Ang II Enhances Atrial Fibroblast Autophagy and Promotes Atrial Remodeling Through the AT1-ERK-mTOR Signaling Pathway


 Background: Atrial remodeling is a common pathological change in atrial fibrillation (AF), while the mechanism of atrial remodeling remains unclear. In the present study, we investigated the autophagy and collagen secretion in atrial fibroblasts in response to renin-angiotensin system (RAS) activation and elucidated the relationship between atrial fibroblast autophagy and atrial remodeling. Methods: Right atrial tissues was obtained from patients that underwent cardiac valve replacement after signed the informed consent form. In vivo, subcutaneous perfusion of angiotensin II (Ang II) was used to mimic activation of the RAS and induce atrial remodeling. Electrical remodeling and AF induction were assessed by electrophysiology and programmed stimulation. In vitro, atrial fibroblasts were isolated and cultured. Autophagic flux changes were assessed by mCherry‑GFP‑LC3 adenovirus transfection. All samples were collected, collagen expression, autophagy changes and atrial remodeling were evaluated by western blot, masson’s trichrome staining, immunohistochemistry and immunoﬂuorescence mainly.Results: Atrial tissue samples from patients with atrial fibrillation showed more collagen deposition and enhanced autophagy than those with sinus rhythm. Chronic subcutaneous Ang II perfusion in mice promoted atrial remodeling and susceptible to AF induction. In cultured atrial fibroblasts, the expression of collagen I (COL-I) and collagen III (COL-III) and autophagy both increased when cells were treated with Ang II, and the autophagic flux was enhanced by Ang II. However, blocking autophagy reduced the expression of COL-I and COL-III. Besides, Ang II induced the phosphorylation of ERK and suppressed the phosphorylation of mTOR. In contrast, inhibition of the angiotensin II type 1 receptor (AT1) or ERK signaling pathway not only suppressed the autophagy induced by Ang II but also reduced COL-I and COL-III expression. Conclusions: In summary, these results suggest that Ang II promotes autophagy in atrial fibroblasts, which aggravates atrial remodeling and increases the susceptibility to AF induction. Autophagy may be a potential target for relieving atrial remodeling and AF after RAS activation.


Introduction
Atrial brillation (AF) is one of the most common tachyarrhythmia in clinical practice, which can induce heart failure and stroke [1]. At present, the treatment of AF focuses on rhythm control, rate control and anti-coagulation. However, both of them only relieve symptoms and decrease the incidence of stroke, while the alteration of atrial substrate is ongoing and leads to AF that is di cult to cure and easy to relapse. The mechanism of AF is complicated, and atrial remodeling is a key point from the perspective of pathophysiology [2]. Electrical remodeling occurs in the early stage of AF, while structural remodeling, such as extracellular matrix (ECM) deposition, occurs as AF progresses. The abnormal ECM production and degradation lead to atrial interstitial brosis, which has been veri ed to aggravate the development of AF [3,4]. The deposition of collagen in the atrial interstitium not only causes atrial structural changes but also affects electrical conduction [5]. Therefore, improving atrial remodeling is a pivotal part in the treatment of AF. cytoskeleton (Denver, Colorado). Phosphatase inhibitors were purchased from Roche (Basel, Switzerland). mCherry-GFP-LC3 was purchased from ViGene (Jinan, China). Antibodies for collagen III (COL-III, ab7778), p62/SQSTM1 (ab109012) and AT1 (ab124734) were purchased from Abcam (Cambridge, UK). Collagen I antibody (COL-I, GTX20292) was purchased from GeneTex (California, USA). Beclin 1 antibody (11306-1-AP) was purchased from Proteintech (Wuhan, China). Vimentin antibody (EM0401) was purchased from Huabio (Hangzhou, China). Phospho-mTOR (2971), mTOR (2972), phospho-ERK (4370) and ERK (4377) antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts). LC3-II antibody (L7543), DMSO and chloroquine diphosphate salt (CQ) were purchased from Sigma (St. Louis, USA). 3methyladenine (3-MA), candesartan cilexetil and PD98059 were from MedChemExpress (Monmouth, NJ). GAPDH antibody (ab011-100), goat anti-rabbit (GAR007) and goat anti-mouse (GAM007) horseradish peroxidase (HRP)-IgG were obtained from Multi Sciences (Hangzhou, China). TRITC-conjugated goat antirabbit antibody (A16115) and Alexa 488-conjugated goat anti-mouse antibody (A-11001) were purchased from Thermo Fisher Scienti c.

Human atrial tissue
Right atrial tissues were obtained from patients who underwent cardiac valve replacement. All patients and at least one family member of each donor signed the informed consent form. The application for collecting and using human samples for science research was approved by the Human Research Ethics Committee of Sir Run Run Shaw Hospital of Zhejiang University. This human sample study conformed to the Declaration of Helsinki. The exclusion criteria included hypertension, heart failure, coronary artery diseases, hyperthyroidism, systemic in ammatory diseases, malignancy and history of angiotensinconverting enzyme inhibitors or angiotensin receptor blockers. Sixteen patients with sinus rhythm and sixteen patients with persistent AF (> 6 months) were enrolled, and their right atrial tissues were obtained. The atrial tissue was collected and cleaned in ice-cold normal saline. Each sample was divided into two parts. One was xed with 4% paraformaldehyde for histological analysis, and the other was saved at -80℃ for RNA and protein extraction. The baseline characteristics of these patients are described in Table 1.

Enzyme-linked immunosorbent assay (ELISA)
The patient's central venous blood was taken and centrifuged immediately. Human serum was saved at -80℃. The concentration of Ang II in serum was measured by ELISA following the manufacturers' instructions.

Echocardiography
Mice were anesthetized by iso urane, and echocardiography was performed using a Vevo 1100 ultrasound system (Visual Sonics, Toronto, Canada) with a 30 MHz transducer as previously described [25]. M-mode images were obtained from a parasternal short-axis view. The parameters measured were as follows: heart rate (HR), interventricular septum at end-diastole (IVS-d left ventricular fractional shortening (LVFS) were calculated. All measurements were averaged from at least three consecutive cardiac cycles.

In vivo electrophysiology and programmed stimulation
The electrophysiology and programmed stimulation in vivo were done as previously described [26,27]. In brief, mice were anesthetized with 0.3% sodium pentobarbital solution and a subcutaneous administration of 0.03 mg/kg buprenorphine hydrochloride to relieve pain. The subdermal needle electrodes were placed in the legs to make a lead II conformation. The right external jugular vein was separated carefully, and a 1.1 F electrophysiology catheter, which included eight electrodes (Transonic Scisense, Canada), was inserted into the right atrium through the jugular vein. The surface electrocardiogram (ECG) and intracardiac ECG were observed. The correct catheter position was ensured by obtaining a sole ventricular signal in the distal lead and a predominant atrial signal in the proximal lead. The atrial effective refractory period (AERP) was determined by applying eight training stimuli (S1) at a cycle length of 100 ms, followed by an extra stimulus (S2). The S1-S2 interval was progressively reduced by 2 ms in each pacing train from 70 ms. AERP was de ned as the longest S1-S2 coupling interval for atria that failed to generate a propagated beat with S2. After each stimulation protocol, there was a recovery period of at least 30 seconds. AF inducibility was tested by a burst pacing protocol in the right atrium. In other words, three trains of 2 s burst pacing were used as follows: the rst 2 s burst was given at a cycle length of 40 ms with a pulse duration of 5 ms. Then, mice stabilized for 3 minutes. The second 2 s burst pacing was set as a cycle length of 20 ms with a pulse duration of 5 ms. Another 3 minutes of stabilization applied. The last 2 s burst pacing was applied at a cycle length of 20 ms with a pulse duration of 10 ms. AF was de ned as irregular R-R intervals without P waves for at least 1 s on the surface electrocardiogram (ECG). All data were acquired by a cardiac electrophysiology stimulator and a multichannel electrophysiological recording system (Transonic Scisense, Canada). All animals were sacri ced by cervical dislocation when the experiment nished.

Masson's trichrome staining
Human and mouse atrial tissues were embedded in para n after xation with 4% paraformaldehyde solution and sliced into 4-µm-thick sections. Tissue sections were stained with Masson's trichrome according to the manufacturer's instructions. Microscopy images were captured by Leica image analysis software (Leica, Germany). The atrial interstitial brotic areas were calculated by Image-Pro Plus software (version 6.0; Media Cybernetics, USA). The collagen volume fraction was calculated as collagen area/total area⋅100%.

Immunohistochemistry
Mouse atrium was harvested and xed with 4% paraformaldehyde solution. Para n-embedded tissues were cut into 4-µm-thick sections. Immunohistochemical staining was performed using the antibodies for COL-I and COL-III (1:200) at 37℃ for 1 hour and then secondary antibody at 37℃ for 30 min. Then, the sections were visualized with a DAB (Gene Tech, China) method. Microscopy images were captured by Leica image analysis software (Leica, Germany).

Immuno uorescence Staining
Atrial tissue sections were stained with antibodies for LC3-II (1:50) and vimentin (1:100) overnight at 4℃. TRITC-conjugated goat anti-rabbit antibody and Alexa 488-conjugated goat anti-mouse antibody (1:500) were applied as secondary antibodies and incubated for 2 hours at 37℃. Cell nuclei were stained with DAPI for 10 min at room temperature. All uorescence images were captured by laser scanning confocal microscopy (Nikon, Japan) under the same conditions.
2.10 Quantitative real-time polymerase chain reaction (Q-PCR) The RNA of human atrial tissues (100 mg) was extracted by using Trizol (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. cDNA was synthesized using a reverse transcription kit (Takara, Dalian, China) and used for subsequent qPCR. All samples were measured in triplicate.

Cell culture
Atrial broblasts were isolated from mouse atria, as previously described [28,29]. Cell culture was followed the methods of Xu et al. 2019 [30]. For chemical stimulations, cells were pretreated with different concentrations of rapamycin, LY294002, CQ, PD98059 or candesartan for 1 hour and induced with Ang II (10 − 6 µM) for 48 hours. For phosphorylated protein measurements, cells were pretreated with PD98059 (10 µM) and candesartan (10 µM) for 1 hour and stimulated with Ang II for 20 min, and cells were harvested for protein detection.
The mixed reagent was added into each well. Negative-control siRNAs were used in the experiment. The effect of siRNA was assessed by q-PCR and Western blot. After transfection, atrial broblasts were stimulated with Ang II similarly to previous experiments in this study.

Ad-mCherry-GFP-LC3 transfection
To observe autophagic ux changes induced by Ang II, atrial broblasts were transfected with mCherry-GFP-LC3 adenovirus as described previously [30]. Cells were pretreated with candesartan, PD98059 or siRNA (targeting AT1) as described above and then stimulated with Ang II. At that time, the cells were xed with 4% paraformaldehyde. Cell nuclei were stained with DAPI for 10 min at room temperature. Images were captured by laser scanning confocal microscopy (Nikon, Japan) under the same conditions. The average numbers of red and yellow puncta (merged channel) from 30 atrial broblasts in each group were counted manually in ve independent experiments. Autophagosomes, which express both green and red uorescence, appeared yellow, and autolysosomes appeared only red because GFP was degraded under acidic conditions. 2.14 Western blot analysis Western blot analysis was followed the methods of Xu et al. 2019 [30] 2.15 Statistical analysis The data analysis was conducted using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). Normal distribution data are expressed as the mean ± standard error of the mean (SEM). Abnormal distribution data are expressed as median and interquartile range by nonparametric test (Mann-Whitney test). Comparisons between two groups were performed with an unpaired student t test. Comparisons more than two groups were performed using one-way ANOVA followed by Bonferroni correction. Fisher's exact test was applied to compare AF inducibility. P < 0.05 was regarded as statistically signi cant.

Enhanced autophagy and more serious interstitial brosis in AF patients' atrial tissues
Persistent activation of RAS promotes atrial brosis and increases the susceptibility to AF. In the present study, we collected the atrial tissue of sinus rhythm and atrial brillation patients. First, we tested the concentration of Ang II in patients' serum. As shown in Fig. 1e, the concentration of Ang II was higher in the AF group than in the SR group. Masson's trichrome staining showed heavier collagen deposition in the atrial interstitial in the AF group, and the collagen volume infraction was approximately 26.7% in the AF group and 19.5% in the SR group (Fig. 1a). We examined the expression of COL-I and COL-III and autophagy markers in two groups of atrial tissues. Both COL-I and COL-III had higher expression in the AF group compared to the SR group at the protein and mRNA levels (Fig. 1b, 1c and 1d), which was consistent with the Masson's trichrome staining results. The expression of LC3-II and Beclin 1 was increased in the AF group, in contrast, the expression of p62 was decreased in the AF group. These changes suggested that autophagy was activated in the AF patients. Further, we found a higher expression of COL-I and COL-III and increased autophagy in AF patients compared to SR patients. Therefore, we speculated that there may be a potential relationship between atrial brosis and autophagy.

Role of autophagy and AT1 in Ang II-induced cardiac remodeling and dysfunction in vivo
Consecutive perfused of Ang II was used to mimic the effect of RAS activation. Cardiac remodeling was evaluated by echocardiography preliminary. The results showed that IVS-d was increased when mice were perfused with Ang II. However, the increased IVS-d was relieved when autophagy was suppressed with 3-MA or AT1 was blocked with candesartan compared to the Ang II group. Similarly, Ang II perfusion with or without rapamycin decreased the LVEF and LVFS, but these were upregulated in groups treated with 3-MA or candesartan, although not signi cantly in the autophagy-inhibited group (3-MA + Ang II) compared to the Ang II group. Additionally, the ratio of heart weight to body weight (HW/BW) was increased in the Ang II and autophagy-enhanced (Rapa + Ang II) groups. 3-MA and candesartan both suppressed the increase in HW/BW induced by Ang II (Table 2). These results indicate that consecutive perfusion with Ang II promoted cardiac remodeling and dysfunction. In contrast, inhibiting autophagy or blocking AT1 both improved the cardiac dysfunction induced by Ang II. 3.3 Inhibiting autophagy or blocking AT1 reduces the susceptibility to AF induction and improves the atrial electrical remodeling induced by Ang II Atrial electrical remodeling occurs in the early stage of AF, which manifests as a reduced AERP, reduced action potential duration and changes in ion channels in cell membrane such as L-type calcium channels, Na + channels and K + channels [31,32]. In this study, we detected AERP and AF inducibility in vivo. As shown in Fig. 2b, the rate of AF inducibility was increased when the mice were perfused with Ang II with or without Rapa. In contrast, the rate of AF inducibility was decreased in the autophagy inhibition group (3-MA + Ang II). We also found that the rate of AF inducibility was reduced when AT1 was inhibited by candesartan (Can). Shortened AERP contributed to the formation of reentry and led to AF occurrence. In this study, the electrophysiological examination showed that AERP was shortened in the Ang II-and autophagy-enhanced groups (Rapa + Ang II); however, this change was reversed when autophagy was repressed by 3-MA or AT1 was blocked by candesartan (Fig. 2c). That is, inhibition of autophagy or AT1 improves the atrial electrical remodeling and reduced the AF inducibility induced by Ang II.
3.4 Inhibiting autophagy or blocking AT1 reverses the deposition of collagen and the expression of COL-I and COL-III in the mouse atrium induced by Ang II.
Collagen deposition in atrial interstitium not only accelerates atrial structural remodeling but also causes abnormal atrial electrical conduction. To investigate collagen deposition in the atrial interstitium induced by Ang II, we detected the collagen volume fraction (CVF) by Masson's trichrome staining. The results showed that CVF was increased in Ang II with or without rapamycin. 3-MA, as an autophagy inhibitor, reduced CVF. That is, blocking autophagy reduced the collagen production induced by Ang II. Additionally, blocking AT1 with candesartan also decreased CVF, which implied that Ang II induced collagen secretion through AT1 (Fig. 3a and 3b). Meanwhile, we analyzed the expression of COL-I and COL-III in the mouse atrium. Similar to CVF, Ang II increased the expression of COL-I and COL-III in the atrium. Enhancing autophagy with Rapa (Rapa + Ang II) also upregulated COL-I and COL-III. However, the expression of COL-I and COL-III were both decreased when mice were treated with 3-MA, an autophagy inhibitor, or candesartan, an AT1 antagonist (Fig. 3c). Taken together, these results suggest that Ang II promoted collagen deposition in atrium. Inhibiting autophagy or blocking AT1 both reduced collagen production and ameliorated atrial remodeling induced by Ang II.
3.5 Candesartan relieves atrial broblast autophagy that is induced by Ang II in mice.
The proportion of heart cells that are broblasts is approximately 40%-60%, and broblasts are the main cell type that secretes collagen [33]. The role of broblasts is important in cardiac brosis and has attracted the attention of researchers [34,35]. To detect changes in atrial broblast autophagy when mice were perfused with Ang II, double immuno uorescence staining of sections from mice atrium was used. Atrial broblasts were labeled with vimentin, a broblast marker, and LC3-II was stained to re ect autophagy. As shown in Fig. 4, the expression of LC3-II was increased in vimentin-positive atrial broblasts in the Ang II group and autophagy-enhanced group (Rapa + Ang II) compared to the control group. However, the increased expression of LC3-II was relieved when autophagy was inhibited by 3-MA or AT1 was blocked by candesartan. These results indicate that Ang II activated atrial broblast autophagy, which was inhibited by candesartan, an AT1 antagonist, and that Ang II-induced atrial broblast autophagy may occur through AT1.

Ang II promotes the expression of COL-I and COL-III and activates autophagy in atrial broblasts.
Ang II is the major effector of RAS, which plays an important role in cardiovascular diseases, including hypertension, cardiac remodeling and interstitial brosis [36]. COL-I and COL-III, the primary ingredient of the extracellular matrix, are regarded as the key biomarkers of cardiac brosis [37]. In this study, the expression of both COL-I and COL-III by atrial broblasts was increased in response to Ang II stimulation ( Fig. 5a and 5b). On the other hand, we found that Ang II also elevated the expression of LC3-II, and decreased the expression of p62 in cultured atrial broblasts ( Fig. 5c and 5d). That is, Ang II activated autophagy in atrial broblasts. These results suggest that Ang II, an effector of RAS, upregulated autophagy and promoted COL-I and COL-III secretion in atrial broblasts.
3.7 Atrial broblast autophagy is positively correlated with the expression of COL-I and COL-III.
The preceding results suggested that Ang II-stimulated COL-I and COL-III expression accompanied increased autophagy in atrial broblasts. To clarify whether atrial broblast autophagy was associated with COL-I and COL-III expression. Rapamycin (Rapa), a mammalian target of rapamycin (mTOR) inhibitor, is widely used as an autophagy inducer. Ang II upregulated the expression of COL-I and COL-III in atrial broblasts. Intriguingly, the expression of COL-I and COL-III was further evaluated when cells were pretreated with rapamycin compared to Ang II stimulation alone ( Fig. 6a and 6d). As expected, COL-I and COL-III was downregulated when autophagy was curbed by CQ, a lysosome acidi cation inhibitor ( Fig. 6b  and 6e). To con rm this effect, LY294002, a PI3-kinase inhibitor, which also inhibits autophagy, was used.
In alignment with the CQ results, the expression of both COL-I and COL-III both decreased when atrial broblasts were pretreated with LY294002 ( Fig. 6c and 6f). Beclin 1 is a pivotal gene in the initial autophagy process. Therefore, we designed a small interfering RNA targeted to Beclin 1 to disturb the process of autophagy. In agreement with the pharmacological experiment, atrial broblast autophagy induced by Ang II was inhibited, and the expression of both COL-I and COL-III was reduced when broblasts were treated with siRNA targeted to Beclin 1 (Fig. 6g, 6 h and 6i). These results con rm that Ang II stimulated COL-I and COL-III secretion by activating autophagy, and this effect was reversed apparently when atrial broblast autophagy was suppressed.

Blocking ERK signaling reduces the expression of COL-I and COL-III and inhibits atrial broblast autophagy induced by Ang II by upregulating the phosphorylation of mTOR.
Extracellular signal-regulated kinase (ERK) is associated with cell proliferation and differentiation, which is a regulator of autophagy [38,39]. We found that the phosphorylation of ERK was increased when atrial broblasts were stimulated with Ang II. PD98059, a speci c inhibitor of ERK, not only reduced the expression of COL-I and COL-III but also suppressed atrial broblast autophagy ( Fig. 7a and 7b). mTOR is the most important target of autophagy regulation, in which it acts as a negative regulator [40]. In the present study, we found that phosphorylation of mTOR was decreased under stimulation with Ang II. Interestingly, this variation was rescued when atrial broblasts were pretreated with PD98059 ( Fig. 7c and   7d). This indicated that enhanced autophagy in atrial broblasts induced by Ang II may be related to the ERK signaling pathway.
3.9 Blocking AT1 reduces the expression of COL-I and COL-III and suppresses atrial broblast autophagy induced by Ang II through the ERK-mTOR signaling pathway.
Ang II affects the cardiovascular system predominantly via AT1. A previous study reported that the angiotensin II receptor acted as a regulator of autophagy in cardiomyocytes [24]. Therefore, we investigated whether Ang II-induced autophagy in atrial broblasts relied on AT1. As expected, the expression of both COL-I and COL-III induced by Ang II was decreased when atrial broblasts were pretreated with candesartan, a speci c AT1 antagonist. Moreover, autophagy was inhibited, as re ected by the decreased expression of LC3-II and increased expression of p62 ( Fig. 8a and 8b). Furthermore, candesartan alleviated the phosphorylation of ERK and enhanced the phosphorylation of mTOR ( Fig. 8c  and 8 g). To further verify the effect of AT, the siRNA targeted to AT1 was designed and transfected to knockdown AT1 (Fig. 8f). Results consistent with the candesartan stimulation were acquired. The knockdown of AT1 reduced COL-I and COL-III production and inhibited broblast autophagy induced by Ang II (Fig. 8e and 8 h). Besides, ERK phosphorylation declined and mTOR phosphorylation elevated when AT1 was silenced ( Fig. 8d and 8i). In summary, Ang II induced collagen secretion and autophagy activation through AT1. Blocking AT1 contributed to improving collagen production and atrial interstitial brosis.
3.10 Ang II induces autophagic ux in atrial broblasts which is reversed by PD98059 and AT1 disturbance.
To further investigate the complex in uences of AT1 and the ERK signaling pathway on autophagy, a special marker, mCherry-GFP-LC3, was utilized in an adenovirus. Cultured atrial broblasts were transfected with the adenovirus for 24 hours. Then, cells were stimulated with Ang II or pretreated with candesartan or PD98059 for 1 hour before stimulation with Ang II. As shown in Fig. 9, yellow puncta represent autophagosomes, and red puncta represent lysosomes in the merged channel. The yellow and red puncta were conspicuously augmented when broblasts were stimulated by Ang II, which represented the enhanced autophagic ux. As expected, the enhanced autophagic ux was suppressed when cells were pretreated with candesartan and PD98059. A similar result was obtained when AT1 was silenced by siRNA. These results re ect the variation of atrial broblast autophagic ux dynamically and directly.
They also verify the autophagy alteration in atrial broblasts.

Discussion
In the present study, we demonstrated the relationship between autophagy and AF, and the principal discoveries were as follows: rst, compared to SR patients, more signi cant collagen deposition and enhanced autophagy were detected in AF patients' atrial tissues. Second, overloaded Ang II aggravates atrial remodeling and AF inducibility in vivo, and enhances broblast autophagy. Meanwhile, blocking autophagy or antagonizing AT1 can relieve atrial remodeling and reduced AF inducibility. Third, Autophagic ux was also increased in response to Ang II. The enhanced autophagy was a positivefeedback response to Ang II stimulation. In contrast, secretion of COL-I and COL-III was reduced when autophagy was restrained. Moreover, the results showed that Ang II induced autophagy through the AT1-ERK-mTOR signaling pathway in atrial broblasts. Antagonists targeted to AT1 and ERK both upregulated the phosphorylation of mTOR, further suppressed autophagy and decreased COL-I and COL-III expression (Fig. 10).
Collagen bers exist in the myocardial interstitium, which helps to maintain the structure of the heart. However, excessive collagen production and deposition in the atrial interstitium result in atrial structural remodeling. Atrial structural remodeling not only gives rise to a lasting atrial tissue injury but also affects atrial electrical conduction [41,42]. Atrial broblasts are easily activated by various stimuli and then proliferate and differentiate into myo broblasts, which secrete amount of collagen that deposits in the atrial interstitium and trigger further atrial remodeling [43]. Numerous studies have documented the link between atrial interstitial brosis and AF [3,4]. Everett et al. reported that atrial remodeling occurred in the chronic canine AF model and that electrical and structural dysfunction aggravated the susceptibility to AF [44].
In the cardiovascular system, the alteration of autophagy was detected in various diseases, such as heart failure, cardiac hypertrophy and ischemia-reperfusion injury [45]. Considering the controversial conclusions about autophagy in cardiovascular diseases [46], it is valuable to illustrate the characteristics of autophagy in AF. In the atrium of persistent AF patients, researchers found that autophagic ux and ATG7 protein, an important regulator of autophagy, both increased. A similar phenomenon was observed in a rabbit model with rapid atrial pacing [47]. Another study also reported that autophagy was induced upon endoplasmic reticulum (ER) stress and that suppression of ER stress contributed to the inhibition of autophagy and protection against atrial remodeling in AF models both in vivo and in vitro [14]. As a consequence, they noted that excessive activation of autophagy occurred in advanced AF. Nevertheless, the mechanisms of autophagy in atrial remodeling are still unclear. In the present study, we further con rmed that heavier collagen deposition was accompanied by upregulated autophagy in AF patients' atrium. In the Ang II-perfused mouse model, atrial remodeling was aggravated by heavier collagen deposition in the atrial interstitium. In addition, atrial broblast autophagy was increased by colocalization of vimentin and LC3 (Fig. 4). In vivo electrophysiological examination revealed a shortened AERP and increased susceptibility to AF induction in response to Ang II. However, collagen deposition was relieved when autophagy was inhibited by 3-MA. Improved AERP and AF inducibility were also observed when autophagy was inhibited (Fig. 2). The autophagic ux in atrial broblasts was also increased in response to Ang II (Fig. 9). In summary, we concluded that suppressing autophagy could ameliorate atrial remodeling induced by Ang II and reduce the susceptibility to AF induction.
Ang II is the principal effector of the RAS, and studies have con rmed that Ang II promotes cardiac interstitial brosis by inducing collagen secretion in rat cardiac broblasts [48,49]. Additionally, Ang II can shorten the AERP, which is reversed by candesartan [50]. In the present study, Ang II was used to mimic the activation effect of the RAS, and it induced COL-I and COL-III expression in atrial broblasts by provoking autophagy (Fig. 5). COL-I and COL-III expression was further augmented when provoking atrial broblast autophagy with an mTOR inhibitor, rapamycin. In contrast, the opposite phenomenon occurred when blocking autophagy by multiple methods (Fig. 6). In summary, the data suggest that atrial autophagy contributes greatly to Ang II-induced atrial remodeling.
It is well recognized that AT1 is the target through which Ang II exerts its effect. Porrello et al. demonstrated that Ang II induced cardiomyocyte autophagy through AT1, and overexpression of AT1 strongly upregulated cardiomyocyte autophagy [24]. That is, AT1 is an upstream regulator of autophagy.
What interested us was whether Ang II induced atrial broblast autophagy also through AT1. The results showed that autophagy and collagen secretion both decreased when AT1 was blocked (Fig. 8). Meanwhile, candesartan, a speci c AT1 inhibitor, restrained atrial broblast autophagy induced by Ang II in vivo (Fig. 4). In addition, disturbing AT1 diminished the autophagic ux in atrial broblasts (Fig. 9). On the whole, these data showed that AT1 is the key molecular that regulates atrial broblast autophagy and collagen secretion induced by Ang II.
It was reported that the ERK signaling pathway was activated in AF patients' atrial tissue and that ACEI inhibited this alteration in ERK [51]. Transgenic mice overexpression the human pro-renin receptor gene exhibited spontaneous AF after 10 months, with serious cardiac brosis. Further study found that ERK phosphorylation was speci cally increased in cardiac broblasts [52]. In brief, these studies suggested that the ERK signaling pathway plays an important role in atrial remodeling and AF. Based on these studies, we found that blocking the ERK signaling pathway not only reduced collagen production, but also restrained autophagy in response to Ang II stimuli (Fig. 7).
Autophagy has a complicated regulatory mechanism involving various signaling molecules. mTOR is a direct negative regulator of autophagy [53]. In vascular smooth muscle cells, autophagy is inhibited by the activated Akt-mTOR signaling pathway [54]. In cardiomyocytes, activated mTOR exerts a protective effect in ischemia/reperfusion injury by restraining autophagy [55]. In summary, mTOR is a pivotal upstream regulator of autophagy in the cardiovascular system. In the current study, Ang II induced broblast autophagy by suppressing the phosphorylation of mTOR. In turn, the prevention of AT1 or ERK both rescued the alterations of mTOR (Figs. 7 and 8). In addition, the autophagic ux induced by Ang II in atrial broblasts was reduced (Fig. 9). In summary, Ang II induced atrial broblast autophagy and further promoted collagen secretion via the AT1-ERK-mTOR signaling pathway in the current study.
From the perspective of translational medicine, the present study identi ed a potential bene t of pharmacological inhibition of autophagy as a therapeutic strategy in clinical AF. Autophagy is a complicated regulatory system that can be either physiologic or pathologic. The absence of a speci c pharmacological inhibitor of autophagy may be an immense de ciency in clinical medicine. The number of enrolled patients was slightly insu cient in this study, but combining previous studies adequately demonstrates that autophagy is upregulated in AF patients' atrial tissues [14,47]. Although the results show that inhibiting autophagy mitigated collagen deposition and improved atrial remodeling, Ang II increased atrial broblast autophagy in vivo. Little is known about the autophagy changes in atrial cardiomyocytes in vivo. Previous study suggested that autophagy exerted protective effects in cardiomyocytes under stress [56]. Therefore, it is very important to clarify the discrepant roles of autophagy in broblasts and cardiomyocytes. Finally, we illustrated the mechanism by which Ang II induced broblast autophagy activation in detail. Nevertheless, we did not clarify the downstream mechanism by which enhanced broblast autophagy promoted COL-I and COL-III expression in the current study. Our future work will focus on this question. The current study demonstrated the role of autophagy in atrial remodeling and AF from the perspective of atrial broblasts. Ang II upregulated broblast autophagy and promoted collagen production, further aggravating atrial remodeling.
In conclusion, the current study focused on atrial remodeling after RAS activation. We found that excessive autophagy was concomitant with AF. Ang II aggravated atrial remodeling and susceptibility to AF induction by stimulating atrial broblast autophagy and further increasing the expression of COL-I and COL-III. We uncovered the mechanism by which Ang II activated autophagy in atrial broblasts (Fig. 10).
Prevention of broblast autophagy may be an effective way to improve atrial remodeling and AF.    Assessment of AERP and susceptibility to AF in mice. a Representative ECG recordings during intracardiac programmed stimulation in mouse right atrium. b Summary of susceptibility to induced AF in mice. Numbers in parentheses indicate the number of animals that entered AF following burst pacing. c Analysis of AERP in mice. Data are expressed as the mean ± SEM, and the most representative pictures are shown; n=9 each group. **P<0.01 vs control group (Con); ***P<0.001 vs control group. &P<0.05 vs Ang II group (Ang II); &&&P<0.001 vs Ang II group.

Figure 3
Expression of COL-I and COL-III in mouse atrial tissues. Mouse atrial tissues were obtained from ve groups: the control group with vehicle (0.9% saline) infused, the Ang II group with Ang II infused (1.6 mg/kg/day), the rapamycin (Rapa) treatment group with intraperitoneal injection of Rapa (1.5 mg/kg/day) and Ang II infused (1.6 mg/kg/day), the 3-MA treatment group with intraperitoneal injection of 3-MA (35 mg/kg/day) and Ang II infused (1.6 mg/kg/day), and the candesartan (Can) treatment group with oral administration of Can (5 mg/kg/day) and Ang II infused (1.6 mg/kg/day). LA: left atrial; RF: right atrial. a Examples of Masson's trichrome staining of mouse atrial tissues. Collagen was stained with blue. b The statistical results of collagen volume infraction. Scale bar: 100 μm and the original magni cation was 100×. c Immunohistochemistry images of COL-I (brown) and COL-III (brown) in mouse atrial tissue. Scale bar: 50 μm and the original magni cation was 200×. Data are expressed as the mean ± SEM, n=9 each group. **P<0.01 vs control group (Con); ***P<0.001 vs control group. &P<0.05 vs Ang II group (Ang II); &&P<0.01 vs Ang II group. Autophagy in mouse atrial broblasts. Mouse atrial tissues were obtained from ve groups: the control group with vehicle (0.9% saline) infused, the Ang II group with Ang II infused (1.6 mg/kg/day), the rapamycin (Rapa) treatment group with intraperitoneal injection of Rapa (1.5 mg/kg/day) and Ang II infused (1.6 mg/kg/day), the 3-MA treatment group with intraperitoneal injection of 3-MA (35 mg/kg/day) and Ang II infused (1.6 mg/kg/day), and the candesartan (Can) treatment group with oral administration Figure 6 Effects of Rapa, CQ, LY294002 and si-Beclin 1 on the expression of COL-1 and COL-III in Ang II-induced atrial broblasts. Cells were pretreated with different concentrations of Rapa, CQ or LY294002 for 1 hour before stimulation with Ang II (10-6 M) for 48 hours or cells pretreated with si-Beclin 1(100 nM) or negative control (NC, 100 nM) for 48 hours before stimulation with Ang II (10-6 M) for another 48 hours. The expression of COL-I, COL-III, p62 and LC3-II was detected by western blot. a, d Atrial broblast Autophagosomes and autophagic ux in atrial broblasts. Cells were transfected with mCherry-GFP-LC3 adenovirus (MOI=100). Then, they were pretreated with Can (10 μM) or PD980559(10 μM) for 1 hour before stimulation with Ang II (10-6 M) for 48 hours. Cells were pretreated with si-AT1(100 nM) for 48 hours before stimulation with Ang II (10-6 M) for another 48 hours. a Representative confocal fluorescence microscopy images (600×) of mCherry-GFP-LC3 localization in atrial broblasts. b Quantitative analysis of green puncta in the green channel and red puncta in the red channel. c, d Quantitative analysis of autophagosomes (yellow puncta in the merged channel) and autolysosomes (red puncta in the merged channel). The average numbers of green, red and yellow puncta were determined by manual counting of uorescent puncta from at least 30 different broblasts in each group. The data are expressed as the mean ± SEM, and the most representative pictures are shown; n=5. Scale bar: 20 μm and the original magni cation was 600×. ***P<0.001 vs control group (Con); &&&P<0.001 vs Ang II group (Ang II).

Figure 10
Schematic diagram of Ang II-induced atrial remodeling. Activation of RAS promotes Ang II secretion. Ang II enhances atrial broblast autophagy through the AT1-ERK -mTOR signaling pathway. Enhanced atrial broblast autophagy aggravates the expression of COL-I and COL-III and further promotes atrial remodeling and susceptibility to AF. RAS: renin-angiotensin system; AT1: angiotensin II receptor type 1; ARB: angiotensin II receptor blocker; Rapa: rapamycin; CQ: chloroquine.