Ion Channels and Atrial Fibrillation: Mitochondrial Recycling as a Key Link

Atrial fibrillation (AF) is a common arrhythmia in clinical practice that often leads to severe complications such as heart failure, myocardial infarction, and stroke. It is associated with increased mortality and a significantly reduced quality of life (Hu et al., 2023; Ruddox et al., 2017). Recent epidemiologic studies confirm that AF remains a significant global public health problem, with notable regional and substantial variations. Over the past 31 years (1990–2021), the global prevalence of AF increased by 1.37-fold, and its incidence rose by 1.24-fold, with 4.48 million new cases reported in 2021, bringing the total number of prevalent cases to 52.55 million (Cheng et al., 2024). Current treatments for AF include risk factor control, medications for rate and rhythm control, and anticoagulation. For refractory cases, interventional procedures like cardiac radiofrequency ablation are used. However, these treatments have limitations, including adverse effects such as bleeding and a significant risk of AF recurrence (Wolfes et al., 2025). Thus, further elucidating the mechanisms of AF development and identifying precise intervention targets are urgently needed.

The pathogenesis of AF has not been fully elucidated, but the core pathological basis for its development and maintenance primarily involves two major mechanisms: atrial electrical remodeling and structural remodeling (Arabia et al., 2024). Electrical remodeling is mainly manifested as abnormal ion channel function in atrial myocytes, resulting in a shortening of action potential duration and increased dispersion of the effective refractory period. This creates a substrate for reentrant arrhythmias. Structural remodeling, on the other hand, involves morphological changes such as atrial fibrosis, myocardial hypertrophy, and dilation, which further promote the persistence and stabilization of AF (Andrade et al., 2014; Nattel and Harada, 2014) Recent studies have confirmed that mitochondrial dysfunction is a central hub driving these remodeling processes. As the energy factories of the cell, mitochondria generate adenosine triphosphate (ATP) through oxidative phosphorylation, providing the necessary energy for sustained contraction, ion pump operation, and electrical signaling in cardiomyocytes. In the AF state, atrial myocytes are subjected to rapid, disorganized, high-frequency electrical excitation. The dramatic increase in energy demand leads to mitochondrial overload and accelerates mitochondrial senescence and damage (Harada et al., 2017; Kohlhaas et al., 2017).

Mitochondrial dysfunction affects intracellular ionic homeostasis and membrane excitability through dual disruptions of energy crisis (ATP insufficiency) and oxidative stress (reactive oxygen species burst). These disruptions directly impair cardiomyocyte ion channel function and expression, driving the onset and progression of AF (Yang et al., 2014; Mason et al., 2020). Insufficient ATP production alters the function of all ATP-dependent enzymes and ion-transport proteins, thereby disrupting normal cellular excitability. Specifically, inhibition of the sodium-potassium pump (Na+/K + -ATPase) leads to an increase in intracellular Na + concentration. This activates the sodium-calcium exchanger in reverse mode, promoting Ca2+ influx and triggering calcium overload (Chung et al., 2024; Chkadua et al., 2024). Simultaneously, ATP deficiency impairs the function of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2a), hindering cytoplasmic calcium reuptake into the SR and further exacerbating calcium overload.

Excess reactive oxygen species (ROS) also play a critical role in this process. They directly cause oxidative modification of key ion channel proteins. Oxidation of ryanodine receptor 2 (RyR2) and inhibition of SERCA activity lead to SR calcium leakage, amplifying calcium overload10. Additionally, ROS inhibit potassium channel function and decrease the expression of Kv currents (IKr, IKs, and IKur), resulting in delayed repolarization, prolonged action potential duration, and a shortened effective refractory period (Cerbai et al., 1991; Li et al., 2008; Nerbonne and Kass, 2005). The increase in the late sodium current (late INa) leads to prolonged action potential duration and increased early afterdepolarizations and aftercontractions, which in turn elevate intracellular Na+ and Ca2+ concentrations, ultimately resulting in electrical and contractile dysfunction (Song et al., 2006). Moreover, high levels of ROS interfere with excitation-contraction coupling, induce cardiac adaptive remodeling via redox-sensitive kinases, and trigger cell death through mitochondrial permeability transition (Bertero and Maack, 2018). Thus, mitochondrial dysfunction is a critical driver in the development of AF, and maintaining mitochondrial functional homeostasis is essential for the prevention and treatment of AF.

Mitophagy, a key mechanism for mitochondrial quality control, selectively removes damaged mitochondria to prevent ROS accumulation and preserve the healthy mitochondrial network. However, chronic AF-related stress (e.g., calcium overload, sustained ROS exposure) can impair mitophagy pathways, resulting in the accumulation of dysfunctional mitochondria (Lin et al., 2024a; Zhu et al., 2022; Sang et al., 2024). Moderate mitophagy maintains cardiomyocyte calcium homeostasis and energy balance, exerting cardioprotective effects (Zhou et al., 2020). Defective mitophagy profoundly affects ion channel function and expression by exacerbating oxidative stress and disrupting calcium homeostasis. This defect constitutes a core pathological link connecting mitochondrial dysfunction to the vicious cycle of AF self-perpetuation ("AF begets AF"). Targeted enhancement of mitophagy or improvement of mitochondrial quality may represent a novel strategy to intervene in the electrical remodeling of AF (Mauriello et al., 2024).

This study combined bioinformatics analysis and experimental validation to uncover key genes and molecular networks underlying the interaction between mitophagy and ion channels in AF. The objective was to elucidate the molecular mechanisms underlying the "mitophagy defects → ion channel dysfunction → electrical remodeling" axis. First, AF-related mitophagy-ion channel genes (AFRMICGs) were identified using differential expression analysis. Functional enrichment analysis was then performed on these AFRMICGs to elucidate their functional characteristics, and immune infiltration analysis was employed to assess their associated immune microenvironment features. Subsequently, multiple machine learning algorithms were applied to identify and characterize Hub gene candidates. Subsequently, an acute electrically remodeled rat model of atrial tachyarrhythmia was constructed to verify, at the in vivo level, mitophagy activity, key ion channel expression/function, and expression changes of the Hub gene candidates. Finally, based on the identified Hub genes, an AF risk prediction nomogram was constructed, and their upstream regulatory networks were thoroughly analyzed, including transcription factor regulation and competitive endogenous RNA (ceRNA) interactions. As gene therapy offers a promising approach to modulate AF's molecular drivers (O'Donnell et al., 2025), this study is expected to identify potential targets for the development of novel antiarrhythmic drugs acting via the mitophagy pathway.

AF remains a major global public health problem, imposing a significant treatment burden due to limited therapeutic options. Therefore, elucidating the pathogenesis of AF to identify precise intervention targets is crucial for improving its management. This study used a bioinformatics approach focusing on mitophagy and ion channels to explore their key genes and potential mechanisms in AF. This study first identified the DEGs in AF and investigated the potential pathogenesis of AF through functional enrichment analysis. The results revealed that the pathogenesis of AF is closely related to inflammation, immune response, ion channels, and various organelles.

Structural and electrical remodeling are currently considered the main pathogenic mechanisms of AF. However, recent studies have highlighted that inflammation and immune response also play significant roles in the development and progression of AF. Inflammation and its associated immune responses have been implicated in the triggering and sustenance of AF episodes. Inflammation-induced AF is perpetuated through a cycle in which it stimulates new inflammatory responses and initiates atrial remodeling. Mediators of the inflammatory response increase susceptibility to AF by altering atrial electrophysiology and structural substrates. Inflammation also disrupts calcium homeostasis and connexin function, thereby increasing triggers for heterogeneous atrial conduction. Additionally, inflammation mediates the activation of fibrotic pathways via fibroblasts, transforming growth factor-β, and matrix metalloproteinases, which promotes myocardial fibrosis (Hu et al., 2015; Harada and Nattel, 2021). A meta-analysis has shown that inflammatory cytokine levels in perioperative patients are significantly associated with the development of postoperative AF (Weymann et al., 2018). Elevated expression of inflammatory factors, such as C-reactive protein, interleukin-6, interleukin-2, interleukin-1β, and tumor necrosis factor-α, has been significantly and positively correlated with the incidence of AF (Hu et al., 2015; Harada and Nattel, 2021). The activation of NLRP3 inflammasomes is observed in cardiomyocytes from patients with AF, contributing to AF by recruiting immune cells and enhancing atrial structural and electrical remodeling39. Immune infiltration analysis in this study revealed significant dysregulation of the immune microenvironment in the left atrial tissues of patients with AF. Specifically, a high level of infiltration of neutrophils and a lower abundance of anti-inflammatory M2 macrophages were observed in the atrial myocardium of these patients. These findings suggest the presence of a chronic inflammatory state in AF patients. Immune cells, including T cells, macrophages, and neutrophils, mediate cardiac inflammation by infiltrating the myocardium from peripheral tissues and inducing localized inflammatory responses (Yao et al., 2018; Li and Brundel, 2020; Yao et al., 2022). In conclusion, inflammation and immune response play a crucial role in the pathogenesis of AF. However, the exact mechanisms through which these processes contribute to the development and progression of AF remain incompletely defined and require further investigation.

Ion channel abnormalities are equally important in the pathogenesis of AF. In the normal cardiac cycle, electrical impulses originating from the sinoatrial node propagate through the atria, atrioventricular node, His bundle, and Purkinje fibers to trigger synchronized ventricular contraction. Myocardial action potentials depend on sequential activation/inactivation of depolarizing (Na⁺, Ca²⁺) and repolarizing (K⁺) ion channels. During depolarization, Ca²⁺ influx via voltage-gated channels triggers RyR2-mediated calcium-induced calcium release (CICR) from the SR. This calcium binds troponin C, initiating cardiomyocyte contraction. Diastole requires membrane repolarization (driven by Kv and Kir channels) and calcium clearance via SERCA and NCX10, (Roden et al., 2002; Heijman et al., 2014). In vivo experiments in this study revealed significant dysregulation of ion channel gene expression in the Ach-CaCl₂-induced rat model of AF. Specifically, sodium channel (SCN5A/Nav1.5), potassium channel (KCNA5/Kv1.5, KCNH2/hERG), and calcium channel (CACNA1C/Cav1.2, CACNA1G/Cav3.1, CACNA1H/Cav3.2) gene expression was significantly downregulated, while SLC8A1/NCX1 and CAMK2/CaMKII gene expression was significantly upregulated. Sodium channel SCN5A (Nav1.5) downregulation reduces the phase 0 depolarization rate and conduction velocity. Potassium channel (KCNA5/Kv1.5, KCNH2/hERG) downregulation prolongs action potential duration and increases repolarization dispersion. Calcium channel (CACNA1C/Cav1.2, CACNA1G/Cav3.1, CACNA1H/Cav3.2) downregulation shortens action potential duration. Upregulated SLC8A1 (NCX1) generates net inward currents, delaying repolarization and increasing electrical instability. Activated CAMK2/CaMKII exacerbates calcium overload (Roden et al., 2002; Heijman et al., 2014). These findings suggest that atrial myocytes in the Ach-CaCl₂-induced rat model of AF undergo typical processes of electrical remodeling and calcium-regulated remodeling. Electrical remodeling is characterized by downregulation of sodium, potassium, and calcium channels, leading to conduction slowing, prolonged action potential duration, and repolarization dissociation. Calcium-regulated remodeling involves upregulation of NCX1, activation of CaMKII, calcium overload, calcium leakage, arrhythmogenic inward currents, and delayed afterdepolarizations.

Mitochondrial dysfunction is a key mechanism driving electrical remodeling, as it promotes AF episodes by impairing cardiomyocyte ion channel function and expression through energy deficiency and ROS bursts. Studies have confirmed that targeting mitochondrial function can effectively alleviate AF (Ge et al., 2025). Mitophagy, a key component of mitochondrial quality control, helps to improve overall mitochondrial function by removing dysfunctional mitochondria. The key proteins of the ubiquitin-dependent pathway are PINK1 and Parkin, constituting PINK1/Parkin-mediated mitophagy. PINK1, a kinase on the mitochondrial outer membrane, is usually degraded after translocation to the inner membrane. When mitochondria are damaged and depolarized, PINK1 accumulates on the outer membrane, dimerizes, and autophosphorylates to activate. Activated PINK1 phosphorylates Parkin, activating its E3 ligase activity to ubiquitinate mitochondrial proteins. This process facilitates the recognition and clearance of damaged mitochondria. In contrast, non-ubiquitin-dependent mitophagy is mediated by receptors like NIX, BNIP3, and FUNDC1 on the mitochondrial outer membrane. Upon damage, these receptors bind to LC3II via their LC3-binding domains, thereby anchoring damaged mitochondria to the autophagosomal membrane and initiating mitophagy (D'Arcy, 2024; Lu et al., 2023). This study found that PINK1, Parkin, FUNDC1, BNIP3, NIX, LC3I/MAP1LC3A, and LC3II/MAP1LC3B genes were downregulated in the Ach-CaCl₂-induced rat model, suggesting a significant impairment of mitophagy. Therefore, moderate activation of mitophagy is considered a potential therapeutic mechanism for AF.

Functional enrichment analysis of AFRMICGs revealed that the PI3K/AKT signaling pathway is a key regulator linking mitophagy to ion channel function in AF. The PI3K/AKT signaling pathway is a critical cellular pathway that modulates a wide range of biological processes, including cell growth, proliferation, survival, metabolism, migration, and apoptosis. The role of the PI3K/AKT pathway in AF has been extensively studied, yet its specific effects appear to vary considerably across different experimental models and species. For instance, in several animal models of AF, such as heart failure-induced AF in rats (Chong et al., 2015) and rabbits (Liu et al., 2016), the spontaneously hypertensive rat model (Wang et al., 2015), the canine model of rapid atrial pacing (Zhao et al., 2021), the diabetes-induced AF rat model (Xue et al., 2020), and the Ach-CaCl₂-induced rat model (Xu et al., 2024; Zhang et al., 2025a), the expression of PI3K and AKT was significantly downregulated. Conversely, in the Angiotensin II (Ang II)-induced AF mouse model, the expression of PI3K and AKT was significantly upregulated (Li et al., 2025; Chen et al., 2024; Ji and Ning, 2024). A finding by Badreldin et al., which reported upregulation of PI3K and AKT in an Ach-CaCl₂-induced rat model, is inconsistent with the majority of previous studies (Badreld et al., 2024).

The role of the PI3K/AKT signaling pathway in regulating mitochondrial function, autophagy, and ion channels, such as calcium and sodium channels, has been partially elucidated in the context of AF. For instance, Xu et al. demonstrated that activation of the PI3K/AKT signaling pathway improved mitochondrial function in the Ach-CaCl₂-induced rat model. This activation restored the activity of Ca²⁺-ATPase and downregulated the expression of p-CaMKII/CaMKII, p-RyR2/RyR2, and NCX1, while upregulating SERCA2 and Cav1.2 expression (Xu et al., 2024). In another study, ibrutinib-treated HL-1 cells exhibited increased susceptibility to AF due to the inhibition of the PI3K/AKT/mTOR signaling pathway, which enhanced autophagy and led to connexin degradation (Qin et al., 2024). Additionally, the PI3K/AKT signaling pathway was shown to prevent cellular remodeling in rapidly paced HL-1 atrial myocytes by inhibiting late sodium currents (Ko et al., 2023). Moreover, laminar shear stress was found to upregulate KCa2.3 expression in H9c2 cardiomyocytes through activation of the PI3K/AKT/p300 pathway, thereby affecting potassium-calcium channel function (Li et al., 2019). In a rabbit model of heart failure-induced AF, activation of the PI3K/AKT pathway led to upregulation of ion channels such as KvLQT1, Cav1.2, and SERCA2a, which improved atrial electrical remodeling and reduced fibrosis (Liu et al., 2016).

Subsequently, 65 machine learning models were constructed, among which glmBoost + Lasso was confirmed as the optimal model based on ROC curves, accuracy, and F1 scores. Through in vivo experimental validation, BAX, MIF, and TLR4 were ultimately identified as Hub genes for AF.

BCL-2-associated X protein (BAX), a key pro-apoptotic member of the BCL-2 family, significantly contributes to the pathophysiology of AF. Studies have demonstrated that BAX expression is markedly upregulated in AF patients and animal models, including rats and dogs (Li et al., 2018; Jia et al., 2017; Yuan et al., 2021). BAX levels correlate positively with the severity of AF, with higher levels observed in permanent AF compared to persistent and paroxysmal AF in patients with rheumatic heart disease and in persistent AF than in paroxysmal AF (Diao et al., 2016). Furthermore, elevated BAX expression in a senescent AF model suggests its potential as a biomarker for aging-related AF (Xu et al., 2013). BAX's interaction with mitophagy primarily involves the regulation of apoptosis. Upon apoptotic stimulation, BAX accumulates on the outer mitochondrial membrane, forming oligomers that enhance mitochondrial outer membrane permeability, leading to the release of pro-apoptotic factors such as cytochrome c, thereby initiating apoptosis. This mitochondrial outer membrane permeability-mediated mitochondrial swelling exposes the inner mitochondrial membrane, activating mitophagy via a PINK1-Parkin-independent pathway (Saunders et al., 2024). While this process may serve as a compensatory mechanism for clearing damaged mitochondria, sustained high BAX expression can disrupt the balance between autophagy and apoptosis. Additionally, BAX may play a role in AF electrical remodeling by affecting calcium cycling. It has been shown to trigger endoplasmic reticulum ryanodine receptors (RyRs), facilitating the release of Ca²⁺ from the endoplasmic reticulum into the mitochondrion (SanMartín et al., 2020). Conversely, BAX deficiency results in decreased mitochondrial Ca²⁺ uptake, highlighting BAX as a crucial regulator of calcium homeostasis (Scorrano et al., 2003).

Macrophage Inflammation Factor (MIF) is a pro-inflammatory cytokine released by activated macrophages, which has been shown to be elevated in patients with AF and correlates with disease severity. Specifically, MIF levels are highest in patients with permanent AF, followed by those with persistent AF, and lowest in those with paroxysmal AF (Wan and Li, 2018; Li et al., 2020). Mendelian randomization analysis has confirmed a causal link between MIF and AF (Wei et al., 2023). MIF modulates calcium homeostasis and ionic currents by affecting L-type and T-type calcium channels (Rao et al., 2013; Rao et al., 2009). In HL-1 cardiomyocytes, an in vitro model, MIF treatment induced enhanced calcium transients, increased SR calcium content, elevated sodium/calcium exchanger efflux rate, calcium leak, transient outward potassium current, and ultra-rapid delayed rectifier potassium current (Cheng et al., 2020). MIF induces abnormal sodium and calcium regulation through activation of the CaMKII signaling pathway and ROS, which contributes to the triggering of AF during inflammation (Chin et al., 2023). MIF also inhibits mitophagy; it has been found that loss of autophagy/mitophagy leads to the accumulation of cytoplasmic ROS and mitochondrial DNA, which activates immune signaling pathways and ultimately leads to the release of MIF (Harris et al., 2018). In a septic kidney injury model, MIF inhibits mitophagy by disrupting the PINK1-Parkin interaction (Li et al., 2024a). However, another study shows that MIF acts as a protective factor against myocardial contractile dysfunction caused by side-stream smoke exposure by promoting mitophagy and the formation of autophagolysosome (Wang et al., 2020). Additionally, MIF appears to exert a certain improvement effect in myocardial fibrosis (Zhao et al., 2025; Zhu et al., 2021). Thus, MIF plays a pivotal role in AF pathogenesis, influencing inflammation, mitophagy, ion channels, and myocardial fibrosis. Targeting MIF and its signaling pathways may offer therapeutic strategies for AF management, potentially alleviating symptoms and improving patient outcomes.

Toll-like Receptor 4 (TLR4), a member of the Toll-like Receptor family and a Pattern Recognition Receptor within the human immune system, has been linked to AF. Studies have reported elevated TLR4 expression in AF patients (Gur et al., 2018; Gurses et al., 2016), with particularly high levels observed in those with persistent AF compared to paroxysmal AF, indicating a potential correlation between TLR4 and AF severity81. TLR4 is emerging as a potential biomarker for new-onset AF following acute myocardial infarction, with significantly increased levels observed in patients experiencing new-onset AF compared to healthy controls and those with myocardial infarction alone (Zhang et al., 2018). In hypertensive rats, silencing TLR4 expression has been shown to reduce left atrial fibrosis and susceptibility to AF (Ge et al., 2022). Furthermore, MD1, a negative regulator of TLR4, when deleted, enhances AF susceptibility and induces atrial remodeling in diabetic cardiomyopathy mice through activation of the TLR4/NF-κB signaling pathway (Shi et al., 2023). TLR4 plays a crucial role in regulating intracellular ionic homeostasis and currents by influencing sodium (Li et al., 2024b), potassium (Bowen et al., 2024), L-type and T-type calcium channels (Li et al., 2017; Zhang et al., 2025b), and calcium homeostasis (Zhu et al., 2024). In a high-fat diet-induced AF model, MD1 knockdown promotes atrial remodeling in AF mice by activating the TLR4 signaling pathway, which modulates the expression of CaMKII, MyD88, and NF-κB, thereby affecting the expression of Kv4.2, Kv4.3, Kv1.5, Kv2.1, and Cav1.2 channel proteins, and regulates potassium-calcium channels (Shuai et al., 2019b; Shuai et al., 2019a). HMGB1 disrupts excitation-contraction coupling in cardiomyocytes by increasing SR Ca2+ leak via the TLR4-ROS signaling pathway (Zhang et al., 2014). TLR4 has been implicated in the regulation of mitophagy in conditions such as atherosclerosis (Chang et al., 2025), heat stroke-induced brain injury (Lin et al., 2024b), and acute pancreatitis-associated splenic injury (Wen et al., 2022), although its specific role in AF is not fully understood. Furthermore, TLR4-mediated inflammatory responses are crucial in regulating myocardial hypertrophic remodeling (Xiao et al., 2020). Studies have shown that cardiac-specific deletion of TLR4 reduces the severity of Ang II-induced myocardial fibrosis (Theobald et al., 2023). This suggests that modulating the TLR4 signaling pathway could be an effective strategy for managing the pathophysiology of cardiac hypertrophy.

This study employed integrated bioinformatics analyses to identify key genes and mechanisms linking mitophagy and ion channel dysfunction in AF. This study's results establish the PI3K/AKT signaling pathway and genes BAX, TLR4, GLUL, and MIF as central regulators (Hub genes). Validation in an Ach-CaCl₂-induced rat model confirmed dysregulation of mitophagy and sodium/potassium/calcium channel-related genes in atrial tissue, with BAX, TLR4, and MIF further identified as core AFRMICG Hub genes. Upstream regulatory networks (transcription factors, ceRNA interactions) for these Hub genes were also explored using public databases. These findings provide valuable mechanistic insights into AF pathogenesis and highlight potential therapeutic targets.

However, this study has several limitations. First, to address the heterogeneity across AF datasets, the analyses were restricted to training sets from a single platform (GPL570) and region (China: GSE41177, GSE79768). This approach may introduce population bias and limit the generalizability of the findings to other populations. Second, only expression differences of the Hub genes were validated in rats, without further functional investigation. Furthermore, the use of exclusively male animals in the experimental validation, though intended to minimize variability from the estrous cycle, represents a significant limitation. This design precludes assessment of potential sex-dependent differences in the mitophagy-ion channel axis, thus limiting the applicability of the results to females. Previous studies have shown that AF patients of different genders exhibit distinct biomarkers (Wu et al., 2024). Additionally, the isoflurane anesthesia used during tissue collection in rats, while standard, is known to modulate various cardiac ion channels. Its potential influence on the electrophysiological state at the time of sampling cannot be entirely ruled out, which may confound the interpretation of ion channel-related findings. Therefore, the changes in these genes in human AF patients, particularly across different sexes, require further validation to confirm their broad clinical relevance. Moreover, the study lacks gain- and loss-of-function experiments to elucidate the causal roles of BAX, MIF, and TLR4 in regulating mitophagy and ion channels. Finally, while the study implicates a mitophagy-ion channel axis, the specific molecular interactions and functional consequences require experimental validation.

In addition, it is important to note a limitation regarding the in vivo model. The Ach-CaCl₂ induction method can produce a spectrum of atrial tachyarrhythmias. While electrocardiogram patterns consistent with AF were observed, characterized by irregular f waves and variable R-R intervals, some recorded episodes also displayed electrocardiographic features of atrial flutter. Both AF and atrial flutter share common pathophysiological substrates, such as electrical and structural remodeling. Therefore, the key findings of this study regarding the dysregulation of mitophagy and ion channels are interpreted in the context of a sustained, rapid atrial tachyarrhythmia model, which robustly recapitulates critical aspects of the clinical arrhythmogenic substrate.

To address these limitations, future research should expand the datasets to include male and female subjects from diverse ethnic and geographic backgrounds, enabling the development of more comprehensive and generalizable models of AF mechanisms. Subsequent studies should conduct sex-stratified analyses to systematically explore potential differences in gene expression, pathway activity, and immune infiltration between sexes. Additionally, gain- and loss-of-function experiments in vitro and in animal models are essential to establish the causal roles of the identified hub genes (BAX, MIF, TLR4) and to dissect the functional interplay within the mitophagy-ion channel axis. Finally, future work should explicitly address the impact and biological implications of sex differences, emphasizing this as a key factor for understanding AF pathophysiology and developing personalized therapeutic strategies.

BAX, MIF, and TLR4 are key genes linking mitophagy and ion channels in AF, which appear to influence the immune microenvironment by modulating immune cell infiltration.