene expression during cardiac differentiation andmorphogenesis by evolutionarily conserved regulatory networks. miRNAs (microRNAs) play important roles in thepost-transcriptional regulation of gene expression, and recent studies have established critical functions for thesetiny RNAs in almost every facet of cardiac development and disease. The realization that miRNAs are amenable totherapeutic manipulation has also generated considerable interest in the potential of miRNA-based drugs for thetreatment of a number of human diseases, including cardiovascular disease. In the present review, I discusswell-established and emerging roles of miRNAs in cardiac development, their relevance to congenital heart diseaseand unresolved questions in the field for future investigation, as well as emerging therapeutic possibilities forcardiac regeneration.

Key words: cardiac regeneration, cardiovascular development, cardiovascular disease, congenital heart disease, microRNA, non-coding RNA

INTRODUCTION

The heart is the first organ to form during mammalian embryogen-esis and its uninterrupted development and function are integralto organismal survival. Heart development begins when a popu-lation of mesodermal stem cells commits to the cardiogenic fateand eventually migrates and fuses to form the linear heart tube [1].Rhythmic contractions begin shortly thereafter. The developingheart subsequently undergoes a series of transformations, includ-ing looping morphogenesis, septation, chamber specification andcardiac valve formation, to form the multi-chambered heart. Awave of cardiomyocyte proliferation subsequently ensures rapidgrowth of the heart prior to birth. Shortly after birth, the majorityof mammalian cardiomyocytes withdraw from the cell cycle andrapid post-natal heart growth is achieved predominantly throughcellular hypertrophy [2].

At the molecular level, complex gene regulatory networksinvolving a number of transcription factors, transcriptional

Abbreviations: AAV9, adeno-associated virus serotype 9; AGTR1, angiotensin II type 1 receptor; Arl2, ADP-ribosylation factor-like 2; ASD, atrial septal defect; AVC, atrioventricularcanal; Cdc42, cell division cycle 42; CHD, congenital heart disease; Chek1, checkpoint kinase 1; DGCR8, DiGeorge syndrome critical region 8; E, embryonic day; ESC, embryonic stemcell; Gata5, GATA-binding protein 5; Hand, heart and neural crest derivatives expressed; hpf, hours post fertilization; I/R, ischaemia/reperfusion; iPSC, induced pluripotent stem cell;lncRNA, long non-coding RNA; MCK, muscle creatine kinase; Mef2, myocyte-enhancer factor 2; miRNA (miR), microRNA; dmiR-1, Drosophila miR-1; αMHC/Myh6, α-myosin heavy chain;βMHC/Myh7, β -myosin heavy chain; myomiR, muscle-specific miRNA; Nkx2.5, Nk2 homeobox 5; P, post-natal day; pre-miRNA, precursor-miRNA; pri-miRNA, primary miRNA; RISC,RNA-induced silencing complex; RISC, RNA-induced silencing complex; Robo, Roundabout; SM22α, smooth muscle cell–specific protein of 22 kDa; SNP, single nucleotidepolymorphism; Sox, SRY (sex determining region Y)-box; SRF, serum-response factor; T3, 3,3′,5-tri-iodothyronine; TAB, thoracic aortic banding; Tbx5, T-box 5; TRH, thyroid hormonereceptor; Thrap1/Med13, TRH-associated protein 1/mediator complex subunit 13; UTR, untranslated region; VSD, ventricular septal defect.

Correspondence: Dr Enzo R. Porrello (email [email protected]).

co-activators and repressors, their corresponding enhancerand promoter elements and chromatin-modifying enzymes co-ordinate heart development. The transcriptional regulation ofheart development is highly conserved across species and het-erozygous mutations in a number of cardiac-expressed transcrip-tion factors underlie several human cardiac malformations [3].The recent identification of profound and unexpected roles fora new class of genes encoding non-protein-coding RNA, knownas miRNAs (microRNAs), has added a new layer of regulatorycomplexity to the gene networks that govern heart developmentand disease. miRNAs can act as context-dependent ‘rheostats’ or‘molecular switches’ of gene expression and are often intertwinedin complex cardiac signalling and transcriptional circuits, wherethey have been found to modulate almost every facet of cardiacbiology [4]. In the present review, I will provide an overviewof recent studies highlighting the important roles of miRNAsin cardiac development, as well as emerging prospects for theirtherapeutic application in the context of regenerative medicine.

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miRNA DISCOVERY, BIOGENESISAND FUNCTION

miRNAs are a class of evolutionarily conserved small (20–26nucleotides in length) non-protein-coding RNAs that negativelyregulate gene expression by affecting mRNA stability and/ortranslation [5]. miRNAs were initially discovered in Caenorhab-ditis elegans in the early 1990s, where the lin-4 gene was foundto encode a small RNA responsible for the regulation of develop-mental timing in nematodes through suppression of LIN-14 pro-tein expression [6,7]. The evolutionary conservation of miRNAsacross all eukaryotes, including vertebrates, was not appreciatedfor almost a decade after their initial discovery in nematodes [8,9].However, it is now recognized that the human genome containsgreater than 1000 miRNAs and it is predicted that more than onethird of all protein-coding genes in the mammalian genome areregulated by at least one miRNA [10]. One of the most excitingrecent developments has been the realization that miRNAs can betherapeutically manipulated, and Santaris Pharma launched thefirst in-human clinical trial of a miRNA therapeutic for hepatitisC virus in 2010, which has since successfully progressed to PhaseIIb [11]. The results of these clinical trials are eagerly awaitedby the miRNA community and may usher in a new era of RNA-based therapeutics for the treatment of a range of human diseaseson the backing of exciting results for many miRNA-based drugsin pre-clinical animal models.

miRNAs are transcribed by RNA polymerase II as long pre-cursor RNAs called pri-miRNAs (primary miRNAs), which aresequentially processed by enzymes in the nucleus and cytoplasmto generate mature miRNAs of ∼22 nucleotides in length. Thepri-miRNAs are cleaved in the nucleus by the RNase III en-donuclease Drosha, which associates with the double-strandedRNA-binding protein DGCR8 (DiGeorge syndrome critical re-gion 8) and other cofactors, to produce ∼70-nucleotide-longpre-miRNAs (precursor-miRNAs) [5]. In some cases, specificmiRNAs, known as mirtrons, can bypass Drosha processing andthe pre-miRNA in this case is generated directly through splicingof intronic sequences [12]. The pre-miRNAs have a character-istic hairpin structure and are exported from the nucleus to thecytoplasm by exportin-5, where Dicer, another RNase III en-donuclease, subsequently cleaves the hairpin to release a ∼22nucleotide miRNA duplex [5]. One strand of the miRNA du-plex becomes the mature miRNA and the other strand is typic-ally, but not always, rapidly degraded [13]. The mature single-stranded miRNA is then loaded into RISC (RNA-induced si-lencing complex), where a multi-protein complex consisting ofArgonaute proteins facilitates interactions between the miRNAand target mRNAs [5]. The miRNA interaction with the targettranscript is mediated by Watson–Crick base pairing with se-quences that are canonically located in the 3′-UTR (untranslatedregion) of target mRNAs, although there is a growing list ofexamples of miRNA interactions with 5′-UTRs, protein-codingsequences and introns [14]. Association of the miRNA with itsmRNA target typically results in mRNA degradation and/or trans-lational inhibition [5], but some rare examples of translationalactivation have also been reported [15]. Current evidence sug-gests that the primary mechanism for miRNA-mediated target

gene repression is via mRNA decay rather than translationalrepression [16].

The primary determinant of miRNA target recognition is thepairing of nucleotides 2–7 at the 5′ end of the mature miRNA(known as the ‘seed’ region) and the target mRNA [5].miRNA ‘families’ are classified on the basis of a common ‘seed’sequence that is shared between all family members and they arebelieved to have largely redundant functions. However, the otherregions of the miRNA are also important for specificity andstabilization of miRNA–mRNA binding [17]. Current estimatesfrom biochemical miRNA target identification studies [e.g. HITS-CLIP (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation)] suggest that a single miRNA caninteract with hundreds of target mRNAs in a cell, although tar-get gene regulation is typically modest with, on average, only20–50 % changes in mRNA expression levels detected for mosttarget transcripts [16,18]. Proteomic analyses similarly indicatethat most targets are modestly repressed (<2-fold) by miRNAs[19,20]. These results indicate that miRNAs probably functionthrough the modest repression of a number of mRNA targets,which act in concert to mediate the biological actions of miRNAs.In addition to miRNA ‘seed’ redundancy, the promiscuity ofmiRNA interactions with target transcripts and the targeting ofindividual transcripts by several different miRNAs [4], as wellas the potential for generation of multiple mature miRNAs fromthe same miRNA precursor (isomirs) [21], allows for enormousregulatory complexity and functional redundancy. Indeed, thereis immense sequence diversity among miRNAs and a large pro-portion of cardiac-expressed miRNAs, including miR-133, haveprevalent 5′ isomirs, which can affect miRNA targeting [21]. Inlight of these findings, it is not entirely surprising that only 10 %of genetic miRNA deletions in C. elegans have revealed grosslyabnormal phenotypes in mutant animals [22]. Similarly, geneticdeletions of miRNAs in mice have, in most cases, failed to revealstriking functions during development or under normal homoeo-static conditions in adulthood [23]. However, major functionsare often unmasked in response to specific injury or stress con-ditions [24]. It is unclear why compensatory mechanisms canovercome the effects of loss-of-function for most of the miRNAstested so far during development, but not in response to specificstress signals. Nevertheless, critical roles for a number of cardiacmiRNAs have emerged and their roles in heart development willbe discussed in the following sections.

miRNAs AND CARDIAC DEVELOPMENT

miRNAs are essential for heart development:lessons from Dicer- and DGCR8-knockout studiesCompelling evidence for a critical role of miRNAs during cardiacdevelopment has been garnered from loss-of-function mutationsof the miRNA-processing enzymes Dicer and Drosha/DGCR8.Although global deletion of miRNA-processing enzymes causeslethality during early gestation [25], conditional knockout ofthese enzymes in specific cell lineages has highlighted essentialroles during heart development. Conditional deletion of Dicer

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microRNAs in cardiac development and regeneration

using Cre recombinase under the control of the endogenousNkx2.5 (Nk2 homeobox 5) regulatory region (Nkx2.5–Cre) ab-olished the processing of pre-miRNAs into their mature form inearly cardiac progenitors at E8.5 (embryonic day 8.5) [26]. Earlydeletion of Dicer in cardiac progenitors led to embryonic leth-ality resulting from cardiac failure at E12.5 [26]. The ventricu-lar myocardium of Nkx2.5–Cre-knockout mice was poorly de-veloped and the heart exhibited signs of pericardial oedema.However, most markers of initial cardiac differentiation and pat-terning, such as Tbx5 (T-box 5), Hand (heart and neural crestderivatives expressed) 1, Hand2 and Mlc2v, were expressed nor-mally [26]. Using a similar approach, but employing an alternat-ive Nkx2.5–Cre line with slightly different spatiotemporal kinet-ics [27], it was demonstrated further that miRNAs are necessaryfor cardiac outflow tract alignment and chamber septation [28].Interestingly, Dicer deletion was associated with a reduced num-ber of apoptotic cells in the outflow tract at E13.5 comparedwith wild-type controls [28]. Although both Nkx2.5–Cre Dicermutant strains died in utero, the extended survival period of thecardiac Dicer-deficient mice generated by Tabin’s group [28] al-lowed for a longer time window for the visualization of the effectsof miRNA on outflow tract morphogenesis and chamber septa-tion. The different phenotypes of these two Nkx2.5–Cre Dicermutants are most likely due to background strain differences anddisparities in the timing of transgene expression between the twoNkx2.5–Cre constructs.

The development of the outflow tract involves the co-ordinatedregulation of cell proliferation, differentiation, migration and ap-optosis of multiple cell types, including those derived from theneural crest. Neural crest cells are required for proper develop-ment of craniofacial structures, as well as development of thegreat arteries and outflow tract septation [29]. Deletion of Dicerfrom the neural crest lineage using Wnt1–Cre causes severe de-fects in craniofacial and cardiovascular structures, including VSD(ventricular septal defect), double outlet right ventricle and type Binterrupted aortic arch [30–32]. Although miRNAs were shownto be dispensable for the survival of cardiac neural crest cells,they were required for migration and patterning of these cellsthroughout the outflow tract [30]. Although the effects of Dicerdeletion in cardiac neural crest were postulated to be due to miR-21- and miR-181a-dependent regulation of ERK (extracellular-signal-regulated kinase) 1/2 signalling [30], it should be notedthat miR-21-knockout mice do not display any overt develop-mental phenotype [33]. Further studies employing combinatorialdeletion of miR-21 and miR-181a in neural crest cells will berequired in order to establish further the relative importance ofthese individual miRNAs in developing neural crest cells.

miRNAs are also essential for the maintenance of post-natal heart function. Deletion of Dicer in cardiomyocytes us-ing an αMHC (α-myosin heavy chain) promoter-driven Cre re-combinase (αMHC–Cre) resulted in the death of mutant micebetween post-natal days 0 and 4 [34]. Post-natal lethality in Dicer-mutant mice was associated with dilated cardiomyopathy andheart failure. Interestingly, αMHC–Cre Dicer-mutant hearts dis-played decreased expression levels of mature miRNAs as early asE14.5, but, in contrast with Nkx2.5–Cre-mutant mice, αMHC–Cre-mutants were born at expected Mendelian frequencies [34].

αMHC–Cre-mediated Dicer deletion did not affect cell num-ber or cardiomyocyte size at P0 (post-natal day 0), but wasassociated with depressed cardiac contractility, which was ac-companied by misexpression of cardiac contractile proteins andprofound sarcomere disarray [34]. Further support for a crit-ical role of miRNAs during neonatal heart development comesfrom genetic deletion of Dgcr8 using a MCK (muscle creatinekinase)-promoter-driven Cre recombinase (MCK–Cre), which isactivated around birth with cardiac transgene activity declining byP10 [35]. Similar to Dicer-mutant hearts, disruption of Dgcr8 inneonatal cardiomyocytes resulted in depressed cardiac contractil-ity and conduction abnormalities with progression to dilated car-diomyopathy [35]. Premature death occurred within 2 monthsafter birth [35]. Collectively, these studies have established thatcanonical miRNA biogenesis is essential for the maintenance ofnormal cardiac function and animal survival during neonatal life.

The miRNA processing machinery has also been disruptedin cardiomyocytes at several different post-natal ages using atamoxifen-inducible αMHC–Cre line. Deletion of Dicer in juven-ile (3-week-old) hearts resulted in mild ventricular remodelling,dramatic atrial enlargement, selective re-activation of fetal car-diac genes and sudden cardiac death within 1 week after the startof tamoxifen treatment [36]. In contrast, cardiac-restricted Dicerdeletion in adult (8-week-old) mice was accompanied by dra-matic ventricular hypertrophy, myofibre disarray, cardiac fibrosisand marked induction of fetal cardiac genes [36]. Similar resultswere obtained in a separate study, which also reported patholo-gical ventricular remodelling and contractile dysfunction follow-ing tamoxifen-inducible Dicer deletion in 6-week-old adult mice[37]. Therefore miRNAs are not only required for normal em-bryonic and neonatal heart development, but are also crucial forcardiac growth and maintenance of cardiac function in juvenileand adult stages.

The development of the vasculature is also under miRNA con-trol. Deletion of Dicer in vascular smooth muscle using SM22α

(smooth muscle cell–specific protein of 22 kDa)–Cre resulted inembryonic lethality around E16–E17 [38]. SM22α–Cre mutantsexhibited thinner blood vessel walls, and reduced smooth musclecell proliferation and differentiation, as well as impaired vas-cular contractility and haemorrhaging. Deletion of Dicer in en-dothelial cells using Tie2 (tunica internal endothelial cell kinase2)–Cre or inducible VE-cadherin–Cre has also established es-sential roles for miRNAs in post-natal angiogenesis [39]. Fur-thermore, Dicer deletion in the developing epicardium usingGata5 (GATA-binding protein 5)–Cre has identified a crucialrole for miRNAs in coronary vessel development [40]. Gata5–Cre-mutant mice die shortly after birth and display defects inepicardial epithelial-to-mesenchymal transition, proliferation anddifferentiation into coronary vascular smooth muscle cells [40].Taken together, these findings have clearly established import-ant roles for Dicer, Drosha/Dgcr8 and, by inference, miRNAs incardiovascular development. However, it is unclear whether thephenotypes resulting from Dicer and Dgcr8 deletion reflectthe critical importance of specific miRNAs in cardiac develop-mental processes or whether they are the consequence of disrup-tion of multiple miRNAs with overlapping roles in multiple es-sential processes. Manipulation of individual miRNAs allows for

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Figure 1 miRNAs regulate multiple stages of cardiac developmentmiRNAs, downstream targets and the developmental processes they regulate are shown. CM, cardiomyocyte; OFT, outflowtract.

Figure 2 Regulation of cardiac regeneration by miRNAsActivation of cardiomyocyte proliferation, stem/progenitor cell differentiation and direct lineage reprogramming representthree approaches towards regeneration of the heart following myocardial infarction. miRNAs that have been implicated inthese processes are shown.

more precise delineation of physiological functions, regulatorycircuits and molecular mechanisms of action. The following sec-tions will discuss the roles of individual miRNAs in cardiomyo-cyte development (Figure 1) and regeneration (Figure 2). Al-though a number of functions for individual miRNAs in vasculardevelopment and pathophysiology have also emerged over recentyears, these findings have been reviewed extensively elsewhere[4,41,42] and will not be a major focus of the present review.

Essential roles of the miR-1/miR-133 bicistroniccluster during heart developmentmiR-1 is the most abundant miRNA in the mammalian heart,accounting for almost 40 % of all miRNAs in the adult mur-ine heart [35]. miR-1 is highly conserved from fruit flies tohumans and it was the first miRNA to be implicated in heartdevelopment. In vertebrates, the genes encoding miR-1 and miR-133 family members are clustered and generated from com-mon bicistronic transcripts. The miR-1-1/miR-133a-2 clusteris located in an intergenic region on human chromosome 20,whereas the miR-1-2/miR-133a-1 cluster is positioned in the an-

tisense orientation within an intron of the Mib1 gene on humanchromosome 18. miR-1 and miR-133a are expressed in bothheart and skeletal muscle, whereas the closely related miR-206/miR-133b cluster is exclusively expressed in skeletal muscle[26,43,44].

The miR-1/miR-133a clusters are embedded within conservedgene regulatory networks involving interactions between a num-ber of transcription factors, as well as upstream and intragenicenhancers. The miR-1/miR-133a clusters are regulated by severalimportant myogenic transcription factors including SRF (serum-response factor), Mef2 (myocyte-enhancer factor 2), MyoD(myogenic differentiation factor D) and Nkx2.5 [26,45,46]. De-letion analysis of miR-1 enhancers in mice revealed that SRF isrequired for cardiac expression of miR-1 [26], whereas Mef2 isnecessary for expression of miR-1 and miR-133a in skeletal mus-culature [26,45]. SRF also regulates cardiac expression of miR-1in Drosophila [47] and Mef2 co-operates with Twist in flies tocontrol the expression of miR-1 in skeletal muscle [48]. Studiesin fruit flies have also uncovered a regulatory loop involving co-operation between miR-1, the Rho-GTPase Cdc42 (cell division



cycle 42) and the Nkx2.5 orthologue tinman [46]. In the fly heart,tinman negatively regulates miR-1 expression and miR-1 itselfdirectly targets Cdc42 for translational repression. The geneticinteraction between Cdc42 and Nkx2.5, as well as the negativeregulation of miR-1 by Nkx2.5, is also conserved in the mouseheart [46]. The highly conserved transcriptional regulation ofmiR-1 and miR-133 by myogenic transcription factors suggeststhat there has been strong evolutionary pressure to maintain ex-pression of these miRNAs during heart development.

Gain- and loss-of-function experiments in the mouse,zebrafish and fruit fly have established critical roles for the miR-1and miR-133 families during heart development. Genetic loss ofmiR-1 in Drosophila (dmiR-1) causes embryonic lethality in thelarval stages after hatching and is associated with a spectrum ofmuscle defects [47,48]. Nearly half of all dmiR-1-mutant fliesdisplayed severe defects in sarcomeric gene expression, indicat-ing a late requirement for miR-1 in muscle differentiation [47].Furthermore, the most severely affected embryos displayed ab-normal cardiac muscle patterning, and cardiac progenitors wereoften arrested in a proliferative state and failed to terminally dif-ferentiate [47]. The Notch ligand Delta, a known regulator ofassymetric cell division of muscle progenitors, was shown to be adirect target of miR-1 in vivo, providing a plausible mechanism bywhich miR-1 influences cardiac progenitor differentiation [47]. Amore recent genome-wide forward genetic screen in Drosophilahas revealed novel signalling pathways and functions for dmiR-1,including a previously unrecognized function for this miRNA inregulating cardiac progenitor cell polarity [49]. The transcriptionfactor kayak, the Drosophila orthologue of mammalian c-Fos,was identified as a direct target of dmiR-1, and c-Fos was sim-ilarly dysregulated upon miR-1 gain- and loss-of-function in themouse heart following TAB (thoracic aortic banding) [49]. Al-though the significance of miR-1-dependent alterations in cardiacprogenitor cell polarity for mammalian heart development is cur-rently unclear, these findings may shed light on the mechanisticbasis for the spectrum of cardiac developmental phenotypes ob-served in miR-1-knockout mice.

Overexpression of miR-1 under the control of the βMHC(β myosin heavy chain) promoter disrupted embryonic mouseheart development and was associated with thin-walled vent-ricles, heart failure and lethality at E13.5 [26]. miR-1 overexpres-sion caused a reduction in the number of proliferating cardiomyo-cytes without affecting the number of apoptotic cells. Inhibitionof cardiogenesis by miR-1 overexpression was proposed to bemediated, at least in part, through translational inhibition of thebasic helix–loop–helix transcription factor Hand2 [26], whichis required for ventricular cardiomyocyte proliferation during theearly stages of cardiac development [50,51]. Interestingly, despitestrong evolutionary conservation of both miR-1 and Hand fromfruit flies to humans, the Drosophila orthologue of mammalianHand does not contain any miR-1-binding sites, suggesting thatthe miR-1/Hand2 regulatory axis appeared later in evolution.

Genetic deletion of miR-1-2 provided the first demonstrationof an essential role for an individual miRNA during cardiogen-esis. Homozygous deletion of miR-1-2 in mice causes lethal-ity, with incomplete penetrance, between E15.5 and birth dueto VSDs [52]. Consistent with previous gain-of-function exper-

iments, which proposed a role for miR-1 as a negative regu-lator of cardiomyocyte proliferation via translational repressionof Hand2, miR-1-2-knockout mice have an increased number ofmitotic cardiomyocytes and higher expression levels of Hand2protein [52]. The miR-1-2-homozygous mice that survive toadulthood display a spectrum of cardiac phenotypes includingconduction abnormalities and ∼15 % of miR-1-2-knockout micedevelop dilated cardiomyopathy and die by 2–3 months of age.Cardiac electrophysiological defects in miR-1-2 mutants appearto be due to de-repression of the miR-1 target Irx5 (iroquoishomeobox 5), a homeodomain transcription factor that regulatesKcnd2 (encoding potassium voltage-gated channel, Shal-relatedsubfamily, member 2), which is involved in cardiac repolarization[52]. Although the underlying basis for the incomplete penetranceof miR-1-2 mutant phenotypes is currently unclear, this could re-flect a potential role for additional epigenetic modifiers, whichmay be able to compensate for miR-1-2 loss-of-function duringheart development. Alternatively, the phenotypic heterogeneityof miR-1-2-mutant mice could reflect intrinsic differences in ge-netic background, as it is unclear whether these mice were inbredto homozygosity on a pure genetic background. Nevertheless, theprofound cardiac abnormalities observed in a subset of miR-1-2mutants, in which miR-1-1 was unaffected, suggests that heart de-velopment is particularly sensitive to the dosage of this miRNA.Future studies employing compound deletions of miR-1-1 andmiR-1-2 will allow for a more comprehensive understanding ofthe functions of miR-1 during cardiogenesis.

In contrast with the sensitivity of miR-1-knockout mice to hap-loinsufficiency, mice lacking either miR-133a-1 or miR-133a-2do not display any overt developmental phenotype [53]. However,deletion of both miR-133a genes causes lethal VSDs in approx-imately half of double-mutant embryos or neonates [53]. ThemiR-133a double mutants that survive to adulthood eventuallysuccumb to dilated cardiomyopathy by 5–6 months of age and atleast half of these mice suffer from sudden cardiac death. As withmiR-1 mutants, the precise basis for the incomplete penetrance ofmiR-133a-double-mutant phenotypes is unclear, but may be re-lated to underlying genetic or epigenetic modifiers that influencestochastic variations in gene expression during critical windowsof cardiac development.

miR-133a-double-mutant hearts are characterized by in-creased cardiomyocyte proliferation and apoptosis, profoundsarcomere disarray and cardiac fibrosis [53]. Consistent withincreased cardiomyocyte proliferation and sarcomere disarray,absence of miR-133a expression is associated with aberrantexpression of muscle contractile genes, ectopic expression ofsmooth muscle genes in the heart and increased expression ofcell-cycle-related genes. These effects were proposed to be dueto direct targeting of SRF and cyclin D2, both of which containfunctional binding sites for miR-133a in their 3′-UTRs [53]. Thepotent ability of miR-133a to regulate cardiomyocyte prolifer-ation was established further by gain-of-function studies whereoverexpression of miR-133a in the embryonic heart under thecontrol of the βMHC promoter led to VSDs, ventricular thinningand a diminution of cardiomyocyte proliferation [53]. Taken to-gether, these findings have established critical roles for miR-133ain the regulation of proliferative and sarcomeric gene expression

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during cardiac development and suggest that miR-133a plays animportant role in guiding cardiomyocyte maturation and prolif-erative arrest during late embryonic stages.

Interestingly, there appears to be significant overlap betweenthe functions of miR-1 and miR-133a during cardiac develop-ment, despite the fact that these miRNAs have different ‘seed’regions and presumably regulate a distinct repertoire of targetgenes. A previous affinity-purification study suggests that somemiR-1/miR-133 target genes may contain binding sites for bothmiRNAs and Hand2, for example, is regulated in vivo by bothmiR-1 and miR-133 in the mouse heart [54]. Similarly, it wasdemonstrated previously that ∼54 % of up-regulated transcriptsin zebrafish muscle Dicer mutants contain 7–8mer binding sitesfor miR-1 and miR-133 [55]. Furthermore, loss of both miR-1 andmiR-133 by morpholino knockdown in zebrafish results in a moresevere sarcomere disorganization phenotype compared with lossof either miRNA alone [55]. It therefore appears that miR-1 andmiR-133 have been co-opted into bicistronic clusters during evol-ution where they act in concert to shape cardiac contractile andproliferative gene expression during development. However, thefunctions of miR-1 and miR-133 may not be entirely overlapping.Although expression of miR-1 and miR-133 in embryonic stemcells suppresses their differentiation into endodermal or ecto-dermal lineages and promotes myogenic differentiation in vitro,miR-1 and miR-133 play opposing roles in this process [56].Further studies employing compound loss-of-function of bothmiR-1 and miR-133 in the mouse heart will be required to furtherestablish the overlapping functions of this bicistronic miRNAcluster during mammalian cardiogenesis and whether or not theyhave any role during gastrulation and early cardiac differentiationin vivo.

The myomiR (muscle-specific miRNA) regulatorynetwork: an intronic miRNA signalling system forthe regulation of myosin expressionThe expression of muscle contractile proteins is tightly regulatedduring heart development and dysregulation of muscle myosingenes is a hallmark feature of pathological cardiac remodelling.In rodents, αMHC/Mhy6, a fast ATPase, is highly expressed inthe adult heart, whereas βMHC/Myh7, a slow ATPase, is the pre-dominant myosin isoform in cardiomyocytes prior to birth [57]. Incontrast, βMHC is the predominant cardiac myosin isoform ex-pressed in large mammals including humans [58], indicating thatthere are important species differences in the regulatory mechan-isms governing myosin isoform expression. In response to cardiacstress and hypothyroidism in both humans and rodents, the heartmodulates MHC isoform content, resulting in an up-regulationof βMHC and a down-regulation of αMHC, which subsequentlycompromises cardiac contractility and function [59]. One of themost interesting and surprising discoveries in recent years hasbeen the finding that these muscle-specific myosin genes areunder the control of a family of intronic miRNAs, known asmyomiRs.

The myomiRs consist of miR-208a, miR-208b and miR-499,which are embedded within the introns of Myh6, Myh7 andMyh7b respectively. The expression of the myomiRs parallels theexpression of their respective host genes during development,

with miR-208a/Myh7 expression levels declining rapidly afterbirth, whereas miR-499/Myh6 and miR-208b/Myh7b expressionlevels are relatively low during embryogenesis and increase post-natally [60–62]. The myomiRs do not exist in invertebrates butare highly conserved in terms of both sequence identity and gen-omic organization from fish to humans. miR-208a and miR-208bhave identical ‘seed’ sequences and miR-499 is highly similarto miR-208 family members, with six overlapping nucleotides inthe ‘seed’ region, suggesting that the myomiRs probably sharecommon target genes and functions. These observations also im-ply that there has been strong selective pressure to maintain thesemiRNAs within myosin gene regulatory networks throughoutvertebrate evolution.

Genetic loss-of-function studies in mice have revealed thatmiR-208a is not essential for cardiac development. miR-208amutants are viable, fertile, have normal cardiac contractile func-tion under baseline conditions and do not display any overt signsof cardiac pathology up to 20 weeks of age [62]. However, car-diac function in miR-208a mutants progressively declines withage (>6 months) owing to abnormalities in sarcomere structure[62]. Although these results suggest that miR-208a is dispensablefor embryonic cardiac development, another study has revealedstriking cardiac conduction abnormalities in 4-month-old miR-208a mutant mice, which completely lacked P waves precedingQRS complexes, indicative of atrial fibrillation [61]. Consistentwith the effects of miR-208a loss-of-function, transgenic over-expression of miR-208a in the mouse heart caused a prolonga-tion of the PR interval, indicative of arrhythmias associated withfirst-degree AV block [61]. The effects of miR-208a on cardiacconduction were proposed to occur through interactions with anumber of target genes including the transcription factors, Hop(homeodomain-only protein) and GATA4, as well as the gap junc-tion protein connexin-40 [61]. In contrast, acute administrationof miR-208a antimiRs in adult mice was not sufficient to induceany obvious electrophysiological abnormalities [63], suggestingthat, although miR-208a may play an important role in the de-velopment of the cardiac conduction system, its function is notrequired for maintenance of electrical conduction in adulthood.

In contrast with the relatively subtle effects of miR-208a loss-of-function on cardiac development, miR-208a is indispensablefor mediating some aspects of the cardiac stress response dur-ing pressure overload or hypothyroidism. In response to TAB ortransgenic expression of activated calcineurin, which both inducecardiac hypertrophy in wild-type mice, miR-208a-mutant anim-als showed no signs of cardiomyocyte hypertrophy or cardiacfibrosis [62]. The blunted hypertrophic response of miR-208amutants was associated with a reduction in cardiac contractilityand a profound inability to up-regulate βMHC/Myh7 expres-sion, whereas expression levels of other cardiac stress markers,such as ANF (atrial natriuretic factor) and BNP (brain natri-uretic peptide), were unaffected. Similarly, mice lacking miR-208a are incapable of up-regulating βMHC/Myh7 in response toinhibition of T3 (3,3′,5-tri-iodothyronine) signalling (i.e. hypo-thyroidism) in the adult heart [62]. Consistent with these find-ings, miR-208a overexpression in transgenic mice is sufficient toinduce βMHC/Myh7 up-regulation prior to the onset of any overtsigns of cardiac hypertrophy and/or pathology [62]. Conversely,



developmental loss of miR-208a does not appear to have anyeffect on βMHC protein levels in the embryonic and neonatalheart [60–62], suggesting that distinct regulatory hierarchiescontrol the expression of βMHC/Myh7 during embryogenesisand under conditions of cardiac stress in adulthood. Completeloss-of-function of miR-208a, miR-208b and miR-499 will berequired to overcome potential issues of functional redundancyand to fully assess the functions of the myomiR network duringembryonic heart development.

The molecular mechanisms by which miR-208a regulatesβMHC expression in the adult heart involve a fascinating in-terplay between multiple members of the myomiR regulatorynetwork and their downstream molecular targets. In the adultheart, miR-208a is essential for expression of Myh7b/miR-499expression, as well as Myh7/miR-208b [60]. Indeed, adult miR-208a-knockout mice can essentially be considered null mutantsfor all of the myomiR members. Through an elegant series ofgenetic deletions and rescue experiments, van Rooij et al. [60]demonstrated that re-expression of miR-499 in the adult heartis sufficient to re-activate Myh7, Myh7b and miR-208b expres-sion, as well as prevent aberrant up-regulation of fast skeletalmuscle troponin isoforms, in hypothyroid miR-208a-mutant mice[60]. These results indicate that miR-499 is a major downstreameffector of the actions of miR-208a in the heart and define apositive-feedback loop whereby miR-208a regulates the expres-sion of Myh7b and its intronic miRNA miR-499 which, in turn,regulates βMHC.

The biological actions of miR-208 and miR-499 on my-osin expression have been reported to occur through a suite ofdownstream target genes including the TRH (thyroid hormonereceptor) co-regulator Thrap1/Med13 (TRH-associated protein1/mediator complex subunit 13) [61,62], as well as several tran-scriptional repressors of slow myosin gene expression, includingSox [SRY (sex determining region Y)-box] 6, Purβ (purine-richelement-binding protein β), Sp3 (specificity protein 3) and HP-1β (heterochromatin protein 1β) [60]. However, βMHC expres-sion levels are similar in wild-type and Thrap1/Med13-cardiac-transgenic mice following inhibition of T3 signalling [64], in-dicating that Thrap1/Med13 may not be a major mediator ofmyosin switching in vivo. In contrast, recent in vivo studiesof Thrap1/Med13-transgenic and -knockout mice have revealedan unexpected role for the miR-208/Thrap1 cardiac signallingcircuit in the regulation of systemic insulin sensitivity and gluc-ose tolerance following high-fat-diet-induced obesity, suggestingimportant roles for the myomiR regulatory network beyond con-trol of myosin expression [64]. With regards to myosin switching,a recent in vitro study by Yeung et al. [65] supports the notionthat miR-499 represses βMHC expression through repression ofSox6 and suggests that the lymphoid transcription factor Ikaros4 (Eos) may be an important upstream activator of Myh7b/miR-499 transcription. However, another study demonstrated that, al-though miR-499 overexpression in the heart was associated withincreased βMHC expression, Sox6 expression was unaffected[66]. Srivastava and co-workers [66] concluded that Sox6 maynot be a direct target of miR-499 in the heart in vivo and an ad-ditional role for miR-499 in the regulation of immediate earlyresponse genes, which are involved in the early cardiac transcrip-

tional response to cardiac stress, was identified. Furthermore,miR-499 also appears to be important for modulating mitochon-drial dynamics, through regulation of the Drp1 (dynamin-relatedprotein 1), and overexpression of miR-499 inhibits apoptosis andis sufficient to blunt the cardiac stress response following I/R(ischaemia/reperfusion) injury [67]. Interestingly, the expressionof miR-499 in the ischemic heart appears to be under the influ-ence of p53, which down-regulates miR-499 independently ofMyh7b expression [67]. Thus miR-499 processing is sometimesuncoupled from Myh7b transcription and the regulatory actionsof the myomiR network are complex, highly context-dependentand remain incompletely understood.

The miR-17∼92 cluster is required forheart developmentThe miR-17∼92 cluster, also known as Oncomir-1, consists ofsix miRNAs, belonging to four miRNA families, that are gen-erated from a common pri-miRNA transcript. Additionally, twoparalogous clusters, miR-106a∼93 and miR-106b∼25, are tran-scribed from genetic loci on separate chromosomes and providea further layer of redundancy among the four miRNA familiesthat comprise the miR-17∼92 cluster. The miR-17∼92, miR-106a∼93 and miR-106b∼25 clusters probably arose through aseries of gene duplication events during vertebrate evolution andthe broad conservation of sequence and genomic organizationacross species implies important functions during vertebrate de-velopment. Loss-of-function studies in the mouse have revealedcritical roles for the miR-17∼92 cluster during heart develop-ment [68]. miR-17∼92 mutants die shortly after birth with VSDsand lung hypoplasia. Loss of the paralogous miR-106a∼93 andmiR-106b∼25 clusters does not affect organismal viability andthese mutants do not display an obvious phenotype. However,compound mutant embryos lacking both miR-17∼92 and miR-106b∼25 die at mid-gestation with an increased severity of car-diac developmental defects, including VSDs, ASDs (atrial septaldefects) and ventricular wall thinning [68]. miRNAs within themiR-17∼92 cluster were proposed to exert their biological effectsvia repression of a number of pro-apoptotic proteins, includingBim, which contains well-conserved 3′-UTR-binding sites formiR-19, miR-25/92/363 and miR-17/20/93/106 [68]. These find-ings highlight the essential and overlapping roles of miR-17∼92and related clusters during heart development.

The miR-17∼92 cluster also appears to influence myocar-dial differentiation of cardiac progenitors in the secondary heartfield, which is required for normal outflow tract development.BMP (bone morphogenetic protein) signalling activates tran-scription of multiple members of the miR-17∼92 cluster, whichin turn repress the expression of the cardiac progenitor genes Isl1(ISL LIM homeobox 1) and Tbx1 (T-box 1) [69]. Studies in thezebrafish have also shown that miR-92 is critical for endodermformation and overexpression of miR-92 reduces endoderm form-ation during gastrulation and causes cardia bifida [70]. The effectsof miR-92 on developing endoderm were found to be due to mod-ulation of Gata5 levels by this miRNA. Gain-of-function studiesin the mouse have revealed further a function for the miR-17∼92cluster in the regulation of organ size, as transgenic mice glob-ally overexpressing miR-17 display growth retardation in multiple

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organs, including the heart [71]. The growth-inhibitory actions ofmiR-17 are thought to be due to repression of fibronectin, which isan extracellular glycoprotein that binds to integrins and mediatescell adhesion and proliferation during tissue development [71].However, in stark contrast with the growth-suppressing effectsof global miR-17 overexpression, transgenic overexpression ofthe entire miR-17∼92 cluster in developing cardiac and smoothmuscle cells induces cardiomyocyte hypertrophy and hyperplasia[72]. These findings may point to non-overlapping functions ofdifferent miRNAs within the miR-17∼92 cluster and further stud-ies will be required to dissect the precise roles of the miR-17∼92cluster during cardiac development.

Regulation of cardiac morphogenesisand patterning by miR-138 and miR-218Cardiac morphogenesis and patterning require exquisite spati-otemporal control of gene expression and function. Recent studiesin zebrafish suggest that miRNAs are integrated into the geneticcircuitries that distinguish chamber-specific gene expression pat-terns and guide early heart morphogenesis. During vertebratedevelopment, the atria and ventricles are separated by a domainknown as the AVC (atrioventricular canal), which eventually givesrise to the valves that ensure proper unidirectional flow of bloodthrough the multi-chambered heart. In zebrafish, the AVC is char-acterized by expression of cspg2 (versican) and notch1b, whichdistinguish the AVC from adjacent cardiac chambers [73]. A rolefor miRNAs in the regulation of AVC-specific gene expressionhas emerged from loss-of-function studies. Knockdown of miR-138 by antagomiRs in one-to-two cell zebrafish embryos results inpericardial oedema, indicative of cardiac dysfunction, impairedcardiomyocyte maturation and cardiac looping defects in 60–80 % of antagomiR-treated embryos [74]. Interestingly, cardiacpatterning was also affected, with genes normally restricted tothe AVC region becoming ectopically expressed in the ventricu-lar chamber following miR-138 knockdown. miR-138 restrictsAVC gene expression in the cardiac ventricle through direct re-pression of the gene encoding versican (cspg2), as well as throughdirect targeting of the retinoic acid synthesis enzyme aldehydedehydrogenase 1a2 (aldh1a2), which itself is a positive regulatorof versican expression [74]. Although these findings have iden-tified an important role for miR-138 in establishing the distinctidentity of cardiac structures in the developing heart, additionalstudies will be required in order to determine the relevance ofthis regulatory circuitry during mammalian heart development.

Further evidence for a role of miRNAs during vertebrate car-diac morphogenesis and patterning comes from recent gain- andloss-of-function studies of miR-218 in zebrafish. The miR-218family consists of three highly conserved members includingmiR-218a-1, miR-218a-2 and miR-218b. In vertebrates, miR-218a-1 and miR-218a-2 are intronically encoded in the slit2and slit3 genes, whereas miR-218b is intergenic. The Slit lig-ands and their Robo (Roundabout) receptors are best known fortheir roles in axon guidance in the nervous system [75], but theyalso provide guidance cues for development of several other or-gan systems, including the heart and vasculature [76,77]. Fishet al. [76] recently reported that knockdown of miR-218 usingtwo different morpholinos in one- to two-cell zebrafish embryos

results in cardiac morphological defects, impaired migration ofheart field progenitors to the midline during heart tube formationand pericardial oedema at 48 hpf (hours post fertilization), butdoes not cause any severe vascular defects [76]. Furthermore, asub-phenotypic dose of robo1 morpholino significantly rescuedthe miR-218 morphant phenotype, suggesting that miR-218 reg-ulates heart field migration and heart tube formation throughmodulation of Robo1 dosage [76]. However, Chiavacci et al. [78]could not reproduce these findings and, in contrast, reported thatmiR-218 knockdown, even following microinjection of very highdoses of one of the identical morpholinos used in the study by Fishet al. [76], did not cause any defects in cardiac development. Chia-vacci et al. [78] argued that a role for miR-218 in the migration ofendocardial and myocardial progenitors is unlikely because miR-218 is not substantially expressed before 24 hpf when the fusionof migrating cardiac cells is almost complete [78]. In support ofthis model, Chiavacci et al. [78] reported that overexpression ofmiR-218 during early stages of zebrafish embryogenesis causedcardiac defects including incomplete looping, as well as impairedventricular and atrial morphogenesis. In addition, overexpressionof miR-218a disrupts cardiac patterning and is associated withectopic expression of the normally endothelial-restricted genetie2 in the atria and ventricles. It was also shown that expressionof miR-218 correlates with Tbx5 during zebrafish heart devel-opment and down-regulation of miR-218 is sufficient to rescuemost of the developmental heart defects associated with Tbx5overexpression [78]. Given that mutations in the TBX5 gene are acommon cause of Holt–Oram syndrome, a condition associatedwith a wide spectrum of congenital heart abnormalities includ-ing VSDs and ASDs, the functions of miR-218 during cardiacmorphogenesis in mammals warrants future investigation.

The miR-15 family and neonatal heart developmentIn mammals, a number of important physiological transitions takeplace during the immediate post-natal period, which prepares theheart for life outside the womb. In rodents, the first 2 weeks ofpost-natal life are characterized by rapid cardiac growth coin-cident with cardiomyocyte cell-cycle withdrawal, cardiomyocytebinucleation, cellular hypertrophy, a switch from glycolytic tofatty acid substrate utilization, increased mitochondrial biogen-esis and maturation, and an increase in myofibril density, as wellas alterations in the composition of contractile proteins includ-ing myosin isoform switching [79]. Genetic deletion of Dicerand DGCR8 in mice has identified essential roles for miRNAsduring neonatal heart development [34,35]. Furthermore, recentexpression profiling studies have revealed dynamic alterations incardiac miRNA expression during neonatal life, including up-regulation of multiple members of the miR-15 family betweenP1 and P10 [80]. The miR-15 family consists of six members,which are clustered on three separate chromosomes. miR-15aand miR-16-1 are intronically encoded within the Dleu2 lncRNA(long non-coding RNA), which is frequently deleted in patientswith chronic lymphocytic leukaemia. miR-15b and miR-16-2 areclustered within an intron of a structural maintenance of chromo-somes gene, Smc4, whereas miR-195 and miR-497 are embeddedwithin an intron of a putative lncRNA, termed the miR-497-195cluster host gene (MIR497HG). Recent gain- and loss-of-function



studies in the mouse indicate that the miR-15 family is involved inthe induction of cardiomyocyte mitotic arrest during the neonatalperiod.

The timing of miR-15 family up-regulation in the neonatalmouse heart coincides with the onset of cardiomyocyte cell-cyclearrest and binucleation [80]. Overexpression of miR-195 in theembryonic heart under the control of the βMHC promoter in-hibits cardiomyocyte proliferation and is associated with VSDsand ventricular hypoplasia in ∼30 % of transgenic mice at birth[80]. The remaining mice, which do not have VSDs, developa slow-onset dilated cardiomyopathy and die between 5 and 18months of age. Overexpression of miR-195 in vivo is associatedwith a reduction in the number of mitotic cardiomyocytes, an in-creased number of multinucleated cardiomyocytes and repressionof mitotic genes [80]. Similarly, overexpression of miR-15 fam-ily members in vitro inhibits cardiomyocyte proliferation [80,81].Consistent with these findings, inhibition of the miR-15 familyin neonatal mice by systemic administration of antimiRs delayscardiomyocyte mitotic arrest [80], suggesting that post-natal up-regulation of the miR-15 family provides an important regulatorybrake on the cardiac cell-cycle machinery.

At the molecular level, the miR-15 family directly targets anumber of cell-cycle genes. In the heart, biochemical analysis ofneonatal miR-195 transgenic mice using next-generation sequen-cing of RISC-associated transcripts (RISC-seq) led to the identi-fication of a number of miR-15 family target genes involved in cellproliferation, including Chek1 (checkpoint kinase 1) [80]. Chek1has many important functions during DNA repair and mitosis,including prevention of genomic instability, co-ordinating pro-gression through G2/M and spindle checkpoints, chromosomesegregation and cytokinesis [82]. Although recent analyses ofmiR-15a/-16-1-knockout mice [83], as well as human cisplatin-resistant cancer cells [84] and follicular lymphomas [85], areconsistent with the notion that Chek1 is an important down-stream mediator of miR-15 family functions, the role of Chek1during cardiac development remains unknown and awaits furtherinvestigation.

The functions of the miR-15 family in cardiomyocytes extendbeyond cell-cycle control and other studies indicate that the miR-15 family also regulates a number of proteins with important rolesin the maintenance of mitochondrial function and cell survival.Inhibition of the miR-15 family in vitro protects cardiomyocytesfrom cell death, and these effects are proposed to occur via regula-tion of Bcl-2 (B-cell CLL/lymphoma 2) [86,87] and Sirt1 (sirtuin1) [87]. Inhibition of the miR-15 family also protects against I/Rinjury in vivo in adult mice, and this effect is associated withde-repression of the putative miR-15 target genes Pdk4 (pyr-uvate dehydrogenase kinase 4) and Sgk1 (serum-glucocorticoid-regulated kinase 1), which regulate mitochondrial function andapoptosis respectively [86]. Additionally, miR-15 family mem-bers have been found to regulate cellular ATP levels in culturedneonatal rat cardiomyocytes, and this involves repression of Arl2(ADP-ribosylation factor-like 2) [88]. Overexpression of miR-15b also causes changes in mitochondrial morphology, includinga reduction in mitochondrial size and mitochondrial degenera-tion, suggesting that the miR-15 family regulates mitochondrialintegrity [88]. Interestingly, in addition to its metabolic func-

tion, Arl2 also acts as a regulator of microtubule biogenesis andcytokinesis [89]. Indeed, inhibition of Arl2 partially rescues thecell-cycle-inhibitory actions of miR-16 overexpression in A549human adenocarcinoma cells, suggesting that Arl2, at least inpart, mediates the effects of miR-15 family members on cell pro-liferation [90]. The functions of Arl2 during cardiac developmentremain to be elucidated, but it is tempting to speculate that themiR-15/Arl2 axis may serve a dual role in cardiomyocyte prolif-erative arrest and mitochondrial maturation during the neonatalperiod. However, it should be noted that all of the in vivo loss-of-function studies of the miR-15 family to date have utilizedantisense oligonucleotides, which inhibit miR-15 family mem-bers in multiple tissues and cell types. Additionally, the miR-15family probably recognizes a distinct repertoire of target genesin the neonatal and adult heart. Therefore delineation of miR-15 targets with specific relevance to developmental comparedwith disease contexts requires further attention. Future stud-ies employing more sophisticated cell-specific loss-of-functionstrategies will be required in order to more fully elucidate thefunctions of the miR-15 family during cardiac development anddisease.

miRNAs AND CHD (CONGENITAL HEARTDISEASE) IN HUMANS

CHD accounts for approximately one third of all major congenitalanomalies and is the leading cause of childhood morbidity andmortality [91]. Genetic mutations in a number of critical genesfor cardiovascular development, such as NKX2.5 and GATA4,have been found to underlie a number of cardiac malformations[91]. Most of the known human heart malformations result fromhaploinsufficiency or heterozygous point mutations in transcrip-tion factors, suggesting that the heart is particularly sensitive tothe dosage of critical developmental pathways [91]. The causesof most congenital heart defects remain undefined and, giventhe emerging role of miRNAs as ‘fine-tuners’ of gene expres-sion during cardiac development, miRNAs may serve as usefuldiagnostic, prognostic and therapeutic targets for CHD. How-ever, despite dozens of miRNA expression profiling studies link-ing specific miRNA expression signatures with cardiovasculardiseases in adults, there is little information available regardingmiRNAs and cardiovascular disease during childhood in humans.Further studies will be required to assess the utility of miRNAs aspotential biomarkers for CHD. Furthermore, given the stabilityof miRNAs in the circulation, miRNA profiling in maternal, fetalor neonatal serum could also be particularly useful for assistingCHD diagnosis and prognosis in the future [92].

SNPs (single nucleotide polymorphisms) or mutations can al-ter miRNA expression, processing, targeting and function. Forexample, SNPs located in the 3′-UTR of the AGTR1 (angiotensinII type 1 receptor) gene affect miRNA-dependent regulation ofAGTR1 by miR-155 and are associated with hypertension [93].SNPs have also been identified in mature miRNA sequences.Of particular relevance to cardiac biology, systematic screeningfor sequence variants in the myomiR network identified a nat-urally occurring, rare, mutation in miR-499 [94]. Interestingly,

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cardiac-specific overexpression of wild-type and mutantmiR-499 revealed misdirected recruitment of a number of miR-499 target genes in mutant mice, despite the fact that this mutationis located in the 3′ end of miR-499, outside of the canonical ‘seed’region thought to determine miRNA target recognition [94]. Al-though the pathophysiological relevance of this naturally occur-ring miRNA mutation is currently unclear, that study providedan elegant proof-of-principle for the existence of functionallyrelevant mutations within miRNAs, even in regions outside ofthe canonical sequences required for target recognition. In ad-dition, a functionally relevant variant of miR-196a-2 has beenassociated with CHD susceptibility in a Chinese population [95].Genotype–phenotype correlation analyses revealed that the miR-196a-2 mutation was associated with increased cardiac expres-sion levels of mature miR-196a and aberrant targeting of HOXB8(homeobox B8), a natural target for miR-196a [95]. However, themechanistic basis for regulation of cardiac development by miR-196a is not clear. Knockdown studies in the developing chick em-bryo do not indicate a central, evolutionarily conserved, role formiR-196 in vertebrate heart development, but this miRNA doesappear to be particularly important for patterning of the axialskeleton [96]. Thus additional studies are required to validatethe broader significance of miR-196a-2 mutations for vertebrateheart development in animal models, and these findings will alsoneed to be replicated in different human populations.

miRNAs AND CARDIAC REGENERATION

miRNAs and cardiomyocyte proliferationThe classic dogma that the heart is terminally differentiated hasbeen overturned in recent years by the identification of low rates ofcardiomyocyte turnover during normal aging [97,98] and follow-ing myocardial infarction [98]. However, the adult mammalianheart cannot undergo appreciable regeneration following injuryand, as a consequence, ischaemic heart disease remains the lead-ing cause of death in adults in the developed world. In contrast,recent studies have identified a robust, yet transient, regenerat-ive capacity of the neonatal mammalian heart [99,100], whichdisplays similarities with regenerative mechanisms in lower ver-tebrates [101]. Cardiomyocyte proliferation appears to be thedominant cellular mechanism driving cardiomyocyte replenish-ment in the regenerating neonatal mouse heart following apicalresection injury [99] and myocardial infarction [100].

Given the importance of the miR-15 family and miR-133for cardiomyocyte proliferation during development, the func-tions of these miRNAs in the context of neonatal mouse andadult zebrafish heart regeneration have been recently assessed.Transgenic mice overexpressing the miR-15 family member miR-195 in the neonatal heart fail to regenerate following myocar-dial infarction and display reduced rates of cardiomyocyte pro-liferation, increased cardiomyocyte size, fibrosis and cardiacdysfunction compared with wild-type littermates [100]. Con-sistent with previously identified roles for the miR-15 familyin cardiac development and disease, miR-195 overexpression wasassociated with repression of a large number of cell-cycle and mi-tochondrial genes [100]. Similar studies in the zebrafish revealed

a critical role for miR-133 in the regulation of cardiomyocyteproliferation and regeneration following apical resection injury[102]. These findings suggest that miR-15 and miR-133 familymembers impair the cardiac regenerative response of neonatalmice and adult zebrafish respectively, through inhibition of car-diomyocyte proliferation.

Loss-of-function experiments in the mouse support a role forthe miR-15 family in post-natal regulation of mammalian cardiacregenerative capacity. Pharmacological inhibition of the miR-15family by systemic administration of antimiRs from an earlypost-natal age until adulthood improves cardiac function follow-ing I/R injury and is associated with induction of cardiomyocyteproliferation in adult mice [100]. Importantly, acute administra-tion of miR-15 inhibitors following I/R injury in adult mice alsoimproves cardiac function [86], implying that the miR-15 familymay be an attractive therapeutic target for ischaemic heart dis-ease. However, acute inhibition of the miR-15 family followingI/R inhibits cell death and reduces infarct size following infarc-tion [86], whereas chronic administration of antimiRs duringneonatal development does not affect cell survival following I/R[100]. These differences probably reflect distinct subsets of targetgenes that are modulated by the miR-15 family in immature andmature cardiomyocytes, as well as potential differences in thesubset of genes regulated during normal development comparedwith injury. Moreover, owing to the non-tissue-specific nature ofsystemic antimiR administration and the potential risk for can-cer following miR-15 loss-of-function [83,103], future studiesshould assess the therapeutic potential of cardiomyocyte-specificinhibition of the miR-15 family in animal models before movingto any potential clinical trials.

Further support for therapeutic strategies based on miRNA-dependent re-activation of cardiac regenerative mechanismscomes from a recent functional screen for miRNAs that inducecardiomyocyte proliferation. Eulalio et al. [81] performed a high-content high-throughput screen representing 988 mature miRNAsfor identification of miRNAs that induce cardiomyocyte prolifera-tion [81]. In addition to validating previously identified repressorsof cardiomyocyte proliferation, such as the miR-15 family, 40additional miRNAs were found to induce DNA synthesis inneonatal rat and mouse cardiomyocytes. Two of these miRNAs,miR-199a and miR-590, were shown to induce cell-cycle re-entryand cytokinesis of cultured adult cardiomyocytes in vitro. In vivodelivery of miR-199a or miR-590 to neonatal mice using the car-diotropic adeno-associated virus, AAV9 (adeno-associated virusserotype 9), resulted in widespread activation of cardiomyocyteproliferation and increased heart size, without inducing cardio-myocyte hypertrophy. Moreover, AAV9-mediated induction ofmiR-199a or miR-590 following myocardial infarction in adultmice induced marked cardiac regeneration, associated withaugmented cardiomyocyte DNA synthesis rates and reducedfibrosis. miR-199a and miR-590 did not affect cardiomyocyteapoptosis following myocardial infarction. However, myocardialviability, using the standard redox indicator tetrazolium chloride,was not assessed in this study and a potential protective effectof these miRNAs on cell viability (possibly through protectionagainst necrotic cell death) cannot be excluded. Analysis ofpotential downstream mRNA targets indicates that Homer1



Figure 3 Regulation of cardiomyocyte proliferation by miRNAsA summary of the molecular mechanisms by which miRNAs regulatecardiomyocyte proliferation. Ccnd2, cyclin D2; CM, cardiomyocyte.

[Homer homologue 1 (Drosophila)] and Hopx (HOP homeobox),previously implicated in the regulation of cardiomyocyte calciumsignalling and embryonic cardiomyocyte proliferation respect-ively, are targeted by both miR-199a and miR-590 [81]. Furtherstudies will be required to assess the endogenous physiologicalfunctions of miR-199a and miR-590 during cardiac developmentand any potential side effects associated with ectopic expressionof these miRNAs in non-cardiomyocytes. Nevertheless, thesestudies provide a powerful demonstration of the potential utilityof miRNA-based therapeutics for induction of cardiomyocyteproliferation and cardiac regeneration following myocardialinfarction. A summary of miRNAs implicated in the regulationof cardiomyocyte proliferation is provided in Figure 3.

miRNAs and stem/progenitor cellsStem cells represent a potentially attractive source of cardiaccells for the treatment of cardiovascular diseases. Over the lastdecade, several subsets of exogenous and endogenous cardiacstem/progenitor cells have demonstrated therapeutic potential inanimal models and clinical trials. However, most of the func-tional benefits reported for stem cell clinical trials of ischaemicheart disease have been modest, short-lived, associated with lowengraftment rates and appear to be due to paracrine effectsrather than differentiation of the applied cells into cardiomyo-cytes [104]. Several barriers will need to be overcome in order toimprove the safety, efficiency and efficacy of stem cell therapiesfor heart disease, including selection of an appropriate cell source,improvement of stem cell homing and survival in the infarct zone,reducing the tumorigenic potential of pluripotent stem cells andenhancing the efficiency of stem cell differentiation towards spe-cific cardiovascular lineages. One avenue towards achieving atleast some of these goals may involve the use of miRNAs tocontrol the gene expression networks underlying self-renewal,pluripotency and differentiation decisions in stem cells.

miRNA expression profiling of human ESC (embryonic stemcell)-derived cardiomyocytes revealed that miR-1, miR-133 andthe myomiRs (miR-208 and miR-499) are significantly inducedduring cardiomyocyte differentiation [56,105,106]. Overexpres-sion of miR-1 induces the expression of mesodermal and cardiac

marker genes in mouse and human ESCs [56], as well as humanESC-derived embryoid bodies [106] and adult cardiac progenitorcells [105]. As well as controlling the expression of sarcomericgenes in differentiating ESCs, miR-1 has also been proposedto regulate the electrophysiological maturation of ESC-derivedcardiomyocytes [105]. In contrast, the bicistronically clusteredmiR-133, which shares many functions with miR-1 during car-diac development in vivo, opposes cardiomyocyte differentiationand represses cardiac gene markers in ESCs in vitro [56]. Further-more, Takaya et al. [107] reported that both miR-1 and miR-133inhibit cardiomyocyte differentiation in a two-dimensional mouseESC culture model, suggesting context-specific functions of miR-1 and miR-133 during cardiac differentiation. Consistent with acrucial role for the myomiR network in cardiac differentiation,overexpression of miR-499 in human cardiac progenitor cells andESCs induces expression of cardiac gene markers and hastensthe formation of beating embryoid bodies, whereas inhibitionof miR-499 blocks cardiac differentiation in vitro [105,106,108].The effects of miR-499 on cardiac differentiation were proposedto occur through repression of the transcription factor Sox6 [105].However, in contrast with miR-1, overexpression of miR-499 doesnot induce the calcium amplitude and kinetics of calcium hand-ling that are characteristic of more mature ventricular cardiomyo-cytes [108]. Therefore miR-1 and miR-499 both appear to be po-tent inducers of cardiomyogenic differentiation of stem cells, butmiR-1 induces a more mature ventricular cardiomyocyte pheno-type than miR-499.

Recent studies also support the translational potential ofmiRNA-mediated manipulation of stem cells for heart disease.Transplantation of ESCs overexpressing miR-1 into the borderzone of infarcted mouse hearts protects the myocardium fromischaemic injury in vivo [109,110]. miR-1 inhibits expressionof PTEN (phosphatase and tensin homologue deleted on chro-mosome 10) in ESCs, which augments PI3K (phosphoinositide3-kinase)/Akt signalling, improves cardiac function, enhancescardiomyocyte differentiation and attenuates apoptotic cell death[109,110]. Similarly, overexpression of miR-499 in c-kit+ cardiacprogenitor cells improves myocardial function, enhances cardio-myocyte differentiation and potentiates restoration of myocar-dial mass following injection into the border zone of infarctedrat hearts [111]. Thus ectopic expression of miRNAs in cardiacstem and progenitor cells can alter the differentiation potential ofengrafted cells in vivo and could be used to improve the efficacyof stem cell therapies for heart disease.

An alternative strategy to exogenous cell replacement ther-apies for heart disease involves harnessing the innate regenerat-ive potential of the mammalian heart. In addition to inductionof cardiomyocyte proliferation (discussed above), several cardiacprogenitor populations have been identified in the adult heart andtheir regenerative potential could potentially be harnessed fortherapeutic purposes. Little is known with regards to the func-tions of miRNAs in the control of self-renewal and differentiationdecisions of endogenous cardiac progenitor cells. Interestingly,c-kit progenitors undergo a maturation process during early post-natal life, which coincides with a significant loss of cardiomyo-genic potential during the neonatal period [112]. Although themolecular mechanisms regulating developmental maturation of

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c-kit progenitors are not understood, a recent expression profilingstudy has identified a number of miRNAs that are development-ally regulated in neonatal compared with adult c-kit progenitors.Among the differentially regulated miRNAs, miR-17, a mem-ber of the miR-17∼92 cluster, was found to be reduced in adultcompared with neonatal progenitors [113]. Overexpression of themiR-17∼92 cluster increased the proliferation of adult c-kit pro-genitors in vivo, suggesting that developmental down-regulationof this miRNA cluster may be an important mechanism regulat-ing the proliferative potential of cardiac progenitor cells. Furtherstudies of the roles of miR-17∼92, as well as other miRNAs, us-ing gain- and loss-of-function strategies in vivo will be requiredin order to more fully elucidate the importance of miRNAs incardiac progenitor cell populations during development and fol-lowing cardiac injury.

miRNAs and direct lineage reprogrammingAs an alternative to cell replacement strategies, some investig-ators have recently turned their attention to lineage reprogram-ming, which involves transforming one somatic cell type intoanother. The most stunning example of lineage reprogramminginvolves the re-induction of pluripotency in somatic cells byintroduction of the so-called Yamanaka factors: Oct4/POU5F1(POU class 5 homeobox 1), Sox2, Klf4 (Kruppel-like factor 4)and c-Myc [v-myc myelocytomatosis viral oncogene homologue(avian)] [114]. Intriguingly, overexpression of the miR-302 clustermembers and miR-367 can completely substitute for the Yaman-aka factors and induces more efficient reprogramming of mouseand human somatic cells than the four Yamanaka factors [115].The subsequent generation of iPSCs (induced pluripotent stemcells) provides an ethically unencumbered source of stem cellsfor disease modelling, drug discovery and regenerative medicine.However, barriers to the use of iPSC technology for cardiac re-generation include very low reprogramming efficiencies, incom-plete or partial cell reprogramming, functional heterogeneity andimmaturity of iPSC-derived cardiomyocytes, low survival andretention of stem cells in the infarct zone, as well as a number ofsafety concerns. In an attempt to overcome some of these barri-ers, researchers have recently turned their attention to direct re-programming of fibroblasts into cardiomyocytes, with the addedbonus that the human heart provides a rich endogenous supply offibroblasts for reprogramming following myocardial infarction.

By systematically screening a number of core componentsof the cardiogenic transcriptional machinery, Ieda et al. [116]provided the first demonstration that a combination of threetranscription factors, Gata4, Mef2c and Tbx5, could repro-gramme mouse fibroblasts directly into cardiomyocytes in vitro.Although this study provided an important proof-of-principle fordirect cardiac reprogramming, the efficiency of reprogrammingwas very low and very few cardiomyocytes showed evidenceof spontaneous beating, a hallmark feature of a fully matureventricular cardiomyocyte. Nevertheless, two recent landmarkpapers by Qian et al. [117] and Song et al. [118] provide evidencethat in vivo reprogramming of fibroblasts into cardiomyocytesis possible and, moreover, direct reprogramming is associatedwith long-lasting improvements in cardiac function followingmyocardial infarction.

As an alternative to transcription-factor-mediated direct re-programming, miRNAs can also induce reprogramming offibroblasts to cardiomyocytes in vitro and in vivo. Lentiviral-mediated delivery of miR-1, miR-133, miR-208 and miR-499 intothe border zone of the mouse myocardium following myocar-dial infarction induces direct conversion of fibroblasts into car-diomyocytes in situ [119]. One potential advantage of miRNA-based approaches for direct reprogramming is the potential to cir-cumvent the use of viral vectors for cardiac transduction by usingchemically synthesized miRNA mimics. However, Jayawardenaet al. [119] did not assess the impact of miRNA-based repro-gramming on cardiac function, so the therapeutic benefits of thisapproach await further investigation.

FUTURE DIRECTIONS

Since the discovery that miRNAs are involved in cardiac develop-ment in 2005 [26], rapid progress has been made in decipheringthe myriad roles for miRNAs in cardiovascular biology. miRNAsnow constitute an important component of the regulatory circuitsthat govern heart development (Figure 1) and this knowledgehas revolutionized our understanding of the diverse roles of non-protein-coding RNA in cardiac biology. However, with the humangenome encoding over 1000 miRNAs, only a handful of whichhave been studied in the heart, much remains to be learned aboutthis new class of regulatory RNA. Indeed, most of the largest andmost abundant cardiac miRNA families, such as let-7 and miR-30, have not yet been extensively characterized, owing to tech-nical difficulties associated with the requirement for compoundgenetic deletions of multiple miRNA family members locatedon multiple different chromosomes. Indeed, miRNA redundancycan also exist across different miRNA families, with multiplemiRNAs recognizing the same target mRNAs and/or impingingon different mRNA targets within a common biological path-way. In light of these multiple layers of functional redundancy,the often-modest effects of miRNAs on individual target genesand the accompanying subtle phenotypes associated with geneticdeletion of individual miRNAs are not entirely surprising. Under-standing the potential for redundancy in the regulatory hierarchyof miRNA interactions will form a critical component of future in-vestigations in the field. Furthermore, current models of miRNAtarget recognition are inadequate and an enhanced understandingof the mechanisms of miRNA action is required. In particular, theimportance of non-seed regions in the mature miRNA may shedlight on functional redundancy outside of miRNA families. Thecontinued development of biochemical approaches for miRNAtarget identification will aid our understanding of miRNA mech-anisms of action, which are often clouded by biased bioinformaticapproaches that are highly susceptible to false discovery.

One of the most promising avenues of miRNA researchcontinues to be the development of diagnostic and therapeuticplatforms for detection and manipulation of miRNAs. The clin-ical utility and safety of these approaches will be closely mon-itored in the coming years. With a growing list of miRNAs beingimplicated in cardiac developmental processes, it will be inter-esting to see whether this work in animal models will translate



into any meaningful clinical benefits for patients with congenitalheart disease. miRNA-based therapeutics could also be harnessedin the context of regenerative medicine (Figure 2), which wouldusher in a new era of miRNA-based drugs for ischaemic heartdisease. All of these possibilities will require ongoing explorationof the roles of miRNAs in cardiac developmental and regenerativeprocesses, as well as the continued refinement of experimentaland therapeutic approaches. Given the rapid progress that hasbeen made in miRNA biology over the last decade, there is goodreason to be optimistic about the future.

ACKNOWLEDGEMENTS

I apologize to those researchers whose work was not cited in thisreview and for the omission of some discussion points related tomiRNA function during cardiovascular development and regenera-tion owing to space constraints.

FUNDING

My own research was funded by the National Health and MedicalResearch Council and National Heart Foundation of Australia [grantnumbers APP1033815 and 635530].

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Received 3 January 2013/11 February 2013; accepted 15 February 2013

Published on the Internet 26 April 2013, doi: 10.1042/CS20130011


micrornas in cardiac development and regeneration...early cardiac progenitors at e8.5 (embryonic day...

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