The epigenetic promise to improve prognosis of heart failure and heart transplantation

    The Epigenetic Promise to Improve Prognosis of Heart Failure and Heart Transplantation Chiara Sabia, Antonietta Picascia, Vincenzo Grimaldi, Cristiano Amarelli, Ciro Maiello, Claudio Napoli PII: DOI: Reference:

S0955-470X(17)30017-4 doi: 10.1016/j.trre.2017.08.004 YTRRE 459

To appear in:

Transplantation Reviews

Please cite this article as: Sabia Chiara, Picascia Antonietta, Grimaldi Vincenzo, Amarelli Cristiano, Maiello Ciro, Napoli Claudio, The Epigenetic Promise to Improve Prognosis of Heart Failure and Heart Transplantation, Transplantation Reviews (2017), doi: 10.1016/j.trre.2017.08.004

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ACCEPTED MANUSCRIPT Transplantation Reviews, Review Article, Revised form THE EPIGENETIC PROMISE TO IMPROVE PROGNOSIS OF HEART FAILURE AND HEART

, Cristiano Amarelli 3, Ciro Maiello

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Claudio Napoli 1,4. 1

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Chiara Sabia 1*, Antonietta Picascia 1, Vincenzo Grimaldi

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TRANSPLANTATION

U.O.C. Division of Clinical Immunology, Immunohematology, Transfusion Medicine and Transplant

Immunology, Department of Internal Medicine and Specialistics, Azienda Ospedaliera Universitaria, Department of Medical, Surgical, Neurological, Aging and Metabolic Sciences, Università degli Studi della Campania “L. Vanvitelli”, Italy.

Department of Sciences and Technologies, University of Sannio, Benevento, Italy.

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Department of Cardiovascular Surgery and Transplants, Monaldi Hospital, Azienda dei Colli, Naples, Italy.

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SDN Foundation, Institute of Diagnostic and Nuclear Development, IRCCS, Via Gianturco 113, 80143

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Naples, Italy

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Chiara Sabia and Antonietta Picascia contributed equally to this work.

*Corresponding author Dr. Chiara Sabia U.O.C. Division of Clinical Immunology, Immunohematology, Transfusion Medicine and Transplant Immunology, Department of Internal Medicine and Specialistics, Azienda Ospedaliera Universitaria, Department of Medical, Surgical, Neurological, Aging and Metabolic Sciences, Università degli Studi della Campania “L. Vanvitelli”, Piazza L. Miraglia 2, 80138 Naples, Italy E-mail address: [email protected]

ACCEPTED MANUSCRIPT Transplantation Reviews, Review Article, Revised form

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INTRODUCTION

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Heart transplantation (HT) remains the gold standard treatment for end-stage heart failure (HF) ensuring the highest long-term survival; however, due to the shortage of organ donors, the left ventricular assist device

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(LVAD) provides a backup strategy for patients as bridge to transplantation or when clinical contraindications are present [1,2]. HF represents the end-stage of several cardiac pathological conditions

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including atherosclerosis, coronary heart disease (CHD) and dilated, hypertrophic and restrictive cardiomyopathies [3-5].

Human genetic studies have identified mutations that are associated with the onset and progression of cardiovascular disease (CVD). A growing body of evidence indicates the involvement of epigenetics in the

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development of CVD. In some cases, epigenetic modifications, a set of heritable and acquired modifications of the genome that alter gene expression without changing the DNA sequence, are stable and inherited by

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future generations, but in other cases many epigenetic changes are dynamic and responsive to environmental

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stimuli. [4,6-8].

Typically, epigenetic modifications regulate gene expression and are grouped into three main classes: DNA

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methylation, histone modifications and RNA-based mechanisms. DNA methylation consists in the addition of a methyl group on the carbon 5 of the cytosine residues (5mC) located in CpG dinucleotides. CpG dinucleotides are distributed throughout the genome, but display a high frequency in particular regions, termed “CpG islands”, that are localized in different genomic sites including gene promoter and body, and intergenic regions with enhancers and repetitive sequences. DNA methylation is catalyzed by a group of enzymes, the DNA methyltrasferase (DNMTs) and S-adenosylmethionine is the donor of the methyl groups. DNA methylation promotes gene silencing either directly, by blocking the binding of transcription factors to DNA, or indirectly, by binding MBPs (methyl-binding proteins) that, in turn, induce gene repression by recruiting co-repressors [9]. Acetylation or deacetylation of lysine residues in selected nucleosomal histone tails promotes gene activation or silencing, respectively [10]. Acetylation is a dynamic process regulated by two groups of enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs), whose expression and activity are

ACCEPTED MANUSCRIPT finely regulated. Also, histone methylation can be associated with either activation or repression of transcription, depending on the degree of methylation of Lys or Arg residues. Furthermore, microRNAs (miRNAs), a class of small endogenous non-coding ribonucleic acids, regulates

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gene expression at post-transcriptional level, through either transcript degradation or translational repression by binding to the 3 'UTR of the target mRNA [11]. MiRNAs originate from a primary transcript that is

for the processing by the endonuclease Dicer [12].

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cleaved in the nucleus by the RNase Drosha, thus resulting in a pre-miRNA that is exported to the cytoplasm

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Epigenetic mechanisms have been involved in the onset and progression of CVD as well as hypertrophic and dilated cardiomyopathies leading to HF, and have been proposed as potential new strategies for treating these diseases.

Here we discuss about epigenetic changes associated with all the clinical conditions leading to HF, and we also report the epigenetic mechanisms involved both in graft outcome and in diagnostic workup during post-

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transplantation period.

EPIGENETIC MODIFICATIONS IN CARDIOVASCULAR DISEASE LEADING TO HEART

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The development of HF implies the complex regulation of gene networks including both re-activation of

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fetal genes and/or re-expression of postnatal genes and leading to cardiac remodeling [13,14]. Even in disorders that recognize important environmental causes, such as CHD and hypertension, the risk of HF depends to some extent on genetic predisposition (Fig. 1). Epigenetic modifications can contribute to exacerbate the various stage of HF, by regulating synthesis of protein structures and by increasing the activity of cardiac fibroblasts with consequences on heart contractility and hypertrophy (Table 1). It is still unclear whether DNA methylation, histone changes, and miRNA synthesis are the cause or the effect of the failing hearts, but they could also represent an ideal target for future therapeutic strategies in HF [38].

ACCEPTED MANUSCRIPT DNA Methylation The effect of DNA hyper and /or hypomethylation on the expression of genes involved in several pathways underlying CVD has been studied in various experimental models (Table 1).

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In vitro studies and animal models

In animal model, a significant global hypomethylation status was observed in aortic samples, before the clinical evidence of atherosclerosis, by supporting the hypothesis that in CVD, some aberrations in DNA

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methylation can be prelude to the manifestation of disease [39]. Several studies have focused on the effects

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in vivo and in vitro of demethylating agents as 5-aza-2′-deoxycytidine (5-AzaD). Recently, in LDL receptor knockout mice (Ldlr-/-), it has been shown that treatment with 5-AzaD reduces inflammation and downregulates genes such as tumor necrosis factor TNF, interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), besides a lowering of macrophage migration that contributes to

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regulating chemotaxis genes [15,16]. The effects of 5-AzaD inhibitors have been evaluated in human primary fibroblast cell line and in a spontaneously hypertensive rat model, by showing an antifibrotic and

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antihypertrophic action due to the lowering of myocardial collagen levels and myocyte size in vivo and the reduction of collagen I, collagen III, and α-smooth muscle actin in vitro[23]. It has been established HF is

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associated with hypermethylation-induced downregulation of different genes, including sarcoplasmic reticular ATPase and Pitx2c [30,31], which are involved in calcium homeostasis and cardiac electrical

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activity [32]. Previously, it has been demonstrated that tissue inhibitor of matrix metalloprotease 4 (TIMP4) is downregulated in volume overload-induced HF [40]; besides, a fine regulation of TIMP4 and matrix metalloprotease 9 (MMP9) was found. TIPM4 is the endogenous inhibitor of MMP9 in the heart and its overexpression can confer cardioprotection [41]. Chaturvedi and colleagues found the presence of 7 methylated CpGi in the TIMP4 promoter during the progression of HF that along with the up-regulated levels of miR-122a led to its epigenetic silencing [33]. Human studies Interestingly, despite the inherent difficulty in obtaining heart tissue from living patients and easy deterioration of these tissues, Movassagh and colleagues demonstrated a differential DNA methylation profile in human cardiac tissue from end-stage cardiomyopathic hearts and normal control hearts [24]. Particularly, using both a methylated DNA immunoprecipitation-chip (MeDIP-chip) and a bisulfite-(BS)

ACCEPTED MANUSCRIPT PCR and high-throughput sequencing, they identified three angiogenesis-related genetic loci (AMOTL2, ARHGAP24 and PECAM1) differentially methylated in left ventricular (LV) tissue from male patients compared to controls [24]. By comparing LV tissues from failed and normal human hearts, differences of

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methylation have been identified in promoter CpGi, in intragenic CpGi and in H3K36Me3 enriched regions [25]. It has been hypothesized that distinct epigenomic patterns control the expression of genes involved in the regulation of human end-stage cardiomyopathy [25]. The high density of gene mutations associated with

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the development of hypertrophic cardiomyopathy led to investigate the cardiac myosin binding protein C

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gene (MYBPC3), the gene most frequently associated with the onset of cardiomyopathies. In pooled samples of human placenta, the methylation level of the CpGi resulted to be gene-specific as demonstrated by comparison of the coding regions of MYBPC3 with those of the skeletal muscle isoform MYBPC2 [26]. Regarding dilated cardiomyopathy, Haas and colleagues observed distinct patterns of altered DNA methylation in four genes with still unclear function, such as Lymphocyte antigen 75 (LY75), Tyrosine

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kinase-type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A

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(ADORA2A) in LV heart tissue in vitro [27]. Recently, a total of 1828 differentially methylated probes have been identified by comparing the methylomes between 18 left ventricles and 9 right ventricles, with

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hypermethylated regions in severely affected LV [28]. A global analysis of gene promoter DNA methylation, performed on failing and non-failing human LV, identified changes in promoters of genes associated with

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CVD and dilated cardiomyopathy [42]. Recently, genome-wide DNA methylation analysis confirmed a differential gene methylation pattern in HF patients compared to normal controls using blood leukocytes [43]. These results strengthen the importance of evaluating DNA methylation changes in CVD and support the therapeutic use of compounds that influence the activity of epigenetic modulators such as DNMTs. Indeed, epigenetic modifiers such as DNA methylation inhibitors could be used for treating cardiac hypertrophy-associated disorders [23].

Histone Modifications In vitro studies and animal models Several in vitro and animal studies suggested the involvement of histone modifications in the onset and progression of CVD. The function of HDAC1, HDAC2 and HDAC3 has been investigated in knockout mice

ACCEPTED MANUSCRIPT that showed cardiac arrhythmias, and dilated and hypertrophic cardiomyopathy [44,45]. Previously, rats with HF showed altered trimethylation on lysine 4 or 9 of histone H3 [46]. In an elegant in vivo experiment, Papait and colleagues analyzed cardiomyocytes isolated from the LV of mice subjected to transverse aortic

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constriction (TAC) and showed different regions of the genome with changes of their histone profile. Indeed, H3K9ac, H3K4me9, H3K79me2 and H3K27me3 showed a differential distribution in hypertrophic heart [47].

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HDAC inhibitors (HDACi) possess a wide range of cytoprotective activities such as anti-inflammatory,

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antioxidant, anti-apoptotic, anti-fibrotic, and antihypertrophic properties, which are beneficial for the treatment of a variety of CVDs. In support of this hypothesis, preclinical studies demonstrated that HDACi, such as trichostatin A (TSA) and the novel MPT0E014 exert a beneficial effect on heart remodeling, thereby diminishing the emergence of HF [48, 49].

The inhibition of HDAC3, as well as the deletion of HDAC9, reduces atherogenic macrophage functions

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through an increased accumulation of total acetylated H3 and H3K9 at the promoters of ATP-binding

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cassette transporter (ABCA1), ABCG1, and peroxisome proliferator-activated receptor γ (PPARγ) [50]. However, clinical implications for the therapeutic use of histone modulating enzymes need to be further

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investigated [51].

In a model of cardiac hypertrophy, treatment with a selective HDACi, magnesium valproate, prevented

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cardiac hypertrophy, improved hemodynamic functions, and also increased survival rate [52]. Human studies

In humans, Lu et al. showed a downregulation of SIRT1, a member of a family of protein deacetylases, in hearts of patients with advanced HF [53]. In patients with abdominal aortic aneurysm, an increased expression of HDACs 1, 2, 4 and 7 was found in abdominal aorta samples compared with those from donors [54]. These finding are coherent with the recognition of the fundamental role of reversible protein acetylation in the pathological remodeling of the failed heart.

ACCEPTED MANUSCRIPT MiRNA In vitro studies and animal models Increasing evidence shows that the expression of specific mRNAs is strongly correlated with the regulation

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of genes involved in onset and progression of CVD leading to HF.

The levels of miRNAs were found to vary along with the development and progression of CVD, modulate the inflammatory response and play also an anti-hypertrophic role (Table 1)

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Many miRNAs, such as miR-195 and miR-1, change their expression pattern during the development of

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cardiac hypertrophy consequent to pressure-overload and their deregulation is an underlying cause of cellular dysfunction and disease in mouse cardiac myocytes [55,56]. Moreover, in hearts of Smad4-deleted mice, TGF-β-regulated miR-27 plays a pivotal role in the development of cardiac hypertrophy and HF and miR27b resulted significantly over-expressed in hearts from transgenic mice with cardiac hypertrophy at 3 months of age [57]. MiR-214 was over-expressed in the presence of hypertrophic cardiomyopathy and

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regulated negatively EZH2, the major histone methyltransferase of Polycomb repressor complex [55,58].

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Kim and colleagues have lately shown that miR-185 was significantly down-regulated in myocardial cells of mice subjected to TAC, indicating its substantial anti-hypertrophic role in failed heart [59].

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Regarding statin administration in case of hypertrophic hearts, atorvastatin downregulated miR-22 in mice [60]. The functional role of miR-22 in cardiac remodeling in response to stress was reported both in cardiac

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hypertrophy and dilated cardiomyopathy. In particular, miR-22 expression was related with reorganization of the sarcomeres, increased size of cardiomyocytes and LV in mouse models [61]. Human studies

MiR-214 over-expression has been observed in hypertrophic cardiac tissue of patients with valvular diseases as well as in HF mouse model, and its inhibition with antisense oligonucleotides (antagomiRNAs), restored the EZH2 expression, thus protecting heart function [62,63]. Barsanti and colleagues observed 13 upregulated and 10 downregulated miRNAs when comparing end-stage HF patients undergoing transplantation with or without a previous LVAD support [64]. The expression level of 7 of these miRNAs correlated with the improvement of cardiac index; several target genes of these miRNAs belonged to the integrin signaling network involved in the remodeling process [64]. In addition, the levels of 186 miRNAs were measured in sera of chronic systolic HF patients respect to healthy controls,

ACCEPTED MANUSCRIPT demonstrating that elevated serum levels of miR-423-5p, miR-320a, miR-22, miR-92b correlate with severe clinical prognostic parameters, being involved in remodeling process following myocardial injury [65]. In a cohort of pediatric patients with dilated cardiomyopathy, several miRNAs resulted differently expressed

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between children who recovered ventricular function and children who died or required HT. Therefore, the circulating miRNA profile of children with severe HF secondary to dilated cardiomyopathy could be a biomarker for outcome prediction [66]. MiR-155, previously shown to be a biomarker of dilated

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cardiomyopathy also in adult population, is implicated in cardiac disease and in the development of

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hypertrophy in vivo. Indeed, reduced levels of inflammation-associated miR-155 and SMC-associated miR145 were found in whole blood and plasma of CHD patients suggesting that the alteration of miR-155 levels is not limited to endothelial or vascular cells. Accordingly, CHD patients exhibit a miRNA-linked alteration in the inflammatory capacity of blood mononuclear cells [67,68].

In some circumstances, the correlation between biomarkers and miRNAs not only depends on the time of

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measurement, but also on the disease state as recently reported in patients with HF at different stage. Several

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miRNAs, identified in HF patients with a worse prognosis, seem to be linked to targets and pathways leading to cardiac disease, including cardiac remodeling/fibrosis, inflammation and angiogenesis [48,69]. As a

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whole, miRNAs are the more effective players in the modulation of key signaling elements and enzymes in

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the pathogenesis and progression of HF [70].

EPIGENETIC INSIGHTS IN HEART-TRANSPLANTED PATIENTS Although HT still represents the last chance for patients with end-stage HF, the outcome of the transplantation and, first of all, patients’ survival are closely related to several post-transplantation immune and non-immune complications such as infections, acute and chronic rejection, hypertension, kidney failure and malignancies [71]. Particularly, cardiac allograft vasculopathy (CAV), a manifestation of chronic injury, is commonly associated with graft deterioration and failure and the invasive allograft biopsy remains the main approach used for rejection surveillance. Over the years, several prognostic markers have been associated with the development of CAV, including the donor specific anti-HLA antibodies that develop despite the immunosuppressive therapies currently used in the management of transplanted patients. Among the non-immunological factors the most relevant are the

ACCEPTED MANUSCRIPT age of the donor, ischemia-reperfusion injury and cytomegalovirus infection [72]. The adoption of epigenetic mechanisms as non-invasive biomarkers for prediction and monitoring of CAV is actively investigated and their involvement in transplantation medicine needs further insight. Until now, few studies have focused on

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the role of DNA methylation, histone modification and miRNA activity in controlling and monitoring the adverse post-transplantation events as well as the immunosuppressive therapies. Furthermore, an open

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that contribute to the graft outcome [73] (Fig. 1).

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question regards the contribute of both donor and recipient epigenetic signatures in the biological processes

DNA methylation

DNA methylation plays a pivotal role in the balanced immune response versus graft, by regulating the immune cells, but a direct link between this epigenetic mechanism and rejection is partially unknown. During immune response, DNA-methylation regulates T cell differentiation as well as instability and

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plasticity which need to be considered if a therapy based on regulatory T cell is carried out [74,75]. In this

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regard, FOXP3+Treg cells play an important role in the suppression of immune response and in the induction of tolerance in organ transplantation, although their contribution in favoring the tolerance or immune

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activation in human is still unclear. Indeed, intragraft FOXP3+ T cells have been associated with graft acceptance and better graft survival in both mice and humans, but higher FOXP3 mRNA expression in

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cardiac biopsies has also been associated with rejection. Likely, the expression of FOXP3 in different cell types could explain these results [76,77]. The preservation of self-tolerance has been further demonstrated in mice models, where two methylation modifiers, 5-AzaD and TSA, modulated the expression of FOXP3 thus promoting the conversion of naïve T cells into regulatory T cells [78] (Table 2).

Histone modification In Table 2 are described some evidence of histone modification involvement in post-transplantation period (Table 2).Therapeutic manipulation of FOXP3 acetylation through HDACi could favor the development and suppressive functions of Treg cells also in experimental models of transplant rejection [79,80].

ACCEPTED MANUSCRIPT The impact of epigenetic modifications on B cell function and its involvement in antibody-mediated rejection is still to be explored. HDACi, including SAHA, are under investigation as potential anti-rejection drugs. They synergize with tacrolimus in the prevention of rejection by promoting T effector cell apoptosis

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and by favoring Treg. Moreover, SAHA treatment attenuates B cell-mediated rejection, prolongs graft survival and reduces serum levels of alloantibodies and IgG deposition [81,82]. Probably, SAHA prevents allograft rejection by balancing the Treg and Th17 ratio as well as by driving the balance towards Treg

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deviation [81]. An additional study in a mouse model of cardiac transplantation evaluated the impact of the

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HDACi, valproic acid, on proliferation, apoptosis, class switch recombination, differentiation and secretion of immunoglobulins on isolated murine B cells. Indeed, valproic acid reduced antibody generation significantly in a dose-dependent manner without altering B cell proliferation and apoptosis, thus suggesting a promising therapeutic role of this agent after organ transplantation [83]. The graft survival time could be further prolonged thanks to HDACi, such as TSA and the newly developed FR276457. This latter compound

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has been administered to a rat transplantation model alone or in combination with tacrolimus as

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immunosuppressive drugs, to improve overall survival time and to prevent the activation of T cells [84]. In humans, curcumin (a HAT inhibitor) in combination with cyclosporine A suppressed Th1 cytokines in ex

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MicroRNA

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vivo peripheral blood lymphocytes from kidney transplantation patients [85].

With the purpose of identifying new biomarkers, much attention has been focused on miRNAs due to their prominent role in the regulation of innate and adaptive immunity. MiRNAs regulate the expression of 30% of human genes, including those involved in cell proliferation, differentiation, DNA repair, metabolism, and apoptosis (Table 2). The expression profile of miRNAs is both cell/tissue/organ specific and disease-stage specific [86]. MiRNA levels are relatively stable unless cell injury or death occurs as after rejection and therefore miRNA profiling can be explored in different samples including biofluids. Several studies in animal models are still needed to translate the results into clinical setting. Recently, miR-223 was associated with rejection, differently from miR-146a, miR-15b, miR-23a, miR-27a, miR-451, miR-101 and miR-148a that were involved in tolerance process in a murine cardiac allograft. These miRNAs seem to be specific for

ACCEPTED MANUSCRIPT heart allograft because their expression pattern showed a different trend compared with hepatic allograft, perhaps for the different number of immune cells and for the tissue-specificity of miRNAs [87]. Most of the upregulated miRNAs are involved in inflammation, innate immune system and fibrosis, which

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are all specific manifestations of rejection. MiRNA profiling of human endomyocardial biopsies of patients with acute cellular rejection and murine allograft, highlighted increased levels of miR-21, miR-142-3p, miR142-5p, miR-146a, miR-146b, miR-155, miR-222, miR-223 and miR-494, and a decreased level of miR-149-

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5p. Interestingly, miR-155, with a well-established role in inflammation and immunity by targeting IL-6 and

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SPl1, has been identified as the most upregulated miRNA after rejection [88]. Moreover, miR-155 can regulate Th1/Th17-related inflammation by ameliorating the tissue injury in murine chronic cardiac rejection [89]. Overall, these results suggest that miR-155 might be considered a responsive target for a novel therapeutic approach also in the post-transplantation period. The levels of certain circulating organ-specific miRNAs might be significantly upregulated during acute rejection, but significantly downregulated during

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chronic or late rejection [90].

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Increased levels of miR-21, together with cardiac fibrosis, were found in murine hearts after transplantation as well as in patients with cardiac rejection. In vitro, miR-21 was induced by IL-6 treatment and it activated a

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fibrotic gene programming by promoting monocyte-to-fibrocyte transition. In addition, inhibition of miR-21 in vivo successfully reduced fibrosis and fibrocyte accumulation in cardiac allografts [91]. MiRNA

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expression profiling in patients with acute biopsy-proven allograft rejection compared with control patients without rejection, both in heart allograft tissue and in serum, highlighted 7 miRNAs differentially expressed, namely miR-10a, miR-21, miR-31, miR-92a, miR-142-3p, miR-155, and miR-451. In particular, miR-10a, miR-31, miR-92a, and miR-155 strongly discriminated patients with allograft rejection from patients without rejection and showed a differential serological expression that correlated with their tissue expression. As reported, these four miRNAs are specific for inflammatory burdens in EC, inflammatory pathways, cardiomyocytes/interstitial cells and EC, respectively [92]. A cross-sectional study on 52 patients undergoing coronary angiography between 5 and 15 years after HT, showed that circulating levels of endotheliumenriched miR-21-5p, miR-92a-3p, miR-126-3p, and miR-126-5p were almost two-fold increased in patients with CAV than patients without CAV [93].

ACCEPTED MANUSCRIPT In the attempt to identify miRNAs as biomarkers for CAV monitoring, a microarray analysis reported miR34a, miR-98, miR-155, miR-204, miR-628-5p as differentially regulated in plasma of patients with CAV. In particular plasma levels of miR-628-5p and miR-155 were significantly increased and a miR628-5p value

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above 1.336 predicted CAV with a sensitivity of 72% and a specificity of 83% [93].

These results support a differential expression of miRNAs not only at the tissue level, but also in serum and

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plasma, thus confirming their potential relevance as non-invasive biomarkers in HT rejection [92,94].

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CONCLUSIONS

Epigenetic mechanisms have a role in the control of the pathological processes occurring at cardiac level thus, their alteration often results in an impaired cardiac function. Several evidences point out the involvement of the different epigenetic modifications in the onset and progression of CVD leading to advanced HF. Concerning the post-transplantation period, few researches so far proved the direct

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involvement of epigenetics in the outcome of HT; accordingly, this field remains largely unexplored (Fig. 1).

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While waiting for a breakthrough in regenerative medicine to treat CVD [95], patients suitable for HT have a poor survival prognosis in the absence of any feasible alternative therapeutic treatment and the outcome of

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HT depends on several post-transplantation immune and non-immune complications. Major efforts are now directed toward the identification of new non-invasive and reliable biomarkers for the prediction of allograft

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rejection and for monitoring immunosuppressive therapy. Differently from other pathological conditions affecting the cardiovascular system [96], the current invasive endomyocardial biopsy represents still a diagnostic procedure not free from life-threatening complications for patients who develop heart insufficiency with reduced LV function. The mechanism of epigenetic regulation is not an independent actor, rather it is involved in a complex network of information exchange implicated at the transcriptional level. In this regard, much attention has been focused on miRNAs, because the levels of several circulating organ-specific miRNAs seem to be fluctuating over the time during acute or chronic rejection both in biological fluids and biopsies. The levels of circulating miRNAs are specific of organ/tissue transplanted, and influenced by organ/tissue mass as well; therefore, a panel of miRNAs rather than only one could be useful to cover the widest range of organ or tissue specificity for rejection diagnosis or for early prediction of the evolution of cardiomyopathy.

ACCEPTED MANUSCRIPT This approach is further confirmed by some ongoing clinical trials that analyze the expression of different miRNAs in heart transplanted patients; those miRNAs involved in the mechanisms of rejection might then be inhibited by the use of antagonists to stop or limit the damage (Table 3) [ https://clinicaltrials.gov ].

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The multi-faceted field of transplantation medicine should be boosted to maximize the donor pool, enhance organ quality, overcome the life limiting complications of immunosuppression and infections, optimize organ allocation. However, a deeper understanding of the mechanisms of immunological tolerance and of the

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epigenetic tags implied in the balance of the immune response toward the graft, could allow the identification

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of patients at risk of rejection with respect to patients who develop tolerance. Epigenetic modifications have the undeniable advantage of being reversible by using pharmacological treatment. Indeed, in the clinical setting, epigenetic drugs are currently used in the treatment of several disease, mostly cancer, but preliminary data on animal models suggest that these drugs, in combination with immunosuppressive therapies, prolong graft survival in cardiac and renal recipients. It would be advisable to

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overcome the model of “one therapy/all patients” and further in-depth studies will evaluate if epigenetic

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mechanisms can be used for designing a more tailored therapy and as a valid strategy to modulate the balancing between tolerance and immunity in HT. Indeed, the opportunity to modulate pharmacologically

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epigenetic targets could allow to improve the immunosuppressive therapies towards the prevention and reduction of their side-effects.

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Although on one side it would be unwise to consider epigenetic promise as the panacea for all failed hearts, on the other side epigenetic mechanisms are emerging more and more convincingly as the right key for interpreting the still unexplained processes in the pre- and post-heart transplantation.

CONFLICT OF INTERESTS The authors declare that they have no conflict of interest concerning this article.

FUNDING This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

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[59] Kim JO, Song DW, Kwon EJ, Hong SE, Song HK, Min CK, et al. MiR-185 plays an antihypertrophic role in the heart via multiple targets in the calcium-signaling pathways. PLoS One 2015;10:e0122509. [60] Tu Y, Wan L, Bu L, Zhao D, Dong D, Huang T, et al. MicroRNA-22 downregulation by atorvastatin in a mouse model of cardiac hypertrophy: a new mechanism for antihypertrophic intervention. Cell Physiol Biochem 2013;31:997-1008. [61] Huang ZP, Chen J, Seok HY, Zhang Z, Kataoka M, Hu X, et al. MicroRNA-22 regulates cardiac hypertrophy and remodeling in response to stress. Circ Res 2013;112:1234-43. [62] Thum T, Condorelli G. Long noncoding RNAs and microRNAs in cardiovascular pathophysiology. Circ Res 2015;116:751-62. [63] Yang T, Gu H, Chen X, Fu S, Wang C, Xu H, et al. Cardiac hypertrophy and dysfunction induced by overexpression of miR-214 in vivo. J Surg Res 2014;192:317-25. [64] Barsanti C, Trivella MG, D'Aurizio R, El Baroudi M, Baumgart M, Groth M, et al. Differential regulation of microRNAs in end-stage failing hearts is associated with left ventricular assist device unloading. Biomed Res Int 2015;2015:592512. [65] Goren Y, Kushnir M, Zafrir B, Tabak S, Lewis BS, Amir O. Serum levels of microRNAs in patients with heart failure. Eur J Heart Fail 2012;14:147-54. [66] Miyamoto SD, Karimpour-Fard A, Peterson V, Auerbach SR, Stenmark KR, Stauffer BL, et al. Circulating microRNA as a biomarker for recovery in pediatric dilated cardiomyopathy. J Heart Lung Transplant 2015;34:724-33. [67] Fichtlscherer S, De Rosa S, Fox H, Schwietz T, Fischer A, Liebetrau C, et al. Circulating microRNAs in patients with coronary artery disease. Circulation Research 2010;107:677-84. [68] Bronze-da-Rocha E. MicroRNAs expression profiles in cardiovascular diseases. Biomed Res Int 2014;2014:985408. [69] Vegter EL, Schmitter D, Hagemeijer Y, Ovchinnikova ES, van der Harst P, Teerlink JR, et al. Use of biomarkers to establish potential role and function of circulating microRNAs in acute heart failure. Int J Cardiol 2016;224:231-9. [70] Zhang X, Schulze PC. MicroRNAs in heart failure: Non-coding regulators of metabolic function. Biochim Biophys Acta 2016;1862:2276-87. [71] Zuckermann A, Barten MJ. Surgical wound complications after heart transplantation. Transpl Int 2011; 24: 627-36. [72] Picascia A, Grimaldi V, Casamassimi A, De Pascale MR, Schiano C, Napoli C. Human leukocyte antigens and alloimmunization in heart transplantation: an open debate. J Cardiovasc Transl Res 2014;7:664-75. [73] Picascia A, Grimaldi V, Napoli C. From HLA typing to anti-HLA antibody detection and beyond: The road ahead. Transplant Rev (Orlando) 2016;30:187-94. [74] Suarez-Alvarez B, Rodriguez RM, Fraga MF, López-Larrea C. DNA methylation: a promising landscape for immune system-related diseases. Trends Genet 2012;28:506-14. [75] Youngblood B, Hale JS, Ahmed R. T-cell memory differentiation: insights from transcriptional signatures and epigenetics. Immunology 2013;139:277-84. [76] Dijke IE, Velthuis JH, Caliskan K, Korevaar SS, Maat AP, Zondervan PE, et al. Intragraft FOXP3 mRNA expression reflects antidonor immune reactivity in cardiac allograft patients. Transplantation 2007;83:1477-84. [77] Waldmann H, Adams E, Fairchild P, Cobbold S. Regulation and privilege in transplantation tolerance. J Clin Immunol 2008;28:716-25. [78] Moon C, Kim SH, Park KS, B.K. Choi, H.S. Lee, J.B. Park, et al. Use of epigenetic modification to induce FOXP3 expression in naive T cells. Transplant Proc 2009;41:1848-54. [79] de Zoeten EF, Wang L, Butler K, U.H. Beier, T. Akimova, H. Sai, et al. Histone deacetylase 6 and heat shock protein 90 control the functions of Foxp31 T-regulatory cells. Mol Cell Biol 2011;31:2066-78.

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[80] Beier UH, Wang L, Han R, Akimova T, Liu Y, Hancock WW. Histone deacetylases 6 and 9 and sirtuin-1 control Foxp3+ regulatory T cell function through shared and isoform-specific mechanisms. Sci Signal 2012;5: ra45. [81] Zhang X, Han S, Kang Y, Guo M, Hong S, Liu F, et al. SAHA, an HDAC inhibitor, synergizes with tacrolimus to prevent murine cardiac allograft rejection, Cell. Mol. Immunol 2012;9:390-8. [82] Zhang X, Guo M, Kang Y, Liu F, Zheng X, Han S, et al. SAHA, an HDAC inhibitor, attenuates antibody-mediated allograft rejection. Transplantation 2013;96:529-37. [83] Ye J, Li J, Zhou M, Xia R, Liu R, Yu L. Modulation of Donor-Specific Antibody Production After Organ Transplantation by Valproic Acid: A Histone Deacetylase Inhibitor. Transplantation 2016;100:2342-51. [84] Kinugasa F, Yamada T, Noto T, Matsuoka H, Mori H, Sudo Y, et al. Effect of a new immunosuppressant histon deacetylase (HDAC) inhibitor FR276457 in a rat cardiac transplant model. Biol Pharm Bull 2008;31:1723-6. [85] Bharti AC, Panigrahi A, Sharma PK, Gupta N, Kumar R, Shukla S, et al. Clinical relevance of curcumin-induced immunosuppression in living-related donor renal transplantation: an in vitro analysis. Exp Clin Transplant 2010;8:161-71. [86] Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004;116:281-97. [87] Fujino M, Zhu P, Cai S, Nishio Y, Zhuang J, Li XK. MicroRNAs Involved in Acute Rejection and Tolerance in Murine Cardiac Allografts. Exp Clin Transplant 2016;14:424-30. [88] Van Aelst LN, Summer G, Li S, Gupta SK, Heggermont W, De Vusser K, et al. RNA Profiling in Human and Murine Transplanted Hearts: Identification and Validation of Therapeutic Targets for Acute Cardiac and Renal Allograft Rejection. Am J Transplant 2016;16:99-110. [89] Zhang A, Wang K, Zhou C, Gan Z, Ma D, Ye P, et al. Knockout of microRNA-155 ameliorates the Th1/Th17 immune response and tissue injury in chronic rejection. J Heart Lung Transplant 2017;36:175-84. [90] Wilflingseder J, Regele H, Perco P, Kainz A, Soleiman A, Mühlbacher F, et al. miRNA profiling discriminates types of rejection and injury in human renal allografts. Transplantation 2013;95:83541. [91] Gupta SK, Itagaki R, Zheng X, Batkai S, Thum S, Ahmad F, et al. miR-21 promotes fibrosis in an acute cardiac allograft transplantation model. Cardiovasc Res 2016;110:215-26. [92] Duong Van Huyen JP, Tible M, Gay A, Guillemain R, Aubert O, Varnous S, et al. MicroRNAs as non-invasive biomarkers of heart transplant rejection. Eur Heart J 2014;35:3194-202. [93] Neumann A, Napp LC, Kleeberger JA, Benecke N, Pfanne A, Haverich A, et al. MicroRNA 6285p as a novel biomarker for cardiac allograft vasculopathy. Transplantation 2017;101:e26. [94] Singh N, Heggermont W, Fieuws S, Vanhaecke J, Van Cleemput J, De Geest B. Endotheliumenriched microRNAs as diagnostic biomarkers for cardiac allograft vasculopathy. J Heart Lung Transplant 2015;34:1376-84. [95] Sommese L, Zullo A, Schiano C, Mancini FP, Napoli C. Possible muscle repair in the human cardiovascular system. Stem Cell Rev 2017;13:170-91. [96] Grimaldi V, Schiano C, Casamassimi A, Zullo A, Soricelli A, Mancini FP et al. Imaging techniques to evaluate cell therapy in peripheral artery disease: state of the art and clinical trials. Clin Physiol Funct Imaging 2016;36:165-78.

ACCEPTED MANUSCRIPT Legend to Fig. 1 Epigenetic mechanisms could contribute to establishing innovative diagnostic and predictive biomarkers, as

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well as ground-breaking therapies for heart failure and heart transplantation rejection. In patients with failed

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heart, for example histone methylation and miRNAs could represent a useful tool for diagnostic purpose. Furthermore, in heart transplanted patients epigenetic tags might also contribute to the balance between

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rejection and tolerance by allowing a tailored therapeutic approach.

ACCEPTED MANUSCRIPT Table 1. Some evidence of epigenetic involvement in cardiovascular disease

Epigenetic Mechanism

Study Mo dels

Target/Effects

Ref

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Cardiac Condition

15 AM

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5-aza-dC treatment downregulated expression of genes involved in inflammation and chemotaxis by attenuating macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques

DNA and histone methylation of KLF2dependent genes by LDL such as thrombomodulin, eNOS and PAI by leading to endothelial activation and dysfunction

17

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16

hypomethylation of two CpG dinucleotides and acetylation of histone 3 by LDL in the human increase in expression of endothelial intercellular adhesion molecule-1 (ICAM1) and decrease in expression of thrombomodulin (TM) by promoting proadhesive and procoagulant features

HC

ED PT CE

histone modification

AC

Atherosclerosis and Coronary Heart Disease

methylation

miRNAs

Cardiomyopathies

hypermethylation of 11 mechanosensitive genes such as HoxA5 and Klf3

methylation

18

Atorvastatin up-regulate ACE2 via epigenetic histone modifications

AM

19

Reduced global trimethylation levels of lysine 27 on histone H3 (H3K27me3) in vessels of advanced atherosclerotic plaques that could reflect a phenotype switching of SMC vessels

HT

20

miR-155 reduce the atherogenesis through a decrease of macrophage inflammation and an enhanced macrophage cholesterol efflux

AM

21

miR-125a mediates lipid uptake and decreases the inflammatory response through the lowering of several cytokines including IL-2, IL-6, TNF- and providing a posttranscriptional regulation of the proinflammatory response

HC

22

Antifibrotic and antihypertrofic action of methylation inhibitor due to the reduction of collagen I, collagen III, and α-smooth muscle

HC/AM

23

Differential DNA methylation profiles in promoter CpGi, in intragenic

HT

24,25

ACCEPTED MANUSCRIPT CpGi and in H3K36Me3 enriched regions, especially in three angiogenesis-related genetic loci (AMOTL2, ARHGAP24 and PECAM1) Differential methylation of CpG sites in two isoforms of myosin binding protein C

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Distinct patterns of altered DNA methylation in four genes whit unclear function like Lymphocyte antigen 75 (LY75), Tyrosine kinase-type cell surface receptor HER3 (ERBB3), Homeobox B13 (HOXB13) and Adenosine receptor A2A (ADORA2A)

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27

Alterations in DNA methylation the cisregulatory regions of cardiac development genes involved in ventricular development (e.g., TBX5 and HAND1)

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HF

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methylation

Histone trimethyl-lysine demethylase JMJD2A promotes cardiac hypertrophy

AM

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histone modification

28

histone modification

miRNAs

29

Hypermethylation of genes including sarcoplasmic reticular ATPase and Pitx2c, involved in calcium homeostasis and cardiac electrical activity

30,31,3 2

Different methylated 7 CpG islands in the TIMP4 promoter

33

Higher levels of HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 whose inhibition improves cardiac function by modulating the cardiac peroxisome proliferatoractivated receptors and inflammatory cytokines

34 AM/cells

Differential expression patterns of HDAC1 and 2 whose inhibition reverses interstitial fibrosis

35

Cardiac-specific overexpression of miR222 induces heart failure and inhibits autophagy in mice

36

miR-155 as an inducer of pathological cardiomyocyte hypertrophy and suggest that inhibition of endogenous miR-155 might have clinical potential to suppress cardiac hypertrophy and heart failure

37

AM: animal model; HC: human cells; HT: human tissue

ACCEPTED MANUSCRIPT

Table 2. In vivo and in vitro epigenetic modifications involved in transplantation tolerance and rejection.

SOURCE

EFFECT/ OUTCOME

REFERENCES

DNA-methylation inhibition (5AzaD)

Mouse splenic naïve T cells

Increased FOXP3 expression that promote the conversion of naïve T cells to regulatory T cells

[78]

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EPIGENETIC SIGNATURE

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HDAC inhibition (TSA)

Mouse cardiac transplantation model

Modulation of Treg suppressive activity and HSP90 acetylation

[79]

HDAC6 inhibition (Tubastatin and ACY-738)

Mouse spleens and somatic lymph nodes

Deacethylation of FOXP3 and improvement of Treg suppressive function

[80]

Mouse cardiac transplantation model

Modulation of inflammatory cytokines and enhancement of Treg function

[81]

Isolated mouse B cells

Immunosuppression of splenic B cells

[82]

HDAC inhibition (VPA)

Mouse cardiac transplantation model

Inhibition of B cells and reduced antibody generation

[83]

HDAC inhibition (FR276457)

Rat

heterotopic cardiac transplantation model

Immunosuppressive effect on T cells proliferation

[84]

MiRNAs

Mouse cardiac transplantation

up-regulated miR-223 associated with rejection and down-regulated

[87]

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HDAC6 inhibition

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HDAC9 inhibition

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HDAC inhibition (SAHA)

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Sirt1 inhibition (EX-527)

ACCEPTED MANUSCRIPT model

miR-146a, -15b, -23a, -27a, -34a, -451, -101a, -101b, and -148a associated with tolerance

up-regulated miR-21, -142-3p, -1425p, -146a, -146b, -155, -222, 223, -494 and down-regulated miR-149-5p in rejection

[88]

Mouse cardiac transplantation model

miR-155-/- resistance to rejection and weakened Th17 cells mediated inflammation

[89]

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Human EMB and serum

Plasma from cardiac transplanted patients

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Human and mouse EMB

differential expression of miR-10a, 31, -92a, and -155 in rejection

[92]

miR-34a, miR-98, miR-155, miR-204, miR-628-5p differentially regulated in patients with CAV

[93]

Increased levels of miR-210-5p, -92a3p, -126-3p, and -126-5p in patients with CAV

[94]

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5AzaD, demethylating agent 5-aza-2′-deoxycytidine; CAV, cardiac allograft vasculopathy; EMB, endomyocardial biopsies; FOXP3, forkhead box P3; HDAC, histone deacetilase; MiRNAs, micro-RNA; SAHA,

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Suberoylanilide hydroxamic acid; Sirt1, sirtuin-1; TSA, trichostatin A; VPA, Valproic acid.

ACCEPTED MANUSCRIPT Table 3. Clinical ongoing trials on miRNA expression in heart-transplanted patients.

Status

Study Type

Outcome Measures

NCT02672683

Not

Observational

Circulating miRNAs measurement in patients with rejection

No

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yet recrui ting

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Number

of enroll ed patien ts

400 (estim ated)

Completed

Randomized

Plasma miR-133b and miR-208a evaluation

60

NCT01848301

Active, not recrui ting

Interventional

Expression of miRNAs and relationship with endothelial function and intimal thickness

12

NCT02602834

Completed

Changes in miRNA levels with interval training compared to moderate training (1 week)

19

NCT03076580

Recruiting

microRNA-seq to determine the correlation of echocardiographic parameters of systolic and diastolic functional entry with circulating molecules (plasma specimen)

2000 (estim ated)

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NCT02149316

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Interventional

Observational

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ACCEPTED MANUSCRIPT

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Figure 1