5. The Cardiovascular Role of Differentially Expressed MicroRNAs during Ventricular Development
Over the last few years, we have witnessed an increasing interest on the contribution of microRNAs to cardiovascular development. Microarray analyses and deep-sequencing studies have increased our knowledge among the microRNA signatures of distinct cardiovascular tissues and conditions. For example, a large number of microRNAs that were initially reported to be upregulated within the developing heart by microarrays [14] have also been reported to be highly expressed in the developing heart by deep sequencing approaches [39]. Vacchi-Suzzi et al. [30] have further provided detailed information about the microRNA hallmarks of different cardiac structures, revealing an abundant expression of miR-125, miR-99 and miR-320 in valve tissues and of miR-133 and miR-30 in the myocardium, among those differentially expressed during mouse ventricular maturation [14]. However, the tissue distribution and the functional role of several other microRNAs, such as miR-320, miR-99, miR-125, miR-93 and miR-322 in the developing and/or diseased heart, remains to be elucidated, apart from being highly and dynamically expressed during cardiogenesis [14,30,39], while the function role of others, such as, for example, miR-23, miR-24, miR-83, miR-25, miR-27 and several members of the let-7 family members, is currently emerging, as stated below.
As we have previously mentioned, the heart is a complex structure in which distinct tissue layers and structures can be delineated. Three distinct layers can be delimited, the endocardium, myocardium and epicardium, the myocardium being the most important functionally. In addition, the heart is composed of interstitial tissue, arterial and atrioventricular valves and its own system of blood perfusion, the coronary vasculature. Understanding of the impact of microRNA regulation in these tissues is progressively emerging. To date, the contribution of microRNAs to myocardium biology is emerging at a quick pace. The role of miR-133, together with miR-1, is one of the most extensively studied in both cardiac and skeletal muscle development and disease [40,41]. Chen et al. [42] describes the opposite roles of miR-1 and miR-133 in skeletal muscle proliferation and differentiation, despite being transcribed from the same polycistronic unit. Surprisingly, genetic deletion of miR-133a or miR-133b displays no cardiac phenotype [22,23], yet deregulation of miR-133 in distinct cardiovascular diseases has been extensively described, such as during myocardial infarction [43,44] and arrhythmias [45,46]. At the molecular level, miR-133 contributes to repression of cardiac hypertrophy by negatively regulating Nfatc4 signaling [34,35], controlling, thus, the metabolic status of cardiac myocytes [36], and it has been proposed as biomarker in the predicted regression of left ventricular hypertrophy after valve replacement [47]. Most importantly, a significant decrease of miR-133 was observed during zebrafish cardiac regeneration [48], and the experimental modulation of miR-133 by either over-expression or deletion demonstrates a pivotal role of this microRNA in cardiomyocyte proliferation during cardiac regeneration. 
Several other microRNAs, such as miR-143, miR-145 and the miR-17-92 cluster, have also been reported to play a role in cardiac muscle. Miyasaka et al. [49] elegantly demonstrate that cardiac development is modulated by hemodynamic inputs controlling miR-143 expression and, thus, cardiac morphogenesis in zebrafish. Knockdown of miR-143 elicits re-expression of retinoic acid signaling components leading to outflow tract and ventricular dysmorphogenesis. Further evidence on the role of miR-143 in heart development has been provided also in zebrafish by Deacon et al. [37], who nicely showed that miR-143 targets adducin3, and if impaired, abnormal growth and elongation of the ventricular chambers occurs, leading to decrease cardiac contractility and, eventually, collapse. Interestingly, in the human adult heart, deep-sequencing of the miRNA transcriptome in the left and right atrial chambers has revealed that miR-143 is the highest expressed microRNA in the atrial chambers [50], yet its functional role in the adult (normal and diseased) heart remains to be uncovered.
miR-145 has been primarily implicated in smooth muscle cells, and particularly, it is highly upregulated within the lungs of both experimentally-induced pulmonary arterial hypertension, as well as in patients with idiopathic and hereditable pulmonary arterial hypertension [51]. While there is no evidence of a functional role of miR-145 in the developing cardiovascular development, impaired miR-145 expression has been reported in several cardiovascular diseased conditions, such as acute cardiac infarction and coronary artery disease [52,53]. A functional link between miR-145 and these diseased statuses has been recently reported by Li et al. [54], demonstrating that miR-145 protects against oxidative stress-induced apoptosis in cardiomyocytes and also regulating Wnt/beta-catenin signaling through targeting of Dab2 in the ischemic heart [38]. 
The precise contribution of miR-17 remains to be discerned, yet systemic deletion of the miR-17-92 cluster leads to lung hypoplasia and ventricular septal defects at birth [55]. It has been also demonstrated that in embryonic cardiomyocytes, miR-17-92 regulates Fog-2 and, thus, cardiomyocyte proliferation [25], while in neonatal cardiac progenitor cells, it regulates Rb12/p130 [26]. In addition to its role during cardiovascular development, miR-17-92 also plays a pivotal role in the adult heart, since it is differentially expressed in the aging heart [56], and overexpression of this microRNA cluster leads to cardiac hypertrophic cardiomyopathy and arrhythmias [57]. Yet to date, the most described functional phenotype of miR-17 relates to pulmonary hypertension [58,59] and extracellular matrix remodeling [60] in this context.
In addition to the role reported for miR-133, miR-143, miR-145 and the miR-17-92, among those microRNAs differentially expressed during cardiac development, initial hints for miR-494 and miR-210 function in myocyte adaptation and survival during hypoxia/ischemia are also emerging, yet with limited understanding [61,62].
While the myocardium is the most relevant tissue layer in the pumping heart, the role of the interstitial cardiac tissue is progressively emerging, since, in fact, it is the most abundant cell type within the adult heart. In this context, several microRNAs, such as miR-15, miR-30 and miR-24, have been reported to play key functions in the cardiac fibroblasts, yet as previously mentioned, they also seem to provide pivotal roles in other cardiovascular tissues.
miR-15 displays a key role governing cardiomyocyte cell cycle withdrawal and binucleation, by controlling Chek1 expression [63], while in cardiac fibroblasts, it modulates collagen deposition [64]. 
miR-30 display a differential expression in experimental pulmonary hypertension [51]. Within the heart, it is has been reported to play a role in extracellular matrix remodeling, by controlling Ctgf expression, in the context of ventricular hypertrophy [31], a role that might also be linked with downregulation of miR-30 in a model of induced atrial fibrillation with fibrosis and inflammation [46].
Wang et al. [65] reported that miR-24 is downregulated after myocardial infarction, correlating with increased extracellular matrix deposition. Forced in vivo miR-24 overexpression decreased fibrosis, by controlling furin expression, which in turn, regulated tgf-beta bioavailability. In addition to its role in the interstitial cardiac tissue, a role on the cardiac endothelium has also been demonstrated [28] in the context of myocardial infarction. These authors demonstrate that miR-24 impairs angiogenesis by controlling gata2 and pak4 expression and blocking miR-24 in vivo leads to decreased endothelial apoptosis, increased vascularization and preservation of cardiac function in a mouse experimental model of myocardial infarction. Importantly, a role for miR-24 in myocardial cells has also been reported. Li et al. [29] reported that ischemia increased miR-24 expression in cardiomyocytes, while forced miR-24 expression in cultured cardiomyocytes increased cell viability and apoptosis, and necrosis rates were reduced. Such effects seemed to be mediated by miR-24 mediated control of the pro-apoptotic gene, Blc2l11. Given these pivotal roles, therapeutic usage of miR-24, in combination with other miRs (miR-21 and miR-221), has provided increased survival and engraftment to cocktail-treated cardiac progenitor cells in a mouse model of coronary artery ligation [66], opening, thus, new therapeutic approaches to heal the diseased heart. 
Apart from microRNAs playing a role in cardiomyocytes and interstitial tissue, two microRNAs have been revealed to play an important role in valve and vascular development. miR-23 has been highlighted in the context of cardiac valve formation. Elegant studies in zebrafish demonstrate that miR-23 controls Has2 expression, therefore regulating the extracellular matrix remodeling during endocardial cushion formation [27]. Additional evidence on the role of miR-23 has been reported in the vascular context, as recently reviewed by Bang et al. [67]. Similarly, miR-126 is predominantly expressed in the vascular tissue [21]. Targeted deletion of miR-126 resulted in impaired angiogenesis and vascular integrity [21,68]. In addition, a role for miR-126 in valve development, controlled by VEGF, has been also reported [69]. Yet, more recently, a role of miR-126 has also been ascribed in the developing cardiomyocytes, being regulated by hypoxia and histone deacetylases (HDAC) inhibitors and, therefore, contributing to cardioprotection [70]. 
For other microRNAs, such as miR-122 and let-7, although there is unequivocal evidence of a determinant role during cardiovascular development, it remains to be elucidating at which tissue level they contribute. miR-122 has been reported to play a pivotal role in several endodermal-derived tissues [71,72,73,74], yet its role in the cardiovascular system is just emerging. Huang et al. [33] recently reported that miR-122 was upregulated in Pax8 null mutants, which display ventricular septal defects. Analyses of putative targets of miR-122 uncovered that cck-8 and caspase-3 are genuine targets, supporting the notion that Pax8-related cardiovascular defects are mediated by miR-122. Similarly, let-7 has been proposed to play a role in cardiac development [39] and differentiation [75,76], yet full mechanistic insights remain to be clarified. Similarly, abnormal expression of let-7 has been identified in a rat model of myocardial injury (doxorubicin treatment), yet it functional role remains unexplored [77].