Necroptosis is required for atrial fibrillation and involved in aerobic exercise‐conferred cardioprotection

Abstract Necroptosis, a novel programmed cell death, plays a critical role in the development of fibrosis, yet its role in atrial fibrillation (AF) remains elusive. Mounting evidence demonstrates that aerobic exercise improves AF‐related symptoms and quality of life. Therefore, we explored the role of necroptosis in AF pathogenesis and exercise‐conferred cardioprotection. A mouse AF model was established either by calcium chloride and acetylcholine (CaCl2‐Ach) administration for 3 weeks or high‐fat diet (HFD) feeding for 12 weeks, whereas swim training was conducted 60 min/day, for 3‐week duration. AF susceptibility, heart morphology and function and atrial fibrosis were assessed by electrophysiological examinations, echocardiography and Masson's trichrome staining, respectively. Both CaCl2‐Ach administration and HFD feeding significantly enhanced AF susceptibility (including frequency and duration of episodes), left atrial enlargement and fibrosis. Moreover, protein levels of necroptotic signaling (receptor‐interacting protein kinase 1, receptor‐interacting protein kinase 3, mixed lineage kinase domain‐like protein and calcium/calmodulin‐dependent protein kinase II or their phosphorylated forms) were markedly elevated in the atria of AF mice. However, inhibiting necroptosis with necrostatin‐1 partly attenuated CaCl2‐Ach (or HFD)‐induced fibrosis and AF susceptibility, implicating necroptosis as contributing to AF pathogenesis. Finally, we found 3‐week swim training inhibited necroptotic signaling, consequently decreasing CaCl2‐Ach‐induced AF susceptibility and atrial structural remodeling. Our findings identify necroptosis as a novel mechanism in AF pathogenesis and highlight that aerobic exercise may confer benefits on AF via inhibiting cardiac necroptosis.


| INTRODUC TI ON
Atrial fibrillation (AF), a highly prevalent arrhythmia in clinical practice associated with increased morbidity and mortality, has become a public health issue. 1 The underlying mechanism of AF pathogenesis is widely explored, and structural remodeling, consisting of elevated atrial myocyte death and resultant fibrosis, as well as compensatory atrial dilation, is thought to be pivotal in this process. 2 Apoptosis and necrosis, two major forms of cell death, are crucial pathological basis of cardiovascular diseases. 3 The role of apoptosis in AF has been intensively explored, 4,5 yet whether necroptosis, a recently defined programmed necrosis, contributes to AF remains elusive.
Necroptosis, combining features of both necrosis and apoptosis, is a newly caspase-independent programmed form of necrosis. 6 It is now attracting more and more attention that necroptosis contributes to the development of various cardiovascular diseases, 7 and especially acts as a molecular target for age-related heart disease. 8 Necroptosis is mediated by a signaling complex called "necrosome," containing the receptor-interacting protein kinase 1-the receptor-interacting protein kinase 3 (RIP1-RIP3) complex and its downstream substrates-the mixed lineage kinase domain-like protein (MLKL), 9 or calcium/calmodulin-dependent protein kinase II (CaMKII), 10 which ultimately leading to cell membrane rupture and cell death. Increasing evidence demonstrates inhibition of necroptotic signaling with either genetic manipulation (RIP3 knockout mice) or pharmacological drugs (necrostatin-1, Nec-1) protect against fibrosis, 11,12 the hallmark of atrial structural remodeling of AF. In line with this, silencing its downstream substrate CaMKII or inhibiting its activity by KN93 is also implicated to reduce transforming growth factorβ (TGFβ)-mediated fibrosis. 13 Therefore, necrotic loss of cardiomyocytes by necroptosis might lead to cardiac fibrosis and extracellular matrix remodeling, 2,14 thereby providing a structural substrate for AF.
Physical exercise is a non-invasive therapeutic approach for prevention and treatment of cardiovascular diseases, such as coronary artery disease, hypertension and diabetes, described as the notion "exercise is medicine." 15 However, in the aspect of AF, the effects of exercise are more complicated and depend on exercise frequency, intensity, time and type (the FITT principle). 16 Both clinical and experimental studies demonstrated high-intensity exercise might raise the risk of AF and are even used as a means to establish an AF model in animal studies. [17][18][19] However, lately, emerging clinical evidence supported low to moderate regular exercise exerted beneficial effects against AF, 20 probably via reversing atrial remodeling, improving risk factors, improving autonomic tone and reducing inflammation. 16

| Swim training protocol
Mice were pre-adapted to swim training with a 10-min session for the first day and then progressively increased to 60 min daily in a week and swam 60 min/day for 3 weeks.

| Non-invasive transthoracic echocardiography
After skin preparation of the chest and upper abdomen, mice were fixed on the operating table in supine position and anaesthetized with 1.5% isoflurane. Non-invasive transthoracic echocardiography was carried out to assess heart morphology and function using the Vevo2100 Imaging system (VisualSonics, Toronto, Ontario, Canada) equipped with a 30 MHz phased-array transducer. Left atrium (LA) diameter was determined from M-mode at end-systole. All these images were processed using Vevolab 3.1 software (VisualSonics). on the high right atrium was performed to induce AF after thoracotomy with atrial ECG recorded. AF was successfully induced when P wave disappeared in combination with irregular RR intervals on surface ECG, and rapid and irregular atrial waves appeared on atrial ECG, lasting for at least 1 s. AF frequency (number of incidences per animal) and AF duration (in seconds) were used to evaluate AF susceptibility.

| Histological evaluation of atrial fibrosis
After fixation in 4% paraformaldehyde overnight, atria were embedded in paraffin and sliced into 5µm-thick sections. Then, atria sections were stained with Masson trichrome to evaluate the degree of atrial fibrosis and images were captured under the fixed microscope illumination settings (Olympus, Tokyo, Japan). Fibrosis was analyzed and quantified using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). Degree of fibrosis was evaluated and calculated by measuring the blue regions (collagen) relative to total tissue area and represented the average of three randomly selected fields (400× magnification) per slide.

| Protein extraction and western blot analysis
Western blot was performed according to standard protocols. Atrial tissues were homogenized, and proteins were isolated using radioimmunoprecipitation assay lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentration was determined using a BCA assay kit (TDY Biotech, Beijing, China).
Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes.
Subsequently, the membranes were blocked with 5% skim milk for 1 h at room temperature. Diluted primary antibodies against RIP1 Band signals were detected with Tanon-5200 Multi chemiluminescent imaging system (Tanon, Shanghai, China) and quantified by densitometry (standardized to the GAPDH band) using Quantity One software (Bio-Rad, Hercules, California, USA).

| Statistical analysis
All values are presented as mean ±SEM and analyzed by SPSS ver-

| Inhibiting necroptosis decreased HFDinduced AF susceptibility and mitigated atrial fibrosis
To further investigate the role of necroptosis in different AF models, we established a HFD-induced mouse AF model by feeding HFD for 12 weeks and inhibited necroptosis via Nec-1 injection in the last 3 weeks from the tail vein. Mice were randomly divided into 4 groups: NFD group (n = 7), HFD group (n = 9), Nec-1 group (n = 10) and HFD+Nec-1 group (n = 7) ( Figure 3A). HFD-fed mice started to meet the standard of obesity at the end of week 9 (body weight increased by 20% compared with NFD group, Figure 3B) and showed apparently enhanced AF susceptibility at the end of Moreover, echocardiography revealed that swim training significantly decreased LA diameter in CaCl 2 -Ach-induced AF mice (Table S1 and Figure 5E,F). In line with this finding, Masson's trichrome staining showed that the degree of fibrosis was significantly lower in CaCl 2 -Ach+Swim group compared with CaCl 2 -Ach+Sed group ( Figure 5G,H). All the findings above demonstrated that swim exercise training could apparently attenuate CaCl 2 -Achinduced AF.

| Swim exercise training inhibited cardiac necroptosis in CaCl 2 -Ach-induced AF mice
To further identify whether necroptosis was involved in swim exercise-conferred benefits on AF, we evaluated necroptotic signaling in CaCl 2 -Ach-induced AF mice with or without 3-week swim exercise training. As shown in Figure 6A

| DISCUSS ION
Necroptosis plays a critical role in the development of cardiovascular diseases, but the role of necroptosis in AF and exercise-mediated cardioprotection against AF is still unknown. In this study, we tend to explore (1) the role of necroptosis in AF pathogenesis and (2) whether 3-week swim exercise training could attenuate AF and the role of necroptosis in this process. Our results showed cardiac necroptosis occurred in CaCl 2 -Ach (or HFD)-induced AF mice, and inhibiting necroptosis attenuated AF susceptibility and reversed atrial structural remodeling in both AF models. Furthermore, 3-week swim exercise training could inhibit cardiac necroptosis, thus protecting the heart against CaCl 2 -Ach-induced AF.
Calcium overload has long been recognized as crucial for triggering AF because it may cause delayed after depolarizations/triggered activity as well as atrial structural remodeling. In line with this, Ach releasing can activate Ach-sensitive potassium current (I KACh ), consequently shortening action potential duration, increasing atria vulnerability and facilitating re-entry formation, thus maintaining AF. 26,27 Early in 1982, Zhao and Guo, 28 reported CaCl 2 -Ach-induced AF in mice. Later, more and more researchers used different dosage of CaCl 2 -Ach mixture to establish an AF model in rats. 24,[29][30][31][32] Therefore, we used CaCl 2 (10 mg/kg)-Ach (66 µg/kg) mixture to establish a mouse AF model via tail vein injection and found increased AF susceptibility only after 3 weeks of CaCl 2 -Ach administration, whereas no significant changes were observed earlier ( Figure S2). Obesity has been linked to AF and recognized as an independent risk factor for AF recently. 33 Obese patients showed cardiac hypertrophy, LA dilation, atrial fibrosis and inflammation, 33 contributing to AF triggering and progression. Growing evidence proves HFD-induced obesity also causes atrial inflammation and fibrosis, mediating atrial remodeling and AF in animal models. [34][35][36][37] Thus, we fed mice HFD containing 60 kcal% fat to mimic obesity and established an obesity-induced mouse AF model. We found mice started to meet the standard of obesity at the end of week 9 ( Figure 3B) and showed enhanced AF susceptibility at the end of week 12 ( Figures 3C-E and S3).
Necroptosis is a regulated necrotic cell death emerging as a potential molecular target for cardiovascular diseases, especially age-related heart disease. 8 AF is an age-related arrhythmia, and its prevalence increases with advancing age, rising from 0.7% in persons aged 55-59 years to 17.8% in those aged ≥85 years. 38 Here we demonstrated that necroptosis was stimulated in CaCl 2 -Ach-induced AF mice (Figure 2), whereas inhibition of necroptosis via Nec-1 attenuated CaCl 2 -Ach-induced AF burden and atrial structural remodeling (Figure 1), implicating the critical role of necroptosis in AF pathogenesis. In line with this, we found increased AF susceptibility and atrial fibrosis in HFD-induced obesity mice and decreased AF susceptibility and atrial fibrosis after Nec-1 administration (Figure 3), further supporting that necroptosis was required for AF pathogenesis independent of AF models. These are consistent with previous reports showing necroptosis and downstream targets play a crucial role in fibrosis, a hallmark of AF. Lee et al. 11 revealed necroptosis was involved in the pathogenesis of lung fibrosis, and RIP3 knockout as well as RIP1 inhibition by Nec-1 attenuated lung inflammation and fibrosis. Wei et al. 12 proved RIP3 deficiency could mitigate liver fibrosis in mice. Moreover, CaMKII is also implicated to activate a set of downstream signaling to promote fibrosis such as TGFβ-mediated collagen and fibronectin genes expression, whereas CaMKII silencing ameliorates TGFβ-mediated fibrosis. 13 To our knowledge, this is the first report of the link between necroptosis and AF pathogenesis, providing a new therapeutic target for prevention and treatment of AF.
Mechanistic link between necroptosis and atrial remodeling or AF pathophysiology has been widely explored. On one hand, compensatory proliferation of fibroblasts could be induced in response to loss of cardiomyocytes caused by necroptosis to maintain tissue homeostasis. On the other hand, necroptotic cells could release cellular contents, resulting in inflammatory 39 and fibrotic responses, 40 consequently leading to the development of myocardial fibrosis in severe cardiac pathological conditions. A growing body of evidence demonstrates the existence of inflammation and fibrosis in AF patients and experimental AF models such as CaCl 2 -Ach-induced AF and HFD-induced AF. 30,36,41 Therefore, necroptosis, and resultant activation of inflammation and fibrosis, might be important pathogenic factors of AF. Of note, up-regulation of necroptosis, accompanied by chronic activation of inflammation and fibrosis, is commonly observed in the elderly, 42 thus raising the possibility that necroptosis is considered as the potential mechanism of age-related diseases such as AF, and providing a therapeutic target for AF prevention and management in the elderly. In addition to inflammation and fibrosis, metabolic syndrome, such as insulin resistance, is also widely implicated in AF pathogenesis. Polina et al. 43 showed that loss of insulin signaling may contribute to AF in type 1 diabetes and Maria et al. 44 showed that insulin treatment reduces susceptibility to AF in type 1 diabetic mice. In line with this, we did observe that both CaCl 2 -Achinduced AF mice and HFD-induced obese mice exhibited impaired glucose tolerance compared with control mice (data not shown).
However, inhibition of necroptosis with Nec-1 showed unchanged glucose tolerance in CaCl 2 -Ach-induced AF mice (data not shown), suggesting impaired insulin sensitivity might occur prior to necroptosis in AF pathogenesis, but not vice versa.
It is widely accepted that exercise is medicine for cardiovascular diseases. Hou et al. 45  whether moderate-intensity swim exercise (60 min/day, once daily for 3 weeks) confers benefits on AF and found this exercise training protocol significantly reduced AF susceptibility and structural remodeling in CaCl 2 -Ach-induced AF mice, without causing apparent cardiac hypertrophy (Table S1). Our researches in this study and others support the notion that the effect of exercise on AF follows F I G U R E 3 Twelve-week HFD feeding promoted AF susceptibility and atrial fibrosis while inhibiting necroptosis reversed these effects. (A) Experimental protocols of Group 2. (B) Body weight apparently increased in both HFD-fed groups compared with NFD group or Nec-1 group at the end of week 8 and met the standard of obesity at the end of week 9. (C)-(E) Representative ECGs and bar graphs showed burst pacing-induced AF with increased AF frequency and duration in HFD group (n = 9) compared with NFD group (n = 5), and less episodes of AF with decreased AF frequency and duration in HFD+Nec-1 group (n = 7) compared with HFD group. (F)-(G) Representative Masson's trichrome staining and quantitative analysis of fibrotic area exhibited more collagen deposition (blue) in HFD group (n = 3) under 400× magnification compared with NFD group (n = 3) and less fibrosis in HFD+Nec-1 group (n = 3) compared with HFD group. (H)-(I) Representative images and quantitative analysis showed elevated levels of fibrosis-related gene: α-SMA and TGFβ in HFD group compared with NFD group but reversed effect after Nec-1 administration. Scale bar: 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. AF, atrial fibrillation; ECG, electrocardiogram; HFD, high-fat diet; Nec-1, necrostatin-1; NFD, normal-fat diet These findings indicated AF patients should be more careful about exercise intensity, and low to moderate exercise intensity may be a preferred exercise modality for AF management without arrhythmogenic effects.
Increasing studies have demonstrated exercise was implicated to reduce cardiac apoptosis and necrosis. 48 High-intensity interval training exercise has previously been shown to prevent cardiac remodeling after myocardial ischemia/reperfusion injury via targeting necroptosis. 23 In line with this, our results showed that 3-week swim exercise training inhibited necroptotic signaling in CaCl 2 -Ach-induced AF mice. These results prompt the question as to how exercise inhibits necroptotic signaling. Previous researches have shown that oxidative stress and AMPK were upstream mechanisms that regulate necroptosis, and autophagy was linked to necroptosis,though we did not detect any significant change in cardiac oxidative stress between sedentary and exercised AF mice (data not shown), AMPK/mTOR-mediated autophagy signaling was apparently enhanced in exercised mice compared with sedentary mice ( Figure S4). Therefore, activated autophagy after swim exercise might lead to decreased necroptosis in AF mice. A further understanding of the molecular mechanisms can help to identify potential targets involved in exercise-conferred cardioprotection and reveal novel therapeutic strategies for AF prevention and treatment.

| CON CLUS ION
In the present study, we investigated the role of necroptosis in AF and exercise-conferred benefits on AF. First, our pharmacological studies demonstrated that necroptosis was attributable to AF pathogenesis. Next, improving physical activity via 3-week swim training (60 min/day, once daily) reduced AF susceptibility in CaCl 2 -Ach-induced AF mice, mechanistically via suppressing necroptosis.
Taken together, our findings identify necroptosis as a potential therapeutical target for AF treatment and highlight that aerobic exercise may confer benefits on AF via inhibiting cardiac necroptosis.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (grant number 81870257 and 31871172).

CO N FLI C T O F I NTE R E S T
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.