Myocardial infarction animal model
The study was approved by the protocols and guidelines of the Laboratory Animal Ethics Committee of Shandong First Medical University Affiliated Qianfoshan Hospital.
Protocol 1
Adult male Sprague Dawley rats (250–300 g) were used as research subjects. Myocardial infarction (MI) surgery was performed following a seven-day acclimatization period. Briefly, rats were anesthetized with 1% pentobarbital sodium (45 mg/kg body weight). Endotracheal intubation was performed carefully. Mechanical ventilation was achieved by connecting the endotracheal tube to a ventilator (HX-300S, TME, Chengdu, China), cycling at 80 breaths/min with a tidal volume of 1.5 ml per 100 g body weight. The left coronary artery was routinely ligated approximately 2–3 mm from its origin, between the pulmonary artery conus and the left atrium. Rats were randomly assigned to ensure an approximately equal number of survivors in each group. The groups were designated as follows: Control, MI, and MI + OT groups (MI rats received OT therapy). OT was delivered subcutaneously using the Alzet mini-osmotic pump (Model 2001) at [125 ng/(kg.h)] for 7 days from chest opening procedure until approximately 24 h before the end of the experiments to eliminate pharmacological actions. Seven days post-MI, the animals were sacrificed by cervical dislocation after anesthesia, and their hearts were collected for further study.
Protocol 2
OT-knockout mice were created by a professor from Shandong University and were kindly gifted by the National Institutes of Biomedical Innovation. This strain was backcrossed with C57BL/6J mice in our laboratory. Homozygous KO and wild-type (WT) mice were used in this study. We created an MI model in 12–13-week-old male KO mice (KO + MI) and WT littermates (WT + MI) by ligating the left coronary artery, as described in protocol 1. All animals were randomly assigned to the following groups: Control, OTR+/- MI, OTR-/- MI groups.
In vivo ECG recording and in vivo electrophysiological experiments
Before the cardiac LAD artery was ligated, an implantable transmitter (Telemetry Research) was embedded in the rat abdomen to record ECG signals. ECG data were collected using PowerLab software. All animals were monitored for seven days with the transmitter, and the data were stored for further analysis. Before sacrifice, a programmed stimulation protocol was employed using electrodes implanted on the epicardial surface of the left ventricle (LV). Arrhythmias were triggered using an electric Bloom stimulator (Chengdu Electronic Machine Company) and then induced through ventricular stimulation at a basic cycle length of 150 ms (S0), with single (S1), double (S2), and triple (S3) extra stimuli delivered after eight paced beats. The pacing protocols were halted if they induced sustained ventricular tachycardia (VT). Ventricular tachyarrhythmias, including VT and fibrillation, were considered non-sustained if their duration lasted < 15 beats and sustained if the duration lasted > 15 beats. The experimental protocols were completed within 10 min. All procedures were performed and recorded using an animal biological function experimental system (LEAD-7000; JJET, China). The VA scores were calculated as described in our previous study (12).
Historical preparation
The rat hearts were frozen in liquid nitrogen immediately after euthanasia. The tissues were then embedded in OCT for sectioning. All slices were 7-µm thick. Sections were fixed in acetone for 10 min at room temperature. Slices were washed with tap water and tissues were stained with 0.5% toluidine blue for 10 min. Ammonia (1%) was used to diminish background staining. The slices were then dehydrated using an alcohol gradient. Neutral balsam was used to cover the tissues for permanent storage. Mast cells were visualized using an optical microscope.
Cell culture
The murine mast cell line (p815) was a gift from Professor Chuanyong Liu of Shandong University. P815 was used to characterize the degranulation of mast cells, as other mast cell lines, such as HMC-1, do not degranulate (13). P815 was planted on PDL (poly-D-lysine)-coated coverslips separately and cultured for 48 h in DMEM with 10% FBS in 5% CO2 and 95% air. Stimulation reagents, including mast cell degranulation trigger C48/80 (6 mg/ml) (14), OT (0.01 ng/mL); PI3K inhibitor, LY294002 (6.4 µM), and a selective OTR antagonist, Atosiban (1 µM) (15).
Immunohistochemistry
The infarct size was evaluated using heart tissue sections stained with Masson’s trichrome (Jiancheng, China) according to standard protocols. The digitized images were analyzed using planimetry, and the infarcted area was expressed as the percentage of stained fibrotic area over the total LV. For cardiac mast cell staining, cardiac tissues were prepared using a cryostat, then stained with 0.05% toluidine blue. Briefly, a 1-% stock solution of toluidine blue in 70% ethanol was dissolved in 0.5% NaCl (pH 2.2–2.3). The slides were immersed in the staining solution for 30 min, washed twice with distilled water, dehydrated using a series of increasing concentrations of ethanol, and finally immersed in butyl acetate ester. Cover slips were applied using Eukitt® mounting medium and the slides were allowed to dry overnight. The number of intact mast cells (IMCs) and degranulated mast cells (DMCs) in the sections were observed and counted, and the degranulation rate of mast cells (MCD = DMCs/(IMCs + DMCs) ×100%) was calculated.
For tissue immunofluorescence, hearts were harvested after the hemodynamics study, immersed in 30% sucrose in phosphate-buffered saline (PBS) overnight, embedded in Tissue-Tek® OCT compound (Sakura Finetek), and frozen in an isopentane bath on dry ice. For cell immunofluorescence, cells were washed thrice using PBS for 5 min each, followed by fixation with 4% paraformaldehyde (PFA) for 20 min at room temperature. The coverslips were washed three times with PBS. Triton X-100 (0.5%) was used to penetrate cell membranes and facilitate antibody entry into cells. Coverslips were blocked with 10% BSA for 30 min. Samples were incubated with anti-TH Ab (Abcam, ab112, 1:500), anti-OTR Ab (Abcam, ab87312, 1:500), and anti-Tryptase Ab (Abcam, ab2378, 1:200) overnight at 4°C, followed by a 2-h incubation with FITC-conjugated rabbit anti-sheep (1:200; Bethyl), Alexa 545-conjugated goat anti-rabbit (1:100; Peprotech) or FITC-conjugated rabbit anti-mouse (1:200; BioLegend) secondary antibodies. The sections were counterstained with DAPI (Life Technologies) to identify nuclei. Quantification of the fraction of sympathetic nerve fibers was expressed as the ratio of the labeled nerve fiber area to the total area, while papillary muscles were excluded from the study because variable sympathetic innervation has been reported.
Ten microscopic fields in a 20× field (0.13 mm2) were randomly selected by a blinded investigator. All images were obtained using an Olympus LCX100 Imaging System. All images were analyzed using ImageJ software (version 1.38 ×; National Institutes of Health).
Free calcium (Ca2+) measurement
P815 cells were activated with or without C48/80, and then with or without OT and LY294002 for 10 min. The medium was discarded and the suspension was incubated in Tyrode’s buffer supplemented with Fluo-3 acetoxymethyl ester (Fluo-3-AM) (Invitrogen) at 37°C for 20 min. After discarding the Fluo-3-AM, the cells were resuspended in Tyrode’s buffer and stimulated with 100 ng/mL DNP-HSA. Baseline calcium levels were recorded at 488 nm excitation/520 nm emission using the Accuri™ C6 flow cytometer (BD Biosciences, USA) (10,000 events), after which the cells were exposed to AgNPs for 2 min and the mean fluorescence intensity was recorded. The FCS Express 4 software was used for data presentation (De Novo Software, Glendale, CA).
Flow cytometry
A FACSTAR equipped with a Consort 30 Data system (Becton Dickinson, Mountain View, CA, USA) was used. The excitation of the argon-ion laser was 488 nm at a power of 150 mW. A nozzle with a diameter of 70 PM was used. Mast cells were identified by size and shape using FSC, and by granularity using SSC. A minimum of 1 × 104 cells from each group (n = 6 per group) were used for each analysis. After identification by FACS, mast cells (1 × 105) were incubated with various treatment concentrations for 3 min at room temperature. The percentage of mast cell degranulation was calculated as previously described(16).
Western blot
P815 cells were treated with C48/80, C48/80 + OT, and C48/80 + OT + Atosiban for 15 min and harvested for western blot analysis. The normal medium Protein Extraction Kit (Beyotime Institute of Biotechnology, Jiangsu, China) was used, and protein concentration was measured using the BCA protein assay reagent kit (Pierce). Equal amounts of total protein (80 µg of protein/lane) were resolved on 5 − 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes using the semi-dry transfer method. The membranes were blocked with 5% non-fat dry milk in PBST (PBS containing 0.05% Tween 20) and incubated overnight at 4 ºC with an anti-pAKT primary antibody (CST, 1:2000). The blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore) and visualized using a FluorChem E Imager (Protein-Simple, Santa Clara, CA, USA). The densities relative to GAPDH were analyzed using ImageJ software (NIH).
Statistical Analysis
All data are presented as mean ± SE, t-tests were used to test for differences between groups. When a statistically significant (P ≤ 0.05) treatment was identified, for all analyses, values of P < 0.05 was accepted as evidence of significance.