This study was conducted in strict accordance with the recommendations in Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Committee governing the Ethics of Animal Experiments of the Central South University approved the protocol. Dogs were anesthetized with 3% sodium pentobarbital and ventilated with a positive-pressure respirator (MAO01746; Harvard Apparatus, Holliston, MA); initial dose was 1 ml/kg and an additional 2 ml/h was administered. All efforts were made to minimize suffering.
24 male beagle dogs (weight, 8-10 kg) were randomly divided into four groups (n=6 each): (a) sham-operated group (normal chow), (b) AF group (AF with normal chow), (c) AF+FO (AF and chow supplemented with fish oil, (d) FO (normal chow supplemented with FO). Alignment with previous reports , daily oral administration of FO (FO, containing 0.6 g/kg/day EPA/DHA) was initiated 1 week before surgery and continued throughout the study period.
Canine Model of atrial fibrillation
AF was induced in canine using long-term RAP . In brief, a programmable pacemaker (AOO, Harbin University of Science and Technology, China) was implanted and used for continuous atria pacing at 400 bpm for 4 weeks to induce AF. The success of this procedure was confirmed by electrocardiography. Sham-operated dogs were implanted with a same instrument but were maintained without pacemaker activation.
Atrial electrophysiological study
Standard ECG limb leads were recorded at baseline and after 4-week RAP. We dissected the right femoral vein and inserted a 4 Fr multielectrode catheter into the high right atrium (HRA). The electrophysiological parameters, such as effective refractory period (ERP), window of vulnerability (WOV) and AF duration were detected at the HRA site. Programmed stimulation of atrial was performed by the computer-based Lab System (Lead 7000; Jingjiang, Chengdu City, China). ERP was determined by programmed pacing with 8 consecutive stimuli (S1-S1=300 ms) followed by a premature stimulus (S1-S2) at 2×threshold, the S1-S2 interval was decreased from 160 ms initially in decrements of 10 to 2 ms when approaching ERP. The difference between the longest and shortest S1-S2 interval, where AF was induced at each bipolar pair, was defined as WOV, which serves as a quantitative measure of AF inducibility . AF was defined as an irregular atrial rate faster than 500 beats/min correlated with irregular atrioventricular conduction lasting 5s . To determine AF vulnerability, 10 consecutive bursts of rapid atrial pacing (cycle length 60 ms) at HRA for 2 s were implemented with 30 s intervals. AF duration induced by burst pacing from all episodes in each dog was analyzed. Operators were performing the electrophysiological study blinded to the treatment group for the dogs.
After 4 weeks RAP, anaesthetized animals were euthanized and hearts were removed. Left atrial posterior wall tissue samples were collected and immersed in 4% paraformaldehyde or snap frozen at −80˚C for further analysis. Fasting blood samples were collected at baseline before pacemaker implantation and before measuring electrophysiological parameters and stored at −80°C for further analysis.
LA samples were fixed in 4% paraformaldehyde, embedded in paraffin and sliced into 5‑μm-thick sections. Masson-stained sections was used to estimate the interstitial fibrosis of LA which was showed as percentage of total area occupied by blue-stained interstitial tissue. For each section, 5 optical fields (400×magnification) were examined using Image-Pro 6.2 software, and the data averaged.
TUNEL (TdT-mediated dUTP Nick-End Labeling) assay
To detect apoptosis, paraffin-embedded LA was cut transversely into 5-µm-thick sections. TUNEL assay was performed with DeadEnd Fluorimetric TUNEL System (Promega), according to the manufacturer’s protocol. Cell nuclei were counterstained with DAPI (4′,6‐diamidino‐2‐phenylindole). The number of TUNEL-positive nuclei was manually counted. Image-Pro 6.2 software was used to determine the total number of nuclei by automatically counting the DAPI.
Western blot analyses
Protein-extracts of snap-frozen LA were prepared using standard procedures. Bicinchoninic acid (BCA) assay (ASPEN, USA) was used to measure the protein concentrations of supernatants. After separating on SDS-polyacrylamide gels, proteins were transferred to PVDF membranes. The primary antibodies, rabbit monoclonal anti-GAPDH antibody (diluted 1:10000), rabbit polyclonal anti-GRP78 antibody (diluted 1:500; Abcam), rabbit monoclonal anti-PERK antibody (diluted 1:1000; Abcam), rabbit monoclonal anti-p-PERK antibody (diluted 1:1000; Bioss), rabbit polyclonal anti-CHOP antibody (diluted 1:1000;Bioss), rabbit polyclonal anti-Caspase12 antibody (diluted 1:500; Abcam), followed by secondary goat anti-rabbit antibody (diluted 1:10000, Abcam) were used. The ECL detection reagent (ASPEN, USA) was used to detect the antibody binding. Bands were quantified with ImageJ software.
Real-time quantitative PCR
According to the manufacturer’s protocol, the RNA extraction of LA tissues proceeded after homogenization in TRIZOL (Invitrogen, Carlsbad, CA). Reverse transcription was performed with RevertAid First strand cDNA Synthesis Kit (Thermo Scientific), and gene-specific primers and SYBR Green PCR Master Mix (Applied Biosystems) were used for quantitative PCR. Real-time qPCR analysis was carried out with the StepOne Real-Time PCR (Life technologies, Alameda, CA). The primer of gene β-actin, IL-1β, IL-6, TNF-α, RyR2, SERCA2a, NCX, CACNA1C was synthesized from GeneCreate (Wuhan, China). We listed the primer sequences used in Table 1 and used the amount of β-actin to determine and normailize the amount of each gene. The differences among groups were evaluated through the relative expression quantity 2-ΔΔCt value.
We used R-3.4.3 (https://www.r-project.org/) for statistical analysis. All values were expressed as mean±SD. Statistically significant differences between means were assessed by ANOVA and Tukey HSD for comparisons between four groups. P<0.05 was considered statistically significant.