In the present study, we demonstrated the relationship between autophagy and AF, and the principal discoveries were as follows: first, compared to SR patients, more significant 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 fibroblast autophagy. Meanwhile, blocking autophagy or antagonizing AT1 can relieve atrial remodeling and reduced AF inducibility. Third, Autophagic flux was also increased in response to Ang II. The enhanced autophagy was a positive-feedback 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 fibroblasts. 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 fibers 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 fibroblasts are easily activated by various stimuli and then proliferate and differentiate into myofibroblasts, which secrete amount of collagen that deposits in the atrial interstitium and trigger further atrial remodeling. Numerous studies have documented the link between atrial interstitial fibrosis 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.
In the cardiovascular system, the alteration of autophagy was detected in various diseases, such as heart failure, cardiac hypertrophy and ischemia-reperfusion injury. Considering the controversial conclusions about autophagy in cardiovascular diseases, it is valuable to illustrate the characteristics of autophagy in AF. In the atrium of persistent AF patients, researchers found that autophagic flux and ATG7 protein, an important regulator of autophagy, both increased. A similar phenomenon was observed in a rabbit model with rapid atrial pacing. 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. 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 confirmed 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 fibroblast 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 flux in atrial fibroblasts 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 confirmed that Ang II promotes cardiac interstitial fibrosis by inducing collagen secretion in rat cardiac fibroblasts[48, 49]. Additionally, Ang II can shorten the AERP, which is reversed by candesartan. 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 fibroblasts by provoking autophagy (Fig. 5). COL-I and COL-III expression was further augmented when provoking atrial fibroblast 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. That is, AT1 is an upstream regulator of autophagy. What interested us was whether Ang II induced atrial fibroblast autophagy also through AT1. The results showed that autophagy and collagen secretion both decreased when AT1 was blocked (Fig. 8). Meanwhile, candesartan, a specific AT1 inhibitor, restrained atrial fibroblast autophagy induced by Ang II in vivo (Fig. 4). In addition, disturbing AT1 diminished the autophagic flux in atrial fibroblasts (Fig. 9). On the whole, these data showed that AT1 is the key molecular that regulates atrial fibroblast 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. Transgenic mice overexpression the human pro-renin receptor gene exhibited spontaneous AF after 10 months, with serious cardiac fibrosis. Further study found that ERK phosphorylation was specifically increased in cardiac fibroblasts . 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. In vascular smooth muscle cells, autophagy is inhibited by the activated Akt-mTOR signaling pathway. In cardiomyocytes, activated mTOR exerts a protective effect in ischemia/reperfusion injury by restraining autophagy. In summary, mTOR is a pivotal upstream regulator of autophagy in the cardiovascular system. In the current study, Ang II induced fibroblast 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 flux induced by Ang II in atrial fibroblasts was reduced (Fig. 9). In summary, Ang II induced atrial fibroblast 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 identified a potential benefit 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 specific pharmacological inhibitor of autophagy may be an immense deficiency in clinical medicine. The number of enrolled patients was slightly insufficient 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 fibroblast 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. Therefore, it is very important to clarify the discrepant roles of autophagy in fibroblasts and cardiomyocytes. Finally, we illustrated the mechanism by which Ang II induced fibroblast autophagy activation in detail. Nevertheless, we did not clarify the downstream mechanism by which enhanced fibroblast 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 fibroblasts. Ang II upregulated fibroblast 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 fibroblast autophagy and further increasing the expression of COL-I and COL-III. We uncovered the mechanism by which Ang II activated autophagy in atrial fibroblasts (Fig. 10). Prevention of fibroblast autophagy may be an effective way to improve atrial remodeling and AF.