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The role of long noncoding RNAs in atrial fibrillation
Journal Pre-proof The Role of Long Non-Coding RNAs in Atrial Fibrillation Savalan Babapoor-Farrokhran, M.D., Deanna Gill, M.D., Roozbeh Tarighati Rasekhi, M.D. PII:
S1547-5271(20)30028-X
DOI:
https://doi.org/10.1016/j.hrthm.2020.01.015
Reference:
HRTHM 8251
To appear in:
Heart Rhythm
Received Date: 20 November 2019 Accepted Date: 14 January 2020
Please cite this article as: Babapoor-Farrokhran S, Gill D, Rasekhi RT, The Role of Long Non-Coding RNAs in Atrial Fibrillation, Heart Rhythm (2020), doi: https://doi.org/10.1016/j.hrthm.2020.01.015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of Heart Rhythm Society.
The Role of Long Non-Coding RNAs in Atrial Fibrillation Short title: Long Non-Coding RNAs in Atrial Fibrillation
Savalan Babapoor-Farrokhran, M.D. 1#* Deanna Gill M.D.2#, Roozbeh Tarighati Rasekhi, M.D. 2
1
Departments of Cardiology, Albert Einstein Medical Center, Philadelphia, PA, 19141, USA 2 Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA # Savalan Babapoor-Farrokhran and Deanna Gill contributed equally to this work.
*Correspondence: Savalan Babapoor-Farrokhran, M.D. Department of Cardiology, Albert Einstein Medical Center 5501 Old York Road, Philadelphia, PA 19141, United States Email: [email protected]
Conflict of Interest: The authors have no conflicts to disclose.
Word count: 5994
Babapoor-Farrokhran et al., 1
Abstract Atrial Fibrillation (AF) is a common arrhythmia with serious clinical sequelae, but despite its significance little is known about the genetic origins. Recently, the untranscribed 98% of the human genome has been increasingly implicated in important processes like cardiac organogenesis, physiology, and pathophysiology. Specifically, long non-coding RNAs (lncRNA) have been shown to interact with the transcriptome in various ways that alter gene expression. Previously, multiple lncRNAs have been identified in disease processes such as heart failure, coronary artery disease, diabetes, and more. Multiple studies now show lncRNAs are involved in each fundamental mechanism leading to the development of AF including structural remodeling, electrical remodeling, renin angiotensin system (RAS) effects, and calcium handling abnormalities. The altered expression of lncRNAs offers genetic targets for the diagnosis and treatment of AF. In this article, we discuss the role of lncRNAs in atrial fibrillation and its pathogenesis.
Keywords: Atrial fibrillation, long non-coding RNAs, cardiovascular disease, structural remodeling, electrical remodeling, renin angiotensin system effects, calcium handling abnormalities.
Introduction Atrial Fibrillation (AF), the most common arrhythmia of clinical significance, affects almost 3 to 5 million people in the United States.1, 2 The number of affected patients is expected to reach more than 8 million by the year 2050.3 AF increases the risk of stroke, heart failure, morbidity,
Babapoor-Farrokhran et al., 2
and contributes to a 2-fold increase in mortality. It is associated with a higher medical cost and reduced quality of life, leaving a heavy burden on the health care system.4 Long noncoding RNAs (lncRNAs) are characterized as a group of RNAs >200 nucleotides that are 5’capped and 3’ polyadenylated. lncRNAs do not code for a functional protein, but they regulate gene expression.5 lncRNAs are conserved within lineages but not between lineages, and many lncRNAs conserved among primates that are not conserved with rodents.5,6,7 LncRNAs play important functional roles and they have been involved in different biological processes such as epigenetic regulation, imprinting, cell stability, cell-cycle control, cell differentiation, splicing, nuclear/cytoplasmic trafficking, and transcription/translation. Due to their involvement in different aspects of biological pathways, lncRNAs may explain lineage differences in development.7,8 Various targeted therapeutics have been introduced for restoring and/or maintaining sinus rhythm in AF. These therapeutic approaches are mainly limited by their side effect profiles and pro-arrhythmic effects and/or by their procedure-related complications. These limitations have necessitated identification of newer therapeutic targets to expand the treatment options. Despite extensive research focused on the pathogenesis of AF, a thorough understanding of various pathways mediating initiation and propagation of AF still remains limited. Research efforts focused on the identification of these pathways and molecular mediators have generated a great degree of interest for developing more targeted therapies. Recently, efforts have been made to understand the link between heart disease and novel non-coding RNAs. Among multiple noncoding RNAs, lncRNA has emerged as a novel therapeutic target in cardiovascular medicine and potentially atrial fibrillation. Multiple studies have explored the role of lncRNAs in cardiovascular disease. But there are few studies focusing on their role in atrial fibrillation. Babapoor-Farrokhran et al., 3
In this article, we discuss the role of lncRNAs in atrial fibrillation and its pathogenesis. Furthermore, we discuss the lncRNAs that are involved in atrial fibrillation and might be a therapeutic target for the treatment and control of this disease.
Junk DNA or Master of the Genome? Since the discovery of the human genome, the advent of new technological advancements and techniques have enabled scientists to explore the human genome in detail and study its implication in the pathophysiology of diseases. In the early 1960s, it was predicted that the human genome consists of at least two million protein-coding genes. However, the introduction of the Human Genome Project dropped this number to 20,500 coded genes, that is only about 2% of the genome.9 The other 98% consists of non-coding genes and was labeled as “genetic junk” or “transcriptional noise” with the thought being that they are not involved in cellular function. Therefore, scientists have ignored these non-coding regions. But non-coding RNAs (ncRNAs) transcribed from these regions are recently emerging as important regulators of cellular processes with many implications in human diseases.10 In fact, the biggest genomic difference between our genome and that of the nematode lies within our non-coding DNA, with the number of coding genes in the nematode approaching that of a human. As we progress from the genome of prokaryotes to humans, it is the non-coding DNA that has been evolving and growing in size from <25% to 98.5% of the genome.11
Mechanisms of LncRNA Functions
Babapoor-Farrokhran et al., 4
According to ncRNA classification, lncRNAs are more than 200 base pairs in length. They are transcribed by RNA polymerase II, polyadenylated, and subjected to splicing. LncRNAs also have promoter structures and are pervasively expressed across the genome.9 LncRNAs are classified into five subclasses, which include: intergenic, intronic, sense overlapping, anti-sense, and bidirectional lncRNAs. Each subclass is categorized by the genomic location of lncRNAs in relation to their target encoding regions.10 Given the variation in location, lncRNAs can act in cis or in trans, and through a multitude of mechanisms. For example, lncRNAs can upregulate transcription by binding transcription factors and targeting them to promoter regions of specific genes by using sequences complementary to the promoter DNA. Alternatively, lncRNAs may use complementary sequences to mRNA to decreases translation by sequestration of the mRNA.12 LncRNAs can sequester other proteins such as miRNAs, giving variable effects on translation and transcription. An example of this is Cardiac Apoptosis Related LncRNA (CARL), which binds miRNA539, whose downstream target gene is prohibitin-2 (PHB2), an apoptosis-inhibitor. MiRNA-539 typically suppresses PHB2, so when CARL sequesters miRNA-539, PHB2 is transcribed and translated into proteins that inhibit cardiac myocyte apoptosis.13 In addition, lncRNAs play a large role in epigenetics, with many of the RNA’s protein partners being chromatin modifiers.14 Aside from gene transcription and translation, lncRNAs have been implicated in the role of cellular stability, for example, by binding and stabilizing the enzyme, telomerase, which is responsible for adding telomeric repeats back to our chromosomes.15
LncRNA in cardiovascular development, physiology, and disease
Babapoor-Farrokhran et al., 5
The heart is the first organ to function in vertebrates. It is becoming more evident that lncRNAs play important roles in its organogenesis. Specifically, recent research has shown that they are involved in cardiac development as key mediators in the transition from mesoderm to cardiac myocytes. Their role in gene regulatory networks during cardiogenesis is only just beginning to be discovered, but important players have already been identified. Possibly, the most well studied example of this is a lncRNA named Braveheart, first identified by Klattenhoff et al. in mice. Through embryonic stem cell differentiation strategies, they determined the role of Braveheart in the progression and maintenance of cardiac myocytes from mesoderm. Klattenhoff’s team found that Braveheart-depleted cells had altered expression in 548 genes compared to controls. One of the genes encodes for a transcription factor involved in cardiac myocyte differentiation, called Mesoderm Posterior 1 (Mesp-1). Mesp-1 is expressed transiently in mesoderm cells that are destined for cardiac lineage.
They also found that Braveheart
interacts with protein complexes involved in epigenetic changes during cardiac myocyte differentiation.16 After cardiogenesis, lncRNAs have ongoing importance for cardiac physiology, specifically with cardiac contractility. Myosin is made up of two myosin heavy chain (MHC) components and four myosin light chain (MLC) components. Two isoforms of MHC exist in humans, alpha and beta. A study by Luther et al. demonstrated that antisense lncRNA exists for both MHC isoforms. In vitro analysis demonstrated that MHC translation was inhibited by antisense lncRNA.17 Therefore, the levels of antisense lncRNA alters the ratio of alpha to beta MHC in cardiac myocytes, which determines the energy efficiency of cardiac contractility. High levels of alpha MHC suggest stronger contractility whereas higher levels of beta MHC is associated with slower and more efficient contractility.
Babapoor-Farrokhran et al., 6
The presence of these antisense
lncRNAs is associated with rapid switching between MHC isoforms through opposing regulation, where the expression of one leads to repression of the other.16 Many of the lncRNAs involved in normal development and physiology mentioned above have also been implicated in pathophysiologic states. Haddad et al. found that diseases such as hypothyroidism and diabetes are associated with isoform switching from predominantly alpha MHC to predominantly beta MHC. In hypothyroidism specifically, the isoforms switch back to mostly alpha MHC with thyroid hormone replacement therapy.18 Further, they also have been connected to genes that confer increased susceptibility to coronary artery disease,19 myocardial infarction,20,
21, 22
and myocardial hypertrophy.23 Specifically, for myocardial infarction, the
lncRNA ZFAS1 has been shown to lead to impaired contractility after myocardial insult. ZFAS1 binds to a sarcoplasmic reticulum calcium transporter, SERCA2a, thereby inhibiting its function leading to decreased calcium release from the sarcoplasmic reticulum (SR) and thus impairing cardiac contractility.24
lncRNAs in Atrial Fibrillation Evidently, lncRNAs have been implicated in many diseases of the heart yet little is known about lncRNA’s role in atrial fibrillation (AF). As mentioned previously, non-coding RNAs (ncRNAs) constitute nearly 98% of human transcripts and have the potential to provide novel insight into the pathogenesis of AF. The fundamental mechanism of AF is complex but can be reduced to four main processes including: structural remodeling (in particular atrial fibrosis), electrical remodeling, Ca2+ handling abnormalities, and effects of the renin angiotensin system (RAS) .25 Many studies have reported that lncRNAs function importantly in the pathogenesis of AF by regulating critical pathways or core proteins. Initially, multiple reports revealed that a number of Babapoor-Farrokhran et al., 7
lncRNAs were differentially expressed in atrial fibrillation.26,27,28,29 Mei et al., reported differentially expressed lncRNAs in the left atrial tissue of patients with rheumatic heart disease and atrial fibrillation. They demonstrated that 182 lncRNAs were either up-regulated or downregulated, reported as greater than 1.5-fold change, in AF patients compared to patients with sinus rhythm.28 Ruan et al. examined the lncRNA expression profiles of atrial tissues by microarray analyses in AF patients and compared them to AF free patients. They discovered 219 differentially expressed lncRNAs. Randomly, they selected 5 upregulated lncRNAs and 5 downregulated lncRNAs to be validated by real time quantitative PCR (qRT-PCR), and they confirmed that 4 out of 5 for both the upregulated and downregulated lncRNAs were correlated with AF associated genes. It was postulated that the lncRNAs identified in this study initiate AF by promoting atrial electrical remodeling and changes in the RAS. The atrial electrical remodeling takes place primarily through calcium signalling. The RAS pathway promotes AF by inducing atrial fibrosis via mitogen-activated protein kinase, but also through electrical remodeling by shortening the atrial effective refractory period.26 Both of these mechanisms will be discussed in further detail later. Similarly, Xu et al. found 177 lncRNAs differentially expressed in patients with AF compared to controls. Additionally, they found mRNAs that were also differentially expressed. Further, they investigated the co-expression pathways of the mRNA and lncRNA, discovering three transcription regulatory elements that play critical roles in the expression of lncRNAs in AF including GATA1, TAF7, and EBF1.27 Both GATA1 and TAF7 already have proven roles in the molecular pathways leading to AF.30,31 Additionally, Ke et al. did a similar study that showed differential expression of lncRNAs in the left and right atria in patients with atrial fibrillation. They linked a gene called Heat Shock Factor 2 (HSF2), an established player in hypertension induced heart failure,32 to the pathogenesis of AF. They Babapoor-Farrokhran et al., 8
identified two lncRNAs, RP11-99E15.2 and RP3-523K23.2, that interacted with HSF2 and its downstream proteins.33 Chen et al. went a step further and compared lncRNA from the left atrial appendage to the left atrial tissue surrounding the pulmonary veins, a common origin of AF. They noted that lncRNAs that had altered expression in the area surrounding the pulmonary veins interacted with genes involved with mitochondrial energy production including Cyp450 and the electron transport chain.34 Although the correlation of the lncRNAs with transcription factors implicated in AF was strong, the actual molecular relationship between these lncRNAs and TFs had yet to be elucidated. Future studies would solidify the interactions of lncRNAs and AF associated genes. As mentioned previously, lncRNAs studied in other diseases act in a variety of ways to alter gene expression. It appears that for AF the most common mechanism of action is for lncRNAs to “sponge” or sequester their targets, usually miRNA, so that they cannot bind to their target gene or transcript. This “competition” between lncRNAs and miRNAs has led to some researchers calling lncRNAs Competing Endogenous RNAs or ceRNAs.35 The initial work that suggested the involvement of lncRNAs in the development of AF and described the molecular mechanism of how lncRNAs function established a foundation that allowed researchers to pinpoint specific targets of lncRNAs and correlate them to the pathogenesis of AF. Our review will touch on each of the fundamental mechanisms of AF development mentioned above and relate them to lncRNAs that have identified involvement.
Structural remodeling To begin, the hallmark of structural remodeling is atrial fibrosis characterized by abnormal proliferation of fibroblasts and excessive extracellular matrix deposition.36 Structural remodeling Babapoor-Farrokhran et al., 9
is a multifactorial process but miRNAs and eventually lncRNAs were identified that interact with factors integral to the development of atrial fibrosis and thus atrial fibrillation such as TGFβ-1.37 While the TGFβ-1/SMAD pathway is most commonly cited for the development of fibrosis, other pathways reliant on lncRNAs are being discovered. For example, Huang et al discovered that a specific microRNA, miRNA-21, was upregulated in mice that underwent sterile pericarditis and experienced atrial fibrosis and atrial fibrillation. This method was meant to mimic post-operative atrial fibrillation in humans, and when the levels of miRNA-21 were decreased through inhibition of specific transcription factors, both the duration of AF and probability of AF induction were decreased.38 While not clearly related to atrial remodeling, later work revealed a likely connection between the pericardium and the development of atrial fibrosis. Zhao et al. analyzed the expression profiles of lncRNA in the epicardial adipose tissue of patients with AF and without. The epicardium, the inner layer of the pericardium, has been implicated as a key player in atrial remodeling, atrial fibrosis, and therefore AF through the action of overlying adipose tissue. There is compelling evidence that lncRNAs secreted from the epicardial adipose tissue passively diffuse to the neighboring myocardium and are modulators of atrial remodeling.39 Wu et al. found, via trichrome staining, that patients with rheumatic valvular disease and atrial fibrillation had increased amounts of adipose tissue on their myocardium. Western blot of this fat demonstrated increased levels of fibrosis-related proteins including TGFβ-1 and Smad2.40 Zhao’s analysis found that 57 lncRNAs were differentially expressed between the 6 AF patients and 6 sinus rhythm patients.39 To confirm, 6 lncRNAs were randomly selected by QT-PCR. Multiple lncRNAs were connected with genes that have proven roles in the pathogenesis of cardiac remodeling and their effect is not trivial. In fact, co-expression analysis showed that one of the identified upregulated lncRNAs was associated with the top five downBabapoor-Farrokhran et al., 10
regulated protein-coding genes. The genes include PDLIM1, NOS3, TTC3, and SP1 which correlate with processes such as inflammatory signaling, lipid metabolism, and TGFβ-1-induced epithelial-mesenchymal transition. Cao et al. determined the specific interaction between lncRNA and SP1. Their research discovered that a lncRNA, plasmacytoma variant translocation 1 (PVT1), binds to miR-128-3p which usually acts as an inhibitor of SP1, which is an activator of TGFβ-1. However, PVT1 acts as a sponge to miR-128-3p, so that SP1 is free to activate TGFβ-1 leading to atrial fibrosis. To confirm, human atrial fibroblasts were transfected with either overexpressed or silenced PVT1. Fibroblasts with overexpressed PVT1 showed increased TGFβ-1 signaling proteins as well as increased collagen I and II. Conversely PVT1 knockdown fibroblasts demonstrated the opposite effect.37 A downstream target of TGFβ is AKL5, a wellknown promoter of cellular proliferation in the field of oncology.41 Recently it has also been shown to have a role in the proliferation of myocardial fibroblasts. A lncRNA known as Growth Inhibitory Specificity 5 (GAS5) inhibits ALK5 in the myocardium. In cells transfected with overexpressed GAS5 cell growth was inhibited and vice versa in GAS5 knockdown transfected cells.42 As mentioned above, inflammatory signaling also plays a role in the development of atrial fibrosis, specifically macrophages. Macrophages exist as one of two phenotypes. M1 macrophages are the first to arrive to damaged sites and facilitate the clearance of necrotic debris. M2 macrophages promote tissue healing. Studies have shown that the inhibition of M1 and promotion of M2 prevent cardiac remodeling and improved function.43 Sun et al. demonstrated that a lncRNA suppresses a gene known as Nuclear Factor of Activated T cells (NFAT). The lncRNA, Non-coding Represser Of NFAT (NRON) is normally recruited to the promoter region of IL-12 in atrial myocytes, which causes the phenotype switch of macrophages from M2 to M1 thus facilitating atrial fibrosis. In this study, NRON was shown to inhibit nuclear Babapoor-Farrokhran et al., 11
transport of NFAT and thus decreasing expression of IL-12 which resulted in decreased M1 phenotype macrophages and finally diminished myocardial fibrosis.43 Also recognizing the importance of the immune system in the development of AF, Yu et al. collected lymphocytes from 6 patients with AF and 6 patients in SR. Via qRT-PCR and Gene Ontology (GO) analysis, co-expression networks were discovered between mRNAs and lncRNAs that were upregulated in the AF patients. These networks were tied to Tumor Necrosis Factor (TNF) signaling pathway, Toll Like Receptor (TLR) signaling pathway, and NF-kappaβ signaling pathway. Collectively, this linked lncRNAs from systemic lymphocytes to processes such as oxidative stress, inflammation, apoptosis, and collagen synthesis, all of which have known roles in the development of atrial fibrosis.44
Electrical Remodeling Although not as robust as structural remodeling, lncRNAs have also been implicated in electrical remodeling. It has been established that electrical remodeling occurs primarily through shortening of the atrial effective refractory period and action potential duration. The Paired-like homeodomain transcription factor 2 (PITX2) gene is involved in the development of both human and murine hearts. Specifically, the transcription factor is localized to the left atrium of the heart. Holmes et al. showed that mice with reduced levels of PITX2 are predisposed to developing AF. The specific function of PITX2 is elusive, but preliminary analysis suggests it is involved in the regulation of ion channel genes and reduced PITX levels are associated with shortened atrial effective refractory periods.45 Eventually Gore et al. identified an lncRNA upstream of PITX2 known as PITX2 Adjacent Non-Coding RNA (PANCR.) PITX2 and PANCR have coordinated increased expression in cardiomyocytes of the left atrium during stem cell differentiation, and Babapoor-Farrokhran et al., 12
PANCR knockdown models showed associated downregulation of PITX2. Unlike previously mentioned lncRNAs that act by “sponging” target mRNAs via complementary sequences, PANCR and PITX2 do not share any areas of complementarity and therefore work through a different, but unknown, mechanism.46 This study did not correlate the effects of PANCR and PITX2 to the development of AF but taken together with the study of Holmes et al. it appears PITX2 is directly involved with the pathophysiology of AF and is regulated by a lncRNA, PANCR.44,45 In a study on AF and non-AF rabbits, Li et al. found that a lncRNA, TCONS_00075467, acts by “sponging” miR-328 similar to how PVT1 acts on miR-128-3p. In vitro silencing of TCONS_00075467 resulted in shortening of the atrial effective refractory period and action potential. It appears that the lack of TCONS_00075467 binding to miR-328 causes the dysregulation of CACNA1C, an L-type calcium channel, and electrical remodeling.47 CACNA1C also appears to be involved in the development of AF through the Renin Angiotensin System (RAS).
Renin Angiotensin System Effects Activation of RAS in hypertension and heart failure results in elevation of left atrial pressure via angiotensin II (AT-II). Increased pressure can cause left atrial dilation which leads to alterations in ion-channels, the hallmark of electrical remodeling. Prolonged activation of RAS also results in high myocardial tissue levels of angiotensin converting enzyme (ACE) and increased density of AT-II receptors triggering inflammation and fibrosis, or structural remodeling.48 Shen et al. found that a lncRNA, KCNQ1 overlapping transcript 1 (KCNQ1OT1), was upregulated in mice hearts with AT-II induced AF. KCNQT1OT1 binds to miR-384, which usually binds to CACNA1C much like miR-328. Since increased KCNQT1OT1 means increased “sponging” of Babapoor-Farrokhran et al., 13
miR-384, this leads to upregulated CACNA1C, which as mentioned above is associated with the development of AF.48 RAS also interacts with the autonomic nervous system (ANS) which has been heavily implicated for its role in both the development and maintenance of AF. Increased sympathetic tone has been recorded in patients before they developed: post operative AF, atrial flutter, and paroxysmal AF during sleep.50 This role of the RAS is sometimes classified as neural remodeling. Yuzong et al. discovered over 500 differentially expressed lncRNAs in canines with AF and isolated two, TCONS_00032546 and TCONS_00026102, implicated in neural remodeling.51 It appears that the RAS can mediate the development of AF independently, via neural remodeling, but also by working through both electrical and structural remodeling.
Calcium Handling Abnormalities Finally, calcium handling is the fourth and final, fundamental mechanism of AF pathogenesis our review will discuss. Calcium handling abnormalities pertains to the dysregulation of calcium storage and release in cardiac myocytes. Very recently, a study by Wang et al. found a relationship between lncRNA, Ryanodine receptors, and AF development. Specifically, the dysregulation of Ryanodine Receptor-2 (RyR2) is thought to lead to inappropriate calcium release and the onset and progression of AF.52 RyR2 is regulated by Junctophillin-2 (JP2), a signaling protein involved in electrical coupling of the sarcoplasmic reticulum. The ratio of JP2:RyR2 has been shown to be reduced in patients with AF, emphasizing the stabilizing effect of JP2 on RyR2. JP2 levels can be decreased by miRNA-24, but miRNA-24’s competitor is lncRNA-LINC00472 which has been shown to downregulate miRNA, therefore upregulating JP2 resulting in stabilized RyR2. LncRNA-LINC00472 contains a CpG island in its promoter region and methylation of this region is inversely correlated with lncRNA-LINC00472 levels. In Babapoor-Farrokhran et al., 14
this new study, miRNA-24 and lncRNA-LINC00472 levels in 125 AF patients were compared to 168 healthy controls. The results showed that the AF patients had higher levels of miRNA-24 and lower levels of lncRNA-LINC00472 compared to the controls. They also found that DNA methylation in the CpG regions of the lncRNA-LINC00472 promoter was more prominent in AF patients compared to the controls.
Discussion Hundreds of lncRNAs with altered expression have been identified in patients with AF. LncRNA interactions with AF associated genes and transcripts are getting stronger by the day. Research so far suggests that lncRNAs are involved with each of the 4 major mechanisms of AF pathophysiology including structural remodeling, electrical remodeling, RAS effects, and calcium handling abnormalities. Structural remodeling has the most robust evidence for lncRNA involvement so far. Cytokines and interleukins such as TGFβ-1 and IL-12 are both major players in structural remodeling and multiple studies have linked them to differentially expressed lncRNAs in patients and animals with AF. LncRNAs are also associated with electrical remodeling e.g. through alterations of ion channels. Finally, inappropriate release of calcium into cardiac myocytes has been a studied cause of AF, and lncRNAs that alter the stability of RyR2 through JP2 have been identified. As of now, these lncRNAs have no clinical significance for patients with AF. However, clinicians and scientists are becoming increasingly aware of how important AF biomarkers are for the diagnosis, risk stratification, treatment, and prognosis after treatment. Even though about 6% of people over 65 are diagnosed with AF, and there are likely even more asymptomatic patients that do not present, there are currently minimal screening tools for physicians to use. Babapoor-Farrokhran et al., 15
Especially at risk are patients with paroxysmal asymptomatic AF, who may or may not be in AF when routine electrocardiographs (ECGs) are performed by their cardiologist or primary care physicians. Of course, the most devastating complication of AF is stroke, and recently researchers have been looking into biomarkers such as brain natriuretic peptide (BNP) and troponin to stratify stroke risk in patients with AF.53 However, as one could imagine the sensitivity and specificity of these biomarkers is low and therefore would be inefficient and unpredictable screening tools. Many of the lncRNAs linked to AF discussed in this paper have been isolated in the left atrium and some have even been isolated specifically in the pulmonary vein region. These studies suggest a promising role for lncRNAs for possible detection and screening tool for AF. LncRNAs can also be targets for treatment in patients with AF. In lung and cervical cancers, a new treatment is being developed called Antisense Oligonucleotide (ASO)-based therapy. ASO therapy has fewer off-target effects and higher specificity while weakening malignant phenotypes via cell-cycle arrest.54 Sugihara et al. utilized ASO-based therapy to reduce Creactive protein levels in patients with AF with the primary endpoint of change in AF burden. Although this study did not achieve its endpoint, it demonstrated a favorable safety and tolerability profile for this novel therapy.55 Dozens of studies have now been published on lncRNAs as they relate to AF. However, little has been done by way of using them in clinical practice. LncRNAs would make fantastic diagnostic tools, but little is known about their behavior in patients with AF and further studies need to be done in order for them to be used as a diagnostic tool. Given how little we know about their behavior in the body, treatment is even further away. However, they show extraordinary promise for the future of how clinicians manage patients with AF and those at risk of developing AF. Babapoor-Farrokhran et al., 16
Their progressing roles in the diagnosis and treatment of cancers as well as other diseases offers much to be excited about pertaining to their role in AF.
Conclusion:
LncRNAs are involved in cardiac development, physiology, and pathophysiology. In the last decade, several lncRNAs with altered expression in disease states such as heart failure, coronary artery disease, and diabetes have been identified. More recently lncRNAs have been implicated in the development and progression of atrial fibrillation. The discovery of lncRNAs involved in AF is still in its infancy, with only one having a clear molecular pathway. However, their presence as regulators to key genes known to play roles in the development of AF makes them promising targets for future therapy (Figure 1).
Funding:
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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30. Firouzi M, Bierhuizen MF, Kok B, et al. The human Cx40 promoter polymorphism− 44G→ A differentially affects transcriptional regulation by Sp1 and GATA4. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression. 2006;1759(10):491-6. doi:10.1016/j.bbaexp.2006.09.002 31. Gegonne A, Devaiah BN, Singer DS. TAF7: traffic controller in transcription initiation. Transcription. 2013;4(1):29-33. doi:10.4161/trns.22842 32. Huang CY, Pai PY, Kuo CH, et al. p53-mediated miR-18 repression activates HSF2 for IGF-IIR-dependent myocyte hypertrophy in hypertension-induced heart failure. Cell Death Disease. 2017;8(8):e2990.doi: 10.1038/cddis.2017.320.
33. Ke ZP, Xu YJ, Wang ZS, Sun J. RNA sequencing profiling reveals key mRNAs and long noncoding RNAs in atrial fibrillation. J Cell Biochem. 2019. doi: 10.1002/jcb.29504. Babapoor-Farrokhran et al., 21
34. Chen, G., Guo, H., Song, Y., Chang, H., Wang, S., Zhang, M., Liu, C.Long non coding RNA AK055347 is upregulated in patients with atrial fibrillation and regulates mitochondrial energy production in myocardiocytes. Mol Med Rep. 2016;14(6):53115317. doi: 10.3892/mmr.2016.5893
35. Qian C, Li H, Chang D, Wei B, Wang Y. Identification of functional lncRNAs in atrial fibrillation by integrative analysis of the lncRNA mRNA network based on competing endogenous
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37. Cao F, Li Z, Ding WM, Yan L, Zhao QY. LncRNA PVT1 regulates atrial fibrosis via miR-128-3p-SP1-TGF-β1-Smad axis in atrial fibrillation. Mol Med. 2019;25(1):7.doi: 10.1186/s10020-019-0074-5.
38. Huang Z, Chen XJ, Qian C, et al. Signal transducer and activator of transcription 3/microRNA-21 feedback loop contributes to atrial fibrillation by promoting atrial fibrosis in a rat sterile pericarditis model. Circ: Arrhythmia Elec. 2016;9(7):e003396. doi:10.1161/CIRCEP.115.003396 39. Zhao L, Ma Z, Guo Z, Zheng M, Li K, Yang X. Analysis of long non-coding RNA and mRNA profiles in epicardial adipose tissue of patients with atrial fibrillation. Biomed Pharmacother. 2020;121:109634. doi: 10.1016/j.biopha.2019.109634 Babapoor-Farrokhran et al., 22
40. Wu J, Han D, Shi R, et al. Identification of atrial fibrillation associated lncRNAs in atria from patients with rheumatic mitral valve disease. Micros Res Tech. 2019. doi: 10.1002/jemt.23261
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42. Lu J, Xu FQ, Guo JJ, et al. Long noncoding RNA GAS5 attenuates cardiac fibroblast proliferation in atrial fibrillation via repressing ALK5. Eur Rev Med Pharmacol Sci. 2019;23(17):7605-10.doi: 10.26355/eurrev_201909_18883.
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46. Gore-Panter SR, Hsu J, Barnard J, et al. PANCR, the PITX2 adjacent noncoding RNA, is expressed in human left atria and regulates PITX2c expression. Circulation: Arrhythmia Elec. 2016;9(1):e003197. doi: 10.1161/CIRCEP.115.003197.
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47. Li Z, Wang X, Wang W, et al. Altered long non-coding RNA expression profile in rabbit atria with atrial fibrillation: TCONS_00075467 modulates atrial electrical remodeling by sponging miR-328 to regulate CACNA1C. J Mol Cell Cardiol. 2017;108:73-85.doi: 10.1016/j.yjmcc.2017.05.009.
48. Nair GM, Nery PB, Redpath CJ, Birnie DH. The role of renin angiotensin system in atrial fibrillation. J Atr Fibrillation. 2014;6(6).doi: 10.4022/jafib.972
49. Shen C, Kong B, Liu Y, et al. YY1-induced upregulation of lncRNA KCNQ1OT1 regulates angiotensin II-induced atrial fibrillation by modulating miR-384b/CACNA1C axis.
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52. Wang LY, Shen H, Yang Q, et al. LncRNA-LINC00472 contributes to the pathogenesis of atrial fibrillation (Af) by reducing expression of JP2 and RyR2 via miR-24. Biomed & Pharmacother. 2019;120:109364. doi:10.1016/j.biopha.2019.109364 53. Ardhianto P, Yuniadi Y. Biomarkers of Atrial Fibrillation: Which One is a True Marker? Cardiol Res Prac. 2019. Doi: 10.1155/2019/8302326
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Figure 1. Schematic showing involvement of lncRNAs in cardiovascular disease and atrial fibrillation.
lncRNAs are involved in cardiovascular pathophysiology including coronary artery disease, myocardial hypertrophy, myocardial contraction, myocardial infarction, and organogenesis of the heart. They play a role in atrial fibrillation by affecting atrial fibrosis, calcium signaling, renin aldosterone system, immune system, and atrial electrical remodeling.
Babapoor-Farrokhran et al., 25
Figure 1.
Atrial fibrillation
Atrial electrical remodeling
Immune system
Coronary artery disease
Myocardial hypertrophy
Atrial fibrosis
lncRNA
Cardiogenesis
Calcium signaling
Renin angiotensin system
Myocardial contractility
Myocardial infarction
S1547-5271(20)30028-X
DOI:
https://doi.org/10.1016/j.hrthm.2020.01.015
Reference:
HRTHM 8251
To appear in:
Heart Rhythm
Received Date: 20 November 2019 Accepted Date: 14 January 2020
Please cite this article as: Babapoor-Farrokhran S, Gill D, Rasekhi RT, The Role of Long Non-Coding RNAs in Atrial Fibrillation, Heart Rhythm (2020), doi: https://doi.org/10.1016/j.hrthm.2020.01.015. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of Heart Rhythm Society.
The Role of Long Non-Coding RNAs in Atrial Fibrillation Short title: Long Non-Coding RNAs in Atrial Fibrillation
Savalan Babapoor-Farrokhran, M.D. 1#* Deanna Gill M.D.2#, Roozbeh Tarighati Rasekhi, M.D. 2
1
Departments of Cardiology, Albert Einstein Medical Center, Philadelphia, PA, 19141, USA 2 Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA # Savalan Babapoor-Farrokhran and Deanna Gill contributed equally to this work.
*Correspondence: Savalan Babapoor-Farrokhran, M.D. Department of Cardiology, Albert Einstein Medical Center 5501 Old York Road, Philadelphia, PA 19141, United States Email: [email protected]
Conflict of Interest: The authors have no conflicts to disclose.
Word count: 5994
Babapoor-Farrokhran et al., 1
Abstract Atrial Fibrillation (AF) is a common arrhythmia with serious clinical sequelae, but despite its significance little is known about the genetic origins. Recently, the untranscribed 98% of the human genome has been increasingly implicated in important processes like cardiac organogenesis, physiology, and pathophysiology. Specifically, long non-coding RNAs (lncRNA) have been shown to interact with the transcriptome in various ways that alter gene expression. Previously, multiple lncRNAs have been identified in disease processes such as heart failure, coronary artery disease, diabetes, and more. Multiple studies now show lncRNAs are involved in each fundamental mechanism leading to the development of AF including structural remodeling, electrical remodeling, renin angiotensin system (RAS) effects, and calcium handling abnormalities. The altered expression of lncRNAs offers genetic targets for the diagnosis and treatment of AF. In this article, we discuss the role of lncRNAs in atrial fibrillation and its pathogenesis.
Keywords: Atrial fibrillation, long non-coding RNAs, cardiovascular disease, structural remodeling, electrical remodeling, renin angiotensin system effects, calcium handling abnormalities.
Introduction Atrial Fibrillation (AF), the most common arrhythmia of clinical significance, affects almost 3 to 5 million people in the United States.1, 2 The number of affected patients is expected to reach more than 8 million by the year 2050.3 AF increases the risk of stroke, heart failure, morbidity,
Babapoor-Farrokhran et al., 2
and contributes to a 2-fold increase in mortality. It is associated with a higher medical cost and reduced quality of life, leaving a heavy burden on the health care system.4 Long noncoding RNAs (lncRNAs) are characterized as a group of RNAs >200 nucleotides that are 5’capped and 3’ polyadenylated. lncRNAs do not code for a functional protein, but they regulate gene expression.5 lncRNAs are conserved within lineages but not between lineages, and many lncRNAs conserved among primates that are not conserved with rodents.5,6,7 LncRNAs play important functional roles and they have been involved in different biological processes such as epigenetic regulation, imprinting, cell stability, cell-cycle control, cell differentiation, splicing, nuclear/cytoplasmic trafficking, and transcription/translation. Due to their involvement in different aspects of biological pathways, lncRNAs may explain lineage differences in development.7,8 Various targeted therapeutics have been introduced for restoring and/or maintaining sinus rhythm in AF. These therapeutic approaches are mainly limited by their side effect profiles and pro-arrhythmic effects and/or by their procedure-related complications. These limitations have necessitated identification of newer therapeutic targets to expand the treatment options. Despite extensive research focused on the pathogenesis of AF, a thorough understanding of various pathways mediating initiation and propagation of AF still remains limited. Research efforts focused on the identification of these pathways and molecular mediators have generated a great degree of interest for developing more targeted therapies. Recently, efforts have been made to understand the link between heart disease and novel non-coding RNAs. Among multiple noncoding RNAs, lncRNA has emerged as a novel therapeutic target in cardiovascular medicine and potentially atrial fibrillation. Multiple studies have explored the role of lncRNAs in cardiovascular disease. But there are few studies focusing on their role in atrial fibrillation. Babapoor-Farrokhran et al., 3
In this article, we discuss the role of lncRNAs in atrial fibrillation and its pathogenesis. Furthermore, we discuss the lncRNAs that are involved in atrial fibrillation and might be a therapeutic target for the treatment and control of this disease.
Junk DNA or Master of the Genome? Since the discovery of the human genome, the advent of new technological advancements and techniques have enabled scientists to explore the human genome in detail and study its implication in the pathophysiology of diseases. In the early 1960s, it was predicted that the human genome consists of at least two million protein-coding genes. However, the introduction of the Human Genome Project dropped this number to 20,500 coded genes, that is only about 2% of the genome.9 The other 98% consists of non-coding genes and was labeled as “genetic junk” or “transcriptional noise” with the thought being that they are not involved in cellular function. Therefore, scientists have ignored these non-coding regions. But non-coding RNAs (ncRNAs) transcribed from these regions are recently emerging as important regulators of cellular processes with many implications in human diseases.10 In fact, the biggest genomic difference between our genome and that of the nematode lies within our non-coding DNA, with the number of coding genes in the nematode approaching that of a human. As we progress from the genome of prokaryotes to humans, it is the non-coding DNA that has been evolving and growing in size from <25% to 98.5% of the genome.11
Mechanisms of LncRNA Functions
Babapoor-Farrokhran et al., 4
According to ncRNA classification, lncRNAs are more than 200 base pairs in length. They are transcribed by RNA polymerase II, polyadenylated, and subjected to splicing. LncRNAs also have promoter structures and are pervasively expressed across the genome.9 LncRNAs are classified into five subclasses, which include: intergenic, intronic, sense overlapping, anti-sense, and bidirectional lncRNAs. Each subclass is categorized by the genomic location of lncRNAs in relation to their target encoding regions.10 Given the variation in location, lncRNAs can act in cis or in trans, and through a multitude of mechanisms. For example, lncRNAs can upregulate transcription by binding transcription factors and targeting them to promoter regions of specific genes by using sequences complementary to the promoter DNA. Alternatively, lncRNAs may use complementary sequences to mRNA to decreases translation by sequestration of the mRNA.12 LncRNAs can sequester other proteins such as miRNAs, giving variable effects on translation and transcription. An example of this is Cardiac Apoptosis Related LncRNA (CARL), which binds miRNA539, whose downstream target gene is prohibitin-2 (PHB2), an apoptosis-inhibitor. MiRNA-539 typically suppresses PHB2, so when CARL sequesters miRNA-539, PHB2 is transcribed and translated into proteins that inhibit cardiac myocyte apoptosis.13 In addition, lncRNAs play a large role in epigenetics, with many of the RNA’s protein partners being chromatin modifiers.14 Aside from gene transcription and translation, lncRNAs have been implicated in the role of cellular stability, for example, by binding and stabilizing the enzyme, telomerase, which is responsible for adding telomeric repeats back to our chromosomes.15
LncRNA in cardiovascular development, physiology, and disease
Babapoor-Farrokhran et al., 5
The heart is the first organ to function in vertebrates. It is becoming more evident that lncRNAs play important roles in its organogenesis. Specifically, recent research has shown that they are involved in cardiac development as key mediators in the transition from mesoderm to cardiac myocytes. Their role in gene regulatory networks during cardiogenesis is only just beginning to be discovered, but important players have already been identified. Possibly, the most well studied example of this is a lncRNA named Braveheart, first identified by Klattenhoff et al. in mice. Through embryonic stem cell differentiation strategies, they determined the role of Braveheart in the progression and maintenance of cardiac myocytes from mesoderm. Klattenhoff’s team found that Braveheart-depleted cells had altered expression in 548 genes compared to controls. One of the genes encodes for a transcription factor involved in cardiac myocyte differentiation, called Mesoderm Posterior 1 (Mesp-1). Mesp-1 is expressed transiently in mesoderm cells that are destined for cardiac lineage.
They also found that Braveheart
interacts with protein complexes involved in epigenetic changes during cardiac myocyte differentiation.16 After cardiogenesis, lncRNAs have ongoing importance for cardiac physiology, specifically with cardiac contractility. Myosin is made up of two myosin heavy chain (MHC) components and four myosin light chain (MLC) components. Two isoforms of MHC exist in humans, alpha and beta. A study by Luther et al. demonstrated that antisense lncRNA exists for both MHC isoforms. In vitro analysis demonstrated that MHC translation was inhibited by antisense lncRNA.17 Therefore, the levels of antisense lncRNA alters the ratio of alpha to beta MHC in cardiac myocytes, which determines the energy efficiency of cardiac contractility. High levels of alpha MHC suggest stronger contractility whereas higher levels of beta MHC is associated with slower and more efficient contractility.
Babapoor-Farrokhran et al., 6
The presence of these antisense
lncRNAs is associated with rapid switching between MHC isoforms through opposing regulation, where the expression of one leads to repression of the other.16 Many of the lncRNAs involved in normal development and physiology mentioned above have also been implicated in pathophysiologic states. Haddad et al. found that diseases such as hypothyroidism and diabetes are associated with isoform switching from predominantly alpha MHC to predominantly beta MHC. In hypothyroidism specifically, the isoforms switch back to mostly alpha MHC with thyroid hormone replacement therapy.18 Further, they also have been connected to genes that confer increased susceptibility to coronary artery disease,19 myocardial infarction,20,
21, 22
and myocardial hypertrophy.23 Specifically, for myocardial infarction, the
lncRNA ZFAS1 has been shown to lead to impaired contractility after myocardial insult. ZFAS1 binds to a sarcoplasmic reticulum calcium transporter, SERCA2a, thereby inhibiting its function leading to decreased calcium release from the sarcoplasmic reticulum (SR) and thus impairing cardiac contractility.24
lncRNAs in Atrial Fibrillation Evidently, lncRNAs have been implicated in many diseases of the heart yet little is known about lncRNA’s role in atrial fibrillation (AF). As mentioned previously, non-coding RNAs (ncRNAs) constitute nearly 98% of human transcripts and have the potential to provide novel insight into the pathogenesis of AF. The fundamental mechanism of AF is complex but can be reduced to four main processes including: structural remodeling (in particular atrial fibrosis), electrical remodeling, Ca2+ handling abnormalities, and effects of the renin angiotensin system (RAS) .25 Many studies have reported that lncRNAs function importantly in the pathogenesis of AF by regulating critical pathways or core proteins. Initially, multiple reports revealed that a number of Babapoor-Farrokhran et al., 7
lncRNAs were differentially expressed in atrial fibrillation.26,27,28,29 Mei et al., reported differentially expressed lncRNAs in the left atrial tissue of patients with rheumatic heart disease and atrial fibrillation. They demonstrated that 182 lncRNAs were either up-regulated or downregulated, reported as greater than 1.5-fold change, in AF patients compared to patients with sinus rhythm.28 Ruan et al. examined the lncRNA expression profiles of atrial tissues by microarray analyses in AF patients and compared them to AF free patients. They discovered 219 differentially expressed lncRNAs. Randomly, they selected 5 upregulated lncRNAs and 5 downregulated lncRNAs to be validated by real time quantitative PCR (qRT-PCR), and they confirmed that 4 out of 5 for both the upregulated and downregulated lncRNAs were correlated with AF associated genes. It was postulated that the lncRNAs identified in this study initiate AF by promoting atrial electrical remodeling and changes in the RAS. The atrial electrical remodeling takes place primarily through calcium signalling. The RAS pathway promotes AF by inducing atrial fibrosis via mitogen-activated protein kinase, but also through electrical remodeling by shortening the atrial effective refractory period.26 Both of these mechanisms will be discussed in further detail later. Similarly, Xu et al. found 177 lncRNAs differentially expressed in patients with AF compared to controls. Additionally, they found mRNAs that were also differentially expressed. Further, they investigated the co-expression pathways of the mRNA and lncRNA, discovering three transcription regulatory elements that play critical roles in the expression of lncRNAs in AF including GATA1, TAF7, and EBF1.27 Both GATA1 and TAF7 already have proven roles in the molecular pathways leading to AF.30,31 Additionally, Ke et al. did a similar study that showed differential expression of lncRNAs in the left and right atria in patients with atrial fibrillation. They linked a gene called Heat Shock Factor 2 (HSF2), an established player in hypertension induced heart failure,32 to the pathogenesis of AF. They Babapoor-Farrokhran et al., 8
identified two lncRNAs, RP11-99E15.2 and RP3-523K23.2, that interacted with HSF2 and its downstream proteins.33 Chen et al. went a step further and compared lncRNA from the left atrial appendage to the left atrial tissue surrounding the pulmonary veins, a common origin of AF. They noted that lncRNAs that had altered expression in the area surrounding the pulmonary veins interacted with genes involved with mitochondrial energy production including Cyp450 and the electron transport chain.34 Although the correlation of the lncRNAs with transcription factors implicated in AF was strong, the actual molecular relationship between these lncRNAs and TFs had yet to be elucidated. Future studies would solidify the interactions of lncRNAs and AF associated genes. As mentioned previously, lncRNAs studied in other diseases act in a variety of ways to alter gene expression. It appears that for AF the most common mechanism of action is for lncRNAs to “sponge” or sequester their targets, usually miRNA, so that they cannot bind to their target gene or transcript. This “competition” between lncRNAs and miRNAs has led to some researchers calling lncRNAs Competing Endogenous RNAs or ceRNAs.35 The initial work that suggested the involvement of lncRNAs in the development of AF and described the molecular mechanism of how lncRNAs function established a foundation that allowed researchers to pinpoint specific targets of lncRNAs and correlate them to the pathogenesis of AF. Our review will touch on each of the fundamental mechanisms of AF development mentioned above and relate them to lncRNAs that have identified involvement.
Structural remodeling To begin, the hallmark of structural remodeling is atrial fibrosis characterized by abnormal proliferation of fibroblasts and excessive extracellular matrix deposition.36 Structural remodeling Babapoor-Farrokhran et al., 9
is a multifactorial process but miRNAs and eventually lncRNAs were identified that interact with factors integral to the development of atrial fibrosis and thus atrial fibrillation such as TGFβ-1.37 While the TGFβ-1/SMAD pathway is most commonly cited for the development of fibrosis, other pathways reliant on lncRNAs are being discovered. For example, Huang et al discovered that a specific microRNA, miRNA-21, was upregulated in mice that underwent sterile pericarditis and experienced atrial fibrosis and atrial fibrillation. This method was meant to mimic post-operative atrial fibrillation in humans, and when the levels of miRNA-21 were decreased through inhibition of specific transcription factors, both the duration of AF and probability of AF induction were decreased.38 While not clearly related to atrial remodeling, later work revealed a likely connection between the pericardium and the development of atrial fibrosis. Zhao et al. analyzed the expression profiles of lncRNA in the epicardial adipose tissue of patients with AF and without. The epicardium, the inner layer of the pericardium, has been implicated as a key player in atrial remodeling, atrial fibrosis, and therefore AF through the action of overlying adipose tissue. There is compelling evidence that lncRNAs secreted from the epicardial adipose tissue passively diffuse to the neighboring myocardium and are modulators of atrial remodeling.39 Wu et al. found, via trichrome staining, that patients with rheumatic valvular disease and atrial fibrillation had increased amounts of adipose tissue on their myocardium. Western blot of this fat demonstrated increased levels of fibrosis-related proteins including TGFβ-1 and Smad2.40 Zhao’s analysis found that 57 lncRNAs were differentially expressed between the 6 AF patients and 6 sinus rhythm patients.39 To confirm, 6 lncRNAs were randomly selected by QT-PCR. Multiple lncRNAs were connected with genes that have proven roles in the pathogenesis of cardiac remodeling and their effect is not trivial. In fact, co-expression analysis showed that one of the identified upregulated lncRNAs was associated with the top five downBabapoor-Farrokhran et al., 10
regulated protein-coding genes. The genes include PDLIM1, NOS3, TTC3, and SP1 which correlate with processes such as inflammatory signaling, lipid metabolism, and TGFβ-1-induced epithelial-mesenchymal transition. Cao et al. determined the specific interaction between lncRNA and SP1. Their research discovered that a lncRNA, plasmacytoma variant translocation 1 (PVT1), binds to miR-128-3p which usually acts as an inhibitor of SP1, which is an activator of TGFβ-1. However, PVT1 acts as a sponge to miR-128-3p, so that SP1 is free to activate TGFβ-1 leading to atrial fibrosis. To confirm, human atrial fibroblasts were transfected with either overexpressed or silenced PVT1. Fibroblasts with overexpressed PVT1 showed increased TGFβ-1 signaling proteins as well as increased collagen I and II. Conversely PVT1 knockdown fibroblasts demonstrated the opposite effect.37 A downstream target of TGFβ is AKL5, a wellknown promoter of cellular proliferation in the field of oncology.41 Recently it has also been shown to have a role in the proliferation of myocardial fibroblasts. A lncRNA known as Growth Inhibitory Specificity 5 (GAS5) inhibits ALK5 in the myocardium. In cells transfected with overexpressed GAS5 cell growth was inhibited and vice versa in GAS5 knockdown transfected cells.42 As mentioned above, inflammatory signaling also plays a role in the development of atrial fibrosis, specifically macrophages. Macrophages exist as one of two phenotypes. M1 macrophages are the first to arrive to damaged sites and facilitate the clearance of necrotic debris. M2 macrophages promote tissue healing. Studies have shown that the inhibition of M1 and promotion of M2 prevent cardiac remodeling and improved function.43 Sun et al. demonstrated that a lncRNA suppresses a gene known as Nuclear Factor of Activated T cells (NFAT). The lncRNA, Non-coding Represser Of NFAT (NRON) is normally recruited to the promoter region of IL-12 in atrial myocytes, which causes the phenotype switch of macrophages from M2 to M1 thus facilitating atrial fibrosis. In this study, NRON was shown to inhibit nuclear Babapoor-Farrokhran et al., 11
transport of NFAT and thus decreasing expression of IL-12 which resulted in decreased M1 phenotype macrophages and finally diminished myocardial fibrosis.43 Also recognizing the importance of the immune system in the development of AF, Yu et al. collected lymphocytes from 6 patients with AF and 6 patients in SR. Via qRT-PCR and Gene Ontology (GO) analysis, co-expression networks were discovered between mRNAs and lncRNAs that were upregulated in the AF patients. These networks were tied to Tumor Necrosis Factor (TNF) signaling pathway, Toll Like Receptor (TLR) signaling pathway, and NF-kappaβ signaling pathway. Collectively, this linked lncRNAs from systemic lymphocytes to processes such as oxidative stress, inflammation, apoptosis, and collagen synthesis, all of which have known roles in the development of atrial fibrosis.44
Electrical Remodeling Although not as robust as structural remodeling, lncRNAs have also been implicated in electrical remodeling. It has been established that electrical remodeling occurs primarily through shortening of the atrial effective refractory period and action potential duration. The Paired-like homeodomain transcription factor 2 (PITX2) gene is involved in the development of both human and murine hearts. Specifically, the transcription factor is localized to the left atrium of the heart. Holmes et al. showed that mice with reduced levels of PITX2 are predisposed to developing AF. The specific function of PITX2 is elusive, but preliminary analysis suggests it is involved in the regulation of ion channel genes and reduced PITX levels are associated with shortened atrial effective refractory periods.45 Eventually Gore et al. identified an lncRNA upstream of PITX2 known as PITX2 Adjacent Non-Coding RNA (PANCR.) PITX2 and PANCR have coordinated increased expression in cardiomyocytes of the left atrium during stem cell differentiation, and Babapoor-Farrokhran et al., 12
PANCR knockdown models showed associated downregulation of PITX2. Unlike previously mentioned lncRNAs that act by “sponging” target mRNAs via complementary sequences, PANCR and PITX2 do not share any areas of complementarity and therefore work through a different, but unknown, mechanism.46 This study did not correlate the effects of PANCR and PITX2 to the development of AF but taken together with the study of Holmes et al. it appears PITX2 is directly involved with the pathophysiology of AF and is regulated by a lncRNA, PANCR.44,45 In a study on AF and non-AF rabbits, Li et al. found that a lncRNA, TCONS_00075467, acts by “sponging” miR-328 similar to how PVT1 acts on miR-128-3p. In vitro silencing of TCONS_00075467 resulted in shortening of the atrial effective refractory period and action potential. It appears that the lack of TCONS_00075467 binding to miR-328 causes the dysregulation of CACNA1C, an L-type calcium channel, and electrical remodeling.47 CACNA1C also appears to be involved in the development of AF through the Renin Angiotensin System (RAS).
Renin Angiotensin System Effects Activation of RAS in hypertension and heart failure results in elevation of left atrial pressure via angiotensin II (AT-II). Increased pressure can cause left atrial dilation which leads to alterations in ion-channels, the hallmark of electrical remodeling. Prolonged activation of RAS also results in high myocardial tissue levels of angiotensin converting enzyme (ACE) and increased density of AT-II receptors triggering inflammation and fibrosis, or structural remodeling.48 Shen et al. found that a lncRNA, KCNQ1 overlapping transcript 1 (KCNQ1OT1), was upregulated in mice hearts with AT-II induced AF. KCNQT1OT1 binds to miR-384, which usually binds to CACNA1C much like miR-328. Since increased KCNQT1OT1 means increased “sponging” of Babapoor-Farrokhran et al., 13
miR-384, this leads to upregulated CACNA1C, which as mentioned above is associated with the development of AF.48 RAS also interacts with the autonomic nervous system (ANS) which has been heavily implicated for its role in both the development and maintenance of AF. Increased sympathetic tone has been recorded in patients before they developed: post operative AF, atrial flutter, and paroxysmal AF during sleep.50 This role of the RAS is sometimes classified as neural remodeling. Yuzong et al. discovered over 500 differentially expressed lncRNAs in canines with AF and isolated two, TCONS_00032546 and TCONS_00026102, implicated in neural remodeling.51 It appears that the RAS can mediate the development of AF independently, via neural remodeling, but also by working through both electrical and structural remodeling.
Calcium Handling Abnormalities Finally, calcium handling is the fourth and final, fundamental mechanism of AF pathogenesis our review will discuss. Calcium handling abnormalities pertains to the dysregulation of calcium storage and release in cardiac myocytes. Very recently, a study by Wang et al. found a relationship between lncRNA, Ryanodine receptors, and AF development. Specifically, the dysregulation of Ryanodine Receptor-2 (RyR2) is thought to lead to inappropriate calcium release and the onset and progression of AF.52 RyR2 is regulated by Junctophillin-2 (JP2), a signaling protein involved in electrical coupling of the sarcoplasmic reticulum. The ratio of JP2:RyR2 has been shown to be reduced in patients with AF, emphasizing the stabilizing effect of JP2 on RyR2. JP2 levels can be decreased by miRNA-24, but miRNA-24’s competitor is lncRNA-LINC00472 which has been shown to downregulate miRNA, therefore upregulating JP2 resulting in stabilized RyR2. LncRNA-LINC00472 contains a CpG island in its promoter region and methylation of this region is inversely correlated with lncRNA-LINC00472 levels. In Babapoor-Farrokhran et al., 14
this new study, miRNA-24 and lncRNA-LINC00472 levels in 125 AF patients were compared to 168 healthy controls. The results showed that the AF patients had higher levels of miRNA-24 and lower levels of lncRNA-LINC00472 compared to the controls. They also found that DNA methylation in the CpG regions of the lncRNA-LINC00472 promoter was more prominent in AF patients compared to the controls.
Discussion Hundreds of lncRNAs with altered expression have been identified in patients with AF. LncRNA interactions with AF associated genes and transcripts are getting stronger by the day. Research so far suggests that lncRNAs are involved with each of the 4 major mechanisms of AF pathophysiology including structural remodeling, electrical remodeling, RAS effects, and calcium handling abnormalities. Structural remodeling has the most robust evidence for lncRNA involvement so far. Cytokines and interleukins such as TGFβ-1 and IL-12 are both major players in structural remodeling and multiple studies have linked them to differentially expressed lncRNAs in patients and animals with AF. LncRNAs are also associated with electrical remodeling e.g. through alterations of ion channels. Finally, inappropriate release of calcium into cardiac myocytes has been a studied cause of AF, and lncRNAs that alter the stability of RyR2 through JP2 have been identified. As of now, these lncRNAs have no clinical significance for patients with AF. However, clinicians and scientists are becoming increasingly aware of how important AF biomarkers are for the diagnosis, risk stratification, treatment, and prognosis after treatment. Even though about 6% of people over 65 are diagnosed with AF, and there are likely even more asymptomatic patients that do not present, there are currently minimal screening tools for physicians to use. Babapoor-Farrokhran et al., 15
Especially at risk are patients with paroxysmal asymptomatic AF, who may or may not be in AF when routine electrocardiographs (ECGs) are performed by their cardiologist or primary care physicians. Of course, the most devastating complication of AF is stroke, and recently researchers have been looking into biomarkers such as brain natriuretic peptide (BNP) and troponin to stratify stroke risk in patients with AF.53 However, as one could imagine the sensitivity and specificity of these biomarkers is low and therefore would be inefficient and unpredictable screening tools. Many of the lncRNAs linked to AF discussed in this paper have been isolated in the left atrium and some have even been isolated specifically in the pulmonary vein region. These studies suggest a promising role for lncRNAs for possible detection and screening tool for AF. LncRNAs can also be targets for treatment in patients with AF. In lung and cervical cancers, a new treatment is being developed called Antisense Oligonucleotide (ASO)-based therapy. ASO therapy has fewer off-target effects and higher specificity while weakening malignant phenotypes via cell-cycle arrest.54 Sugihara et al. utilized ASO-based therapy to reduce Creactive protein levels in patients with AF with the primary endpoint of change in AF burden. Although this study did not achieve its endpoint, it demonstrated a favorable safety and tolerability profile for this novel therapy.55 Dozens of studies have now been published on lncRNAs as they relate to AF. However, little has been done by way of using them in clinical practice. LncRNAs would make fantastic diagnostic tools, but little is known about their behavior in patients with AF and further studies need to be done in order for them to be used as a diagnostic tool. Given how little we know about their behavior in the body, treatment is even further away. However, they show extraordinary promise for the future of how clinicians manage patients with AF and those at risk of developing AF. Babapoor-Farrokhran et al., 16
Their progressing roles in the diagnosis and treatment of cancers as well as other diseases offers much to be excited about pertaining to their role in AF.
Conclusion:
LncRNAs are involved in cardiac development, physiology, and pathophysiology. In the last decade, several lncRNAs with altered expression in disease states such as heart failure, coronary artery disease, and diabetes have been identified. More recently lncRNAs have been implicated in the development and progression of atrial fibrillation. The discovery of lncRNAs involved in AF is still in its infancy, with only one having a clear molecular pathway. However, their presence as regulators to key genes known to play roles in the development of AF makes them promising targets for future therapy (Figure 1).
Funding:
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Figure 1. Schematic showing involvement of lncRNAs in cardiovascular disease and atrial fibrillation.
lncRNAs are involved in cardiovascular pathophysiology including coronary artery disease, myocardial hypertrophy, myocardial contraction, myocardial infarction, and organogenesis of the heart. They play a role in atrial fibrillation by affecting atrial fibrosis, calcium signaling, renin aldosterone system, immune system, and atrial electrical remodeling.
Babapoor-Farrokhran et al., 25
Figure 1.
Atrial fibrillation
Atrial electrical remodeling
Immune system
Coronary artery disease
Myocardial hypertrophy
Atrial fibrosis
lncRNA
Cardiogenesis
Calcium signaling
Renin angiotensin system
Myocardial contractility
Myocardial infarction