Gallic Acid Ameliorates Angiotensin II-Induced Atrial Fibrillation by Inhibiting Immunoproteasome- Mediated PTEN Degradation in Mice

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and is a major cause of stroke and heart failure. We and others have found that gallic acid (GA) plays a beneficial role in cardiac hypertrophic remodeling and hypertension. However, the effect of GA on angiotensin II (Ang II)-induced AF and atrial remodeling as well as the underlying mechanisms remain unknown. AF was induced in mice by Ang II infusion (2000 ng/kg/min) for 3 weeks. Blood pressure was measured using the tail-cuff method. Atrial volume was evaluated by echocardiography. Atrial remodeling was studied using hematoxylin and eosin, Masson’s trichrome, and immunohistochemical staining. Atrial oxidative stress was assessed by dihydroethidium staining. The gene expression of fibrotic and inflammatory markers and protein levels of signaling mediators were measured by quantitative real-time PCR and western blot analysis. In mice, GA administration significantly attenuated Ang II-induced elevation of blood pressure, AF incidence and duration, atrial dilation, fibrosis, inflammation, and oxidative stress compared with the vehicle control. Furthermore, GA downregulated Ang II-induced activity and expression of immunoproteasome subunits (β2i and β5i), which reduced PTEN degradation and led to the inactivation of AKT1 and downstream signaling mediators. Importantly, blocking PTEN activity by VO-Ohpic markedly reversed the GA-mediated protective effects on Ang II-induced AF and atrial remodeling. Therefore, our results provide novel evidence that GA exerts a cardioprotective role by inhibiting immunoproteasome activity, which attenuates PTEN degradation and activation of downstream signaling, and may represent a promising candidate for treating hypertensive AF.


INTRODUCTION
Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia and is associated with an increased risk of stroke, heart failure, and all-cause mortality (Staerk et al., 2017). AF is typically preceded by conduction abnormalities and structural remodeling, which is characterized by increased atrial fibrosis and dilation (Andrade et al., 2014). The renin-angiotensin system (RAS) plays an important role in the development of AF. As the main effector of the RAS, Ang II activates AKT1-mTOR, TGF-β1-Smad2/3, NF-κB, and NADPH oxidase through Ang II type 1 receptor (AT1R), which in turn promotes fibrosis, inflammation, production of reactive oxygen species, and an abnormal ion channel function in the atrium, thereby leading to the occurrence of AF (Gao and Dudley, 2009;Hu et al., 2015). Therefore, AT1R is the central mediator of atrial remodeling and AF, and thus represents a key target for early treatment of AF. Recent studies have demonstrated that phosphatase PTEN (a phosphatase and TENsin homolog deleted from chromosome 10) exhibits negative regulation of AT1R-induced signaling pathways and AF (Li et al., 2018. Importantly, we and others have revealed that the activity of PTEN is restored by natural compounds such as resveratrol and indole-3-carbinol Lee et al., 2019;Zou et al., 2019). Thus, regulation of PTEN represents a promising strategy for treating cardiovascular disease.
Gallic acid (GA) is a natural phenolic acid that is abundant in several plants. Accumulating evidence has demonstrated that GA plays a beneficial role in apoptosis, hypertension, cardiac hypertrophy, and fibrosis (Ryu et al., 2016;Jin et al., 2017Jin et al., , 2018a. GA improved isoproterenol-induced cardiomyocyte hypertrophy and fibrosis by inhibiting the JNK2-Smad3 signaling pathway (Ryu et al., 2016). Moreover, GA also reduced cardiac oxidative stress and hypertension by regulating GATA4-NOX signaling in spontaneously hypertensive rats (Jin et al., 2017). Recently, we found that administration of GA attenuated pressure overload-induced cardiac hypertrophic remodeling and heart failure, which are associated with the autophagy-dependent degradation of hypertrophic mediators (EGFR, gp130, and calcineurin A) and the suppression of downstream signaling cascades . Moreover, GA ameliorated hypertension and vascular remodeling in Ang II-treated mice by suppressing the immunoproteasome-dependent degradation of endothelial nitric oxide synthase (Yan et al., 2020). However, the cardioprotective effect of GA against Ang II-induced AF and the underlying mechanism remain unknown.
In this study, we revealed that GA significantly reduced AF incidence and duration as well as atrial structural remodeling in Ang II-treated mice. Mechanistically, the protective effect is associated with suppression of the activity and expression of the immunoproteasome subunits β2i and β5i, which inhibit PTEN degradation and AKT1 activation, resulting in the downregulation of the TGF-β1-Smad2/3 and NF-κB signaling pathways. Collectively, our results suggest that GA is a novel regulator of the immunoproteasome and PTEN stabilization, and may provide a potential treatment option for hypertensive AF.

Animal Models and Experimental Protocols
C57BL/6 mice (male, 8-10 weeks of age, n = 200) were used to establish the model of AF by subcutaneous infusion of Ang II (2000 ng/kg/min; Sigma-Aldrich, St. Louis, MO, United States) or saline for 3 weeks as described previously (Li et al., 2018Zhang et al., 2020). The tail-cuff system (Softron BP98A; Softron Tokyo, Japan) was used to assess the systolic blood pressure (SBP) as previously described (Wang et al., 2016Zhang et al., 2020). All animal studies were approved by the Animal Care and Use Committee of Dalian Medical University (No. LCKY2016-31) and conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Dosage Information
Animals were orally gavaged with vehicle or GA (5 or 20 mg/kg BW/day, Sigma-Aldrich, United Kingdom) 1 day before Ang II infusion. The specific PTEN inhibitor VO-OHpic (Selleck, Houston, TX, United States) was intraperitoneally administered (10 mg/kg/day per mouse) in mice beginning 1 day before Ang II infusion .

AF Induction
Mice were anesthetized with 2.5% tribromoethanol (0.02 mL/g; Sigma-Aldrich, United Kingdom) by intraperitoneal injection. A Millar 1.1 F octapolar EP catheter (Scisense, NY, United States) was guided into the right atrium and ventricle. AF Inducibility was gauged by applying a 5-s burst using the automated stimulator as described previously (Li et al., 2018Zhang et al., 2020). The 1-lead body surface ECG and ≤4 intracardiac bipolar electrograms were recorded by the 15 A computer-based data acquisition system (GY6328B; HeNan HuaNan Medical Science and Technology, Co., Ltd.). A series of bursts was repeated three times after 5-min stabilization.

Proteasome Activity Assay
The activity of proteasome in the atria was gauged by specific luminogenic peptide substrates, including Z-nLPnLD-aminoluciferin, Z-LRR-aminoluciferin, and Suc-LLVY-aminoluciferin for the caspase-like, trypsin-like, and chymotrypsin-like activities according to the manufacturer's instructions (Li et al., 2018Chen et al., 2019).

Western Blot Analysis
Total proteins were extracted from snap-frozen atrium samples using RIPA buffer plus protease inhibitors (Solarbio Science Technology Co., China). The protein lysates were separated by electrophoresis in 8-12% SDS-PAGE gels, transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with appropriate primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies. The signal intensities of immunoblots were detected by the ECL Plus chemiluminescent system (Bio-Rad, CA, United States) and were analyzed with a Gel-pro 4.5 Analyzer (Media Cybernetics, United States) .

Statistical Analysis
The normality test (Shapiro-Wilk) was utilized to verify whether the data were normally distributed. The student t-test was performed to evaluate the significant difference between two groups in normal distribution. For the data that were not normally distributed, the Mann-Whitney test was used. For the other comparisons, the significance of the difference between means of the groups was determined by one-way ANOVA following Newman-Keuls multiple comparison test from GraphPad Prism 5 (GraphPad Prism Software). The values of P < 0.05 were considered statistically significant.

GA Inhibits Ang II-Induced AF in Mice
We first tested whether administration of GA could reduce AF. GA treatment significantly improved the elevation of SBP in Ang II-treated mice in a dose-dependent manner ( Figure 1A). Moreover, the incidence and duration of Ang II-induced AF were significantly reduced in GA-treated mice compared with the vehicle-treated mice (Figures 1B-D). Furthermore, Ang II infusion resulted in a marked increase in LA dilation in mice, which was attenuated after GA treatment ( Figure 1E). There was no significant difference in SBP, AF inducibility and duration, or LA dilation between the two groups after saline infusion (Figures 1A-E). Thus, these data suggest that GA administration attenuates Ang II-induced AF.

GA Suppresses Ang II-Induced Atrial Fibrosis
We next examined whether GA attenuates the formation of atrial fibrosis. After 3 weeks of Ang II infusion, the atrial fibrotic area, percentage of α-SMA + myofibroblasts, and mRNA expression of α-SMA, collagen I, and collagen III were increased in mice, and these effects were significantly abrogated by GA administration (Figures 2A-C). Moreover, Ang II infusion markedly upregulated the key signaling mediators of atrial fibrosis, including TGF-β1 and p-Smad2/3, while this effect was diminished in GA-treated mice ( Figure 2D). Accordingly, these results indicate that GA treatment effectively improves adverse atrial structural remodeling after Ang II infusion.

GA Attenuates Ang II-Induced Atrial Inflammation and Oxidative Stress
The administration of GA dose-dependently inhibited Ang IIinduced infiltration of inflammatory cells (as indicated by Mac-2-positive macrophages) and the mRNA expression levels of IL-1β, IL-6, TNF-α, and MCP-1 compared with the vehicle (Figures 3A,C). Furthermore, Ang II infusion increased atrial superoxide formation (demonstrated by DHE staining) and the gene expression of NOX2 and NOX4 (Figures 3B,D). These effects were reduced in GA-treated mice (Figures 3B,D). We next determined the effect of GA on NF-κB signaling and gap junction gene expression. In agreement with our recent studies (Li et al., 2018Zhang et al., 2020), the protein expression levels of p-p65 and connexin 43 (Cx43) were upregulated in Ang II-treated atria, and this upregulation was reversed after GA treatment ( Figure 3E).

GA Inhibits Immunoproteasome Activity and PTEN Degradation
Recent studies have demonstrated that PTEN and its downstream signaling are involved in Ang II-induced AF (Li et al., 2018, therefore next we investigated the effect of GA on proteasome-mediated PTEN degradation in the atria. The administration of GA significantly and dose-dependently suppressed Ang II-induced trypsin-like and chymotrypsinlike activities ( Figure 4A). Because constitutively expressed βsubunits (β1, β2, and β5) and immunosubunits (β1i, β2i, and β5i) have proteasome activity, RT-PCR and western blotting analysis were utilized to evaluate subunit expression. Ang II infusion increased the mRNA expression levels of β2i and β5i compared with saline control (Figure 4B). Furthermore, GA treatment dose-dependently reduced the gene expression of the immunosubunits β2i and β5i but not β1i and constitutively expressed β-subunits (β1, β2, and β5) in the atria after Ang II infusion ( Figure 4B). In addition, the Ang II-induced increase in protein expression levels of β2i and β5i was also downregulated in GA-treated atria tissues ( Figure 4C). In line with our previous findings (Li et al., 2018, Ang II infusion for 3 weeks increased PTEN degradation and p-AKT1 protein expression, and these effects were dose-dependently reversed by the administration of GA (Figure 4C). Taken together, these results demonstrate that GA treatment reduces PTEN degradation likely via suppressing the expression and activity of immunoproteasome subunits in Ang II-treated atria.

Blockage of PTEN Activity Reduces the GA-Mediated Protective Effect on Ang II-Induced AF
To identify the role of PTEN in Ang II-induced atrial remodeling and AF after GA treatment, mice were treated with the PTEN specific inhibitor VO-OHpic with or without administration of GA. Three weeks after Ang II infusion, mice treated with GA exhibited a significant reduction of SBP, AF inducibility and duration, and LA dilation compared with the vehicle-treated mice (Figures 5A-E); these effects were abolished by VO-OHpic treatment (Figures 5A-E). Furthermore, the GA-mediated decrease in atrial fibrotic area, number of α-SMA-positive myofibroblasts, Mac-2 + macrophage infiltration, superoxide production, and mRNA expression levels of α-SMA, collagen I, and collagen III were reversed by VO-OHpic in GA-treated mice (Figures 6A-D and Supplementary Figure 1). Accordingly, GAinduced reduction of PTEN degradation and protein expression of p-AKT1, TGF-β1, and p-p65 in Ang II-treated mice were blocked after VO-OHpic treatment ( Figure 6E). Collectively, these results suggest that GA inhibits Ang II-induced atrial remodeling and AF via reducing PTEN degradation.

DISCUSSION
Here, we revealed for the first time that the administration of GA inhibits Ang II-induced AF incidence and atrial dilation. Specifically, GA blocks the activity and expression of the immunoproteasome catalytic subunit β2i and β5i, which reduces PTEN degradation and AKT activation. This leads to the suppression of downstream signaling mediators (TGF-β1-Smad2/3, NF-κB, and Cx43), which improves atrial fibrosis, inflammation, and oxidative stress (Figure 7). Therefore, our results provide new evidence that GA serves as an effective inhibitor of the immunoproteasome FIGURE 4 | GA reduces the activity and expression of immunoproteasome subunits β2i and β5i and PTEN degradation in the atria after Ang II infusion. (A) The proteasome activities in the atria after Ang II infusion in the presence or absence of GA treatment (n = 4). (B) qPCR analyses of the mRNA expression levels of β1, β2, β5, β1i, β2i, and β5i (n = 4). (C) Representative immunoblotting analyses of the protein expression levels of β2i, β5i, PTEN, p-AKT1, and AKT1 (left), and quantification of the relative protein levels (right, n = 4). GAPDH as an internal control. *P < 0.05 versus saline, # P < 0.05 versus Ang II. and is a promising agent for treating AF and atrial structural remodeling.
Atrial structural remodeling is the hallmark of the development and progression of AF, which subsequently causes LA enlargement and conduction abnormalities (Andrade et al., 2014). This process consists of several mechanisms, including atrial fibrosis, inflammation, and oxidative stress (Dzeshka et al., 2015). Ang II has been demonstrated to significantly increase SBP, left ventricular hypertrophy, and dysfunction, which are established clinical risk factors for AF (Purohit et al., 2013;Jansen et al., 2018). Current therapeutic agents for AF, which include oral anticoagulation, angiotensin-converting enzyme inhibitors, and antiarrhythmic drugs, focus on the common symptoms and complications. However, adverse events with these agents may increase mortality (Kirchhof, 2017). Therefore, new options targeting atrial structural remodeling, atrial dilation, and AF are urgently needed. GA is a natural polyphenol compound that exerts a key role in protecting against cardiac hypertrophy, hypertension, and fibrosis in several animal models (Jin et al., 2017(Jin et al., , 2018b. Recently, we demonstrated that GA administration improved pressure overload-induced myocardial hypertrophic remodeling (including hypertrophy, fibrosis, inflammation, and oxidative stress)  and Ang II-induced hypertension and vascular dysfunction (Yan et al., 2020), suggesting that GA may exert a protective effect on AF. In agreement with this, we demonstrated that GA not only markedly reduced the elevation blood pressure but also attenuated atrial fibrosis, inflammation, and ROS production in Ang II-treated mice (Figures 2, 3), which are involved in the pathogenesis of AF. Thus, our results indicate that GA markedly reduced AF development at least in part through blocking Ang II/hypertension-induced atrial remodeling.
Proteasomal degradation is the vital pathway for intracellular protein turnover in most mammalian cells and organs, including the heart (Lyon et al., 2013). After stimulation by inflammatory cytokines, H 2 O 2 , and heat shock, the immunoproteasome is induced and exhibits immune and non-immune functions, such as antigen presentation, inflammation, oxidative stress, and cell signaling (Angeles et al., 2012). Increasing evidence demonstrates that adverse stimuli including pressure overload, isoproterenol, deoxycortone acetate/salt, and Ang II increase the catalytic activity and expression of the immunoproteasome, leading to cardiac hypertrophy and AF (Depre et al., 2006;Drews et al., 2010;Li et al., 2015;Yan et al., 2017). In contrast, the proteasome inhibitor bortezomib effectively ameliorates Ang II-induced myocardial hypertrophy and contractile function (Li et al., 2015). Moreover, we recently found that the activity and expression of β2i and β5i are upregulated in the atria of Ang II-treated mice and the serum of patients with AF (Li et al., 2018. Knockout of β2i or β5i attenuates Ang II-induced atrial remodeling and development of AF (Li et al., 2018. Interestingly, several natural products, including quercetin and resveratrol, have been shown to inhibit proteasome activity, thereby ameliorating atherosclerosis and cardiac hypertrophy (Pashevin et al., 2011;Chen et al., 2019). In the present study, we have provided novel evidence that GA administration significantly suppresses the activity and expression of the immunoproteasome catalytic subunits β2i and β5i in the atria of Ang II-treated mice.
PTEN plays a role in Ang II-induced AF and atrial structural remodeling by ameliorating AKT1 and downstream signaling mediators (TGF-β1-Smad2/3, NF-κB, and Cx43) (Li et al., 2018. Thus, the regulation of PTEN activity and/or protein expression is considered as a promising therapeutic strategy for heart disease. PTEN activity is indeed modulated by posttranslational modifications, especially ubiquitination-mediated proteasomal degradation (Lee et al., 2018). We and others demonstrated that proteasomal dysfunction induced by knockout of the immunoproteasome subunits β1i, β2i, or β5i in mice attenuated PTEN degradation, leading to the downregulation of AKT-associated signaling pathways in ischemic hearts and AF (Cai et al., 2008;Li et al., 2018Li et al., , 2019. Interestingly, several natural compounds have been identified as potent inhibitors of PTEN degradation. Indole-3-carbinol, derived from cruciferous vegetables, directly blocks E3 ubiquitin ligase WWP1 and subsequently suppresses the K27-linked ubiquitination of PTEN (Lee et al., 2019). Moreover, we recently showed that resveratrol represents a novel inhibitor of the immunoproteasome and restores PTEN stability in pressure overload-induced cardiac hypertrophy and fibrosis Zou et al., 2019).
In the present study, we extended our previous findings and demonstrated that administration of GA markedly decreased PTEN degradation and activation of AKT1, leading to inhibition of AF and atrial remodeling in Ang II-treated mice. These effects were reversed by the specific PTEN inhibitor VO-OHpic. Collectively, our novel findings suggest that PTEN plays a role in the GA-mediated cardioprotective effects on Ang II-treated atria. Several studies have explored the bioavailability of GA in humans (Manach et al., 2005). After the oral administration of pure tablets or black tea (each containing 50 mg GA), GA was rapidly absorbed and the plasma concentrations of GA reached 1.83 µmol/L (Shahrzad et al., 2001). Interestingly, our recent study revealed that the dosage of GA used in mice (5 or 20 mg/kg BW) was equivalent to 0.41 or 1.63 mg kg/BW in humans , which is consistent with the results of others. Moreover, we found that primary cardiomyocytes treated with GA (1-200 µmol) showed no significant toxic effect . Thus, GA may serve as a safe and effective natural product for the prevention and treatment of cardiovascular diseases. Further investigations are needed to verify the bioavailability of different forms of GA in foods.

CONCLUSION
In summary, in this study we demonstrated for the first time that GA administration significantly attenuated Ang II-induced AF and atrial structural remodeling in mice. Moreover, GA suppressed the activity and expression of the immunoproteasome subunits β2i and β5i, which ameliorated PTEN degradation leading to inactivation of AKT1 and downstream signaling pathways (TGF-β1-Smad2/3, NF-κB, and Cx43). Thus, these findings identify GA as a novel inhibitor of the immunoproteasome and suggest a potential approach to AF prevention and treatment.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care and Use Committee of Dalian Medical University (No. LCKY2016-31) and conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.

AUTHOR CONTRIBUTIONS
DH, Q-YZ, Y-LZ, and XH conducted the experiments. DH, FT, and XY analyzed the data. S-BG, XY, and H-HL designed the study. XY and H-HL drafted the manuscript, provided the funding for the study, and had primary responsibility for the final content. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by grants from the China Postdoctoral Science Foundation (2020M670384 to XY) and the National Natural Science Foundation of China (81703217 to XY, 81630009 to H-HL).