Diabetes causes oxidative stress and contractile dysfunction in the heart. Previous studies have confirmed that autophagy is inhibited by diabetes, which in turn increases endoplasmic reticulum stress and phosphorylation of pro-hypertrophic signaling molecules [12]. Advanced glycation end products (AGE) play a role in diabetic cardiac dysfunction. Autophagy is inhibited by knocking out the AGE receptor, endothelial-to-mesenchymal transition is suppressed, and thereby cardiac fibrosis are attenuated [13]. Defective autophagy and NLRP3 (NOD-like receptor pyrin domain-containing-3) inflammatory vesicle activation caused by diabetes mellitus, cardiac remodeling after myocardial infarction exacerbated by excessive secretion of pro-inflammatory cytokines [14]. Impaired autophagy is associated with cardiac metabolic dysregulation and oxidative stress in diabetic patients and is an important cause of cell death, fibrosis, and cardiac dysfunction [15].

Autophagy has been shown to be involved in the mechanisms of occurrence in a range of age-related diseases, including cardiovascular disease. Autophagic activity declines with aging, which may be one of the causes of age-related cardiac dysfunction [16]. Autophagy is involved in the organismal lifespan; impaired autophagy is associated with age-related mitochondrial dysfunction and the resulting accumulation of reactive oxygen species (ROS) [17]. Autophagy has been shown to be an important component of intracellular homeostasis, limiting ROS production by degrading dysfunctional mitochondria [18]. Impaired mitochondrial autophagic clearance has multiple consequences, including increased risk of arrhythmia, reducing contractile reserve, and increasing inflammation; age-related dysregulation of autophagy is central to the link between autophagy, ROS, and aging [16].

Inflammation is associated with many mechanisms related to the development of multiple diseases, including cardiovascular disease. Autophagy has been shown to regulate the degree of inflammation. Autophagy is accompanied by activation of inflammatory vesicles and moderates inflammation by eliminating active inflammatory vesicles [19]. Stimulation of interferon gene over-expression may mitigate cardiac inflammation by pressure overload through inhibition of autophagy [20]. Interventions, including the use of autophagy inhibitors, can lead to higher interleukin-1$\beta$ (IL-1$\beta$) levels [21]. Autophagy and inflammation are interdependent processes.

Gap junctions and ion channels form a pathway for electrical conduction between cardiomyocytes (Fig. 2). Changes in the structure, function, and characteristics of the atrial ion channels are thought to be pivotal mechanisms of AER [3]. Connexin (Cx) forms gap junctions and is responsible for the propagation of cardiac action potentials between cardiomyocytes [4]. Cx40 and Cx43 are the most important members of the connexin family. Reduced numbers or abnormal distribution of Cx43 may result in different impedances and conduction velocities between cardiomyocytes, in turn resulting in micro-reentrant and AF susceptibility [30]. Connexins have an unexpectedly short half-life of only 1–5 h, raising the question of how cardiomyocytes regulate connexin degradation so quickly. In recent years, several studies have demonstrated that autophagy is involved in connexin degradation. Several studies on different tissues, primary cultured cells, and cell lines have shown that gap junctions are influenced by constitutive and induced autophagy. Ultrastructural analyses have revealed cytoplasmic annular gap junctions inside phagophores, indicating that autophagy is a connexin-degradation pathway [31]. Chen et al. [32] found that the mammalian target of rapamycin (mTOR) inhibitor rapamycin promoted autophagy, further inhibiting the protective effect of apelin-13 on Cx43. Their finding demonstrated that increased autophagy inhibited cardiac Cx43 expression [32]. Thus, regulation of autophagic flux to preserve Cx43 remodeling may represent a new strategy for AF management.

Fig. 2.

Schematic diagram of autophagy involved in atrial electrical remodeling (AER). Rapid atrial rates increase the influx of Ca${}^{2+}$ into atrial cells at each action potential, leading to calcium overload, and then inducing atrial fibrillation (AF). AF can also lead to calcium overload. Activated autophagy induced by rapid atrial rates and ERS induces a decrease in action potential duration (APD) and a shortened atrial effective refractory period (AERP) via ubiquitin-dependent selective degradation of Ical. Shortened APD and atrial fibrillation reinforce each other in a vicious cycle. Activated autophagy promotes connexin degradation, leading to different impedances and conduction velocities between cardiomyocytes, resulting in micro-reentrant and inducing AF. ERS, endoplasmic reticulum stress; Ical, L-type calcium channel; Cx, connexin.

Cardiac depolarization activates voltage-dependent calcium channels on the myocardial membrane and calcium ions enter the cell, inducing ryanodine receptor 2 (RyR2) opening on the sarcoplasmic reticulum, thereby triggering a calcium transient. In the AF model of the right atrium in fast-pacing dogs, the openness of the RyR2 receptor was significantly increased in the atrial-fibrillation group. Therefore, the increased openness of the RyR2 receptor is speculated to lead to the leakage of sarcoplasmic reticulum Ca${}^{2+}$ into the cytoplasm during diastole, thereby causing AF [33]. Autophagy markers and the key Atg7 of cardiomyocytes are significantly increased in patients with AF. Inhibition of autophagy by lentivirus-mediated Atg7 knockdown, or the autophagy inhibitor chloroquine (CQ), restores the shortened AERP and alleviates the AF vulnerability caused by tachypacing in rabbits [34]. High-frequency electrical stimulation of fast-pacing atria also activates autophagy of cardiomyocytes and promotes autophagy-mediated degradation of the L-type calcium channel protein by ubiquitin protein, further shortening the APD and AERP. These processes make AF more likely to occur and persist [34]. Endoplasmic reticulum stress (ERS)-associated autophagy can induce AER, which appears as L-type calcium channel reduction [35]. By fluorescent immunostaining, we found a significant increase in the expression of the autophagy marker LC3 in the left atrial tissue of rats after myocardial infarction, and a significant reduction in protein expression of the L-type calcium channel $\alpha$1c, voltage-gated sodium channel 1.5, and voltage-activated A-type potassium ion channel 4.3; however, this was completely reversed by the small-molecule integrated stress-response inhibitor [36].

Angiotensin II (Ang II) and transforming growth factor-$\beta$1 (TGF-$\beta$1) are important molecules for atrial fibrotic remodeling in AF [37]. Ang II is a major effector of the renin-angiotensin system and plays an important role in the regulation of collagen deposition and interstitial fibrosis. Ang II increases collagen secretion from atrial fibroblasts; enhanced fibroblast autophagy is also observed. When the autophagy inhibitors 3-methyladenine or chloroquine (CQ) were used to block autophagy, collagen secretion was significantly reduced, suggesting that activated autophagy may promote myocardial fibrosis [40]. Homoplastically, primary human atrial myofibroblasts were treated with TGF-$\beta$1 to assess fibrogenic and autophagic responses. The results showed that TGF-$\beta$1 led to an elevation of autophagic markers and fibronectin, and pharmacological inhibition of autophagy decreased the fibrotic response, these results support that TGF$\beta$1-induced autophagy increases the fibrogenetic response [41]. Inflammatory processes lead to renin-angiotensin-aldosterone system activation and electrical and structural remodeling. Previous studies have shown that this inflammatory condition is often accompanied by releasing of ROS, which is closely related to the development and progression of cardiac fibrosis [42]. A recent study demonstrated that excessive ROS increases autophagosome formation by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mTOR signaling pathway, and eventually enhanced cardiomyocyte apoptosis [43]. Therefore, autophagy may be considered as a potential mechanism for the formation of atrial fibrosis.

Excessive extracellular matrix deposition in the cardiac interstitium is characteristic of cardiac fibrosis. Inhibition of myocardial fibrosis is a potential approach to prevent and treat AF. Increasingly, studies have shown that promoting autophagy can inhibit myocardial fibrosis and improve cardiac function. Ang II has been established as an effective inducer of extracellular matrix protein. Autophagy also promotes excessive collagen degradation, which attenuates Ang II-induced cardiac fibrosis [44]. Adiponectin (APN), also known as adipocyte complement-related protein, has been shown to possess anti-inflammatory properties and to attenuate myocardial fibrosis. The inhibitory effect of APN on cardiac inflammation and fibrosis may be achieved by enhancing autophagy in macrophages [45]. In addition, in AF model cells and rat myocardial tissues, isoprenaline induces myocardial fibrosis, whereas quercetin-activated autophagy reverses this process [46].

Previous studies have shown that autophagy is critical for the regulation of extracellular matrix homeostasis. Autophagy is activated by many cellular signaling factors such as oxidative stress, volume overload, and inflammation, and it directly degrades collagen and fibronectin. The autophagic degradation of collagen and fibronectin induces adverse cardiac remodeling [47]. Overactive adrenergic stimulation has been demonstrated to stimulate fibroblast autophagic procollagen digestion, which contributes to reduced fibrosis by regulating the extracellular matrix [48]. Autophagy-associated collagen degradation is a compensatory mechanism for myocardial fibrosis. In contrast, autophagy suppresses cardiac fibrosis by regulating the secretion of pro-fibrotic factors. TGF-$\beta$1 stimulates fibroblast growth and extracellular matrix production, resulting in dysfunction of atrial tissues. The secretion of TGF-$\beta$1 strictly depends upon secretory autophagosome intermediates, in fibroblasts and macrophages, as carriers. Notably, all treatments interfering with autophagosome formation inhibited TGF-$\beta$1 release [49]. Osteopontin (OPN), which contributes to various tissue fibrosis processes, is highly expressed in the circulation of patients with AF. It promotes atrial fibrosis in vitro, and further increases with the progression of AF. In vitro studies in human atrial fibroblasts demonstrated that OPN activates AKT, a suppressor of autophagy, which suppresses autophagy and increases atrial collagen I and fibronectin production. However, this process was reversed by rapamycin, an autophagy inducer [50].

Proper protein folding is a key mechanism for maintaining cell and tissue function; the strict regulation of the production and folding of these proteins is known as proteostasis. Cells have a precise cellular protein quantity control (PQC) network. However, if the protein is unable to fold correctly, the body will digest and degrade the proteins through the ubiquitin-proteasome system or autophagosome-lysosome pathway, which prevents downstream dysfunction [54]. Autophagy maintains cellular proteostasis by regulating PQC. On the one hand, autophagy mitigates the accumulation of misfolded proteins by degrading potentially toxic molecules and organelles in cardiomyocytes. On the other hand, autophagy acts as a cellular recycling program to recycle amino acids, lipids, and other molecular building blocks liberated from substrates with the help of lysosomal acid hydrolases [55]. The critical role of proteostasis in AF has been extensively demonstrated in numerous studies [56]. Endoplasmic reticulum stress is a protective stress response to the accumulation of misfolded and unfolded proteins and has been observed to increase autophagy and to promote atrial remodeling in animal models and in the cardiac tissue of patients with AF (Fig. 4A). In vivo treatment with the chemical chaperone 4-phenyl butyrate, an inhibitor of ERS, prevents autophagy activation, protects atrial-tachypacing canine cardiomyocytes from electrical remodeling, and attenuates AF progression [35]. In a H9c2 cardiomyoblast model of ERS, TGF-$\beta$ activates ERS autophagy and cell injury, whereas an autophagy inhibitor protects H9c2 cardiomyoblasts from myocardial fibrosis by attenuating ERS autophagy [57]. Notably, similar results were obtained after treatment with another autophagy inhibitor. Tetrameric DsRed (a red fluorescent protein) causes proteinopathy and cellular injuries, and it has been shown to promote severe fibrosis. In transgenic mice expressing tetrameric DsRed, the ubiquitin-proteasome system and autophagy-lysosome systems were impaired and overburdened [58]. The derivation of proteostasis leading to atrial remodeling is an important cause of the self-sustained characteristics of AF, and autophagy may be the pivotal mechanism for maintaining protein homeostasis.

Fig. 4.

Schematic diagram of autophagy involved in proteostasis and mitochondrial function. (A) Autophagy protects cardiomyocytes from atrial fibrillation (AF) by degrading unfolded and misfolded proteins. But there are other views that ERS-induced autophagy also increases AF susceptibility by aggravating myocardial fibrosis. (B) Rapid atrial pacing leads to mitochondrial fragmentation and impaired function, which in turn leads to increased mt-ROS and decreased ATP. Impaired mitophagy causes a decrease in dysfunctional mitochondria as the potential mechanisms. mt-ROS, mitochondrial reactive oxygen species; TGF-$\beta$1, transforming growth factor-$\beta$1; ATP, adenosine-triphosphate; ERS, endoplasmic reticulum stress.

Atrial structural remodeling is rooted in derailment of the homeostasis of production, function, and breakdown of proteins, including degradation of structural and contractile proteins. Decreased levels of acetylated $\alpha$-microtubulin in atrial tissue of patients with paroxysmal and persistent atrial fibrillation coincide with increased histone deacetylase-6 (HDAC6) expression and activity [59]. Restoration of the microtubule network is the key to recovery of structural and functional reconstruction after rapid pacing [60]. HDACs modulate atrial cardiomyocytes Ca${}^{2+}$ signaling and contractility by regulating protein acetylation status of $\alpha$-tubulin. Activated HDAC6 was observed in tachypacing HL-1 atrial cardiomyocytes, and the same transformation was observed in atrial tissue from patients with AF. Fast pacing induces HDAC6-specific activation leading to $\alpha$-microtubulin deacetylation, depolymerization, and degradation [61]. McLendon et al. [62] showed that hyperacetylated tubulin is an adaptive response that can enhance autophagy in the face of proteotoxicity. In addition, Song et al. [63] also found that ɑ-tubulin hyperacetylation protected cardiomyocytes against doxorubicin-induced acute damage by recovering impaired autophagy flux. AF induces remodeling partly through HADC6 activation; derailment of ɑ-tubulin proteostasis may be a potential mechanisms and therapeutic target for AF.

Fatty acid oxidation is the main energy source of cardiomyocytes, and elevated levels of serum-free fatty acids are strong risk factors and outcome predictors of AF and AF-related stroke [64]. Increased expression of genes related to atrial fatty acid metabolism in patients with AF positively correlates with atrial structural- and electrical-remodeling levels [65]. Thus, abnormalities in atrial fatty acid metabolism are involved in structural and electrical remodeling of the atria, which may contribute to the future development or recurrence of atrial fibrillation. Autophagic vesicles selectively degrade excess intracellular lipids to maintain the balance of fatty acid metabolism; this is called “lipophagy” [66]. Autophagy prevents the accumulation of toxic fatty acid metabolites (e.g., diacylglycerols) in cells by degrading intracellular lipids [65]. Lipophagy, a unique form of selective autophagy, is a fundamental mechanisms of lipid clearance [53]. Previous research has shown that high-fat intake aggravates cardiac remodeling, including hypertrophy and interstitial fibrosis. Mitochondrial aldehyde dehydrogenase (ALDH2) serves an indispensable role in protecting against cardiac anomalies from high-fat-induced obesity by enhancing autophagy [67]. Modifying autophagy-mediated protein clearance is a potential new strategy for regulating cardiac lipoprotein lipase levels, which regulate fatty acid levels in the heart [68]. Dysfunction of energy metabolism in the atrial tissue may be one of the pathogenic mechanisms of AF.

The relationship between diabetes mellitus and AF is well established. Enhanced p62 levels and decreased levels of autophagy-related gene 7 (ATG7) were observed in cardiac cells treated with high levels of glucose, indicating impaired autophagy, whereas ALDH2 alleviated diabetes-induced cardiac mechanical and geometric changes, which are mediated by the regulation of autophagy [69]. Significant deposition of collagen fibers, significant increase of senescent cells in myocardial tissue, significant inhibition of cellular autophagy, increased aging of myocardial cells, and increased fibrosis in diabetic myocardium were observed in myocardial tissue of diabetic rats [69]. Autophagy is activated in diabetic rats during cardiac fibrosis, and autophagic activity is correlated with cardiac fibrosis [70]. In a rat model of type 2 diabetes, trimetazidine therapy enhanced cardiac autophagy and reduced myocardial fibrosis and cardiomyocyte apoptosis [71].

The source of adenosine-triphosphate (ATP) in cardiac tissue is critically dependent on mitochondrial oxidative phosphorylation. Mitochondria are organelles of cardiomyocytes associated with energy metabolism. Accumulation of dysfunctional mitochondria significantly increases the production of mitochondrial reactive oxygen species (mt-ROS), initiating the endogenous cell death process and resulting in myocardial cell damage. Mitochondrial dysfunction leads to reduced ATP production, metabolic dysregulation, increased ROS production, and the activation of apoptotic cascades, which are associated with the development of arrhythmogenic atrial electroanatomical remodeling [72]. Increasing evidence suggests that mitochondrial dysfunction is associated with AF. Rapid atrial pacing created an animal model of AF, and AERP, AF induction rate, and mitochondrial DNA were determined [73]. Reduced mitochondrial DNA content and downregulation of mitochondrial respirators were observed in rapidly paced atria, demonstrating a dysfunction of mitochondrial energy metabolism [73]. Atrial biopsies from patients with AF also exhibited aberrant ATP levels, upregulation of mitochondrial stress chaperones, and fragmentation of the mitochondrial network [74]. Montaigne et al. [75] found that patients with impaired mitochondrial function in the atrial myocardium had a higher incidence of AF after coronary artery bypass graft surgery, indicating that damaged mitochondrial accumulation may be a potential factor for the occurrence of AF.

Selective autophagic removal of mitochondria (mitophagy) is a crucial homeostatic mechanism that promotes proper functioning of the mitochondrial network. The elimination of dysfunctional mitochondria through mitophagy could contribute to maintaining normal function and the number of mitochondria [72]. Mitochondrial quality and content are properly regulated by mitochondrial autophagy during cell differentiation. Inhibition of B-cell lymphoma-2 (BCL2) interacting protein 3 like (BNIP3L)- and FUN14 domain containing 1 (FUNDC1)-mediated mitochondrial division during differentiation leads to sustained mitochondrial fission and donut-shaped impaired mitochondria [76]. However, both excessive reduction and increase in mitochondrial clearance contribute in part to cardiovascular dysfunction. Mitophagy is involved in metabolic remodeling of several cardiovascular diseases, such as cardiac hypertrophy, myocardial infarction, ischemia/reperfusion, heart failure, hypertension, and diabetic cardiomyopathy [77]. Mitochondrial autophagy is essential for maintaining the balance between mitochondrial degradation and accumulation. Increasing evidence has revealed that mitochondrial dysfunction provides an arrhythmogenic substrate for the development and perpetuation of AF [57, 74, 75]. Based on these findings, it is reasonable to suppose that impaired mitophagy may contribute to AF (Fig. 4B). Patients with chronic AF with defective cardiac autophagy and an increased number of dysfunctional mitochondria provided convincing evidence that impaired mitophagy is a potential mechanism of human chronic AF [5].