A narrative review of non-coding RNAs in atrial fibrillation: potential therapeutic targets and molecular mechanisms.

OBJECTIVE: This review summarizes the advances in the study of ncRNAs and atrial remodeling mechanisms to explore potential therapeutic targets and strategies for AF.
BACKGROUND: Atrial fibrillation (AF) is one of the most common arrhythmias, and its morbidity and mortality rates are gradually increasing. Non-coding ribonucleic acid RNAs (ncRNAs) are transcribed from the genome and do not have the ability to be translated into proteins. A growing body of evidence has shown ncRNAs are extensively involved in the pathophysiological processes underlying AF. However, the precise molecular mechanisms of these associations have not been fully elucidated. Atrial remodeling plays a key role in the occurrence and development of AF, and includes electrical remodeling, structural remodeling, and autonomic nerve remodeling. Research has shown that ncRNA expression is altered in the plasma and tissues of AF patients that mediate cardiac excitation and arrhythmia, and is closely related to atrial remodeling.
METHODS: Literatures about ncRNAs and atrial fibrillation were extensively reviewed to discuss and analyze.
CONCLUSIONS: The biology of ncRNAs represents a relatively new field of research and is still in an emerging stage. Recent studies have laid a foundation for understanding the molecular mechanisms of AF, future studies aimed at identifying how ncRNAs act on atrial fibrillation to provide potentially promising therapeutic targets for the treatment of atrial fibrillation. 2021 Annals of Translational Medicine. All rights reserved.

Introduction

Non-coding RNAs (ncRNAs) do not encode proteins, and include transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). The common features among these RNAs are that they can be transcribed from the genome but are not translated into proteins, and can perform their respective biological functions at the RNA level. NcRNAs play a key role in the physiology and normal development of the cardiovascular system, but have also been implicated in the development of cardiovascular diseases (1).

Atrial fibrillation (AF) is common type of arrhythmia that arises due to serious disorder in the atrial electrical activity (2). It is induced by rapid and disordered AF that replaces regular physiological atrial electrical activity. AF shares strong links with other cardiovascular diseases, such as coronary artery disease, valvular heart disease, and hypertension (3), which are referred to as upstream risk factors. The association between comorbid cardiovascular disease and AF is complex and not completely understood; the accurate mechanism by which cardiovascular risk factors induce AF is not fully understood either and is currently being investigated. Catecholamine excess, hemodynamic stress, atrial ischemia, atrial inflammation, metabolic stress, and neurohumoral cascade activation may promote AF.

To date, there have been several proposed hypotheses regarding the mechanism of AF, including the multiple wavelet and automatic focus hypotheses (4). The focal origin of AF is supported by several experimental models, indicating that AF persists only in isolated regions of atrial myocardium, and the pulmonary veins appear to be the most frequent source of these automatic foci (5). The multiple wavelet hypothesis proposes that fractionation of wave fronts propagating through the atria results in self-perpetuating “daughter wavelets”. In this model, the number of wavelets is determined by the refractory period, conduction velocity, and mass of atrial tissue. Increased atrial mass, shortened atrial refractory period, and delayed intra-atrial conduction increase the number of wavelets and promote sustained AF (6). One study reported on the complex relationship between trigger and trigger-substrate substrates in patients with AF, and suggested an initiating mechanism for the occurrence of AF (7). The onset and maintenance of paroxysmal AF is associated with ectopic activity; consistency within and between patients in trigger-substrate interaction during the development of AF has also been reported (7).

Following the occurrence of AF, the loss of effective atrial contraction and diastole due to the rapid and disordered rhythm, combined with the disordered ventricular rate caused by decremental conduction of the atrioventricular node in response to rapid atrial excitation, often leads to impaired cardiac function and atrial appendage thrombosis (2). With the increasing aging population, the incidence of AF has gradually increased, leading to significant research advances for understanding its mechanisms, diagnosis, and treatment. In recent years, with the increase and depth of ncRNA research, it might be regarded as a biomarker of atrial fibrillation (8,9). This review set out to describe some of the more recent developments on ncRNAs in AF to develop a better and comprehensive understanding of the interaction between them and explore potential therapeutic targets and strategies for AF, which will likely lead to a more translational approach for preventing and treating AF. We present the following article in accordance with the Narrative Review reporting checklist (available at https://dx.doi.org/10.21037/atm-21-4483).

miRNAs in AF

Changes to miRNA levels in patients with AF

Previous studies have reported changes in the transcription levels of miRNAs in patients with AF. Due to the large heterogeneity in the design of previous studies, we focused on studies in which the control group was a healthy population.

In circulation, increased expression of miR-9, miR-152, miR-374a, miR-454, and miR-664 has been observed in patients with AF (10,11). One study that screened patient plasma samples revealed that the concentration of miR-150 in patients with paroxysmal or persistent AF was significantly lower than that in a control group with normal cardiac rhythm (4), which was also observed in two additional studies (12,13). Reduced plasma levels of miR-29b have also been observed in AF patients with or without congestive heart failure (14). Another study that included healthy controls, well-controlled AF patients, and acute new-onset AF patients showed increased expression of miR-133b, miR-328, and miR-499 in patients with acute new-onset AF compared to the healthy controls, as well as decreased expression of miR-21 in patients with well-controlled AF (15). However, a large cohort study subsequently contradicted this, and found that levels of miR-328 were lower in patients with AF after adjusting for confounding factors (16). This discrepancy reflects the complex regulation of miRNA expression, as well as the temporal variation in circulating miRNA levels in AF patients. Moreover, recent studies have also shown significant elevation of miR-223-3p and miR-320a-3p in patients with AF (17,18).

Various differentially expressed miRNAs have also been identified at the tissue level. In these analyses, samples were mostly obtained from atrial tissue in coronary artery bypass grafting (CABG) or valve replacement, and control groups were comprised of patients who did not have AF but had coronary or valvular disease. A study on myocardium-specific miRNA expression in the right atrial appendage of patients with postoperative AF showed upregulation of miR-1 expression after CABG (19). Although other continuous studies screened a range of miRNAs that were differentially expressed in the atrial tissue of patients with AF and sinus rhythm, the miRNA most consistently upregulated in these studies was miR-21, while miR-26 and miR-29 were consistently found to be downregulated (20-23). Notably, some studies have compared miRNA expression levels between different atrial tissues and found that the left and right atria are both affected by the development of AF. However, in patients with normal rhythm or AF, miRNAs may also be differentially expressed between the left and right atria (24-27).

The role of miRNAs in the mechanism of AF

Although the mechanism of AF has not been fully elucidated, the occurrence and development of AF are closely related to atrial remodeling, which mainly includes electrical remodeling, structural remodeling, and autonomic nerve remodeling (ANR).

Electrical remodeling is associated with intercellular ion channels, including potassium (K+), calcium (Ca2+), and sodium (Na2+) channels. Potassium channel-related miRNAs include miR-26, miR-1, miR-30d, and miR-499, which can shorten the action potential duration (APD) and atrial effective refractory period (AERP) by upregulating the atrial slow delayed rectifier potassium current (IKs) and enhancing the inward rectifier potassium current (IK1) (28). Calcium channel-related miRNAs include miR-328, miR-208a/b, miR-21, and miR-106b-25 clusters, which can weaken the L-type calcium current (LTCC) and hyperphosphorylate ryanodine receptor type 2 (RyR2), causing Ca2+ leakage during diastole (29). At present, miR-192-5p is the only miRNA reported to be associated with sodium channel remodeling (30). Structural remodeling is characterized by atrial dilatation and fibrosis. In this process, there is a vicious cycle in which dilated and fibrotic atria induce AF, which can in turn promote atrial dilatation and fibrosis (31,32). Multiple miRNAs are involved in structural remodeling by promoting the deposition of extracellular matrix (ECM) and increasing collagen I/III (33). Similarly, dysregulation of the autonomic nervous system plays an important role in the onset and maintenance of AF (34). The mechanism underlying the roles of miRNAs in atrial remodeling is shown in .

Figure 1

miRNAs in the remodeling of atrial fibrillation. miRNAs, microRNAs.

Electrical remodeling of AF

MiR-1 is the most abundantly expressed miRNA in the myocardium and plays an important role in promoting the occurrence and development of AF. In a rabbit model of AF with rapid atrial pacing, the expression of miR-1 was significantly increased, whereas KCNE1 and KCNB2 were downregulated at both the messenger RNA (mRNA) and protein expression levels. Meanwhile, IKs was significantly enhanced, and APD and AERP were shortened, promoting the occurrence of AF (35). Furthermore, analysis using luciferase revealed that KCNE1 and KCNB2 were direct targets of miR-1, and downregulation of miR-1 prevented downregulation of KCNE1 and KCNB2, thereby reducing the susceptibility and occurrence of AF (36).

Research on the changes in miR-1 expression in humans has produced contradicting results. Biliczki et al. observed downregulation of miR-1 and increased Ik1 in the atrial tissue of patients with AF (37). Downregulation of miR-1 has also been observed in patients with AF in other studies (19,37). Based on these studies, we speculated that downregulation of miR-1 regulates KCNJ2, thus enhancing Ik1 in patients with AF. However, the mechanistic role of miR-1 in AF is likely complex, as both upregulation and unaltered expression of miR-1 have been observed in studies of human AF (23,29,38). Terentyev et al. not only observed increased miR-1 in human AF but also found that cellular Ca2+ influx was increased as a result of enhanced LTCC. The sarcoplasmic reticulum Ca2+ channel RyR2 is hyperphosphorylated and activated by calmodulin-dependent protein kinase II (CaMKII) (39). However, studies on miR-1 and Ca2+ dynamics in patients with AF have also been contradictory.

Shan et al. found that when miR-1 was overexpressed, Ca2+ influx in atrial myocytes increased, leading to AF (40), while another study found that miR-1 was downregulated in AF patients, which suppressed the expression of the Cavbeta2 subunit of the LTCC. Ultimately, a reduction in the intracellular Ca2+ concentration inhibits the occurrence of AF (13). Feng et al. found that miR-1 was upregulated and the connexin Adam O protein was downregulated, which suppressed the expression of myofibrils; thus, intercellular electrical conduction was slowed, thereby increasing susceptibility to AF (41).

Downregulation of miR-26 in the atrial tissue of AF patients is associated with an increase in KCNJ2 and potassium channel 2.1 (Kir2.1) (28). Inhibition of miR-26a in mice was found to enhance IK1 and increase the occurrence of AF, whereas the overexpression of miR-26a reversed this trend in humans (28). Qi et al. knocked out miR-26a in vitro with locked nucleic acid (LNC)-based drugs in canine fibroblasts to induce an increase in IK1, thereby hyperpolarizing the resting membrane potential (RMP) and enhancing fibroblast proliferation, which was consistent with the above conclusion (42). In addition, a study based on patients with AF and canine models found that miR-26 acted on KCNJ2 to activate T cell nuclear factor and increase K+ influx, thereby shortening APD and causing AF (43). Similarly, Harada et al. found that miR-26a was downregulated in canine and rat models of AF, and transient receptor potential channel 3 (TRPC3) protein increased (33).

Increased expression of miR-328 has been reported in both AF patients and animal models; the target genes of miR-328 are CACNA1C and CACNB1, which encode the cardiac LTCC subunits Cav1.2 and Cavβ1, respectively (29). In atrial myocytes, miR-328 targets the genes encoding L-type Ca2+ channel proteins to decrease L-type calcium channel (ICaL) density and shorten APD and AERP, thereby enhancing the sensitivity of AF. Conversely, downregulation of miR-328 by gene knockout can reduce its sensitivity (29). Karnabi et al. found that when only the α1 subunit of LTCC was knocked out, miR-328 was overexpressed and AF did not develop, suggesting that miR-328 is involved in the development of AF by regulating LTCC (44). Furthermore, a study by Guo et al. showed that miR-328 targeted the 3’ non-coding region of the LTCCα1 sequence region, thereby exerting a negative regulatory effect on LTCC and participating in the development of AF (45). Li et al. also found that when miR-328 was overexpressed, the expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) decreased and intracellular Ca2+ influx increased, suggesting that miR-328 could regulate intracellular calcium dynamics through the sarcoplasmic reticulum (46).

Structural remodeling of AF

The structural remodeling of AF is characterized by atrial enlargement and fibrosis, of which the latter is considered a hallmark of the pathological process. Fibrosis can promote the maintenance of re-entry, which plays a key role in the development of AF (47,48). MiRNAs participate in structural remodeling during AF, and miR-21 has been intensively investigated in various studies. MiR-21 could increase the deposition of ECM and enhance the expression of collagen I and III to promote fibrosis, and several regulatory mechanisms have been proposed. One study found that miR-21 could target and suppress the expression of SPRY1, which is associated with the negative regulation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. Thus, the expression of connective tissue growth factor (CTGF) was enhanced, while fibroblasts caused fibrosis. Furthermore, knocking out miR-21 inhibited fibrosis and decreased the occurrence of AF in vivo (49). Another signaling pathway associated with the role of miR-21 in AF was reported by He et al., who found that miR-21 could repress the expression of Smad7, which was associated with an increase in collagen I and III. Inhibition of miR-21 upregulates Smad7 and suppresses atrial fibrosis (50). MiR-21 can also promote atrial fibrosis via an inflammation-related pathway. Downregulation of miR-21 in rats with pericarditis and AF could suppress the phosphorylation of transcription activator 3 (STAT3), thereby downregulating the expression of fibrosis-related genes and reducing the susceptibility to AF (51,52).

Previous studies have shown that miR-133 and miR-590 are associated with AF via multiple mechanisms. Nicotine has been shown to significantly downregulate miR-133 and miR-590 and enhance the expression of TGF-β1 and TGF-β receptor II genes. An in vitro experiment showed that transfection of miRNA-133 and miRNA-590 into cultured atrial fibroblasts could reduce the expression of collagen, and that TGF-β1 and TGF-β receptor II were also downregulated (53).

MiR-1 is not only involved in atrial electrical remodeling but also in the structural remodeling of AF. Karakikes et al. found that exogenous miR-1 intervention could reverse cardiac hypertrophy and fibrosis caused by pressure loads. With the upregulation of miR-1, the mRNA expression of Fbln-2 was downregulated, and myocardial fibrosis was suppressed by inhibiting the increase in ECM deposition (54).

MiR-29 can target various genes associated with fibrous proteins, including elastin, collagen, and fibrillin. Downregulation of miR-29 promotes the expression of these fibrous proteins and atrial fibrosis. Decreased miR-29b expression was observed in an AF animal model, and after knocking down of miR-29b in vivo, the expression of collagen increased (14).

MiR-26 also promotes structural remodeling by regulating the ECM. Harada et al. found that in canine and rat models of AF, miR-26a was downregulated and transient receptor potential channel 3 (TRPC3) protein was upregulated. The upregulation of TRPC3 was positively correlated with ERK phosphorylation and promoted the expression of ECM-related genes, which could stimulate the proliferation, differentiation, and activation of fibroblasts (33).

MiR-30 and miR-133 could directly target connective tissue growth factor, and recovery of the normal levels of these miRNAs in myocardial cells may control structural remodeling (55). MiR-208 is required for cardiomyocyte hypertrophy and fibrosis, and can promote atrial remodeling by inhibiting gap junction protein 40 (56,57). In addition, miR-499 is significantly upregulated in patients with AF, which could suppress the expression of CACNB2 and contribute to electrical remodeling (58).

ANR of AF

ANR refers to changes in the density, morphology, and spatial distribution of autonomic nerve fibers after myocardial ischemia, injury, and necrosis. ANR is characterized by nerve regeneration and disordered distribution of nerve fibers that cooperate and promote each other with electrical and structural remodeling, forming a vicious circle, which plays an important role in the pathological process of AF (59).

A study using high-throughput sequencing found that the expression levels of miR-206 were significantly increased in AF animal models, while superoxide dismutase 1 (SOD1) was reported to be the target gene of miR-206. Furthermore, studies have also found that miR-206 can mediate the oxidative stress response via the SOD1-ROS and GCH1-BH4-NO pathways, and promote ANR as well as the occurrence of AF (14,60). However, besides miR-206, no other miRNAs have been directly associated with ANR.

With the continuous in-depth study of miRNAs and the deep understanding of the functions of new miRNAs, significant progress has been made in the fields of miRNAs and AF. The molecular biological mechanism of the occurrence and development of AF has been improved, which relates to the diagnosis and treatment of AF, while prevention provides new strategies. However, most of the current literature lacks comprehensive research, and there remain several gaps in the understanding of the gene regulation of miRNA in AF. In-depth investigation of the expression level and specific mechanism of miRNA in patients with AF as well as targeted regulation of miRNA to prevent and reverse AF will improve AF atrial remodeling and provide new insights into the diagnosis and treatment of AF. summarizes the known miRNAs that participate in atrial remodeling and AF development.

Table 1

miRNAs related to AF-associated remodeling

miRNA	Target gene	Remodeling type	References
miRNA-26	KCNJ2	Electrical remodeling	(28)
miRNA-1	KCNE1, KCNB2, KCNJ2		(35)
miRNA-499	KCNN3		(61)
miRNA-328	CACNA1C, CACNB1		(29)
miRNA-208a/b	Unknown		(57)
miRNA-21	SPRY1, TGF-β1	Structure remodeling	(49,62)
miRNA-133	TGF-β1, TGF-βR, CTGF		(53)
miRNA-590	TGF-β1, TGF-βR		(53)
miRNA-208	GJP40		(33)
miRNA-206	SOD1	Autonomic nerve remodeling	(63)

LncRNAs in AF

LncRNAs play an important role in cardiovascular diseases, including AF. Several lncRNAs are not conserved between humans and mice; however, the underlying mechanism of their role in the pathological process of AF remains unclear (64).

Changes in the transcription level of lncRNAs in AF patients

Using microarray and high-throughput sequencing technology to compare the expression of lncRNAs in the cardiac tissue of patients with AF and sinus rhythm, numerous studies have found differentially expressed lncRNAs in the cardiac tissue of patients with AF (65-68). Microarray analysis has shown that, compared with patients with sinus rhythm, there were 182 and 219 differentially expressed lncRNAs in the left atrial tissue samples of AF patients, respectively. Another study analyzed the co-expression profile of lncRNAs and mRNAs, and found 177 differentially expressed lncRNAs in AF patients, of which GATA1, EBF1, and TAF7 were thought to be involved in the regulation of AF (66). Another study showed that GATA1 and TAF1 play an important role in the occurrence and development of AF (69,70). Ke et al. also analyzed the differential expression of lncRNAs in the left and right atria of patients with AF and found that two AF-related lncRNAs (RP3-523K23.2, RP11-99E15.2) regulated heat shock factor 2 (HSF2), which is associated with hypertensive heart failure (71). In addition, Chen et al. tested the expression profile of lncRNAs in the left atrial appendages and left heart tissues around the pulmonary veins and found 94 differentially expressed lncRNAs. Among them, the most significant changes occurred in AK055347, and knock outing could suppress the expression of mitochondrial genes Cyp450, ATP synthase, and MASS51, which were associated with the decreased viability of H9C2 cardiomyocytes (72). These findings suggest that AK055347 may act as a regulator of AF by affecting mitochondrial energy production. In summary, by establishing the relationship between the expression of lncRNA and the expression of AF-related gene networks, lncRNAs have been confirmed to be involved in AF. Therefore, we will now focus on lncRNAs related to structure and electrical remodeling in AF.

AF remodeling-related lncRNAs and pathogenic mechanism

LncRNAs are also involved in the remodeling mechanism of AF, and are mainly involved in structural and electrical remodeling. Through pathway enrichment analysis, differentially expressed lncRNAs may induce AF by increasing electrical remodeling and changing the renin-angiotensin system (RAS). However, the mechanisms involved in electrical remodeling are not as clear as those in structural remodeling, which is a chronic pathological process mainly reflected in atrial fibrosis at the level of pathological tissue.

Inflammation putatively plays an important role in the process of fibrosis; in particular, macrophages are known to be involved in atrial fibrosis. The classical classification of macrophages divides them into M1- and M2-type macrophages. M1 macrophages phagocytose cell debris, while M2 macrophages promote tissue repair and healing. Studies have shown that the conversion of M1 to M2 macrophages can prevent cardiac remodeling and improve cardiac function (73). Non-coding repressor of NFAT (NRON) is a lncRNA that can bind to the interleukin 12 (IL-12) promoter to alleviate atrial fibrosis. M2 type macrophages are stimulated by IL-12 to induce their conversion to M1 type macrophages, thus leading to atrial fibrosis, which can play a role in inhibiting the nuclear localization of NFAT. This in turn inhibits the expression of IL-12 and IL-12, which cannot reverse the polarization of macrophages, thereby reducing atrial fibrosis (73). Yu et al. compared lncRNAs related to immune signals in lymphocytes from AF and normal sinus rhythm patients and found that there are signaling pathways, such as toll-like receptors (TLR) and tumor necrosis factor (TNF), between upregulated mRNA and lncRNA co-expression network (74). Through bioinformatics analysis, the authors concluded that lncRNAs from lymphocytes are related to oxidative stress, collagen synthesis, apoptosis, inflammation, and other cellular processes, which are all related to the occurrence of atrial fibrosis.

Studies have shown that lncRNAs can enter the myocardium from the epicardial fat layer by diffusion. The authors collected the epicardial adipose tissue of patients with sinus rhythm and AF patients and found 57 differentially expressed lncRNAs through microarray analysis. These lncRNAs may play a role in inducing atrial fibrosis (75). Meanwhile, Zhao et al. (75) found multiple lncRNAs that play a role in atrial fibrosis and are related to protein-coding genes. Of these lncRNAs, the expression of plasmacytoma variant translocation 1 (plasmacytoma variant translocation 1, PVT1) is linked to inflammation, lipid metabolism, and TGF-cad1-induced epithelial-mesenchymal transition (EMT)-related genes such as TTC3, PDLIM1, NOS3, and SP1 (75). TGF-cad1, a key factor related to the development of atrial fibrosis and AF, often interacts with lncRNAs. Overexpression of PVT1 can lead to increased fibrosis, while inhibition of PVT1 can reverse fibrosis. The main molecular mechanism involves PVT1 acting as a sponge of miR-128-3p to regulate the miR-128-3p/Sp1/TGF-cads1/Smad axis, which activates the Sp1-mediated TGF-cads1/Smad pathway and increases the production of collagen I and II. TGFβ type I receptor kinase (ALK5), the downstream target of TGF-cad1, regulates cell proliferation (76), including cardiac fibroblasts. Growth inhibitory specificity (GAS5) is a lncRNA in cardiomyocytes that can inhibit ALK5 expression (77). GAS5 expression is reduced in AF atrial tissues in vivo. Inhibition of GAS5 expression in vitro promotes cell growth, while its overexpression inhibits cell growth. In addition to its role in myocardial hypertrophy and fibrosis, myocardial infarction association transcript (MIAT) has recently been shown to participate in AF by inhibiting miR-133-3p (78). In a rat AF model, the expression of MIAT increased in the atrial tissue, whereas the expression of miR-133-3p decreased. Knockdown of MIAT can inhibit cardiomyocyte apoptosis, indirectly enhance atrial function, and shorten the onset time of AF, thereby reducing AF (79). Another mechanism through which MIAT knockdown can alleviate AF is to inhibit AF-induced atrial fibrosis. In addition to PVT1, another lncRNA found in AF structural remodeling and fibrosis is long non-coding RNA predicting cardiac remodeling (lnc LICPAR) (80). In the atrial muscle tissues of patients, LICPAR and TGF-β1 were upregulated and positively correlated, where LIPCAR regulates atrial fibrosis via modulating TGF-β/Smad pathway (81).

LncRNAs are also related to the electrical remodeling of AF, but this causal relationship is not as clear as its involvement in structural remodeling. The main driving factors of atrial electrical remodeling are the shortening of the AERP and APD. One gene found in AF is PITX2, which is also involved in heart development. Studies have also found that PITX2 levels in mice prone to AF are relatively low. PITX2 can affect heart ion channels and change the AERP (82). In addition, we discovered an upstream lncRNA PITX2 adjacent noncoding RNA (PANCR) targeting PITX2 (83). PANCR is not directly related to the development of AF; however, due to the role of PITX2 in AF, PANCR may be an AF-related lncRNA (74). A previous study has tested lncRNAs in rabbits with and without AF, and found that silencing lncRNA TCONS_00075467 can cause AERP and shorten the APD (84). This may be due to its role as a sponge of miR-328, which impedes its inhibitory effect on target mRNA. Due to the loss of this lncRNA, the expression of miR-328 increases, resulting in the downregulation of the type 1 calcium channel, CACNA1C. In fact, dysregulation of CACNA1C has been shown to participate in the development of AF by regulating the RAS (29,85). RAS activation is implicated in hypertension and heart failure through AngII, which increases left arterial pressure. In addition, prolonged RAS activation can result in high levels of angiotensin-converting enzyme (ACE) and AngII receptors, leading to an immune response and eventually, fibrosis or structural remodeling (86). In vascular-induced AF mice, upregulated lncRNA and KCNQ1 overlapping transcript 1 (KCNQ1OT1) have been observed. KCNQ1OT1 can target and silence CACNA1C through its sponge function acting on miR-384. Excessive KCNQ1OT1 inhibits the silencing effect of miR-384, leading to increased levels of CACNA1C and the occurrence of AF (87). A previous study analyzing RNA-seq data from the left and right atrial appendages of patients with AF and sinus rhythm identified key lncRNAs associated with AF (71). The authors suggested that lncRNAs could potentially regulate adjacent genes encoding proteins, and found that lncRNA NPPA-AS1, rp11-99 e15.2, and rp3-523 k23.2 may be related to NPPA, ITGB3, HSF2, and in particular NPPA-AS1, which was found to interact with NPPA. The co-expression of six contractile genes, including PLCE1, TNNC1, TACR1, GSTO1, and TNN1, suggests that NPPA-AS1 participates in the pathogenesis of AF by regulating cardiac contraction.

In addition to structural remodeling and electrocardiographic remodeling, nerve remodeling may also be a pathological mechanism of lncRNA-mediated AF. Interestingly, RAS is associated with the autonomic nervous system and participates in nerve remodeling. A previous study has analyzed the lncRNAs in the heart fat pad of dogs with and without AF and found that the abnormally expressed lncRNAs are related to the development, differentiation, and degeneration of neurons (88). The identification of lncRNAs indicates that the inhibition of two lncRNAs related to neural remodeling (TCONS_00032546 and TCONS_00026102) in vivo as well as shortening or prolonging the atrial refractory period leads to an increase in the occurrence and prevention of AF. Moreover, the expression of these lncRNAs was negatively correlated with FGF19, FGF4, CCND1, FGF3, and SLC25A4 expression, and these lncRNA genes are adjacent. In summary, these studies indicate that lncRNAs may be involved in the occurrence of AF through RAS-mediated neural remodeling. summarizes the known lncRNAs that participate in atrial remodeling and AF development.

Table 2

LncRNAs related to AF-associated remodeling

LncRNA	Target gene	Remodeling type	Reference
PANCR	PITX2	Electrical remodeling	(83)
TCONS_00075467	CACNA1C		(84)
KCNQ1OT1	CACNA1C		(87)
NPPA-AS1	Modulating cardiac contraction genes		(71)…
lncRNA-HBL1	miR-1		(89)
PVT1	miR-128-3p/Sp1/TGF-β1/Smad	Structural remodeling	(75)
GAS5	ALK5		(77)
LICPAR	TGF-β1/Smad		(81)
MIAT	miR-133-3p		(78)
NRON	IL-12		(73)
TCONS_00032546	Related to RAS-mediated neuronal remodeling	Nerve remodeling	(88)
TCONS_00026102	Related to RAS-mediated neuronal remodeling		(88)

Transcription factors (TFs) and ncRNA

A growing number of evidence suggests that putative gene regulatory networks (GRNs), including cardiac-enriched transcription factors (TFs) and its target genes, can play a potentially important role in the process of adaptive and maladaptive atrial rhythm remodeling (90-92). In addition, the human genome-wide association studies (GWAS) have successfully identified more than 100 genetic sites associated with atrial fibrillation, including genes encoding cardio-enriched TFs (93,94). Most of the GWAS variants associated with AF are located in the non-coding genome (95).

Recently, several studies have documented that cardiac GRNs are controlled by an interlaced network of regulatory ncRNAs, including miRNAs and lncRNAs. The mature miR transcript (approximately 22 nucleotides long) acts as an inhibitor of target gene expression by promoting mRNA degradation or inhibiting translation. In this case, lncRNA (more than 200 nucleotides in length) can activate or inhibit gene expression by adjusting chromatin conformation and TF binding or by isolating miRNA from its target mRNA. Previous research has indicated that ncRNAs are dysregulated in many forms of adult heart disease in patients and animal models (96,97). In particular, a growing body of evidence suggests that ncRNAs may form an additional key layer of complex regulator structure for controlling atrial gene expression (84,95,98,99). The perturbation of the expression balance between TF and ncRNA networks can promote the development of AF (100). Therefore, before considering translational therapy strategies, it is necessary to understand the future research of pathways regulated by the atrial TF-miRNA circuit in various AF environments.

summarizes the interplay between known cardiac transcription factors and non-coding RNAs in AF.

Table 3

ncRNAs and TFs in AF

TFs	Cardiac phenotype	Related ncRNAs	Reference
PITX2	Regulates left-right differentiation, lack of which leads to structural and electrical remodeling	miR-17-92, miR-106b-25, miR-21, miR-1, miR-29a, PANCR	(83,85,101-103)
TBX5	Essential for the development of the interventricular septal and atv-ventricular conduction system and the maintenance of the atrial ventricular bundle. TBX5 point mutations are known to cause AF	miR-19, miR-10a/b, RACER	(104-106)
ZFHX3	As a transcriptional inhibitor of myogenic differentiation highly expressed in adult mouse hearts and human stem cell-derived cardiomyoblasts	miR-1	(107)
SHOX2	Controls the development and function of sinoatrial node located in the adult right atrium	miR-92b-5p	(108)

CircRNAs in AF

CircRNAs are ncRNAs that form a closed-loop structure. Like other ncRNAs, circRNAs play an important role in gene regulation. CircRNAs have been implicated to be closely associated with AF (109). Hu et al. found 108 differentially expressed circRNAs in the atrial tissue of patients with persistent AF compared to the myocardial tissue of controls without AF. Among these, 51 circRNAs were upregulated in AF, while 57 were downregulated (110). Genome-wide analysis of AF patients and healthy controls found a total of 14,215 circRNAs, of which 28 were differentially expressed (111).

Shangguan et al. analyzed circRNA expression in the atrial tissue of canines with rapid atrial pacing, and found that differentially expressed circRNAs interacted with AF-related miRNAs and mRNAs. This provided a basis for further research into the potential mechanistic roles of circRNAs in AF (112). Zhang et al. subsequently proposed a circRNA-related competitive endogenous RNA network in non-valvular persistent AF to better understanding its pathogenesis (113).

Among the circRNAs found to interact with miRNAs, circRNA19591, circRNA19596, and circRNA16175 interacted with 36, 28, and 18 different miRNAs, respectively. Additionally, the expression of miR-29b-1-5p and miR-29b-2-5p is associated with the downregulation of 12 circRNAs (110). CircRNAs have been hypothesized to play a critical role in AF via a sponge regulatory mechanism of miRNAs. In the sponge regulatory technique, the tandem repeat (6× or 8×) miRNA-binding domain is cloned into a vector regulated by the CMV promoter, and the miRNA sponge is transcribed into the cytoplasm in the form of an mRNA, which induces miRNA binding with a similar binding domain in the cytoplasm. It performs sponge-like miRNA absorption and competes with RNA-induced silencing complex (RISC) to bind to free miRNA with a specific RNA-binding domain in the cytoplasm, which can simultaneously inhibit miRNA and the RNA-binding domain (114). In addition, Zhang et al. found that hsa-circ-0000075 and has-circ-0082096 may be involved in the pathogenesis of AF through the TGF-β signaling pathway by targeting the GDF7 and IFNG genes, which are upregulated in the pathway (111).

Conclusion and perspectives

NcRNAs, including miRNAs, lncRNAs, and circRNAs, play an important role in the development of AF. Recent studies have laid a foundation for understanding the molecular mechanisms of AF, and identified ncRNAs that could serve as targets for diagnosis and treatment in the future. The use of ncRNA to treat disease is a promising strategy and may offer new treatment options to patients with AF.

Substantial progress has been made thus far in the area of ncRNAs as they relate to AF. Multiple ncRNAs have been implicated in the pathological processes underlying AF, including electrical remodeling, structural remodeling, and ANR. Furthermore, with the development of microarray and sequencing technology, the popularity of weighted gene co-expression network analysis (WGCNA) and lncRNA-mRNA co- expression (ceRNA) network analysis, we can identify key modules and hub genes associated with atrial fibrillation more deeply (115,116). We can associate ncRNA with clinical features, which is predicted that there may be small molecule drugs with similar lncRNA or targeted lncRNA therapeutic function in the treatment of AF. When these ncRNAs were employed in the treatment of AF patients, few have achieved a clinical transformation. Many aspects of this research area remain in a preliminary, exploratory stage, such as the emerging roles of circRNAs in AF. However, most of the research has focused only on the relevance of RNA. Moreover, the available data in this area are conflicting, likely due to the complexity of RNA and its transcriptional control, as well as a lack of mechanistic by how RNA acts directly. And because the etiology of AF is very different between human and animal models, it is necessary to use human cells or tissues to develop appropriate disease models (117). Although the AF disease model constructed by human pluripotent stem cells (hPSCs) is becoming one of the basic model of AF and provides a more reliable platform for the study of ncRNA function in AF, there are still many challenges in establishing a mature and stable model to develop new therapies for atrial fibrillation and other cardiovascular diseases and research on ncRNA translation function (118). Thus, further comprehensive research is warranted to address the discrepancies in the literature and the knowledge gaps in this field.

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