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

doi:10.21203/rs.3.rs-3246284/v1

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

https://doi.org/10.21203/rs.3.rs-3246284/v1

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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

In organisms, an infection can induce a dysregulated systemic inflammatory response, resulting in the development of sepsis¹. 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 complications⁴. 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 sepsis⁵.

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 AF⁷. 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' lives². 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 myogenesis⁸. 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 injury⁹. 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 AF¹². 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.

2.1 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 programs¹⁰, 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.

3.1 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. 1and 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, APD₅₀, APD₉₀, and AERP of CLP rats, and APD₁₀, APD₅₀, APD₉₀, and AERP of the Sham + NRG-1 rats were reduced when compared with those of Sham rats (Table 1). Meanwhile, NRG-1 administration reduced APD₁₀ 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. 2A&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. 3A-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. 3A-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. 3A-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. 4A). 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. 4B&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. 6A&B). Interestingly, the peak current density of I_Na was also reduced in the Sham + NRG-1 and CLP + NRG-1 groups (Fig. 6A&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. 6C).

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. 6D 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. 6E and Table 2). Compared with Sham rats, septic and NRG-1 treated rats showed a prolonged recovery time following I_Na inactivation (Fig. 6F 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. 7A&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. 7C).

As shown in Fig. 7D 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. 7E&F). Administration of NRG-1 caused a rightward shift in I_{Ca, L} inactivation (Fig. 7E), and recovery curves of Sham + NRG-1 and CLP + NRG-1 groups (Fig. 7F). 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. 8A&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. 8D). 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. 8C 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. 8A-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

4.1 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 prognosis¹³.

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 factors¹⁴. 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 myocytes¹⁴. 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 rats¹⁰. 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 AF¹⁴. 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 sepsis¹⁷.

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 Ca²⁺ 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 Kv1.5¹⁹. 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 al²³. 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 AF¹². 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 pathways¹⁴. Administration of NRG-1 can significantly reduce the levels of circulating inflammatory mediators in septic rats and improve myocardial injury in sepsis²⁴. 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.

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.

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.

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

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.

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

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Abstract

Purpose

Methods

Results

Conclusion

Figures

1 Introduction

2 Materials and methods

2.1 Ethics approval

2.2 Animals and specimen collection

2.3 In vivo electrophysiology

2.4 Histology and immunohistochemistry

2.5 Enzyme-linked immunosorbent assays (ELISA)

2.6 Whole-cell patch-clamp experiments

2.7 Western blot analysis

2.8 Statistical analysis

3 Results

3.1 Sepsis-induced changes in atrial electrophysiology and the effect of NRG-1

3.2 Changes in the atrial organization after sepsis and the effect of NRG-1

3.3 Sepsis-induced changes in atrial autonomic nerves and the effect of NRG-1

3.4 Sepsis-induced changes in serum CRP, NE, and Ach and the effect of NRG-1

3.5 Sepsis-induced changes in INa of atrial myocytes and Nav1.5 protein expression in atrial tissue and the effect of NRG-1

3.6 Sepsis-induced changes in ICa, L of atrial myocytes, and atrial Cav1.2 protein expression and the effect of NRG-1

3.7 Sepsis-induced changes in Ikur of atrial myocytes and atrial Kv1.5 protein expression and the effect of NRG-1

4 Discussion

4.1 Major findings

4.2 The possible mechanisms of AF in sepsis

4.3 Effect of NRG-1 on AF and its underlying mechanism

5 Conclusions

6 Limitations

Declarations

Acknowledgements

Funding

Availability of data and material

Author Contributions

Ethics approval

Consent for publication

Conflict of Interest

References

Additional Declarations

Supplementary Files

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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

3.6 Sepsis-induced changes in I_{Ca, L} of atrial myocytes, and atrial Cav1.2 protein expression and the effect of NRG-1

3.7 Sepsis-induced changes in I_kur of atrial myocytes and atrial Kv1.5 protein expression and the effect of NRG-1