Human Studies.
The study protocol was approved by the Research Ethics Committees of The First Affiliated Hospital of Harbin Medical University (Harbin, Heilongjiang, China) and The First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, Shaanxi, China) and performed in accordance with the Declaration of Helsinki. The participants all provided written informed consent. We retrospectively reviewed the oral glucose tolerance test (OGTT) records of the patients (n=12,012) of Biobank at The First Affiliated Hospital of Xi'an Jiaotong University between January 2018 and September 2019. The clinical information was shown in Supplemental Table-1(Additional File 1). In addition, the following samples were collected from the qualified participants. First, the plasma samples of patients (18-75 years; n=1,152) who underwent physical examination in The First Affiliated Hospital of Harbin Medical University between September 2019 and November 2019 were collected. The clinical information was shown in Supplemental Table-2 (Additional File 1). Second, the normal colon tissue samples from young (18-40 years; n=10) and old patients (over 65 years; n=10) who underwent the radical operation for left hemicolon cancer in The First Affiliated Hospital of Harbin Medical University were collected. The clinical information was shown in Supplemental Table-3 (Additional File 1). Finally, the right atrial appendages of young (18~40 years; n=6) and elderly (over 65 years; n=6) patients who underwent open-heart surgery for valve replacement in The First Affiliated Hospital of Harbin Medical University were collected. The clinical information was shown in Supplemental Table-4 (Additional File 1).
The general exclusion criteria of patients were a known history of diabetes (except OGTT analysis), treatment with antibiotics or probiotics within the past three months, or cold within the past month. In addition, other specific exclusion criteria were also applied. First, patients who had medical history of cancer, inflammatory bowel disease major gastrointestinal surgery within the past five years, invasive medical intervention within the past three months, or significantly altered diet compositions in the week of blood collection were excluded. Second, patients who had other intestinal inflammatory diseases except for colon cancer and major gastrointestinal surgery within the past five years were excluded. Finally, patients with end-stage heart failure, cancer, or inflammatory bowel diseases were also excluded.
Experimental Animals.
The animal experiments in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Harbin Medical University. Both aged male Sprague Dawley (SD) rats (22-24 months old) and young male SD rats (2-3 months old) were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd (Beijing, China). The rats were individually housed under 12:12-h light-dark cycles and fed with food and water available ad libitum. In anti-lipopolysaccharide (LPS) experiments, rats received oral LPS-RS (a potent LPS antagonist; InvivoGen, CA, USA) at 1 mg/Kg once per week for six weeks. In anti-NLRP3 experiments, rats received MCC950 (a small-molecule selective inhibitor of NLRP3; Sigma, San Jose, CA, USA; cat. no.: PZ0280-5MG) via intraperitoneal injection at 15 mg/Kg once per week for six weeks.
Electrophysiological study.
Atrial fibrillation (AF) was induced with essentially the same protocol as described previously in detail[14]. In brief, rats were anesthetized with 1% sodium pentobarbital (30 mg/kg) through peritoneal injection. After open-chest surgery, a 1.9-F octapolar catheter (Transonic Systems Inc., New York, USA) was placed on the right atrium to deliver programmed stimuli. To assess the inducibility of atrial arrhythmias, 50-Hz burst pacing was applied for 3 s with 12 bursts separated by a 2-s interval. AF was defined as >1 s of irregular atrial electrograms (>800 bmp) with irregular ventricular response. AF duration was defined as the mean duration of all AF episodes within 60 s in each rat.
Gut Microbiota Profiling.
The sequencing of 16S ribosomal RNA (rRNA)-encoding gene of fecal samples was performed as described previously[15]. Microbiota in fecal samples, representative of that in the proximal colon, was collected from rats and immediately frozen and stored. Bacteria taxa were analyzed by amplifying the V3 to V4 hypervariable regions of the 16S rRNA gene and sequenced with Illumina HiSeq 2500 platform (Illumina, San Diego, CA). Quality filter of 16S rRNA sequences was conducted by using Quantitative Insights Into Microbial Ecology (QIIME) and custom scripts. Resulting sequences were then clustered into Operation Taxonomic Unit (OTU) by searching against Greengenes reference database of 16S rRNA gene sequences and clustered at 97% by Uclust algorithm[16]. The differences of Genus level and Family level between treatment groups were calculated by using pairwise Wilcoxon rank-sum tests.
Collect Fecal Sample and Microbiota Transplantation.
Feces from 3 aged rats (22-24 months old) with increased AF susceptibility residing and from young rats (2-3 months old) were collected and the samples were pooled by group. Each feces sample was diluted in sterile PBS (100 mg feces in 2 ml buffer) and homogenized for 5 min. After vortexed for 1 min and centrifuged at 800g for 3 min, the supernatant was collected.
The protocol of fecal microbiota transplantation (FMT) was performed as described by García-Lezana et al.[17] In order to facilitate re-colonization of transplanted gut microbiota, rats were administrated with omeprazole (50 mg/Kg/d) for 3 days before FMT for decontamination. To facilitate intestinal emptying, 1 ml and 2 ml of CitraFleet (sodium picosulfate, 0.16 mg/ml and magnesium oxide 51.2 mg/ml) along with 2 ml of water were administrated to the animals 24h and 12h prior to FMT, respectively. A study reported by Manichanh et al.[18] showed that the transplanted microbiota can be acquired following a single fecal transplant and can last for at least three months. We therefore adapted a single oral gavage for 6 weeks for re-colonization of transplanted gut microbiota. The young rats gavaged with autogenous fecal were referred to the Young-FMT group and the young rats gavaged with fecal from aged rats to the Aged-FMT group.
For the experiments involving long-term young FMT rescuing study, we collected feces from 3 young without AF rats (2-3 months old). The aged recipient rats (16-18 months old) were administrated by autologous fecal microbiota transplantation (Aged+AgedFMT group) and microbiota transplantation from young rats (Aged+YoungFMT group), respectively, once a week for 6 months.
Nuclear and Cytoplasmic Protein Extraction.
Pre-frozen atrial tissue was cut into small pieces and washed with PBS, followed by centrifugation at 10000r/min for 5 min, according to the subcellular structure nuclear and cytoplasmic protein extraction kit protocol (Wuhan, China). Homogeneous cell suspension was collected by vortex after mixed with cytoplasmic protein extraction reagent A in a tube. Cytoplasmic protein extraction reagent B was then added into the tube on ice. Finally, the tube was centrifuged at 16000g for 5 min, and the supernatant was removed, followed by addition of the nucleoprotein extraction reagent to the remaining insoluble cell debris containing nuclei. After 40 min vortex and 5 min centrifugation, the supernatant was transferred into a clean tube containing nuclear proteins.
Isolation of Rat Cardiomyocytes.
Neonatal rat cardiomyocytes (NRCMs) were isolated from 1 to 3-day-old Sprague-Dawley (SD) neonatal rats as previously described[19]. Briefly, neonatal rat hearts were finely minced and placed together in 0.25 % trypsin. Cell suspension was collected, centrifuged and re-suspended in DMEM supplemented with 10 % FBS and 100 U/ml penicillin and 100 μg/ml streptomycin. The re-suspension was plated onto a culture flask, and cardiac fibroblasts (CFs) were obtained after differential adhesion for 90 min leaving cardiac myocytes (CMs) in suspension portion. Then the culture medium was replaced to purify CFs, and the CMs were seeded at a density of 1 × 106 cells per well in a six-well culture plate. Cell cultures were incubated at 37°C in a humidified atmosphere with 5 % CO2 and 95 % air. In vitro experiments, the CFs and CMs were cultured with LPS (100 ng/mL)[20] and different concentrations of glucose: 5.5 mmol/L glucose (control) and 30 mmol/L glucose (HG) [21]for 72 h at 37°C with 5% CO2, respectively.
Histology.
Fresh atrial and proximal colon tissues were cut into 5-μm sections, fixed in 4% paraformaldehyde and embedded with paraffin. Then, the tissues were stained with hematoxylin and eosin (HE), Masson’s trichrome and TUNEL staining according to our previous study[22]. The HE staining was used to determine atrial and proximal colon structure. Subsequently, the fibrotic area was measured by Masson’s trichrome staining and quantified by using software (Image-pro plus 6.0, Meida Cybernetics LP). Collagen volume fraction was calculated as collagen area/total area × 100%. The cell apoptosis was assessed by TUNEL staining and semi-quantified as the number of apoptotic cells per field.
Western Blots (WBs).
Western blotting procedures in the present study were essentially the same as described in a previous study[22]. Briefly, proteins were separated by electrophoresis on 8% to 12% SDS-polyacrylamide gels and transferred moist to polyvinylidene difluoride membranes. The membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 h and then incubated with anti-Claudin 4 (Proteintech, 1:500, 16195-1-AP), anti-ZO-1 (Proteintech, 1:500, 21779-1-AP), anti-Occludin (Abcam, 1:1000), anti-TLR4 (Abcam, 1:500, ab1356), anti-NLRP3 (Proteintech, 1:500, 19771-1-AP), anti-Caspase 1 p20 (Invitrogen, 1:200, Prod#PA5-78915), anti-ASC (Abcam, 1:200, ab175449), anti-IL-1β (Abcam, 1:500, ab9722), and anti-NF-κB P65 (NOVUS, 1:500, NB100-56721), anti-NF-κB P65 of Phospho-Ser536 (NOVUS, 1:500, NB100-82088), anti-MyD88 (Proteintech, 1:1000, 66660-1-Ig), anti-Bcl2 (Abcam, 1:100, ab7973), anti-BAX (Proteintech, 1:500, 60267-1-Ig), anti-TGF-β1 (Proteintech, 1:1000, 21898-1-AP), anti-α-SMA (Arigo, 1:4000, SQab1735), anti-CD68 (Santa Cruz Biotechnology, 1:100, sc-70761), ISG15 (Abcom, 1:500, ab227541), S100A8 (Proteintech, 1:500, I5792-I-AP), or S100A9 (Proteintech, 1:500, I4226-I-AP), and GAPDH (Zsbio, 1:1000, TA-08) and β-tublin (CST, 1:1000, #2148) or β-actin (Zsbio, 1:1000,TA-09). After washing, the membranes were incubated with the secondary antibody (Santa Cruz Biotechnology, Dallas, USA) for 1 h. The membranes were exposed to ECL buffer after another three washes, and the blots were detected by ChemiDoc XRS gel documentation system (Bio-Rad, Hercules, CA, USA).
FITC-Dextran Permeability Assay.
Following 6 h of fasting, rats received a single i.v. dose of 4kDa FITC-dextran (44mg/100g, Sigma Aldrich, catalog #FD4). After 4 h feeding, blood samples wwere collected and centrifuged. FITC fluorescence was quantified using the standard curve method according to manufacturer’s instructions.
Plasma and Fecal LPS Levels.
The levels of serum and fecal lipopolysaccharide (LPS) from rats were measured by using a quantitative chromogenic limulus amoebocyte lysate (LAL) QCL-1000 test kit (Lonza Bioscience, Switzerland) according to the manufacturer’s instructions. First, the serum and fecal samples were pretreated with pyrogen-free water provided in the kit. Then, the dilute samples were deactivated in 75°C water bath for 10 min. After incubation with 50 μl of LAL reagent at 37°C for 10 min, 100 μl of LAL chromogenic substrate was added into the samples for 6 min. Finally, 100 mg/ml of SDS was added to terminate the reaction with yellow color indicating cleavage of the substrate. The OD value of LPS level was measured by spectrophotometrically at 405 nm.
LC-MS/MS Analysis.
After proximal colon protein extraction, the protein solution was reduced with 5 mM dithiothreitol and 11 mM iodoacetamide for digestion[23]. The tryptic peptides were dissolved in proper sequence with 0.1% formic acid (solvent A) and different concentrations of solvent B (0.1% formic acid in 98% acetonitrile), followed by centrifugation with an EASY-nLC 1000 UPLC system. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific, US) coupled online to the UPLC. Then, the peptides were selected for MS/MS using NCE setting of 28 and the fragments were detected in the Orbitrap. A data-dependent procedure was performed by alternating between one MS scan, which was followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.
Statistical Analyses.
The statistical analyses were performed with GraphPad Prism 6.0 software (GraphPad Software, Inc, La Jolla, CA). Data are all expressed as mean ± SEM. Two-group comparisons were performed using Student’s non-paired t-test or Kruskal-Wallis H test. Data with more than two groups were analyzed by one-way ANOVA, followed by Tukey tests. AF incidence rates were compared with Fisher’s exact test. The statistics regarding microbiome analysis in the subsections on microbiome analysis was described in gut microbiota profiling of methods. Differences were considered as statistically significant when P < 0.05.