Cardiac-specific miRNA in cardiogenesis, heart function, and cardiac pathol-
ogy (with focus on myocardial infarction)

Dimitry A. Chistiakov, Alexander N. Orekhov, Yuri V. Bobryshev

PII: S0022-2828(16)30062-1
DOI: doi: 10.1016/j.yjmcc.2016.03.015
Reference: YJMCC 8363

To appear in: Journal of Molecular and Cellular Cardiology

Received date: 20 October 2015

Revised date: 9 February 2016
Accepted date: 24 March 2016

Please cite this article as: Chistiakov Dimitry A., Orekhov Alexander N., Bobryshev
Yuri V., Cardiac-specific miRNA in cardiogenesis, heart function, and cardiac pathology
(with focus on myocardial infarction), Journal of Molecular and Cellular Cardiology (2016),
doi: 10.1016/j.yjmcc.2016.03.015

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 ACCEPTED MANUSCRIPT

JMCC9652R.2
Review

Cardiac-specific miRNA in cardiogenesis, heart function, and

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cardiac pathology (with focus on myocardial infarction)

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*
Dimitry A. Chistiakov a, Alexander N. Orekhov b, c,d ,Yuri V. Bobryshev b,e,f,

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a
Department of Molecular Genetic Diagnostics and Cell Biology, Division of Laboratory Medicine,

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Institute of Pediatrics, Research Center for Children's Health, 119991 Moscow, Russia;
b
Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, Moscow
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125315, Russia;
c
Department of Biophysics, Biological Faculty, Moscow State University, Moscow 119991, Russia;
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Institute for Atherosclerosis Research, Skolkovo Innovative Center, Moscow 121609, Russia;
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Faculty of Medicine, School of Medical Sciences, University of New South Wales, Sydney, NSW
2052, Australia;
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School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia.

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Short title: MicroRNAs in myocardial infarction

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* Corresponding author:
Yuri V. Bobryshev, Ph.D.
School of Medical Sciences
Faculty of Medicine
University of New South Wales
Sydney, NSW 2052
Australia
Tel/Fax: + 612 93851217
E-mail: y.bobryshev@unsw.edu.au

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ABSTRACT

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Cardiac miRNAs (miR-1, miR133a, miR-208a/b, and miR-499) are abundantly expressed in the
myocardium. They play a central role in cardiogenesis, heart function and pathology. While miR-1

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and miR-133a predominantly control early stages of cardiogenesis supporting commitment of
cardiac-specific muscle lineage from embryonic stem cells and mesodermal precursors, miR-208

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and miR-499 are involved in the late cardiogenic stages mediating differentiation of cardioblasts to
cardiomyocytes and fast/slow muscle fiber specification. In the heart, miR-1/133a control cardiac

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conductance and automaticity by regulating all phases of the cardiac action potential. miR-208/499
located in introns of the heavy chain myosin genes regulate expression of sarcomeric contractile
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proteins. In cardiac pathology including myocardial infarction (MI), expression of cardiac miRNAs
is markedly altered that leads to deleterious effects associated with heart wounding, arrhythmia,
increased apoptosis, fibrosis, hypertrophy, and tissue remodeling. In acute MI, circulating levels of
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cardiac miRNAs are significantly elevated making them to be a promising diagnostic marker for
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early diagnosis of acute MI. Great cardiospecific capacity of these miRNAs is very helpful for
enhancing regenerative properties and survival of stem cell and cardiac progenitor transplants and
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for reprogramming of mature non-cardiac cells to cardiomyocytes.

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Keywords: Heart; Myocardial infarction; miR-1; miR-133a; miR-208a/b; miR-499

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Contents

1. Introduction
2. miR-1

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2.1. miR-1 expression regulators
2.2. miR-1 is involved in control of differentiation of embryonic stem cells to mesodermal

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precursors

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2.3. miR-1 regulates differentiation of mesodermal precursors to cardiomyocytes
2.4. miR-1 drives reprogramming of other cell types to cardiomyocytes

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2.5. miR-1 in cardiac depolarization
2.6. miR-1 in cardiac repolarization
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2.7. miR-1 and calcium cycling in cardiomyocytes
2.8. miR-1 and conduction between cardiac muscle cells
2.9. Proapoptotic role of miR-1 in myocardial infarction
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2.10. Arrhythmogenic potential of miR-1

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2.11. miR-1 aggravates oxidative stress in cardiomyocytes

2.12. Therapeutic potential of miR-1 in cardiovascular pathology
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3. miR-133a
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3.1. Role of miR-133a in cardiogenesis

3.2. miR-133a in cardiomyocyte-specific reprogramming
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3.3. miR-133a in cardiac conductance

3.4. miR-133a in myocardial infarction
3.5. Therapeutic potential of miR-133a
4. miR-208a/b
4.1. miR-208 in cardiogenesis
4.2. miR-208 in heart function
4.3. miR-208 as a diagnostic marker of early myocardial infarction
4.4. Therapeutic potential of miR-208
5. miR-499
5.1. miR-499 in cardiogenesis
5.2. miR-499 in acute myocardial infarction

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5.3. Therapeutic potential of miR-499

Conclusion
Sources of funding
Disclosures

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References

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1. Introduction

Myocardial infarction (MI) is the most frequent end-point of cardiovascular pathology that

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leads to higher mortality in economically developed countries. MI is resulted from acute cardiac
injury caused by ischemic and hypoxic conditions accompanied with oxidative stress and

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inflammation in the infarcted area [1]. Chronic loss of a proper signaling in cardiac muscle leads to

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expanding infarct region, amplifying reactive oxygen species (ROS) production, cardiac muscle cell
death, and remodeling of the survived functional cardiomyocytes. Initially, cardiac remodeling

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starts as an adaptive response against heart damage but this process becomes maladaptive in
pathogenic conditions and generally results in cardiac fibrosis, dilated cardiomyopathy, and heart
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failure (HF) [2]. Post-infarction remodeling is initiated due to poor blood supply in the infarcted
myocardium [3] and goes along with pathological changes in intracardiac signaling [2].
Cardiac remodeling is a complex process, with the involvement of many molecular
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components. At present, investigating the contribution of endogenous microRNAs (miRNAs) to

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cardiovascular pathogenesis is on a cutting edge of studies focused on dissecting molecular

mechanisms of HF and post-infarction complications [4]. MiRNAs belong to a class of small non-
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coding RNAs (an average size of 22 nucleotides), which negatively regulate expression of target
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genes through binding to the 3’untranslated region of miRNA targets. MiRNAs play a crucial role
in the control of gene expression since approximately 2/3 of known human genes are regulated by
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miRNAs [5]. MiRNAs are critically involved in heart function and heart dysfunction in
physiological and pathophysiological conditions respectively [6]. Da Costa Martins et al. [7]
showed that genetic deletion of DICER, a key nuclease involved in miRNA biogenesis and
function, results in spontaneous start of myocardial remodeling. This observation hence underlines a
role of miRNAs in the regulation of a proper heart organogenesis and function.
MiR-1, miR-133a, miR-208a/b, and miR-499 are believed to belong to cardiac-specific or
cardiac-enriched miRNAs. These miRNAs are involved in differentiation of medodermal precursors
and transdifferentiation/reprogramming of adult fibroblasts/myofibroblasts to mature heart
myocytes and maintaining functionality and survival of cardiac muscle cells [8]. In the heart,
activity and function of these miRNAs is strictly regulated to ensure proper cardiac contractility and
conduction. In pathologic conditions, deregulation of expression of cardiac miRNAs may lead to

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progressive HF associated with arrhythmia, hypoxia, ischemia, left ventricular dilatation, fibrosis,
myocardial necrosis, and other destructive processes. In this review, we characterize a role of these
miRNAs in cardiac biology and pathobiology.

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2. miR-1

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Human miR-1 has two isomers (miR-1-1 and miR-1-2) that have identical sequences but are

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encoded by distinct genes. miR-1-1 is encoded by the miR1-1/miR-133a-2 gene cluster located on
chromosome 20q13.3 and transcribed as a common precursor for both miRNAs. A precursor for

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miR-1-1 serves as a source for mature miR-1-3p and miR-1-5p. Accordingly, miR-1-2 is encoded
by the miR-133a-1/miR-1-2 gene cluster located in intron 12 of the mindbomb ubiquitin-protein
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ligase E3 (MIB1) on chromosome 18q11 [9]. In contrast to the miR-1-1 precursor, the only miR-1-
3p is generated from the precursor for miR-1-2. Duplication of the bicistronic miR-1/miR-133a
cluster is evolutionary preserved in vertebrates [10]. A single copy of this cluster was found in
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ascidians, which are ancestors of chordates [11].

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MiR-1 is highly expressed in cardiac and skeletal muscles. This miRNA plays a key role in
differentiation and proliferation of muscle cells [12].
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2.1. miR-1 expression regulators

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Serum response factor (SRF), MyoD, myocardin, and myocyte enhancer factor-2 (Mef2) are
primary transcriptional activators of miR-1 expression [13-15]. Myogenin enhances expression of
miR-1/miR-133 by binding to the enhancer region upstream the target gene [16]. In contrast,
myostatin acts as a negative regulator of expression of miR-1 and other myomiRs [17].
Mammalian target of rapamycin (mTOR), a key regulator of myogenesis (Yoon and Chen
2013)[18], is involved in indirect stimulation of miR-1 expression by up-regulation of MyoD
through increasing stability of this factor [19]. miR-1 suppress histone deacetylase 4 (HDAC4) that
inhibits expression of follistatin involved in the regulation of myocyte fusion, an essential stage in
heart development [20]. On the other hand, follistatin activates mTOR-dependent signaling through
stimulating mothers against decapentaplegic homolog 3 (Smad3) [21]. Indeed, miR-1 and mTOR
could cooperate in myogenesis.

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In myoblasts, the bioavailability of miR-1 and miR-206 is regulated by TAR DNA-binding

protein TDP43, a transcription repressor that is able to interact with both DNA and RNA [22]. The
assembling of miR-1 with TDP43 limits incorporation to the RNA-induced silencing complex
(RISC) essential for functional activity of miRNAs. Muscleblind-like 1 (MBNL1), an RNA-binding

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protein, could bind to miR-1 preventing its interaction with LIN28 involved in miRNA biogenesis
[23]. KH-type splicing regulatory protein (KHSRP), another RNA-binding factor, could modulate

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processing miR-1-1 from the primary transcript [24]. Number of known miR-1 expression

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regulators constantly increases. Further studies are required for understanding the complexity of
regulatory networks involving miR-1 in muscle development.

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2.2. miR-1 is involved in control of differentiation of embryonic stem cells to mesodermal
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precursors

In mice, targeted deletion of miR1-1 leads to abnormalities in heart development (such as

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ventricular septal defect and myocyte cell cycle aberrations) and cardiac function including heart
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arrhythmia and disturbances in heart conduction [25]. Knockout of miR1-2 results in the same
phenotype but animals generally survive due to the expression of miR-1 from the remaining miR1-1
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gene [26]. Mice lacking both miR-1 genes die soon after birth due to serious developmental defects.
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In muscles of miR-1-deficient animals, mitochondrial abnormalities along with defective

sarcomeres were observed [27]. These findings suggest for a profound role of this miRNA in the
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regulation of cardiac muscle development.

In developing myocardium, overproduction of miR-1 leads to reduced proliferation of
ventricular cardiac muscle cells since miR-1 inhibits translation of heart and neural crest derivatives
expressed transcript 2 (Hand2), an embryonic transcription factor involved in expansion of
ventricular myocytes [13]. In addition, overexpression of miR-1 increases cardiac excitation-
contraction and promotes arrhythmogenesis through down-regulation of the regulatory subunit
B56α of protein phosphatase PP2A [28]. Reduced PP2A activity decreases Ca2+ sensitivity of
miofilaments [29]. miR-1 overproduction also increases phosphorylation of the ryanodine receptor
(RyR2) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) that enhances Ca2+ release from
the sarcoplasmic reticulum (SR) and induces spontaneous arrhythmogenic oscillations in

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cardiomyocytes [28]. Indeed, expression and activity of miR-1 should be strictly controlled to
support normal cardiac development and function.
miR-1 is involved in generation of mesodermal precursors from embryonic stem cells
(ESCs) by inhibiting ESC differentiation to endodermal and neuroectodermal precursors as

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reflected by induction of Nk2 homeobox 5 (Nkx2.5), a early marker of cardiac mesoderm [30].
Nkx2.5 is a transcription factor that induces expression of Mef2 and therefore expression of Mef2-

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dependent downstream targets [31]. In ESCs, SRF primes induction of miR-1 expression that

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followed by arrest of expression of non-muscle genes by miR-1-dependent translational repression
of the Notch ligand Delta-like 1 (Dll-1), a factor involved in mediating cell fate decisions [30].

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miR-1 also represses transcription factor Hes-1 [32] that drives differentiation of stem cells towards
the ectoderm fate [33]. In addition, miR-1 up-regulates cyclin-dependent kinase-9 (Cdk9) [34], a
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core component of the p300/GATA-4 complex involved in myocardial cell differentiation [35].
miR-1 also stimulates myogenesis via suppressing HDAC4 that inhibits Mef2-dependent expression
of muscle genes at transcriptional level [12, 36]. Furthermore, Schneider et al. [37] visualized
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colocalization of miR-1 with myosin-positive cells (cardiomyocytes) during cardiomyogenesis from

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embryoid bodies. These observations indeed suggest for the important role of miR-1 in the
commitment of the muscle cell lineage in early stages of cardiogenesis (Figure 1).
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2.3. miR-1 regulates differentiation of mesodermal precursors to cardiomyocytes

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Global transcriptome analyses showed continuous increase in miR-1 expression in

cardiogenesis, with the highest levels in fetal and especially in the adult heart [38, 39]. Furthermore,
genome-wide screening of signaling networks in Drosophila revealed conserved pathways that are
influenced by miR-1 in the regulation of the polarity of cardiac progenitor cells [40]. miR-1 levels
were observed to be increased in human cardiomyocytes but not in endothelial cells (ECs) or
smooth muscle cells produced from ESC-derived multipotent cardiovascular progenitors (MCPs).
In MCPs, miR-1 overexpression leads to the inhibition of Wnt and fibroblast growth factor (FGF)
signaling via targeting of Frizzled-7 and fibroblast growth factor receptor substrate 2 (FRS2)
respectively. This in turn determines miR-1-dependent fate switching of MCPs towards formation
of cardiac muscle cells [41].

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Myocardin (encoded by the MyoCD gene) serves as a myocyte-specific transcriptional

coactivator of SRF [42]. Myocardin-dependent induction of miR-1 supports the commitment of the
muscle cell lineage [43, 44]. Interestingly, miR-1 could support differentiation of ESCs to vascular
smooth muscle cells (VSMCs) by inhibiting Kruppel-like factor 4 (KLF4), a transcription factor

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involved in supporting stemness of ESCs. Furthermore, miR-1 expression was shown to be
increased during ESC differentiation to VSMCs [44].

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In embryonic heart, overexpression of myocardin, a key regulator of smooth muscle-specific

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transcriptional program, leads to the arrest of cardiomyocytes in immature state. In cooperation with
miR-133, miR-1 down-regulates myocardin levels thereby providing a negative feedback loop in

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response to myocardin-dependent activation [45]. miR-1-dependent suppression of myocardin
results in inhibiting expression of telokin (a transcriptional target of myocardin), a smooth muscle-
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restricted repressor of the regulatory myosin light chain-2 (MLC2) phosphorylation [27]. Indeed,
miR-1-mediated suppression of myocardin favors preferential differentiation of mesoderm towards
skeletal and cardiac-specific phenotype.
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2.4. miR-1 drives reprogramming of other cell types to cardiomyocytes

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Cardiac muscle cells could be produced through the mechanism of transdifferentiation, i.e.
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conversion of other differentiated cell type to another by avoiding the pluripotent stage.
Jayawardena et al. [46] showed that transient tranfection of murine myofibroblasts with four
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exogenic cardiac miRNA (miR-1, miR-133, miR-208, and miR-499) leads to the generation of
functional cardiomyocyte-like cells characterized by expression of myocardial-specific markers,
sarcomere formation, and induction of spontaneous Ca2+flux. These miRNA as transgenic lentiviral
constructs were then injected to fibroblast-specific protein 1-Cre mice/tandem dimer mice with
experimental cardiac injury. The treatment resulted in improved cardiac function associated with
induction of cardiac markers and sarcomere organization in cardiomyocyte-like cells [47].
Human adult and neonatal foreskin fibroblasts could be also converted to cardiomyocytes by
treatment of a cocktail consisting of four transcription factors (GATA4, myocardin, Hand2, and T-
box5) and two myomiRs (miR-1 and miR-133) [48]. After 11 week of incubation, fibroblasts
acquired cardiomyocyte-like properties such as sarcomere-like structures, voltage-gated Ca2+
channels, with a small subpopulation of spontaneously contractile cells [48]. These experiments

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suggest for a promising potential of miRNA-based strategies for regeneration of myocardial tissue
after heart injury by reprogramming autologous non-muscle cells.

2.5. miR-1 in cardiac depolarization

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Cardiac muscle has a unique capacity to contract automatically and rhythmically due to self-

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exciting. The heartbeating is a cyclic and constitutive process that supports uninterrupted supply of

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the body with blood. Indeed, the tight coordination of the cardiac contraction cycle (that includes
excitation, impulse propagation, and repolarization) is a vitally important. Disturbances in the

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control of heart automaticity lead to cardiac arrhythmias whose manifestation is a major contributor
to cardiovascular mortality by significant increasing the risk of sudden death [49].
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The role of miRNAs in the regulation of cardiac contraction and conduction is actively
studying. To date, the involvement of miRNAs in the regulation of cardiac depolarization was
shown [50]. miR-1 targets the human (not mouse) CaV1.2 (or CACNA1C) gene encoding the α1c-
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subunit of the cardiac L-type Ca2+ channel involved in the plateau phase (ICaL) (a stage between the
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early depolarization and late repolarization) [23]. In ventricular myocytes, the CaV1.2 channel
controls potential duration and excitation-contraction coupling [51]. Among Ca2+conductances, L-
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type Ca2+ channels play the most profound role in supporting Ca2+ flow in cardiac muscle cells.
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Changes in membrane density and permeability of these channels contribute to the development of
cardiac pathologies such as cardiac hypertrophy and HF [52].
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Myotonic dystrophy is the most frequent type of muscular dystrophy in adults. Along with
serious muscular and neuropsychiatric impairments, patients with muscular dystrophy exhibit
cardiac defects associated with dysfunctional conduction between the heart atria and ventricles (i.e.
by atrioventricular block). The disorder is caused by expansion of CTG and CCTG motifs in the
DMPK and ZNF9 gene respectively [23]. Mutant mRNA binds and sequesters MBNL1, a protein
involved in processing of miR-1 from the precursor in the heart [23]. This leads to the cardiac
deficiency of mature miR-1 that in turn leads to the increase in levels of its target (i.e. CaV1.2) in
ventricular myocytes and development of the atrioventricular block [53]. Indeed, myotonic
dystrophy studies suggest for the important role of miR-1 in the regulation of atrioventricular
conduction and changes in miR-1 levels could lead to atrial and ventricular tachyarrhythmias.

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2.6. miR-1 in cardiac repolarization

Cardiac depolarization is followed by repolarization in which the membrane potential

returns to the resting state. Outward K+ currents (such as IK1, Ito, IKr, and IKs) are responsible for

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repolarization of heart muscle cells [54]. Zhao et al. [55] first showed the involvement of miR-1 in
the regulation of myocardial depolarization. Targeted deletion of miR-1-2 in mice led to

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conductance defects in the ventricular septum and increased mortality due to the sudden death.

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Iroquois homeobox protein 5 (Irx5), a transcriptional regulator, was shown to be targeted by miR-1-
2. Irx5 inhibits expression of the potassium voltage-gated channel D2 (KCND2) gene that encodes

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the Kv4.2 subunit involved in transient outward K+ current (Ito) [56]. Ito is the major current
contributing to the repolarization gradient in ventricular wall. Indeed, higher Irx5 levels cause
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reduction of KCND2 expression that disturbs ventricular repolarization and increases risk of
arrhythmias.
miR-1 was shown to down-regulate expression of the KCNJ2 gene, which encodes the
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Kir2.1 (inward rectifier potassium channel) subunit [57]. Overproduction of miR-1 slowed heart
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conduction due to the inhibition of Kir2.1 responsible for keeping membrane potential. Up-
regulation of miR-1 was observed in ischemic hearts of mice with experimental MI that exhibited
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enhanced arrhythmia [57]. Interestingly, Shan et al. [58] reported that arsenic trioxide, an anticancer
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agent used for treatment of acute promyelocytic leukemia, also up-regulates miR-1 (and miR-133)
in the heart. Higher cardiac levels of miR-1 correlated with reduced levels of its target Kir2.1.
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Indeed, this led to heart rhythm disturbances (i.e. delayed late repolarization (IK1) and QT
prolongation). These observations at least in part explain adverse cardiotoxic effects of arsenic
trioxide in leukemia subjects who experience cardiac rhythm alterations (i.e. QT prolongation and
ventricular arrhythmia) [59]. Indeed, increase in heart miR-1 levels predisposes to ventricular
arrhythmogenesis.
In contrast, in atrial fibrillation (AF), miR-1 is significantly down-regulated while Kir2.1
levels are increased [60]. This in turn results in IK1 alterations (i.e. increase in late repolarization) in
the atrial tissue. In summary, these data again indicate the key role of miR-1 in controlling
atrioventricular conduction through repolarization mechanism and importance of maintaining
myocardial miR-1 concentrations within a proper range in order to avoid arrhythmogenesis.

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2.7. miR-1 and calcium cycling in cardiomyocytes

In the myocardium, Ca2+ cycling plays a central role in heart excitability and conductance.
Ca2+ enters the cardiac muscle cell via several pathways of which L-type Ca2+ current (ICaL) is the

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most profound. The entrance of extracellular Ca2+into the sarcoplasm induces Ca2+ release from the
SR through activation of ryanodine receptor 2 (RyR2) [61]. CaMKII and PKA phosphorylate Ca2+

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channels and increase their permeability. Sarcoplasmic Ca2+ content is reduced by Ca2+ efflux from

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the cell via the activity of sarcolemmal Ca2+ pump and forward-mode Na+-Ca2+ exchanger NCX1.
SR Ca2+-ATPase (SERCA) also contributes to depletion of sarcoplasmic Ca2+by pumping Ca2+

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back to the SR stores [62].
NCX1 is a target of miR-1 that represses its mRNA [63]. This Na+/Ca2+ exchanger is
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involved in cardiac muscle relaxation. In failing heart characterized by decreased levels of miR-1,
NCX1 expression is up-regulated [64]. Thus leads to the abnormal overactivity of this channel and
delayed afterdepolarization that contributes to the induction of cardiac arrhythmia.
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HF is characterized by multiple aberrations in Ca2+ cycling including spontaneous diastolic

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Ca2+ release [65], reduced SR Ca2+ content, and up-regulation of Na+-Ca2+ exchanger [66]. In dogs
with chronic heart failure, cardiomyocytes have delayed afterdepolarizations associated with
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enhanced Ca2+ concentration in the sarcoplasm due to CaMKII-dependent hyperphosphorylation of

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RyR2 and spontaneous Ca2+ leakage from SR stores [67]. Hyperphosporylated state of RyR2 is
maintained because of reduced levels of PP2A involved in RyR2 dephosphorylation. miR-1 (and
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miR-133) were elevated in HF myocytes [68]. Terentyev et al. [28] reported that miR-1 negatively
regulates expression of the PP2A regulatory subunit and indeed could be responsible dissociating
PP2A activity from RyR2, diastolic spontaneous Ca2+ vaves, and arrhythmia in HF myocytes.
Interestingly, Kumarswamy et al. [69] showed that HF could be healed by recovery of miR-
1 expression in failing cardiac muscle cells through overproduction of sarcoplasmic reticulum
calcium ATPase 2a (SERCA2a) delivered by adenoviral vector. Overexpression of SERCA2a leads
to the dephosphorylation (i.e. activation) of Akt and transcription factor FoxO3A, both are
impоrtant in driving miR-1 expression. Elevated levels of NCX1 could be also negated by
SERCA2a overproduction.

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In overall, by targeting CaV1.2 and the regulatory B56α subunit of PP2A, miR-1 enhances
heart excitation-contraction coupling through increasing phosphorylation of the L-type Ca2+ and
RyR2 channels.

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2.8. miR-1 and conduction between cardiac muscle cells

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Intercellular gap junctions are involved in transfer of current from one myocyte to another.

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This process is essential for the electrical heart activation. Gap junctions between cardiomyocytes
are constituted by connexin-43 that is abundantly present in the myocardium and by connexin-40

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that is located in the artia and conduction system [70]. In cardiac pathology, remodeling of the gap
junction architecture is a common event [71, 72]. The pathologic remodeling alters the distribution
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and expression levels of connexin 43 in junctions that could impair conduction and lead to
arrhythmia [73].
Gene GJA1 encoding connexin-43 (also known as gap junctional protein-α1) is a molecular
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target for miR-1 [57]. Indeed, in various cardiomyopathies, heart expression of miR-1 is altered. For
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example, in viral myocarditis, miR-1 expression was found to be up-regulated. Accordingly, protein
levels of connexin-43 were significantly reduced whereas connexin-43 mRNA expression was
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altered slightly only [74]. Indeed, viral myocarditis, miR-1 suppresses connexin-43 at post-
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translational level [75].

In post-MI hearts, hypoxic conditions stimulate miR-1 expression that leads to decrease in
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Cx43 protein expression and arrhythmogenesis [76, 77]. Implication of β-blockers such as
propranolol and tanshinone IIa had cardioprotective effect by suppressing cardiac miR-1 and
improving heart function. Indeed, heart rhythm abnormalities caused by overstimulation with
epinephrine could be mediated by miR-1 through β-adrenoceptor-cAMP-protein kinase A (PKA)
mechanism [76]. Up-regulation of miR-1 leads to impaired heart conductance and miR-1-mediated
alterations in connexin-43 levels and junctional distribution are involved in induction of heart
rhythm defects.
In murine hypertrophic hearts, miR-1 was shown to be down-regulated. In contrast,
junctional connexin-43 amounts and its phosphorylation by mitogen-activated protein kinase Erk1/2
were elevated leading to connexin-43 displacement from the junctions [78]. In cardiac hypertrophy,

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impaired miR-1 and connexin-43 activities interconnect resulting in reorganization of gap junctions
and generation of tachyarrhythmias [78].
In summary, miR-1, which is abundantly expressed in the heart, is crucially involved in
cardiac development and exhibits a robust cardiogenic potential that supports commitment of

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cardiomyocyte-specific lineage from EMCs and reprogramming of mature non-cardiac cells to heart
muscle cells. miR-1 regulates key heart functions related to excitation-contraction coupling. In

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cardiac pathology, both these mechanisms are usually impaired or even uncoupled. Deregulated

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expression of miR-1 contributes to this impairment.

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2.9. Proapoptotic role of miR-1 in myocardial infarction

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Acute MI refers to acute coronary syndrome (ACS), a late complication of coronary
atherosclerosis commonly associated with plaque rupture and severe thrombosis that could lead to
complete arterial occlusion and rapid necrosis of downstream myocardium. Heart ischemia resulted
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from limited myocardial blood supply and chronic hypoxia strongly predisposes to MI. Electrical
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conduction of post-MI injured cardiac tissue is slower than that of the normal myocardium. The
difference in conduction velocity between infarcted and non-infarcted tissue causes reentrant
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arrhythmias, an abnormality when an electrical impulse recurrently moves in a tight circle within
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the heart rather than moving from one end of the heart to the other [79]. Reentrant arrhythmias are
responsible for more dangerous and life-threatening arrhythmias such as ventricular tachycardia and
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increase risk of sudden death.

Since miR-1 is critically involved in the regulation of cardiac contractility, alterations in
expression of this miRNA could be involved in the pathogenesis of MI. However, it should be noted
that expression levels of circulating miR-1 cannot directly reflect its actual expression in
cardiomyocytes. Meanwhile, blood levels of miR-1 were shown to be significantly elevated in
patients with acute MI [67, 80, 81]. In rodent models of acute MI caused by myocardial ischaemia/
reperfusion (I/R) injury, levels of circulating miR-1 were also dramatically up-regulated (up to 200-
fold) [82, 83]. In mouse models, miR-1 was reported to augment cardiac I/R injury [84].
Hydrogen peroxide (H2O2) was found to strongly up-regulate miR-1 in rat cardiomyocytes
suggesting for a stimulating role of oxidative stress in miR-1 expression. Overexpression of miR-1
was shown to induce apoptosis of cardiomyocytes especially in response to H2O2 [85]. This miRNA

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down-regulates many anti-apoptotic genes including B-cell lymphoma-2 (Bcl-2), heat shock protein
(HSP)-60, HSP-70, and insulin-like growth factor-1 (IGF-1) [85-88]. Interestingly, IGF-1 and miR-
1 reciprocally inhibit expression of each other. IGF-1-dependent signaling suppresses miR-1
through up-regulating transcription factor Forkhead box O3 (Foxo3), a negative miR-1 regulator

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[89, 90]. In cardiomyocytes, IGF-1 exhibits anti-apoptotic effects by preventing miR-1-mediated
apoptosis induced by oxidative stress (Li et al. 2012)[91] and higher glucose [87]. These findings

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could be especially actual in context of diabetes and diabetes-related glucotoxicity, both are strong

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cardiovascular risk factors.

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2.10. Arrhythmogenic potential of miR-1

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The arrhythmogenic potential and a positive role of miR-1 in post-MI cardiac electrical
remodeling were in part discussed above. By altering expression levels of connexin-43 and
components of L-type Ca2+ current and outward K+ currents, miR-1 could affect both repolarization
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and depolarization phases of the cardiac action potential and impair electric conduction either in
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the cardiomyocyte or between the cardiomyocytes [92]. Along with Kir2.1 and NCX1, miR-1 was
shown to modulate expression of other K+ channels such as K+/Na+ hyperpolarization-activated
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cyclic nucleotide-gated ion channel (HCN)-2 and HCN-4 regulated by cAMP and located in the
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cardiac pacemaker, a region that controls the heart rate [92]. Furthermore, cardiac channels HCN-2
and HCN-4 were observed to be permeable for Ca2+ and therefore could conduct Ca2+ efflux and
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contribute to the pacemaker function (If) upon hyperpolarization [93]. Thus, changes in HCN
expression, activity, and sarcolemmal density could be implicated in arrhythmogenesis in
pathologic conditions [94]. Recently, Myers et al. [95] found that inducible cAMP early repressor
(ICER), a blocker of cAMP-dependent signaling, is targeted by miR-1 in cardiomyocytes. ICER
inhibition stimulates β-adrenoreceptor/cAMP/PKA mechanism, an arrhythmogenic signaling
pathway that also leads to the activation of miR-1 expression. Indeed, miR-1 could mediate
arrhythmogenic effects of increased sympatho-adrenergic stimulation of the ischemic myocardium
[96].

2.11. miR-1 aggravates oxidative stress in cardiomyocytes

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As mentioned above, oxidative stress up-regulates cardiac expression of miR-1. In miR-1-

trangenic rat cardiomyocytes, miR-1 overexpression was shown to correlate with increased ROS
generation and reduced resistance against H2O2-induced oxidative stress due to inhibited production
of antioxidant enzymes such as Cu2+/Zn2+-dependent superoxide dismutase (SOD1) and catalytic

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subunit of glutamate-cysteine ligase (GCLC) [97]. In the heart, miR-1 suppresses insulin- and IGF-
1-dependent signaling that protects against miR-1-induced injury under oxidative stress [91, 98].

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Heme oxygenase-1 (HMOX1), a cytoprotective an antioxidant enzyme involved in detoxification of

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heme by its oxidation and thereby preventing heme deposition in the atherosclerotic plaque, down-
regulated miR-1 expression on myoblasts [99]. Indeed, these data suggest that up-regulation of

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miR-1 in heart muscle cells exhibits prooxidant properties and promotes oxidative stress thereby
contributing to the development of cardiovascular pathology.
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2.12. Therapeutic potential of miR-1 in cardiovascular pathology
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Since circulating miR-1 levels are markedly increased in patients with acute MI, this
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miRNA was proposed to be used as a selective biomarker for early diagnosis of acute MI and
distinguishing between acute MI and other cardiac events such as angina pectoris [81, 100], non-
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acute MI [67], and other cardiovascular diseases [101]. Furthermore, in patients with ASC or acute
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MI, serum miR-1 levels highly correlated with circulating troponin T, an established marker of
cardiac damage [102, 103]. However, the giagnostic value of miR-1 and other cardiac-specific
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miRNAs (miR-133a, miR-208b, and miR-499) was not superior compared with troponin T [103].
Studies in ASC and MI patients failed to show significance of miR-1 as a prognostic marker for
prediction of risk of mortality or HF [102, 104]. Additional studies are required to evaluate
prognostic value of miR-1.
Because miR-1 shows deteriorating effects in heart pathology, it represents a potential
therapeutic target in the treatment of cardiovascular disease. In ischemic/hypoxic cardiomyocytes,
inhibition of miR-1 with antisense oligonucleotides was cardioprotective by reducing apoptosis,
increasing resistance to oxidative stress [85], and attenuating spontaneous arrhythmogenic
oscillations [28]. Indeed, cardiac-specific down-regulation of miR-1 would be beneficial for
treatment of heart ischemia and post-MI complications.

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Ischemic preconditioning (IPC) is an experimental approach focused on achieving tissue

resistance to hypoxic and ischemic conditions. In the cardiac IPC, repeated short ischemic episodes
protect the myocardium from a subsequent ischemic insult [105]. In a myocardial I/R model,
implementation of IPC was showed to have a cardioprotective effect by reducing apoptosis and

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infarct size area, preserving mitochondrial function, and decreasing expression of pro-apoptotic
markers in part due to suppressing miR-1 expression [106-108].

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In contrast to ischemic and infarcted hearts, miR-1 expression is down-regulated in cardiac

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hypertrophy due to the IGF-1-dependent inhibition [109]. Indeed, due to suppressing effects of
miR-1 on pro-hypertophic IGF-1 signaling, restoration of miR-1 levels was suggested to be helpful

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in regressing hypertrophy and preserving heart against adverse remodeling. In a rat model of
pressure overload-induced hypertrophy, cardiac-specific adenoviral-based delivery of miR-1
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resulted in markedly decreased cardiac fibrosis and apoptosis and improved cardiac function.
Fibullin-2 (Fbln2) that is involved in extracellular matrix remodeling is a target of miR-1 [110].
A prominent role of miR-1 in cardiac development could be applicable for improvement of
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cardiac-specific regenerative properties of stem cells in cell therapy of post-IM heart in order to
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intensify and enhance the efficiency of cardiac tissue repair. Glass and Singla [111, 112] reported
that miR-1-overproducing ESCs engrafted to post-IM hearts protected host myocardium from
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apoptosis, oxidative stress, and fibrosis and promoted cardiac regeneration. Finally, as already
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discussed, miR-1 exerts a great reprogramming capacity that could be used for generation of
cardiomyocyte-like cells from differentiated non-cardiac cells and further use of these
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cardiomyocytes in regenerative medicine and developing cell panels for high-throughput screening
of potential cardiac drugs.

3. miR-133a

Human miR-133 has two isoforms, miR-133a and miR-133b. miR-133b has a sequence that
is highly similar with that of miR-133a-1/2 but is different at the 3’-terminal base, i.e. guanidine
(mir-133a) and adenosine (miR-133b) [15]. miR-133b is clustered with miR-206. The miR-
133b/miR-206 cluster is located on chromosome 6p12. As already mentioned, miR-133a exists in
two identical isomers (miR-133a-1 and miR-133a-2) encoded by two distinct genes clustered with
miR-1-1 and miR-1-2 respectively. In the miR-1/miR-133 clusters, each miRNA gene has its own

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transcriptional regulators [14]. Indeed, expression of miR-1 and miR-133 is independent on each
other and is differently regulated.
Like miR-1, miR-133a is the most abundant cardiac-specific miRNA [113]. Myogenic
transcription factor such as Mef2, MyoD, myocardin, and SRF are involved in the regulation of

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miR-133 expression in muscle cells and their precursors [14, 114]. Brahma-related Gene 1 (Brg1)
also known as ATP-dependent helicase SMARCA4 is a part of the large ATP-dependent chromatin

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remodeling complex SWI/SNF [115]. Brg1 was shown to cooperate with MyoD in activating miR-

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133a expression [116].
In contrast to miR-133a, the miR-133b isoform is not expressed in cardiomyocytes since

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expression of the miR-133b/miR-206 cluster is specific for skeletal muscle only [45, 117].
Furthermore, Boettger et al. [118] found that this cluster is dispensable for development, function,
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and regeneration of skeletal muscle since mice deficient for both miRNAs did not exhibit any
obvious abnormalities in myogenesis and skeletal muscle activity. Likely, this could be explained
by overlapping functions with the miR-1/miR-133a clusters that are also highly expressed in the
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skeletal muscle [119].

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3.1. Role of miR-133a in cardiogenesis

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Deletion of either miR-133a-1 or miR-133a-2 in mice does not significantly alter

cardiogenesis due to the compensatory activity of the remaining miR-133 gene copy. However,
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knockout of both miR-133 genes leads to the formation of the immature heart phenotype
characterized by lethal ventricular septal abnormalities, increased apoptosis and proliferation,
sarcomere disorganization, and aberrant expression of smooth muscle genes likely due to the role of
SRF and cyclin D2, a mitogenic cell cycle activator that is targeted by miR-133a [120].
Together with miR-1, miR-133a is crucially involved in the cardiac muscle cell
development and proliferation [12]. Both cardiac miRNAs cooperate in the control of early
mesodermal and future cardiac fate of ESCs [121]. miR-133a was shown to down-regulate
epidermal growth factor receptor (EGFR) thus preventing ectodermal differentiation [122]. The
preferential medoserm formation was characterized by down-regulation of endodermal markers
such as α-fetoprotein and hepatocyte nuclear factor 4 (HNF4) and early neurogenic transcription
factors Neurod4, Phox2b, and Myt1 [30]. miR-133a promotes myogenesis by inhibiting SRF [12].

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However, in the commitment of muscle-specific lineage from mesodermal precursors, miR-1 and
miR-133a play opposite roles [121].
miR-133a is abundantly expressed in myoblasts [12] but plays a contradictory role in further
differentiation of myoblasts to myocytes. In ESCs, overexpression of miR-133a was observed to

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down-regulate expression of cardiac markers [30, 34]. On the other hand, miR-133a was found to
suppress myocardin whose activation could be responsible to aberrant expression of smooth muscle

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genes in the developing heart of mice lacking both copies of the miR-1/miR-133a cluster [45]. miR-

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133a could promote myoblast proliferation through targeting SRF and Cyclin D2 [12]. However,
Zhang et al. [123] reported the inhibitory effect of miRNA-133a on myoblast proliferation by

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targeting Sp1, a transcription factor that stimulates cyclin D2 expression. It seems that controversial
functional effects of mi-133a at the cardiac (and skeletal) muscle-specific lineage commitment are
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greatly influenced by concomitant expression of miR-1 that could share some targets with miR-
133a but exhibit opposite effects on their expression.
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3.2. miR-133a in cardiomyocyte-specific reprogramming

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A cardiogenic potential of miR-133a was successfully applied for reprogramming of

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fibroblasts and cardiomyocytes to cardiomyocytes [46-48]. Furthermore, adding of miR-133a alone

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to the cocktail of myogenic transcriptional factors (GATA4+Mef2c+Tbx5+Mesp1+myocardin)

increased yield of cardiomyocytes from human and murine fibroblasts via suppression of Snai1, a
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key regulator of epithelial-to-mesenchymal transition by inhibiting expression of ectodermal genes

in the mesoderm [124]. Indeed, miR-133a-specific targeting Snai1 could also play a role in the
preferential formation of mesoderm from ESCs during cardiogenesis. Supplementation of the basic
reprogramming mixture (i.e. GATA4+Mef2c+Tbx5) with mesoderm posterior 1 homolog (Mesp1)
and myocardin enchanced cardiac-specific effect [125].

3.3. miR-133a in cardiac conductance

The catalytic subunit of PP2A, which is involved in dephosphorylation of RyR2, is targeted

by miR-133a [68]. As mentioned above, decreased cardiac PP2A levels and activity observed in
failing heart lead to maintaining of CaMKII-dependent hyperphosphorylated state of RyR2 and

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altered Ca2+ cycling (i.e. spontaneous Ca2+ leak from the SR, accumulation of Ca2+in the
sarcoplasm, creased frequency of diastolic Ca2+ waves and afterdepolarizations) [126]. Since miR-1
targets the regulatory subunit of PP2A [28], miR-1 and miR-133a play a key role in coordinated
regulation of heart Ca2+ cycling in PP2A-dependent manner.

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Several miR-133a targets are involved in the repolarization of the heart action potential.
miR-133a targets KCNQ1 mRNA which encodes Kv7.1, a pore-forming subunit of the voltage-

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gated K+ channel responsible for the IKs (i.e. slow delayed rectifying cardiac K+ current) [127]. As

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miR-1, miR-133a down-regulates HCN-2 [128], responsible for the pacemaker current (If) that
plays a key role in determining heart automaticity. In cardiac hypertrophy, myocardial expression of

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miR-133a is reduced while expression of HCN-2 and HCN-4 is elevated that leads to electrical
remodeling and contributes to arrhythmogenesis and pacemaker dysfunction in hypertrophic hearts
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[127].
In the heart of diabetic rabbits, Xiao et al. [129] observed up-regulation of miR-133 along
with SRF, a transactivator of this miRNA whereas expression of the ether-a-go-go related gene
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(HERG), a target for miR-133, was depressed. The HERG gene encodes an α-subunit (Kv11.1) of
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the K+ channel involved in the IKr current, and abnormalities in its function contribute to impaired
repolarization phase, arrhythmia, and prolonged QT interval observed in diabetic heart [130].
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Pathologic overactivation of miR-133 could therefore contribute to diabetic HERG K+ dysfunction.

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Matkovich et al. [131] found that overexpression of the miR-133a transgene in mice with
transverse aortic constriction (an experimental model for studying pathogenic mechanisms of HF
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and cardiac hypertrophy) prevented decrease of Ito,f and caused prolongation of QT interval in
ventricular cardiomyocytes. Both mRNA and protein levels of the fast component (Kv channel-
interacting protein 2, or Kcnip2) of Kv4 responsible for transient outward K+ current (Ito,f) were
decreased. However, miR-133a does not directly target Kcnip2 and therefore modulates
repolarization through another mechanism. Increased cardiac expression of miR-133a did not
influence hypertrophy extension but improved diastolic function and diminished myocardial fibrosis
probably through inhibiting connective tissue growth factor (CTGF) [132, 133].
Dong et al. [134] showed that miR-133a and calcineurin could reciprocally inhibit it other in
cardiac hypertophy. In hypertophic heart, expression of calcineurin is increased while expression of
miR-133a is decreased. In cultured primary cardiomyocytes, treatment with cyclosporin A, an
inhibitor of calcineurin, prevented miR-133 suppression. Furthermore, silencing targeting nuclear

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factor of activated T cells transcription factor (NFAT), a calcineurin downstream effector, increased
miR-133 expression in the cells. Indeed, these data suggest that miR-133a and calcineurin are
involved in the regulation of hypertrophy in opposite ways. Furthermore, NFATc4 is a direct target
for miR-133a in cardiomyocytes that in turn down-regulates NFAT-dependent prohypertrophic

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signaling in response to hypertophic stimuli [135]. Calcineurin-NFAT signaling plays a key role in
cardiac hypertophy [136] and miR-133a can inhibit hypertrophy by targeting both key components

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of this pathway.

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In thyroid hormone (TH)- induced heart hypertophy mediated in part by type 1 angiotensin
II receptor (AT1R), miR-133a levels were reduced while expression of SERCA2a and calcineurin

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(both are miR-133a targets) were increased.. MiR-133 mimic blunted TH-dependent cardiomyocyte
hypertrophy suggesting for the suppressive effect of this miRNA in TH-mediated hypertrophy
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[137].
By targeting K+-conducting channels involved in IKr and IKs, miR-133a plays a role in the
regulation of cardiac depolarization. miR-133a-mediated reduction of the slow depolarization phase
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could be compensated by increase in IKr, a mechanism that maintains repolarization reserve and
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prevents arrhythmic induction [138]. Along with miR-1 that controls repolarization and
depolarization phases of the action potential, miR-133 is crucially involved in the regulation of a
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proper heart conductance, contraction, and automaticity. Indeed, interplay between these mRNAs
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should play a crucial role in maintaining normal cardiac beating rhythm Alterations in expression of
these cardiac miRNAs could lead to life-threatening arrhytmias observed in various cardiac
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pathologies.

3.4. miR-133a in myocardial infarction

Cardiac levels of miR-133a levels were shown to be down-regulated in MI patients [139,

140] and in a rat model of I/R-induced acute myocardial injury [84]. In contrast, various studies
showed marked elevation in circulating miR-133a plasma/serum levels in patients with acute MI
[82, 141, 142], indicating cardiac damage [100]. A meta-analysis showed a significance of
circulating miR-133a as a diagnostic marker for acute MI [141]. However, for acute MI diagnosis,
the value of circulating miR-133a was lower than that of the high-sensitivity cardiac troponin T test

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[143]. Accordingly, the prognostic accuracy of miR-133a in predicting mortality of acute MI

subjects was moderate [143].
In contrast to the pro-apoptotic action of miR-1, miR-133a exhibits anti-apoptotic properties
in injured cardiomyocytes. In in vitro hypoxia-reoxygenation injury model and an in vivo rat model

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of I/R injury, overexpression of exogenous miR-133 greatly attenuated cardiac cell apoptosis [144].
In muscle cells, miR-133a was found to inhibit many pro-apoptotic genes such as caspase-9 [86,

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145], apoptotic protease activating factor 1 (APAF1) [146], death-associated protein kinase 2

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(DAPK2), BCL2-like 11 (BCL2L11), and Bcl-2-modifying factor (BMF) [147], thereby
suppressing cell apoptosis. DAPK2 is abundantly expressed in heart and skeletal muscle and could

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be activated in a Ca2+/CaM-dependent manner [148]. Suppression of miR-133a in infarcted hearts
therefore could be associated with overactivation of pro-apoptotic pathways.
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In post-MI hearts, miR-133a displays cardioprotective function by repressing myocardial
fibrosis and fibrotic remodeling of injured myocardium. Chen et al. [149] reported that cardiac-
specific miR-133a overexpression diminished fibrosis in diabetic murine hearts associated with
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down-regulation of apoptotic markers. In HF rats, using of miR-133a mimic and miR-133a

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overproduction in the heart resulted in improved cardiac function and decreased fibrosis [150].
miR-133a exhibits anti-fibrotic effects through direct blocking of expression of pro-fibrotic genes
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such as CTGF and collagen 1A1 [151] and inhibition of Akt-dependent signaling mechanism that is
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up-regulated in failing hearts [151, 152].

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3.5. Therapeutic potential of miR-133a

Due to the key role in heart function, cardiogenesis, and functional alterations in cardiac
pathology, miR-133a represents a promising target in cardiovascular therapy. Clinical
measurements showed a practical value of circulating miR-133a as a diagnostic biomarker of acute
MI. Accordingly, as already considered, a cardiac-specific reprogramming capacity of miR-133a
could be useful for generation of cardiomyocytes from non-cardiac cell sources. Anti-fibrotic and
anti-apoptotic properties of miR-133a will be beneficial in the development of therapeutic tools
focused on cardioprotection and regeneration of cardiac injury.
Protective effects of ischemic post-conditioning against myocardial I/R injury could be
explained in part by anti-apoptotic and regenerative potential of miR-133a whose expression is

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recovered in the treated cardiac muscle cells [145] (Figure 2). Izarra et al. [147] showed that miR-
133a-transgenic cardiac progenitor cells (CPCs) have increased survival compared with non-
transgenic CPCs due to the miR-133-mediated protection against apoptosis. In a rat MI model,
transplantation of miR-133a-overexpressing CPCs led to improved heart function, suppressing

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hypertrophy and fibrosis, and increasing angiogenesis and myocyte proliferation.
Up-regulation of miR-133a had positive effects on human mesenchymal stem cells (MSCs)

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enhancing their regenerative and cardiogenic properties, survival, and resistance against apoptosis.

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Transfection of MSCs with miR-133a increased expression of cardiac-specific genes via miR-133a-
dependent targeting EGFR [122]. miR-133a-overexpressing MSC transplants had increased survival

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and engraftment rate in rat hearts subjected to MI [146]. Indeed, development of stem cells via
cardiac-specific miRNA manipulation has a promising potential to improve the therapeutic outcome
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in subjects undergoing stem cell-based cardiotherapy for MI.

4. miR-208a/b
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miR-208 is a cardiac-enriched miRNA, what is also expressed in the skeletal muscle (Huang
and Li 2015) [153]. This miRNA is presented in two isoforms: miR-208a and miR-208b. Murine
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miR-208 isoforms have a high degree of identity while human isoforms are not [154]. Both human
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miR-208 genes are located on chromosome 14q11.2. The miR-208a gene is located in intron 29 of
the MYH6 gene that encodes α-cardiac muscle myosin heavy chain (MHC) (fast myosin) while
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miR-208b resides in intron 31 of the MYH7 gene encoding β-cardiac muscle MHC (slow myosin).
Interestingly, human miR-208a is exclusively expressed in the heart whereas miR-208b is expressed
in the heart and skeletal muscle [154]. Compared with rodents,
MYH6 and MYH7 are organized in tandem [155]. Both genes have the identical genome
organization, and their sequence has a high homology. These genes arise from the duplication of a
common ancestor that was present in the ancestor chordate [156]. As miR-208a, α-MHC is cardiac-
specific. Both genes are concurrently expressed in cardiogenesis suggesting that their expression is
driven by the common regulatory element [157]. SRF and MEF2 regulate expression of sarcomeric
and other skeletal muscle-specific genes in cooperation with myogenic basic helix-loop-helix
(bHLH) factors such as myogenin and MyoD [158, 159].

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4.1. miR-208 in cardiogenesis

While miR-1 and miR-133 have a prominent role in early cardiogenesis, miR-208 seems to
be involved in late stages of cardiac development related to the commitment of cardiomyocytes

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from myoblasts [160]. miR-208 regulates cardiac MHC expression.
In the heart muscle, the MHC is the major contractile protein comprising α- and β-isoforms.

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The proportion of isoforms could change during the heart development. β-MHC is mainly

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expressed in late fetal life while α-MHC is preferentially expressed in the adult [155]. In rodents,
Myh6 and Myh7 are differentially expressed in adult hearts. Accordingly, cardiac expression of

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miR-208a and miR-208b could also vary in parallel with α-MHC and β-MHC expression [161-163].
miR-208b is predominantly expressed in the fetal myocardium while its expression drops in the
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adult heart. In contrast, miR-208a expression rises during cardiogenesis, with achieving top levels
in the adulthood. The switch from the predominant miR-208b expression to preferential expression
of miR-208a occurs post-natally suggesting that both miRNAs share the same targets [163].
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Compared with rodents, miR-208a is the predominant miR-208 isoform that is expressed in humans
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and large animals [160].

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4.2. miR-208 in heart function

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In mice, knockout of miR-208a results in lack of capacity to up-regulate expression of β-

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MHC and induce hypertrophy in the adult heart under stressful conditions providing evidence that
this miRNA controls expression of Myh7 and therefore miR-208b [163]. Expression of Myh7b
encoding the third (slow) MHC, which is closely related to α- and β-chains, was also decreased in
miR-208a-deficient mice. Indeed, miR-208a also regulates Myh7b expression. Since the miR-499
gene is located in intronic sequence of Myh7b, miR-499 expression is also regulated by miR-208a
[162, 163]. In cardiogenesis, the role of miR-208b and miR-499 is redundant and is focused on the
stimulating slow and down-regulating fast muscle fiber transcription program [162].
Interestingly, in the antisense chain of the MYH7 gene, the MHRT gene encoding the long
non-coding MHC-associated transcript is located. This cardiac-specific regulatory RNA is involved
in epigenetic inhibition of MYH7 expression thereby preventing cardiac hypertrophy [164]. Indeed,
both miR-208a/MHRT and miR-208b/miR-499 tandems are involved in the reciprocal regulation of

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the fast/slow muscle fiber specification. Since MHRT leads to β-MHC silencing, it is likely that
MHRT could also suppress miR-208b. However, it is necessary to study the likelyhood of this
phenomenon.
Lack of miR-208a is accompanied with up-regulated cardiac expression of fast myofibers

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that normally not produced in the myocardium [165-167]. Expression of THRAP1 that encodes
cofactor of the thyroid hormone nuclear receptor was also enhanced. THRAP1 (a target for miR-

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208a) is a negative regulator of the thyroid hormone-dependent signaling that up-regulates cardiac

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β-MHC production [163, 168] and induces hypertrophy. In hyperthyroidism, cardiac levels of miR-
208a are up-regulated leading to the development of heart hypertophy [169].

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Overexpression of miR-208a in cardiomyocytes leads to hypertrophy and is associated with
increased β-MHC expression and miR-208-dependent down-regulation of THRAP1 and myostatin,
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which both act as hypertrophy repressors [163, 170]. In addition, miR-208a up-regulation in
hypertrophic hearts leads to the abnormalities in cardiac rhythm and fibrosis and is associated with
reduced expression of cardiac-specific transcription factors (GATA and homeodomain-only protein
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(HOPX)) and junctional protein connexin-40 involved in intercellular conductance [163]. These
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findings suggest that miR-208 is crucially involved in heart contraction and conduction but
pathogenic miR-208 overactivity possesses prohypertrophic, profibrotic, and arrythmogenic
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properties.
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4.3. miR-208 as a diagnostic marker of early myocardial infarction

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As for other cardiac-specific miRNAs, expression of miR-208 is reduced in the infarcted

heart suggesting for depressed cardiac function [100, 140]. However, plasma/serum levels of miR-
208 (especially miR-208b) were detected to be significantly elevated in patients who suffered from
acute coronary events such as acute MI and ASC. miR-208a could not be detected in the blood of
normal or non-acute MI patients affected with stroke, trauma, or kidney injury [154]. MiR-208a
could be diagnosed in the blood 1h post-infarction in 90% of acute MI patients, even earlier than
troponin T, a gold standard for diagnosis of myocardial damage (plasma troponin could be detected
as early as 4-8 h post-MI, with reaching pick levels 18 h post-MI) [171]. In addition, plasma miR-
208a allow detecting acute MI with 90.9% sensitivity at 100% specificity [101], the best value
compared with other cardiac miRNAs (miR-1, miR-133a, and miR-499). This makes miR-208a as a

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profound diagnostic marker for acute MI that could be potentially better than troponin T for earliest
acute MI diagnosis. In addition, miR-208a is superior to cardiac troponin in specific diagnosis of
acute MI in patients with kidney damage since heart troponins are excreted by kidney and their
levels are frequently increased in subjects with chronic renal failure [172].

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Interestingly, in a model of acute isoproterenol-induced heart injury, circulating miR-208a
levels were superior to cardiac troponin T even after 24 h post-injury [173]. Indeed, persistently

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increased concentrations of miR-208a in the bloodsteam may be a result of miR-208b interference

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due to the high homology between these miRNAs in mice [163].
In patients with acute MI, circulating levels of miR-208b were shown to be greatly up-

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regulated. For example, Gidlöf et al. [104] reported 3000-fold increase in plasma miR-208b 12 h
post-infarction in subjects with acute ST-segment elevation MI compared with healthy subjects.
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Corsten et al. [174] observed 1600-fold elevation in plasma miR-208b concentration in acute AMI
individuals compared to non-affected controls. In acute myocardial injury, plasma miR-208b
showed good correlation with cardiac troponin T levels [103, 104, 175, 176] and area under the
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ROC (receiver operating characteristic) curve (AUC) of 0.94 indicating that this miRNA is a useful
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diagnostic marker for acute MI [174]. However, the diagnostic value of miR-208b for this disease
was not superior compared with troponin T [103].
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In studies in which the diagnostic value of miR-208 in distinguishing acute MI and ASC
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from other cardiovascular pathology was accessed, less profound results were obtained. Widera et
al. [102] found significant increase in miR-208 levels in ACS patients compared with unstable
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angina and showed a prognostic potential of miR-208 for prediction of mortality. However, when
adjusted for high sensitivity cardiac troponin T levels, association of miR-208 with death prediction
was lost. Nabiałek et al. [177] failed to observe significant differences in miR-208a levels between
acute MI and coronary artery disease (CAD). In contract, De Rosa et al. [178] showed significant
plasma miR-208a elevation in ASC compared with CAD. Probably, Nabiałek et al. [177] could not
detect differences in miR-208a levels because they start measuring too late, i.e. 6 h post-MI. In
patients undergoing transcoronary ablation of septal hypertrophy (a clinical model of acute MI)
Liebetrau et al. [179] found increase in blood miR-208a at 105 min post-procedure with subsequent
rapid decline to basic levels.

4.4. Therapeutic potential of miR-208

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As discussed above, miR-208a (and miR-208b in a less content) could serve as a robust and
sensitive marker for early diagnosis of acute MI. To date, molecular approaches are developed that
allow manufacturing synthetic oligonucleotids (miRmimics or antagomiRs) that respectively mimic

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or inhibit mature miRNA targets. This strategy provides an option to manipulate with clinically
significant miRNAs whose expression is altered in pathological conditions [180]. In mice with

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hypertension-induced HF, Montgomery et al. [181] produced down-regulated miR-208a expression

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by injection of miR-208a-specific antigomiR. This resulted in block of pathological cardiac
remodeling, prevention of pathological myosin switch, inhibition of perivascular fibrosis, and

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improved heart function. This experiment shows the utility of targeted inhibition of miR-208a in
cardiopathy accompanied with up-regulated tissue miR-208a levels such as HF and cardiac
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hypertrophy. In human dilated cardiomyopathy, endomyocardial levels of miR-208 and miR-208b
were also up-regulated. miR-208 expression also correlated with β-MHC mRNA levels and
myocardial collagen volume. Furthermore, in a prospective study (a mean follow-up of 517 days),
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Satoh et al. [182] showed a prognostic value of elevated miR-208 levels as a predictor of adverse
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clinical outcomes in dilated cardiomyopathy.

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5. miR-499
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Human miR-499 is located in intron 19 of the MYH7B (also known as MYH14) gene
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(chromosome 20q11.2) encoding an ancient ventricular-specific MHC, a component of slow-twitch

myosin. miR-499 is composed of two genes (miR-499a and miR-499b) that are located in sense and
anti-sense DNA chains of the same region and transcribed in anti-parallel directions [183]. In fact,
miR-499b is an antisense miR-499a. It is unknown whether miR-499b is expressed but expression
of miR-499a was recently detected [184-186]. In the future, if miR-499b will be detected, it would
be interesting to investigate the expression and regulation pattern of the miR-499 locus. If miR-
499b exists, it is important to know how expression of miR-499a/b is separated spatially and
temporally and could miR-499b be involved in self-regulation of the miR-499 locus.

5.1. miR-499 in cardiogenesis

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Like miR-208, miR-499 regulates late cardiogenesis and is responsible for terminal
differentiation of myoblasts to cardiomyocytes. miR-499 down-regulates myocyte enhancer factor
2C (MEF2C) [180] that is involved in the commitment of muscle cells and cardiogenesis but could
also support expression of smooth muscle genes through activation of myocardin [187]. Indeed, by

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inhibiting MEF2C, miR-499 favors switching myogenic differentiation preferentially towards
cardiomyocytes.

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In human fetal cardiomyocyte progenitor cells (CMPCs), miR-499 is up-regulated and

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cooperates with miR-1 to drive differentiation of CMPCs and ESCs to cardiomyocytes [188].
Interestingly, miR-499 was shown to be transferred from differentiated myocytes to progenitor cells

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through gap junctions and regulates differentiation in a paracrine manner [188].
Low expression levels of human MYH7B were detected in the embryonic heart and skeletal
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muscle. After birth, MYH7B expression disappears from most fibers except for slow-tonic fibres
[157]. Expression of both MYH7B and miR-499 is coordinated and is regulated by muscle-specific
transcription factors such as myogenic regulatory factors (MRFs) (MyoD, myogenin, and myogenic
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factor Mif5) and Ikaros, a lymphoid transcription factor that forms complex with MyoD [189].
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miR-499 targets SRY box-6 (Sox-6), a conserved transcription repressor that inhibits slow-
twitch myofiber-specific expression program in zebrafish [190]. Sox-6 is also down-regulated by
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PR domain zinc finger protein 1 (Prdm1), a transcription repressor of the slow-twitch myofiber-
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specific program [191, 192]. By derepressing slow-twitch fiber program, Prdm1 also induces miR-
499 expression [191]. Indeed, Sox-6/Prdm1 and miR-499/miR-208a regulatory tandems that play
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opposite roles in the control of expression of slow/fast MHC are crucially involved in determining
specification of cardiomyocytes toward either fast or slow myofibers.
Interestingly, MYH7B transcript could be alternatively spliced that leads to the generation
of the premature stop-codon [193]. This abolishes MYH7B expression but does not affect
transcription of miR-499 [193]. miR-499 can be formed even when MYH7B expression is lacking
[194]. Indeed, in mammals, there are two mechanisms (i.e. coordinated transcription and alternative
splicing) that uncouple expression levels of MYH7B and miR-499 when their coexpression is not
necessary [194].
In human miR-499, Dorn et al. [195] identified a naturally exising rare mutation (U17C) at
the 3’terminus. This mitation does not affect the seed sequence but impairs miRNA binding to
mRNA. This results in changes of a spectrum of cardiac genes targeted by mutant miR-499

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compared to the gene repertoire targeted by wild-type miRNA. In mice, cardiac-specific

overexpression of the miR-499 transgene led to the development of cardiac hypertrophy and heart
dysfunction by altering expression of contractile (MYH7B and skeletal muscle α-actin (ACTA1))
and conductivity (KCNH6 and Kv11.1) [196].

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5.2. miR-499 in acute myocardial infarction

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miR-499 deregulation was shown to be primarily involved in the pathogenesis of heart
hypertrophy due to the pronounced role in the regulation of expression of sarcomeric genes [197].

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The appearance of this miRNA in the bloodstream could serve as an indicator of heart damage that
happened in acute MI. Plasma miR-499 levels are elevated in subjects with acute MI but are below
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the detection level in ASC, congestive HF, and normal controls that may be helpful for
distinguishing acute MI from these cardiopathies [176, 198]. In acute AMI, the magnitude of
plasma miR-499 elevation is less dramatic than that of miR-288b (probably due to the relatively
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low cardiac expression of this miRNA) and typically varies between 100-300-fold increase [104,
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199, 200]. Several studies reported that miR-499 has a superior diagnostic accuracy for acute MI
compared with conventional cardiac troponin T assay (AUC of 0.79-0.92 vs. 0.70) [198, 199, 201]
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while other studies failed to show this [103, 104]. However, compared with troponins, miR-499 has
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an advantage for early acute MI diagnosis because this miRNA could be detected in the blood <4 h
post-MI while detectable levels of troponins appear later [202]. Thus, miR-499 could substantially
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improve the accuracy of troponin T for diagnosis of acute MI and hence be a reliable marker for
early diagnosis of acute MI.
MiR-499 was shown to have a cardioprotective effect by protecting heart muscle cells
against H2O2-induced apoptosis [203]. Overproduction of miR-499 in rat cardiomyocytes increased
cell survival by inhibiting the mitochondrial apoptotic mechanism. MiR-499 targets several
proapoptotic regulators such as dual specificity tyrosine-phosphorylation-regulated kinase 2
(Dyrk2), programmed cell death protein 4 (Pdcd4), and phosphofurin acidic cluster sorting protein 2
(Pasc2). Dyrk2 promotes apoptosis by phosphorylation of p53, a key regulator of apoptosis [204].
Thus, miR-499-dependent Dyrk2 repression prevents translocation of activated p53 to mitochondria
where it interacts with proapoptotic factors (Bak, Bax, Bid) and induces apoptosis [205]. miR-499
could also inhibit apoptosis of cardiomyocytes via inhibition of calcineurin-dependent

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dephosphorylation of dynamin-related protein-1 (Drp1), which in turn leads to reducing Drp1

accumulation in mitochondria [206] and Drp1-mediated activation of the mitochondrial fission
program [207] (Figure 3). Interestingly, H2O2-induced oxidative stress up-regulates miR-499
expression in cardiac cells (as a part of stress-induced response) through phosphorylation of c-Jun

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that then associates with c-Fos to form transcription factor AP-1 and prime miR-499 expression
from the MYH7B promoter [203].

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miR-499 could inhibit cardiac cell apoptosis through down-regulating Sox-6, which

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suppresses cell proliferation and enhances apoptosis [208]. Sox-6 inhibition by miR-499 results in
derepression of the cyclin D1 promoter and activation of cyclin D-mediated cell growth and

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proliferation [209]. Anti-apoptotic and proliferative effects of miR-499 are important during heart
development and may be helpful in therapeutic applications of this miRNA.
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5.3. Therapeutic potential of miR-499
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The crucial involvement of miR-499 in cardiac development suggests for their utility in
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using for obtaining heart muscle cells through reprogramming of other mature cell types. As already
discussed, cardiac miRNAs including miR-499 could be used with success for generation of
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cardiomyocytes in vitro [46] and in vivo [47] via reprogramming strategies that provides new
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promises for recovering injured cardiac tissue after MI.

Cardiospecific regenerative capacity of miR-499 could promote preferential differentiation
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of cardiac stem cells (CSCs) to mature functional cardiomyocytes. Usually, CSC transplants to
post-MI myocardium leads to generation of a large number of cardiomyocyte-like cells with
immature phenotype that failed to differentiate to cardiac muscle cells with adult properties. In
human CSCs, overexpression of the miR-499 transgene resulted in improved regenerative potential
of miR-499-overexpressing cell grafts, increased myocardial tissue repair, and enhanced restoration
of heart mass [210].

6. Conclusions

The contribution of cardiac miRNAs to cardiac developmental program is well coordinated and
organized. MiR-1 and miR-133a are predominantly involved in the induction and maintaining

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cardiac-specific myogenesis whereas miR-208 and miR-499 regulate terminal differentiation of

cardiomyocytes. Despite a remarkable progress in studying a role of miRNA in heart function and
biology, it is necessary to continue discovery of new cardiac-specific targets and investigate
functional consequences of their targeting by cardiac miRNAs. This should expand our knowledge

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about heart defects and functional/structural aberrations caused by pathological disregulation of
expression of cardiac miRNAs. A significant value of cardiac miRNA as diagnostic biomarkers for

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early diagnosis of acute MI was demonstrated. However, a prognostic significance of these miRNA

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in prediction of death in patients with acute MI is widely underexplored to date. Indeed, more
follow-up studies involving independent and large patient cohorts should be performed.

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Cardiospecific regenerative potential of cardiac miRNAs should be studied in details. This
should expand our options to manipulate with expression of miRNA and their cardiac targets in
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order to improve heart function and enhance repair of post-MI cardiac injury. The utility of cardiac
miRNAs in cardiac-specific reprogramming of fibroblasts/cardiac fibroblasts was shown. However,
it is necessary to optimize reprogramming techniques for increasing quantitative and qualitative
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cadiomyocyte yield.
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Disclosures
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None declared.
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Sources of funding

The work was supported by the Russian Scientific Foundation (grant 14-15-00112), Russian
Federation for support of our work.

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Figure legends

Figure 1. Role of cardiac miRNAs in the differentiation of embryonic stem cells to

cardiomyocytes.

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Figure 2. Anti-apoptotic effects of miR-133a in cardiac muscle cells. The main apoptotic

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mechanism that is suppressed by miR-133a is the mitochodria-dependent pathway. MiR-133a

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targets two major components (caspase-9 (CASP9) and apoptotic protease activating factor 1
(APAF1) that together with cytochrome C form a cell death complex that irreversibly leads to

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apoptosis through the activation of caspase 3 (CASP3). MiR-133a can also inhibit the mitochondrial
apoptotoc path through direct targeting proaptotic Bcl-2-like protein 11 (BCL2211) and Bcl-2-
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modifying factor (BMF), both are involved in the inhibition of the function of BCL2, an inhibitor of
mitochondria-dependent apoptosis. Finally, miR-133a suppresses DAPK2, a death-associated
protein kinase 2, which is involved in tumor necrosis factor (TNF)-related apoptosis-inducing
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ligand (TRAIL) through activating two death receptors (DR)4 and DR5.
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Figure 3. The anti-apoptotic role of miR-499 in cardiomyocytes. MiR-499 targets several

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proapoptotic genes including Dyrk2 (dual specificity thyrosine-phosphorylation–regulated kinase

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2), Pdcd4 (programmed cell death protein 4), Pacs2 (phosphorylation acidic cluster sorting protein
2), and DRP1 (dynmaic related protein-1). Dyrk2 kinase stimulates p53, a main proapoptotic
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regulator. MiR-499-mediated Dyrk2 down-regulation arrests transfer of activated p53 to the

mitochondria where it binds to various proapoptotic proteins such as Bcl-2 homologous
antagonist/killer (Bak), Bcl-2-associated X protein (Bax) and BH3 interacting-domain death agonist
(Bid) and thereby initiates a mitochondria-dependent pathway of apoptosis. Acccordingly, miR-
499-dependent inhibition of Pacs2 also prevents the mitochondrial apoptotic mechanism by
suppression of Bid, a proapoptotic protein. Pacs2-mediated recruitment of PDCD4 inhibits the
activity of activator protein-1 (AP-1), a transcription factor that drives expression of a plethora of
genes responsible for cell growth and proliferation. DRP1 is involved in mitochondrial fission and
apoptosis via interaction with several proteins including mitochondrial fission 1 protein (FIS1),
mitofusin-2 (MFN2), and mitochondrial fusion factor (MFF).

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Highlights

 Cardiac miRNAs are abundantly expressed in the myocardium

 Contribution of cardiac miRNAs to cardiac developmental program is well

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coordinated
 Significant value of cardiac miRNA as diagnostic biomarkers for early diagnosis

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of acute myocardial infarction is appreciated.

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