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

    Cardiac-specific miRNA in cardiogenesis, heart function, and cardiac pathology (with focus on myocardial infarction) Dimitry A. Chistiakov, Alexander N. Orekhov, Yuri V. Bobryshev PII: DOI: Reference:

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

To appear in:

Journal of Molecular and Cellular Cardiology

Received date: Revised date: Accepted date:

20 October 2015 9 February 2016 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

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

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

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

Institute of Pediatrics, Research Center for Children's Health, 119991 Moscow, Russia; Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, Moscow

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125315, Russia;

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

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2052, Australia;

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School of Medicine, University of Western Sydney, Campbelltown, NSW 2560, Australia.

Short title: MicroRNAs in myocardial infarction

* 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: [email protected]

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ABSTRACT

Cardiac miRNAs (miR-1, miR133a, miR-208a/b, and miR-499) are abundantly expressed in the

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myocardium. They play a central role in cardiogenesis, heart function and pathology. While miR-1 and miR-133a predominantly control early stages of cardiogenesis supporting commitment of

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cardiac-specific muscle lineage from embryonic stem cells and mesodermal precursors, miR-208 and miR-499 are involved in the late cardiogenic stages mediating differentiation of cardioblasts to

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cardiomyocytes and fast/slow muscle fiber specification. In the heart, miR-1/133a control cardiac conductance and automaticity by regulating all phases of the cardiac action potential. miR-208/499

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located in introns of the heavy chain myosin genes regulate expression of sarcomeric contractile 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,

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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 early diagnosis of acute MI. Great cardiospecific capacity of these miRNAs is very helpful for

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enhancing regenerative properties and survival of stem cell and cardiac progenitor transplants and for reprogramming of mature non-cardiac cells to cardiomyocytes.

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

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ACCEPTED MANUSCRIPT Contents

1. Introduction

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2. miR-1 2.1. miR-1 expression regulators

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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

2.3. miR-1 regulates differentiation of mesodermal precursors to cardiomyocytes

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2.4. miR-1 drives reprogramming of other cell types to cardiomyocytes 2.5. miR-1 in cardiac depolarization

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2.6. miR-1 in cardiac repolarization

2.7. miR-1 and calcium cycling in cardiomyocytes 2.8. miR-1 and conduction between cardiac muscle cells

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2.9. Proapoptotic role of miR-1 in myocardial infarction

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2.10. Arrhythmogenic potential of miR-1 2.11. miR-1 aggravates oxidative stress in cardiomyocytes

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

3.1. Role of miR-133a in cardiogenesis 3.2. miR-133a in cardiomyocyte-specific reprogramming 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 3

ACCEPTED MANUSCRIPT 5.3. Therapeutic potential of miR-499 Conclusion Sources of funding

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Disclosures

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References

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1. Introduction

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Myocardial infarction (MI) is the most frequent end-point of cardiovascular pathology that leads to higher mortality in economically developed countries. MI is resulted from acute cardiac

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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 expanding infarct region, amplifying reactive oxygen species (ROS) production, cardiac muscle cell

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death, and remodeling of the survived functional cardiomyocytes. Initially, cardiac remodeling starts as an adaptive response against heart damage but this process becomes maladaptive in

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pathogenic conditions and generally results in cardiac fibrosis, dilated cardiomyopathy, and heart 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].

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

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mechanisms of HF and post-infarction complications [4]. MiRNAs belong to a class of small noncoding RNAs (an average size of 22 nucleotides), which negatively regulate expression of target 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 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 5

ACCEPTED MANUSCRIPT 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

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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 encoded by distinct genes. miR-1-1 is encoded by the miR1-1/miR-133a-2 gene cluster located on

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chromosome 20q13.3 and transcribed as a common precursor for both miRNAs. A precursor for miR-1-1 serves as a source for mature miR-1-3p and miR-1-5p. Accordingly, miR-1-2 is encoded

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by the miR-133a-1/miR-1-2 gene cluster located in intron 12 of the mindbomb ubiquitin-protein ligase E3 (MIB1) on chromosome 18q11 [9]. In contrast to the miR-1-1 precursor, the only miR-13p is generated from the precursor for miR-1-2. Duplication of the bicistronic miR-1/miR-133a

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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]. MiR-1 is highly expressed in cardiac and skeletal muscles. This miRNA plays a key role in

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differentiation and proliferation of muscle cells [12].

2.1. miR-1 expression regulators

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. 6

ACCEPTED MANUSCRIPT 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

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(RISC) essential for functional activity of miRNAs. Muscleblind-like 1 (MBNL1), an RNA-binding protein, could bind to miR-1 preventing its interaction with LIN28 involved in miRNA biogenesis

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[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 regulators constantly increases. Further studies are required for understanding the complexity of

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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 precursors

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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 arrhythmia and disturbances in heart conduction [25]. Knockout of miR1-2 results in the same

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phenotype but animals generally survive due to the expression of miR-1 from the remaining miR1-1 gene [26]. Mice lacking both miR-1 genes die soon after birth due to serious developmental defects. 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 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 excitationcontraction 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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ACCEPTED MANUSCRIPT 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

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(ESCs) by inhibiting ESC differentiation to endodermal and neuroectodermal precursors as reflected by induction of Nk2 homeobox 5 (Nkx2.5), a early marker of cardiac mesoderm [30].

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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 followed by arrest of expression of non-muscle genes by miR-1-dependent translational repression

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of the Notch ligand Delta-like 1 (Dll-1), a factor involved in mediating cell fate decisions [30]. miR-1 also represses transcription factor Hes-1 [32] that drives differentiation of stem cells towards

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the ectoderm fate [33]. In addition, miR-1 up-regulates cyclin-dependent kinase-9 (Cdk9) [34], a 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

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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 embryoid bodies. These observations indeed suggest for the important role of miR-1 in the

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commitment of the muscle cell lineage in early stages of cardiogenesis (Figure 1).

2.3. miR-1 regulates differentiation of mesodermal precursors to cardiomyocytes

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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ACCEPTED MANUSCRIPT 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

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smooth muscle cells (VSMCs) by inhibiting Kruppel-like factor 4 (KLF4), a transcription factor

increased during ESC differentiation to VSMCs [44].

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involved in supporting stemness of ESCs. Furthermore, miR-1 expression was shown to be

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In embryonic heart, overexpression of myocardin, a key regulator of smooth muscle-specific transcriptional program, leads to the arrest of cardiomyocytes in immature state. In cooperation with

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miR-133, miR-1 down-regulates myocardin levels thereby providing a negative feedback loop in response to myocardin-dependent activation [45]. miR-1-dependent suppression of myocardin

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results in inhibiting expression of telokin (a transcriptional target of myocardin), a smooth musclerestricted repressor of the regulatory myosin light chain-2 (MLC2) phosphorylation [27]. Indeed, miR-1-mediated suppression of myocardin favors preferential differentiation of mesoderm towards

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skeletal and cardiac-specific phenotype.

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2.4. miR-1 drives reprogramming of other cell types to cardiomyocytes

Cardiac muscle cells could be produced through the mechanism of transdifferentiation, i.e. 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 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 Tbox5) 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 9

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

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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 the body with blood. Indeed, the tight coordination of the cardiac contraction cycle (that includes

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excitation, impulse propagation, and repolarization) is a vitally important. Disturbances in the control of heart automaticity lead to cardiac arrhythmias whose manifestation is a major contributor

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to cardiovascular mortality by significant increasing the risk of sudden death [49]. 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

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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 early depolarization and late repolarization) [23]. In ventricular myocytes, the CaV1.2 channel

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controls potential duration and excitation-contraction coupling [51]. Among Ca2+conductances, Ltype Ca2+ channels play the most profound role in supporting Ca2+ flow in cardiac muscle cells. Changes in membrane density and permeability of these channels contribute to the development of cardiac pathologies such as cardiac hypertrophy and HF [52]. 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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ACCEPTED MANUSCRIPT 2.6. miR-1 in cardiac repolarization

Cardiac depolarization is followed by repolarization in which the membrane potential

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returns to the resting state. Outward K+ currents (such as IK1, Ito, IKr, and IKs) are responsible for repolarization of heart muscle cells [54]. Zhao et al. [55] first showed the involvement of miR-1 in

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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. Iroquois homeobox protein 5 (Irx5), a transcriptional regulator, was shown to be targeted by miR-1-

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2. Irx5 inhibits expression of the potassium voltage-gated channel D2 (KCND2) gene that encodes the Kv4.2 subunit involved in transient outward K+ current (Ito) [56]. Ito is the major current

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contributing to the repolarization gradient in ventricular wall. Indeed, higher Irx5 levels cause reduction of KCND2 expression that disturbs ventricular repolarization and increases risk of arrhythmias.

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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 conduction due to the inhibition of Kir2.1 responsible for keeping membrane potential. Up-

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regulation of miR-1 was observed in ischemic hearts of mice with experimental MI that exhibited enhanced arrhythmia [57]. Interestingly, Shan et al. [58] reported that arsenic trioxide, an anticancer 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. 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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ACCEPTED MANUSCRIPT 2.7. miR-1 and calcium cycling in cardiomyocytes In the myocardium, Ca2+ cycling plays a central role in heart excitability and conductance.

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Ca2+ enters the cardiac muscle cell via several pathways of which L-type Ca2+ current (ICaL) is the most profound. The entrance of extracellular Ca2+into the sarcoplasm induces Ca2+ release from the

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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 the cell via the activity of sarcolemmal Ca2+ pump and forward-mode Na+-Ca2+ exchanger NCX1.

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SR Ca2+-ATPase (SERCA) also contributes to depletion of sarcoplasmic Ca2+by pumping Ca2+ back to the SR stores [62].

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NCX1 is a target of miR-1 that represses its mRNA [63]. This Na+/Ca2+ exchanger is 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

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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 Ca2+ release [65], reduced SR Ca2+ content, and up-regulation of Na+-Ca2+ exchanger [66]. In dogs

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with chronic heart failure, cardiomyocytes have delayed afterdepolarizations associated with enhanced Ca2+ concentration in the sarcoplasm due to CaMKII-dependent hyperphosphorylation of 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 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 miR1 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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ACCEPTED MANUSCRIPT 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

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2.8. miR-1 and conduction between cardiac muscle cells

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RyR2 channels.

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Intercellular gap junctions are involved in transfer of current from one myocyte to another. This process is essential for the electrical heart activation. Gap junctions between cardiomyocytes

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are constituted by connexin-43 that is abundantly present in the myocardium and by connexin-40 that is located in the artia and conduction system [70]. In cardiac pathology, remodeling of the gap

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junction architecture is a common event [71, 72]. The pathologic remodeling alters the distribution and expression levels of connexin 43 in junctions that could impair conduction and lead to arrhythmia [73].

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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 example, in viral myocarditis, miR-1 expression was found to be up-regulated. Accordingly, protein

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levels of connexin-43 were significantly reduced whereas connexin-43 mRNA expression was altered slightly only [74]. Indeed, viral myocarditis, miR-1 suppresses connexin-43 at posttranslational level [75].

In post-MI hearts, hypoxic conditions stimulate miR-1 expression that leads to decrease in 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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ACCEPTED MANUSCRIPT 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

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cardiac development and exhibits a robust cardiogenic potential that supports commitment of cardiomyocyte-specific lineage from EMCs and reprogramming of mature non-cardiac cells to heart

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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

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

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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 conduction of post-MI injured cardiac tissue is slower than that of the normal myocardium. The

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difference in conduction velocity between infarcted and non-infarcted tissue causes reentrant arrhythmias, an abnormality when an electrical impulse recurrently moves in a tight circle within 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 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 200fold) [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 14

ACCEPTED MANUSCRIPT 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 miR1 reciprocally inhibit expression of each other. IGF-1-dependent signaling suppresses miR-1

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through up-regulating transcription factor Forkhead box O3 (Foxo3), a negative miR-1 regulator [89, 90]. In cardiomyocytes, IGF-1 exhibits anti-apoptotic effects by preventing miR-1-mediated

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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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2.10. Arrhythmogenic potential of miR-1

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cardiovascular risk factors.

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

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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 the cardiomyocyte or between the cardiomyocytes [92]. Along with Kir2.1 and NCX1, miR-1 was

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shown to modulate expression of other K+ channels such as K+/Na+ hyperpolarization-activated cyclic nucleotide-gated ion channel (HCN)-2 and HCN-4 regulated by cAMP and located in the 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 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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ACCEPTED MANUSCRIPT As mentioned above, oxidative stress up-regulates cardiac expression of miR-1. In miR-1trangenic 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

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of antioxidant enzymes such as Cu2+/Zn2+-dependent superoxide dismutase (SOD1) and catalytic subunit of glutamate-cysteine ligase (GCLC) [97]. In the heart, miR-1 suppresses insulin- and IGF-

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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 heme by its oxidation and thereby preventing heme deposition in the atherosclerotic plaque, down-

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regulated miR-1 expression on myoblasts [99]. Indeed, these data suggest that up-regulation of miR-1 in heart muscle cells exhibits prooxidant properties and promotes oxidative stress thereby

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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 miRNA was proposed to be used as a selective biomarker for early diagnosis of acute MI and

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distinguishing between acute MI and other cardiac events such as angina pectoris [81, 100], nonacute MI [67], and other cardiovascular diseases [101]. Furthermore, in patients with ASC or acute 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 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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ACCEPTED MANUSCRIPT 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,

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implementation of IPC was showed to have a cardioprotective effect by reducing apoptosis and infarct size area, preserving mitochondrial function, and decreasing expression of pro-apoptotic

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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 hypertrophy due to the IGF-1-dependent inhibition [109]. Indeed, due to suppressing effects of

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miR-1 on pro-hypertophic IGF-1 signaling, restoration of miR-1 levels was suggested to be helpful in regressing hypertrophy and preserving heart against adverse remodeling. In a rat model of

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pressure overload-induced hypertrophy, cardiac-specific adenoviral-based delivery of miR-1 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].

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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 intensify and enhance the efficiency of cardiac tissue repair. Glass and Singla [111, 112] reported

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that miR-1-overproducing ESCs engrafted to post-IM hearts protected host myocardium from apoptosis, oxidative stress, and fibrosis and promoted cardiac regeneration. Finally, as already 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 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 miR133b/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 17

ACCEPTED MANUSCRIPT 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

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transcription factor such as Mef2, MyoD, myocardin, and SRF are involved in the regulation of miR-133 expression in muscle cells and their precursors [14, 114]. Brahma-related Gene 1 (Brg1)

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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 miR133a expression [116].

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In contrast to miR-133a, the miR-133b isoform is not expressed in cardiomyocytes since expression of the miR-133b/miR-206 cluster is specific for skeletal muscle only [45, 117].

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Furthermore, Boettger et al. [118] found that this cluster is dispensable for development, function, 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

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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

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, 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]. 18

ACCEPTED MANUSCRIPT 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

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differentiation of myoblasts to myocytes. In ESCs, overexpression of miR-133a was observed to down-regulate expression of cardiac markers [30, 34]. On the other hand, miR-133a was found to

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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]. miR133a could promote myoblast proliferation through targeting SRF and Cyclin D2 [12]. However,

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Zhang et al. [123] reported the inhibitory effect of miRNA-133a on myoblast proliferation by targeting Sp1, a transcription factor that stimulates cyclin D2 expression. It seems that controversial

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functional effects of mi-133a at the cardiac (and skeletal) muscle-specific lineage commitment are greatly influenced by concomitant expression of miR-1 that could share some targets with miR-

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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 fibroblasts and cardiomyocytes to cardiomyocytes [46-48]. Furthermore, adding of miR-133a alone 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 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 19

ACCEPTED MANUSCRIPT 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 regulation of heart Ca2+ cycling in PP2A-dependent manner.

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targets the regulatory subunit of PP2A [28], miR-1 and miR-133a play a key role in coordinated

Several miR-133a targets are involved in the repolarization of the heart action potential.

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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 miR-1, miR-133a down-regulates HCN-2 [128], responsible for the pacemaker current (If) that

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plays a key role in determining heart automaticity. In cardiac hypertrophy, myocardial expression of miR-133a is reduced while expression of HCN-2 and HCN-4 is elevated that leads to electrical

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remodeling and contributes to arrhythmogenesis and pacemaker dysfunction in hypertrophic hearts [127].

In the heart of diabetic rabbits, Xiao et al. [129] observed up-regulation of miR-133 along

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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 the K+ channel involved in the IKr current, and abnormalities in its function contribute to impaired

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repolarization phase, arrhythmia, and prolonged QT interval observed in diabetic heart [130]. Pathologic overactivation of miR-133 could therefore contribute to diabetic HERG K+ dysfunction. 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 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 channelinteracting 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 20

ACCEPTED MANUSCRIPT 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

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for miR-133a in cardiomyocytes that in turn down-regulates NFAT-dependent prohypertrophic signaling in response to hypertophic stimuli [135]. Calcineurin-NFAT signaling plays a key role in

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cardiac hypertophy [136] and miR-133a can inhibit hypertrophy by targeting both key components

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of this pathway.

In thyroid hormone (TH)- induced heart hypertophy mediated in part by type 1 angiotensin

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II receptor (AT1R), miR-133a levels were reduced while expression of SERCA2a and calcineurin (both are miR-133a targets) were increased.. MiR-133 mimic blunted TH-dependent cardiomyocyte

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hypertrophy suggesting for the suppressive effect of this miRNA in TH-mediated hypertrophy [137].

By targeting K+-conducting channels involved in IKr and IKs, miR-133a plays a role in the

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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 prevents arrhythmic induction [138]. Along with miR-1 that controls repolarization and

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depolarization phases of the action potential, miR-133 is crucially involved in the regulation of a proper heart conductance, contraction, and automaticity. Indeed, interplay between these mRNAs 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 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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ACCEPTED MANUSCRIPT [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

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in injured cardiomyocytes. In in vitro hypoxia-reoxygenation injury model and an in vivo rat model of I/R injury, overexpression of exogenous miR-133 greatly attenuated cardiac cell apoptosis [144].

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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 (DAPK2), BCL2-like 11 (BCL2L11), and Bcl-2-modifying factor (BMF) [147], thereby

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suppressing cell apoptosis. DAPK2 is abundantly expressed in heart and skeletal muscle and could be activated in a Ca2+/CaM-dependent manner [148]. Suppression of miR-133a in infarcted hearts

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therefore could be associated with overactivation of pro-apoptotic pathways. 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-

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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 overproduction in the heart resulted in improved cardiac function and decreased fibrosis [150].

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

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 22

ACCEPTED MANUSCRIPT recovered in the treated cardiac muscle cells [145] (Figure 2). Izarra et al. [147] showed that miR133a-transgenic cardiac progenitor cells (CPCs) have increased survival compared with nontransgenic CPCs due to the miR-133-mediated protection against apoptosis. In a rat MI model,

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transplantation of miR-133a-overexpressing CPCs led to improved heart function, suppressing hypertrophy and fibrosis, and increasing angiogenesis and myocyte proliferation.

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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. Transfection of MSCs with miR-133a increased expression of cardiac-specific genes via miR-133a-

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dependent targeting EGFR [122]. miR-133a-overexpressing MSC transplants had increased survival and engraftment rate in rat hearts subjected to MI [146]. Indeed, development of stem cells via

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cardiac-specific miRNA manipulation has a promising potential to improve the therapeutic outcome in subjects undergoing stem cell-based cardiotherapy for MI.

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4. miR-208a/b

miR-208 is a cardiac-enriched miRNA, what is also expressed in the skeletal muscle (Huang

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and Li 2015) [153]. This miRNA is presented in two isoforms: miR-208a and miR-208b. Murine miR-208 isoforms have a high degree of identity while human isoforms are not [154]. Both human 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 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 cardiacspecific. 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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ACCEPTED MANUSCRIPT 4.1. miR-208 in cardiogenesis

While miR-1 and miR-133 have a prominent role in early cardiogenesis, miR-208 seems to

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be involved in late stages of cardiac development related to the commitment of cardiomyocytes from myoblasts [160]. miR-208 regulates cardiac MHC expression.

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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 expressed in late fetal life while α-MHC is preferentially expressed in the adult [155]. In rodents,

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Myh6 and Myh7 are differentially expressed in adult hearts. Accordingly, cardiac expression of miR-208a and miR-208b could also vary in parallel with α-MHC and β-MHC expression [161-163].

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miR-208b is predominantly expressed in the fetal myocardium while its expression drops in the 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

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of miR-208a occurs post-natally suggesting that both miRNAs share the same targets [163].

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and large animals [160].

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Compared with rodents, miR-208a is the predominant miR-208 isoform that is expressed in humans

4.2. miR-208 in heart function

In mice, knockout of miR-208a results in lack of capacity to up-regulate expression of βMHC 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 24

ACCEPTED MANUSCRIPT 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.

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Lack of miR-208a is accompanied with up-regulated cardiac expression of fast myofibers that normally not produced in the myocardium [165-167]. Expression of THRAP1 that encodes

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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 β-MHC production [163, 168] and induces hypertrophy. In hyperthyroidism, cardiac levels of miR-

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208a are up-regulated leading to the development of heart hypertophy [169]. Overexpression of miR-208a in cardiomyocytes leads to hypertrophy and is associated with

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increased β-MHC expression and miR-208-dependent down-regulation of THRAP1 and myostatin, 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

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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 findings suggest that miR-208 is crucially involved in heart contraction and conduction but

properties.

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pathogenic miR-208 overactivity possesses prohypertrophic, profibrotic, and arrythmogenic

4.3. miR-208 as a diagnostic marker of early myocardial infarction

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 miR208 (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 miR208a 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 25

ACCEPTED MANUSCRIPT 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

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levels are frequently increased in subjects with chronic renal failure [172]. Interestingly, in a model of acute isoproterenol-induced heart injury, circulating miR-208a

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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 due to the high homology between these miRNAs in mice [163].

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In patients with acute MI, circulating levels of miR-208b were shown to be greatly upregulated. For example, Gidlöf et al. [104] reported 3000-fold increase in plasma miR-208b 12 h

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post-infarction in subjects with acute ST-segment elevation MI compared with healthy subjects. 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

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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 diagnostic marker for acute MI [174]. However, the diagnostic value of miR-208b for this disease

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was not superior compared with troponin T [103]. In studies in which the diagnostic value of miR-208 in distinguishing acute MI and ASC 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 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 26

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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

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allow manufacturing synthetic oligonucleotids (miRmimics or antagomiRs) that respectively mimic or inhibit mature miRNA targets. This strategy provides an option to manipulate with clinically

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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 by injection of miR-208a-specific antigomiR. This resulted in block of pathological cardiac

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remodeling, prevention of pathological myosin switch, inhibition of perivascular fibrosis, and improved heart function. This experiment shows the utility of targeted inhibition of miR-208a in

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cardiopathy accompanied with up-regulated tissue miR-208a levels such as HF and cardiac 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

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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.

5. miR-499

Human miR-499 is located in intron 19 of the MYH7B (also known as MYH14) gene (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 miR499b 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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ACCEPTED MANUSCRIPT 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

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also support expression of smooth muscle genes through activation of myocardin [187]. Indeed, by inhibiting MEF2C, miR-499 favors switching myogenic differentiation preferentially towards

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cardiomyocytes.

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In human fetal cardiomyocyte progenitor cells (CMPCs), miR-499 is up-regulated and cooperates with miR-1 to drive differentiation of CMPCs and ESCs to cardiomyocytes [188].

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Interestingly, miR-499 was shown to be transferred from differentiated myocytes to progenitor cells through gap junctions and regulates differentiation in a paracrine manner [188].

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Low expression levels of human MYH7B were detected in the embryonic heart and skeletal 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

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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]. miR-499 targets SRY box-6 (Sox-6), a conserved transcription repressor that inhibits slow-

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twitch myofiber-specific expression program in zebrafish [190]. Sox-6 is also down-regulated by PR domain zinc finger protein 1 (Prdm1), a transcription repressor of the slow-twitch myofiberspecific program [191, 192]. By derepressing slow-twitch fiber program, Prdm1 also induces miR499 expression [191]. Indeed, Sox-6/Prdm1 and miR-499/miR-208a regulatory tandems that play 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 28

ACCEPTED MANUSCRIPT 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))

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and conductivity (KCNH6 and Kv11.1) [196].

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5.2. miR-499 in acute myocardial infarction

miR-499 deregulation was shown to be primarily involved in the pathogenesis of heart

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hypertrophy due to the pronounced role in the regulation of expression of sarcomeric genes [197]. The appearance of this miRNA in the bloodstream could serve as an indicator of heart damage that

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happened in acute MI. Plasma miR-499 levels are elevated in subjects with acute MI but are below 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

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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, 199, 200]. Several studies reported that miR-499 has a superior diagnostic accuracy for acute MI

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compared with conventional cardiac troponin T assay (AUC of 0.79-0.92 vs. 0.70) [198, 199, 201] while other studies failed to show this [103, 104]. However, compared with troponins, miR-499 has 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 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 29

ACCEPTED MANUSCRIPT 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

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expression in cardiac cells (as a part of stress-induced response) through phosphorylation of c-Jun that then associates with c-Fos to form transcription factor AP-1 and prime miR-499 expression

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from the MYH7B promoter [203].

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miR-499 could inhibit cardiac cell apoptosis through down-regulating Sox-6, which suppresses cell proliferation and enhances apoptosis [208]. Sox-6 inhibition by miR-499 results in

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derepression of the cyclin D1 promoter and activation of cyclin D-mediated cell growth and proliferation [209]. Anti-apoptotic and proliferative effects of miR-499 are important during heart

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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 using for obtaining heart muscle cells through reprogramming of other mature cell types. As already

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discussed, cardiac miRNAs including miR-499 could be used with success for generation of cardiomyocytes in vitro [46] and in vivo [47] via reprogramming strategies that provides new promises for recovering injured cardiac tissue after MI. Cardiospecific regenerative capacity of miR-499 could promote preferential differentiation 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 30

ACCEPTED MANUSCRIPT 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

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functional consequences of their targeting by cardiac miRNAs. This should expand our knowledge about heart defects and functional/structural aberrations caused by pathological disregulation of

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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 in prediction of death in patients with acute MI is widely underexplored to date. Indeed, more

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follow-up studies involving independent and large patient cohorts should be performed. Cardiospecific regenerative potential of cardiac miRNAs should be studied in details. This

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should expand our options to manipulate with expression of miRNA and their cardiac targets in 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,

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it is necessary to optimize reprogramming techniques for increasing quantitative and qualitative

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cadiomyocyte yield.

Disclosures

None declared.

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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ACCEPTED MANUSCRIPT References

[1] Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial

PT

infarction. Cardiovasc Res 2002;53:31-47.

[2] Jessup M, Brozena S. Heart failure. N Engl J Med 2003;348:2007-18.

RI

[3] Waller C, Hiller KH, Pfaff D, Gattenlöhner S, Ertl G, Bauer WR. Functional mechanisms of

SC

myocardial microcirculation in left ventricular hypertrophy: a hypothetical model of capillary remodeling post myocardial infarction. Microvasc Res 2008;75:104-11.

NU

[4] Duygu B, de Windt LJ, da Costa Martins PA. Targeting microRNAs in heart failure. Trends Cardiovasc Med 2015; pii:S1050-1738(15)00153-X.

MA

[5] Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009;19:92-105. [6] Topkara VK, Mann DL. Role of microRNAs in cardiac remodeling and heart failure.

D

Cardiovasc Drugs Ther 2011;25:171-82.

TE

[7] da Costa Martins PA, Bourajjaj M, Gladka M, Kortland M, van Oort RJ, Pinto YM, Molkentin JD, De Windt LJ. Conditional dicer gene deletion in the postnatal myocardium provokes

AC CE P

spontaneous cardiac remodeling. Circulation 2008;118:1567-76. [8] Piubelli C, Meraviglia V, Pompilio G, D'Alessandra Y, Colombo GI, Rossini A. MicroRNAs and cardiac cell fate. Cells 2014;3:802-23. [9] Li J, Dong X, Wang Z, Wu J. MicroRNA-1 in Cardiac Diseases and Cancers. Korean J Physiol Pharmacol 2014;18:359-63. [10] Tani S, Kuraku S, Sakamoto H, Inoue K, Kusakabe R. Developmental expression and evolution of muscle-specific microRNAs conserved in vertebrates. Evol Dev 2013;15:293304. [11] Kusakabe R, Tani S, Nishitsuji K, Shindo M, Okamura K, Miyamoto Y, Nakai K, Suzuki Y, Kusakabe TG, Inoue K. Characterization of the compact bicistronic microRNA precursor, miR-1/miR-133, expressed specifically in Ciona muscle tissues. Gene Expr Patterns 2013;13:43-50. [12] Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM, Conlon FL, Wang DZ. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 2006;38:228-33. 32

ACCEPTED MANUSCRIPT [13] Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 2005;436:214-20. [14] Liu N, Williams AH, Kim Y, McAnally J, Bezprozvannaya S, Sutherland LB, Richardson JA,

PT

Bassel-Duby R, Olson EN. An intragenic MEF2-dependent enhancer directs muscle-specific expression of microRNAs 1 and 133. Proc Natl Acad Sci USA 2007;104:20844-9.

RI

[15] Mitchelson KR, Qin WY. Roles of the canonical myomiRs miR-1, -133 and -206 in cell

SC

development and disease. World J Biol Chem 2015;6:162-208. [16] Liu N, Olson EN. MicroRNA regulatory networks in cardiovascular development. Dev Cell

NU

2010;18:510-25.

[17] Rachagani S, Cheng Y, Reecy JM. Myostatin genotype regulates muscle-specific miRNA

MA

expression in mouse pectoralis muscle. BMC Res Notes 2010;3:297. [18] Yoon MS, Chen J. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis. Mol Biol Cell 2013;24:3754-63.

D

[19] Sun Y, Ge Y, Drnevich J, Zhao Y, Band M, Chen J. Mammalian target of rapamycin regulates

TE

miRNA-1 and follistatin in skeletal myogenesis. J Cell Biol 2010;189:1157-69. [20] Iezzi S, Di Padova M, Serra C, Caretti G, Simone C, Maklan E, Minetti G, Zhao P, Hoffman

AC CE P

EP, Puri PL, Sartorelli V. Deacetylase inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin. Dev Cell 2004;6:673-84. [21] Winbanks CE, Weeks KL, Thomson RE, Sepulveda PV, Beyer C, Qian H, Chen JL, Allen JM, Lancaster GI, Febbraio MA, Harrison CA, McMullen JR, Chamberlain JS, Gregorevic P. Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin. J Cell Biol 2012;197:997-1008. [22] King IN, Yartseva V, Salas D, Kumar A, Heidersbach A, Ando DM, Stallings NR, Elliott JL, Srivastava D, Ivey KN. The RNA-binding protein TDP-43 selectively disrupts microRNA1/206 incorporation into the RNA-induced silencing complex. J Biol Chem 2014;289:1426371. [23] Turner C, Hilton-Jones D. Myotonic dystrophy: diagnosis, management and new therapies. Curr Opin Neurol 2014;27:599-606. [24] Trabucchi M, Briata P, Garcia-Mayoral M, Haase AD, Filipowicz W, Ramos A, Gherzi R, Rosenfeld MG. The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature 2009;459:1010-4. 33

ACCEPTED MANUSCRIPT [25] Tao G, Martin JF. MicroRNAs get to the heart of development. Elife 2013;2:e01710. [26] Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, Muth AN, Tsuchihashi T, McManus MT, Schwartz RJ, Srivastava D. Dysregulation of cardiogenesis, cardiac conduction,

PT

and cell cycle in mice lacking miRNA-1-2. Cell 2007;129:303-17. [27] Heidersbach A, Saxby C, Carver-Moore K, Huang Y, Ang YS, de Jong PJ, Ivey KN,

RI

Srivastava D. microRNA-1 regulates sarcomere formation and suppresses smooth muscle

SC

gene expression in the mammalian heart. Elife 2013;2:e01323. [28] Terentyev D, Belevych AE, Terentyeva R, Martin MM, Malana GE, Kuhn DE, Abdellatif M,

NU

Feldman DS, Elton TS, Györke S. miR-1 overexpression enhances Ca(2+) release and promotes cardiac arrhythmogenesis by targeting PP2A regulatory subunit B56alpha and

MA

causing CaMKII-dependent hyperphosphorylation of RyR2. Circ Res 2009;104:514-21. [29] Wijnker PJ, Boknik P, Gergs U, Müller FU, Neumann J, dos Remedios C, Schmitz W, Sindermann JR, Stienen GJ, van der Velden J, Kirchhefer U. Protein phosphatase 2A affects

D

myofilament contractility in non-failing but not in failing human myocardium. J Muscle Res

TE

Cell Motil 2011;32(3):221-33.

[30] Ivey KN, Muth A, Arnold J, King FW, Yeh RF, Fish JE, Hsiao EC, Schwartz RJ, Conklin BR,

AC CE P

Bernstein HS, Srivastava D. MicroRNA regulation of cell lineages in mouse and human embryonic stem cells. Cell Stem Cell 2008;2:219-29. [31] Skerjanc IS, Petropoulos H, Ridgeway AG, Wilton S. Myocyte enhancer factor 2C and Nkx25 up-regulate each other's expression and initiate cardiomyogenesis in P19 cells. J Biol Chem 1998;273:34904-10.

[32] Huang F, Tang L, Fang ZF, Hu XQ, Pan JY, Zhou SH. miR-1-mediated induction of cardiogenesis in mesenchymal stem cells via downregulation of Hes-1. Biomed Res Int 2013;2013:216286. [33] Hatakeyama J, Bessho Y, Katoh K, Ookawara S, Fujioka M, Guillemot F, Kageyama R. Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation. Development 2004;131:5539-50. [34] Takaya T, Ono K, Kawamura T, Takanabe R, Kaichi S, Morimoto T, Wada H, Kita T, Shimatsu A, Hasegawa K. MicroRNA-1 and MicroRNA-133 in spontaneous myocardial differentiation of mouse embryonic stem cells. Circ J 2009;73:1492-7.

34

ACCEPTED MANUSCRIPT [35] Kaichi S, Takaya T, Morimoto T, Sunagawa Y, Kawamura T, Ono K, Shimatsu A, Baba S, Heike T, Nakahata T, Hasegawa K. Cyclin-dependent kinase 9 forms a complex with GATA4 and is involved in the differentiation of mouse ES cells into cardiomyocytes. J Cell Physiol

PT

2011;226:248-54.

[36] Lu J, McKinsey TA, Zhang CL, Olson EN. Regulation of skeletal myogenesis by association

RI

of the MEF2 transcription factor with class II histone deacetylases. Mol Cell 2000;6:233-44.

SC

[37] Schneider M, Andersen DC, Silahtaroglu A, Lyngbæk S, Kauppinen S, Hansen JL, Sheikh SP. Cell-specific detection of microRNA expression during cardiomyogenesis by combined in

NU

situ hybridization and immunohistochemistry. J Mol Histol 2011;42:289-99. [38] Synnergren J, Améen C, Lindahl A, Olsson B, Sartipy P. Expression of microRNAs and their

Genomics 2011;43:581-94.

MA

target mRNAs in human stem cell-derived cardiomyocyte clusters and in heart tissue. Physiol

[39] Synnergren J, Améen C, Jansson A, Sartipy P. Global transcriptional profiling reveals

D

similarities and differences between human stem cell-derived cardiomyocyte clusters and

TE

heart tissue. Physiol Genomics 2012;44:245-58. [40] King IN, Qian L, Liang J, Huang Y, Shieh JT, Kwon C, Srivastava D. A genome-wide screen

AC CE P

reveals a role for microRNA-1 in modulating cardiac cell polarity. Dev Cell 2011;20:497-510. [41] Lu TY, Lin B, Li Y, Arora A, Han L, Cui C, Coronnello C, Sheng Y, Benos PV, Yang L. Overexpression of microRNA-1 promotes cardiomyocyte commitment from human cardiovascular progenitors via suppressing WNT and FGF signaling pathways. J Mol Cell Cardiol 2013;63:146-54.

[42] Wang D, Chang PS, Wang Z, Sutherland L, Richardson JA, Small E, Krieg PA, Olson EN. Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell 2001;105:851-62. [43] Chen J, Yin H, Jiang Y, Radhakrishnan SK, Huang ZP, Li J, Shi Z, Kilsdonk EP, Gui Y, Wang DZ, Zheng XL. Induction of microRNA-1 by myocardin in smooth muscle cells inhibits cell proliferation. Arterioscler Thromb Vasc Biol 2011;31:368-75. [44] Xie C, Huang H, Sun X, Guo Y, Hamblin M, Ritchie RP, Garcia-Barrio MT, Zhang J, Chen YE. MicroRNA-1 regulates smooth muscle cell differentiation by repressing Kruppellike factor 4. Stem Cells Dev 2011;20:205-10.

35

ACCEPTED MANUSCRIPT [45] Wystub K, Besser J, Bachmann A, Boettger T, Braun T. miR-1/133a clusters cooperatively specify the cardiomyogenic lineage by adjustment of myocardin levels during embryonic heart development. PLoS Genet 2013;9:e1003793.

PT

[46] Jayawardena TM, Egemnazarov B, Finch EA, Zhang L, Payne JA, Pandya K, Zhang Z, Rosenberg P, Mirotsou M, Dzau VJ. MicroRNA-mediated in vitro and in vivo direct

RI

reprogramming of cardiac fibroblasts to cardiomyocytes. Circ Res 2012;110:1465-73.

SC

[47] Jayawardena TM, Finch EA, Zhang L, Zhang H, Hodgkinson CP, Pratt RE, Rosenberg PB, Mirotsou M, Dzau VJ. MicroRNA-mediated in vitro and in vivo direct reprogramming of

NU

cardiac fibroblasts to cardiomyocytes. Circ Res 2015;116:418-24. [48] Nam YJ, Song K, Luo X, Daniel E, Lambeth K, West K, Hill JA, DiMaio JM, Baker LA,

MA

Bassel-Duby R, Olson EN. Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors. Proc Natl Acad Sci USA 2013;110:5588-93. [49] Marbán E. Cardiac channelopathies. Nature 2002;415:213-8.

D

[50] Kim GH. MicroRNA regulation of cardiac conduction and arrhythmias. Transl Res

TE

2013;161:381-92.

[51] Klugbauer N, Welling A, Specht V, Seisenberger C, Hofmann F. L-type Ca2+ channels of the

AC CE P

embryonic mouse heart. Eur J Pharmacol 2002;447:279-84. [52] Goonasekera SA, Hammer K, Auger-Messier M, Bodi I, Chen X, Zhang H, Reiken S, Elrod JW, Correll RN, York AJ, Sargent MA, Hofmann F, Moosmang S, Marks AR, Houser SR, Bers DM, Molkentin JD. Decreased cardiac L-type Ca²+ channel activity induces hypertrophy and heart failure in mice. J Clin Invest 2012;122:280-90. [53] Furling D. Misregulation of alternative splicing and microRNA processing in DM1 pathogenesis. Rinsho Shinkeigaku 2012;52:1018-22. [54] Nerbonne JM, Dorn GW 2nd. MicroRNA-133a protects against myocardial fibrosis and modulates electrical repolarization without affecting hypertrophy in pressure-overloaded adult hearts. Circ Res 2010;106:166-75. [55] Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, Muth AN, Tsuchihashi T, McManus MT, Schwartz RJ, Srivastava D. Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell 2007 129:303-17. [56] Costantini DL, Arruda EP, Agarwal P, Kim KH, Zhu Y, Zhu W, Lebel M, Cheng CW, Park CY, Pierce SA, Guerchicoff A, Pollevick GD, Chan TY, Kabir MG, Cheng SH, Husain M, 36

ACCEPTED MANUSCRIPT Antzelevitch C, Srivastava D, Gross GJ, Hui CC, Backx PH, Bruneau BG. The homeodomain transcription factor Irx5 establishes the mouse cardiac ventricular repolarization gradient. Cell 2005;123:347-58.

PT

[57] Yang B, Lin H, Xiao J, Lu Y, Luo X, Li B, Zhang Y, Xu C, Bai Y, Wang H, Chen G, Wang Z. The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by

RI

targeting GJA1 and KCNJ2. Nat Med 2007;13:486-91.

SC

[58] Shan H, Zhang Y, Cai B, Chen X, Fan Y, Yang L, Chen X, Liang H, Zhang Y, Song X, Xu C, Lu Y, Yang B, Du Z. Up-regulation of microRNA-1 and microRNA-

NU

133 contributes to arsenic-induced cardiac electrical remodeling. Int J Cardiol 2013;167:2798805.

MA

[59] Barbey JT, Pezzullo JC, Soignet SL. Effect of arsenic trioxide on QT interval in patients with advanced malignancies. J Clin Oncol 2003;21:3609-15. [60] Girmatsion Z, Biliczki P, Bonauer A, Wimmer-Greinecker G, Scherer M, Moritz A, Bukowska

D

A, Goette A, Nattel S, Hohnloser SH, Ehrlich JR. Changes in microRNA-1 expression and

TE

IK1 up-regulation in human atrial fibrillation. Heart Rhythm 2009;6:1802-9. [61] Yamakage M, Namiki A. Calcium channels--basic aspects of their structure, function and gene

AC CE P

encoding; anesthetic action on the channels - a review. Can J Anaesth 2002;49:151-64. [62] Aronsen JM, Swift F, Sejersted OM. Cardiac sodium transport and excitationcontraction coupling. J Mol Cell Cardiol 2013;61:11-9. [63] Tritsch E, Mallat Y, Lefebvre F, Diguet N, Escoubet B, Blanc J, De Windt LJ, Catalucci D, Vandecasteele G, Li Z, Mericskay M. An SRF/miR 1 axis regulates NCX1 and annexin A5 protein levels in the normal and failing heart. Cardiovasc Res 2013;98:372-80. [64] DiPolo R, Beaugé L. Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions. Physiol Rev 2006;86:155-203. [65] Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: Roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness. Circ Res 2001;88:1159-67. [66] Pogwizd SM, Bers DM. Calcium cycling in heart failure: the arrhythmia connection. J Cardiovasc Electrophysiol 2002;13:88-91.

37

ACCEPTED MANUSCRIPT [67] Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM. Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ Res 2005;97:1314-22.

PT

[68] Belevych AE, Sansom SE, Terentyeva R, Ho HT, Nishijima Y, Martin MM, Jindal HK, Rochira JA, Kunitomo Y, Abdellatif M, Carnes CA, Elton TS, Györke S, Terentyev D.

RI

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase

SC

activity from RyR2 complex. PLoS One 2011;6:e28324.

[69] Kumarswamy R, Lyon AR, Volkmann I, Mills AM, Bretthauer J, Pahuja A, Geers-Knörr C,

NU

Kraft T, Hajjar RJ, Macleod KT, Harding SE, Thum T. SERCA2a gene therapy restores microRNA-1 expression in heart failure via an Akt/FoxO3A-dependent pathway Eur Heart J

MA

2012;33:1067-75.

[70] Stroemlund LW, Jensen CF, Qvortrup K, Delmar M, Nielsen MS. Gap junctions - guards of excitability. Biochem Soc Trans 2015;43:508-12.

D

[71] Yao JA, Hussain W, Patel P, Peters NS, Boyden PA, Wit AL. Remodeling of gap junctional

TE

channel function in epicardial border zone of healing canine infarcts. Circ Res 2003;92:43743.

AC CE P

[72] Cabo C, Yao J, Boyden PA, Chen S, Hussain W, Duffy HS, Ciaccio EJ, Peters NS, Wit AL. Heterogeneous gap junction remodeling in reentrant circuits in the epicardial border zone of the healing canine infarct. Cardiovasc Res 2006;72:241-9. [73] Nattel S, Maguy A, Le Bouter S, Yeh YH. Arrhythmogenic ion-channel remodeling in the heart: heart failure, myocardial infarction, and atrial fibrillation. Physiol Rev 2007;87:425-56. [74] Xu HF, Ding YJ, Shen YW, Xue AM, Xu HM, Luo CL, Li BX, Liu YL, Zhao ZQ. MicroRNA-1 represses Cx43 expression in viral myocarditis. Mol Cell Biochem 2012;362:141-8. [75] Klotz LO. Posttranscriptional regulation of connexin-43 expression. Arch Biochem Biophys 2012;524:23-9 [76] Lu Y, Zhang Y, Shan H, Pan Z, Li X, Li B, Xu C, Zhang B, Zhang F, Dong D, Song W, Qiao G, Yang B. MicroRNA-1 downregulation by propranolol in a rat model of myocardial infarction: a new mechanism for ischaemic cardioprotection. Cardiovasc Res 2009;84:434-41.

38

ACCEPTED MANUSCRIPT [77] Zhang Y, Zhang L, Chu W, Wang B, Zhang J, Zhao M, Li X, Li B, Lu Y, Yang B, Shan H. Tanshinone IIA inhibits miR-1 expression through p38 MAPK signal pathway in postinfarction rat cardiomyocytes. Cell Physiol Biochem 2010;26:991-8.

PT

[78] Curcio A, Torella D, Iaconetti C, Pasceri E, Sabatino J, Sorrentino S, Giampà S, Micieli M, Polimeni A, Henning BJ, Leone A, Catalucci D, Ellison GM, Condorelli G, Indolfi C.

RI

MicroRNA-1 downregulation increases connexin 43 displacement and induces ventricular

SC

tachyarrhythmias in rodent hypertrophic hearts. PLoS One 2013;8:e70158. [79] Allessie MA, Bonke FI, Schopman FJ. Circus movement in rabbit atrial muscle as a

NU

mechanism of tachycardia. II. The role of nonuniform recovery of excitability in the occurrence of unidirectional block, as studied with multiple microelectrodes. Circ Res

MA

1976;39:168-77.

[80] Long G, Wang F, Duan Q, Chen F, Yang S, Gong W, Wang Y, Chen C, Wang DW. Human circulating microRNA-1 and microRNA-126 as potential novel indicators

D

for acute myocardial infarction. Int J Biol Sci 2012;8:811-8.

TE

[81] Li C, Fang Z, Jiang T, Zhang Q, Liu C, Zhang C, Xiang Y. Serum microRNAs profile from genome-wide serves as a fingerprint for diagnosis of acute myocardial infarction and angina

AC CE P

pectoris. BMC Med Genomics 2013;6:16. [82] D'Alessandra Y, Devanna P, Limana F, Straino S, Di Carlo A, Brambilla PG, Rubino M, Carena MC, Spazzafumo L, De Simone M, Micheli B, Biglioli P, Achilli F, Martelli F, Maggiolini S, Marenzi G, Pompilio G, Capogrossi MC. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur Heart J 2010;31:2765-73. [83] Cheng Y, Tan N, Yang J, Liu X, Cao X, He P, Dong X, Qin S, Zhang C. A translational study of circulating cell-free microRNA-1 in acute myocardial infarction. Clin Sci (Lond) 2010;119:87-95. [84] Pan Z, Sun X, Ren J, Li X, Gao X, Lu C, Zhang Y, Sun H, Wang Y, Wang H, Wang J, Xie L, Lu Y, Yang B. miR-1 exacerbates cardiac ischemia-reperfusion injury in mouse models. PLoS One 2012;7:e50515. [85] Tang Y, Zheng J, Sun Y, Wu Z, Liu Z, Huang G. MicroRNA-1 regulates cardiomyocyte apoptosis by targeting Bcl-2. Int Heart J 2009;50:377-87.

39

ACCEPTED MANUSCRIPT [86] Xu C, Lu Y, Pan Z, Chu W, Luo X, Lin H, Xiao J, Shan H, Wang Z, Yang B. The musclespecific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes. J Cell Sci 2007;120:Pt 17:3045-52.

PT

[87] Yu XY, Song YH, Geng YJ, Lin QX, Shan ZX, Lin SG, Li Y. Glucose induces apoptosis of cardiomyocytes via microRNA-1 and IGF-1. Biochem Biophys Res Commun 2008;376:548-

RI

52.

SC

[88] Shan ZX, Lin QX, Deng CY, Zhu JN, Mai LP, Liu JL, Fu YH, Liu XY, Li YX, Zhang YY, Lin SG, Yu XY. miR-1/miR-206 regulate Hsp60 expression contributing to glucose-mediated

NU

apoptosis in cardiomyocytes. FEBS Lett 2010;584:3592-600. [89] Elia L, Contu R, Quintavalle M, Varrone F, Chimenti C, Russo MA, Cimino V, De Marinis L,

MA

Frustaci A, Catalucci D, Condorelli G. Reciprocal regulation of microRNA-1 and insulin-like growth factor-1 signal transduction cascade in cardiac and skeletal muscle in physiological and pathological conditions. Circulation 2009;120:2377-85.

D

[90] Kumarswamy R, Lyon AR, Volkmann I, Mills AM, Bretthauer J, Pahuja A, Geers-Knörr C,

TE

Kraft T, Hajjar RJ, Macleod KT, Harding SE, Thum T. SERCA2a gene therapy restores microRNA-1 expression in heart failure via an Akt/FoxO3A-dependent pathway. Eur Heart J

AC CE P

2012;33:1067-75.

[91] Li Y, Shelat H, Geng YJ. IGF-1 prevents oxidative stress induced-apoptosis in induced pluripotent stem cells which is mediated by microRNA-1. Biochem Biophys Res Commun. 2012;426:615-9.

[92] Suffredini S, Stillitano F, Comini L, Bouly M, Brogioni S, Ceconi C, Ferrari R, Mugelli A, Cerbai E. Long-term treatment with ivabradine in post-myocardial infarcted rats counteracts f-channel overexpression. Br J Pharmacol 2012;165:1457-66. [93] Michels G, Brandt MC, Zagidullin N, Khan IF, Larbig R, van Aaken S, Wippermann J, Hoppe UC. Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations. Cardiovasc Res 2008;78:466-75. [94] Michels G, Er F, Khan I, Südkamp M, Herzig S, Hoppe UC. Single-channel properties support a potential contribution of hyperpolarization-activated cyclic nucleotide-gated channels and If to cardiac arrhythmias. Circulation 2005;111:399-404.

40

ACCEPTED MANUSCRIPT [95] Myers R, Timofeyev V, Li N, Kim C, Ledford HA, Sirish P, Lau V, Zhang Y, Fayyaz K, Singapuri A, Lopez JE, Knowlton AA, Zhang XD, Chiamvimonvat N. Feedback mechanisms

Electrophysiol 2015;8:942-50.

PT

for cardiac-specific microRNAs and cAMP signaling in electrical remodeling. Circ Arrhythm

[96] Schömig A, Richardt G, Kurz T. Sympatho-adrenergic activation of the ischemic myocardium

RI

and its arrhythmogenic impact. Herz 1995;20:169-86.

SC

[97] Wang L, Yuan Y, Li J, Ren H, Cai Q, Chen X, Liang H, Shan H, Fu ZD, Gao X, Lv Y, Yang B, Zhang Y. MicroRNA-1 aggravates cardiac oxidative stress by post-transcriptional

NU

modification of the antioxidant network. Cell Stress Chaperones 2015;20:411-20. [98] Chen T, Ding G, Jin Z, Wagner MB, Yuan Z. Insulin ameliorates miR-1-induced injury in

MA

H9c2 cells under oxidative stress via Akt activation. Mol Cell Biochem 2012;369:167-74. [99] Kozakowska M, Ciesla M, Stefanska A, Skrzypek K, Was H, Jazwa A, Grochot-Przeczek A, Kotlinowski J, Szymula A, Bartelik A, Mazan M, Yagensky O, Florczyk U, Lemke K, Zebzda

D

A, Dyduch G, Nowak W, Szade K, Stepniewski J, Majka M, Derlacz R, Loboda A, Dulak J,

TE

Jozkowicz A. Heme oxygenase-1 inhibits myoblast differentiation by targeting myomirs. Antioxid Redox Signal 2012;16:113-27.

AC CE P

[100] Kuwabara Y, Ono K, Horie T, Nishi H, Nagao K, Kinoshita M, Watanabe S, Baba O, Kojima Y, Shizuta S, Imai M, Tamura T, Kita T, Kimura T. Increased microRNA-1 and microRNA133a levels in serum of patients with cardiovascular disease indicate myocardial damage. Circ Cardiovasc Genet 2011;4:446-54. [101] Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, Qin YW, Jing Q. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 2010;31:659-66. [102] Widera C, Gupta SK, Lorenzen JM, Bang C, Bauersachs J, Bethmann K, Kempf T, Wollert KC, Thum T. Diagnostic and prognostic impact of six circulating microRNAs in acute coronary syndrome. J Mol Cell Cardiol 2011;51:872-5. [103] Li YQ, Zhang MF, Wen HY, Hu CL, Liu R, Wei HY, Ai CM, Wang G, Liao XX, Li X. Comparing the diagnostic values of circulating microRNAs and cardiac troponin T in patients with acute myocardial infarction. Clinics (Sao Paulo) 2013;68:75-80. [104] Gidlöf O, Andersson P, van der Pals J, Götberg M, Erlinge D. Cardiospecific microRNA plasma levels correlate with troponin and cardiac function in patients with ST elevation 41

ACCEPTED MANUSCRIPT myocardial infarction, are selectively dependent on renal elimination, and can be detected in urine samples. Cardiology 2011;118:217-26. [105] Heusch G, Bøtker HE, Przyklenk K, Redington A, Yellon D. Remote ischemic conditioning. J

PT

Am Coll Cardiol 2015;65:177-95.

[106] Duan X, Ji B, Wang X, Liu J, Zheng Z, Long C, Tang Y, Hu S. Expression of microRNA-

RI

1 and microRNA-21 in different protocols of ischemic conditioning in an isolated rat heart

SC

model. Cardiology 2012;122:36-43.

[107] Brandenburger T, Grievink H, Heinen N, Barthel F, Huhn R, Stachuletz F, Kohns M, Pannen

NU

B, Bauer I. Effects of remote ischemic preconditioning and myocardial ischemia on microRNA-1 expression in the rat heart in vivo. Shock 2014;42:234-8.

MA

[108] Slagsvold KH, Rognmo O, Høydal M, Wisløff U, Wahba A. Remote ischemic preconditioning preserves mitochondrial function and influences myocardial microRNA expression in atrial myocardium during coronary bypass surgery. Circ Res 2014;114:851-9.

D

[109] Hua Y, Zhang Y, Ren J. IGF-1 deficiency resists cardiac hypertrophy and myocardial

2012;16:83-95.

TE

contractile dysfunction: role of microRNA-1 and microRNA-133a. J Cell Mol Med

AC CE P

[110] Karakikes I, Chaanine AH, Kang S, Mukete BN, Jeong D, Zhang S, Hajjar RJ, Lebeche D. Therapeutic cardiac-targeted delivery of miR-1 reverses pressure overload-induced cardiac hypertrophy and attenuates pathological remodeling. J Am Heart Assoc 2013;2:e000078. [111] Glass C, Singla DK. MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart. Am J Physiol Heart Circ Physiol 2011;301:H2038-49. [112] Glass C, Singla DK. ES cells overexpressing microRNA-1 attenuate apoptosis in the injured myocardium. Mol Cell Biochem 2011;357:135-41. [113] Yu H, Lu Y, Li Z, Wang Q. microRNA-133: expression, function and therapeutic potential in muscle diseases and cancer. Curr Drug Targets 2014;15:817-28. [114] Chen M, Herring BP. Regulation of microRNAs by Brahma-related gene 1 (Brg1) in smooth muscle cells. J Biol Chem 2013;288:6397-408. [115] Zhang M, Chen M, Kim JR, Zhou J, Jones RE, Tune JD, Kassab GS, Metzger D, Ahlfeld S, Conway SJ, Herring BP. SWI/SNF complexes containing Brahma or Brahma-related gene 1 play distinct roles in smooth muscle development. Mol Cell Biol 2011;31:2618-31. 42

ACCEPTED MANUSCRIPT [116] Mallappa C, Nasipak BT, Etheridge L, Androphy EJ, Jones SN, Sagerström CG, Ohkawa Y, Imbalzano AN. Myogenic microRNA expression requires ATP-dependent chromatin remodeling enzyme function. Mol Cell Biol 2010;30:3176-86.

PT

[117] Porrello ER. microRNAs in cardiac development and regeneration. Clin Sci (Lond) 2013;125:151-66.

RI

[118] Boettger T, Wüst S, Nolte H, Braun T. The miR 206/133b cluster is dispensable for

SC

development, survival and regeneration of skeletal muscle. Skelet Muscle 2014;4:23. [119] Koutsoulidou A, Mastroyiannopoulos NP, Furling D, Uney JB, Phylactou LA. Expression of

NU

miR-1, miR-133a, miR-133b and miR-206 increases during development of human skeletal muscle. BMC Dev Biol 2011;11:34.

MA

[120] Liu N, Bezprozvannaya S, Williams AH, Qi X, Richardson JA, Bassel-Duby R, Olson EN. microRNA-133a regulates cardiomyocyte proliferation and suppresses smooth muscle gene expression in the heart. Genes Dev 2008;22:3242-54.

D

[121] Izarra A, Moscoso I, Cañón S, Carreiro C, Fondevila D, Martín-Caballero J, Blanca V,

TE

Valiente I, Díez-Juan A, Bernad A. miRNA-1 and miRNA-133a are involved in early commitment of pluripotent stem cells and demonstrate antagonistic roles in the regulation of

AC CE P

cardiac differentiation. J Tissue Eng Regen Med 2014. doi: 10.1002/term.1977. [Epub ahead of print].

[122] Lee SY, Ham O, Cha MJ, Song BW, Choi E, Kim IK, Chang W, Lim S, Lee CY, Park JH, Lee J, Bae Y, Seo HH, Choi E, Jang Y, Hwang KC. The promotion of cardiogenic differentiation of hMSCs by targeting epidermal growth factor receptor using microRNA133a. Biomaterials 2013;34:92-9. [123] Zhang D, Li X, Chen C, Li Y, Zhao L, Jing Y, Liu W, Wang X, Zhang Y, Xia H, Chang Y, Gao X, Yan J, Ying H. Attenuation of p38-Mediated miR-1/133 Expression Facilitates Myoblast Proliferation during the Early Stage of Muscle Regeneration. PLoS One 2012; 7: e41478. [124] Muraoka N, Yamakawa H, Miyamoto K, Sadahiro T, Umei T, Isomi M, Nakashima H, Akiyama M, Wada R, Inagawa K, Nishiyama T, Kaneda R, Fukuda T, Takeda S, Tohyama S, Hashimoto H, Kawamura Y, Goshima N, Aeba R, Yamagishi H, Fukuda K, Ieda M. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures. EMBO J 2014;33:1565-81. 43

ACCEPTED MANUSCRIPT [125] Christoforou N, Chellappan M, Adler AF, Kirkton RD, Wu T, Addis RC, Bursac N, Leong KW. Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-

One 2013;8:e63577.

PT

inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming. PLoS

[126] Currie S. Cardiac ryanodine receptor phosphorylation by CaM Kinase II: keeping the balance

RI

right. Front Biosci (Landmark Ed) 2009;14:5134-56.

SC

[127] Xiao L, Xiao J, Luo X, Lin H, Wang Z, Nattel S. Feedback remodeling of cardiac potassium current expression: a novel potential mechanism for control of repolarization reserve.

NU

Circulation 2008;118:983-92.

[128] Luo X, Lin H, Pan Z, Xiao J, Zhang Y, Lu Y, Yang B, Wang Z. Down-regulation of miR-

MA

1/miR-133 contributes to re-expression of pacemaker channel genes HCN2 and HCN4 in hypertrophic heart. J Biol Chem 2008;283:20045-52. [129] Xiao J, Luo X, Lin H, Zhang Y, Lu Y, Wang N, Zhang Y, Yang B, Wang Z. MicroRNA miR-

D

133 represses HERG K+ channel expression contributing to QT prolongation in diabetic

TE

hearts. J Biol Chem 2007;282:12363-7. [130] Zhang Y, Xiao J, Wang H, Luo X, Wang J, Villeneuve LR, Zhang H, Bai Y, Yang B, Wang

AC CE P

Z. Restoring depressed HERG K+ channel function as a mechanism for insulin treatment of abnormal QT prolongation and associated arrhythmias in diabetic rabbits. Am J Physiol Heart Circ Physiol 2006;291:H1446-55. [131] Matkovich SJ, Wang W, Tu Y, Eschenbacher WH, Dorn LE, Condorelli G, Diwan A, Nerbonne JM, Dorn GW 2nd. MicroRNA-133a protects against myocardial fibrosis and modulates electrical repolarization without affecting hypertrophy in pressure-overloaded adult hearts. Circ Res 2010;106:166-75. [132] Duisters RF, Tijsen AJ, Schroen B, Leenders JJ, Lentink V, van der Made I, Herias V, van Leeuwen RE, Schellings MW, Barenbrug P, Maessen JG, Heymans S, Pinto YM, Gidlöf O, Smith JG, Miyazu K, Gilje P, Spencer A, Blomquist S, Erlinge D. Circulating cardio-enriched microRNAs are associated with long-term prognosis following myocardial infarction. BMC Cardiovasc Disord 2013;13:12. [133] Abdellatif M. The role of microRNA-133 in cardiac hypertrophy uncovered. Circ Res 2010;106:16-8.

44

ACCEPTED MANUSCRIPT [134] Dong DL, Chen C, Huo R, Wang N, Li Z, Tu YJ, Hu JT, Chu X, Huang W, Yang BF. Reciprocal repression between microRNA-133 and calcineurin regulates cardiac hypertrophy: a novel mechanism for progressive cardiac hypertrophy. Hypertension 2010;55:946-52.

PT

[135] Li Q, Lin X, Yang X, Chang J. NFATc4 is negatively regulated in miR-133a-mediated cardiomyocyte hypertrophic repression. Am J Physiol Heart Circ Physiol 2010;298:H1340-7.

RI

[136] Jeong D, Kim JM, Cha H, Oh JG, Park J, Yun SH, Ju ES, Jeon ES, Hajjar RJ, Park WJ.

SC

PICOT attenuates cardiac hypertrophy by disrupting calcineurin-NFAT signaling. Circ Res 2008;102:711-9.

NU

[137] Diniz GP, Lino CA, Guedes EC, Moreira Ldo N, Barreto-Chaves ML. Cardiac microRNA133 is down-regulated in thyroid hormone-mediated cardiac hypertrophy partially via type 1

MA

angiotensin II receptor. Basic Res Cardiol 2015;110:49. [138] Roden DM. Repolarization reserve: a moving target. Circulation. 2008;118(10):981-2. [139] Bostjancic E, Zidar N, Glavac D. MicroRNA microarray expression profiling in human

D

myocardial infarction. Dis Markers 2009;27:255-68.

TE

[140] Bostjancic E, Zidar N, Stajer D, Glavac D. MicroRNAs miR-1, miR-133a, miR-133b and miR-208 are dysregulated in human myocardial infarction. Cardiology 2010;115:163-9.

AC CE P

[141] Cheng C, Wang Q, You W, Chen M, Xia J. MiRNAs as biomarkers of myocardial infarction: a meta-analysis. PLoS One 2014;9:e88566. [142] Wang F, Long G, Zhao C, Li H, Chaugai S, Wang Y, Chen C, Wang DW. Plasma microRNA-133a is a new marker for both acute myocardial infarction and underlying coronary artery stenosis. J Transl Med 2013;11:222. [143] Devaux Y, Mueller M, Haaf P, Goretti E, Twerenbold R, Zangrando J, Vausort M, Reichlin T, Wildi K, Moehring B, Wagner DR, Mueller C. Diagnostic and prognostic value of circulating microRNAs in patients with acute chest pain. J Intern Med 2015;277:260-71. [144] Li S, Xiao FY, Shan PR, Su L, Chen DL, Ding JY, Wang ZQ. Overexpression of microRNA133a inhibits ischemia-reperfusion-induced cardiomyocyte apoptosis by targeting DAPK2. J Hum Genet 2015;60:709-16. [145] He B, Xiao J, Ren AJ, Zhang YF, Zhang H, Chen M, Xie B, Gao XG, Wang YW. Role of miR-1 and miR-133a in myocardial ischemic postconditioning. J Biomed Sci 2011;18:22.

45

ACCEPTED MANUSCRIPT [146] Dakhlallah D, Zhang J, Yu L, Marsh CB, Angelos MG, Khan M. MicroRNA-133a engineered mesenchymal stem cells augment cardiac function and cell survival in the infarct heart. J Cardiovasc Pharmacol 2015;65:241-51.

PT

[147] Izarra A, Moscoso I, Levent E, Cañón S, Cerrada I, Díez-Juan A, Blanca V, Núñez-Gil IJ, Valiente I, Ruíz-Sauri A, Sepúlveda P, Tiburcy M, Zimmermann WH, Bernad A. miR-133a

RI

enhances the protective capacity of cardiac progenitors cells after myocardial infarction. Stem

SC

Cell Reports 2014;3:1029-42.

[148] Kawai T, Nomura F, Hoshino K, Copeland NG, Gilbert DJ, Jenkins NA, Akira S. Death-

NU

associated protein kinase 2 is a new calcium/calmodulin-dependent protein kinase that signals apoptosis through its catalytic activity. Oncogene 1999;18:3471-80.

MA

[149] Chen S, Puthanveetil P, Feng B, Matkovich SJ, Dorn GW 2nd, Chakrabarti S. Cardiac miR133a overexpression prevents early cardiac fibrosis in diabetes. J Cell Mol Med 2014;18:41521.

D

[150] Sang HQ, Jiang ZM, Zhao QP, Xin F. MicroRNA-133a improves the cardiac function

TE

and fibrosis through inhibiting Akt in heart failure rats. Biomed Pharmacother 2015;71:185-9. [151] Castoldi G, Di Gioia CR, Bombardi C, Catalucci D, Corradi B, Gualazzi MG, Leopizzi M,

AC CE P

Mancini M, Zerbini G, Condorelli G, Stella A. MiR-133a regulates collagen 1A1: potential role of miR-133a in myocardial fibrosis in angiotensin II-dependent hypertension. J Cell Physiol 2012;227:850-6.

[152] Chaanine AH, Hajjar RJ. AKT signalling in the failing heart. Eur J Heart Fail 2011;13:825-9. [153] Huang Y, Li J. MicroRNA208 family in cardiovascular diseases: therapeutic implication and potential biomarker. J Physiol Biochem 2015;71:479-86. [154] Sayed AS, Xia K, Yang TL, Peng J. Circulating microRNAs: a potential role in diagnosis and prognosis of acute myocardial infarction. Dis Markers 2013;35:561-6. [155] Mahdavi V, Chambers AP, Nadal-Ginard B. Cardiac alpha- and beta-myosin heavy chain genes are organized in tandem. Proc Natl Acad Sci USA 1984;81:2626-30. [156] McGuigan K, Phillips PC, Postlethwait JH. Evolution of sarcomeric myosin heavy chain genes: evidence from fish. Mol Biol Evol 2004;21:1042-56. [157] Malizia AP, Wang DZ. MicroRNAs in cardiomyocyte development. Wiley Interdiscip Rev Syst Biol Med 2011;3:183-90.

46

ACCEPTED MANUSCRIPT [158] McKinsey TA, Zhang CL, Olson EN. Signaling chromatin to make muscle. Curr Opin Cell Biol 2002;14:763-72. [159] Potthoff MJ, Olson EN. MEF2: a central regulator of diverse developmental programs.

PT

Development 2007;134:4131-40.

[160] Oliveira-Carvalho V, Carvalho VO, Bocchi EA. The emerging role of miR-208a in the heart.

RI

DNA Cell Biol 2013;32:8-12.

SC

[161] van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stressdependent cardiac growth and gene expression by a microRNA. Science 2007;316:575-9.

NU

[162] van Rooij E, Quiat D, Johnson BA, Sutherland LB, Qi X, Richardson JA, Kelm RJ Jr, Olson EN. A family of microRNAs encoded by myosin genes governs myosin expression and

MA

muscle performance. Dev Cell 2009;17:662-73.

[163] Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, Chen JF, Deng Z, Gunn B, Shumate J, Willis MS, Selzman CH, Wang DZ. MicroRNA-208a is a regulator of cardiac

D

hypertrophy and conduction in mice. J Clin Invest 2009;119:2772-86.

TE

[164] Han P, Li W, Lin CH, Yang J, Shang C, Nurnberg ST, Jin KK, Xu W, Lin CY, Lin CJ, Xiong Y, Chien HC, Zhou B, Ashley E, Bernstein D, Chen PS, Chen HS, Quertermous T, Chang

AC CE P

CP. A long noncoding RNA protects the heart from pathological hypertrophy. Nature 2014;514:102-6.

[165] van Rooij E, Olson EN. MicroRNAs: powerful new regulators of heart disease and provocative therapeutic targets. J Clin Invest 2007;117:2369-76. [166] van Rooij E, Olson EN. microRNAs put their signatures on the heart. Physiol Genomics 2007;31:365-6.

[167] van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN. Control of stressdependent cardiac growth and gene expression by a microRNA. Science 2007;316:575-9. [168] Dillmann W. Cardiac hypertrophy and thyroid hormone signaling. Heart Fail Rev 2010;15:125-32. [169] Diniz GP, Takano AP, Barreto-Chaves ML. MiRNA-208a and miRNA-208b are triggered in thyroid hormone-induced cardiac hypertrophy - role of type 1 Angiotensin II receptor (AT1R) on miRNA-208a/α-MHC modulation. Mol Cell Endocrinol 2013;374:117-24.

47

ACCEPTED MANUSCRIPT [170] Dóka G, Radik M, Krenek P, Kyselovic J, Klimas J. 1A.02: MicroRNA-208a and its host gene cardiac myosin heavy chain MYH6 are involved in hypertrophic heart dysfunction. J Hypertens 2015;33;Suppl 1:e1.

PT

[171] Wu AH, Feng YJ, Moore R, Apple FS, McPherson PH, Buechler KF, Bodor G. Characterization of cardiac troponin subunit release into serum after acute myocardial

RI

infarction and comparison of assays for troponin T and I. American Association for Clinical

SC

Chemistry Subcommittee on cTnI Standardization. Clin Chem 1998;44:6 Pt 1:1198-208. [172] Abbas NA, John RI, Webb MC, Kempson ME, Potter AN, Price CP, Vickery S, Lamb EJ.

NU

Cardiac troponins and renal function in nondialysis patients with chronic kidney disease. Clin Chem 2005;51:2059-66.

MA

[173] Liu L, Aguirre SA, Evering WE, Hirakawa BP, May JR, Palacio K, Wang J, Zhang Y, Stevens GJ. miR-208a as a biomarker of isoproterenol-induced cardiac injury in Sod2+/- and C57BL/6J wild-type mice. Toxicol Pathol 2014;42:1117-29.

D

[174] Corsten MF, Dennert R, Jochems S, Kuznetsova T, Devaux Y, Hofstra L, Wagner DR,

TE

Staessen JA, Heymans S, Schroen B. Circulating MicroRNA-208b and MicroRNA499 reflect myocardial damage in cardiovascular disease. Circ Cardiovasc Genet 2010;3:499-

AC CE P

506.

[175] Ji X, Takahashi R, Hiura Y, Hirokawa G, Fukushima Y, Iwai N. Plasma miR-208 as a biomarker of myocardial injury. Clin Chem 2009;55:1944-9. [176] Adachi T, Nakanishi M, Otsuka Y, Nishimura K, Hirokawa G, Goto Y, Nonogi H, Iwai N. Plasma microRNA 499 as a biomarker of acute myocardial infarction. Clin Chem 2010;56:1183-5.

[177] Nabiałek E, Wańha W, Kula D, Jadczyk T, Krajewska M, Kowalówka A, Dworowy S, Hrycek E, Włudarczyk W, Parma Z, Michalewska-Włudarczyk A, Pawłowski T, Ochała B, Jarząb B, Tendera M, Wojakowski W. Circulating microRNAs (miR-423-5p, miR-208a and miR-1) in acute myocardial infarction and stable coronary heart disease. Minerva Cardioangiol 2013;61:627-37. [178] De Rosa S, Fichtlscherer S, Lehmann R, Assmus B, Dimmeler S, Zeiher AM. Transcoronary concentration gradients of circulating microRNAs. Circulation 2011;124:193644.

48

ACCEPTED MANUSCRIPT [179] Liebetrau C, Möllmann H, Dörr O, Szardien S, Troidl C, Willmer M, Voss S, Gaede L, Rixe J, Rolf A, Hamm C, Nef H. Release kinetics of circulating muscle-enriched microRNAs in patients undergoing transcoronary ablation of septal hypertrophy. J Am Coll Cardiol

PT

2013;62:992-8.

[180] van Rooij E, Marshall WS, Olson EN. Toward microRNA-based therapeutics for heart

RI

disease: the sense in antisense. Circ Res 2008;103:919-28.

SC

[181] Montgomery RL, Hullinger TG, Semus HM, Dickinson BA, Seto AG, Lynch JM, Stack C, Latimer PA, Olson EN, van Rooij E. Therapeutic inhibition of miR-208a improves

NU

cardiac function and survival during heart failure. Circulation 2011;124:1537-47. [182] Satoh M, Minami Y, Takahashi Y, Tabuchi T, Nakamura M. Expression of microRNA-208 is

MA

associated with adverse clinical outcomes in human dilated cardiomyopathy. J Card Fail 2010;16:404-10.

[183] Wilson KD, Shen P, Fung E, Karakikes I, Zhang A, Rahatloo KI, Odegaard J, Sallam K,

D

Dawis RW, Lui GK, Ashley EA, Scharfe K, Wu JC. A rapid, high-quality, cost-effective,

TE

comprehensive, and explandable targeted next-generation sequencing assay for inherited heart diseases. Circ Res 2015;117:603-11.

AC CE P

[184] Kakimoto Y, Kamiguchi H, Ochiai E, Satoh F, Osawa M. MicroRNA Stability in postmortem FFPE tissues: Quantitative analysis using autoptic samples from acute myocardial infarction patients. PLoS One 2015;10:e0129338. [185] Miki K, Endo K, Takahashi S, Funakoshi S, Takei I, Katayama S, Toyoda T, Kotaka M, Takaki T, Umeda M, Okubo C, Nishikawa M, Oishi A, Narita M, Miyashita I, Asano K, Hayashi K, Osafune K, Yamanaka S, Saito H, Yoshida Y. Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches. Cell Stem Cell 2015;16:699-711. [186] Xu Z, Han Y, Liu J, Jiang F, Hu H, Wang Y, Liu Q, Gong Y, Li X. MiR-135b-5p and MiR499a-3p promote cell proliferation and migration in atherosclerosis by directly targeting MEF2C. Sci Rep 2015;5:12276. [187] Pagiatakis C, Gordon JW, Ehyai S, McDermott JC. A novel RhoA/ROCK-CPI-17MEF2C signaling pathway regulates vascular smooth muscle cell gene expression. J Biol Chem 2012;287:8361-70.

49

ACCEPTED MANUSCRIPT [188] Sluijter JP, van Mil A, van Vliet P, Metz CH, Liu J, Doevendans PA, Goumans MJ. MicroRNA-1 and -499 regulate differentiation and proliferation in human-derived cardiomyocyte progenitor cells. Arterioscler Thromb Vasc Biol 2010;30:859-68.

PT

[189] Yeung F, Chung E, Guess MG, Bell ML, Leinwand LA. Myh7b/miR-499 gene expression is transcriptionally regulated by MRFs and Eos. Nucleic Acids Res 2012;40:7303-18.

RI

[190] Wang X, Ono Y, Tan SC, Chai RJ, Parkin C, Ingham PW. Prdm1a and miR-

SC

499 act sequentially to restrict Sox6 activity to the fast-twitch muscle lineage in the zebrafish embryo. Development 2011;138:4399-404.

NU

[191] Jackson HE, Ingham PW. Control of muscle fibre-type diversity during embryonic development: the zebrafish paradigm. Mech Dev 2013;130:447-57.

MA

[192] Warkman AS, Whitman SA, Miller MK, Garriock RJ, Schwach CM, Gregorio CC, Krieg PA. Developmental expression and cardiac transcriptional regulation of Myh7b, a third myosin heavy chain in the vertebrate heart. Cytoskeleton (Hoboken) 2012;69:324-35.

D

[193] Ai J, Zhang R, Li Y, Pu J, Lu Y, Jiao J, Li K, Yu B, Li Z, Wang R, Wang L, Li Q, Wang N,

TE

Bell ML, Buvoli M, Leinwand LA. Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping. Mol Cell Biol 2010;30:1937-45.

AC CE P

[194] Bhuiyan SS, Kinoshita S, Wongwarangkana C, Asaduzzaman M, Asakawa S, Watabe S. Evolution of the myosin heavy chain gene MYH14 and its intronic microRNA miR-499: muscle-specific miR-499 expression persists in the absence of the ancestral host gene. BMC Evol Biol 2013;13:142.

[195] Dorn GW 2nd, Matkovich SJ, Eschenbacher WH, Zhang Y. A human 3' miR499 mutation alters cardiac mRNA targeting and function. Circ Res 2012;110:958-67. [196] Shieh JT, Huang Y, Gilmore J, Srivastava D. Elevated miR-499 levels blunt the cardiac stress response. PLoS One 2011;6:e19481. [197] Matkovich SJ, Hu Y, Eschenbacher WH, Dorn LE, Dorn GW 2nd. Direct and indirect involvement of microRNA-499 in clinical and experimental cardiomyopathy. Circ Res 2012;111:521-31. [198] Zhang L, Chen X, Su T, Li H, Huang Q, Wu D, Yang C, Han Z. Circulating miR-499 are novel and sensitive biomarker of acute myocardial infarction. J Thorac Dis 2015;7:303-8. [199] Olivieri F, Antonicelli R, Lorenzi M, D'Alessandra Y, Lazzarini R, Santini G, Spazzafumo L, Lisa R, La Sala L, Galeazzi R, Recchioni R, Testa R, Pompilio G, Capogrossi MC, Procopio 50

ACCEPTED MANUSCRIPT AD. Diagnostic potential of circulating miR-499-5p in elderly patients with acutenon STelevation myocardial infarction. Int J Cardiol 2013;167:531-6. [200] Xiao J, Shen B, Li J, Lv D, Zhao Y, Wang F, Xu J. Serum microRNA 499 and microRNA-

PT

208a as biomarkers of acute myocardial infarction. Int J Clin Exp Med 2014;7:136-41. [201] Devaux Y, Vausort M, Goretti E, Nazarov PV, Azuaje F, Gilson G, Corsten MF, Schroen B,

RI

Lair ML, Heymans S, Wagner DR. Use of circulating microRNAs to diagnose acute

SC

myocardial infarction. Clin Chem 2012;58:559-67.

[202] Chen X, Zhang L, Su T, Li H, Huang Q, Wu D, Yang C, Han Z. Kinetics of plasma

NU

microRNA-499 expression in acute myocardial infarction. J Thorac Dis 2015;7:890-6. [203] Wang J, Jia Z, Zhang C, Sun M, Wang W, Chen P, Ma K, Zhang Y, Li X, Zhou C. miR-499

RNA Biol 2014;11:339-50.

MA

protects cardiomyocytes from H2O2-induced apoptosis via its effects on Pdcd4 and Pacs2.

[204] Taira N, Nihira K, Yamaguchi T, Miki Y, Yoshida K. DYRK2 is targeted to the nucleus and

D

controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage. Mol Cell

TE

2007;25:725-38.

[205] Castrogiovanni C, Vandaudenard M, Waterschoot B, De Backer O, Dumont P. Decrease of

AC CE P

mitochondrial p53 during late apoptosis is linked to its dephosphorylation on serine 20. Cancer Biol Ther 2015;16:1296-307. [206] Wang JX, Jiao JQ, Li Q, Long B, Wang K, Liu JP, Li YR, Li PF. miR-499 regulates mitochondrial dynamics by targeting calcineurin anddynamin-related protein-1. Nat Med 2011;17:71-8.

[207] Estaquier J, Arnoult D. Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis. Cell Death Differ 2007;14:1086-94. [208] Li X, Wang J, Jia Z, Cui Q, Zhang C, Wang W, Chen P, Ma K, Zhou C. MiR-499 regulates cell proliferation and apoptosis during late-stage cardiac differentiation via Sox6 and cyclin D1. PLoS One 2013;8:e74504. [209] Iguchi H, Urashima Y, Inagaki Y, Ikeda Y, Okamura M, Tanaka T, Uchida A, Yamamoto TT, Kodama T, Sakai J. SOX6 suppresses cyclin D1 promoter activity by interacting with betacatenin and histone deacetylase 1, and its down-regulation induces pancreatic beta-cell proliferation. J Biol Chem 2007;282:19052-61.

51

ACCEPTED MANUSCRIPT [210] Hosoda T, Zheng H, Cabral-da-Silva M, Sanada F, Ide-Iwata N, Ogórek B, Ferreira-Martins J, Arranto C, D'Amario D, del Monte F, Urbanek K, D'Alessandro DA, Michler RE, Anversa

AC CE P

TE

D

MA

NU

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mircrine mechanism. Circulation 2011;123:1287-96.

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P, Rota M, Kajstura J, Leri A. Human cardiac stem cell differentiation is regulated by a

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

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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 targets two major components (caspase-9 (CASP9) and apoptotic protease activating factor 1

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(APAF1) that together with cytochrome C form a cell death complex that irreversibly leads to apoptosis through the activation of caspase 3 (CASP3). MiR-133a can also inhibit the mitochondrial

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apoptotoc path through direct targeting proaptotic Bcl-2-like protein 11 (BCL2211) and Bcl-2modifying 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

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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 proapoptotic genes including Dyrk2 (dual specificity thyrosine-phosphorylation–regulated kinase 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 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, miR499-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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ACCEPTED MANUSCRIPT Highlights Cardiac miRNAs are abundantly expressed in the myocardium

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Contribution of cardiac miRNAs to cardiac developmental program is well

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coordinated

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of acute myocardial infarction is appreciated.

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Significant value of cardiac miRNA as diagnostic biomarkers for early diagnosis

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