Atrial fibrillation (AF) and non-alcoholic fatty liver disease (NAFLD) exhibit substantial epidemiological prevalence and impact on health. AF, known for its high mortality, disability, and recurrence rates, heightens the risk of stroke, myocardial infarction, and heart failure (Joseph et al., 2021). Concurrently, NAFLD is globally the leading liver and metabolic disorder (Zhou et al., 2020). By 2018, nearly 25% of the world population, i.e., 1.7 billion people, were affected by NAFLD (Cai et al., 2019b), exceeding the combined total of obese (0.65 billion) and diabetic (0.4 billion) populations (The Lancet Public, 2018). Research has demonstrated a solid link between NAFLD and elevated AF risk, independent of other traditional cardio-metabolic comorbidities (Chen et al., 2021). NAFLD’s contribution to AF onset and recurrence post-ablation is evident, even when adjusted for age, sex, body mass index (BMI), ejection fraction, and AF type (Donnellan et al., 2020). However, despite its recognition as a new cardiac arrhythmia risk factor, NAFLD-related AF remains under-explored, and its underlying mechanisms are unclear.

Emerging studies have shed light on pathophysiological alterations associated with NAFLD that could induce cardiac structural, electrical, and autonomic neural remodeling, potentially leading to cardiac arrhythmias, with some highlighting the key role of the liver-heart inflammation axis in hepatogenic cardiomyopathy development (Matyas et al., 2020). Nevertheless, NAFLD’s role as a novel arrhythmia risk factor is largely underestimated, underplaying its impact on patient morbidity. For example, systemic inflammation, a hallmark of NAFLD, could contribute to atrial structural remodeling, a known AF precursor (Cai et al., 2019a). Metabolic stress in NAFLD leads to hepatic lipid accumulation and subsequent lipotoxicity, with the release of danger-associated molecular patterns, while recent insights point to mitochondrial dysfunction—marked by disrupted energy metabolism and increased oxidative stress—as a factor that not only exacerbates systemic inflammation but also may underlie the arrhythmic tendencies observed in AF (Liu et al., 2020). These molecules may trigger sterile inflammation pathways, boosting local inflammatory responses. The activation of hepatic immune signaling results in the release of pro-inflammatory cytokines and chemokines (Zhang et al., 2017). Importantly, these inflammatory mediators can enter the systemic circulation via the hepatic sinusoidal blood flow, augmenting a systemic pro-inflammatory environment (Cai et al., 2018). Elevated systemic inflammatory markers and inducers have been identified in both NAFLD patients and experimental models (Mohammed et al., 2021; Wang et al., 2021). Research also shows associations between hepatic fat and inflammatory markers, independent of obesity and diabetes, suggesting that NAFLD could directly trigger immune activation and systemic inflammation, possibly affecting distant organs like the heart, thereby increasing AF susceptibility (Käräjämäki et al., 2018). Additionally, the convergence of systemic inflammation and mitochondrial dysfunction in NAFLD may amplify the risk of developing AF, indicating a potential target for therapeutic intervention. Sodium glucose co-transporter 2 inhibitor (SGLT2i) is a new type of hypoglycemic agent, which also shows good application prospects in the cardiovascular diseases (Panico et al., 2023). In recent years, numerous studies have confirmed that SGLT2i could reduce the risk of new-onset AF in patients with Type 2 diabetes mellitus (T2DM), increase the clinical benefit of patients with T2DM combined with AF, and reduce the recurrence of AF after AF ablation (Zinman et al., 2015; Zelniker et al., 2020; Zhao et al., 2023). NAFLD and T2DM are metabolic abnormalities with many common pathophysiological bases, such as glucose metabolism abnormality, insulin resistance, systemic inflammation, and mitochondrial dysfunction (Muzurović et al., 2021; Kosmalski et al., 2023). Studies have shown that SGLT2i could improve not only glycemic control but also liver fatty infiltration and fibrosis in patients with NAFLD and T2DM (Arai et al., 2021; Akuta et al., 2022). In addition, recent studies also found that the direct effect of SGLT2i on NAFLD, that SGLT2i could attenuate high-fat diet-induced steatosis in mice (Nasiri-Ansari et al., 2021; Radlinger et al., 2023). Therefore, we hypothesized that SGLT2i may be a potential therapeutic agent for NAFLD-related AF.

CIBERSORT, a widely used tool for deconvoluting human immune cell subtype expression matrices based on linear support vector regression, provides gene expression feature sets for 22 immune cell subtypes using known reference datasets: LM22 (Newman et al., 2015). CIBERSORT has been primarily used in cancer, but recent years have seen its application in non-cancerous diseases like atherosclerosis, myocardial infarction, and ischemic stroke (Huang B. et al., 2022; Xie et al., 2023). However, CIBERSORT’s use in NAFLD and AF is less frequent, with no studies reporting the immune profile of NAFLD-related AF patients.

In this study, we aim to analyze the common genes in NAFLD and AF patients from the GEO database and validate our findings with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) on patients’ peripheral blood leukocyte samples. We then performed immune infiltration analysis with CIBERSORT on tissue microarrays for each condition, selecting genes associated with specific immune cells. Last but not least, we explored the theoretical feasibility of SGLT2i for the treatment of NAFLD-related AF by molecular docking studies. Our ultimate goal is to find the association between NAFLD and AF, especially the possible mechanisms by which NAFLD increases susceptibility to AF, and identify biological markers and potential targets for intervention associated with NAFLD-related AF.

We retrieved datasets GSE89632 and GSE75092 from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). GSE89632 (platform: GPL14951) includes liver tissue specimens from 24 healthy controls (HC) and 39 NAFLD patients. GSE75092 (platform: GLP16956) comprises blood samples from 3 AF patients and 3 sinus rhythm (SR) controls. As shown in Figure 1, we first calculated the differentially expressed genes (DEGs) of GSE89632 and GSE75092, then study the potential relationship between NAFLD and AF. The HC presence of a normal liver (no steatosis or cirrhosis) on imaging and/or histology, while the NAFLD patients were diagnosed by liver biopsy, as indicated elsewhere (Arendt et al., 2015). The AF patients in this study were all diagnosed with paroxysmal AF by electrocardiograph (ECG), whereas the SR patients had no clinical evidence or history of AF (Su et al., 2018).

In this study, we identified eight co-upregulated and 31 co-downregulated DEGs in the two datasets. Through validation of the upregulated genes and hub genes identified by PPI analysis of the co-downregulated DEGs, we found that AMOT, PDE11A, TYMS, TMEM98, and PTGS2 exhibited good diagnostic potential for NAFLD-related AF. CIBERSORT analysis revealed elevated levels of CD8 T cells, γδ T cells, and M2 macrophages in the livers of NAFLD patients, linking systemic inflammation to NAFLD and AF. Additionally, several hub genes like DGAT1, TYMS, and PTGS2 closely related to mitochondrial function, suggesting that mitochondrial disturbances may underpin the systemic inflammation in NAFLD, which possibly exacerbating AF. Last but not least, molecular docking analysis revealed a strong binding affinity between empagliflozin and DGAT, TYMS, and PTGS2, suggesting the potential of empagliflozin as a therapeutic option for NALFD-related AF.

The concurrent rise in the prevalence of NAFLD and AF in recent times has prompted considerable research interest. NAFLD is characterized by an inflamed liver state that can potentially trigger AF (Chen et al., 2021). This highly active hepatic condition induces various pro-inflammatory cytokines, which can infiltrate the heart, leading to immune cell infiltration and inflammation—key elements of the liver-heart axis that could culminate in AF (Peiseler et al., 2022). Upon reviewing the literature, we have collectively validated five co-upregulated DEGs (DGAT1, AMOT, PDE11A, TYMS, and TMEM98) that were elevated in patients with AF, NAFLD, and in those with NAFLD-related AF compared to the HC group. Notably, the latter four genes were particularly elevated in patients with NAFLD and concurrent AF. The DGAT1 gene encodes a protein functioning as a key metabolic enzyme. Extensive research corroborates that DGAT1 exacerbates lipid storage in hepatocytes (de la Rosa Rodriguez et al., 2021), and drugs targeting this protein can reduce liver fibrosis and inflammation in a murine NASH model (Cheng et al., 2022). Additionally, DGAT1 is essential in maintaining mitochondrial autophagy and metabolic integrity, with studies showing that deferiprone-induced iron depletion—prompting PINK1/PARKIN-independent mitochondrial autophagy—also triggers DGAT1-dependent lipid droplet biogenesis, indicating a synergistic interaction between metabolic reprogramming and mitochondrial autophagy following chelation therapy (Glenn et al., 2011; Long and McWilliams, 2023). No studies to date have established a link between DGAT1 and AF. However, most studies have shown that DGAT1 is associated with inflammation. Huang et al. suggest that DGAT1 inhibitors can protect pancreatic β cells from palmitate-induced apoptosis (Huang et al., 2021). While Graham et al. propose that DGAT1 can mediate autoimmune encephalomyelitis by inhibiting the formation of retinol-dependent regulatory T cells (Graham et al., 2019). Some studies also indicate that DGAT1-dependent triglyceride storage in macrophages can protect mice from diet-induced obesity insulin resistance and inflammation (Koliwad et al., 2010). The AMOT gene belongs to the angiomotin family of angiostatin-binding proteins, which may mediate the inhibitory effect of angiostatin on angiogenesis, and no research thus far has associated this gene with AF or NAFLD. AMOT plays a crucial role in regulating Hippo pathway activity, which is implicated in lipid metabolism, inflammation, and fibrogenesis, all processes intrinsic to the pathogenesis of NAFLD (Zhang et al., 2010). The Hippo pathway also relates to cardiac development, repair, and homeostasis, and its dysregulation can lead to myocardial hypertrophy, fibrosis, and dysfunction, known risk factors for AF (Ikeda et al., 2019). PDE11A is an enzyme-degrading cyclic nucleotide (cAMP and cGMP), and no studies have associated this gene with AF or NAFLD to date. However, research indicates that the cAMP/PKA signaling pathway is a potential therapeutic target in metabolic diseases, including obesity, type 2 diabetes, and related complications (London and Stratakis, 2022). TYMS encodes thymidylate synthase, which is crucial for DNA replication and repair. While the relationship between TYMS and cancer has been extensively studied (Zhao and Kaldis, 2023), its role in NAFLD, AF, or inflammation requires further exploration. TYMS, crucial for maintaining deoxythymidine triphosphate (dTTP) levels within mitochondria for accurate DNA replication, may play a role in NAFLD-AF pathogenesis through its impact on cellular and mitochondrial integrity (Anderson et al., 2011). The TMEM98 gene encodes a transmembrane protein. Intriguingly, it can also be secreted via exosomes. Under antigen-non-specific and antigen-specific Th1-skewing conditions, eukaryotic recombinant TMEM98 promotes Th1 cell differentiation and stimulates IFNγ secretion (Fu et al., 2015). Although there is no direct connection with AF or NAFLD yet, transmembrane proteins are often crucial in biological processes and disease states due to their role in cell signaling, transport, and communication, meriting further research.

We also identified 31 co-downregulated genes and selected the six most central genes for validation (FOS, PTGS2, SOCS3, ZFP36, TLR2, and BTG2). Ultimately, only PTGS2 demonstrated substantial diagnostic value for patients with NAFLD in conjunction with AF. Enrichment analysis of the 31 downregulated genes reveals a downregulation of the NF-κB and TNF pathways, which seems contrary to our understanding of NAFLD and AF’s association with chronic systemic inflammation. However, enriched within these two pathways are genes such as PTGS2, a cyclooxygenase that serves as a key enzyme in prostaglandin biosynthesis. Although COX2 (PTGS2) is usually considered to induce inflammation, previous studies have found that COX-2 deficient mice, or those treated with COX inhibitors, display exaggerated inflammatory responses in the lungs, leading to the development of asthma (Peebles et al., 2002). PTGS2, also known to modulate mitochondrial function and lipid metabolism pathways, plays a crucial role in ferroptosis during cardiac and pulmonary disease processes, challenging the traditional view of its sole pro-inflammatory role (Chen et al., 2022; Lv et al., 2023). These pathways are also enriched with SOCS3 (suppressor of cytokine signaling 3), an essential molecule for negatively regulating cellular signaling pathways, by inhibiting inflammatory cytokines such as interleukin-6 (IL-6) and interferon (IFN) (Zhang et al., 2020). Thus, SOCS3 downregulation could exacerbate inflammation. Additionally, SOCS3 can affect the differentiation of immune cells, particularly regulating the Th1/Th2 balance of T cells and the polarization of macrophages towards the M2 phenotype, thereby influencing the course of immune responses (Huang W. et al., 2022). Recent studies have found an increase in SOCS3 expression in the hepatocytes of patients with NAFLD, which may be related to inflammation and insulin resistance in these conditions (Zhang et al., 2021). Conversely, miR-455-5p mimics can accelerate the progression of AF by activating the STAT3 signaling pathway through direct binding to SOCS3 (Li et al., 2021). SOCS3 not only modulates cellular signaling by inhibiting inflammatory cytokines but also interacts with proteins like KEAP1 to directly regulate mitochondrial-induced apoptosis, and may indirectly affect mitochondrial function by influencing the stability and activity of NRF2, underscoring its significance as a potential target for research and therapeutic intervention (Li et al., 2022; Meng et al., 2023). Herefore, even if these two genes are enriched in downregulated NF-κB and TNF pathways, this does not necessarily signify a downregulation of inflammation. Given these insights, further study into these genes and pathways is certainly warranted.

Mitochondrial dysfunction and systemic inflammation may bridge the gap between NAFLD and AF. Literature and our previous study found that mitochondrial dysfunction is involved in the pathogenesis of NAFLD and AF (Chen et al., 2021; Liu et al., 2021). Under metabolic stress conditions, ROS production increases in mitochondria, and once ROS levels exceed the neutralization capacity of its own antioxidant system, it damages the mitochondrial membrane and mitochondrial DNA, causing mitochondrial dysfunction and inducing hepatic inflammation via the nuclear factor-κB (NF-κB) and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP 3) pathways (Chen et al., 2020). When cardiomyocyte mitochondrial dysfunction occurs, ΔΨm decreases, ATP production decreases, leading to the opening of membrane ATP-sensitive potassium channels (sarcKATP), slowing of myocardial localized electrical activity conduction, increasing inhomogeneity, and ease of formation of refractoriness, which induces AF (Lkhagva et al., 2021). In addition, myocardial mitochondria store a large amount of Ca2+, which is a reservoir of intracellular Ca2+, and when mitochondrial dysfunction occurs, the intracellular calcium balance is disturbed, which promotes the occurrence of AF (Liu et al., 2021). It is believed that NAFLD potentially contribute to structural, functional, electrical, and autonomic remodeling of the heart by induce metabolic disorders, inflammation, oxidative stress and promote epicardial adipose tissue (EAT) expansion, which ultimately leading to increased susceptibility to AF (Chen et al., 2021). Inflammation and immune activation are important bases for the development of AF (Coppini et al., 2019). In our study, we revealed an elevation in the populations of CD8 T cells, γδ T cells, and M2 macrophages in the liver of NAFLD patients through CIBERSORT analysis. This information sheds light on the immunological landscape of NAFLD, potentially linking liver inflammation with the systemic inflammatory state observed in these patients. CD8 T cells, also known as cytotoxic T cells, are typically associated with the direct killing of infected or transformed cells. However, in the context of NAFLD, their elevation may contribute to the hepatic inflammatory environment that characterizes this disease, possibly through the secretion of pro-inflammatory cytokines such as TNF-alpha and interferon-gamma (Sutti and Albano, 2020). Niema and others, through fluorescence-activated cell sorting, demonstrated that CD8 + CD28null T lymphocytes are a potent and independent predictive factor for the occurrence of postoperative atrial fibrillation (POAF) after elective cardiac surgery (Kazem et al., 2020). As an unconventional subset of T cells, γδ T cells possess innate-like characteristics and can rapidly produce a variety of cytokines in response to pathogenic or inflammatory stimuli (Lee et al., 2014). Further, certain studies analyzing atrial samples from patients with permanent AF using CIBERSORT have revealed a significant increase in the proportion of γδ T cells (Stone et al., 2021). M2 macrophages, known for their roles in wound healing and tissue repair, have been implicated in the progression of NAFLD, particularly in the development of fibrosis. Their increased numbers may reflect an attempt to control liver inflammation and repair tissue damage, but could also contribute to fibrosis via the production of pro-fibrotic factors (Wei et al., 2022). Research by Chris and his team found an association between the increase in pro-fibrotic M2 macrophages in human atrial tissue and an elevated expression of pre-collagen and anti-fibrotic BNP genes (Watson et al., 2020). Building upon these findings, we propose that the immune cells in NAFLD could release a plethora of inflammatory mediators into the circulation, fostering a state of systemic chronic inflammation. This inflammatory milieu could potentially create a pro-arrhythmic environment, predisposing individuals with NAFLD to the development of AF (Figure 6). In a word, NAFLD promotes AF development as a result of multiple mechanisms, in which mitochondrial dysfunction and systemic inflammation play an important role, but the specific mechanisms need to be explored in more subsequent studies.

Both NAFLD and AF are metabolic-related disorders, underscoring the possibility of intervention through metabolic modulation. Recent studies have revealed that the beneficial effects of SGLT2i on NAFLD seem to be achieved by directly modulating multiple processes, including endoplasmic reticulum stress, oxidative stress, low-grade inflammation, autophagy, and cell apoptosis (Nasiri-Ansari et al., 2021; Feijóo-Bandín et al., 2022; Radlinger et al., 2023). These effects have been confirmed in vitro studies, animal models, and clinical trials (Androutsakos et al., 2022; Feijóo-Bandín et al., 2022). Moreover, real-world studies have found that SGLT2i can reduce the risk of new-onset AF in patients with T2DM and improve clinical benefits in patients with T2DM and AF (Zinman et al., 2015; Zelniker et al., 2020; Hsiao et al., 2022). Randomized controlled trial findings also suggest that SGLT2i can decrease the recurrence rate after AF ablation (Kishima et al., 2022; Zhao et al., 2023). In this study, empagliflozin has been shown to bind well with DGAT1, TYMS, and PTGS2. This suggests a potential avenue for therapeutic interventions targeted at these molecular interactions in the context of NAFLD and AF. Prior studies have indicated that SGLT2i could reduce renal lipid deposition through the inhibition of DGAT1 (Wang et al., 2017). This mechanism may also provide a promising therapeutic approach for NAFLD, given the central role of lipid accumulation in the pathogenesis of this disease. Concurrently, an effective reduction in lipid accumulation could indirectly attenuate the onset or progression of AF, considering the shared metabolic underpinnings of these conditions (Lavie et al., 2017). While there is currently no research specifically examining the interaction of empagliflozin with TYMS and PTGS2, these molecules represent potential therapeutic targets for managing NAFLD and NAFLD-related AF. Given that DGAT1, TYMS, and PTGS2 all play roles in mitochondrial function, the ability of SGLT2 inhibitors like empagliflozin to interact with these molecules suggests they might also exert beneficial effects on NAFLD and AF by modulating mitochondrial dysfunction. While current literature on the direct impact of SGLT2i on mitochondrial function is limited, the implication of these interactions indicates a potential pathway where SGLT2i could improve AF outcomes by addressing mitochondrial dysfunction. Further investigation is required to elucidate this mechanism and its clinical relevance.

This study possesses certain limitations. Firstly, the sample size used for the AF bioinformatic analysis was small. Secondly, the samples we used for analysis and validation were all peripheral blood from patients with AF, not atrial tissue as it's hard to get. Thirdly, the software tools we used for molecular docking studies used simplified models that do not explicitly account for water molecules, ion concentrations, or pH values in the docking process, and can not get the information about their interaction sites, and more advanced simulations that can take more factors into account are needed in the future. Lastly, the absence of animal experiments in our study design presents another limitation. Thus, the role of hub genes and mechanisms needs to be further identified through in vitro or vivo study.

In summary, we firstly discovered that AMOT, PDE11A, TYMS, TMEM98, and PTGS2 are associated with NAFLD-related AF and hold strong diagnostic values. Our study also indicates that mitochondrial dysfunction and inflammation may be potential mechanisms bridging NAFLD and AF. Additionally, we identified empagliflozin as a potentially effective therapeutic agent for NAFLD-related AF at the molecular structure level. These novel insights contribute to the further understanding, diagnosis, and intervention of NAFLD-related AF.