CYP2J2/EET reduces vulnerability to atrial fibrillation in chronic pressure overload mice

Abstract Growing evidence has well established the protective effects of CYP2J2/EET on the cardiovascular system. The aim of the present study was to determine whether CYP2J2/EET has a preventive effect on atrial fibrillation (AF) and to investigate the underlying mechanisms. Wild‐type mice were injected with or without AAV9‐CYP2J2 before abdominal aortic constriction (AAC) operation. After 8 weeks, compared with wild‐type mice, AAC mice display higher AF inducibility and longer AF durations, which were remarkably attenuated with AAV9‐CYP2J2. Also, AAV9‐CYP2J2 reduced atrial fibrosis area and the deposit of collagen‐I/III in AAC mice, accompanied by the blockade of TGF‐β/Smad‐2/3 signalling pathways, as well as the recovery in Smad‐7 expression. In vitro, isolated atrial fibroblasts were administrated with TGF‐β1, EET, EEZE, GW9662, SiRNA Smad‐7 and pre‐MiR‐21, and EET was demonstrated to restrain the differentiation of atrial fibroblasts largely dependent on Smad‐7, due to the inhibition of EET on MiR‐21. In addition, increased inflammatory cytokines, as well as activated NF‐κB pathways induced by AAC surgery, were also significantly blunted by AAV9‐CYP2J2 treatment. These effects of CYP2J2/EET were partially blocked by GW9662, the antagonist of PPAR‐γ. In conclusion, this study revealed that CYP2J2/EET ameliorates atrial fibrosis through modulating atrial fibroblasts activation by disinhibition of MiR‐21 on Smad‐7, and attenuates atrial inflammatory response by repressing NF‐κB pathways, reducing the vulnerability to AF, and CYP2J2/EET exerts its role at least partially through PPAR‐γ activation. Our findings might provide a novel upstream therapeutic strategy for AF.

Smad-7 expression. In vitro, isolated atrial fibroblasts were administrated with TGF-β1, EET, EEZE, GW9662, SiRNA Smad-7 and pre-MiR-21, and EET was demonstrated to restrain the differentiation of atrial fibroblasts largely dependent on Smad-7, due to the inhibition of EET on MiR-21. In addition, increased inflammatory cytokines, as well as activated NF-κB pathways induced by AAC surgery, were also significantly blunted by AAV9-CYP2J2 treatment. These effects of CYP2J2/EET were partially blocked by GW9662, the antagonist of PPAR-γ. In conclusion, this study revealed that CYP2J2/EET ameliorates atrial fibrosis through modulating atrial fibroblasts activation by disinhibition of MiR-21 on Smad-7, and attenuates atrial inflammatory response by repressing NF-κB pathways, reducing the vulnerability to AF, and CYP2J2/ EET exerts its role at least partially through PPAR-γ activation. Our findings might provide a novel upstream therapeutic strategy for AF.

K E Y W O R D S
atrial fibrillation, atrial fibrosis, epoxyeicosatrienoic acid, inflammation, Smad-7 of atrial fibrosis are the hallmark of structural remodelling in AF and are considered to be the substrate for AF perpetuation. 3 Advanced atrial fibrosis is associated with more frequent paroxysms of AF, transformation of the arrhythmia into a permanent type and reduced effectiveness of anti-arrhythmic therapy. 3,4 Atrial fibrosis causes intra-and inter-atrial inhomogeneity in conduction, thus creating a substrate for local re-entry and contributing to the maintenance and progression of AF. Besides that, emerging evidence indicated enhanced inflammatory responses in patients with AF and accompanied by increased circulating levels of pro-inflammatory cytokines. 5,6 Further, NLRP3 inflammasome activation was recently shown to be involved in the pathogenesis of AF. 7 Because AF is able to exacerbate inflammation and atrial fibrosis that further perpetuates the arrhythmia, reduction of inflammation and reversal of structural remodelling have increasingly become the focus of new therapeutic strategies for the prevention of AF. 3,6 CYP2J2 is a cytochrome P450 epoxygenase that is abundantly expressed in the human heart. CYP2J2 converts arachidonic acid to the four regioisomeric epoxyeicosatrienoic acids (5,8,11,, and EETs are further metabolized by the soluble epoxide hydrolase enzyme to form the corresponding 1,2diols, hydroxyeicosatetraenoic acids (DHET) with diminished bioactivity. 8 EET is a pleiotropic cytokine that regulates a wide range of cellular actions and pathophysiological processes; in particular, the studies of EET in the cardiovascular system have become increasingly appreciated in recent years. 9 Notably, CYP2J2/EET was recently demonstrated to prevent cardiac fibrosis. 10,11 Meanwhile, the anti-inflammatory properties of CYP epoxygenase-derived EET have been well evidenced 12,13 ; further, we found that CYP2J2/EET inhibited TNF-α-induced cardiac injury by attenuation of nuclear factor-kappaB activation. 14 These protective effects might be mediated by peroxisome proliferator-activated receptor gamma (PPAR-γ), an important effector of EET. 15 Due to the features of EET with antifibrosis and anti-inflammation, EETs derived from cytochrome P450 epoxygenase were speculated to have the potential to prevent AF.
Westphal et al 16 firstly reported that transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 enhanced cardiac EET biosynthesis, reducing ventricular tachyarrhythmia and AF susceptibility during maladaptive cardiac hypertrophy. Additionally, treatment with soluble epoxide hydrolase inhibitor (sEHI) by blocking the degradation of EET to DHET significantly reduces inflammation, oxidative stress, atrial structural and electric remodelling. 17 However, the precise mechanisms of EET modulate AF remained obscure.
Here, we found that abdominal aortic constriction (AAC) operation enhanced the atrial fibrosis and inflammation response, resulting in increased AF occurrence and AF duration, whereas cardiac-specific overexpression of CYP2J2 via adeno-associated virus 9 (AAV9) vector reduced the susceptibility of AF, as well as ameliorated atrial fibrosis and inflammation in mice with AAC. Our study further elucidated that CYP2J2/EET exerts its effects by PPAR-γ activation, thereby inhibiting atrial fibrosis through targeting MiR-21/ Smad-7 and diminishing inflammatory response by inactivation of NFκB pathways, leading to the reduction in AF vulnerability.

| Ethics statement
All animal protocols in this study were approved by the Animal

| Production of recombinant adeno-associated virus 9
The rAAV vectors (Serotype 9) containing CYP2J2 or GFP were produced by triple plasmid cotransfection in HEK293 cells as previously described. [18][19][20] The detailed description about the construction and preparation of the AAV vector is demonstrated in Figure S1. Purified rAAV9-CYP2J2 (1 × 10 11 pfu) was injected through tail vein 14 days prior to AAC operation. CYP2J2 expression and its metabolite EETs were measured Western blot and DHET kit according to previous methods, 21 and the results are shown in Figure S2A,B.

| Animal model
All mice were in the C57BL/6J background. The animal model of vulnerable AF was induced by pressure overload via AAC, 22 and died mice resulted from operation will be excluded. Animal experiment 1: Eight-week-old male mice were randomly divided into 4 equal groups (10 mice/group): (a) WT mice undergoing a sham operation, (b) WT mice undergoing an AAC operation, (c) WT mice undergoing an AAC operation after AAV9-CYP2J2 delivery for 14 days and (d) WT mice undergoing an AAC operation after AAV9-GFP delivery for 14 days.
Animal experiment 2: Eight-week-old male mice were randomly divided into four equal groups (6 mice/group): (a) WT mice undergoing a sham operation, (b) WT mice undergoing an AAC operation, (c) WT mice undergoing an AAC operation after AAV9-CYP2J2 delivery for 14 days and (d) WT mice undergoing an AAC operation after AAV9-CYP2J2 delivery for 14 days, and GW9662 was given a dose of 0.35 mg/kg per day in drinking water. After constriction with 8 weeks, all mice are killed and the left atriums of the heart were removed. All the mice were allowed free access to the regular chow and drinking water ad libitum.

| Programmed electrical stimulation
Programmed electrical stimulation was performed according to previous study. 23 Briefly, a transesophageal stimulation catheter (EPR-800, Millar Instruments) was connected to a triggered stimulus isolation unit with variable current output. Inducibility of AF was tested by applying 2-second bursts, through the automated stimulator. The first 2-second burst had a cycle length (CL) of 40 ms, decreasing in each successive burst with a 2-ms decrement down to a CL of 20 ms. AF was defined as a period of rapid irregular atrial rhythm lasting at least 2 seconds. If 2 or more bursts in the 3 series of bursts evoked an AF episode, AF was considered to be inducible in that animal.

| Masson trichrome staining for collagen
Left atrial tissue samples were embedded in paraffin and sliced into 4-µm-thick sections, which underwent Masson's trichrome staining to highlight fibres. The percentage of fibrosis was measured as fibrosis areas/total given field areas × 100%.

| Cell culture and treatment of atrial fibroblasts
Isolation and culture of mouse atrial fibroblasts was performed as previously described. 24 These cultures contained N95% atrial fi- pre-miR-21 precursor or FAM3™ Dye-Labeled pre-miR miRNA precursor negative control #1 (Ambion), according to a previous study. 25

| Western blot
Western blot was performed as described previously. 27 The cytoplasm protein and nuclear protein were extracted according to the instructions of the kit (Beyotime). The protein contents of extracts were determined using the Bradford method. Extracts were resolved using SDS-PAGE and transferred to polyvinylidene difluoride filter membranes. Proteins were detected by immunoblotting and visualized using enhanced chemiluminescence.

| Statistical analysis
All data are represented as mean ± SEM or percentage. Comparisons between two groups were analysed with unpaired t tests. The differences between multiple groups were performed by one-way ANOVA analysis followed by a Newman-Keuls test. A value of P < .05 was considered statistically significant.

| CYP epoxygenase 2J2 reduces the vulnerability to atrial fibrillation in mice with AAC
To test whether CYP epoxygenase 2J2 is sufficient to reduce AF susceptibility, we first develop an AAV9 vector delivery cardio-specific expression of CYP2J2. Then, mice injected with AAV9-2J2 were performed AAC surgery, which exhibited a high inducibility of AF result from heart failure. Programmed electrical stimulation experiments were made at 8 weeks post-AAC via transesophageal catheter to assess susceptibility to AF. Figure 1A shows a representative example of electrocardiogram after programmed electrical stimulation. AF occurred after the termination of the burst pacing and persisted more than 2 seconds, and the non-induced AF mice still displayed sinus rhythm or AF persisted less than 2 seconds after the burst ( Figure 1A). AF inducibility was higher in the mice with AAC (8/10, 80%) than in the sham mice (1/10, 10%, Figure 1B).
Accordingly, as a result of high AF inducibility, the mean duration time of AF in AAV9-GFP-treated mice is substantially more than the AAV9-CYP2J2 group (6.703 ± 2.133s, P < .05; Figure 1C). These findings clearly suggest that CYP2J2 significantly reduces AF susceptibility in mice with AAC.

| CYP epoxygenase 2J2 reduce atrial fibrosis in mice subject to AAC
Next, we sought to explore the mechanisms that CYP2J2 prevents the vulnerability of AF. Thus, we measured interstitial fibrosis by Masson's staining in the left atria of wild-type and AAC mice treated with AAV9-2J2 or AAV9-GFP. Summary data showed a marked increase in atrial interstitial fibrosis in AAC mice compared with sham mice (8.100 ± 0.6277% vs 22.74 ± 1.787%; P < .05; Figure 2A,B), whereas treatment with AAV9-2J2 greatly reduced the percentage of fibrosis area (12.26 ± 0.5316%; P < .05; Figure 2A,B). In addition, we detected that increased deposition of α-smooth muscle actin (α-SMA) and collagen-I and collagen-III in the left atria of AAC mice were also mitigated by AAV9-2J2 gene delivery ( Figure 2C,D).
To determine the mechanism that CYP2J2 inhibits atrial fibrosis, we further examined the fibrosis-related TGF-β/Smad pathways, and the data showed enhanced TGF-β and phosphorylation of Smad-2/3 with unchanged total Smad-2/3 in the left atrium of mice with AAC surgery, whereas AAV9-2J2 suppressed the phosphorylation of

| Epoxyeicosatrienoic acid suppress TGF-βinduced atrial fibroblasts activation
In agreement with previous study, 10,11 AAV9-2J2 improved the worsened cardiac function in AAC mice, as demonstrated in Figure   S2C,D (heart weight/body weight ratio (HW/BW) and ejection fraction (EF)); therefore, the effects of CYP2J2 observed in the atria may be secondary to the improvement in cardiac function.

Relative expression (VS Control)
-- Col-I   Figure 3A,B). Because myofibroblasts derive from cardiac fibroblasts and play a particularly significant role in cardiac fibrosis, they have a ∼2-fold higher capacity to synthesize collagen. Indeed, TGF-β exposure enhanced the synthesis of collagen-I and collagen-III from atrial fibroblasts/ myofibroblasts, which were also largely ameliorated by the treatment with EET ( Figure 3C,D). In the line with the fibrotic signalling in vivo, we found that the application of TGF-β1 caused marked phosphorylation of Smad-2/3 in atrial fibroblasts, whereas EET substantially repressed Smad-2/3 phosphorylation ( Figure 3C,D).
Consistently, the reduction of Smad-7 caused by TGF-β stimulation was also recovered by EET treatment (Figure 3C,E). However, these effects of EET on transdifferentiation of atrial fibroblasts and TGF-β/Smad pathways were blunted by pretreatment with EEZE, GW9662 and Smad-7 SiRNA, respectively. Taken together, these results establish that EET suppresses the activity of atrial fibroblasts by inhibition of TGF-β/Smad-2/3 through Smad-7, via PPAR-γ activation.

| CYP epoxygenase 2J2 inhibits atrial inflammations in mice with AAC
It was well accepted that inflammation promotes the development and perpetuation of AF. 31 Pro-inflammatory cytokine levels were elevated with heart failure induced by TAC. 17 Therefore, to evaluate whether CYP2J2/EET counteracts the inflammatory response in AAC mice, we measured important pro-inflammatory cytokines and chemokines including TNF-α, IL-6 and MCP-1, and our data demonstrate the serum level of TNF-α, IL-6 and MCP-1 were significantly increased in mice with AAC ( Figure 5A). In contrast, treatment with AAV9-2J2 significantly decreased these cytokine levels.
Besides that, we further found that enhanced expression of TNF-α and MCP-1 in the left atria of AAC mice, which were substantially attenuated by AAV9-2J2 treatment ( Figure 5B

| GW9662 blocks the effect of CYP 2J2 on atrial fibrillation in mice with AAC
Finally, to verify the role of PPAR-γ, GW9662, the PPAR-γ antagonist, was co-treated with AAV9-2J2 in AAC mice, and programmed electrical stimulation experiments were performed to test the inducibility of AF. Figure 6A shows a representative example of electrocardiogram after programmed electrical stimulation. Similar to the above data, AF inducibility was higher in the mice with AAC (5/6, 83.3%) than in the sham mice (0/6, 0%, Figure 6B). Treatment with AAV9-2J2 remarkably decreased the AF inducibility in AAC mice (1/6, 16.6%, Figure 6B), whereas GW9662 moderately abolished the effect of AAV9-CYP2J2 in AAC mice (3/6, 50%, Figure 6B).

| D ISCUSS I ON
In the present study, we inquired whether EET-induced alteration in atrial fibrosis and inflammation contributes to the prevention of AF. Hence, the major findings from our study include the fol- Over decades, a large body of studies supported the pivotal role of the cytochrome P450 epoxygenase and its metabolites EET in the cardiovascular system, 8 producing the hypothesis about anti-arrhythmic potency of EET. Therefore, firstly, cardiac-specific overexpression of CYP2J2 was shown to reduce the AF inducibility in pathologic cardiac hypertrophy, and the mechanism may be attributed to decreased atrial fibrosis. 16 Thereafter, enhanced EET level with sEHI treatment was also showed to reduce atrial F I G U R E 4 CYP2J2/EET inhibits atrial fibrosis and atrial fibroblasts activation by targeting MiR-21/Smad-7. A, The effects of AAV9-CYP2J2 on MiR-21 expression in mice with or without AAC; *P < .05, compare with control group; # P < .05, compare with AAC group; & P < .05, compare with AAC + CYP2J2 group, n = 10/group. B, The effects of EET on MiR-21 expression in atrial fibroblasts with or without TGFβ1 stimulation, n = 5/group. C, Representative images of atrial fibroblasts transdifferentiation with TGFβ1, EET, pre-MiR-21 (α-SMA staining, scale bar: 250 μm). D, Statistical analysis of transdifferentiation ratio in atrial fibroblasts (10 visible areas/group; five independent experiments). E, Representative immunoblot of collagen-I, collagen-III, Smad-2/3 phosphorylation and Smad-7. F, Statistical analysis of relative expression of collagen-I, collagen-III and Smad-2/3 phosphorylation. G, Statistical analysis of relative expression of Smad-7. Three independent experiments for Western blot, *P < .05, compare with atrial fibroblasts without TGFβ1 stimulation; # P < .05, compare atrial fibroblasts with TGFβ1 stimulation; & P < .05, compare with atrial fibroblasts with TGFβ1 stimulation and EET treatment arrhythmia susceptibility in TAC mice, by antagonizing inflammatory response. 17 Likewise, we observed the susceptibility of AF in AAC mice was significantly decreased with AAV9-CYP2J2 gene delivery, confirming that CYP2J2/EET is sufficient to prevent AF.
A vulnerable substrate is necessary arrhythmogenic mechanism for AF, including the structural remodelling and electrical remodelling. 32 Atrial fibrosis is considered to be the arrhythmogenic substrate for AF perpetuation and also emerges as an important pathophysiological contributor and link to AF recurrences and resistance to therapy. 3 Fibrotic transformation of atrium causes the deterioration of atrial conduction, promoting re-entry in the atrium, which may be directly related to the mechanisms responsible for maintaining AF. 33 Those drugs that prevent the left atrial fibrosis demonstrated potent suppression in AF susceptibility, 3 implying the efficiency of targeting at the fibrotic substrate in managing AF. Similar to a previous study, 16 compared with AAC mice, the AAV9-CYP2J2-treated AAC mice exhibited a marked reduction in atrial fibrosis area and the deposition of collagen, and decreased AF susceptibility, which is also in response to TGF-β or fibrotic stress, but Smad-7 acts as a major inhibitory Smad protein, in turn, restrains fibrotic response through forming stable complexes with TGF-β type I receptors and thereby blocking the phosphorylation of R-Smads, or recruiting ubiquitin E3 ligases, leading to the ubiquitination and degradation of the activated type I receptors. 36 Accordingly, rapid atrial pacing induces AF and fibrosis, associated with a marked increase in the expression of TGF-β/p-Smad2/3, but decreased Smad-7 expression, and overexpression of Smad-7 could blunt collagen synthesis. 37 In addition, Smad-7 gene transfer by a non-invasive ultrasound microbubble was shown to reduce cardiac fibrosis via blocking TGF-β/Smad-3. 38 Notably, TGF-β1 transgenic mice exhibited a selective atrial fibrosis with increased vulnerability to AF. 23  Because EET was well known for exerting anti-inflammation actions in the cardiovascular system, associated with inhibition of on NF-κB cascade, a key signalling transduction of inflammation. 8,14 Meanwhile, growing evidence implies a significant role of inflammation in the pathogenesis of AF, including elevated serum levels of inflammatory biomarkers in AF subjects, the expression of inflammatory cytokines in cardiac tissues of AF patients and animal models of AF, and beneficial effects of anti-inflammatory drugs in experimental AF paradigms. 6 Moreover, heart failure impaired the atrial structure and enhanced inflammation response, promoting the initiation, progression and persistence of AF, which is one of the most common comorbidities leading to AF. 33 In this study, we also PPAR-γ activator, pioglitazone, has been shown to attenuate atrial interstitial fibrosis and AF promotion in different animal models. 39,44,45 More importantly, EET is viewed as an imagined ligand of PPAR-γ, 15,46 and blocking PPAR-γ can reverse the protection of EET on cardiac remodelling and inflammatory injury. 10,14 Therefore, in this study, GW9662 administration partially abolished the inhibitory effect of CYP2J2 on AF susceptibility, despite that AAV9-CYP2J2 effectively restored the expression of PPAR-γ in left atria of AAC mice, suggesting that PPAR-γ, in part, mediates the action of CYP2J2/EET on AF.
In addition to pro-inflammatory response, NF-κB also plays an important role in cardiac hypertrophy, 10,17 which may increase AF occurrence. Moreover, overexpression of Smad-7 was also demonstrated to restrain NF-κB-driven cardiac inflammation. 38 Hence, the regulation of EET on NF-κB may need further evaluation. Besides that, EET modulates the electrophysiological properties of the heart by regulating L-type Ca 2+ , Na + and ATP-sensitive K + (K ATP ) channel activities, 46,47 which probably influence the electrical remodelling.
Intriguingly, emerging evidence indicated that eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) were also generated via the cytochrome P450 (CYP) epoxygenase pathway, and EPA/DHA were well known to possess anti-inflammatory and anti-arrhythmic properties and exert pleiotropic beneficial effects on cardiovascular function, 48,49 which may contribute to the inhibition of CYP2J2 on AF. Also, oxidative stress is viewed as an important inducer to atrial fibrosis and AF, and the modulation of EET on oxidative stress may be involved in suppressing AF. 10 Furthermore, because of the limitations of the experimental AF model, and AAV9mediated CYP2J2 overexpression is not restricted to the atria, the reduction in AF susceptibility is inseparable from the blockade of CYP2J2/EET on cardiac remodelling, cardiac dysfunction and proinflammatory cytokine formation. It should be noted that we only detected the effect of CYP2J2/EET on AF associated with heart failure induced by chronic overpressure via modulating atrial fibrosis and inflammation in the present study, and further studies are needed to verify our results in other animal models or AF patients.
Taken together, our data provide compelling evidence for the preventive effect of CYP2J2/EET on AF. Meanwhile, there are at least dual mechanisms mediated the inhibition of CYP2J2/EET on atrial fibrillation via activating PPAR-γ ( Figure 6D); on the one hand, CYP2J2/EET reduced atrial fibrosis through modulating atrial fibroblasts by eliminating the inhibition of MiR-21 on Smad-7; on the other hand, CYP2J2/EET ameliorated atrial inflammatory response by repressing NF-κB pathways.

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation project (Nos. 81300090, 81400245, 81570293, 81870245).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict interests in this work.