The Role of MicroRNA in the Myocarditis: a Small Actor for a Great Role

Purpose of Review Myocarditis is an inflammation of the myocardium secondary to a variety of agents such as infectious pathogens, toxins, drugs, and autoimmune disorders. In our review, we provide an overview of miRNA biogenesis and their role in the etiology and pathogenesis of myocarditis, evaluating future directions for myocarditis management. Recent Findings Advances in genetic manipulation techniques allowed to demonstrate the important role of RNA fragments, especially microRNAs (miRNAs), in cardiovascular pathogenesis. miRNAs are small non-coding RNA molecules that regulate the post-transcriptional gene expression. Advances in molecular techniques allowed to identify miRNA’s role in pathogenesis of myocarditis. Summary miRNAs are related to viral infection, inflammation, fibrosis, and apoptosis of cardiomyocytes, making them not only promising diagnostic markers but also prognostics and therapeutic targets in myocarditis. Of course, further real-world studies will be needed to assess the diagnostic accuracy and applicability of miRNA in the myocarditis diagnosis.


Introduction
Myocarditis is an inflammation of the myocardium secondary to a variety of agents such as infectious pathogens, toxins, drugs and autoimmune disorders [1]. Myocarditis can self-resolve, cause sudden cardiac death, or lead to dilated cardiomyopathy [2][3][4][5][6]. The real disease burden is unknown due to the challenge of achieving a definite diagnosis in many patients [3]. Myocarditis can often initially simulate an a acute myocardial infarction with non-obstructive coronary arteries (MINOCA), which occurs in approximately 10-20% of patients with an initial diagnosis of myocardial infarction [1]. Frequently, the diagnosis of myocarditis is performed using the Lake Louise criteria at cardiac magnetic resonance (CMR), which is not yet widely available [7]. Endomyocardial biopsy is the gold standard for diagnosis, but it is not always available and employed [2]. Therefore, broadly available and accurate diagnostic means for the prompt detection of acute myocarditis are not currently available. Advances in genetic manipulation techniques allowed to demonstrate the important role of RNA fragments, especially microR-NAs, in cardiovascular pathogenesis [8]. MicroRNAs (miR-NAs) are small non-coding RNA molecules that regulate the post-transcriptional expression of genes [9]. They are epigenetic regulators of cardiac function, participate in almost all aspects of cardiac physiology and pathology, and are involved in both the etiology and pathogenesis of myocarditis [10]. Advances in molecular techniques allowed to identify myocarditis miRNAs and their mechanisms of action [11]. miRNAs in myocarditis are related to viral infection, inflammation, fibrosis, and apoptosis of myocardiocytes, making them promising diagnostic markers and prognostic and therapeutic targets in myocarditis [12]. In our review, we provide an overview of miRNA biogenesis and their role in the etiology and pathogenesis of myocarditis. Finally, some current perspectives and future directions for myocarditis management are suggested.

MicroRNA Biogenesis and Function
In recent years, the role of miRNAs, mainly in posttranscriptional regulation, was increasingly studied [12]. miRNAs are non-coding RNAs of 16-26 nucleotides and can bind to the 3′-untranslated region (UTR) of target mRNAs to control their translation [12]. Over 2600 miRNAs were studied recently in human beings, although their function was not fully understood [13]. More than 60% of human protein-coding genes are regulated by miRNAs [13]. miRNAs play a key role in all tissues and in all stages of development by regulating cell differentiation, proliferation, and survival [13]. The miRNA has an important role not only in post-transcriptional regulation but also in the epigenetic modulation of gene expression under the control of the argonaute 2 (AGO2) protein [14]. miRNAs are found in intergenic regions, introns, and polycistronic sites and can be transcribed in clusters or alone ( Fig. 1) [15]. Polymerase II transcribes primary miRNAs (pri-miRNAs) with a stem-loop structure (5′ cap and a 3′ poly A tail) like mRNAs [15]. Pri-miRNAs are cleaved by RNase III Drosha into smaller miRNA precursors of 60-70 nucleotides (pre-miRNAs), transported into the cytoplasm by exportin [16]. Here, RNase III dicer further cleaves the pre-miRNAs into short miRNA, which binds to the AGO2 protein, creating RNA-induced silencing complex (RISC) [17]. RISC binding with its 5′ end to the 3′ UTR, 5′ UTR or even to the coding region leads to post-transcriptional gene silencing by inhibition of translational expression or the mRNA degradation [18]. One miRNA influences several miRNAs, and a single mRNA can also be controlled by several miRNAs [18].

MicroRNAs and Myocarditis
In our review, we classified as intracellular the miRNAs detected in cardiac biopsies in patients with myocarditis, and extracellular the miRNAs detected in blood samples. Figure 2 resumes the miRNAs involved in myocarditis and their targets.

Myocardium MicroRNAs
miRNA-1 is the most frequent miRNA within the cardiac cell, accounting for 40% of all miRNAs and is transcribed along with miRNA-133, co-regulating myocardial cell proliferation and differentiation [19]. Indeed, miRNA-1 and miRNA-133 favor mesoderm formation from embryonic cells but have opposite roles during subsequent differentiation into cardiac muscle progenitors [19]. Furthermore, miRNA-1 and miRNA-133 play a key role in the repression of non-muscle genes during embryonic cell differentiation. miRNA-1 and 133 also play an important role in cardiomyopathies such as myocarditis [20][21][22][23]. Indeed, miRNA-1 has been shown to inhibit post-transcriptional expression of connexin 43 (Cx43), the main cardiac gap junction that modulates cardiac conduction, promoting the myocardiocyte apoptosis and triggering arrhythmias [24]. miRNA-1, miRNA-133a, and miRNA-133b also play an important role in chronic Chagas disease cardiomyopathy by regulating cyclin D1, a cell cycle regulator of cardiomyocyte proliferation [20]. In patients with inflammatory cardiomyopathy, endomyocardial miRNA-133a levels correlate with myocardial inflammation and improved left ventricular function [21]. Indeed, miRNA-133a was associated with reduced fibrosis and myocyte necrosis on endomyocardial biopsy and left ventricle functional recovery, malignant arrhythmias, and heart failure admissions during a mean follow-up of 3 years [21][22][23]. These effects of miRNA-133a are caused by repression of transforming growth factor-β 1 (TGF-β1), TGF-β receptor type II (TGF-βRII), connective tissue growth factor (CTGF), or collagen 1A1 (Col1A1) [25]. Myosin is the key regulator of muscle strength and contractility and it is expressed by Myh6, Myh7, and Myh7b, which encode related miRNAs that regulate myosin transcription and performance [26]. In adult myocardiocytes, the Myh6 gene co-expresses miRNA-208a, regulating expression of the other two myosins and their miRNAs (Myh7/miR-208b and Myh7b/miR-499) [27]. In viral myocarditis, the level of miRNA-208a was consistently increased in the acute phase compared to the subacute and resolution/chronic phases [27]. In addition, heart-associated miRNA-208b levels during the subacute phase correlated with recovery of systolic left ventricular function in the resolution/chronic phase [27].

Viral Infection-Related MicroRNAs
miRNAs associated with myocarditis include miRNAs directly involved in viral myocarditis that regulate viral replication or virulence by acting directly on the virus genome or by regulating host responses to viral infection [28]. The star strand of miRNA-10a (miRNA-10a*) could significantly up-regulate the biosynthesis of group B type 3 coxsackievirus (CVB3) through binding to the nt6818-nt6941 sequence of the viral DNA promoting the onset of viral myocarditis [29]. Furthermore, miRNA-10a* was detectable in the Balb/c suckling murine model myocardium, highlighting the influence of miRNA-10a* on CVB3 replication in the myocardiocytes [29]. In addition, miRNA-20b influences the action of zinc finger protein-148 (ZFP-148), a transcription factor that plays an essential role in regulating virus replication, by binding directly to the 3′-UTR and inhibiting its expression [30]. Instead, CVB3 activates ERK1/2 through phosphorylation of the transcription factors ETS-1 and ETS-2, resulting in overexpression of miRNA-126 [31]. Upregulation of miRNA-126 reduces the sprouty-related EVH1 domain containing 1 (SPRED1) by activating ERK1/2 resulting in a positive feedback loop [31]. In addition, miRNA-126 induces GSK-3β, which promotes virus-induced cell death, leading to viral release and virus spread [31]. Instead, miRNA-590-5p is the most highly expressed miRNA in viral vesicles, which promotes viral replication by inhibiting apoptosis through suppression of the antiviral sprouty-1 (Spry1) [32]. In fact, myocardiocytes with increased miRNA-590-5p levels were at a higher risk of infection [32]. The expression of miRNA-223-3p was reduced in the mouse model of experimental autoimmune myocarditis (EAM) compared to the wild type [33]. miRNA-223-3p inhibited pyrin domain-containing-3 (NLRP3) inflammasome formation and promoted regulatory T-cell activation by inhibiting dendritic cell (DC) activation [33]. Administration of DCs with increased expression of miRNA-223-3p was protective against EAM [33]. miRNA-21, miRNA-146b, and miRNA-155 are increased in acute viral myocarditis (VMC) and in the mouse model with CVB3 or T. cruzi myocarditis [34•, 35]. Indeed, miRNA-21 and -146b antagonists inhibit Th17 and RORγt expression and reduce myocardial inflammation [36]. Similarly, miRNA-155 antagonists alter Th17/Treg balance by reducing Th17 activation, thereby reducing myocardial inflammatory damage [37]. miRNA-98 promotes the onset of myocarditis by inhibiting IL-10 release, but at the same time, miRNA-98 acts on FAS/FASL by inhibiting myocardial apoptosis [38,39].

MicroRNAs and T cells
miRNAs play an important role in T-cell development and function. Indeed, miRNA deficiency in the early phase of T-cell formation reduces thymocyte survival, and, in the subsequent phases of development, leads to a reduction in the number of peripheral CD4 + T-cells and blocks the development of CD8 + T-cells [43,44]. Decreased miRNA expression in CD4 + T cells results in a phenotypic switch to Th1 instead of Th2, leading to a reduction in T-reg cells [45,46]. Indeed, miRNA-21 drives Th1 cell differentiation through modulation of IL-12 release, preserving the Th1/Th2 balance [47]. Downregulation of miRNA-21 induces Th1 cell differentiation, while upregulation of miRNA-21 leads to Th2 cell differentiation [48]. Furthermore, miRNA-21 enhances Th17 differentiation through SMAD-7, a TGF β pathway inhibitor [48]. Conversely, miRNA-155 favors the maintenance of Treg cells and enhances Th1-and Th17-cell-mediated inflammation [49]. Lack of miRNA-155 impairs humoral and T-cell-dependent cellular immunity as well as induces Th2 polarization [50][51][52][53]. Blanco-Domínguez et al. found a new miRNA, mmu-miR-721, as a marker of myocarditis in the mouse model, and its human homolog, has-miR-Chr8:96 [54]. Mmu-miR-721 is an upregulated miRNA in mouse models with myocarditis and is produced by Th17 lymphocytes, affecting myocardial inflammation. The diagnostic ability of the detection of hsa-miR-Chr8:96 in plasma to discriminate myocarditis from other conditions was evaluated in different cohorts of patients with different comparators, including myocardial infarction, MINOCA, and autoimmune diseases, and compared to healthy people [54]. The miRNA had a very good diagnostic efficacy in discriminating patients with acute myocarditis from those with myocardial infarction (area under the curve [AUC]: 0.93; 95% CI: 0.88-0.98), from healthy patients (AUC: 0.99; 95% CI: 0.97-1.00), and from MINOCA (AUC: 0.83; 95% CI: 0.72-0.94) together with patients with other Th17-related diseases (rheumatoid arthritis, spondyloarthritis, psoriasis, and multiple sclerosis) (AUC: 0.99; 95% CI: 0.98-1.00) [54]. miRNA maintained its diagnostic value in models after adjustment for age, gender, ejection fraction, and serum troponin level.

Extracellular Signaling
miRNAs do not only play an intracellular but also an intercellular signalling role via extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies, or as part of protein/lipoprotein complexes [55••, 56]. miRNAs can be released into circulation by active secretion or passive leak from cell membranes, thus constituting the fraction of circulating miRNAs [57,58]. These miRNAs are remarkably resistant to degradation by RNase and directly influence various physiological and pathological processes [59,60]. Circulating miRNAs in myocarditis correlate with disease severity and predict prognosis. So far, no single miRNA has been identified as a specific marker of chronic myocarditis. Corsten et al. found increased levels of miRNA-208b and miRNA-499-5p in the plasma of 14 patients with acute CMV, which correlated directly with troponin T levels [27]. Goldberg et al., in a pediatric population, showed that miRNA-21 and miRNA-208a were increased in the acute phase of VMC. Furthermore, miRNA-208a levels in the subacute phase correlated with the degree of recovery of left ventricular systolic function in the resolution phase [28].

Future Perspectives
Myocarditis is a complex disease in terms of etiology, diagnosis, and pathogenesis. Recent studies have shown that miRNAs play a key role in the pathogenesis of myocarditis and will become increasingly important in the diagnostic and prognostic management of myocarditis. Myocarditis is often diagnosed according to the Lake Louise criteria on CMR, which is not yet widely available. As the gold standard for diagnosis, endomyocardial biopsy is not always available and employed. Hence, the myocarditis diagnostic criteria should be reassessed in order to make them widely available to centers that do not have access to CMR and endomyocardial biopsy. Although most miRNAs suffer from non-specificity for myocarditis, the mi-RNA has-miR-Chr8:96, discovered by Blanco-Dominìguez et al., and is the first miRNA discovered so far to be specific for myocarditis compared to myocardial infarction, MINOCA, autoimmune inflammatory disease, and healthy subjects [54]. Therefore, has-miR-Chr8:96 might represent a true new diagnostic test for myocarditis that is also accessible to centers without CMR and endomyocardial biopsy that could be included in the diagnostic workup of myocarditis. Of course, further studies will be needed to assess the diagnostic accuracy and applicability of miRNAs in the diagnosis of myocarditis in the real world. Furthermore, there may be numerous obstacles to the widespread use of miRNAs for the diagnosis of myocarditis, such as cost, local expertise, and the impossibility of analysis in all laboratories. The advantage of inter-hospital networks for miRNA analysis could be the solution by allowing secondary centers to take a blood sample to be sent to a reference center for miRNA analysis (Fig. 3). Indeed, miRNA assessment can be performed either on patient' s plasma or serum and biological samples can be easily collected locally and stored at − 80 °C until the shipping to the referral lab. No specific preparation of samples is necessary, and the results can be available in few days. It can be hypothesized that a blood sample will be collected as soon as the clinical suspicion of myocarditis is done and immediately shipped to the lab, so that the result will be available soon after patient admission to the hospital.

Conclusions
The recent literature has highlighted the important role that miRNAs play in the pathology of myocarditis and their possible role in diagnosis and prognosis. However, the precise and accurate roles of miRNAs in myocarditis have not yet been fully elucidated. Guidelines on the most appropriate design and analysis methods to increase the replicability of studies on circulating miRNAs are scarce. Likewise, more multicenter studies on large cohorts are needed to confirm the reliability of miRNAs as diagnostic and prognostic markers that would allow for more evidence than the preliminary data we currently have.
Funding Open access funding provided by Università degli Studi di Roma La Sapienza within the CRUI-CARE Agreement.

Conflict of Interest
The authors declare no competing interests.

Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.
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