Mechanisms associated with the development of atrial fibrillation after sepsis and the role of neuregulin-1

Mechanisms associated with the development of atrial fibrillation after sepsis and the role of neuregulin-1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanisms associated with the development of atrial fibrillation after sepsis and the role of neuregulin-1 Wen Kang, Jingru Deng, Zheru Fan, Fang Zhou, Xi Wang, Kang Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3246284/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose To explore the mechanisms involved in the development of atrial fibrillation (AF) after sepsis and examine the effect of neuregulin-1 (NRG-1) on AF and related mechanisms. Methods We used cecal ligation and puncture (CLP) to establish the sepsis model. NRG-1 was administered via the tail vein at a dose of 0.01µg/g 12 and 24 h postoperatively to determine its effect on AF after sepsis. Results Compared with Sham rats, septic rats exhibited enhanced AF inducibility, atrial fibrosis, norepinephrine (NE), and C-reactive protein (CRP) levels, reduced action potential duration (APD), atrial effective refractive period (AERP), acetylcholine (Ach) levels, expression of Nav1.5, Cav1.2, and Kv1.5, and significantly decreased I Na , I Ca, L , and I kur current densities. We observed that NRG-1 could reduce APD, atrial fibrosis, levels of CRP and NE, I Na and I Ca, L current densities, and expression levels of Nav1.5 and Cav1.2, however, it failed to prevent the onset of AF. Compared with the Sham group, the Sham + NRG-1 group rats showed a reduction in APD, AERP, I Na and I Ca, L current densities, Nav1.5 and Cav1.2 expression levels, elevated AF inducibility, Ach levels, I kur current density, and Kv1.5 expression. Conclusion Sepsis can induce tissue and electrical remodeling in the atria and promotes the development of AF. NRG-1 could attenuate the degree of atrial fibrosis and organismal inflammation in sepsis while promoting the development of AF in Sham rats, impacting atrial electrophysiology and ionic currents. Biological sciences/Cell biology Biological sciences/Drug discovery Biological sciences/Immunology Biological sciences/Molecular biology Health sciences/Cardiology Sepsis Neuregulin-1 Atrial fibrillation ion currents Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction In organisms, an infection can induce a dysregulated systemic inflammatory response, resulting in the development of sepsis 1 . Along with an aging population and increased complications, sepsis has become a major cause of death and critical illness worldwide and causes an estimated 11 million deaths worldwide each year 2 , 3 . In patients with sepsis, morbidity and mortality are significantly associated with the occurrence of complications, with cardiac injury particularly common among the observed complications 4 . Cardiac injury in sepsis has been associated with cardiac dysfunction, as well as with the development of multiple arrhythmias, including atrial fibrillation (AF), ventricular fibrillation, supraventricular tachycardia, and ventricular tachycardia. Furthermore, the development of arrhythmias is closely associated with high mortality and prolonged hospital stays in patients with sepsis 5 . AF, the most common arrhythmic disease in patients with sepsis, occurs nearly six times more frequently in these patients than in non-septic patients, impacting approximately one-fifth of all patients with sepsis 4 , 6 . Following the onset of sepsis, changes in pathophysiological processes such as systemic pro-inflammatory cytokine release, high levels of stress hormone secretion, intravascular volume shifts, electrolyte dysregulation, autonomic imbalance, and cardiovascular damage can predispose individuals to the development of AF 7 . Persistent AF may be a specific marker of the severity of sepsis. Moreover, the occurrence of AF in patients with sepsis may lead to new-onset heart failure and thromboembolism, as well as ischemic stroke and systemic embolism, which can seriously endanger patients' lives 2 . Therefore, carefully monitoring sepsis development, and early prevention and treatment of septic AF using diverse measures will reduce the mortality among these patients and markedly improve their prognosis. Neuregulin-1 (NRG-1) is produced by endocardial and myocardial microvascular endothelial cells and binds to the epidermal growth factor receptor (ErbB) on the surface of adjacent cardiac myocytes to activate tyrosine kinases, which reportedly play a key role in the induction of embryonic development such as cardiac development and myogenesis 8 . In addition, NRG-1 may exert multifaceted cardioprotective effects by comprehensively activating the NRG-1/ErbBs signaling pathway and regulating the level of fibrosis in response to metabolism, inflammation, and cardiac injury 9 . NRG-1 was shown to protect the vascular endothelium and cardiomyocytes by inhibiting immune-inflammatory responses and suppressing excessive activation of the renin-angiotensin-aldosterone system, thereby improving cardiac function and significantly increasing the survival of septic rats 10 , 11 . Furthermore, serum NRG-1 levels were found to be closely associated with the occurrence of paroxysmal AF 12 . However, the effect of NRG-1 on AF after sepsis remains poorly explored. Accordingly, in the present study, we aimed to provide a more theoretical basis for the treatment of post-sepsis AF by elucidating in-depth mechanisms underlying the occurrence of post-sepsis AF and the related role of NRG-1. 2 Materials and methods 2.1 Ethics approval The animal management and welfare were reviewed and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (Date:2020-07-24/No.20200705, Wuhan, China). All methods are reported in accordance with ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. 2.2 Animals and specimen collection After one week of acclimation, we used 64 male Sprague Dawley rats (weight 200 ‑ 300 g; age, 6–8 weeks; Hunan SJA Laboratory Animal Co., Ltd., Hunan, China) in the present study. The animals were randomly divided into four groups (n = 16/group): Sham, Sham + NRG-1, cecal ligation and puncture (CLP), and CLP + NRG-1. To establish the sepsis model, rats in the CLP and CLP + NRG-1 groups underwent laparotomy and ligation of 50% of the cecum. For rats in the Sham and Sham + NRG-1 groups, the abdomen was opened under identical conditions, but no cecal ligation was performed. Rats in all four groups were subcutaneously administered warm saline (50 mL/kg) for fluid resuscitation immediately post-surgery. The wound was treated with 5% lidocaine ointment for analgesia, and rats were placed on a blanket to maintain body temperature during recovery. Subsequently, rats were provided with standard drinking water and diet. According to the previous research programs 10 , the Sham + NRG-1 and CLP + NRG-1 groups were administered recombinant human neuregulin-1 (rhNRG‑1) (cat. no. 10658‑H08H; Sino Biological Inc, Wayne, PA, USA) via the tail vein at a dose of 0.01 µg/g at 12 and 24 h postoperatively. The Sham and CLP groups were injected with equal amounts of saline via the tail vein. 48 h later, 40 rats (n = 10 in each group) were used for electrophysiological recording. At the end of atrial electrophysiology, blood samples were collected via the inferior vena cava and transferred to sealed tubes. After standing for 15 ‑ 20 min, blood samples were centrifuged at 1,500x g for 15 min at 4˚C and stored at ‑80˚C until measurement. After blood sample collection, cardiac tissue samples were harvested for biochemical and histopathological analyses. For histopathological and immunofluorescence analysis, the left atrial myocardium was removed and fixed at 37˚C for at least 48 h in a 10% paraformaldehyde solution. The remaining 24 rats (n = 6 in each group) were used to conduct the whole-cell patch-clamp experiments. 2.3 In vivo electrophysiology Forty-eight hours post-surgery, rats were deeply anesthetized using 3% sodium pentobarbital (60 mg/kg, intraperitoneally). In vivo , atrial electrophysiology was recorded using an electrophysiological recorder and passed through a bioelectric amplifier to the LabChart software for recording and analysis. Data collected included heart rate (HR), P wave duration (PWD), atrial action potential duration (AAPD), atrial effective refractive period (AERP), and AF inducibility. The AAPD was recorded by S1S1 stimulation, the S1S1 stimulation was composed of ten stimuli (S1) stimulation drives, with the cycle length (CL) of the S1 train fixed at 150 ms. AERP was recorded by S1S2 stimulation, and the S1S2 stimulation was composed of eight stimuli (S1) stimulation drives, followed by a ninth extra-stimulus (S2) drive. The CL of the S1 train was fixed at 120 ms, and S2 stimuli gradually decreased from 80 ms at 10 ms or 2 ms step until no action potential duration occurred after the S2 or atrial arrhythmias. AF was induced by six consecutive Burst stimulations (5V, 50Hz, 2s). The incidence of AF was the number of animals with AF in each group as a percentage of the total number of animals. Successful evocation of AF was defined as one of the six consecutive Burst stimuli that produced irregular electrical activity in the atria lasting more than 1 s. The limb II lead electrocardiogram (ECG) showed f waves of varied sizes, intervals, and forms instead of P waves. 2.4 Histology and immunohistochemistry The degree of interstitial collagen deposition in the left atria was assessed using Masson's trichrome staining, and the degree of atrial fibrosis was compared by evaluating the atrial collagen volume fraction. Immunofluorescence staining was performed to examine tyrosine hydroxylase (TH) and acetylcholinesterase levels in atrial tissues. All four groups of myocardial histological specimens were fixed in 4% paraformaldehyde embedded in paraffin and sliced into sections of 4 µm thick sections from the paraffin block of the left atrial. The sections were stained with TH (1:1000, Abcam, England), and choline acetyltransferase (ChAT) (1:1000, Abcam, England) and then incubated with a secondary antibody corresponding to the first antibody. We determined density using computer-assisted Image J software (version 1.52a, USA). Each slide was examined to select three fields. TH-positive areas appeared red, while ChAT-positive areas appeared green, and the computer automatically measured the positive areas. The density was defined as the ratio of the red or green areas to the total detected area. The mean density in the three selected fields were used to present the average density. 2.5 Enzyme-linked immunosorbent assays (ELISA) Rat serum levels of norepinephrine (NE), acetylcholine (Ach), and C-reactive protein (CRP) were measured using ELISA kits in accordance with the manufacturer's instructions (Elabscience, Wuhan, China). 2.6 Whole-cell patch-clamp experiments Protocols for myocyte isolation and ion currents recording are detailed in the Supplementary material. 2.7 Western blot analysis Total protein in the left atrium was extracted with RIPA lysate (cat.no P0013B, Beyotime, China) and phenylmethylsulfonyl fluoride (PMSF) (cat.no P105539, Aladdin Inc, China), and samples (40 µg protein) were run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel after protein measurement, followed by blotting on polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk, PVDF membranes were subjected to overnight incubation at 4°C with the following primary antibodies: GAPDH (1:1000, Servicebio Inc., China), Nav1.5 (1:1000, Alomone Labs, Israel), Cav1.2 (1:1000, Alomone Labs, Israel), and Kv1.5 (1:1000, Alomone Labs, Israel). The membranes were then treated with secondary horseradish peroxidase (HRP)-labeled antibodies (goat anti-rabbit) for 1 h at room temperature after rinsing three times with 1 × TBST. Image J software was used for digitizing and evaluating the band optical intensity, normalized to GAPDH. 2.8 Statistical analysis Data are presented as mean ± standard error of the mean (SEM). Multiple groups were analyzed using a two-way analysis of variance with Bonferroni's post hottest. AF inducibility was expressed as the percentage of the AF rats and analyzed using the chi-square test. Data analysis and image rendering were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) and Origin 2021b software. A two-tailed P value < 0.05 was deemed statistically significant. 3 Results 3.1 Sepsis-induced changes in atrial electrophysiology and the effect of NRG-1 During stimulation by Burst, AF incidences of 50 and 0% were noted in the CLP and Sham groups, respectively, on administering NRG-1, AF incidences of 70 and 60% were documented in the CLP + NRG-1 and Sham + NRG-1 groups, respectively (Fig. 1 and Table 1 ). On analyzing the limb Ⅱ leads ECG, we found that, compared with the Sham group, the rats in the CLP group exhibited an elevated HR, and prolonged PWD, while the Sham + NRG-1 group had a slower heart rate, and longer PWD (Table 1 ). After S1S1 and S1S2 pacing, APD 50 , APD 90 , and AERP of CLP rats, and APD 10 , APD 50 , APD 90 , and AERP of the Sham + NRG-1 rats were reduced when compared with those of Sham rats (Table 1 ). Meanwhile, NRG-1 administration reduced APD 10 in the CLP group (Table 1 ). Table 1 Electrophysiological examination parameters and AF induction in the four groups Parameters Sham (n = 10) Sham + NRG-1 (n = 10) CLP (n = 10) CLP + NRG-1 (n = 10) AF inducibility (Burst pacing) 0 (0/10) 60% (6/10)** 50% (5/10)** 70% (7/10) HR (bpm) 370.33 ± 11.50 335.31 ± 11.05** 418.98 ± 27.66** 393.15 ± 20.96 PWD (ms) 15.92 ± 0.75 19.20 ± 0.84** 19.52 ± 0.92** 19.35 ± 0.88 APD10 (ms) 14.79 ± 2.53 10.83 ± 1.72* 14.22 ± 3.46 9.13 ± 1.77## APD50 (ms) 26.89 ± 3.81 20.52 ± 2.41** 20.24 ± 3.52** 17.61 ± 2.67 APD90 (ms) 57.03 ± 4.42 43.83 ± 2.09** 46.54 ± 3.92* 40.87 ± 9.44 AERP (ms) 45.43 ± 2.06 30.29 ± 4.71** 38.00 ± 3.00** 36.86 ± 3.68 AF: atrial fibrillation; HR: heart rate; PWD: P wave duration; APD: action potential duration; AERP: atrial effective refractive period. * P < 0.05 vs Sham; ** P < 0.01 vs Sham; ## P < 0.01 vs CLP 3.2 Changes in the atrial organization after sepsis and the effect of NRG-1 At 48 h postoperatively, Masson staining results revealed substantial CLP-induced atrial myocardial fibrosis in rats with sepsis, accompanied by disordered left atrial myocytes. Administration of NRG-1 effectively attenuated atrial myocardial fibrosis after sepsis, and left atrial myocytes appeared neatly arranged and ordered (Fig. 2 A&B). 3.3 Sepsis-induced changes in atrial autonomic nerves and the effect of NRG-1 Compared with the Sham group, the density of TH-positive sympathetic nerves was increased, whereas that of ChAT-positive vagus nerves was decreased in the CLP group, as determined by immunofluorescent staining of atrial autonomic nerves (Fig. 3 A-D). Compared with the CLP group, the density of TH-positive sympathetic nerves decreased, and that of ChAT-positive vagus nerves increased in the CLP + NRG-1 group (Fig. 3 A-D). Compared with the Sham group, the density of ChAT-positive vagus nerves was increased in the Sham + NRG-1 group, while the density of TH-positive sympathetic nerves did not differ significantly between groups (Fig. 3 A-D). 3.4 Sepsis-induced changes in serum CRP, NE, and Ach and the effect of NRG-1 Figure 4 represents altered CRP, NE, and Ach serum levels in the four groups. As an important inflammatory marker, CRP is frequently employed to predict and assess the condition and prognosis of critical patients and guide therapy. Compared with the Sham group, the CLP group displayed a significant increase in CRP, and treatment with NRG-1 decreased CRP levels in the CLP + NRG-1 group (Fig. 4 A). Serum NE and Ach levels can simultaneously reflect autonomic function. We found that the septic rats had higher levels of serum NE, and lower levels of Ach than the Sham rats, administration of NRG-1 decreased NE levels in the CLP + NRG-1 group and increased Ach levels in the Sham + NRG-1 and CLP + NRG-1 groups (Fig. 4 B&C). 3.5 Sepsis-induced changes in I Na of atrial myocytes and Nav1.5 protein expression in atrial tissue and the effect of NRG-1 To further investigate the effect of NRG-1 on atrial electrophysiology, we performed whole-cell patch-clamp experiments using acutely isolated atrial myocytes (Fig. 5 ). We first investigated the effect of NRG-1 on I Na in atrial myocytes. In atrial myocytes isolated from CLP rats, sepsis decreased the peak current density of I Na (Fig. 6 A&B). Interestingly, the peak current density of I Na was also reduced in the Sham + NRG-1 and CLP + NRG-1 groups (Fig. 6 A&B). In addition, we observed that the protein expression level of Nav1.5 showed a downregulated trend in the CLP and Sham + NRG-1 groups when compared with that in the Sham group, however, there was no difference in the protein expression level of Nav1.5 between the CLP and CLP + NRG-1 groups (Fig. 6 C). To evaluate the detailed electrophysiological properties of I Na , we measured activation curves, inactivation curves, and recovery time from inactivation. Considering the four groups, we detected no statistically significant differences in the activation kinetic parameters of I Na (Fig. 6 D and Table 2 ), however, both septic, and NRG-1 treated rats, showed a rightward shift and delayed the deactivation rate of the I Na inactivation curve when compared with those of Sham rats. In the CLP + NRG-1 group, NRG-1 administration caused a rightward shift and delayed the deactivation rate of the I Na inactivation curve (Fig. 6 E and Table 2 ). Compared with Sham rats, septic and NRG-1 treated rats showed a prolonged recovery time following I Na inactivation (Fig. 6 F and Table 2 ). Table 2 Changes in the kinetics of I Na after sepsis and the effect of NRG-1 Kinetic parameters Sham ( n = 6) Sham + NRG-1 ( n = 6) CLP ( n = 6) CLP + NRG-1 ( n = 6) V 1/2act (mV) -75.04 ± 0.00 -75.08 ± 0.00 -75.07 ± 0.00 -75.07 ± 0.00 k act 1.50 ± 0.00 1.50 ± 0.00 1.50 ± 0.00 1.50 ± 0.00 V 1/2inact (mV) -79.59 ± 1.14 -72.12 ± 1.46 ** -75.23 ± 1.73 ** -72.44 ± 1.46 # k inact 5.50 ± 1.03 7.04 ± 1.30 8.52 ± 1.57 ** 7.16 ± 1.30 τ (ms) 5.98 ± 0.48 17.19 ± 1.61 ** 8.31 ± 0.69 ** 9.61 ± 1.00 V 1/2act : half maximum activation potential; V 1/2inact : half maximum inactivation potential; k act : slope factor of activation curve; k inact : slope factor of inactivation curve; τ: recovery time constant. ** P < 0.01 vs Sham; # P < 0.05 vs CLP 3.6 Sepsis-induced changes in I Ca, L of atrial myocytes, and atrial Cav1.2 protein expression and the effect of NRG-1 Compared with the Sham group, the peak current density of I Ca, L was significantly decreased in atrial myocytes of the CLP and Sham + NRG-1 groups, and NRG-1 reduced the peak current density of I Ca, L in the CLP + NRG-1 rat atrial myocytes (Fig. 7 A&B). Furthermore, the protein expression level of Cav1.2 was decreased in CLP rats when compared with that in the Sham rats, treatment with NRG-1 decreased the expression level of Cav1.2 in the atrial muscle of Sham and CLP rats (Fig. 7 C). As shown in Fig. 7 D and Table 3 , the activation curves shifted significantly rightward in Sham + NRG-1 and CLP groups when compared with those of the Sham group. Moreover, treatment with NRG-1 shifted the activation curve significantly to the right in the CLP + NRG-1 group. Compared with the Sham group, the activation rate was decelerated in the CLP group while accelerated in the Sham + NRG-1 group, and similar activation rates were noted in the CLP + NRG-1 and CLP groups. Considering I Ca, L inactivation curves, compared with the Sham group, the CLP group showed modest rightward shifts in I Ca, L inactivation, and recovery curves (Fig. 7 E&F). Administration of NRG-1 caused a rightward shift in I Ca, L inactivation (Fig. 7 E ) , and recovery curves of Sham + NRG-1 and CLP + NRG-1 groups (Fig. 7 F). The Sham + NRG-1 group, CLP group, and CLP + NRG-1 group displayed rapid inactivation rates (Table 3 ). Table 3 Changes in the kinetics of I Ca, L after sepsis and the effect of NRG-1 Kinetic parameters Sham ( n = 6) Sham + NRG-1 ( n = 6) CLP ( n = 6) CLP + NRG-1 ( n = 6) V 1/2act (mV) -22.85 ± 0.63 -8.40 ± 0.64 ** -8.11 ± 0.97 ** -5.73 ± 0.41 ## k act 16.61 ± 0.73 9.63 ± 0.64 ** 19.01 ± 1.27 ** 9.15 ± 0.39 ## V 1/2inact (mV) -37.54 ± 3.50 -30.02 ± 2.96 ** -28.93 ± 1.24 ** -32.27 ± 0.89 k inact 18.86 ± 2.01 13.48 ± 2.30 ** 12.71 ± 1.01 ** 11.57 ± 0.63 τ (ms) 140.43 ± 14.06 281.73 ± 12.13 ** 188.32 ± 3.81 ** 287.60 ± 10.64 ## V 1/2act : half maximum activation potential; V 1/2inact : half maximum inactivation potential; k act : slope factor of activation curve; k inact : slope factor of inactivation curve; τ: recovery time constant. ** P < 0.01 vs Sham; ## P < 0.01 vs CLP 3.7 Sepsis-induced changes in I kur of atrial myocytes and atrial Kv1.5 protein expression and the effect of NRG-1 I kur , which predominantly exists in atrial myocytes and is absent in ventricular myocytes, the current is generally inactivated. Compared with the Sham group, the peak current density of I kur was significantly decreased in atrial myocytes of the CLP group and increased in those of the Sham + NRG-1 group (Fig. 8 A&B). Furthermore, the protein expression levels of Kv1.5 showed a down-regulated trend in the CLP group and up-regulated trend in the Sham + NRG-1 group (Fig. 8 D). Moreover, the activation curve of I kur shifted left in atrial myocytes of the CLP and Sham + NRG-1 groups, the activation rate was decelerated in Sham + NRG-1 group but accelerated in the CLP group (Fig. 8 C and Table 4 ). Compared with the CLP group, the activation curve of the CLP + NRG-1 group shifted right, and no statistically significant differences were observed in peak current density, the Kv1.5 expression level, and activation rate (Fig. 8 A-D and Table 4 ). Table 4 Changes in the kinetics of I kur after sepsis and the effect of NRG-1 Kinetic parameters Sham ( n = 6) Sham + NRG-1 ( n = 6) CLP ( n = 6) CLP + NRG-1 ( n = 6) V 1/2act (mV) 39.66 ± 3.10 21.46 ± 0.54 ** 25.89 ± 0.97 ** 36.12 ± 2.43 ## k act 21.95 ± 1.80 18.32 ± 1.74 ** 25.63 ± 0.98 ** 25.45 ± 1.66 V 1/2act : half maximum activation potential; k act : slope factor of activation curve; ** P < 0.01 vs Sham; ## P < 0.01 vs CLP 4 Discussion 4.1 Major findings In the present study, we explored mechanisms that contribute to the development of AF after sepsis. To the best of our knowledge, we first report the effect of NRG-1 on AF in sepsis. In the rat model of sepsis, the incidence of AF was increased, accompanied by severe myocardial fibrosis, and significant changes were documented in cardiac electrophysiology and ionic currents when compared with those in the Sham group. NRG-1 could attenuate the degree of atrial myocardial fibrosis and organismal inflammation in sepsis. Moreover, NRG-1 could promote the development of AF in Sham rats, affecting atrial electrophysiology and ionic currents. 4.2 The possible mechanisms of AF in sepsis Sepsis is a complex multi-organ dysfunction syndrome that frequently causes severe myocardial injury 4 , 10 . Reportedly, 6–20% of patients with severe sepsis can develop new-onset AF in the setting of septic heart injury. Furthermore, the development of AF can lead to high mortality and prolonged hospital stays in patients with sepsis, and AF-induced thromboembolic dislodgement events are likely to worsen patient prognosis 13 . As an independent risk factor for death in patients with sepsis and septic shock, AF is typically considered to occur in two steps 1): atrial tissue remodeling and electrical remodeling as the basis for the development of AF and 2): triggering of AF by arrhythmogenic factors 14 . Atrial tissue remodeling at the onset of AF is often attributed to the development of atrial myocardial fibrosis, although we observed enlarged atrial myocytes and severe myocardial fibrosis in septic rats in the present study. AF is frequently accompanied by changes in atrial electrophysiology, primarily manifested by a decrease in AAPD and a shortening of the effective inactivity period. The results of the present study revealed that the AAPD and effective expiration period were significantly shortened in sepsis. In addition, elevated inflammatory markers in patients with sepsis increase the risk of AF, and the inflammatory response may contribute to the development of arrhythmias via the direct infiltration of inflammatory factors or oxidative damage to atrial myocytes 14 . Levels of serum inflammatory mediators tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and macrophage migration inhibitory factor (MIF) are known to be significantly increased in septic rats 10 . CRP, as an important inflammatory marker, is often used to predict and assess the condition and prognosis of critically ill patients and guide appropriate treatment protocols. Herein, our results revealed increased serum CRP levels in septic rats. Sepsis, chronic heart failure, hypertension, valvular disease, and myocardial infarction can lead to activation of the renin-angiotensin system, autonomic dysfunction, and production of reactive oxygen species, thereby inducing atrial tissue remodeling and thus promoting the development of AF 14 . During autonomic imbalance, sympathetic activation causes the release of NE from sympathetic neuron terminals, which acts on β-adrenergic receptors, causing corresponding changes in ion channels in various atrial myocytes, triggering atrial ectopic electrical activity and promoting the development of AF. Likewise, vagal activation can alter atrial myocyte membrane ionic currents, shortening the effective atrial expiration period and action potential time course and promoting the formation of refractory circuits. In turn, AF can induce autonomic remodeling, leading to overproduction and uneven distribution of neurotransmitters, resulting in ion channel modulation from normal functional feedback regulation in the early stage to causing a shortening of the effective atrial expiration period. This results in the downregulation of ion channel gene transcription and protein expression through a series of signal transduction cascades in the later stage, ultimately leading to reduced channel protein expression and ion current density and maintaining AF. Based on our findings, septic rats exhibited elevated serum NE levels and enhanced atrial sympathetic nerve activity, suggesting that organismal stress is enhanced during sepsis. Accordingly, sepsis could contribute to the development of AF by affecting atrial sympathetic nerve activity. Changes in transmembrane ion flow are the most important determinants in the development of AF. As the main ion flow in phase 0 of the atrial myocyte action potential, I Na plays a crucial role in phase 0 depolarization of atrial myocytes 15 , 16 , and changes in I Na density and conduction velocity of atrial myocytes are inextricably linked to the development and maintenance of AF. Moreover, changes in conduction velocity are persistently accompanied by changes in I Na . Meanwhile, the decreased cardiac excitability secondary to decreased I Na may significantly contribute to reduced contractility in a rat model of sepsis 17 . In the present study, we found that the peak current density of I Na in atrial myocytes was reduced, and the expression of Nav1.5 was decreased in CLP rats. Sepsis may influence the development of AF by affecting inactivation and recovery properties after I Na inactivation. L-type calcium channels are pivotal for the myocardial excitation-contraction coupling mechanism, mainly maintaining the plateau phase 2 of the action potential in cardiac myocytes, participating in the opening of the channel and Ca 2+ inward flow, determining the length of the plateau phase and the duration of the action potential. The reduced I Ca, L density in atrial myocytes can lead to accelerated repolarization and shortened APD, which may be the primary mechanism underlying atrial electrical remodeling 15 , 18 . Atrial myocytes exhibited reduced I Ca, L peak current density, and Cav1.2 expression during sepsis. Accordingly, sepsis impacts I Ca, L activation, inactivation, and recovery properties after inactivation, which, in turn, contribute to the development of AF. I kur , predominantly present in atrial myocytes and absent in ventricular myocytes, promotes rapid repolarization of phase 1 action potentials in atrial myocytes, and I kur -targeted treatment may prevent the development of AF without increasing the risk of ventricular arrhythmias. I kur density was found to be significantly reduced owing to the decreased expression of Kv1.5 protein in patients with AF. Downregulated Kv1.5 expression during AF reportedly attenuates I kur prolongation of APD and ERP. However, APD and ERP are known to be reduced during AF, which may be attributed to the continuous activation of the Kv1.5 channel. Cardiomyocytes may eventually prevent electrical remodeling and shortening of APD and ERP by reducing the expression of Kv1.5 19 . In essence, it is an adaptive response of cardiomyocytes to excessive atrial contraction. Thus, both increased and decreased Kv1.5 expression may increase susceptibility to AF. In the present study, the peak I kur current density and Kv1.5 expression were decreased in the atrial myocytes of septic rats. Likewise, sepsis can lead to altered I kur activation properties, resulting in AF. 4.3 Effect of NRG-1 on AF and its underlying mechanism NRG-1, a peptide produced by endocardial and myocardial microvascular endothelial cells, reportedly exerts a multifaceted cardioprotective by comprehensively activating the NRG-1/ErbBs pathway to regulate the level of fibrosis in response to metabolism, inflammation, and cardiac injury. Importantly, NRG-1 is considered a potential therapeutic agent for cardiovascular disease. The activity of NRG-1 is mainly attributed to ErbB receptor activation and Ach receptor expression 9 , 20 , 21 . However, current studies on the effects of NRG-1 on cardiac arrhythmias remain controversial. NRG-1 pretreatment can promote myocardial regeneration, effectively alleviate cardiac dysfunction in rats with myocardial infarction, and improve ventricular myocyte electrophysiology, thereby reducing the incidence of ventricular arrhythmias. Moreover, NRG-1 activation can improve atrial electrophysiological stability through ErbB4 receptor-related signaling pathways, thereby inhibiting the development of AF 20 , 22 . In contrast, Ford et al 23 . have shown that NRG-1 modulates cardiac parasympathetic tone and may be involved in the pathogenesis of arrhythmias and heart failure. Furthermore, NRG-1 levels were found to be significantly correlated with the presence of paroxysmal AF 12 . The effects of NRG-1 on septic AF need to be examined in future investigations. Herein, our findings showed that NRG-1 did not significantly impact the occurrence of AF in septic rats, but could increase the susceptibility of normal rats to AF. In addition, administering NRG-1 aggravated the shortening of the atrial action potential in septic and Sham rats, which reduced the effective expiration period of the atrial muscle in normal rats and promoted the occurrence of AF. Considering the mechanisms associated with the development of AF in sepsis, we speculate that NRG-1 may influence the development of AF by affecting the level of inflammation and circulating hormones in the body, and the autonomic function of the atria and ion channels in atrial myocytes. The inflammatory response and autonomic dysfunction can lead to the development of arrhythmias t via diverse pathways 14 . Administration of NRG-1 can significantly reduce the levels of circulating inflammatory mediators in septic rats and improve myocardial injury in sepsis 24 . Herein, we found that NRG-1 could reduce atrial myocardial fibrosis and decrease serum CRP levels in rats with sepsis, which was beneficial for inhibiting the inflammatory response to sepsis, reducing atrial tissue remodeling, and decreasing the occurrence of AF. NRG-1 enhanced serum Ach levels and atrial vagal nerve activity in rats with sepsis. Meanwhile, serum Ach levels and atrial vagal nerve activity were enhanced in Sham rats administered NRG-1, suggesting that NRG-1 contributes to atrial autonomic nerve dysfunction by affecting atrial vagal nerve activity. Changes in transmembrane ion flow are the most critical determinants in the development of AF. In the present study, administering NRG-1 reduced the peak current density of I Na in atrial myocytes in the CLP and Sham groups, and there were no differences in channel activation kinetics, altered inactivation kinetics, and prolonged recovery time after inactivation. We observed that Nav1.5 expression was reduced in the Sham + NRG-1 group, with no change detected in the CLP + NRG-1 group. NRG-1 reduced the I Ca, L peak current density, and Cav1.2 expression in the CLP and Sham groups, with I Ca, L depolarized in the Sham + NRG-1, and CLP + NRG-1 groups. Moreover, I Ca, L inactivation mechanics were altered, and the recovery time after inactivation was prolonged in the Sham, CLP, and Sham + NRG-1 groups. Furthermore, NRG-1 increased the peak current density of I kur and Kv1.5 expression in atrial myocytes of the Sham + NRG-1 group. NRG-1 administration could alter the activation characteristics of I kur in atrial myocytes of normal and septic rats, suggesting that NRG-1 may impact electrophysiological and ion current characteristics of atrial myocytes and promote the occurrence of AF. 5 Conclusions In summary, the occurrence of AF in septic rats can be associated with elevated levels of inflammation, sympathetic excitation, atrial tissue remodeling induced by left atrial myocardial fibrosis, shortening of the ERP, APD, and altered I Na , I Ca, L , I kur properties of atrial myocytes leading to atrial myocyte electrical remodeling. NRG-1 can increase the incidence of AF in normal rats, which was associated with NRG-1 excitation of the vagus nerve, affecting I Na , I Ca, L , and I kur properties of atrial myocytes. 6 Limitations This study has several limitations. Herein, we only observed the effects of tail vein NRG-1 injection on myocardial ion channels in septic rats, the direct effect of NRG-1 on sepsis-injured cardiomyocytes needs to be determined. This study only explored the effect of NRG-1 treatment on AF after sepsis, the effects of NRG-1 preconditioning on AF after sepsis, as well as, on other types of arrhythmias, need to be further explored. Declarations Acknowledgements We thank Yuting Chen and Teng Wang for excellent technical support of electrophysiology and patch clamp. Funding The present work was supported by the National Natural Science Foundation of China (Grant No. 81772044), and Hainan Provincial Personnel Support Project (No. 2019RC368), and Hubei Province Key Laboratory Project (No. 2021KFY035). Availability of data and material The datasets used and analysed during the current study available from the corresponding author on reasonable request. Author Contributions All authors contributed to the study’s conception and design. Wen Kang and Jingru Deng contributed equally to the study. The study was conceptualized and planned by Wen Kang, Xi Wang, Fang Zhou, Kang Liu, and Long Wang. Wen Kang, Jingru Deng and Zheru Fan participated in the experimental study. Wen Kang and Jingru Deng contributed to the preparation and review of the text as well as the analysis of the experimental data. All authors critically reviewed and approved the final version for submission. Ethics approval The animal management and welfare were reviewed and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (Date:2020-07-24/No.20200705, Wuhan, China). All methods are reported in accordance with ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Conflict of Interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. References Walkey, A. J. et al. Atrial fibrillation among Medicare beneficiaries hospitalized with sepsis: incidence and risk factors. Am Heart J 165 , 949-955 e943 (2013). https://doi.org:10.1016/j.ahj.2013.03.020 Aibar, J. & Schulman, S. New-Onset Atrial Fibrillation in Sepsis: A Narrative Review. Semin Thromb Hemost 47 , 18-25 (2021). https://doi.org:10.1055/s-0040-1714400 Rudd, K. E. et al. Global, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the Global Burden of Disease Study. Lancet 395 , 200-211 (2020). https://doi.org:10.1016/S0140-6736(19)32989-7 Bashar, S. K., Hossain, M. B., Ding, E., Walkey, A. J., McManus, D. D. & Chon, K. H. Atrial Fibrillation Detection During Sepsis: Study on MIMIC III ICU Data. IEEE J Biomed Health Inform 24 , 3124-3135 (2020). https://doi.org:10.1109/JBHI.2020.2995139 Shahreyar, M., Fahhoum, R., Akinseye, O., Bhandari, S., Dang, G. & Khouzam, R. N. Severe sepsis and cardiac arrhythmias. Ann Transl Med 6 , 6 (2018). https://doi.org:10.21037/atm.2017.12.26 Fernando, S. M. et al. New-onset atrial fibrillation and associated outcomes and resource use among critically ill adults-a multicenter retrospective cohort study. Crit Care 24 , 15 (2020). https://doi.org:10.1186/s13054-020-2730-0 Afzal, B., Ali, S. A. & Jamil, B. Outcome of Atrial Fibrillation in Patients With Sepsis. Cureus 13 , e19159 (2021). https://doi.org:10.7759/cureus.19159 Jie, B., Zhang, X., Wu, X., Xin, Y., Liu, Y. & Guo, Y. Neuregulin-1 suppresses cardiomyocyte apoptosis by activating PI3K/Akt and inhibiting mitochondrial permeability transition pore. Mol Cell Biochem 370 , 35-43 (2012). https://doi.org:10.1007/s11010-012-1395-7 Geissler, A., Ryzhov, S. & Sawyer, D. B. 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Ann Pharmacother 47 , 1266-1271 (2013). https://doi.org:10.1177/1060028013500938 Bosch, N. A., Cimini, J. & Walkey, A. J. Atrial Fibrillation in the ICU. Chest 154 , 1424-1434 (2018). https://doi.org:10.1016/j.chest.2018.03.040 Zhang, K., Ma, Z., Song, C., Duan, X., Yang, Y. & Li, G. Role of ion channels in chronic intermittent hypoxia-induced atrial remodeling in rats. Life Sci 254 , 117797 (2020). https://doi.org:10.1016/j.lfs.2020.117797 Wallace, C. H., Baczko, I., Jones, L., Fercho, M. & Light, P. E. Inhibition of cardiac voltage-gated sodium channels by grape polyphenols. Br J Pharmacol 149 , 657-665 (2006). https://doi.org:10.1038/sj.bjp.0706897 Koesters, A., Engisch, K. L. & Rich, M. M. Decreased cardiac excitability secondary to reduction of sodium current may be a significant contributor to reduced contractility in a rat model of sepsis. Crit Care 18 , R54 (2014). https://doi.org:10.1186/cc13800 Aoki, Y. et al. Role of ion channels in sepsis-induced atrial tachyarrhythmias in guinea pigs. Br J Pharmacol 166 , 390-400 (2012). https://doi.org:10.1111/j.1476-5381.2011.01769.x Brundel, B. J. et al. Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation. Circulation 103 , 684-690 (2001). https://doi.org:10.1161/01.cir.103.5.684 Rao, P. et al. Pretreatment with neuregulin-1 improves cardiac electrophysiological properties in a rat model of myocardial infarction. Exp Ther Med 17 , 3141-3149 (2019). https://doi.org:10.3892/etm.2019.7306 Falls, D. L. Neuregulins: functions, forms, and signaling strategies. Exp Cell Res 284 , 14-30 (2003). https://doi.org:10.1016/s0014-4827(02)00102-7 Zhou, X. et al. Regulation of the NRG1/ErbB4 Pathway in the Intrinsic Cardiac Nervous System Is a Potential Treatment for Atrial Fibrillation. Front Physiol 9 , 1082 (2018). https://doi.org:10.3389/fphys.2018.01082 Ford, B. D. et al. Neuregulin-1 suppresses muscarinic receptor expression and acetylcholine-activated muscarinic K+ channels in cardiac myocytes. Biochem Biophys Res Commun 308 , 23-28 (2003). https://doi.org:10.1016/s0006-291x(03)01319-6 Zhou, Q. et al. Effect of neuregulin-1 on heart function and inflammatory mediators in rats with sepsis. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue 30 , 140-144 (2018). https://doi.org:10.3760/cma.j.issn.2095-4352.2018.02.009 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMateriald1.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3246284","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":226263843,"identity":"ab0e3ce7-e92b-4091-940f-202f69b8f4c0","order_by":0,"name":"Wen Kang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Kang","suffix":""},{"id":226263844,"identity":"f669b6dc-3fc4-40f0-932b-684db1481a1d","order_by":1,"name":"Jingru Deng","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingru","middleName":"","lastName":"Deng","suffix":""},{"id":226263845,"identity":"244a7ab5-fe68-4b4a-953a-c1b4ec17af5c","order_by":2,"name":"Zheru Fan","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zheru","middleName":"","lastName":"Fan","suffix":""},{"id":226263846,"identity":"d84944e5-92a1-4e88-b910-11cf48ad422c","order_by":3,"name":"Fang Zhou","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Zhou","suffix":""},{"id":226263847,"identity":"2f8eb610-8884-48cf-8647-8d606bbbdd93","order_by":4,"name":"Xi Wang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Wang","suffix":""},{"id":226263848,"identity":"a9ed4942-aee4-4ac7-9605-8c783ca73ab5","order_by":5,"name":"Kang Liu","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Liu","suffix":""},{"id":226263849,"identity":"f040c520-7b63-4fe2-beb5-d3bfbd3ce318","order_by":6,"name":"Long Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYLCCByBCgoHxQUJFDZFaEiBamA0enDlGmhY2yYctzIRVGxw/e/hFQsUduw23e8wqEhvYGPjbuxPwazmTl2aRcOZZ8oY7Z8xuJO6QYZA4c3YDXi1mB3LMDBLbDieb3cgBajnDxmAgkUtAy/k3CC0FiW3MRGi5kWP8AKjFDqSFgSgt9jfemDEknDmcYH8jrVgi4cwxHoJ+kezPMf7woeKwveSM5I0ff1TUyPG39+LXAgRsEkAisQHK4yGkHASYP4AcSIzKUTAKRsEoGKEAAHg8UO1bwrEVAAAAAElFTkSuQmCC","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2023-08-08 16:44:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3246284/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3246284/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":41773773,"identity":"7902fc6a-1c52-4c96-9ac1-542f6606d6d1","added_by":"auto","created_at":"2023-08-18 15:36:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":13944201,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative limb Ⅱ leads ECG recording of burst pacing in the four groups\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/ff9dbc9ec0d3607b31d9aa46.png"},{"id":41773789,"identity":"3dc5d7d9-d726-4e78-a9ab-ecbb7869888a","added_by":"auto","created_at":"2023-08-18 15:36:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30575928,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the atrial organization after sepsis and the effect of NRG-1\u003cstrong\u003e (A) \u003c/strong\u003eRepresentative Masson staining of atrial tissue of four groups (original magnification, 400 ×) of LA, bars=20 µm. \u003cstrong\u003e(B) \u003c/strong\u003eLevels of atrial fibrosis in each group. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs Sham; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs CLP\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/3c525629b773b377a46a103c.png"},{"id":41775332,"identity":"98f36da8-99a4-46c3-9ff5-2199ebadc3df","added_by":"auto","created_at":"2023-08-18 15:44:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3213739,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in atrial autonomic nerves after sepsis and the effect of NRG-1\u003cstrong\u003e (A) \u003c/strong\u003eRepresentative images of TH immunofluorescence staining (original magnification, 400 ×) of the left atrium, bars=50 µm. Blue: Nucleus of cardiomyocyte; Red: TH positive. \u003cstrong\u003e(B) \u003c/strong\u003eThe statistical bars of TH in each group. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative images of ChAT immunofluorescence staining (original magnification, 400 ×) of the left atrium, bars=50 µm. Blue: Nucleus of cardiomyocyte; Green: ChAT positive.\u003cstrong\u003e (D) \u003c/strong\u003eThe statistical bars of ChAT in each group. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05 vs Sham; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs Sham; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs CLP. TH: tyrosine hydroxylase; ChAT: choline acetyltransferase\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/0f6898eb89cd2900a7be0be7.png"},{"id":41773766,"identity":"d0a8d8fb-6f5e-4653-b904-24316e413a52","added_by":"auto","created_at":"2023-08-18 15:36:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":172931,"visible":true,"origin":"","legend":"\u003cp\u003eSepsis-induced changes in serum CRP, NE, and Ach and the effect of NRG-1 \u003cstrong\u003e(A)\u003c/strong\u003e Statistical analysis of serum CRP levels in four groups of rats. \u003cstrong\u003e(B)\u003c/strong\u003e Statistical analysis of serum NE levels in four groups of rats. \u003cstrong\u003e(C)\u003c/strong\u003e Statistical analysis of serum Ach levels in four groups of rats.\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs Sham; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs CLP. CRP: C-reactive protein; NE: norepinephrine; Ach: acetylcholine\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/234881e7d43385426935aa40.png"},{"id":41773771,"identity":"047694ae-4b2b-411b-82b7-d2d0e329248c","added_by":"auto","created_at":"2023-08-18 15:36:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3453142,"visible":true,"origin":"","legend":"\u003cp\u003eThe process of atrial myocyte isolation \u003cstrong\u003e(A) \u003c/strong\u003eState of the atria when digestion is complete \u003cstrong\u003e(B) \u003c/strong\u003eIsolated atrial myocytes for diaphragm clamp experiments\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/de724992a2839cda232b8a27.png"},{"id":41773767,"identity":"da91d077-4221-424f-8eb5-9db185c7ff58","added_by":"auto","created_at":"2023-08-18 15:36:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":718147,"visible":true,"origin":"","legend":"\u003cp\u003eSepsis-induced changes in I\u003csub\u003eNa\u003c/sub\u003e of atrial myocytes and Nav1.5 protein expression in atrial tissue and the effect of NRG-1 \u003cstrong\u003e(A)\u003c/strong\u003e Current density curve of each group of I\u003csub\u003eNa.\u003c/sub\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(B) \u003c/strong\u003eStatistical analysis of maximum I\u003csub\u003eNa\u003c/sub\u003e density in each group. \u003cstrong\u003e(C)\u003c/strong\u003e Nav1.5 protein expression in the atrial and the comparison of quantified Nav1.5 expression levels in the atrial across the four groups. \u003cstrong\u003e(D) \u003c/strong\u003eComparison of activation curves of I\u003csub\u003eNa\u003c/sub\u003e between four groups. \u003cstrong\u003e(E) \u003c/strong\u003eComparison of inactivation curves of I\u003csub\u003eNa\u003c/sub\u003e between four groups. \u003cstrong\u003e(F) \u003c/strong\u003eComparison of recovery curves of I\u003csub\u003eNa\u003c/sub\u003e between four groups. Data are presented as the mean ± SEM. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs Sham; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs Sham; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs CLP\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/88daede64f6f666347d54dd7.png"},{"id":41773769,"identity":"5d2d78e5-085b-4277-99fc-44e29445d0c3","added_by":"auto","created_at":"2023-08-18 15:36:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":742386,"visible":true,"origin":"","legend":"\u003cp\u003eSepsis-induced changes in I\u003csub\u003eCa, L\u003c/sub\u003e of atrial myocytes, and atrial Cav1.2 protein expression and the effect of NRG-1 \u003cstrong\u003e(A)\u003c/strong\u003e Current density curve of each group of I\u003csub\u003eCa, L.\u003c/sub\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(B) \u003c/strong\u003eStatistical analysis of maximum I\u003csub\u003eCa, L\u003c/sub\u003e density in each group. \u003cstrong\u003e(C)\u003c/strong\u003e Cav1.2 protein expression in the atrial and the comparison of quantified Cav1.2 expression levels in the atrial across the four groups. \u003cstrong\u003e(D) \u003c/strong\u003eComparison of activation curves of I\u003csub\u003eCa, L\u003c/sub\u003e between four groups. \u003cstrong\u003e(E) \u003c/strong\u003eComparison of inactivation curves of I\u003csub\u003eCa, L\u003c/sub\u003e between four groups. \u003cstrong\u003e(F) \u003c/strong\u003eComparison of recovery curves of I\u003csub\u003eCa, L\u003c/sub\u003e between four groups. Data are presented as the mean ± SEM. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs Sham;\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs CLP; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs CLP\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/fc5986a61f3fdb8a7845cd96.png"},{"id":41773765,"identity":"551e1e2a-baca-4c8f-ae3b-856bdaff180e","added_by":"auto","created_at":"2023-08-18 15:36:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":555425,"visible":true,"origin":"","legend":"\u003cp\u003eSepsis-induced changes in I\u003csub\u003ekur\u003c/sub\u003e of atrial myocytes and atrial Kv1.5 protein expression and the effect of NRG-1 \u003cstrong\u003e(A)\u003c/strong\u003e Current density curve of each group of I\u003csub\u003ekur.\u003c/sub\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(B) \u003c/strong\u003eStatistical analysis of maximum I\u003csub\u003ekur\u003c/sub\u003e density in each group. \u003cstrong\u003e(C)\u003c/strong\u003e Comparison of activation curves of I\u003csub\u003ekur\u003c/sub\u003e between four groups. \u003cstrong\u003e(D) \u003c/strong\u003eKv1.5 protein expression in the atrial and the comparison of quantified Kv1.5 expression levels in the atrial across the four groups.\u003cstrong\u003e \u003c/strong\u003eData are presented as the mean ± SEM. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs Sham\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/9cf009cff6f19bb739b21a22.png"},{"id":44249437,"identity":"6abcabe8-586d-4cbf-9df7-f1d9996a9cec","added_by":"auto","created_at":"2023-10-08 07:22:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5341228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/6660fb05-3958-4065-876c-ef4270a5b420.pdf"},{"id":41773768,"identity":"3aa8a9f5-ee3f-4256-ba24-bd07ae25873d","added_by":"auto","created_at":"2023-08-18 15:36:53","extension":"doc","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":40448,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMateriald1.doc","url":"https://assets-eu.researchsquare.com/files/rs-3246284/v1/685c50437c63abe3f01f82fa.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanisms associated with the development of atrial fibrillation after sepsis and the role of neuregulin-1","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eIn organisms, an infection can induce a dysregulated systemic inflammatory response, resulting in the development of sepsis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Along with an aging population and increased complications, sepsis has become a major cause of death and critical illness worldwide and causes an estimated 11\u0026nbsp;million deaths worldwide each year\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In patients with sepsis, morbidity and mortality are significantly associated with the occurrence of complications, with cardiac injury particularly common among the observed complications\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Cardiac injury in sepsis has been associated with cardiac dysfunction, as well as with the development of multiple arrhythmias, including atrial fibrillation (AF), ventricular fibrillation, supraventricular tachycardia, and ventricular tachycardia. Furthermore, the development of arrhythmias is closely associated with high mortality and prolonged hospital stays in patients with sepsis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAF, the most common arrhythmic disease in patients with sepsis, occurs nearly six times more frequently in these patients than in non-septic patients, impacting approximately one-fifth of all patients with sepsis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Following the onset of sepsis, changes in pathophysiological processes such as systemic pro-inflammatory cytokine release, high levels of stress hormone secretion, intravascular volume shifts, electrolyte dysregulation, autonomic imbalance, and cardiovascular damage can predispose individuals to the development of AF\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Persistent AF may be a specific marker of the severity of sepsis. Moreover, the occurrence of AF in patients with sepsis may lead to new-onset heart failure and thromboembolism, as well as ischemic stroke and systemic embolism, which can seriously endanger patients' lives\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Therefore, carefully monitoring sepsis development, and early prevention and treatment of septic AF using diverse measures will reduce the mortality among these patients and markedly improve their prognosis.\u003c/p\u003e \u003cp\u003eNeuregulin-1 (NRG-1) is produced by endocardial and myocardial microvascular endothelial cells and binds to the epidermal growth factor receptor (ErbB) on the surface of adjacent cardiac myocytes to activate tyrosine kinases, which reportedly play a key role in the induction of embryonic development such as cardiac development and myogenesis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In addition, NRG-1 may exert multifaceted cardioprotective effects by comprehensively activating the NRG-1/ErbBs signaling pathway and regulating the level of fibrosis in response to metabolism, inflammation, and cardiac injury\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. NRG-1 was shown to protect the vascular endothelium and cardiomyocytes by inhibiting immune-inflammatory responses and suppressing excessive activation of the renin-angiotensin-aldosterone system, thereby improving cardiac function and significantly increasing the survival of septic rats\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Furthermore, serum NRG-1 levels were found to be closely associated with the occurrence of paroxysmal AF\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the effect of NRG-1 on AF after sepsis remains poorly explored.\u003c/p\u003e \u003cp\u003eAccordingly, in the present study, we aimed to provide a more theoretical basis for the treatment of post-sepsis AF by elucidating in-depth mechanisms underlying the occurrence of post-sepsis AF and the related role of NRG-1.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethics approval\u003c/h2\u003e \u003cp\u003e The animal management and welfare were reviewed and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (Date:2020-07-24/No.20200705, Wuhan, China). All methods are reported in accordance with ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals and specimen collection\u003c/h2\u003e \u003cp\u003eAfter one week of acclimation, we used 64 male Sprague Dawley rats (weight 200 ‑ 300 g; age, 6\u0026ndash;8 weeks; Hunan SJA Laboratory Animal Co., Ltd., Hunan, China) in the present study. The animals were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;16/group): Sham, Sham\u0026thinsp;+\u0026thinsp;NRG-1, cecal ligation and puncture (CLP), and CLP\u0026thinsp;+\u0026thinsp;NRG-1. To establish the sepsis model, rats in the CLP and CLP\u0026thinsp;+\u0026thinsp;NRG-1 groups underwent laparotomy and ligation of 50% of the cecum. For rats in the Sham and Sham\u0026thinsp;+\u0026thinsp;NRG-1 groups, the abdomen was opened under identical conditions, but no cecal ligation was performed. Rats in all four groups were subcutaneously administered warm saline (50 mL/kg) for fluid resuscitation immediately post-surgery. The wound was treated with 5% lidocaine ointment for analgesia, and rats were placed on a blanket to maintain body temperature during recovery. Subsequently, rats were provided with standard drinking water and diet. According to the previous research programs\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, the Sham\u0026thinsp;+\u0026thinsp;NRG-1 and CLP\u0026thinsp;+\u0026thinsp;NRG-1 groups were administered recombinant human neuregulin-1 (rhNRG‑1) (cat. no. 10658‑H08H; Sino Biological Inc, Wayne, PA, USA) via the tail vein at a dose of 0.01 \u0026micro;g/g at 12 and 24 h postoperatively. The Sham and CLP groups were injected with equal amounts of saline via the tail vein. 48 h later, 40 rats (n\u0026thinsp;=\u0026thinsp;10 in each group) were used for electrophysiological recording. At the end of atrial electrophysiology, blood samples were collected via the inferior vena cava and transferred to sealed tubes. After standing for 15 ‑ 20 min, blood samples were centrifuged at 1,500x g for 15 min at 4˚C and stored at ‑80˚C until measurement. After blood sample collection, cardiac tissue samples were harvested for biochemical and histopathological analyses. For histopathological and immunofluorescence analysis, the left atrial myocardium was removed and fixed at 37˚C for at least 48 h in a 10% paraformaldehyde solution. The remaining 24 rats (n\u0026thinsp;=\u0026thinsp;6 in each group) were used to conduct the whole-cell patch-clamp experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 In vivo electrophysiology\u003c/h2\u003e \u003cp\u003eForty-eight hours post-surgery, rats were deeply anesthetized using 3% sodium pentobarbital (60 mg/kg, intraperitoneally). \u003cem\u003eIn vivo\u003c/em\u003e, atrial electrophysiology was recorded using an electrophysiological recorder and passed through a bioelectric amplifier to the LabChart software for recording and analysis. Data collected included heart rate (HR), P wave duration (PWD), atrial action potential duration (AAPD), atrial effective refractive period (AERP), and AF inducibility. The AAPD was recorded by S1S1 stimulation, the S1S1 stimulation was composed of ten stimuli (S1) stimulation drives, with the cycle length (CL) of the S1 train fixed at 150 ms. AERP was recorded by S1S2 stimulation, and the S1S2 stimulation was composed of eight stimuli (S1) stimulation drives, followed by a ninth extra-stimulus (S2) drive. The CL of the S1 train was fixed at 120 ms, and S2 stimuli gradually decreased from 80 ms at 10 ms or 2 ms step until no action potential duration occurred after the S2 or atrial arrhythmias. AF was induced by six consecutive Burst stimulations (5V, 50Hz, 2s). The incidence of AF was the number of animals with AF in each group as a percentage of the total number of animals. Successful evocation of AF was defined as one of the six consecutive Burst stimuli that produced irregular electrical activity in the atria lasting more than 1 s. The limb II lead electrocardiogram (ECG) showed f waves of varied sizes, intervals, and forms instead of P waves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Histology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eThe degree of interstitial collagen deposition in the left atria was assessed using Masson's trichrome staining, and the degree of atrial fibrosis was compared by evaluating the atrial collagen volume fraction. Immunofluorescence staining was performed to examine tyrosine hydroxylase (TH) and acetylcholinesterase levels in atrial tissues. All four groups of myocardial histological specimens were fixed in 4% paraformaldehyde embedded in paraffin and sliced into sections of 4 \u0026micro;m thick sections from the paraffin block of the left atrial. The sections were stained with TH (1:1000, Abcam, England), and choline acetyltransferase (ChAT) (1:1000, Abcam, England) and then incubated with a secondary antibody corresponding to the first antibody. We determined density using computer-assisted Image J software (version 1.52a, USA). Each slide was examined to select three fields. TH-positive areas appeared red, while ChAT-positive areas appeared green, and the computer automatically measured the positive areas. The density was defined as the ratio of the red or green areas to the total detected area. The mean density in the three selected fields were used to present the average density.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Enzyme-linked immunosorbent assays (ELISA)\u003c/h2\u003e \u003cp\u003e Rat serum levels of norepinephrine (NE), acetylcholine (Ach), and C-reactive protein (CRP) were measured using ELISA kits in accordance with the manufacturer's instructions (Elabscience, Wuhan, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Whole-cell patch-clamp experiments\u003c/h2\u003e \u003cp\u003eProtocols for myocyte isolation and ion currents recording are detailed in the Supplementary material.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western blot analysis\u003c/h2\u003e \u003cp\u003eTotal protein in the left atrium was extracted with RIPA lysate (cat.no P0013B, Beyotime, China) and phenylmethylsulfonyl fluoride (PMSF) (cat.no P105539, Aladdin Inc, China), and samples (40 \u0026micro;g protein) were run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel after protein measurement, followed by blotting on polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk, PVDF membranes were subjected to overnight incubation at 4\u0026deg;C with the following primary antibodies: GAPDH (1:1000, Servicebio Inc., China), Nav1.5 (1:1000, Alomone Labs, Israel), Cav1.2 (1:1000, Alomone Labs, Israel), and Kv1.5 (1:1000, Alomone Labs, Israel). The membranes were then treated with secondary horseradish peroxidase (HRP)-labeled antibodies (goat anti-rabbit) for 1 h at room temperature after rinsing three times with 1 \u0026times; TBST. Image J software was used for digitizing and evaluating the band optical intensity, normalized to GAPDH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Multiple groups were analyzed using a two-way analysis of variance with Bonferroni's post hottest. AF inducibility was expressed as the percentage of the AF rats and analyzed using the chi-square test. Data analysis and image rendering were performed using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) and Origin 2021b software. A two-tailed \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003e0.05\u003c/em\u003e was deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Sepsis-induced changes in atrial electrophysiology and the effect of NRG-1\u003c/h2\u003e\n\u003cp\u003eDuring stimulation by Burst, AF incidences of 50 and 0% were noted in the CLP and Sham groups, respectively, on administering NRG-1, AF incidences of 70 and 60% were documented in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 and Sham\u0026thinsp;+\u0026thinsp;NRG-1 groups, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eand Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). On analyzing the limb Ⅱ leads ECG, we found that, compared with the Sham group, the rats in the CLP group exhibited an elevated HR, and prolonged PWD, while the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group had a slower heart rate, and longer PWD (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). After S1S1 and S1S2 pacing, APD\u003csub\u003e50\u003c/sub\u003e, APD\u003csub\u003e90\u003c/sub\u003e, and AERP of CLP rats, and APD\u003csub\u003e10\u003c/sub\u003e, APD\u003csub\u003e50\u003c/sub\u003e, APD\u003csub\u003e90\u003c/sub\u003e, and AERP of the Sham\u0026thinsp;+\u0026thinsp;NRG-1 rats were reduced when compared with those of Sham rats (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Meanwhile, NRG-1 administration reduced APD\u003csub\u003e10\u003c/sub\u003e in the CLP group (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eElectrophysiological examination parameters and AF induction in the four groups\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eParameters\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAF inducibility\u003c/p\u003e\n\u003cp\u003e(Burst pacing)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0 (0/10)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e60% (6/10)**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e50% (5/10)**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e70% (7/10)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHR (bpm)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e370.33\u0026thinsp;\u0026plusmn;\u0026thinsp;11.50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e335.31\u0026thinsp;\u0026plusmn;\u0026thinsp;11.05**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e418.98\u0026thinsp;\u0026plusmn;\u0026thinsp;27.66**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e393.15\u0026thinsp;\u0026plusmn;\u0026thinsp;20.96\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePWD (ms)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAPD10 (ms)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14.22\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77##\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAPD50 (ms)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.52**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e17.61\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAPD90 (ms)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e57.03\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e43.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e46.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.92*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40.87\u0026thinsp;\u0026plusmn;\u0026thinsp;9.44\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAERP (ms)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e45.43\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30.29\u0026thinsp;\u0026plusmn;\u0026thinsp;4.71**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e38.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.00**\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.68\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eAF: atrial fibrillation; HR: heart rate; PWD: P wave duration; APD: action potential duration; AERP: atrial effective refractive period. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs Sham; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs Sham; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs CLP\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Changes in the atrial organization after sepsis and the effect of NRG-1\u003c/h2\u003e\n\u003cp\u003eAt 48 h postoperatively, Masson staining results revealed substantial CLP-induced atrial myocardial fibrosis in rats with sepsis, accompanied by disordered left atrial myocytes. Administration of NRG-1 effectively attenuated atrial myocardial fibrosis after sepsis, and left atrial myocytes appeared neatly arranged and ordered (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026amp;B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Sepsis-induced changes in atrial autonomic nerves and the effect of NRG-1\u003c/h2\u003e\n\u003cp\u003eCompared with the Sham group, the density of TH-positive sympathetic nerves was increased, whereas that of ChAT-positive vagus nerves was decreased in the CLP group, as determined by immunofluorescent staining of atrial autonomic nerves (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). Compared with the CLP group, the density of TH-positive sympathetic nerves decreased, and that of ChAT-positive vagus nerves increased in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). Compared with the Sham group, the density of ChAT-positive vagus nerves was increased in the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group, while the density of TH-positive sympathetic nerves did not differ significantly between groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Sepsis-induced changes in serum CRP, NE, and Ach and the effect of NRG-1\u003c/h2\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e represents altered CRP, NE, and Ach serum levels in the four groups. As an important inflammatory marker, CRP is frequently employed to predict and assess the condition and prognosis of critical patients and guide therapy. Compared with the Sham group, the CLP group displayed a significant increase in CRP, and treatment with NRG-1 decreased CRP levels in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Serum NE and Ach levels can simultaneously reflect autonomic function. We found that the septic rats had higher levels of serum NE, and lower levels of Ach than the Sham rats, administration of NRG-1 decreased NE levels in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 group and increased Ach levels in the Sham\u0026thinsp;+\u0026thinsp;NRG-1 and CLP\u0026thinsp;+\u0026thinsp;NRG-1 groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026amp;C).\u003c/p\u003e\n\u003ch2\u003e3.5 Sepsis-induced changes in I\u003csub\u003eNa\u003c/sub\u003e of atrial myocytes and Nav1.5 protein expression in atrial tissue and the effect of NRG-1\u003c/h2\u003e\n\u003cp\u003eTo further investigate the effect of NRG-1 on atrial electrophysiology, we performed whole-cell patch-clamp experiments using acutely isolated atrial myocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). We first investigated the effect of NRG-1 on I\u003csub\u003eNa\u003c/sub\u003e in atrial myocytes. In atrial myocytes isolated from CLP rats, sepsis decreased the peak current density of I\u003csub\u003eNa\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026amp;B). Interestingly, the peak current density of I\u003csub\u003eNa\u003c/sub\u003e was also reduced in the Sham\u0026thinsp;+\u0026thinsp;NRG-1 and CLP\u0026thinsp;+\u0026thinsp;NRG-1 groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026amp;B). In addition, we observed that the protein expression level of Nav1.5 showed a downregulated trend in the CLP and Sham\u0026thinsp;+\u0026thinsp;NRG-1 groups when compared with that in the Sham group, however, there was no difference in the protein expression level of Nav1.5 between the CLP and CLP\u0026thinsp;+\u0026thinsp;NRG-1 groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eTo evaluate the detailed electrophysiological properties of I\u003csub\u003eNa\u003c/sub\u003e, we measured activation curves, inactivation curves, and recovery time from inactivation. Considering the four groups, we detected no statistically significant differences in the activation kinetic parameters of I\u003csub\u003eNa\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), however, both septic, and NRG-1 treated rats, showed a rightward shift and delayed the deactivation rate of the I\u003csub\u003eNa\u003c/sub\u003e inactivation curve when compared with those of Sham rats. In the CLP\u0026thinsp;+\u0026thinsp;NRG-1 group, NRG-1 administration caused a rightward shift and delayed the deactivation rate of the I\u003csub\u003eNa\u003c/sub\u003e inactivation curve (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared with Sham rats, septic and NRG-1 treated rats showed a prolonged recovery time following I\u003csub\u003eNa\u003c/sub\u003e inactivation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eChanges in the kinetics of I\u003csub\u003eNa\u003c/sub\u003e after sepsis and the effect of NRG-1\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKinetic parameters\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eV\u003csub\u003e1/2act\u003c/sub\u003e (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-75.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-75.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-75.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-75.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ek\u003csub\u003eact\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eV\u003csub\u003e1/2inact\u003c/sub\u003e (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-79.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-72.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-75.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-72.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ek\u003csub\u003einact\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e7.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e8.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e7.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026tau; (ms)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e5.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e17.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e8.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e9.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eV\u003csub\u003e1/2act\u003c/sub\u003e: half maximum activation potential; V\u003csub\u003e1/2inact\u003c/sub\u003e: half maximum inactivation potential; k\u003csub\u003eact\u003c/sub\u003e: slope factor of activation curve; k\u003csub\u003einact\u003c/sub\u003e: slope factor of inactivation curve; \u0026tau;: recovery time constant. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs Sham; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs CLP\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e3.6 Sepsis-induced changes in I\u003csub\u003eCa, L\u003c/sub\u003e of atrial myocytes, and atrial Cav1.2 protein expression and the effect of NRG-1\u003c/h2\u003e\n\u003cp\u003eCompared with the Sham group, the peak current density of I\u003csub\u003eCa, L\u003c/sub\u003e was significantly decreased in atrial myocytes of the CLP and Sham\u0026thinsp;+\u0026thinsp;NRG-1 groups, and NRG-1 reduced the peak current density of I\u003csub\u003eCa, L\u003c/sub\u003e in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 rat atrial myocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026amp;B). Furthermore, the protein expression level of Cav1.2 was decreased in CLP rats when compared with that in the Sham rats, treatment with NRG-1 decreased the expression level of Cav1.2 in the atrial muscle of Sham and CLP rats (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the activation curves shifted significantly rightward in Sham\u0026thinsp;+\u0026thinsp;NRG-1 and CLP groups when compared with those of the Sham group. Moreover, treatment with NRG-1 shifted the activation curve significantly to the right in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 group. Compared with the Sham group, the activation rate was decelerated in the CLP group while accelerated in the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group, and similar activation rates were noted in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 and CLP groups.\u003c/p\u003e\n\u003cp\u003eConsidering I\u003csub\u003eCa, L\u003c/sub\u003e inactivation curves, compared with the Sham group, the CLP group showed modest rightward shifts in I\u003csub\u003eCa, L\u003c/sub\u003e inactivation, and recovery curves (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026amp;F). Administration of NRG-1 caused a rightward shift in I\u003csub\u003eCa, L\u003c/sub\u003e inactivation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e, and recovery curves of Sham\u0026thinsp;+\u0026thinsp;NRG-1 and CLP\u0026thinsp;+\u0026thinsp;NRG-1 groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF). The Sham\u0026thinsp;+\u0026thinsp;NRG-1 group, CLP group, and CLP\u0026thinsp;+\u0026thinsp;NRG-1 group displayed rapid inactivation rates (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eChanges in the kinetics of I\u003csub\u003eCa, L\u003c/sub\u003e after sepsis and the effect of NRG-1\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKinetic parameters\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eV\u003csub\u003e1/2act\u003c/sub\u003e (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-22.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-8.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-8.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-5.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ek\u003csub\u003eact\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e16.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e9.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e19.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e9.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eV\u003csub\u003e1/2inact\u003c/sub\u003e (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-37.54\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-30.02\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-28.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e-32.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ek\u003csub\u003einact\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e18.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e13.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e12.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e11.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026tau; (ms)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e140.43\u0026thinsp;\u0026plusmn;\u0026thinsp;14.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e281.73\u0026thinsp;\u0026plusmn;\u0026thinsp;12.13\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e188.32\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e287.60\u0026thinsp;\u0026plusmn;\u0026thinsp;10.64\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eV\u003csub\u003e1/2act\u003c/sub\u003e: half maximum activation potential; V\u003csub\u003e1/2inact\u003c/sub\u003e: half maximum inactivation potential; k\u003csub\u003eact\u003c/sub\u003e: slope factor of activation curve; k\u003csub\u003einact\u003c/sub\u003e: slope factor of inactivation curve; \u0026tau;: recovery time constant. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs Sham; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs CLP\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e3.7 Sepsis-induced changes in I\u003csub\u003ekur\u003c/sub\u003e of atrial myocytes and atrial Kv1.5 protein expression and the effect of NRG-1\u003c/h2\u003e\n\u003cp\u003eI\u003csub\u003ekur\u003c/sub\u003e, which predominantly exists in atrial myocytes and is absent in ventricular myocytes, the current is generally inactivated. Compared with the Sham group, the peak current density of I\u003csub\u003ekur\u003c/sub\u003e was significantly decreased in atrial myocytes of the CLP group and increased in those of the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026amp;B). Furthermore, the protein expression levels of Kv1.5 showed a down-regulated trend in the CLP group and up-regulated trend in the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD). Moreover, the activation curve of I\u003csub\u003ekur\u003c/sub\u003e shifted left in atrial myocytes of the CLP and Sham\u0026thinsp;+\u0026thinsp;NRG-1 groups, the activation rate was decelerated in Sham\u0026thinsp;+\u0026thinsp;NRG-1 group but accelerated in the CLP group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared with the CLP group, the activation curve of the CLP\u0026thinsp;+\u0026thinsp;NRG-1 group shifted right, and no statistically significant differences were observed in peak current density, the Kv1.5 expression level, and activation rate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA-D and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eChanges in the kinetics of I\u003csub\u003ekur\u003c/sub\u003e after sepsis and the effect of NRG-1\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eKinetic parameters\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSham\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCLP\u0026thinsp;+\u0026thinsp;NRG-1\u003c/p\u003e\n\u003cp\u003e(\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eV\u003csub\u003e1/2act\u003c/sub\u003e (mV)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e39.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e21.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e25.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e36.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ek\u003csub\u003eact\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e21.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e18.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.74\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e25.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e25.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eV\u003csub\u003e1/2act\u003c/sub\u003e: half maximum activation potential; k\u003csub\u003eact\u003c/sub\u003e: slope factor of activation curve; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs Sham; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs CLP\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Major findings\u003c/h2\u003e \u003cp\u003eIn the present study, we explored mechanisms that contribute to the development of AF after sepsis. To the best of our knowledge, we first report the effect of NRG-1 on AF in sepsis. In the rat model of sepsis, the incidence of AF was increased, accompanied by severe myocardial fibrosis, and significant changes were documented in cardiac electrophysiology and ionic currents when compared with those in the Sham group. NRG-1 could attenuate the degree of atrial myocardial fibrosis and organismal inflammation in sepsis. Moreover, NRG-1 could promote the development of AF in Sham rats, affecting atrial electrophysiology and ionic currents.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.2 The possible mechanisms of AF in sepsis\u003c/h2\u003e \u003cp\u003eSepsis is a complex multi-organ dysfunction syndrome that frequently causes severe myocardial injury\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Reportedly, 6\u0026ndash;20% of patients with severe sepsis can develop new-onset AF in the setting of septic heart injury. Furthermore, the development of AF can lead to high mortality and prolonged hospital stays in patients with sepsis, and AF-induced thromboembolic dislodgement events are likely to worsen patient prognosis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs an independent risk factor for death in patients with sepsis and septic shock, AF is typically considered to occur in two steps 1): atrial tissue remodeling and electrical remodeling as the basis for the development of AF and 2): triggering of AF by arrhythmogenic factors\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Atrial tissue remodeling at the onset of AF is often attributed to the development of atrial myocardial fibrosis, although we observed enlarged atrial myocytes and severe myocardial fibrosis in septic rats in the present study. AF is frequently accompanied by changes in atrial electrophysiology, primarily manifested by a decrease in AAPD and a shortening of the effective inactivity period. The results of the present study revealed that the AAPD and effective expiration period were significantly shortened in sepsis.\u003c/p\u003e \u003cp\u003eIn addition, elevated inflammatory markers in patients with sepsis increase the risk of AF, and the inflammatory response may contribute to the development of arrhythmias via the direct infiltration of inflammatory factors or oxidative damage to atrial myocytes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Levels of serum inflammatory mediators tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and macrophage migration inhibitory factor (MIF) are known to be significantly increased in septic rats\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. CRP, as an important inflammatory marker, is often used to predict and assess the condition and prognosis of critically ill patients and guide appropriate treatment protocols. Herein, our results revealed increased serum CRP levels in septic rats.\u003c/p\u003e \u003cp\u003eSepsis, chronic heart failure, hypertension, valvular disease, and myocardial infarction can lead to activation of the renin-angiotensin system, autonomic dysfunction, and production of reactive oxygen species, thereby inducing atrial tissue remodeling and thus promoting the development of AF\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. During autonomic imbalance, sympathetic activation causes the release of NE from sympathetic neuron terminals, which acts on β-adrenergic receptors, causing corresponding changes in ion channels in various atrial myocytes, triggering atrial ectopic electrical activity and promoting the development of AF. Likewise, vagal activation can alter atrial myocyte membrane ionic currents, shortening the effective atrial expiration period and action potential time course and promoting the formation of refractory circuits. In turn, AF can induce autonomic remodeling, leading to overproduction and uneven distribution of neurotransmitters, resulting in ion channel modulation from normal functional feedback regulation in the early stage to causing a shortening of the effective atrial expiration period. This results in the downregulation of ion channel gene transcription and protein expression through a series of signal transduction cascades in the later stage, ultimately leading to reduced channel protein expression and ion current density and maintaining AF. Based on our findings, septic rats exhibited elevated serum NE levels and enhanced atrial sympathetic nerve activity, suggesting that organismal stress is enhanced during sepsis. Accordingly, sepsis could contribute to the development of AF by affecting atrial sympathetic nerve activity.\u003c/p\u003e \u003cp\u003eChanges in transmembrane ion flow are the most important determinants in the development of AF. As the main ion flow in phase 0 of the atrial myocyte action potential, I\u003csub\u003eNa\u003c/sub\u003e plays a crucial role in phase 0 depolarization of atrial myocytes\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and changes in I\u003csub\u003eNa\u003c/sub\u003e density and conduction velocity of atrial myocytes are inextricably linked to the development and maintenance of AF. Moreover, changes in conduction velocity are persistently accompanied by changes in I\u003csub\u003eNa\u003c/sub\u003e. Meanwhile, the decreased cardiac excitability secondary to decreased I\u003csub\u003eNa\u003c/sub\u003e may significantly contribute to reduced contractility in a rat model of sepsis\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the present study, we found that the peak current density of I\u003csub\u003eNa\u003c/sub\u003e in atrial myocytes was reduced, and the expression of Nav1.5 was decreased in CLP rats. Sepsis may influence the development of AF by affecting inactivation and recovery properties after I\u003csub\u003eNa\u003c/sub\u003e inactivation.\u003c/p\u003e \u003cp\u003eL-type calcium channels are pivotal for the myocardial excitation-contraction coupling mechanism, mainly maintaining the plateau phase 2 of the action potential in cardiac myocytes, participating in the opening of the channel and Ca\u003csup\u003e2+\u003c/sup\u003e inward flow, determining the length of the plateau phase and the duration of the action potential. The reduced I\u003csub\u003eCa, L\u003c/sub\u003e density in atrial myocytes can lead to accelerated repolarization and shortened APD, which may be the primary mechanism underlying atrial electrical remodeling\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Atrial myocytes exhibited reduced I\u003csub\u003eCa, L\u003c/sub\u003e peak current density, and Cav1.2 expression during sepsis. Accordingly, sepsis impacts I\u003csub\u003eCa, L\u003c/sub\u003e activation, inactivation, and recovery properties after inactivation, which, in turn, contribute to the development of AF.\u003c/p\u003e \u003cp\u003eI\u003csub\u003ekur\u003c/sub\u003e, predominantly present in atrial myocytes and absent in ventricular myocytes, promotes rapid repolarization of phase 1 action potentials in atrial myocytes, and I\u003csub\u003ekur\u003c/sub\u003e-targeted treatment may prevent the development of AF without increasing the risk of ventricular arrhythmias. I\u003csub\u003ekur\u003c/sub\u003e density was found to be significantly reduced owing to the decreased expression of Kv1.5 protein in patients with AF. Downregulated Kv1.5 expression during AF reportedly attenuates I\u003csub\u003ekur\u003c/sub\u003e prolongation of APD and ERP. However, APD and ERP are known to be reduced during AF, which may be attributed to the continuous activation of the Kv1.5 channel. Cardiomyocytes may eventually prevent electrical remodeling and shortening of APD and ERP by reducing the expression of Kv1.5\u003csup\u003e19\u003c/sup\u003e. In essence, it is an adaptive response of cardiomyocytes to excessive atrial contraction. Thus, both increased and decreased Kv1.5 expression may increase susceptibility to AF. In the present study, the peak I\u003csub\u003ekur\u003c/sub\u003e current density and Kv1.5 expression were decreased in the atrial myocytes of septic rats. Likewise, sepsis can lead to altered I\u003csub\u003ekur\u003c/sub\u003e activation properties, resulting in AF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Effect of NRG-1 on AF and its underlying mechanism\u003c/h2\u003e \u003cp\u003eNRG-1, a peptide produced by endocardial and myocardial microvascular endothelial cells, reportedly exerts a multifaceted cardioprotective by comprehensively activating the NRG-1/ErbBs pathway to regulate the level of fibrosis in response to metabolism, inflammation, and cardiac injury. Importantly, NRG-1 is considered a potential therapeutic agent for cardiovascular disease. The activity of NRG-1 is mainly attributed to ErbB receptor activation and Ach receptor expression\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, current studies on the effects of NRG-1 on cardiac arrhythmias remain controversial. NRG-1 pretreatment can promote myocardial regeneration, effectively alleviate cardiac dysfunction in rats with myocardial infarction, and improve ventricular myocyte electrophysiology, thereby reducing the incidence of ventricular arrhythmias. Moreover, NRG-1 activation can improve atrial electrophysiological stability through ErbB4 receptor-related signaling pathways, thereby inhibiting the development of AF\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In contrast, Ford et al\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. have shown that NRG-1 modulates cardiac parasympathetic tone and may be involved in the pathogenesis of arrhythmias and heart failure. Furthermore, NRG-1 levels were found to be significantly correlated with the presence of paroxysmal AF\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The effects of NRG-1 on septic AF need to be examined in future investigations.\u003c/p\u003e \u003cp\u003eHerein, our findings showed that NRG-1 did not significantly impact the occurrence of AF in septic rats, but could increase the susceptibility of normal rats to AF. In addition, administering NRG-1 aggravated the shortening of the atrial action potential in septic and Sham rats, which reduced the effective expiration period of the atrial muscle in normal rats and promoted the occurrence of AF. Considering the mechanisms associated with the development of AF in sepsis, we speculate that NRG-1 may influence the development of AF by affecting the level of inflammation and circulating hormones in the body, and the autonomic function of the atria and ion channels in atrial myocytes.\u003c/p\u003e \u003cp\u003eThe inflammatory response and autonomic dysfunction can lead to the development of arrhythmias t via diverse pathways\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Administration of NRG-1 can significantly reduce the levels of circulating inflammatory mediators in septic rats and improve myocardial injury in sepsis\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Herein, we found that NRG-1 could reduce atrial myocardial fibrosis and decrease serum CRP levels in rats with sepsis, which was beneficial for inhibiting the inflammatory response to sepsis, reducing atrial tissue remodeling, and decreasing the occurrence of AF. NRG-1 enhanced serum Ach levels and atrial vagal nerve activity in rats with sepsis. Meanwhile, serum Ach levels and atrial vagal nerve activity were enhanced in Sham rats administered NRG-1, suggesting that NRG-1 contributes to atrial autonomic nerve dysfunction by affecting atrial vagal nerve activity.\u003c/p\u003e \u003cp\u003eChanges in transmembrane ion flow are the most critical determinants in the development of AF. In the present study, administering NRG-1 reduced the peak current density of I\u003csub\u003eNa\u003c/sub\u003e in atrial myocytes in the CLP and Sham groups, and there were no differences in channel activation kinetics, altered inactivation kinetics, and prolonged recovery time after inactivation. We observed that Nav1.5 expression was reduced in the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group, with no change detected in the CLP\u0026thinsp;+\u0026thinsp;NRG-1 group. NRG-1 reduced the I\u003csub\u003eCa, L\u003c/sub\u003e peak current density, and Cav1.2 expression in the CLP and Sham groups, with I\u003csub\u003eCa, L\u003c/sub\u003e depolarized in the Sham\u0026thinsp;+\u0026thinsp;NRG-1, and CLP\u0026thinsp;+\u0026thinsp;NRG-1 groups. Moreover, I\u003csub\u003eCa, L\u003c/sub\u003e inactivation mechanics were altered, and the recovery time after inactivation was prolonged in the Sham, CLP, and Sham\u0026thinsp;+\u0026thinsp;NRG-1 groups. Furthermore, NRG-1 increased the peak current density of I\u003csub\u003ekur\u003c/sub\u003e and Kv1.5 expression in atrial myocytes of the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group. NRG-1 administration could alter the activation characteristics of I\u003csub\u003ekur\u003c/sub\u003e in atrial myocytes of normal and septic rats, suggesting that NRG-1 may impact electrophysiological and ion current characteristics of atrial myocytes and promote the occurrence of AF.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eIn summary, the occurrence of AF in septic rats can be associated with elevated levels of inflammation, sympathetic excitation, atrial tissue remodeling induced by left atrial myocardial fibrosis, shortening of the ERP, APD, and altered I\u003csub\u003eNa\u003c/sub\u003e, I\u003csub\u003eCa, L\u003c/sub\u003e, I\u003csub\u003ekur\u003c/sub\u003e properties of atrial myocytes leading to atrial myocyte electrical remodeling. NRG-1 can increase the incidence of AF in normal rats, which was associated with NRG-1 excitation of the vagus nerve, affecting I\u003csub\u003eNa\u003c/sub\u003e, I\u003csub\u003eCa, L\u003c/sub\u003e, and I\u003csub\u003ekur\u003c/sub\u003e properties of atrial myocytes.\u003c/p\u003e"},{"header":"6 Limitations","content":"\u003cp\u003eThis study has several limitations. Herein, we only observed the effects of tail vein NRG-1 injection on myocardial ion channels in septic rats, the direct effect of NRG-1 on sepsis-injured cardiomyocytes needs to be determined. This study only explored the effect of NRG-1 treatment on AF after sepsis, the effects of NRG-1 preconditioning on AF after sepsis, as well as, on other types of arrhythmias, need to be further explored.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Yuting Chen and Teng Wang for excellent technical support of electrophysiology and patch clamp.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe present work was supported by the National Natural Science Foundation of China (Grant No. 81772044), and Hainan Provincial Personnel Support Project (No. 2019RC368), and Hubei Province Key Laboratory Project (No. 2021KFY035).\u003c/p\u003e\n\u003ch2\u003eAvailability of data and material\u003c/h2\u003e\n\u003cp\u003eThe datasets used and analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study\u0026rsquo;s conception and design. Wen Kang and Jingru Deng contributed equally to the study. The study was conceptualized and planned by Wen Kang, Xi Wang, Fang Zhou, Kang Liu, and Long Wang. Wen Kang, Jingru Deng and Zheru Fan participated in the experimental study. Wen Kang and Jingru Deng contributed to the preparation and review of the text as well as the analysis of the experimental data. All authors critically reviewed and approved the final version for submission.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eThe animal management and welfare were reviewed and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (Date:2020-07-24/No.20200705, Wuhan, China). All methods are reported in accordance with ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWalkey, A. J.\u003cem\u003e et al.\u003c/em\u003e Atrial fibrillation among Medicare beneficiaries hospitalized with sepsis: incidence and risk factors. \u003cem\u003eAm Heart J\u003c/em\u003e \u003cstrong\u003e165\u003c/strong\u003e, 949-955 e943 (2013). https://doi.org:10.1016/j.ahj.2013.03.020\u003c/li\u003e\n\u003cli\u003eAibar, J. \u0026amp; Schulman, S. 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D.\u003cem\u003e et al.\u003c/em\u003e Neuregulin-1 suppresses muscarinic receptor expression and acetylcholine-activated muscarinic K+ channels in cardiac myocytes. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e308\u003c/strong\u003e, 23-28 (2003). https://doi.org:10.1016/s0006-291x(03)01319-6\u003c/li\u003e\n\u003cli\u003eZhou, Q.\u003cem\u003e et al.\u003c/em\u003e Effect of neuregulin-1 on heart function and inflammatory mediators in rats with sepsis. \u003cem\u003eZhonghua Wei Zhong Bing Ji Jiu Yi Xue\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 140-144 (2018). https://doi.org:10.3760/cma.j.issn.2095-4352.2018.02.009\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sepsis, Neuregulin-1, Atrial fibrillation, ion currents","lastPublishedDoi":"10.21203/rs.3.rs-3246284/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3246284/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo explore the mechanisms involved in the development of atrial fibrillation (AF) after sepsis and examine the effect of neuregulin-1 (NRG-1) on AF and related mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe used cecal ligation and puncture (CLP) to establish the sepsis model. NRG-1 was administered via the tail vein at a dose of 0.01\u0026micro;g/g 12 and 24 h postoperatively to determine its effect on AF after sepsis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompared with Sham rats, septic rats exhibited enhanced AF inducibility, atrial fibrosis, norepinephrine (NE), and C-reactive protein (CRP) levels, reduced action potential duration (APD), atrial effective refractive period (AERP), acetylcholine (Ach) levels, expression of Nav1.5, Cav1.2, and Kv1.5, and significantly decreased I\u003csub\u003eNa\u003c/sub\u003e, I\u003csub\u003eCa, L\u003c/sub\u003e, and I\u003csub\u003ekur\u003c/sub\u003e current densities. We observed that NRG-1 could reduce APD, atrial fibrosis, levels of CRP and NE, I\u003csub\u003eNa\u003c/sub\u003e and I\u003csub\u003eCa, L\u003c/sub\u003e current densities, and expression levels of Nav1.5 and Cav1.2, however, it failed to prevent the onset of AF. Compared with the Sham group, the Sham\u0026thinsp;+\u0026thinsp;NRG-1 group rats showed a reduction in APD, AERP, I\u003csub\u003eNa\u003c/sub\u003e and I\u003csub\u003eCa, L\u003c/sub\u003e current densities, Nav1.5 and Cav1.2 expression levels, elevated AF inducibility, Ach levels, I\u003csub\u003ekur\u003c/sub\u003e current density, and Kv1.5 expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSepsis can induce tissue and electrical remodeling in the atria and promotes the development of AF. NRG-1 could attenuate the degree of atrial fibrosis and organismal inflammation in sepsis while promoting the development of AF in Sham rats, impacting atrial electrophysiology and ionic currents.\u003c/p\u003e","manuscriptTitle":"Mechanisms associated with the development of atrial fibrillation after sepsis and the role of neuregulin-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-18 15:36:48","doi":"10.21203/rs.3.rs-3246284/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0147dcb-d403-4b8b-810f-e59f8d57780d","owner":[],"postedDate":"August 18th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":24026223,"name":"Biological sciences/Cell biology"},{"id":24026224,"name":"Biological sciences/Drug discovery"},{"id":24026225,"name":"Biological sciences/Immunology"},{"id":24026226,"name":"Biological sciences/Molecular biology"},{"id":24026227,"name":"Health sciences/Cardiology"}],"tags":[],"updatedAt":"2023-10-08T07:14:18+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-18 15:36:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3246284","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3246284","identity":"rs-3246284","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}