2.1. Experimental Animals
Adult male Sprague-Dawley rats (body weight 200–250 g) were purchased from SiPeiFu (Beijing) Biotechnology Co., Ltd. (Beijing, China). All animal studies were conducted in compliance with the Animal Care and Use Committee of Capital Medical University and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (the 8th Edition, NRC 2011).
2.2. Osmotic mini-pump implantation, drug administration, and blood pressure measurement
All SD rats were anesthetized with isoflurane (5% induction; 2–3% maintenance). An osmotic pump (model 2ML1, Alzet 2004, USA) preloaded with AngⅡ was aseptically implanted in rats subcutaneously to induce atrial fibrosis by the continuous release of AngⅡ (750 ng/kg/min) for 28 days. Later, all rats were assigned to three groups to receive corn oil, valsartan (VAL, 48 mg/kg body weight, n = 6), or sacubitril/valsartan (SAC/VAL, 60 mg/kg body weight, n = 6) for 28 days. All drugs were dissolved in corn oil and administrated by gavage. Systolic blood pressure (SBP), diastolic blood pressure (SBP), and mean blood pressure (MBP) were measured at the end of experiment by the tail-cuff method as previously described[23].
2.3. Electrophysiological study
The electrophysiological study (EPS) was performed as previously described before the rats were sacrificed[24]. Briefly, rats were anesthetized with isoflurane (5% induction; 2–3% maintenance) and placed on the heated pad to maintain body temperature at 37 °C. An electrode catheter (1.1 F, Science) was introduced into the right atrium via the right jugular vein then released a train of electrical stimuli at a pacing cycle length of 100 ms to test the susceptibility to atrial arrhythmias. Electrical stimulation was performed ten times in the same manner, and the number of successful AF induction within these ten times was recorded. The duration of AF is determined by the time from the end of the burst pacing to the first sinus P wave after the atrial rhythm. AF in this study was defined as a rapid and irregular atrial rhythm with an irregular R-R interval lasting more than 1000 ms.
2.4. Echocardiography
Before and 28 days after osmotic mini-pump containing AngⅡ implantation, rats were examined with M-mode echocardiography (Vevo 770, Visual Sonics, Inc., Toronto, Ontario, Canada). Inhalational anesthesia with 2% isoflurane and a heated pad were adopted during the process of image acquisition. Left atrial diameter (LAD), LV end-diastolic diameter (LVEDD), and ejection fraction (EF) were measured and averaged over three cardiac cycles. Mitral valve flow was assessed by using pulsed-wave Doppler ultrasonography. The early (E) and atrial (A) peaks are measured and averaged over three cardiac cycles.
2.5. Enzyme-linked immunosorbent assay
The serum concentration of atrial natriuretic peptide (ANP), N-terminal pro-brain natriuretic peptide (NT-proBNP) and AngⅡ were detected by ELISA according to the manufacturer's instructions. Kits with Catalog E02A0204, E02A0493, and E02N0008 were purchased from Shanghai BlueGene Biotech Co., Ltd.. The optical density (OD) at 450 nm was measured by an ELISA reader. The results were expressed as pg/ml or ng/ml.
2.6. Histological, immunohistochemical, and immunofluorescence staining
Left atrial tissue samples from rats were fixed with 4% paraformaldehyde, embedded in paraffin, transversely cut into 5 µm thickness. Masson staining was performed as previously described to highlight the fibers, and the extent of interstitial fibrosis was determined by fibrosis area / total administration area × 100%. Immunohistochemical staining were performed using antibodies against collagen I (Abcam, ab34710) and collagen III (Abcam, ab7778) as previously described. Immunofluorescence staining were performed using the methods described previously with antibodies against α-smooth muscle actin (α-SMA, Abcam, ab5694) and vimentin (Abcam, ab137321). Images acquisition and analysis were used by Image-Pro Plus.
2.7. Cell migration and proliferation evaluation
Rat atrial fibroblasts (AFBs, Cat NO.: CP-R074) were purchased from Procell Life Science&Technology Co.,Ltd (Wuhan, China) for cell proliferation and migration evaluation. The AFBs were divided into three groups and pretreated with dimethyl sulfoxide (MedChem Express, control group), valsartan (10 µmol/L, MedChem Express, VAL group), and LBQ657 + valsartan (10 µmol/L, 1:1 ratio, MedChem Express, SAC/VAL group) for 24 h respectively. AFBs in these three groups were then stimulated with AngⅡ (100 nmol/L) for another 24 h. Transwell assay was performed to evaluate cell migration. The nonmigratory cells remaining in the upper chamber were removed, and the membranes containing AFBs were fixed and then stained with crystal violet. Five fields at × 200 magnification were randomly selected under the light microscope to count the cells migrating to the lower compartment. Cell proliferation was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) assay (Sigma, St. Louis, MO, USA). The OD value at a wavelength of 490 nm of each well was detected using a spectrophotometer (BioTek, Elx800, Winooski, VT, USA).
2.8. Western Blotting Analysis
RIPA lysis buffer containing protease and phosphatase inhibitors were added to the proteins extracted from the left atrium of rats and quantified using the BCA protein analysis kit, as described previously. The following primary antibodies against phospho-Smad1/5/9 (CST, 13820), phospho-Smad2/3 (CST, 8828), p38 MAPK (CST, 8690S), phospho-p38 MAPK (CST, 9216S), ERK1/2 (CST, 4695S), phospho-ERK1/2 (CST, 9101S), JNK (CST, 9252S), phospho-JNK (CST, 4668S), and GAPDH (CST, 5174) were used for western blotting. Gel Imaging System (Tanon 5200) was used to detect the bands and the results were quantified by densitometry software (Image-Pro Plus).
2.9. Statistical Analysis
All data were represented as \(\stackrel{-}{x}\pm SD\) or percentage and analyzed using GraphPad Prism (version 6.0). One-way ANOVA followed by Tukey's post-hoc was used to determine statistical significance for multiple groups. A P value < 0.05 was considered statistically significant.