microRNA perspective on cardiomyocyte development and 1 cardiovascular diseases 2

cardiovascular diseases 2 Jose Francisco Islas, Jorge Eugenio Moreno-Cuevas 3 4 Grupo Estrategico de Enfoque en Bioingenieria y Medicina Regenerativa, Escuela de Medicina, 5 Tecnológico de Monterrey, Av Morones Prieto No. 3000, Colonia Los Doctores. Monterrey Nuevo 6 León, México. 64710. 7 Corresponding Author: jemoreno@itesm.mx 8 9 1. Abstract 10 Study of micro-RNA regulatory networks (known as miRNA’s or miR’s), during development and 11 in known pathologies have been the basis of study over the past decades. Herein, we recapitulate 12 these findings in order to highlight the best underlying mechanisms found to date. We also seek to 13 elucidate how miRNA dysregulation can be associated with many cardiovascular diseases. 14 Furthermore, we discuss miR regulation mechanism during in early development in vivo and 15 invitro. Since many of the miR’s are precursors to transcriptional regulation, we relate back to 16 their molecular control as we can then look together at the fundamental disease they might be 17 exacerbating by this dysregulation. 18 19 2. miRNA’s 20 Initial studies by Ruvkon et al., and Ambros et al., in the last decade of the 20th century, 21 demonstrated that a group of small non-coding RNA influenced the development of C. elegans by 22 regulating translation by a process of base pairing (inhibiting translation) to the 3’UTR and in a 23 few cases 5’UTR of mRNA [1,2]. These small RNA were over time shown to be conserved in 24 many cellular eukaryotic species including human cells [2–5]. 25 MicroRNAs (miRNAs), commonly designated as mir for the precursor product or miR for 26 the mature product, are a set of 18-24 nucleotides, which when processed (a process of 27 complementary base pairing to mRNA) can silence or downregulate expression; either by 28 destabilizing and/or cleaving the mRNA or by reducing the efficiency for which it can be 29 processed [6,7]. Unlike other small RNA’s, miR’s have a predictable hairpin loop structure from 30 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2018 doi:10.20944/preprints201805.0154.v1

their precursor transcript [8].The small region required for miRNA regulation is only about eight nucleotides This small area possibly supports the idea that one miR could regulate as much as 60% of available mRNAs, although this has been found not to be the case [9].Suggesting, that miRNA translation regulation is a tightly regulated mechanism that is controlled in a spatialtemporal manner, including how a handful of miR's can be secreted in specific organs and are uptaken far from their origin [5,6,10].

miRNA Biogenesis
Briefly, mir's are initially transcribed from the genomic region by pol II (up to 3kb) as a combined primary-precursor mir's (pri-mir).Further, processing by Drosha/DGCR8 permits the cleavage into a ~70-100bp stem-loop hairpin or precursor-mir (pre-mir).The pre-mir can now be exported to the Cytoplasm via complex to Exportin-5/Ran-GTPase.Once exported, it is at this stage where further enzymatic cleavage by Dicer/TRBP complex produces the commonly known miR duplexes (22 bp).These duplexes now serve as the guides that come into pair with mRNA; this is achieved initially by a dissociation of the duplexes followed by the assimilation of one of the miR strands with Argonaute (AGO), thus forming the RNA-induced Silencing Complex (RiSC), which consequently binds to the complementary sequence of the mRNA.Typically, complementation is on the 3' UTR of the mRNA promoting posttranslational degradation or downregulate expression [1, [11][12][13][14], Fig. 1.
Given this brief overview, it's no surprise that miR's are such important regulatory molecules.In many instances self-regulating by co-expression with target genes, therefore many labs have dedicated a considerable amount of time and resources to study and profile miR's to development, as well as, to underlying diseases which they can incur into by mis-expression, errors in transcription, promoter defects such as hypo-or hyper-methylation and other noticeable conditions [5,8,[15][16][17][18].

miRNA's in Cardiomyocytes
Fundamentally the adult heart has little potential to regenerate when given an insult by injury or disease.Normally cardiomyocytes are lost leading to heart failure and in more complicated cases to death [19][20][21].Hence, understanding cardiomyocyte development has been key in trying to understand repair.

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Studies of individual miR's using developmental models of the heart, have led to demonstrate that miR-1/miR-133 are fundamental in the control of proliferation and the muscle transcriptional network; SRF, MEF2c, MyoD, Hand2 and Myocardin.Interestingly, SRF has been found to be a requirement for miR-1 expression during development.This double mechanism is particularly evident as SRF/miR-1 also come together to regulate the sodium calcium exchanger NCX1 promoter [22].Additionally miR-1 overexpression blocks ventricle myocyte expansion [23].Meanwhile, miR-1 was linked to NOTCH1 receptor.Dlk-1, a critical factor involved in specification thought asymmetric division [24].Mouse studies by Wu et al., found additional evidence of miR-34a is a repressive regulator to NOTCH1, while upregulating Jagged1, Hey2 and Hes [25].A variant of miR-1(-1) is miR-1-2, has been found to have ~50% of embryonic lethality, while ~20% of survivors have major cardiac defects.Deletion miR-1-2 has been reported to repress Kcnd2, crucial factor in the repolarization of the heart [26][27][28].In adult rats experiments miR-1 has been also revealed to target KCNQ1 and KCNE1 [18] The miR-17-92 cluster is has also been implicated in cardiac proliferation by negatively regulating PTEN tumor suppressor gene [9,29,30].Additional reports suggest the implication of the miR-17-92 cluster overexpression may cause Tsc1 repression.Thereby, causing mTOR mediated hypertrophy, proposing that the conditional downregulation relation of Tsc1 is a negative regulator of mTOR and downstream target of PTEN [31].Meanwhile the miR-15 family, balances proliferation by repressing cell cycle regulators, in particular decreasing HSP-20, targeting Bcl2, and repressing TGF activity through [30].
Finally, a distinguishing trait of cardiomyocytes is their contraction.This is provided in part by a delicate balance of  MHC chiefly controlled by miR-208.Principally, miR-208a was found to regulate GATA4 and CX40, thereby partially regulating the conduction system.Additional to this, Callis et al., states that both isoforms of miR-208 (a/b) target TRAP1 and myostatin, whom are important negative regulators of muscle growth and hypertrophy [32,33].(A synopsize of the miR regulation thought development, Table 1)

miRNA's in Stem Cells to Cardiomyocyte Differentiation
In order to best bypass limitations for cardiomyocyte regeneration, direct differentiation of somatic cells into, induced pluripotent stem (IPS) cells and embryonic stem (ES) cells has been the focus for many groups during the past 2 decades and continues to be a great field of study within organ repair [33,34].
Nonetheless, Dazu's group began to show the potential of miR-1, by demonstrating its overexpression was sufficient to convert cells [38].
A result consistent with the expression of RYR2 L-type channel [43].miR-499 has been implicated in expression of MHC6, MHC7, MLC2, and TNNT2.
Poon et al., recently described the importance of miR-200c using ES cells, denoting GATA4, TBX5, and SRF to be its targets.They also noted that knockouts in ES cells altered Ca + , Na + , and K + ion channels (CACNA1C, KCNJ2 and SCN5A) increasing contractility.Additionally, conduction seems to also be altered if deleting miR-1-2, by dysregulating the expression of Irx 4, 5 and Kcnd2.Meanwhile, transcription factor MESP1, once described as the key regulator to heart by Bondue and Blanpain, 2010, regulates Nkx2.5, Tbx5, Hand2, FoxH1, Isl1 and others, while at the same time induces mesoderm lineage by blocking Bry, Fgf6, FoxA2, Sox17, and Gsc.Thereby sitting atop of the hierarchy of cardiomyocyte formation and regulation [46].At a step earlier, we can find that transactivation is succeeded by miR-322/503, this by targeting RNA binding factor Celf1, which would else induce a neural fate [47].Regulation via the most important miR's during differentiation can be viewed in Fig. 2. CVD's, the leading cause of morbidity and mortality amid developed countries.Over the past couple of decades much effort has been put into finding both the physiological fundamentals and molecular mechanisms of control.Research done over miR's in CVD's has pointed out the specificity of certain miR's and clusters to certain conditions.Here we put emphasis on most important and clinically relevant miR's for conditions such as congenital heart disease (CHD), atherosclerosis, myocardial infarction (MI), severe coronary artery disease (CAD), and heart failure (HF) [4,9,15,33,48].Fig. 3 and Supplementary Tables 1 and 2.

miRNA's in Cardiovascular Diseases (CVD's)
CHD's are the leading cause of prenatal deaths (~ 40%), while at the same time comprising the majority of all congenital malformations [49].Reports form the Euro Heart Survey suggests, around 20% of patients with CHD undergo surgery or a catheter-based intervention, resulting in major economic burden for the patient [15,33,50,51].
Accounting for 30 ~ 40% of CHD's is the ventricular septal defect (VSD), and in a smaller proportion is the atrial septal defect (ASD).VSD (ASD) can be viewed as a discontinuation in the septal wall dividing the left and right ventricles (or atria) of the heart.VSD can produce left ventricle overload resulting in pulmonary hypertension [52,53].miR-1/181c regulates the expression of BMPR2 [54,55].During VSD conditions, elevated levels of GJA1 and SOX9 overlap with reduced expression of miR-1-1, and elevate miR-181c [15].Additional data form Li et al., showed that let-7e-5p, miR-222-3p and miR-433 maybe the underlying cause for abnormalities since they target NOTCH1, HAND1, GATA3 , and ZFPM2 resulting in altered morphogenesis and VSD [27].
DiGeroge syndrome is a direct result of deletion in region 8 of chr:22, thereby producing loss-of-function mutation on TBX1 culminating in haploinsufficiency.TBX1 is has a role in differentiation of the neural crest cells, where mutated TBX1 hinders correct formation of the outflow track [15].In addition, this condition leads to DGCR8 downregulation and promotes an accumulation of both pri-miR's and pre-miR's [56].
Recently, studies in Down syndrome (CHD expectancy 50~60%) have confirmed 5 miR's to be directly correlated miR-99a, let-7c, miR-125b-2, miR-155 and miR-802, all linked to over expression in the heart [57].miR-99a has been associated with repression of cardiogenesis when expressed at early stage by regulating Smarca5, let-7c was found to induce it, but only if expressed during mesoderm formation, thereby repressing the activity of Ezh2 [58].Additional studies in cancer biology have concluded that the loss of the let-7 family contributes to the upregulation of Romano's group led to the mechanics of understating the cascade activation of miR-126-5p by Lipoxin A4.A response via pro-inflammatory endothelial microvesicles packed with miR-126-5p.
An antagonizing effect to TNF, leading to the upregulation of VCAM1 and the downregulation of SPRED1 [64].Another component of atherosclerosis mentioned is VSMC differentiation.miR-145 deficiency has shown to reduce the medial layer in arteries.In addition, differentiation genes myocardin, KLF4, KLF5, calmodulin kinase, cholesterol transporter ABCA1 were found to be a direct target of miR-145 [67].
miR-33a and miR-33b have been shown to be in regulation of ABCA1 and ABCG1as they control the sterol regulatory element-binding proteins, hence their control can be a useful tool in potential therapies for dyslipidemia and atherosclerosis [68,69].Hypertension or high blood pressure can lead to atherosclerosis due to the added force at the artery walls/ miR-145/-143 seems to play an important role in high blood pressure, mainly, having as target the angiotensin converting enzyme [4].. In addition, inhibition of miR-145 might improve diabetic resistance via nitric oxide [70].We should reference that, mouse studies have enlighten the role of miR-21, demonstrating reduce blood pressure in inverse correlation with miR-130a and miR-195, whom have been positively shown to be upregulated in serum [71].
MI described as severe CAD or a myocardial cell death due to sustained ischemia.Patients with MI have shown to have a heavy upregulation of miR-1, miR-133, miR-208 and miR-499 [72].
The dysregulation of all 4 miR's has been linked to MI [18,33,72,73].miR-208 by itself has been shown to be sufficient to induce heart hypertrophy as a response to overload, while inducing-MHC expression [74]. to the notion of specific spatial-time regulation, since many (if not all) of these miR's are involve in other conditions.In addition, protective signaling to reduce damage in the heart can be achieved by expression of miR-873 and miR-2861 [75].The finesse required to precisely achieve the protection in the mist of so much disruption, can tell us a bit more about the recurrent selfprotective and pro-survival mechanisms present in the heart.As it was mentioned MI constitutes severe cell death, and cell death itself comes in 3 "flavors"; Necrosis (Necroptosis), Autophagy , and Apoptosis [75][76][77].Each flavor comes with a set of miR regulators acting on specific targets to both promote and inhibit each process.
Necrosis is known a form of death due to exacerbation in cellular or pathogenic damage.
In cardiomyocytes, necrotic death induced by -O2, elevates levels of miR-103 and miR-107, whom act on the Fas-associated protein with death domain [78] mitochondria and long-lived macromolecules via a double-membrane structure, to the lysosomes for degradation [76].When activated miR-188-3p, miR-290 and miR-375 acts as mediators reducing autophagitic activity by activating ATG7.ATG7 acts thought a thiol-ester bond on the E1 activator to free Ubiquitin molecules beginning degradation [82].At a molecular level, master regulators for autophagy are mTOR and AMPK, both regulatable by miR-155 and miR-17-92 complex.Previously, it was mentioned that miR-155 could repress the activation of the RIP complex by inhibition with PTEN, as well as interfering with the Wnt/-catenin and the Akt-pro survival pathway [29,60].Additionally miR 17-92 complex results in mTOR negative regulation [31].FOXO3a a pro-autophagitic factor, is negatively regulated by miR-212/132.Over expression of miR-212/132 significantly disturbs autophagy and results in drastic cardiac hypertrophy and heart failure [62,83].Additionally, energy sensing pathway of AMPK can be blocked in particular by the disruption given to the 1 subunit (AMPK1), activation of miR-148b directly inhibits its expression [84], therefore blocking the full AMPK assembly.
Apoptosis or programed death is a process driven by cell death receptors.Cascading signals mediated by many pro-and anti-apoptotic signals; caspases, Bcl-2 family and p53 [76,85].
An important study by the American Heart Association / American Stroke Association have been on Cavernous malformations, which are best defined as circumscribed vascular lesions with thin-walled sinusoidal spaces lined with endothelial tissue and containing intravascular or intervascular calcifications [91].Developmentally, these malformations control cardiac development via endothelial signaling of MEKK and KLF [92].PDCD10 a major role-playing factor in this condition, is heavily involved in cardiomyocyte autophagy.Has been shown to be susceptible to regulation by miR-613 by acting over LXR [93], while PDCD4 is a direct target miR-155 [94].Yan et al., describe HF as a terminal stage of most types of cardiovascular diseases, which always leads to a negative prognosis.Their study on the clinical relevance of using circulating levels of miR-423-5p as a potential biomarker, they note that the standard marker is B type natriuretic peptide (BNP) [95], similarly BNP in MI miR-208b and miR-499are not the optimal but are under great scrutiny [75].Meanwhile, research is conducted to use miR-1and miR-30a as they play crucial roles in cardiac hypertrophy and apoptosis they target key molecules in the signaling pathways that govern cardiac fibrosis, hypertrophy and apoptosis [96].Nonetheless no defined biomarkers for HF stage.

Future directions
The clinical potential underlying miR's can be seen as a great area of opportunity both for their targeting and as biomarkers.miR's themselves are clearly expressed in a tightly regulated fashion throughout development and during organ maintenance.At the same time miR's can show peculiarities in expression during pathological altered states.For the clinic, knowing and understanding these in-balances gives us a step up in the game.
Important elements should be summarized for the usage or target of miR's, as certain elements can be contemplated as strengths or weaknesses.Let's consider the size for the mature miR, this can vary around 22bp and can be feasibly seen as a target by an antagonizing sequence (antagomiR or anti-miR); analogous to the mRNA outcompeting and thereby not inhibiting transcription [24,60,68].The remarkable feature here, is that a single upstream target can determine the fate of a whole signaling pathway.Instead of targeting individual factors by knockdowns or having to obliterate by fully knocking out a gene; a not always viable solution.
Counterintuitively, pairing a 22bp fragment is to an extent easy, yet, we need to consider the dynamics as targeting of miR to their mRNA.This beings with the "seed", a 5bp region at the 5' end + up to 2 more adjacent bp, using the seed alone in not a great method for pairing, so it is common for non-canonical pairing to occur [1].Hence having the potential for silencing off targets exist so sequences need to be fully vetted by bioinformatics systems [23].Another aspect to consider is these microRNA's have a short half-life, hence modifications or high quantities are considered as a possibility when intended for therapeutics, this might led to toxicity issues and alterations of biological properties [97].Numerous miRNA inhibitors have been designed with different adjustments in particular at the 2 ′ position of the.Moreover, 2 ′ -MOE (2 ′ -O-methoxyethyl) and 2 ′ -fluoro are the most commonly used modification.While an alternative LNA (locked nucleic acid) a miR mimetic, uses O, 4 ′ -C methylene bridge to lock the furanose ring backbone [98].
Attempts to try and direct mimetics and antagomiRs to a specific organ poses a fascinating challenge, a direct attempt to inject the antagomiR to the organ is usually the best shot and delivery system range from the use of liposome vesicles, polymers, and other viral particles, yet for the patient this could lead to potential high costs [101].The first set of experiments using antagomiR was deemed to observe the systematic reduction of miR's.Intravenous administrations was done against miR-16, miR-122, miR-192 and miR-194 resulting in a striking decline of the resultant miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals [102].Further studies have revealed the value of miR's to downregulate miRNAs in primates, using LNA modification [103], the first set of clinical trials (~20) are currently underway using diverse delivery systems [17] .
Previously it was mentioned that miR's can have many potential targets, taking this notion let's consider the well documented case of miR-29.miR-29 can regulate fibrosis in the heart by targeting a whole set of different components such as elastins, fibrillins and collagens; all components of the ECM.Therefore by using a well design antagomiR to miR-29, the group was able to protect cardiomyocytes [98].Furthermore, during MI experiments it was found that antagomiRs for miR-92a and miR-320 could reduce infarct size by contributing to recovery of blood vessels and reduction of apoptotic signals [48].Meanwhile, Huang et al., considered the use of mesenchymal stem cells expressing miR-1.Elsewhere mentioned miR-1 is sufficient to lead conversion of stem cell to cardiomyocytes.This experiment overall improved both cardiac function and reduce overall infarcted size [104].Another set of experiments looked at miR-21.
Dong et al., upregulated the expression of miR-21 by ischemic preconditioning, before MI, demonstrating considerable reduction in infarcted area.Moreover, the effect was even greater when injecting alongside of miR-1 and miR-24 [48].Experiments in Atherosclerosis using AntagomiRs (miR-33) showed a substantial elevation in HDL and reduction in VDL.Additionally, LNA-AntagomiR (miR-122) also showed promise in cholesterol reduction while 2'-Omethoxyethyl phosphorothioate (miR-122) enhanced liver steatosis [99].Promoting a reduction of plaque size and enhanced vasculature, AntagomiR (miR-145) was used rodents.Results showed a marked reduction in plaque in aortic sinuses, necrotic core, increase collagen promoting contractile VSMC [67,99].These studies demonstrated how using the antagomiR or the mirmimic, it's possible to achieve a preferred outcome.
Since their initial discovery, small non-coding RNA's, have played an instrumental role in deciphering the nature and mechanics of human biology and its conditions.As we have seen in this overview of CVD's they are instrumental both in the way they orchestrate through positive and negative feedbacks, and direct control.Ultimately, we see that they work in groups, many of these belonging to a family or super family of miR's, which ultimately in the right balance are responsible for the correct cardiac environment.We can look forward into the next couple of years, where technological advances, will take us further in understanding and uncovering more insights into miR's, mimics and antagomiRs, it's very likely that their local use will be a way of personalized treatment in many illnesses we face.In addition, we expect an even faster uptake of the clinics in using screening methods for identifying miR level in patients as a way to easily access disease information.
Figure 1. mRNA biogenesis.Transcription of pri-miR by pol II, followed by cleavage Figure1. mRNA biogenesis.Transcription of pri-miR by pol II, followed by cleavage (Drosha/DGCR8 complex) to pre-miR.pre-miR is later exported to the cytoplasm via exportin-5/Ran-GTP, where now it can be further cleaved by Dicer/TRBP complex and unwind into a mature form.This mature form is further packed by AGO2 into the RISC complex (with mRNA).The consequence of such loading is either a transcriptional suppression or transcript degradation.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2018 doi:10.20944/preprints201805.0154.v1
[65,66]s.A multidimensional problem, not only dependent on the amount of circulating fat, but also on factors such as endothelial cell (EC) dysfunction, vascular smooth muscle cell (VSMC) differentiation and inflammation.These buildups can lead to partial or full blockage, and thus restricting blood flow, nutrition, and/or oxygen.Consequently, being the initiator of many diseases such as CHD, angina, carotid artery disease amongst others[63].A major component of atherosclerosis, are the EC dysfunction as response to sheer stress.Schober et al., deciphered that mir-126 directly affected vascular integrity, leading to the notion that miR-126-5p was mainly responsible for EC repair by inhibiting NOTCH1 and Dlk1,[64].Additionally, a second isoform miR-126-3p, is responsible for reducing inflammation signaling by promoting VCAM1.By blocking these 2 mechanisms atherosclerosis protection at the EC level is reduced[65,66].

Table 1 .
Principal miRNA during Heart development and ES differentiation Supplemental Table 1.Principal miRNA dysregulation during Cardiovascular Diseases