Ethical approval
This study was approved by the Animal Use and Management Ethics Committee of the First Affiliated Hospital of Harbin Medical University. The use of animals and all procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication 2011; eighth edition). Twenty-one New Zealand white rabbits weighing 2.5-3.0kg were raised in individual cages in the animal center of the First Affiliated Hospital of Harbin Medical University. The temperature in the room was maintained at 23°C under a 12-h light-dark cycle. To determine the effect of eplerenone on a model of atrial fibrillation established by rapid right atrial pacing, the rabbits were randomly categorized into three groups: (1) sham-operated group (sham, n=7) with sutured electrodes and no pacing; (2) rapid atrial pacing group (P , n=7), with sutured electrodes and atrial rapid pacing at 600 beats/min for three weeks; (3) eplerenone treatment group (P+E, n=7) with eplerenone-administered p.o. (Pfizer, Inc., New York, NY, USA) at a dose of 50 mg /day for 28 days and pacing the right atria for three weeks, beginning on the eighth day.
Rabbit AF model
The AF model was established based on our previous studies [17]. The rabbits were anesthetized by the intravenous injection of ketamine (30-35mg/kg; Sigma Aldrich, St Louis, MO, USA) and xylazine (5mg/kg, Sigma Aldrich). After anesthesia, the rabbits were intubated through the nasal cavity and connected to the ventilator. After the pericardium was opened, one end of the unipolar epicardial electrode (Medtronic, US) was sutured to the right atrial appendage and the other side was attached to the pacemaker (Fudan University, Shanghai, China) that was placed in a subcutaneous pocket in the backs of rabbits. After one week of recovery, the pacemaker began to work to make the right atrial rapid pacing (RAP). Electrocardiogram was randomly checked during the pacemaker operation to ensure the working of a pacemaker. After three weeks of pacemaker operation, atrial electrophysiological tests and sampling were performed. All atrial tissues and serum were stored at -80°C.
Cell culture
To determine whether ERK1/2 MAPK plays a critical role in the autonomic remodeling associated with AF, we used an HL-1 cell model of AF, which was established by using YC-2 stimulator (Chengdu, China). The experimental procedure is based on our previous studies[17].
To investigate the role of eplerenone in the regulation of GAP-43, ChAt and ERK1/2 expression induced by tachypacing in vitro, HL-1 cells were categorized into three groups: (1) control group: cultured for 24 h, no rapid field stimulation or other drug interventions; (2) pacing group (P): rapid field stimulation for 24 h as aforementioned; (3) eplerenone and pacing group (P+E): rapid field stimulation for 24 h with eplerenone (4μm/L, CAS No; 107724-20-9, MedChemExpress, eplerenone concentration screening in Supplementary Materials).
To evaluate the effects of MAPK inhibitors on the expression of GAP-43 and ChAt were also evaluated in cells. The HL-1 cells were divided into four groups: (1) control group: cultured for 24 hours, no rapid field stimulation or other drug interventions; (2) pacing group: rapid field stimulation for 24 hours as aforementioned; (3) pacing and U0126 group: rapid field stimulation for 24 hours with U0126 (10μm/L, an ERK1/2 inhibitor; CAS No;109511-58-2, MedChemExpress, U0126 concentration screening in Supplementary Materials) pretreatment for 1 h; and (4) U0126 group:cultured for 24 h with U0126.
Biochemical measurements
Laboratory measurements were performed under blinded conditions. Three sets of blood samples were collected from the hearts of rabbits after three weeks of rapid atrial pacing. The samples were centrifuged at 3500×g for 15 min. Norepinephrine (NE), acetyl choline (ACH) and aldosterone (ALD) level measurement kits were purchased from Jiancheng Biological Technical Institute (China) and measurements were performed according to the manufacturer’s instructions.
Atrial electrophysiological measurements
The electrophysiological measurements were performed before the collection of blood samples according to the method described in our previous study[17]. After anesthetization, intubation and mechanical ventilation, left side thoracotomy was performed. A four-electrode was sutured to the free wall of the right atrial appendage. The tail end of the electrode was connected to the Prucka32 lead electrophysiological recorder to record the body surface and intracardiac electrocardiogram synchronously. AF was induced by burst stimulation applied to the right atrium with 10 Hz, 2-ms stimuli for 1–10 s. If atrial fibrillation lasted for 30 s, synchronous direct current cardioversion was performed. The duration of atrial fibrillation was recorded for 30 s, and the electrophysiological test was continued after 1 minutes.
Immunohistochemical analysis
Rabbit atrial tissue specimens were fixed using 10% formalin and sliced into sections of 3-5 μm thick and then embedded in paraffin. The specimens were stained with hematoxylin and eosin (HE) and Masson's stains and examined under a light microscope. Cardiomyocytes appear red and collagen appears blue in the Massen's staining. Myocardial collagen volume fraction (CVF) was analyzed and measured using a digital medical image analysis system (HPISA-1000, Olympus, Shinjuku, Japan). CVF was calculated as myocardial collagen area/total area of the field, and values of this parameter were randomly selected from each of five tissue sections. HE and Masson's staining was performed to evaluate fibrotic deposition in rabbit atria.
To observe the remodeling of the atrial autonomic nerve, atria tissue wax block was incubated with anti-growth-associated protein 43 (GAP-43, Abcam), anti-tyrosine hydroxylase (TH, Abcam), anti-nerve growth factor (NGF, Abcam) and anti-choline acetyltransferase (ChAT, Abcam) overnight at 4℃. The atrial tissue was rinsed three times, and then peroxidase-conjugated goat anti-rabbit IgG (1:1000, Abcam) was added and incubated for 10 minutes. Finally, a diaminobenzidine solution was used to visualize the specimen. The immunohistochemical analysis of the atrial autonomic nerve was performed using a digital medical image analysis system (HPISA-1000, Olympus, Shinjuku, Japan).
Real-time quantitative polymerase chain reaction
After atrial tissue was homogenized, total RNA was extracted using TRIzol (CWbio. Co. Ltd, Beijing, China) and a real-time quantitative polymerase chain reaction was performed according to a previous study[18].
Western blotting
Proteins were extracted from rabbit atria tissue specimens or HL-1 cells. The protein content was determined by using the Bradford method according to our previous study[19]. The same amount of protein (40μg) was loaded per lane and separated by 8–12% sodium dodecy1 sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene difluoride membranes. The membranes were incubated overnight at 4°C with primary antibodies against ChAT (1:500, Abcam), GAP-43 (1:500, Abcam), Pan-actin (1:1000, sigma) and ERK1/2(1:500, CST). The bands were visualized using Super-Signal West Femto Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) and quantified by scanning densitometry(Chemi-DOC, Bio-Rad, Laboratories, Hercules, CA, USA).
Data and statistical analyses
Quantitative data are presented as mean ± standard error of the mean. One-way analysis of variance was performed for comparing continuous parameters among three or more groups and Tukey’s post-hoc test was performed to test significant differences. A P value of <0.05 was considered statistically significant. Figures were constructed using the GraphPad Prism 8.0 software. The data and statistical analyses comply with the recommendations on experimental design and analysis in pharmacology.