Introduction
Atrial fibrillation (AF), a surging global health care burden, affects nearly 1–4% of the adult population in the USA, and this number is expected to exceed 13% for individuals older than 80 years of age [
1]. Valvular heart disease (VHD) is also a common disease worldwide [
2]. The common causes of VHD are degeneration and some modifiable risk factors (such as elevated blood pressure and lipid profiles) in higher income countries, whereas rheumatic heart disease is a common cause in developing regions [
3]. Importantly, AF patients with concomitant VHD are at higher risk of stroke and systemic embolism than those without VHD [
4].
Autophagy is a self-digesting mechanism that maintains cellular homeostasis by eliminating unnecessary or dysfunctional cellular components [
5,
6]. The Human Autophagy Database (HADb) is a web-based resource, that provides a comprehensive and up-to-date list of human genes and proteins involved in autophagy [
7]. Previous studies have reported that autophagy is a potential novel mechanistic contributor to the pathological processes of AF genesis [
8,
9]. Nakano et al. showed that AF patients displayed a significant decrease in the expression level of mitochondrial ALDH2, which regulates cardiac autophagy [
10]. These findings suggest that autophagy and AF may have underlying relationships. Additionally, immune cell infiltration in the atrial myocardium is common in patients with either lone AF or valvular AF [
11,
12]. A previous study observed that activated T lymphocytes (CD3
+ and HLA-DR
+) were significantly up-regulated in the peripheral blood of AF patients compared with individuals with sinus rhythm (SR) (36% vs 27%;
P < 0.001) [
13]. Moreover, this up-regulation was reversed when SR was maintained after cardioversion at follow-up [
13]. These findings suggested that immune infiltration may play an essential role in AF development. In addition, cross talk between components of autophagy and immunity has also been reported; regulators of autophagy control regulators of inflammation, and vice-versa [
14]. However, the communicative regulatory mechanisms of autophagy and immunity in the initiation and maintenance of AF remain unknown.
In this study, a protein–protein interaction (PPI) network of differentially expressed autophagy-related genes (DEARGs) was constructed, and hub genes were revealed. To determine the functions of DEARGs in valvular AF, we constructed a valvular AF-related TF/mRNA/miRNA network by integrating all TF-DEARG and miRNA-DEARG interactions. In addition, we used the CIBERSORT algorithm to estimate fractions of the immune cell subpopulations in VHD samples [
15]. Finally, we conducted co-expression analysis with DEARGs and immune cells to identify the underlying regulatory mechanisms in AF genesis.
Discussion
In the present study, we applied network analysis and the CIBERSROT algorithm to identify biologically significant DEARGs and immune cells related to AF genesis. We revealed the key correlated regulators of six DEARGs (BECN1, GAPDH, ATG7, MAPK3, BCL2L1, and MYC) and three immune cell subpopulations (T cells CD4 memory resting, T cells follicular helper, and neutrophils) that were unique to patients who developed AF after VHD.
Through meta-analysis of associated genome-wide association studies, Ellinor et al. identified six AF susceptibility loci (PRRX1, CAV1, SYNE2, FBP1/2, HCN4, and SYNPO2L-MYOZ1) involved in cardiac electrical and structural remodeling [
22]. In addition, performing a bioinformatics analysis, Zou et al. found four co-expressed genes (ZNF566, PDZK1IP1, ZFHX3, and PITX2) significantly associated with AF-related stroke [
23]. In this study, we identified six autophagy-related genes (BECN1, GAPDH [
24], ATG7, MAPK3 [
25], BCL2L1, and MYC [
26,
27]) associated with AF genesis. Currently, numerous studies have revealed that cardiovascular diseases are associated with autophagic genes, both positively [
28] and negatively [
29]. For example, a recent study demonstrated that FAK-mediated phosphorylation of BECN1 negatively regulated cardiomyocyte autophagy, thereby initiating hypertrophic cardiac growth [
30]. Down-regulation of GAPDH reduced H9C2 cardiomyoblast death following acute hypoxia and reoxygenation injury [
31]. A study by Yuan et al. [
32] showed that ATG7 expression was up-regulated in the atria of AF patients and rabbit models of rapid atrial pacing. In addition, lentivirus-mediated ATG7-knockdown in rabbits was found to protect against atrial electrical remodeling in intracardiac experiments [
32]. In an acute myocardial infarction rat model, miRNA-15b was demonstrated to deteriorate cardiomyocyte apoptosis by post-transcriptionally down-regulating the expression of BCL-2 and MAPK3 [
33]. High-dose administration of chlorpromazine led to an elevated expression level of BCL2L1 and various cardiovascular disorders, such as arrhythmia and myocardial fibrosis [
34]. The up-regulation of c-MYC has been shown to be a central component in the Wnt/β-catenin/c-MYC axis mediated cardiac remodeling abnormalities in heart failure [
35].
Currently, pharmacologic therapy of AF is based on anti-arrhythmic drugs, and the interventional treatment is mostly radiofrequency catheter ablation [
36]. Other effective preventive medications are needed to curb the occurrence of AF, and some drugs have been reported to induce apoptotic effects in various diseases targeting these identified DEARGs. For instance, Wei et al. described apogossypol derivatives that inhibit antiapoptotic Bcl-2 family proteins [
37]. Meanwhile, Germann et al. showed that ulixertinib, targeting the MAPK3 signaling pathway, reduced the proliferation and enhanced the caspase activity of sensitive cancer cells [
38].
To comprehensively investigate the biological function of the DEARGs in valvular AF, we performed functional enrichment analysis. The results showed that autophagy genes were significantly enriched in autophagic and inflammatory signaling pathways. These results were consistent with a previous study, which demonstrated that the DEGs identified between the AF-VHD and SR-VHD groups were primarily associated with inflammatory responses [
23]. The function of TFs and miRNAs is to regulate gene expression, which is closely involved in the genesis and progression of valvular AF. In our study, the TF/mRNA/miRNA network analysis revealed that most hub genes were associated with two TFs (MYC and TP53), and 35 miRNAs mainly targeted 3 DEARGs (ATG7, BCL2L2 and MYC). We hypothesized that TFs and miRNAs might be critical for AF development by regulating hub gene expression. Additional studies are needed to explore the specific mechanism of TFs and miRNAs in valvular AF.
Previous studies have reported that autophagy and immune infiltration are closely linked to the development and progression of AF [
9,
39,
40]. However, no systematic investigation or research has been conducted to elucidate the communicative functions of autophagy and immune infiltration in VHD patients who develop AF. Thus, we comprehensively analyzed the potential mechanisms of DEARGs and immune infiltration cells in AF-VHD, which has rarely been the foci of prior studies.
We also found that three immune cell subpopulations (T cells CD4 memory resting, T follicular helper cells, and neutrophils) were related to AF genesis in VHD patients. Previous studies reported that neutrophils constitute the majority of the inflammatory cells in AF patients undergoing pericardiotomy, atriotomy, or catheter ablation [
41,
42]. Moreover, an elevated neutrophil–lymphocyte ratio in postoperative AF patients was highly consistent with the correlation results in our study. The level of neutrophils was inversely correlated with that of CD4 T cells. In addition, CD4 T cells (T cells CD4 memory resting and T follicular helper cells) have different roles during chronic inflammation, and their activation might be mediated through interactions with Toll-like receptor 2 (TLR2) and TLR4 [
40,
43]. However, little is known about the specific mechanism of CD4 T cells in the pathogenesis of AF. To confirm our conclusions, experimental mechanistic research should be carried out both in vitro and in vivo in future studies.
The role of autophagy can be seen in a range of cell types involved in immunity, such as lymphocytes, dendritic cells (DCs) and myeloid cells, which contribute to inflammatory responses in diverse pathophysiological processes [
14]. BECN1 knockdown in mesenchymal stem cells lead to autophagy suppression, inducing inhibitory effects on T lymphocyte infiltration [
44]. In mice and humans, an immunomodulatory drug, dimethyl fumarate, inactivates the catalytic cysteine of GAPDH, which activates myeloid and lymphoid cells [
45]. IL-17A was reported to positively impact microglial autophagy and inflammation by promoting the essential autophagy gene ATG7 [
46]. Compared to the DCs of MAPK3(
+/
+) mice, the DCs of MAPK3 (
−/
−) mice possessed a superior capacity to activate and prime naïve T cells into a functional phenotype [
47]. Autophagy also plays an essential role in maintaining Treg cells. Inhibition of c-MYC following autophagy deficiency causes Treg cell apoptosis and lineage instability [
48]. In summary, we inferred that DEARGs might have significant roles in the occurrence of AF by regulating innate and adaptive immunity through these immune cells. More research directly investigating molecular mechanisms is required to validate the communication between those DEARGs and immune cells.
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