Introduction

MicroRNAs (miRNAs) are endogenous, single-stranded, small (approximately 22 nucleotides in length), noncoding RNAs. miRNAs are generally regarded as negative regulators of gene expression by inhibiting translation and/or promoting mRNA degradation by base pairing to complementary sequences within the 3′ UTR region of protein-coding mRNA transcripts [1–3]. However, recent studies have suggested that miR-binding sites are also located in 5′ UTRs or ORFs, and the mechanism(s) of miR-mediated regulation from these sites has not been defined [4–7]. The first miRNA assigned to a specific function was lin-4, which targets lin-14 during temporal pattern formation in Caenorhabditis elegans [8]. Subsequently, a variety of miRNAs have been discovered. More than 500 miRNAs have been cloned and sequenced in humans, and the estimated number of miRNA genes is as high as 1000 in the human genome [9]. Each miRNA regulates dozens to hundreds of distinct target genes; thus, miRNAs are estimated to regulate the expression of more than a third of human protein-coding genes [10]. On the other hand, accumulating evidence suggests that miRNAs are regulated by various mechanisms, including epigenetic changes [11]. Thus, the full picture of miRNA-associated regulation remains quite complex.

Cardiovascular disease is the leading cause of morbidity and mortality in developed countries. The pathological process of the heart is associated with an altered expression profile of genes that are important for cardiac function. Much of our current understanding of cardiac gene expression indicates that it is controlled at the level of transcriptional regulation, in which transcription factors associate with their regulatory enhancer/promoter sequences to activate gene expression [12]. The regulation of cardiac gene expression is complex, with individual genes controlled by multiple enhancers that direct very specific expression patterns in the heart. miRNAs have reshaped our view of how cardiac gene expression is regulated by adding another layer of regulation at the post-transcriptional level.

The implications of miRNAs in the pathological process of the cardiovascular system have recently been recognized, and research on miRNAs in relation to cardiovascular disease has now become a rapidly evolving field. Here, we review the available published studies that show the involvement of miRNAs in different aspects of the cardiovascular system.

miRNAs have been reviewed recently in several specific systems, including cardiovascular development, cardiac fibrosis and arrhythmia [13–15]. As is common to all new and rapidly moving fields, it is relatively hard to obtain an overview of the available knowledge from reviews. In this minireview, we summarize the current understanding of miRNA function in the heart and outline details of what is known about their putative targets. In addition, we review several aspects of the regulation of miR expression and their roles in cell signaling that have not been addressed in a cardiovascular context in the accompanying minireviews [11,16].

Cardiac development

One approach for studying the comprehensive requirements of miRNAs during vertebrate development has been to create mutations in the miRNA processing enzyme, Dicer. Several study groups have disrupted the gene for Dicer in mice and reported that the loss of Dicer resulted in embryonic lethality at embryonic day (E)7.5, before body axis formation, as a result of either a loss of pluripotent stem cells [17] or impaired angiogenesis in the embryo [18]. Dicer1 hypomorphic expression mice also exhibited corpus luteum insufficiency and infertility as a result of impaired angiogenesis [19]. To understand the role of miRNAs in the developing heart, cardiac-specific deletion of Dicer was generated using Cre recombinase expressed under the control of endogenous Nkx2.5 regulator elements. Nkx2.5-Cre is active from E8.5, during heart patterning and differentiation, although only after the initial commitment of cardiac progenitors. These embryos showed cardiac failure as a result of a variety of developmental defects, including pericardial edema and underdevelopment of the ventricular myocardium, which resulted in embryonic lethality at E12.5. These phenotypes are consistent with the defects during heart development observed in zebrafish embryos devoid of Dicer function [20]. It will be important to determine whether Dicer is required for earlier stages of cardiogenesis before E8.5. Dicer activity is also required for normal functioning of the mature heart because adult mice lacking Dicer in the myocardium have a high incidence of sudden death, cardiac hypertrophy and reactivation of the fetal cardiac gene program [21].

Recently, Rao et al. [22] generated mice with a muscle-specific deletion of the DiGeorge syndrome critical region gene 8 (DGCR8), which is another component of the miRNA biogenesis pathway, by the use of muscle creatine kinase-Cre mice and a conditional floxed allele of the DGCR8 [22]. Because endogenous muscle creatine kinase expression reportedly peaks around birth and declines to 40% of peak levels by day 10, these mice can be used to determine the importance of the miRNA pathway in muscle homeostasis. The phenotypic outcome was similar to the cardiac-specific Dicer deficient mice, showing a critical role for miRNAs in maintaining cardiac function in mature cardiomyocytes. It was also reported [22] that miR-1 was quite enriched and accounted for almost 40% of all known miRNAs in the adult heart by the deep sequencing of a small RNA library. Because this result is quite different from the findings obtained in other studies [36–42], additional experiments using a high-throughput analyzer are required.

Two widely conserved miRNAs that display cardiac- and skeletal-muscle-specific expression during development and in adults are miR-1 and miR-133, which are derived from a common precursor transcript [23,24]. miR-1 has been shown to regulate cardiac differentiation [23,25–27] and control heart development in mice by regulation of the cardiac transcription factor Hand2 [23]. The importance of miR-1 in cardiogenesis was shown in mice lacking miR-1-2 [26]. Although miR-1-1, which targets the same sequences as miR-1-2, is still expressed in miR-1-2-deficient mice, these mice had a spectrum of abnormalities, including ventricular septal defects in a subset that suffer early lethality, cardiac rhythm disturbances in those that survive, and a striking myocyte cell-cycle abnormality that leads to hyperplasia of the heart with nuclear division persisting postnatally. With regard to miR-133, mice lacking either miR-133a-1 or miR-133a-2 are normal, whereas deletion of both miRNAs causes lethal ventricular septal defects in approximately half of the double-mutant embryos or neonates [28]. miR-133a double mutant mice that survive to adult succumb to dilated cardiomyopathy and heart failure. Dysregulation of cell cycle control genes and aberrant activation of the smooth muscle gene program were observed in double-mutant mice, which may be attributable to the upregulation of the miR-133a mRNA targets, cyclin D2 and serum response factor (SRF).

Previous studies have indicated that miRNAs are broadly important for proper organ development. However, their individual temporal and spatial functions during organogenesis are largely unknown. The heart has been a particularly informative model for such organ patterning, with numerous transcriptional networks that establish chamber-specific gene expression and function [29]. Zebrafish have a two-chambered heart containing a single atrium and ventricle separated by the atrioventricular canal [30]. miR-138 is specifically expressed in the ventricular chamber of the zebrafish heart. Temporal-specific knockdown of miR-138 in zebrafish by morpholino and antagomiR led to expansion of atrioventricular canal gene expression into the ventricular chamber and failure of ventricular cardiomyocytes to fully mature, indicating that miR-138 is required for cardiac maturation and pattering in zebrafish [31]. It is noteworthy that miR-138 is required during a discrete developmental window, 24–34 h post-fertilization. Transcriptional networks that establish chamber-specific gene expression are highly conserved and miR-138 is also conserved across species, ranging from zebrafish to humans; thus, it will also be interesting to determine whether miR-138 plays similar roles in the patterning of the mammalian four-chambered heart.

Cardiac hypertrophy

Because cardiac hypertrophy (i.e. an increase in heart size) is associated with almost all forms of heart failure, it is of clinical importance that we understand the mechanisms responsible for cardiac hypertrophy. It has two forms: (a) physiological, where the heart enlarges in healthy individuals subsequent to heavy exercise and is not associated with any cardiac damage, and (b) pathological, where the size of the heart initially increases to compensate for the damage to cardiac tissue, but subsequently leads to a decline in left ventricular function [32].

In the model of physiological hypertrophy, only one study [33] has demonstrated that rats subjected to exercise training and transgenic mice with selective cardiac overexpression of a constitutively active mutant of the Akt kinase had reduced levels of the muscle-specific miRNAs, miR-1 and miR-133. In line with this finding, miR-1 and miR-133 were found to be downregulated in the plantaris muscle of mice in response to functional overload [34].

Pathological hypertrophy is mainly caused by hypertension, loss of myocytes subsequent to ischemic damage and genetic alterations that lead to cardiomyopathy. Moreover, metabolic abnormality or stress can also lead to hypertrophy [35]. Pathological hypertrophy is the phenotypic endpoint that has been mostly studied in relation to miRNAs of the heart to date. In animal models of cardiac hypertrophy, whole arrays of miRNAs have indicated that separate miRNAs are upregulated, downregulated or remain unchanged with respect to their levels in a normal heart [36–42]. In these studies, some miRNAs have been more frequently reported as being differentially expressed in the same direction in contrast to others, indicating the possibility that these miRNAs might have common roles in hypertrophy pathogenesis. For example, miR-21, miR-23a, miR-24, miR-125, miR-129, miR-195, miR-199, miR-208 and miR-212 have often been found to be upregualted with hypertrophy, whereas miR-1, miR-133, miR-29, miR-30 and miR-150 have often been found to be downregualted. Interestingly, the forced expression of individual miRNAs, such as miR-23a, miR-23b, miR-24, miR-195, miR-199a and miR-214, found to be upregulated with cardiac hypertrophy, was sufficient to induce hypertrophic growth. More specifically, miR-195 was sufficient to drive pathological cardiac growth when overexpressed in transgenic mice [36]. Despite the interesting phenotype of these mice, neither targets, nor mechanisms underlying the mechanism of action for miR-195 have been discovered. By contrast to miR-195, in vitro overexpression of miR-150 and miR-181b, which are downregulated in cardiac hypertrophy, resulted in reduced cardiomyocyte cell size [36]. The role of miR-21 in hypertrophy is controversial [43,44]. The ability of individual miRNAs to modulate cardiac phenotypes suggests that regulated expression of miRNAs is a cause rather than simply a consequence of cardiac remodeling.

Although the levels of many miRNAs have been demonstrated to be altered in cardiac hypertrophy by a series of high-throughput miRNA microarray analyses, the transcriptional machinery that regulates the expression of miRNAs during cardiac hypertrophy and the molecular mechanisms responsible for individual miRNA-mediated effects on cardiac hypertrophy need to be studied in more detail.

Transcriptional regulation of miRNAs is well studied for miR-1/miR-133. SRF is a cardiac-enriched transcription factor responsible for the regulation of organized sarcomeres in the heart [45]. SRF interacts synergistically with myocardin to activate miR-1-1 and miR-1-2 by binding to the upstream SRF-binding consensus element known as the CArG box [23]. Myocyte enhancer factor (MEF)2 also activates transcription of the bicistronic precursor RNA encoding miR-1-2 and miR-133a-1 via an intragenic muscle-specific enhancer [46]. It was reported that nuclear factor of activated T cells isoform 3 (NFATc3), which is well-documented as playing a key role in mediating the hypertrophic signal of calcineurin, as well as other stimuli [47], regulates the expression of miR-23a. NFATc3 can bind directly to the promoter region of miR-23a and activate its expression, which may convey the hypertrophic signal by suppressing the translation of muscle specific ring finger protein 1 [48]. It appears that different miRNAs have distinct mechanisms in regulating hypertrophy. miR-1 negatively regulates the expression of hypertrophy-associated calmodulin, MEF2a and GATA4, and attenuates calcium-dependent signaling through the calcineurin-NFAT pathway [49]. miR-133 inhibits hypertrophy through targeting RhoA and Cdc42 [33]. It was reported that targets of miR-208 include thyroid hormone receptor-associated protein 1 [50,51], suggesting that miR-208 initiates cardiomyocyte hypertrophy by regulating triiodothyronine-dependent repression of β-myosin heavy chain. miR-27a also regulates β-myosin heavy chain gene expression by targeting TRβ1 in cardiomyocytes [52].

An miRNA may have multiple targets and the currently available results do not exclude the involvement of any other molecules and/or pathways that can be regulated by miRNAs with reported functions.

Myocardial infarction and cell death

It is well established that acute myocardial infarction (MI) is a complex process in which multiple genes have been found to be dysregulated [53]. Therefore, it is reasonable to hypothesize that miRNAs could be involved in MI.

Cardiomyocyte death/apoptosis is a key cellular event in ischemic hearts. Ren et al. [54] applied a mouse model of cardiac ischemia/reperfusion (I/R) in vivo and ex vivo to determine the miRNA expression signature in ischemic hearts, and found that miR-320 expression was consistently dysregulated in ischemic hearts. They identified heat-shock protein 20, a known cardioprotective protein, as a target of for miR-320. Knockdown of endogenous miR-320 provides protection against I/R-induced cardiomyocyte death and apoptosis by targeting heat-shock protein 20. The miRNA expression signature in rat hearts at 6 h after MI revealed that miR-21 expression was significantly downregulated in infracted areas but upregulated in boarder areas [55]. Adenoviral transfer of miR-21 in vivo decreased cell apoptosis in the border and infracted areas through its target gene, programmed cell death 4, and activator protein 1 pathway.

In vitro experiments showed that miR-1 and miR-133 produced opposing effects on apoptosis induced by oxidative stress in H9c2 rat ventricular cells, with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic. Post-transcriptional repression of HSP60 and HSP70 by miR-1 and of caspase-9 by miR-133 contributes significantly to their opposing actions. miR-1 is also associated with the cell death pathway by inhibiting the translation of insulin-like growth factor-1 [56,57].

Early ischemia or hypoxia preconditioning is an immediate cellular reaction to brief hypoxia/reoxygenation cycles that involve de novo protein, but not mRNA synthesis [58]. It is described as a mechanism that protects the heart against subsequent prolonged ischemia or I/R induced damage [59]. A recent study by Rane et al. [60] revealed a unique function of miR-199a, serving as a molecular switch that triggers an immediate drop in gene expression in response to a decline in oxygen tension, possibly through selective miRNA stability and processing of the stem-loop. They showed that miR-199a directly targets and inhibits translation of hypoxia-inducible-factor-1α and Sirtuin1. Hif-1α regulates hypoxia-induced gene transcription and is regulated by a post-transcriptional oxygen-sensitive mechanism that triggers its prompt expression subsequent to a drop in oxygen levels. These results indicate that miR-199a is a master regulator of a hypoxia-triggered pathway and can be utilized for preconditioning cells against hypoxic damage. Because this result demonstrates a functional link between 2 key molecules that regulate hypoxia preconditioning and longevity, it would be of interest to examine the precise regulatory mechanism of miR-199a.

Recent studies have shown that some miRNAs are present in circulating blood and that they are included in exosomes and microparticles [61,62]. The levels of circulating miRNAs have been reported for several disease conditions [63,64]. In the cardiovascular diseases, studies on circulating miRNAs have been shown in a rat model of myocardial injury [65]. Recently, circulating miRNAs have been reported in patients with myocardial infarction [15]. Accordingly, it has been hypothesized that miRNAs in systemic circulation may reflect tissue damage and, for this reason, they can be used as a biomarker of myocardial infarction [66–68].

Cardiac fibrosis

Cardiac fibrosis is an important contributor to the development of cardiac dysfunction in diverse pathological conditions, such as MI, ischemic, dilated and hypertrophic cardiomyopathies, and heart failure and can be defined as an inappropriate accumulation of extracellular matrix proteins in the heart [69–74]. Cardiac fibrosis leads to an increased mechanical stiffness, initially causing diastolic dysfunction, and eventually resulting in systolic dysfunction and overt heart failure. In addition, fibrosis causes electrical connection disruption between cardiac myocytes, and hence increases the chance of arrhythmias. Finally, the enhanced diffusion distance for cardiac substrates and oxygen to the cardiac myocytes, caused by fibrosis, negatively influences the myocardial balance between energy demand and supply [71,72].

The miR-29 family, which is fibroblast enriched, targets mRNAs encoding a multitude of extracellular matrix-related proteins involved in fibrosis, including multiple collagens, fibrillins and elastin [75]. miR-29 is dramatically repressed in the border zone flanking the infracted area in the mouse model of MI. Downregulation of miR-29 would be predicted to counter the repression of these mRNAs and enhance the fibrotic responses. Therefore, it is tempting to speculate that upregulation of miR-29 may be a therapeutic option for MI.

miR-21 is among the most strongly upregulated miRNAs in response to a variety of forms of cardiac stress [16,36,75]. Recently, Thum et al. showed that miR-21 is upregulated in cardiac fibroblasts in the failing heart, where it represses the expression of Sprouty homolog1, a negative regulator of the extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway [76]. Upregulation of miR-21 in response to cardiac injury was shown to enhance extracellular signal-regulated kinase/mitogen-activated protein kinase signaling, leading to fibroblast proliferation and fibrosis. Phosphatase and tensin homolog (PTEN) has also been demonstrated to be a direct target of miR-21 in cardiac fibroblasts [77]. Previous reports characterize PTEN as a suppressor of matrix metalloprotease-2 expression [78,79]. I/R in the heart induced miR-21 in cardiac fibroblasts in the infracted region. Thus, I/R-induced miR-21 limits PTEN function and causes activation of the Akt pathway and increased matrix metalloprotease-2 expression in cardiac fibroblasts.

Connective tissue growth factor (CTGF), a key molecule involved in fibrosis, was shown to be regulated by two miRNAs; miR-133 and miR-30, which are both consistently downregulated in several models of pathological hypertrophy and heart failure [80]. miR-133 and miR-30 are downregulated during cardiac disease, which inversely correlates with the upregulation of CTGF. In vitro experiments designed to overexpress or inhibit these miRNAs can effectively repress CTGF expression by interacting directly with the 3′ UTR region of CTGF mRNA.

Taken together, these data indicate that miRNAs are important regulators of cardiac fibrosis and are involved in structural heart disease.

Arrhythmia

The electrical activities of the heart (i.e. the rate and force of contraction of the heart) are orchestrated by multiple categories of ion channels, which are transmembrane proteins that control the movement of ions across the cytoplasmic membrane of cardiomyocytes. Each heartbeat is initiated by a pulse of electrical excitation that begins in a group of specialized pacemaker cells and subsequently spreads throughout the heart. At rest, the membrane is selectively permeable to K⁺, and the electrochemical potential inside the myocyte is negative with respect to the outside. During electrical excitation, the membrane becomes permeable to Na⁺ and the electrochemical potential reverses or depolarizes. Thus, Na⁺ channels determine the rate of membrane depolarization. Connexin43 (Cx43) is critical for the ventricular gap junction communication, being responsible for inter-cell conduction of excitatory signals. L-type Ca²⁺ channels are mediators of Ca²⁺ influx and account for excitation-contraction coupling. L-type Ca²⁺ channels are located in sarcolemma, including the T-tubes facing the sarcoplasmic reticulum junction, and are activated by membrane depolarization. I_caL is important in heart function because it modulates action potential shape and contributes to pacemaker activities in the sinoatrial and atrioventricular nodal cells. When K⁺ channels open during repolarization, K⁺ exits from the cell because the channels allow the passive movement of ions down their respective concentration gradients. Thus, K⁺ channels govern the membrane potential and the rate of membrane repolarization. Pacemaker channels, which carry the nonselective cation currents, are critical in generating the sinus rhythm and ectopic heart beats. Because the heart beat is so dependent on the proper movement of ions across the surface membrane, disorders of ion channels, or channelopathies, which may result from genetic alterations in ion channel genes or aberrant expression of these genes, can render electrical disturbances predisposing to cardiac arrhythmias [81].

Recently, using luciferase reporter activity and western blot analysis, it was established that gap junction protein α1 (GJA1) (encoding Cx43) and potassium inwardly-rectifying channel, subfamily J, member 2 (KCNJ2) (encoding the K⁺ channel subunit Kir2.1) are target genes for miR-1 [82]. Cx43 is critical for inter-cell conductance of excitation [83–85] and Kir2.1 governs the cardiac membrane potential [86,87], both of which are important determinants for cardiac excitability. It was shown that miR-1 levels are increased in individuals with coronary artery disease and that, when miR-1 is overexpressed in normal and infracted rat hearts, this results in a slowed conduction velocity, excessively prolonged repolarization and the induction of PVCs and arrhythmias. On the other hand, blocking miR-1 function with antisense oligoribonucleotides was found to normalize the expression of Cx43 and Kir2.1, prevent QRS and QT prolongation, and reduce arrhythmias after MI.

Zhao et al. [26] demonstrated that one of the miR-1-2 targets is the cardiac transcription factor iroquois homeobox (Irx)5, which represses potassium voltage-gated channel, Shal-related subfamily, member 2 (KCND2), a potassium channel subunit (Kv4.2) responsible for transient outward K⁺ current (I_to) by use of a targeted deletion technique. The increase in Irx5 and Irx4 protein levels in miR-1-2 mutants corresponded well with a decrease in KCND2 expression. It is suggested that the combined loss of Irx5 and Irx4 disrupts mouse ventricular repolarization with a predisposition to arrhythmias when miR-1 levels are enhanced.

To date, the cardiac ion channel genes that have been confirmed experimentally to be targets of miR-1 or miR-133 include gap junction protein α1/Cx43/I_J [82], KCNJ2/Kir2.1/I_K1 [82], potassium voltage-gated channel, subfamily H (eag-related), member 2 (KCNH2)/ human ether-à-go-go-related gene (HERG)/I_Kr [88], potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1)/KvLQT1/I_Ks [89] and potassium voltage-gated channel, Isk-related family, member 1 (KCNE1)/mink/I_Ks [89]. The fact that altered expression of miRNAs can deregulate expression of cardiac ion channels provided novel insight into the molecular understanding of cardiac excitability.

However, considering the inherent capacity of miRNAs to target a broad range of proteins, the link between miR-1 and arrhythmia is far from clear, and more miR-1 targets may be involved. Terentyev et al. [90] investigated the effects of increased expression of miR-1 on excitation contraction coupling and Ca²⁺ cycling in rat ventricular myocytes using cellular electrophysiology and Ca²⁺ imaging. They found that the protein phosphatase PP2A regulating subunit B56α is potentially an important target for miR-1 in the heart and, through translational inhibition of this mRNA, miR-1 causes Ca/calmodulin kinase II-dependent hyperphosphorylation of the ryanodine receptor (RyR2), enhances RyR2 activity, and promotes arrythmogenic sarco(endo)plasmic reticulum Ca²⁺ release.

Thus, miR-1 may have important pathophysiological functions in the heart, and may be a potential anti-arrythmic target.

Angiogenesis and vascular diseases

Recently, a few specific miRNAs that regulate endothelial cell functions and angiogenesis have been described. Pro-angiogenic miRNAs include let7f and miR-27b [91], miR-17-92 cluster [92], miR-126 [93,94], miR-130a [95], miR-210 and miR-378, [96,97]. MiRNAs that exert anti-angiogenic effects include miR-15/16 [98,99], miR-20a/b [98], miR-92a [100] and miR-221/222 [101,102].

Inflammation not only comprises an important part of the host defenses against infection and injury, but also contributes to the initiation and progression of atherosclerosis [103,104]. The response-to-injury hypothesis proposed that endothelial dysfunction caused by, for example, elevated low density lipoproteins, free radicals, hypertension, diabetes mellitus and/or other factors, represents an early step in atherosclerosis [103].

Adhesion molecules expressed by activated endothelial cells play a key role in regulating leukocyte trafficking to sites of inflammation. Resting endothelial cells normally do not express adhesion molecules; however, cytokines activate endothelial cells to express adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), which mediate leukocyte adherence to endothelial cells. Harris et al. [105] showed that endothelial cells predominantly express miR-126, which inhibits VCAM-1 expression. On the other hand, transfection of endothelial cells with an oligonucleotide that decreases miR-126 permitted an increase in tumor necrosis factor-α stimulated VCAM-1 expression and increased leukocyte adherence to endothelial cells.

Recently, Ji et al. [106] revealed miRNAs that are aberrantly expressed in the vascular walls after balloon injury. Modulating an aberrantly overexpressed miR-21, via antisense-mediated depletion, had a significant negative effect on neointimal lesion formation. It was also demonstrated that PTEN and Bcl-2 were involved in miR-21-mediated cellular effects. Liue et al. [107] also revealed that miR-221 and miR-222 expression levels were elevated in rat carotid arteries after angioplasty. Moreover, p27 (Kip1) and p57 (Kip2) were found to be the two target genes that were involved in miR-221- and miR-222-mediated effects on vascular smooth muscle cell (VSMC) growth. Knockdown of miR-221 and miR-222 resulted in decreased VSMC proliferation both in vitro and in vivo.

Another study demonstrated that the angiotensin II type 1 receptor (AT1R) and miR-155 are coexpressed in endothelial cells and VSMCs, and that miR-155 translationally represses the expression of AT1R [108]. The gene for AT1R is highly polymorphic. In particular, a single nucleotide polymorphism has been described in which there is an A/C transversion at position 1166 in the 3′ UTR of this gene. The increased frequency of the +1166 allele has been associated with essential hypertension, cardiac hypertrophy and MI [109–111], probably mediated by enhanced AT1R activity. Interestingly, the presence of the +1166 C-allele interrupts base pairing complementarity within the 3′ UTR of AT1R, and thereby, decreases translational repression of human AT1R by miR-155 [108].

Thus, miR-21, miR-155, miR126, miR-221 and miR-222 might be important modulators of vascular disease and vessel remodeling.

Heart failure

Because cardiac hypertrophy, fibrosis, arrhythmia, and coronary artery disease can cause heart failure, all of the miRNAs discussed so far are associated with this disease entity.

It is well known that heart failure is characterized by left ventricular remodeling and dilatation associated with activation of a fetal gene program triggering pathological changes in the myocardium associated with progressive dysfunction. Consistent with the reactivation of the fetal gene program during heart failure, an impressive similarity has been found between the miRNA expression pattern occurring in human failing hearts and that observed in the hearts of 12–14-week-old fetuses [42]. Indeed, more than 80% of the induced and repressed miRNAs were regulated in the same direction in fetal and failing heart tissue compared to healthy adult control left ventricle tissue. The most consistent changes were upregulation of miR-21, miR-29b, miR-129, miR-210, miR-211, miR-212 and miR-423, with downregulation of miR-30, miR-182 and miR-526. Interestingly, gene expression analysis revealed that most of the upregulated genes were characterized by the presence of a significant number of the predicted binding sites for downregulated miRNAs and vice versa.

Recently, many profiling studies have been conducted and revealed a large number of miRNAs that are differentially expressed in heart failure, pointing to the new mode of regulation of cardiovascular diseases [2,38,40,41,49,80]. Horie et al. [112] indicated that miR-133 may fine-tune glucose transporter 4 via targeting kruppel-like factor-15 in heart failure and that there may be many other miRNA functions in specific disease settings. Nishi et al. [113] suggested that four different miRNAs, which have the same seed sequence, regulate mitochondrial membrane potential during the transition from cardiac hypertrophy to failure.

miRNA exerts its role in the treatment with chemotherapeutic agent. It is suggested that the upregulation of miR-146a after Dox treatment is involved in acute Dox-induced cardiotoxicity by targeting ErbB4 [114]. Inhibition of both ErbB2 and ErbB4 signalling may be one of the reasons why those patients who receive concurrent therapy with Dox and trastuzumab suffer from congestive heart failure.

Metabolic syndrome and cholesterol regulation

Recent studies have indicated that miR-33 controls cholesterol homeostasis based on knockdown experiments using antisense technology [115–117]. miR-33 deficient mice were generated and the critical role for miR-33 in the regulation of ATP-binding cassette transporter A1 expression and high density lipoprotein (HDL) biosynthesis was confirmed in vivo [118].

In humans, sterol regulatory element binding protein (SREBP)1 and SREBP2 encode miR-33b and miR-33a, respectively [117]. It is well known that hypertriglycemia in metabolic syndrome is caused by the insulin-induced increase in SREBP1c mRNA and protein levels [119,120]. Low HDL often accompanies this situation and it is possible that the reduction in HDL is caused by a decrease in ATP-binding cassette transporter A1 because of the increased production of miR-33b from the insulin-induced induction of SREBP1c. Although it is impossible to prove this in animal models that lack miR-33b, antagonizing miR-33 could be a promising way to raise HDL levels when the transcription of both SREBPs is upregulated. Thus, a combination of silencing of endogenous miR-33 and statins may be a useful therapeutic strategy for raising HDL and lowering low density lipoprotein levels, especially for metabolic syndrome subjects.

The potential binding sites of miRNAs (included in the TargetScan datbase; http://www.targetscan.org/) associated with cardiovascular diseases are summarized in Table 1. Single nucleotide polymorphism information is derived from the Single Nucleotide Polymorphism Database (http://www.ncbi.nlm.nih.gov/projects/SNP/).

Conclusions

Recent studies provide clear evidence that miRNAs modulate a diverse spectrum of cardiac functions with developmental, pathophysiological and clinical implications. The biology of miRNAs in cardiovascular disease represents a young research area and is still an emerging field. Identifying the gene targets and signaling pathways responsible for their cardiovascular effects is critical for future studies.

Taken together, the recent evidence shows that miRNAs play powerful roles in cardiovascular systems and are sure to open the door to previously unappreciated medical therapies.