Noncoding RNAs as regulators of cardiomyocyte proliferation and death

YJMCC-08012; No. of pages: 9; 4C: 3, 7 Journal of Molecular and Cellular Cardiology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

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Review article

Noncoding RNAs as regulators of cardiomyocyte proliferation and death Maria-Teresa Piccoli a,b, Shashi Kumar Gupta a, Thomas Thum a,b,c,⁎ a b c

Institute of Molecular and Translational Therapeutic Strategies (IMTTS), IFB-Tx, Hannover Medical School, Hannover, Germany Excellence Cluster REBIRTH, Hannover Medical School, Hannover, Germany National Heart and Lung Institute, Imperial College London, UK

a r t i c l e

i n f o

Article history: Received 7 November 2014 Received in revised form 15 January 2015 Accepted 1 February 2015 Available online xxxx Keywords: MicroRNAs Cardiomyocyte Proliferation Apoptosis Necrosis Autophagy

a b s t r a c t Cardiovascular diseases are currently the main cause of morbidity and mortality worldwide. Ischemic heart disease, in particular, is responsible for the majority of cardiac-related deaths. Given the negligible regenerative potential of the human myocardium, there is a strong need for therapeutic strategies aiming at enhancing cardiomyocyte survival and proliferation following injury or at inhibiting their death. MicroRNAs (miRNAs) are small non-coding RNA molecules regulating gene expression at a post-transcriptional level with important functions in cardiovascular physiology and disease. It has been demonstrated that miRNAs can influence the ability of cardiomyocytes to enter the cell cycle and/or escape from death pathways. Additionally, long non coding-RNAs could be involved in such pathways. This review summarizes recent evidences on noncoding RNAs regulating proliferation and death of cardiomyocytes representing a future therapeutic for the treatment of heart diseases. This article is part of a Special Issue entitled SI: Non-coding RNAs. © 2015 Elsevier Ltd. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. MicroRNAs and the proliferative potential of cardiomyocytes . . . . . 3. MicroRNAs regulating cardiomyocyte death . . . . . . . . . . . . 4. Autophagy and microRNAs: how non-dividing cells renew themselves 5. Long noncoding RNAs: new players in cardiomyocyte biology . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Abbreviations: miRNAs, microRNAs; lncRNA, long non-coding RNA; LAD, left anterior descending coronary artery; hCMPCs, human Cardiomyocyte Progenitor Cells; CSCs, Cardiac Stem Cells; MI, Myocardial Infarction; MFN1, Mitofusin 1; I/R, Ischemia/ Reperfusion; BNIP3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3; mPTP, mitochondrial Permeability Transition Pore; CMPCs, Cardiomyocyte Progenitor Cells; VSMCs, Vascular Smooth Muscle Cells; T3, Triiodothyronine; Atg5, Autophagy related gene 5; Atg9, autophagy related gene 9; LDL, low-density lipoprotein; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of rapamycin complex 2; ARC, apoptosis repressor with caspase recruitment domain; EPCs, endothelial progenitor cells; DM, diabetes mellitus; TSC1, tuberous sclerosis complex 1; HCM, hypertrophic cardiomyopathy; AAV, adeno-associated virus; CARL, cardiac apoptosis-related LncRNA ⁎ Corresponding author at: Hannover Medical School, Institute of Molecular and Translational Therapeutic Strategies, OE 8886, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.: +49 511 532 5272. E-mail address: [email protected] (T. Thum).

Heart diseases remain the worldwide leading cause of morbidity and mortality. Globally, cardiovascular and cerebrovascular diseases caused, in 2001, 12.45 million deaths out of ~ 56 million, with ischemic heart disease being, in particular, the main cause of worldwide mortality and accounting for more than 7 million deaths, in both developed and developing countries [1,2]. Research conducted in the last decades have revealed that miRNAs are important regulators of cardiac development and play essential roles in many cardiovascular diseases [3,4]. MiRNAs are a class of small non-coding RNAs of 22–23 nucleotides in length, which can negatively regulate the expression of many different genes [5,6]. The effects of miRNAs are mainly due to inhibition of protein translation or to target mRNA degradation [7]. Complete sequence recognition between the mature miRNA and the complementary 3′UTR

http://dx.doi.org/10.1016/j.yjmcc.2015.02.002 0022-2828/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002

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region of the target mRNA is followed by cleavage and degradation of the latter. On the other hand, inhibition of protein translation occurs when there is only a partial complementarity between the two RNA species. The biogenesis of miRNAs is under tight spatial and temporal control [8] and it is known that several functionally related mRNAs can be targeted by one miRNA [7]. Similarly, one mRNA can be recognized by several different miRNAs. Therefore, changes in expression of a small number of miRNAs can simultaneously result in the perturbation of complex networks of intracellular and extracellular signals, which is a basic and common characteristic of both disease and developmental programs. As mentioned, ischemic heart disease is a major cause of mortality all over the world and often leads to the sudden loss of several millions of myocytes in the heart. Replacing these cells is a critical key point for the functional recovery of the heart and therapeutic strategies aiming at inducing proliferation of adult cardiomyocytes or inhibiting their death are of great clinical interest. In this regard, recent studies, which will be reviewed here, have started to reveal that many miRNAs are involved in the regulation of cardiomyocyte proliferation and death pathways. The possibility to modulate the expression of such miRNAs in vivo is, therefore, a promising therapeutic option in regenerative medicine.

2. MicroRNAs and the proliferative potential of cardiomyocytes Early cardiac development is characterized in several species by intense proliferation of cardiomyocytes, which is responsible for the morphological changes leading to the formation of a four-chambered heart [9]. However, it is known that cardiac growth is achieved after birth by means of hypertrophic processes rather than proliferation of cardiomyocytes, which are still involved in the de novo synthesis of DNA during adult life without, however, undergoing cellular duplication [10]. The mechanisms allowing the transition of cardiomyocytes from a highly proliferative phenotype to a quiescent one are poorly understood and comprehension of this event from a molecular point of view could pave the way for new therapeutic interventions capable of re-inducing cardiomyocyte division. In this regard, recent studies are pointing at miRNAs as novel regulators of this process [11–13]. MiR-499, as an example, was found to be expressed during late cardiomyocyte differentiation together with SOX6. This transcription factor represses cyclin D1 and causes cell cycle exit and arrest of cardiomyocyte proliferation [11]. Interestingly, the role of miR-499 is to keep the expression of SOX6 to appropriate levels in order to avoid triggering of apoptotic pathways [11]. Overexpression of this miRNA results in increased DNA synthesis and hyperplastic growth of cardiomyocytes during terminal differentiation [11]. Recently, Porrello E. R. et al. compared miRNA expression profiles of mouse cardiac ventricles at postnatal day 1 and 10 to identify miRNAs involved in cell cycle withdrawal [12]. A member of the miR-15 family, miR-195, emerged as the most upregulated miRNA during postnatal heart development and was confirmed to be upregulated specifically in cardiomyocytes between P7 and P14. Transgenic mice overexpressing miR-195 in the embryonic heart developed congenital heart abnormalities due to cardiomyocyte hypoplasia and died prematurely. No changes in cell size or apoptosis were observed in comparison to wild type mice, while reduction of cell mitosis and increased numbers of multinucleated myocytes were reported. Overexpression of miR-195 in vitro in neonatal rat cardiomyocytes led to cellular hypertrophy and binucleation, with arrest in the G2 phase of cell cycle (Fig. 1). Further confirmation of the role of miR-15 family in the regulation of cardiomyocyte proliferation was obtained through postnatal inhibition of all family members, leading to higher numbers of cardiomyocytes showing disorganized sarcomeric structures and undergoing mitosis without, however, being able to complete cytokinesis [12].

The miR-15 family also plays a role in regulating the regenerative capacity of the neonatal mammalian myocardium after ischemic injury [14]. Indeed, Porrello E. R. et al. have shown that neonatal mouse hearts are able to achieve regeneration after myocardial infarction (MI) at postnatal day 1, before the withdrawal of cardiomyocyte from cell cycle, in a manner similar to the one observed in zebrafish after cryocauterization, with restored perfusion to the infarcted zone [14, 15]. The regeneration of the infarcted myocardium occurs in this model of left anterior descending coronary artery (LAD) ligation by means of proliferation of preexisting cardiomyocytes rather than a stem cell population and it is not observed when LAD ligation is performed after 1 week of postnatal life. The overexpression of miR-195 is sufficient to impair regeneration of the heart in 1-day old mice, leading to the formation, after MI, of an adult-like fibrotic scar tissue. Conversely, the inhibition of the miR-15 family by administration of locked nucleic acid anti-miRs promoted myocardial regeneration after ischemia reperfusion injury at postnatal day 21 [14] (Fig. 1). Interestingly, the miR-15 family was reported to be upregulated in the infarct region of mice and pigs after ischemia-reperfusion injury [16]. Inhibition of the miR-15 family increased cell survival and viability of cardiomyocytes after hypoxia in vitro, which was associated to higher levels of BCL-2 [16]. Furthermore, in vivo therapeutic targeting of miR-15 family through the use of anti-miR oligonucleotides in mice ameliorated cardiac function following MI and reduced infarct size [16] (Fig. 1). To identify miRNAs involved in cardiomyocyte withdrawal from cell cycle, Zhang Y. et al. compared the expression levels of around 600 miRNAs in cultured 3-day, 6-day and 1-year-old rat cardiomyocytes versus 1-day-old myocytes [13]. Among the others, miR-29, miR-30 and miR-141 were upregulated in 6-day and 1-year old cardiomyocytes [13] and, in accord with these data, expression of such miRNAs is reported to be downregulated in dedifferentiated cardiomyocytes which have regained the ability to proliferate in culture [17]. Several cell cycle regulators are among the identified targets of these miRNAs and treatment of neonatal rat cardiomyocytes with anti-miRs against miR-29, miR-30 and miR-141 in quiescent cardiomyocytes led to the upregulation of cyclin A2 and to increased numbers of cells in the S or M cell cycle phase [13]. The notion that terminally differentiated cardiomyocytes could still maintain a limited capacity of re-entering the cell cycle is relatively old [18]. In addition, more recent studies have shown that a slow turnover of cardiomyocytes actually occurs during lifetime in mammalians [19], even though this regards 0,45% to 1% of the human myocardium per year [20]. Several experimental models, including genetic modification of primary cardiomyocyte cultures or exposition to exogenous agents, as well as creation of transgenic and knockout mouse models, have been used in the past years to elucidate which molecules are capable of increasing proliferation of terminally differentiated cardiomyocytes and the pathways involved [21]. Simultaneously, several reports have linked miRNAs to the regulation of proliferation in several cell types, including mature cardiomyocytes as well as their progenitors. In this regard, miR-10a, has been shown to negatively regulate the proliferation of human cardiomyocyte progenitor cells (hCMPCs), by targeting GATA6, a zinc-finger protein involved in heart development [22]. In particular, the use of miR-10a mimics in hCMPCs led to decreased DNA synthesis and inhibition of G1/S transition, together with decreased expression of specific positive cell cycle regulators such as E2F-1, cyclin B, cyclin D1, cyclin E1 [22]. Conversely, the same regulators appeared to be increased following inhibition of miR-10a. Moreover, growing expression levels of miR-10a in developing hearts at different time-points, peaking at P0, indicate that this miRNA is an important regulator of proliferation in hCMPCs during heart development [22]. As already mentioned, the traditional view of the heart as a postmitotic organ has been recently challenged by many studies [19, 20,23]. However, the field is still controversial and the genetic evidence

Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002

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Fig. 1. Involvement of the miR-15 family in the regulation of cardiomyocyte proliferation. Upregulation of miR-195 between P7 and P14 leads to withdrawal of mouse cardiomyocytes from cell cycle, cellular hypertrophy and binucleation. Involvement of the lncRNA Uc.283 + A in the regulation of miR-195 levels during postnatal development of cardiomyocytes is currently unknown. Inhibition of miR-195 in vivo restores the ability of preexisting cardiomyocytes to replicate themselves and regenerate the myocardium after LAD ligation at P21. Additionally, members of the miR-15 family are upregulated in the infarct region of mice and pigs after I/R injury, leading to decreased levels of BCL-2 and cell death. In vivo targeting of the miR-15 family protects against cardiac ischemic injury, increasing cardiac function and decreasing infarct size.

still points to the adult heart as a largely non-renewable organ [19,24]. According to some reports, the human heart harbours cardiac stem cells (CSCs) of uncertain origin that can generate every day different type of new cells, including cardiomyocytes, endothelial cells and vascular smooth muscle cells [25]. Recently, miRNAs expressed in CSCs of adult mouse hearts have been partially profiled and compared to those of embryonic heart cells and bone marrow progenitor cells, in order to identify miRNAs that likely have a role in the maintenance of a low-proliferative and undifferentiated state with limited potential [26]. Among the results of this comparison, the downregulation of the miR-17/92 cluster appeared to be the main difference between CSCs and their two potential progenitor populations. MiR-17/92 cluster is involved in a variety of human cancers as an oncogene [27] and it was recently reported by Chen J. et al. to regulate proliferation of embryonic, postnatal and adult cardiomyocytes ex vivo and in vitro [28]. The tissue-specific knockout of the miR-17–92 cluster has been shown to be partially lethal in mouse embryos and lead to impaired cardiac development visible in the postnatal phase. Postnatal hearts of conditional knockout mice showed reduced proliferation of cardiomyocytes with no change in apoptotic rates, while adult mice showed a substantial decrease in the total number of cardiomyocytes and a certain degree of compensatory hypertrophy. Overexpression of the miR-17–92 cluster in transgenic mice led to increased heart size, ventricular wall thickness and cardiomyocyte hyperplasia in embryonic and postnatal hearts. Interestingly, inducible overexpression of the miRNA cluster, specifically in adult hearts, led to increased cardiac size, heart/ body weight ratio and ventricular wall thickness, together with increased cell numbers, specifically of mono-nucleate cardiomyocytes, proliferation rate and decreased cell size. Overexpression of the miR-17–92 cluster protected adult hearts from MI and doxorubicin-induced injury. Finally,

the ability of the miRNA cluster to induce proliferation of cardiomyocytes was confirmed in vitro in neonatal rat cardiomyocytes and suggested to occur through targeting of PTEN [28]. Another member of the miR-17–92 family, miR-19b, was shown to promote cell proliferation and DNA synthesis when overexpressed in P19 cells [29], a multipotent mouse cell strain which can be induced to differentiate into cardiomyocyte in the presence of DMSO and which is often used as a study model for cardiomyocyte differentiation. Even though the mammalian myocardium could retain a certain degree of an endogenous regenerative potential, this is certainly not enough to ensure cardiac repair after acute injuries like MI. miRNAs, on the other hand, have been implicated in the capability of other species, and in particular of adult zebrafish, to regenerate cardiac muscle after injury [30]. The source of regeneration in zebrafish is represented by differentiated cardiomyocytes that retain the ability to enter the cell cycle and replace the lost myocardium by proliferation [31]. Comparison of miRNA profiles between uninjured and regenerating zebrafish ventricles allowed the identification of the cardiomyocytespecific miR-133 as one of the most downregulated miRNAs during cardiac regeneration [30]. Additionally, miR-133 was reported to be significantly downregulated in mouse cardiomyocytes during pathological hypertrophy [32]. Modulation of miR-133 levels in zebrafish shortly after cardiac injury leads to changes in the proliferation index of cardiomyocytes, with increased proliferation near the injury site observed after miR-133 depletion, indicating that miR-133 expression negatively influences proliferation of cardiomyocytes [30]. Long-term overexpression of miR-133 following partial ventricular resection inhibits cardiac regeneration leading to scar formation as alternative repair process, while depletion of miR-133 enhances cardiomyocyte proliferation and cardiac regeneration [30].

Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002

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The role of miR-133a in the regulation of cardiomyocyte proliferation was also described in mice by Liu N. et al. in a double knockout model for miR-133a-1/miR-133a-2. These two members of the miR-133 family are specifically expressed in cardiac and skeletal muscles [33] and mice lacking both miRNAs died prematurely between postnatal days 0 and 1 due to cardiac defects. Moreover, cardiomyocytes from dKO mice showed an increase in proliferation compared to wild type animals, while overexpression of miR-133a-1/2 in the developing hearts of transgenic mice resulted in decreased cardiomyocyte proliferation and premature death. In addition, dKO mice showed enhanced cardiac expression of several positive regulators of cell cycle, such as cyclin D1, cyclin D2 (which was confirmed to be a target of miR-133a) and cyclin B1 [34]. The results of the first screening for miRNAs with the ability of enhancing cardiomyocyte proliferation were published in 2012 by Eulalio A. et al. The authors performed a high-throughput functional screening in neonatal rat cardiomyocytes with a library of around 1000 miRNAs and identified 204 miRNAs enhancing cardiomyocyte proliferation and 331 miRNAs showing the opposite effect [35]. Among the pro-proliferative miRNAs, 40 molecules were also able to induce higher proliferation rates in mouse cardiomyocytes, which usually have a lower proliferative capacity than rat cardiomyocytes, thus confirming the conserved effect of such miRNA candidates. Analysis of the expression of markers for late G2/mitosis and cytokinesis, confirmed the ability to induce karyokinesis and cell divisions for 10 top candidates in mouse and rat cardiomyocytes. Most importantly, transfection of cardiomyocytes isolated from adult rats with the top candidates miR-590-3p and miR-199a-3p, led to a re-entry of cells into cell cycle with a consequent increase in cell number [35]. Among the identified targets or miR-590-3p and miR-199a-3p, the authors validated HOMER1, HOPX and CLIC5, whose knockdown by means of siRNAs led to increased proliferation levels. Furthermore, the authors were able to show that short- and long-term overexpression of miR-590-3p and miR-199a-3p increased cardiomyocyte proliferation in vivo without affecting cardiac fibroblast proliferation and, thus, without induction of fibrosis [35]. Moreover, viral delivery of miR-590-3p and miR-199a-3p shortly after permanent LAD ligation significantly preserved cardiac function and reduced infarct size [35]. Finally, another miRNA potentially regulating cell cycle re-entry of cardiomyocytes is miR-29a. Comparison of miRNA expression profiles between P2 and P4W rats revealed miR-29a to be upregulated in 4-week-old vs. 2-day-old rat cardiomyocytes [36]. Functional studies were conducted in H9c2 cell line and neonatal rat cardiomyocytes, to confirm the ability of miR-29a to suppress cell proliferation by modulating cell cycle progression, likely through the downregulation of CCND2 [36]. 3. MicroRNAs regulating cardiomyocyte death Cardiomyocytes can undergo mainly two different types of cell death, specifically apoptosis and necrosis, and both are observed in the progression of heart diseases [37]. Apoptosis, or programmed cell death, is a highly regulated process that plays a fundamental homeostatic role in healthy animals as well as a causative role in a plethora of pathological conditions. Programmed cell death can be the result of the activation of two different pathways, named “extrinsic” and “intrinsic”, both converging on a family of cysteine proteases, called caspases [38]. The extrinsic pathway relies on the activity of extracellular death molecules, such as TNF-alpha or FAS-ligand, which are able to activate an intracellular cascade of signals leading to cellular death [38]. Conversely, the intrinsic apoptotic pathway is mediated by mitochondria [39] and it is regulated by a family of proteins called BCL-2, which is made up of both pro-apoptotic and anti-apoptotic members [40]. The pro-apoptotic member BAX is normally retained in the cytoplasm and transported into mitochondria, following cellular

stress, to form heterodimers with BCL-2 and promote cell death [41]. Moreover, the translocation of BAX leads to the release of cytochrome C from the mitochondria, which is in turn a trigger for the activation of caspase-3 and caspase-9 and for a series of downstream apoptotic events [41]. Importantly, mitochondria are constantly subjected to fusion and fission and both processes are needed for maintaining the integrity of these organelles [42]. It was shown that abnormalities in fusion and fission of mitochondria can influence the regulation of apoptosis [43], with fusion involved in the inhibition of apoptosis and fission, conversely, associated to the initiation of programmed cell death [44]. Mitofusin 1 (MFN1) is a protein involved in the regulation of mitochondrial fusion which can inhibit mitochondrial fission and apoptosis in cardiomyocytes [45]. Apoptotic stimulation of cardiomyocytes by hydrogen peroxide and doxorubicin treatment results in the downregulation of MFN1 and the concomitant upregulation of miR-140, which can directly target MFN1 expression by binding to its 3′UTR. Furthermore, overexpression of MFN1 is able to suppress doxorubicin and hydrogen peroxideinduced mitochondrial fission and apoptosis as well as downregulation of miR-140, indicating that both molecules are involved in the apoptotic program driven by mitochondrial fission. In a mouse model of ischemia/ reperfusion (I/R) injury, inhibition of miR-140, by using specific antimiR, resulted in lower levels of cardiomyocyte apoptosis and reduction of infarct size, which were concomitant with an attenuation of MFN1 reduction and mitochondrial fission. These effects on I/R in vivo were reversed by MFN1 downregulation [46]. On the other hand, when exposed to excessive stress, due, for example, to the lack of oxygen and nutrients, cardiomyocyte can undergo necrosis. Necrosis has several characteristics which are profoundly different from those of programmed cell death and autophagy, such as complex morphological changes, increase in cell volume, organelles swelling, disruption of membrane integrity and loss of ATP [47]. Moreover, following necrosis, cardiomyocyte cellular contents are released in the extracellular environment, triggering a robust inflammatory reaction, with severe pathological consequences [37]. Obviously, one important cause of cardiomyocyte necrosis is MI. Indeed, ischemic insults lead to increased levels of intracellular calcium, depletion of ATP, oxidative stress and acidosis. These conditions result in necrotic death of cardiomyocytes, while apoptosis of cardiac cells is mainly linked to the reperfusion phase [48]. In line with these findings, necrosis inhibitors proved to be effective in reducing cell death and infarct size in animal models [49]. However, cell death in the reperfusion phase is of great clinical importance since reperfusion is the definitive treatment for acute MI. As anticipated, reperfusion has the potential to make tissue injury worse. The critical damaging event which induces massive apoptosis of cardiomyocytes during I/R is the excessive production of reactive oxygen species [50]. BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3), in this case, is an important sensor of oxidative stress and player of intrinsic apoptosis. BNIP3 is found in the outer membrane of the mitochondria and can transduce apoptotic signals leading to activation of apoptotic proteins, inhibition of antiapoptotic proteins, and depolarization of the mitochondrial membrane together with formation of the mitochondrial permeability transition pore (mPTP) [51]. The traditional view of necrosis as a passive process caused by a strong external damaging event has changed with time and it is now known that necrosis, similar to apoptosis, is a controlled process with several pathways involved. Furthermore, necrosis can also occur in the form of “programmed” cell death [52]. In this case, it is referred to as “necroptosis”, a regulated cell death pathway which is mainly controlled by TNF-alpha mediated activation of RIP1 and RIP3 kinases [53]. Additionally, necrosis can be triggered by the disruption of mitochondrial integrity as well [54]. While the key event in intrinsic apoptosis is the permeabilization of the mitochondrial outer membrane, triggered by BCL-2 pro-apoptotic members, during necrosis opening of

Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002

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the mPTP in the inner membrane occurs, causing the dissipation of the membrane potential and the subsequent ATP depletion, ROS production, organelle damage and swelling [55]. Current knowledge on miRNAs regulating necrosis or necroptosis is still very limited. miR-155 was shown by Liu J. et al. to be a promising therapeutic target in cardiomyocyte progenitor cells (CMPCs), since its modulation could improve survival after implantation of these cells in the infarcted myocardium. Indeed, overexpression of miR-155 was achieved by Liu J. et al. in vitro in CMPCs resulting in decreased cell death, specifically necrotic cell death, after exposure to hydrogen peroxide, with no change in apoptosis levels and cardiomyogenic differentiation potential. Furthermore, the ability of miR-155 to induce anti-necrotic responses in CMPCs was shown to occur through repression of RIP1 [56]. Furthermore, Wang K. et al. have recently provided new insights in the pathogenesis of myocardial necrosis. Through microarray analysis, they identified miR-874 as highly upregulated following hydrogen peroxide-induced necrotic cell death of cardiomyocytes. The knockdown of miR-874 in vitro led to decreased cardiomyocyte necrosis and proved to be cardioprotective in a mouse model of MI. Analysis of miR-874 potential targets led to the validation of caspase-8 as direct target, and further investigations showed that the regulation of necrosis by miR-874 occurs through targeting of caspase-8, which is able to inhibit necrosis thanks to the catalytic cleavage of RIP3 and RIP1. The transcription factor FOXO3A was shown to be involved in the regulation of miR-874 expression through inhibition of its transcription and use of in vivo transgenic and knockout models confirmed the hypothesis that FOXO3A is an anti-necrotic transcription factor which can inhibit miR874 expression, leading to increased caspase-8 activity and decreased levels of necrosis, potentially due to the ability of caspase-8 to cleave the RIP3 and RIP1 kinases [57]. Differently from necrosis, the literature investigating the role of miRNAs in the regulation of cardiomyocyte apoptosis is much larger. In addition to the already mentioned miR-140 and among many others, miR-20a, miR-145, miR-24, miR-17–92 cluster, miR-30a and miR-133 have been implicated in the regulation of cardiomyocyte apoptosis [58–63]. Progressive cardiac remodelling following MI or pressure/volume overload is characterized by excessive levels of apoptosis, which increases more than 100-fold in comparison to healthy human hearts [64]. Continuous biomechanical stress is, indeed, a strong activating stimulus for cardiomyocyte apoptosis in vitro and in vivo [65,66]. In a study from Frank D. et al., biaxial mechanical stretch significantly deregulated the expression of several microRNAs, including miR-20a, in neonatal rat cardiomyocytes [58]. The upregulation of miR-20a was observed both in this experimental setting and in an in vitro I/R assay, while no change was observed after stimulation with phenylephrine, indicating that miR-20a does not regulate cardiomyocyte hypertrophy. Furthermore, overexpression of miR-20a showed a marked dosedependent anti-apoptotic effect in neonatal rat cardiomyocyte after I/ R. Conversely, knockdown of miR-20a induced cardiomyocyte apoptosis already 48 h after transfection of specific antimiR targeting miR-20a. Bioinformatic prediction followed by luciferase assay and antimiRmediated knockdown identified the pro-apoptotic prolyl hydroxylase EGLN3/PHD3 and E2F protein family as targets of the anti-apoptotic miR-20a [58]. Another miRNA, miR-145, has been shown to regulate apoptosis in tumour cells [67] and is aberrantly expressed in vascular smooth muscle cells (VSMCs) in response to oxidative stress induced by hydrogen peroxide [68]. Furthermore, it appears to be significantly downregulated in the myocardium after I/R injury and in cultured cardiomyocytes after hydrogen peroxide treatment [59]. Involvement of miR-145 in the regulation of hydrogen peroxide-induced apoptosis was shown through overexpression and inhibition experiments, resulting in, respectively, inhibition and induction of apoptosis of cardiomyocytes. Furthermore, the anti-apoptotic effect of miR-145 can be rescued by overexpression

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of its direct target BNIP3 [59]. MiR-145 is indeed able to decrease the rates of mitochondrial apoptosis, as shown by the decreased expression of key molecules in the mitochondrial apoptotic pathways after both hydrogen peroxide treatment and miR-145 overexpression [59]. Similarly, morphology of mitochondria after hydrogen peroxide treatment is largely preserved when miR-145 is concomitantly overexpressed [59]. Members of the already described miR-17–92 cluster are involved not only in the regulation of cardiomyocyte proliferation, but also in cardiomyocyte apoptosis. Du W. et al. described a reciprocal change in the expression of miR-17-5p and STAT3 in mouse myocardium after I/ R, with miR-17-5p being upregulated [61]. Authors observed the same trend in vitro after treatment of neonatal rat cardiomyocytes with hydrogen peroxide. Interestingly, overexpression of miR-17-5p alone was not sufficient to affect cell viability in vitro, but increased hydrogen peroxide-induced damage of cardiomyocytes. Conversely, inhibition of miR-17-5p was able to rescue, at least partially, the apoptosis induced by hydrogen peroxide. Also, administration of LNA-anitmiR-17-5p prior to I/R in mice reduced infarct size and TUNEL positivecardiomyocytes [61]. Among the direct targets of miR-17-5p, Stat3 is known to be a cell survival factor which is increased in response to oxidative stress and has an anti-apoptotic role in cardiomyocytes [69]. In vitro and in response to oxidative stress, overexpression of miR-17-5p resulted in decreased p-STAT3 levels, an effect which was eliminated when co-transfecting cells with miR-17-5p inhibitors [61]. Finally, cardioprotective agents such as triiodothyronine (T3) and carvedilol were also reported to achieve their anti-apoptotic effects through modulation of miRNA-mediated pathways [62,70]. A recent publication by Forini F. et al. attributes the cardioprotective effects of T3 administration after I/R to the prevention of miR-30a loss in the myocardium, which in turn limits the activation of p53-induced death pathways [71]. Authors described a decrease in levels of serum free and myocardial T3 hormone after I/R in mice and developed an effective delivery system of T3 to the damaged myocardium. The latter resulted in improved cardiac function after I/R, as shown by the decreased end systolic LV diameter and improved contractility, as well as by a small increase in heart rate. Furthermore, administration of T3 provided retainment of mitochondrial activity and integrity with less superoxide accumulation, which was associated to a decreased infarct size and TUNEL positivity. T3 administration abrogated the increase in the expression, at the infarct site, of p53 and BAX and, importantly, prevented loss of miR-30a in the infarct area. In vitro, the link between T3, miR-30a and p53 was shown by exposing rat cardiomyocytes to hypoxia and later on to T3. T3 treatment was able to restore miR-30a levels after 24 h hypoxia and led to a reduction in p53 activation. Furthermore, the knockdown of miR-30a during hypoxia exaggerated p53 activation and abolished the T3 beneficial effects [62]. On the other hand, the anti-apoptotic potential of carvedilol had been already described by Yeh C.H. et al. [63], even though the exact mechanisms by which these protective effects are achieved are unknown. Recently, Xu C. et al. have proposed a miRNA-based mechanism for this cardioprotective potential [70]. According to the authors, carvedilol treatment in a rat model of MI results in the upregulation of miR-133, which was already described as a cardioprotective microRNA and a negative regulator of caspase-9 [72,73]. Furthermore, treatment of cardiomyocytes in vitro with hydrogen peroxide results in loss of miR-133, which can be reversed by carvedilol. Xu C. et al. showed that the beneficial effects of carvedilol are achieved through relief from oxidative stress and downregulation of caspase-9 and can be abolished by antisense oligonucleotides against miR-133 [70]. 4. Autophagy and microRNAs: how non-dividing cells renew themselves Autophagy is a self-degradative process in which cells degrade and recycle their non-functional organelles and proteins to maintain cellular

Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002

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homeostasis and it is reported to play a role in ageing, caloric restriction, inflammation, tumour metabolism, neurodegenerative and cardiovascular diseases [74]. Even though autophagy is generally accepted as a cell survival phenomenon, it has also been described as a cell death phenomenon, due to the presence of autophagic vacuoles in dying cells and to the close inter-link of autophagy and apoptosis pathways [75]. In cardiomyoctyes, the process of autophagy maintains the renewal of mitochondria, and, thus, the energy demand of the heart [75,76]. Cardiomyocytes, which constitute the bulk of the heart, are highly dependent on autophagy for the renewal of non-functional mitochondria, proteins and other organelles, because of their non-dividing nature. Autophagic process is regulated by several Atg genes together with other genes, a majority of which still remain to be identified. Cardiac deletion of autophagy related gene 5 (Atg5) leads to ageing phenotype in the heart, characterized by cardiomyocyte hypertrophy, fibrosis and reduced function, all features depicting the importance of autophagy in the heart [76]. Recently, miRNAs were demonstrated to regulate cardiac autophagy in vitro and in vivo [77]. Mammalian target of rapamycin (mTOR) is the central regulator of autophagy and inhibitors of mTORC1 like rapamycin, everolimus and inhibitors of other downstream mediators have shown a beneficial effect in MI model by activation of autophagy [78]. In line with these observations, miRNA mediated inhibition of mTOR by miR-99a demonstrated increased survival, improved cardiac function, decreased infarct size and decreased myocardial remodelling [79]. MiR-99a was downregulated following MI and hypoxia in vitro and overexpression of miR-99a results in activation of autophagy, which mediates the beneficial effect. Chronic exposure to inhibitors of mTORC1 affect mTORC2 and causes several other side effects and thus exploration of specific inhibition mediated by miRNAs could be an alternative strategy to activate autophagy in disease settings. Contrary to mTOR, activation of autophagy by Beclin-1 was found to induce cell death in cardiac ischemia reperfusion model. MiR-325 expression is induced in hearts exposed to ischemia reperfusion, which in turn downregulates its target apoptosis repressor with caspase recruitment domain (ARC), an inhibitor of Beclin-1 [80]. It increases freely available Beclin-1, which activates autophagy and induces cell death. A MiR-325 transgenic heart shows higher accumulation of autophagosomes, enlarged infarct and severe cardiac dysfunction after ischemia reperfusion injury. Activation of autophagy by inhibition of mTOR had been established to inhibit cardiomyocyte hypertrophy by several studies, on the other hand activation of autophagy by Beclin-1 or Atg9 shows opposite effects. It has been reported that increased autophagy in cardiomyocytes in response to Angiotensin II leads to downregulation of miR-30 and miR-34a and upregulation of their targets Beclin-1 and Atg9a respectively [81,82]. Overexpression of Atg9a resulted in increased autophagy with concomitant upregulation of hypertrophy genes and cardiomyocyte cell size, while its upstream negative regulator miR-34a overexpression had reversed effects. MiR-30a also reverses the effects of Angiotensin II by inhibiting cardiomyocyte autophagy. In addition, another study reported miR-30a to be downregulated in model of pressure overload induced cardiac hypertrophy and miR-30a was shown to inhibit hypertrophy and autophagy. Although these studies indicated inhibition of autophagy via either miR-30a or miR-34 to inhibit cardiac hypertrophy, in vivo evidence of the same still remains to be investigated. On the contrary, a very detailed study from our group identified the miR212/132 cluster as pro-hypertrophic and anti-autophagic. The miR-212/ 132 family increases in murine hearts following pressure overload as well as in human failing hearts. Cardiac specific miR-212/132 family overexpression leads to maladaptive remodelling with enormous hypertrophy, fibrosis and reduced cardiac function, and eventually cardiac failure. The transgenic hearts also show inhibition of autophagy at the basal level and in response to starvation. A null mice line for miR-212/ 132 family shows higher basal autophagic activity and reduced hypertrophy in response to pressure overload. MiR-212/132 induced hypertrophy and inhibited autophagy by post-transcriptional regulation of

FOXO3. Although a direct link was not established between development of hypertrophy and autophagy, some support for inhibitory autophagy and hypertrophy is provided [75,77]. In another disease model of hypertrophic cardiomyopathy, characterized by cardiac hypertrophy, miR-451 was found to be downregulated [83]. MiR-451 inhibition activated autophagy and induced hypertrophy in cardiomyocytes by targeting tuberous sclerosis complex 1 (TSC1). Higher autophagic activity was depicted in human hypertrophic cardiomyopathy biopsies compared to control. Although in a different study, utilizing a mice model of hypertrophic cardiomyopathy (HCM) with MybpC3 mutation, autophagic flux was reported to be inhibited in HCM [84]. In the latter study, accumulation of Beclin-1, LC3 and p62 was reported in HCM condition which is in agreement with human HCM Table 1 miRNAs and lncRNAs regulating proliferation, apoptosis and autophagy of cardiomyocytes. Proliferation

Apoptosis

Autophagy

miR-499 ➢ Induces proliferation in P19 multipotent cells ➢ Targets SOX6 ➢ Ref. [11]

miR-140 ➢ Induces apoptosis ➢ Targets MFN1 ➢ Ref. [46]

miR-195 ➢ Inhibits proliferation in mouse cardiomyocytes ➢ Targets CHEK1 ➢ Ref. [12,15]

miR-155 ➢ Decreases necrosis in cardiomyocyte progenitor cells ➢ Targets RIP1 ➢ Ref. [56] miR-874 ➢ Promotes necrosis ➢ Targets caspase-8 ➢ Ref. [57]

miR-99a ➢ Activates beneficial autophagy ➢ Targets mTOR ➢ Ref. [79] miR-325 ➢ Activates apoptotic autophagy ➢ Targets ARC ➢ Ref. [80] miR-30a ➢ Inhibits hypertrophic autophagy ➢ Targets Beclin-1 ➢ Ref. [81] miR-34 ➢ Inhibits hypertrophic autophagy ➢ Targets ATG9A ➢ Ref. [82] miR-212/132 ➢ Inhibits aiding autophagy ➢ Targets FOXO3A ➢ Ref. [77] miR-451a ➢ Inhibits hypertrophic autophagy ➢ Targets TSC1 ➢ Ref. [83]

miR-29, 30, 141 ➢ Inhibits proliferation in rat cardiomyocytes ➢ Targets cyclin A2 ➢ Ref. [13]

miR-10a ➢ Inhibits proliferation of hCMPCs ➣Targets GATA6 ➢Ref. [23]

miR-20a ➢ Anti-apoptotic effects in rat cardiomyocytes ➢ Targets PHD3 and E2F ➢ Ref. [58]

miR-17/92 cluster ➢ Induces proliferation in adult mice cardiomyocytes ➢ Targets PTEN ➢ Ref. [28]

miR-145 ➢ Decreases mitochondrial apoptosis ➢ Targets BNIP3 ➢ Ref. [59]

miR-19b ➢ Promotes cell proliferation in P19 multipotent cells ➢ Inhibits differentiation of P19 cells into cardiomyocytes ➢ Targets Wnt1 ➢ Ref. [29] miR-133a ➢ Inhibits cardiomyocyte proliferation in zebrafish and mouse cardiomyocytes ➢ Targets cyclin B2, cx43 ➢ Ref. [30,34] miR-590-3p, miR-199a-3p ➢ Induces cell cycle re-entry in adult mice cardiomyocytes ➢ Targets HOMER1, HOPX, CLIC5 ➢ Ref. [35] Uc.283 + A ➢ Negatively regulates the biogenesis of miR-195 ➢ Ref. [93]

miR-17–92 cluster ➢ Promotes apoptosis ➢ Targets STAT3 ➢ Ref. [61]

miR-30a ➢ Anti-apoptotic ➢ Targets p53 ➢ Ref. [62]

miR-133 ➢ Anti-apoptotic ➢ Targets caspase-9 ➢ Ref. [70] CARL ➢ Sponges miR-539 inhibiting mitochondrial apoptosis ➢ Ref. [94]

Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002

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samples, except for p62 which was not analysed. Evaluation of p62 is very important to determine the inhibition or activation of autophagic flux and thus measurement of p62 protein level in human HCM samples can clarify the autophagic state of HCM hearts. It appears that activation of autophagy by fine-tuning mTOR pathway is beneficial while Beclin-1 mediated activation of autophagy could have detrimental effects and thus modulation of autophagy for therapeutic usage needs to check both the pathways simultaneously. 5. Long noncoding RNAs: new players in cardiomyocyte biology The world of noncoding RNAs has expanded in the last two decades with the identification of functional RNA molecules which share many biological properties with mRNAs but have very limited or no coding potential [85,86]. These so called long noncoding RNAs (lncRNAs) are generally distinguished from small noncoding RNAs, such as microRNAs, because of their length, and are defined as transcripts of N200 nucleotides in length. LncRNAs are located throughout the genome, in intergenic, exonic and intronic positions and in sense and antisense orientations [87] relative to coding genes. After being transcribed, lncRNAs can reside in the nucleus or in the cytoplasm and interact with nucleic acids or proteins, acting as activating or inhibiting signal molecules, decoys for microRNAs, guides for transcription factors, and scaffolds for three-dimensional structures [88]. In this way, lncRNAs can virtually regulate every cellular process and, as a matter of fact, they have been recently implicated in a plethora of human diseases [89]. It is known that lncRNAs can regulate proliferation and death pathways in different cell types [90–92], however, knowledge regarding lncRNAs involved in proliferation, survival and death of cardiomyocytes is still very limited. Recently, a long noncoding RNA (lncRNA) regulating the biogenesis of miR-195 has been described [93]. This lncRNA, called Uc.283+A, is transcribed from an ultraconserved region and regulates miR-195 post-transcriptionally at the level of Drosha cleavage, by binding to the complementary lower stem region of pri-miR-195 and inhibiting its recognition by DGCR8. Evidence for this regulatory mechanism being functional in cardiomyocytes is still missing; however, regulation of miR-195 biogenesis by a lncRNA transcribed from an ultraconserved region may provide new explanations for the postnatal exit of cardiomyocytes from cell cycle (Fig. 1) [93]. So far, the only lncRNA which was described as an inhibitor of cardiomyocyte apoptosis is called CARL (cardiac apoptosis-related lncRNA) and acts as a sponge for miR-539 [94]. Wang K. et al. have shown that PHB2, a subunit of the prohibitin complex, is able to inhibit mitochondrial fission and apoptosis of cardiomyocyte and can be downregulated by miR-539. Therefore, miR-539 can provoke mitochondrial fission and apoptosis by targeting PHB2. In this context, CARL is able to regulate miR-539 expression and activity by binding to miR-539 in vivo and inhibiting cardiomyocyte apoptosis. Therefore, the modulation of

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CARL levels could represent a future approach for the treatment of myocardial infarction.

6. Conclusions MiRNAs have been shown to be easily targetable by antisense oligonucleotide technology, which gave an unprecedented access to researchers to study their function in vitro and in vivo. Based on the studies presented in this review, miRNAs demonstrate enormous potential to modulate cardiomyocyte viability and survival by regulating their proliferation, apoptosis, necrosis and autophagy (Table 1, Fig. 2) in vitro and in animal models; given the prevalence and mortality rates associated with cardiovascular diseases, clinical studies evaluating the performance of miRNAs as therapeutic targets in humans could lead in the future to significant improvements in the current care standards. There are several anti-miR chemistries available today for inhibition of miRNA functions [95] and they have a high potential for becoming a new class of therapeutics, especially if the expression of their target miRNA is restricted to a specific tissue. In this regard, one key issue that is hardly ever addressed in the preclinical studies reviewed here, is the effects of miRNA inhibition in organs different from the heart. Off-target effects of miRNA modulation are complex and harder to study in comparison to traditional targeted therapies, due to the fact that one single miRNA can regulate a whole network of coding genes, which can differ according to the cell type where miRNA inhibition is achieved. A deeper understanding of pharmacodynamic and pharmacokinetic properties of anti-miR molecules is therefore essential for their entrance into the clinical practice. In this regard, safety and efficacy data following phase 2 clinical trials on Miravirsen, an inhibitor of miR-122 for the treatment of HCV infections, support the awareness that miRNA-based therapeutics can actually become a reality in medicine. In the field of cardiovascular disease, inhibition of miR-15 family members by use of LNA-modified oligonucleotides family proved to be protective in a mouse and pig model of MI [16] and additional data describing the role of miR-195 in promoting cardiomyocyte exit from cell cycle [12] make this miRNA family a promising candidate for future clinical investigations on the possibility to promote cardiac regeneration after ischemic injury. On the other hand, strategies for restoration of downregulated miRNAs are currently limited and mostly rely on the use of adenoassociated viruses (AAV). AAVs have already been successfully used in humans for cardiac gene delivery [96] and data from Eulalio et al. [35] on the potential of miR-590-3p and miR-199a-3p to bring back cardiac function to almost normal levels after MI strongly encourage future studies aimed at the exogenous administration of miRNAs to achieve the golden goal of cardiac regeneration.

Fig. 2. miRNAs regulating proliferation, apoptosis and autophagy of cardiomyocytes. Proliferating cardiomyocyte are coloured in green (left side), apoptotic cardiomyocytes in orange (centre) and autophagy is indicated in pink (right side). miRNAs promoting or inhibiting these processes are depicted respectively in yellow (underlined) and red (italicized).

Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002

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Please cite this article as: Piccoli M-T, et al, Noncoding RNAs as regulators of cardiomyocyte proliferation and death, J Mol Cell Cardiol (2015), http://dx.doi.org/10.1016/j.yjmcc.2015.02.002