Pivotal role of microRNAs in cardiac physiology and heart failure

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Pivotal role of microRNAs in cardiac physiology and heart failure Q1

Andre´ M. Leite-Moreira, Andre´ P. Lourenc¸o, Ineˆs Falca˜o-Pires and Adelino F. Leite-Moreira Department of Physiology and Cardiothoracic Surgery, Faculty of Medicine, University of Porto, Portugal

Cardiac hypertrophy is a hallmark of heart failure (HF), a highly prevalent, debilitating and deadly condition in Western countries. Pronounced changes in molecular pathways governing cardiac physiology underlie hypertrophy and progression to HF. MicroRNAs, small nucleotide sequences that coordinate gene expression, may have a central role in orchestrating these changes since the hypertrophic and HF myocardium clearly shows disturbed microRNA profiles. Experimental interventions targeting miR disturbances have been shown beneficial in animal models of cardiac hypertrophy and HF. This short review discusses exciting potential diagnostic and therapeutic applications of microRNAs to cardiac hypertrophy and HF, highlighting the underlying molecular pathways.

Introduction Since Crick postulated the ‘central dogma of molecular biology’ describing the flow of biologic information, numerous complex regulatory systems, such as post-translational modifications, transcriptional regulation, epigenetic mechanisms and, more recently, microRNAs (miRs) have been uncovered [1]. MiRs are highly conserved short single-stranded non-coding RNAs that target specific complementary sequences mostly in the 30 untranslated regions and inactivate mRNAs through association with a large, multiprotein RNA-induced silencing complex. Individual miRs regulate various mRNAs usually involved in coordinated responses leading to major transcription pattern changes whilst each mRNA can in turn be regulated by multiple miRs in intertwined regulatory networks. Moreover, although mRNAs are mainly downregulated, miRs can indirectly upregulate participants of intracellular signalling pathways and, under particular conditions, may also upregulate mRNA expression [2]. Compared with transcriptional regulation and epigenetic mechanisms, miRs are more frequently involved in the fine tuning of gene expression and are thus more suited to precisely regulate homeostatic mechanisms, but several recent works Corresponding author:. Leite-Moreira, A.M. ([email protected]), ([email protected])

emphasise the strong interrelationship between miRs and epigenetic mechanisms [3]. Ongoing research seeks to elucidate miR biology and its repercussions in heart development and cardiovascular disease. MiRs participate not only in the initial adaptive hypertrophic response to cardiac stress but also in the later pathophysiological mechanisms that lead to functional deterioration and extensive myocardial and extracellular matrix (ECM) remodelling. Their pathophysiological involvement opens ways to clinical applications not only as biomarkers but also as potential therapeutic targets [4].

Overview of miR biology Computational sequence analysis identified over 1000 miRs in the human genome. MiRs are 18–25 nucleotides in length and they are transcriptionally regulated by either the parent protein-coding genes when found within introns (‘miRtrons’) or independently when located outside protein-coding regions. Beyond transcriptional regulation, post-transcriptional control mechanisms are also important. Initially transcribed as long primary transcripts (pri-miR) by RNA polymerase II, most miRs are subsequently first spliced by the Drosha RNase III-cofactors complex, notably DiGeorge syndrome critical region gene eight (DGCR8) cofactor, into 60–100-nucleotide-long hairpin folded precursors (pre-miRs).

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After extrusion from the nucleus by exportin-5 [5], pre-miRs are then cleaved by Dicer RNA-processing ribonuclease III, into small double-stranded miR-miR* duplexes. The complementary nonfunctional miR* passenger strand is degraded whereas the mature functional miR strand is incorporated into the RISC where argonaute proteins have a crucial role in mRNA silencing by either degradation or translational inhibition [6]. Some miRtrons, however, are spliced with the parent coding gene bypassing the canonical Drosha pathway [7]. MiR processing is crucial for cardiac development and homeostasis, DGCR8 knockouts develop lethal heart failure (HF) due to impaired miR synthesis [8] whereas Dicer ablation either leads to cardiac hypoplasia in utero [9] or to cardiomyopathy in cardiac-selective adult knockouts [10]. Recent evidence further increased our grasp of the complexity of miR synthesis, supporting regulatory roles for transcription factors such as Smads, tumour suppressor genes such as p53 and oestrogen receptors [11].

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TABLE 1

Currently identified miRs most relevant to heart failure progression Pathologic myocardial hypertrophy Anti-hypertrophic Ca -dependent pathways miR-1 [19] miR-9 [23] miR-26 [20] miR-133 [21,22]

miR-22 [32] miR-23a [33]

Cytokine pathways miR-142 [38]

miR-199a [35–37]

Cytoskeleton-related pathways miR-1 [39] miR-142 [38] MyomiRs

Cardiac hypertrophy can occur either naturally and reversibly during growth, pregnancy and exercise, as a physiological process that has no morbid consequences, or in response to sustained pathological stimuli such as chronic haemodynamic overload, ischaemic injury or neuroendocrine activity. Under the influence of the latter cardiac hypertrophy is initially an adaptive process that preserves function and normalises wall tension under stress, but ultimately leads to cell death, impaired vascularisation, inflammation, fibrosis, reactivation of the foetal gene program and metabolic shift, therefore becoming a pathological process that constitutes an independent risk factor and worse prognosis predictor in HF [12]. In the next chapters we will resume current evidence on miR involvement in hypertrophy and HF signalling pathways. A summary table is given (Table 1).

Myocardial fibrosis

Cyclic calcium (Ca2+) transients responsible for electro-mechanical coupling are unable to modulate signalling pathways, but small, sustained increases in Ca2+ in the perinuclear microdomain can [13]. Nuclear microdomain Ca2+ levels are regulated by humoural signals such as endothelin-1 (ET-1) and angiotensin II (Ang II) coupled to Gq proteins which activate perinuclear endoplasmic reticulum type 2 IP3 receptors [14]. Ca2+ levels can also increase by Na+/H+ exchanger activation due to both mechanical stress and humoural activity, which refrains Ca2+ extrusion through the Na+/Ca2+ antiporter, [15] and by b-adrenergic stimulation, through Gs, of adenylate cyclase, protein-kinase A (PKA), phosphorylation of phospholamban, and ultimately enhanced sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity [16]. Hypertrophy signalling pathways are mediated by the Ca2+/calmodulin (CaM)-dependent protein phosphatase calcineurin (CN) and by Ca2+/CaM-dependent protein kinase (CaMK) [13]. Briefly, CN dephosphorylates nuclear factor of activated T-cells (NFATs), which translocate to the nucleus and induce genes associated with pathological hypertrophy, after interacting with other transcription factors, notably GATA4 and myocyte enhancer factor 2a (MEF2a) [17], whereas CaMK II phosphorylates histone deacetylase 4 (HDAC4) favouring nuclear exit and derepression of hypertrophic genes [18]. 2

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miR-23a [25] miR199b [24]

PI3K/Akt pathways miR-1 [30,31] miR-133a [30]

Role of miRNAs in cardiomyocyte hypertrophy pathways

Calcium-dependent pathways

Pro-hypertrophic

2+

miR-208a [41], mR-208b [40], miR-499 [42] Inhibitors

Promoters

miR-29 [47] miR-30 [44] miR-133 [44]

miR-21 [45,46]

The table summarises references supporting a role for miRs in specific aspects of heart failure progression, details can be found along the text.

Various miRs have been shown to refrain Ca2+-dependent pathways. These are summarised in Fig. 1. Indeed, miR-1, the most abundant healthy heart miR, which is mostly expressed in myocytes, attenuates cardiac hypertrophy in vivo through negative regulation of CaM, MEF2a and GATA4 in mice. Adenoviral induced overexpression decreased these proteins, whereas miR-1 knockdown upregulated them [19]. miR-26 overexpression in mice cardiomyocytes reduced GATA4-dependent transcription and ET-1-induced hypertrophy whilesensitising cells to apoptosis [20]. Hypertrophy can also be antagonised through CN and NFATc4. Indeed, adenoviral treatment of mice neonatal cardiomyocytes with miR-133 disrupted the CN-NFAT pathway and prevented a1-adrenergic induced hypertrophy [21]. Moreover, CN overexpressing hypertrophy models showed low miR-133 levels, which were reversed by CN inhibitor cyclosporine A while, conversely, CN was downregulated by miR133 transfection, but not following cotransfection with its antisense oligoribonucleotide [22]. Another miR, miR-9, also attenuates cardiac hypertrophy and improves myocardial function by decreasing the levels of the transcriptional coactivator myocardin a downstream target of NFAT which is poorly expressed under physiologically conditions but strongly activated during hypertrophic stimulation by the CN/NFAT pathway [23]. Contrarily, some miRs promote hypertrophy by CN/NFAT pathway activation. Costa Martins et al. recently described miR-199b involvement in an autoamplification loop promoting CN/NFAT signalling by targeting an hypertrophy inhibitor that acts by negative nuclear regulation of NFATs, the dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Indeed, increased sensitivity to CN/NFAT signalling was found in mutant mice made either haploinsufficient for

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FIGURE 1

Calcium (Ca2+)-dependent hypertrophy pathway modulation by microRNAs (miRs). MiRs with pro-hypertrophic and anti-hypertrophic roles are signalled in black and white, respectively. Abbreviations: Ang II, angiotensin II; CaM, calmodulin; CaMK, Ca2+/CaM-dependent protein kinase; CN, calcineurin; ET-1, endothelin-1; GpCRs, G protein coupled receptors; HDAC4, histone deacetylase 4; NFAT, nuclear factor of activated T-cells; MEF2a, myocyte enhancer factor 2a; GATA4, a member of the GATA family of zinc finger transcription factors; MuRF1, muscle ring finger 1; Dyrk1a, dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a; P, phosphate.

Dyrk1a or overexpressing miR-199b. By contrast, in vivo inhibition of miR-199b normalised Dyrk1a expression reduced NFAT activity, and led to significant inhibition and even reversal of hypertrophy and fibrosis in murine models of HF. Modulation of Dyrk1a by miR199b thus constitutes a feed forward mechanism that enhances pathological cardiomyocyte hypertrophy processes [24]. This is also the case of miR-23a, its transcription is governed by NFATs while its role is to target muscle ring finger 1 (MuRF1), a negative regulator of hypertrophy as well. Not surprisingly, miR-23a overexpression greatly reduced MuRF1 levels and aggravated hypertrophy in mice, both in vitro and in vivo, while its silencing had opposite effects [25].

Phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathways One of the major hypertrophy promoting pathways is induced by insulin-like growth factor-1 (IGF-1). The catalytic tyrosine kinase IGF-1 receptor (IGF-1R) phosphorylates insulin receptor substrates which, in turn, activate PI3K and the small monomeric G protein Ras, which mediates intricate cross talking downstream pathways [26]. PI3K phosphorylates membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) converting it into phosphatidylinositol 3,4,5-trisphosphate (PIP3) which then recruits Akt and its activator, 3-phosphoinositide dependent kinase-1 (PDK-1). Upon colocalization and phosphorylation by PDK-1 activated Akt mediates hypertrophic responses mainly by turning on mammalian

target of rapamycin (mTOR) and inhibiting anti-hypertrophic regulators such as glycogen synthase kinase-3 (GSK-3), but also by inactivating members of the Forkhead box-O class of transcription factors (FOXOs), promoting their translocation to the cytosol [27]. As for Ras, in its active guanosine triphosphate (GTP) bound form it recruits mitogen-activated protein kinase (MAPK) to the plasma membrane and is one of the major initiators of the MAPK cascade. MAPKs constitute one of the core signal transduction pathways responsible for coupling a wide range of extracellular signals with intracellular processes such as differentiation, movement, proliferation and death and, not surprisingly, are therefore co-activated by many other pathways such as PKA, PKC and Janus kinase (JAK). MAPK, the ultimate effector of the MAPK cascade, phosphorylates various cytoplasmic and nuclear proteins, many of which are transcription factors, such as MEF2 family members [28] and GATA4 [29]. Despite its central involvement in cardiac hypertrophy and fibrosis, the intricate nature of the MAPK pathway has made it hard to decipher the role of miR modulation. Still, upstream targets of PI3K/Akt signalling have already been identified and are schematically presented in Fig. 2. Hua et al. showed IGF-1 is directly regulated by both miR-1 and miR-133a [30] and Elia et al. also validated the IGF-1R as a target of miR-1 through gain-of-function studies [31]. Moreover, miR-1 and IGF-1 are reciprocally regulated in experimental cardiac hypertrophy and PI3K/Akt pathway hyperactivity induced by either transverse www.drugdiscoverytoday.com

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FIGURE 2

Phosphatidylinositol 3-kinase (PI3K)/Akt signalling hypertrophy pathway modulation by microRNAs (miRs). MiRs with pro-hypertrophic and anti-hypertrophic roles are signalled in black and white, respectively. Abbreviations: Akt, serine/threonine-specific protein kinase also known as protein kinase B; FOXOs, forkhead box-O class of transcription factors; GATA4, a member of the GATA family of zinc finger transcription factors; GSK-3, glycogen synthase kinase-3; IGF-1, insulin-like growth factor 1; IGF-1 R, IGF-1 receptor; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; MAPKKK, kinase of MAPK kinase; MEF2a, myocyte enhancer factor 2a; mTOR, mammalian target of rapamycin; PDK-1, 3 phosphoinositide dependent kinase-1; Ras, one of the small monomeric G proteins; PTEN, phosphatase and tensin homolog; P, phosphate.

aortic constriction or Akt overexpression. Indeed, FOXO3a induces miR-1 transcription but ceases to do so when inactivated by Akt during myocardial hypertrophy [31]. Low levels of miR-1 expression accompany myocardial hypertrophy not only in experimental settings but also in IGF-1 overproducing acromegalic patients [31]. By bioinformatic analysis, Xu et al. identified phosphatase and tensin homolog (PTEN), a tumour suppressant protein that inhibits PI3K/Akt pathway by dephosphorylating PIP3 back into PIP2 (reversing PI3K action), as a putative target of miR-22 [32]. To support their hypothesis, they then showed that miR-22 was upregulated in hypertrophy induced by either an a-agonist or ang II, miR-22 could suppress PTEN expression, miR-22 overexpression markedly downregulated PTEN in cardiomyocytes and, finally, that miR-22 ablation attenuated hypertrophy [32]. Beyond its connection with the CN/NFAT pathway, miR-23a is also a potential trigger for myocardial hypertrophy through a reciprocal regulation with FOXO3a that further strengthens its role. FOXO3a directly represses it by binding to its promoter region, while miR23a gain and loss-of-function studies conversely decrease and increase FOXO3a levels, respectively [33].

Cytokine signalling pathways Some of the most important promoters of myocardial hypertrophy are cytokines, most of these belong to the interleukin-6 (IL-6) family. They bind to tyrosine kinase-associated receptors composed 4

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of a common glycoprotein 130 (gp130) subunit and a second subunit that confers ligand specificity. Downstream signalling is mediated by three pathways: PI3K/Akt, the MAPK cascade and JAK/ signal transducers and activators of transcription (STAT) [34]. JAK/ STAT signalling is initiated upon ligand binding by receptor conjugation with JAKs, phosphorylation of the intracellular domain of gp130, STAT family cytokine-specific member recruitment phosphorylation and activation by JAK. STATs then detach, dimerise, translocate to the nucleus and initiate transcription of several genes, including GATA4 [34]. The overall effects on cardiac hypertrophy can be either beneficial or detrimental depending on which STATs are activated. Although many specific effects of STATs remain controversial, some seem to lead to myocyte apoptosis, depression of contractility, disturbed function of ion channels, derangements of sarcomeric structure and induction of the foetal gene program, whilst others antagonise these same actions [34]. Notably, STAT3 has mainly stress protective effects, preserving cardiac integrity and function in the postnatal heart, which was well illustrated in knockout mice. Curiously, STAT3 knockouts also showed increased levels of pro-hypertrophic miR-199a [35] at the same time as STAT3 blocked its overexpression in cardiomyocytes preventing not only hypertrophy but also sarcomere disruption [36]. As for miR-199a, during hypoxia preconditioning its downregulation upregulates hypoxia-inducible transcription factor 1-a (HIF-1a) and histone deacetylase sirtuin (Sirt1) [37] whilst its

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FIGURE 3

Cytokine signalling pathways modulated by microRNAs (miRs). MiRs with pro-hypertrophic and anti-hypertrophic roles are signalled in black and white, respectively. Inhibitory and stimulatory actions upon pathways and factors are marked with ‘’ and ‘+’, respectively, whereas ill-defined actions are indicated by ‘?’. Abbreviations: IL-6, interleukin-6; gp130, glycoprotein 130, or CD130, a transmembrane protein cytokine receptor; MAPK, mitogen-activated protein kinase; JAK, Janus kinase; PI3K, phosphatidylinositol 3-kinase; UCEs (E2), ubiquitin-conjugating enzymes; Ub, ubiquitin; STAT, signal transducers and activators of transcription; HIF-1a, hypoxia-inducible factor-1a; Sirt1, protein deacetylase sirtuin (silent mating type information regulation 2 homolog) 1; GATA4, a member of the GATA family of zinc finger transcription factors; p300, protein co-activator of gene transcription; P, phosphate.

overexpression by cardiomyocyte transfection reduces ubiquitinconjugating enzyme levels [35]. Finally, downregulated ubiquitinconjugating enzymes lead to sarcomeric disorganisation and decreased synthesis of myosin heavy chains (MHC), culminating in cardiomyocyte dysfunction. The pathophysiological path whereby impaired STAT3 activity increases miR-199a, and subsequently disturbs the ubiquitin-proteasome system, ultimately disrupting sarcomere structure (Fig. 3) and emphasises how one of the best described JAK/STAT pathways courses with cardioprotection through inhibition of miRs [35]. Conversely, JAK/STAT pathways may be targeted by miRs. Activation of the survival pathway transduced through the IL6 receptor, gp130 and STAT3 is essential during mechanical loading. Acetyltransferase p300 whose levels increase during overload and HF is a crucial mediator of this response. Along with the MAPK cascade, p300 downregulates miR-142 expression, enabling adaptive cytokine-mediated survival signalling during haemodynamic stress. Sharma et al. recently observed repression of cytokine signalling due to negative regulation of gp130 and nuclear factor k-light-chain-enhancer of activated B cells (NF-kB) pathway by miR-142, through gain- and loss-of-function studies. Indeed, its overexpression not only inhibits growth but also induces cell death and cardiac dysfunction by targeting p300 and gp130 [38].

Cytoskeleton-related signalling pathways Some miRs have been linked to regulatory cytoskeletal proteins. miR-1 suppresses the inhibitor of actin polymerisation twinfilin-1 (Twf1), therefore its downregulation during hypertrophy raises Twf1, prevents actin polymerisation and elicits hypertrophy by cytoskeletal modulation [39]. Also miR-142 profoundly downregulates a-actinins, which in turn have been implicated in cardiomyopathy, cell growth and apoptosis. High miR-142 levels during hypertrophy may thus impair structural integrity and contribute to apoptosis [38].

Involvement of miRNAs in myofilament synthesis, myocardial function and hypertrophy The cardiac muscle myosin heavy chain (MHCs) encoding genes, responsible for the coding of fast-twitch a and slow-twitch b-isoforms also contain miR-208a, miR-208b and miR-499 in the intronic sequences of Myh6, Myh7 and Myh7b, respectively. The functional relevance of these so-called ‘myomiRs’ is still poorly understood, but since the replacement of a by bMHC is an important part of the foetal gene reprogramming in pathological hypertrophy concomitant changes in miRs are most probably at least as important [40]. Myh6 codes for fast a-isoforms but coexpresses miR-208a which in turn downregulates the slow MHC www.drugdiscoverytoday.com

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genes. Conversely, slow Myh7 and Myh7b genes co-express miR208b and -499, respectively, which activate slow and repress fast myofiber gene programs [40]. Cardiac specific overexpression of miR-208a in the adult heart under aMHC promoter control can induce hypertrophy [41]. Similarly, elevated miR-499 levels also induce cardiomyocyte hypertrophy, cardiac dilation and contractile dysfunction [42]. Reviews  POST SCREEN

Role of miRs in fibrosis Fibroblasts, the most prevalent heart cells, produce the ECM that embeds and imparts structural support and consistency to the myocardium. In HF, however, excessive or disturbed deposition of ECM under the coordination of fibroblasts, which transdifferentiate into myofibroblasts, and increase their migratory, proliferative and secretory activities, is a major contributor to progressive deformation and increased stiffness [43]. MiRs modulate the ECM not only by direct fibroblast action but also by interfering with cardiomyocyte paracrine biology [44]. Increased cardiac expression of miR-21 is mainly ascribed to fibroblasts and promotes survival and activation through the MAPK and PI3K/Akt pathways in HF models. Furthermore, miR21 also represses PTEN and Sprouty-1, which are negative regulators of PI3K/Akt and the MAPK cascade, respectively [45,46]. Other miRs do not interfere with fibroblast activation but rather regulate ECM genes. miR-29 inhibits fibrosis by negatively regulating ECM component mRNAs. Upregulation of transforming growth factor beta (TGF-b) in response to myocardial infarction (MI) downregulates miR-29 levels and hence stops holding back the synthesis of collagen, fibrillin and other ECM proteins [47]. Yet another mechanism of miR action is the modulation of pro-fibrotic paracrine-acting growth factors such as TGF-b. Two anti-fibrotic miRs, namely miR-30 and miR-133, are important negative regulators of TGF-b. Both are repressed during pathological hypertrophy but, while the latter is exclusive of cardiomyocytes, the former is also found in fibroblasts [44].

Potential clinical applications of miRs in HF MiRs as disease markers Many studies have established by now that miRs can be easily quantified in body fluids, including plasma and serum, with high sensitivity. Because of their potential major role in disease pathogenesis, strong correlations have been observed with disease in acute MI, coronary artery disease and HF [48]. Another contributing characteristic is their remarkable stability. While they are typically highly susceptible to degradation by nucleases, most of circulating miRs are protected in vesicles or exosomes which shield the RNA. Isolation of exosomes yields higher miR levels, but the fraction within exosomes is variable which adds to the complexity of determinations. In vitro reports suggest exosomes may mediate transfer of mRNAs and miRs between cells supporting a potential paracrine regulatory role [49], but although miRs in body fluids may be markers of disease, their origin and whether they serve any concrete functions remains to be established [50]. Other questions remain to be answered, namely the comparison with current biomarkers, the correlation with disease stage and response to therapy, the combination of several miRs to develop more accurate cumulative scores. 6

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Therapeutic approaches MiR targeting falls into the RNA interference therapeutic approach. It is the ultimate evolution from the realm of antisense oligonucleotides and small interference RNAs. The involvement in integrated physiological responses suggests an added value of specificity but precludes targeting specific mRNAs. New molecular developments may allow the inhibition of miR-mRNA binding and an additional step towards specificity. Regarding pharmacology and pharmacokinetics, RNA-based therapy is limited by molecular instability, complex and expensive synthesis, immunomodulation, off-target effects and difficulties in achieving long-lasting effects through endogenous expression [51]. Most of these limitations are circumvented in miR-targeting. Antagomirs are antisense oligonucleotides that cancel target mature miRs upon binding. Various chemical modifications have been developed to enhance their stability, protect them from nucleic acid-degrading enzymes and frustrate cellular uptake. SPC3649 (Miravirsen), a selective inhibitor of the liver specific miR-122 that is required for Hepatitis C virus replication had promising efficacy and safety results in primates [52] and in previously untreated hepatitis C patients (Phase 2a clinical trial; ClinicalTrials.gov Identifier: NCT01200420). An ongoing study now evaluates miR-122 antagonist by subcutaneous route in null responders to standard therapy (ClinicalTrials.gov Identifier: NCT01727934). Such progresses are still far from accomplished in the cardiovascular field, and it is difficult to envisage such a specific and straightforward approach in the complex pathophysiology of HF. Nevertheless, it is foreseeable that administration of miR antagonists (antagomirs) may offset or counteract the disturbances observed in myocardial hypertrophy and HF and perhaps alter the course of disease, at least based on preliminary animal studies [53]. Another approach to therapeutic modulation of miRs and widespread target mRNA gene silencing is the miR-mimic technology. The miR-mimic functions in a manner similar to endogenous miRs and, unlike antagomirs, cannot be stabilised. Higher doses are needed for effectiveness, which may hinder clinical application. Nonetheless, miR-mimics have already been used in vitro and topically injected in rodents’ hearts in vivo [54]. The complex and multifaceted pathophysiology of HF has baffled most therapeutic approaches for decades, the multifarious orchestrating role played by miRs may pose a unique opportunity to target HF pathophysiology at its core. Further preclinical developments and small scale clinical trials are warranted.

Concluding remarks Growing knowledge on miRs is dramatically changing our perspective on cardiac physiology and HF pathogenesis. Many of the intricate underlying molecular disturbances are partly orchestrated by miRs and understanding this gives way to new focused therapeutic strategies. Further studies are warranted but miR-based diagnostic and therapeutic tools are probably to become a possibility in a near future, increasing lifespan and quality of life of patients afflicted with HF. Although regrettably many functions of miRs are yet to be deciphered and substantial gaps must be filled to fully understand their interrelationships with the molecular pathways on which they operate, demanding a word of caution for potential side effects and need for organ or tissue selectivity, currently miRs probably constitute the best opportunity for targeting the foundations of HF therapeutically.

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