Male C57BL/6 J WT mice (7-week-old; body weight, 20–25 g) were received from Japan Clea, Co. (Tokyo, Japan). Mice were housed under 12 h light/dark cycles at room temperature. All mice had free ad libitum access to normal diet and water. Animal care procedure and all experiments were performed in accordance with the Tokyo Medical and Dental University Guide for the Care and Use of Laboratory Animals (Permit Number: A2022–096C and G2019-038C5) and Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Mice were deeply anesthetized with an intraperitoneal injection of a cocktail (medetomidine-midazolam-butorphanol (MMB); 0.1 mL per 10 g) comprising medetomidine (Domitor; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan; 0.75 mg·kg-1), midazolam (Dormicum; Maruishi Pharmaceutical, Osaka, Japan; 4 mg·kg-1), and butorphanol (Vetrophale; Meiji Seika Pharma CO., Ltd., Tokyo, Japan; 5 mg·kg-1). Euthanasia was performed by intraperitoneally administering an overdose of MMB.
Genetically modified mice
The gRNA for the mouse Vamp8 gene, donor oligo containing K47A (AAG to GCG) mutation sites, and Cas9 mRNA were introduced into mouse eggs to produce vesicle-associated membrane protein 8 (VAMP8)-K47A mutants. F0 founder animals were detected using polymerase chain reaction (PCR) followed by sequence analysis using the following primers: 5’-CAAGGGAGGAGTGACACCTGACCAC-3’ and 5’-ATCCAACCACTCACTGGGCTCTCTAC-3’.
To generate Tg-GFP-LC3-RFP-LC3ΔG mice (Tg-tfLC3ΔG mice), frozen sperm from C57BL/6J-Tg (CAG-GFP/LC3/RFP/LC3 < *>deltaG)2Nmz mice was provided by the RIKEN RBC through the National BioResource Project of the MEXT/AMED, Japan. We obtained the first generation of GFP-LC3-RFP-LC3ΔG heterozygous mice by external fertilization. The transgenic strains were identified by performing PCR using the following primers: 5’-CCTACAGCTCCTGGGCAACGTGC-3’; 5’-GTACCACCACACTGGGATCCTTAG-3’; 5’-CTAGGCCACAGAATTGAAAGATCT-3’; and 5’-GTAGGTGGAAATTCTAGCATCATCC-3’.
To generate VAMP8-K47A;Tg-tfLC3ΔG mice, VAMP8-K47A KI mice were bred in house with Tg-tfLC3ΔG mice.
The WTP.g. strain ATCC33277 (WTP.g.) and gingipain-deficient P.g. strain KDP981 (ΔP.g.) were kindly provided by Dr. Keiko Sato. They were cultured on blood agar plates in the AnaeroPack system (Mitsubishi Gas Chemical. Co., Ltd. Tokyo, Japan) and were incubated at 37 °C for 2–3 days. Bacterial cells were then added to peptone yeast extract and incubated for 1 week. The bacterial concentration was estimated using a spectrophotometer at a wavelength of 660 nm and was standardized to 109 CFU·mL-1.46
Inoculation of periodontal pathogens
To evaluate the effects of P.g. on systemic organs other than periodontal tissue in mice, we employed the P.g. oral gavage method.47 This approach is widely used to obtain a less artificial infectious animal model, ensuring a more realistic representation of the biological response. Mice were administered with trimethoprim (0.4 mg·mL-1) and sulfamethoxazole (0.7 mg·mL-1) solutions (FUJIFILM Wako Pure Chemical. Co., Ltd. Tokyo, Japan), which were prepared by mixing the drugs with drinking water for 10 days, and only water was administered on the following 2 days. The mice were then inoculated once every 2 days with 0.1 mL of P.g. (1 × 109 CFU·mL-1) dissolved in 2.5% carboxymethylcellulose (CMC; Nacalai Tesque, Inc., Kyoto, Japan) via oral gavage. Noninoculated mice were administered with 0.1 mL of 2.5% CMC alone.48 The level of anti-P.g.–specific IgG in the plasma before sacrificing the mice was determined using enzyme-linked immunosorbent assay (ELISA).49
Extraction of gingipains from P.g
The A7A1–28 strain of P.g., which potentially produces gingipains,50 was used. Briefly, P.g. was inoculated into enriched tryptic soy broth and incubated at 37 °C under anaerobic conditions until the bacterial cells were sedimented. P.g. cells were centrifuged at 20 000 × g for 20 min at 4 °C. The supernatant was separated, and the precipitated protein was collected by centrifugation at 25 000 × g for 30 min at −10 °C. The suspension was then added to dialysis bags (MWCO, 12 000–14 000; Thermo Fisher Scientific, Waltham, MA, USA), which were placed in dithiopyridine disulfide buffer at 4 °C, followed by two changes gel filtration buffer at 4 °C. The dialyzed fraction was obtained by performing ultracentrifugation for 1 h at 100 000 × g at 4 °C and separated by ultrafiltration using an Amicon PM-30 membrane (Merk Millipore, Burlington, MA, USA).
Experimental MI model
MI was induced as previously described.51 Mice were deeply anesthetized with an intraperitoneal injection of MMB (0.1 mL per 10 g). Overall, the mice were intubated using a small rodent ventilator (MiniVent 845, Harvard Apparatus, Holliston, MA). Left lateral thoracotomy was performed, and the LAD coronary artery was ligated using an 8/0 nylon suture after removing the pericardium. The chest was closed, antipamezole (0.75 mg·kg−1; Antisedan; Nippon Zenyaku Kogyo Co., Ltd.) was administered for reversal, and then the mice were removed from the respirator. The mice were allowed to recover on a warm surface. Sham mice underwent all procedures except for actual LAD occlusion. Some mice were examined until 28 days after MI to analyze survival. The other mice were examined until 7 days after MI and then sacrificed to obtain samples. All mice were necropsied to obtain evidence regarding post-MI cardiac rupture or heart failure as described previously.52
Hemodynamic measurements and echocardiography
A tail-cuff system (BP-98A; Softron Co., Tokyo, Japan) was used to measure the arterial blood pressure and heart rate. Transthoracic echocardiography was carried out on mice that were administered MMB intraperitoneally (0.08 mL per 10 g body weight). The left ventricle function was assessed using an echocardiographic machine with a 14-MHz transducer (Toshiba, Tokyo, Japan). A two-dimensional targeted M- and B-modes echocardiogram was received, and left ventricular end-diastolic (LVDd) and end-systolic (LVDs) dimensions and left ventricular fractional shortening (LVFS) (LVFS = (LVDd − LVDs)/LVDd) were computed over three cardiac cycles according to the leading edge method described by the American Society for Echocardiography. The average of measurements from three consecutive cardiac cycles, performed off-line by two independent investigators, was calculated.
The hearts were harvested immediately after sacrificing the mice on day 7 or 28 after MI induction. Midventricular slices of the heart were stained using hematoxylin and eosin stain, Mallory’s trichrome stain, and lectin obtained from Triticum vulgars (WGA; Sigma-Aldrich, St. Louis, MO, USA). The area of fibrosis represented by blue-stained collagen fibers was calculated using ImageJ. The average infarct size was defined as the average circumference of the infarcted portion and the normal area from the consecutive myocardial sections.32
Measurement of plasma levels of NT-proBNP
At 7 days after MI, blood samples were collected and plasma NT-proBNP levels were measured using a mouse NT-proBNP ELISA kit (Novus Biologicals, Centennial, CO, USA) according to the manufacturer’s instructions.
Preparation of primary neonatal rat cardiomyocytes (NRCMs)
Ventricular cardiomyocytes were isolated from 1- or 2-day-old neonatal Wistar rats using the Pierce Primary Cardiomyocyte Isolation Kit (Thermo Fisher Scientific), following a previously described method53 with minor modifications. To achieve NCRM purification, centrifugation was performed using a discontinuous Percoll (Sigma-Aldrich) gradient. The NRVM cultures contained >97–99% cardiomyocytes, as confirmed by immunofluorescence staining using the MF20 monoclonal antibody against sarcomeric myosin (Sigma-Aldrich). NCRMs were then incubated in Eagle’s minimum essential medium (Sigma-Aldrich) enriched with 5% calf serum (JRH Biosciences, Lenexa, KS, USA) for 24 h at 37 °C. NRCMs were treated with WTP.g., ΔP.g., or gingipains for 4 h at 37 °C.
Human embryonic kidney (HEK) 293 T cells and H9c2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) enriched with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U·mL-1 penicillin, and 100 mg·mL-1 streptomycin (Nacalai tesque, Kyoto, Japan) at 37 °C. For the starvation treatment, cells were rinsed with phosphate buffer saline (PBS) and were put in amino acid-free DMEM (FUJIFILM Wako Pure Chemical Co., Ltd., Osaka, Japan) without serum.
Protein extraction from the myocardium and the cells
The infarct area of MI heart specimens was removed on day 7 after MI and homogenized in radioimmunoprecipitation assay (RIPA) buffer (containing 50 mmol·L-1 Tris-HCl, pH 6.8, 150 mmol·L-1 NaCl, 1 mmol·L-1 PMSF, 2 ng·mL–1 aprotinin, 1% Triton X-100, 1% SDS, and 1% sodium deoxycholate). HEK 293 T and H9c2 cells were washed with PBS and lysed with IGEPAL CA-630 buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). The samples were centrifuged and supernatant was transferred to new tubes. Protein concentration was calculated using bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Unused proteins were stored at −80 °C until further use.
Plasmid DNA of FLAG-VAMP8, FLAG-syntaxin 17 (Stx17), and FLAG-synaptosoma–associated protein 29 (SNAP29) was purchased from Addgene (Watertown, MA, USA). Plasmid DNA of FLAG-VAMP8-K47A was purchased from Vector Builder (Chicago, IL, USA). HEK 293 T cells (10 mL) were plated in a 10-cm dish 2 days before transfection. 1 mL Opti-MEM (Gibco, Billings, MT, USA), 10 μg plasmid DNA, and 20 μL FuGENE (Promega, Madison, WI, USA) were properly mixed and incubated for 15 min at room temperature. The medium of the HEK 293 T cells was aspirated. Next, the transfection complex and 5 mL DMEM were added dropwise to the cells and incubated at 37 °C. After 24 h, another 5 mL of DMEM was added and the cells were incubated for 24 h at 37 °C. The transfected cells were treated with either WTP.g. or ΔP.g. for 4 h at 37 °C.
Equal amounts of protein from the infarcted area of hearts after 7 days of inducing MI, as well as NRCMs, HEK 293 cells, or H9c2 cells were separated using AnykDTM precast sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The separated proteins were transferred onto polyvinylidene difluoride (Immobilon-P; Merck Millipore, Burlington, MA, USA) or nitrocellulose (Bio-Rad) membranes, which were then incubated overnight with primary antibodies (anti-VAMP8, Abcam, ab76021, 1:1 000; anti-endobrevin, Santa Cruz Biotechnology [Dallas, TX, USA], sc-166820, 1:1 000; anti-p62, MBL [Nagoya, Japan], M162-3, 1:1 000; anti-LC3, MBL, M186-3, 1:1 000; anti-troponin I, Cell Signaling, #4002, 1:1 000; anti-SNAP29, Proteintech [Rosemont, IL, USA], 12704-1-AP, 1:500; and STX17, Proteintech, 17815-1-AP, 1:500). The membranes were then incubated with a secondary antibody (anti-rabbit or anti-mouse horseradish peroxidase [HRP]-conjugated antibodies, Cell Signaling) for 2 h, and the catalysis was promoted using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Enhanced chemiluminescence using iBright Imaging systems was used to detect the proteins (Thermo Fisher Scientific).25
Pull-down binding assays
Initially, VAMP8-modified 293 cells were washed with PBS and lysed with IGEPAL CA-630 buffer containing protease and phosphatase inhibitors. His-tagged Lys-gingipain recombinant protein (enQuireBioReagents, Littleton, CO, USA) was incubated with cobalt resin (Thermo Fisher Scientific) and IGEPAL CA-630 buffer at 4 °C for 2 h. The supernatant was removed and gingipain activation buffer54 (200 mmol·L-1 HEPES [pH 8.0], 5 mmol·L-1 CaCl2, and 20 mmol·L-1 L-cysteine, HCl solution) were added and incubated at 37 °C for 30 min. The samples were washed thrice with IGEPAL CA-630 buffer and 1× SDS sample buffer and processed for immunoblotting.
Protein cleavage assay
VAMP8-, VAMP8-K47A-, or STX17-modified 293 cells were lysed with IGEPAL CA-630 buffer containing protease and phosphatase inhibitors. Protein concentration was determined using BCA assay, and the unused samples were stored at −80 °C until further use. Anti-FLAG M2 magnetic beads (Sigma-Aldrich) were rinsed thrice with IGEPAL CA-630 buffer and incubated with the samples for 2 h at 4 °C. The samples were washed thrice with high salt IGEPAL CA-630 buffer. The supernatants were then replaced with gingipain activation buffer. Gingipains were added and incubated for 30 min at 37 °C. After washing the sample thrice with IGEPAL CA-630 buffer, 1× SDS sample buffer was added to the beads. The obtained samples were then subjected to SDS–PAGE.
NRCMs were lysed using RIPA buffer containing protease and phosphatase inhibitors. Next, VAMP8- and VAMP8-K47A-modified 293 cells and H9c2 cells were plated in a 10 mL volume in a 10-cm dish and incubated at 37 °C. After 2 days, the cells were rinsed with PBS, starved in amino acid-free DMEM, and then treated with either WTP.g. or ΔP.g. for 4 h at 37 °C. The cells were lysed with IGEPAL CA-630 buffer. Primary antibody (VAMP8) was covalently immobilized on protein A/G agarose (Thermo Scientific) for 1 h at 4 °C. Samples were incubated with immobilized antibody beads overnight at 4 °C. After immunoprecipitation, the samples were washed thrice with IGEPAL CA-630 buffer, followed by adding 1× SDS sample buffer. The obtained samples were then subjected to SDS–PAGE using specific primary antibodies and a conformation-specific secondary antibody that recognizes only native IgG (Cell Signaling, #5127, 1:2 000).
NDP52 gingipains assay
GST–agarose beads were incubated with the GST–NDP complex (CALCOCO2 Recombinant Protein; Abnova [Taipei, Taiwan]) and then incubated with gingipains at 37 °C for 30 min. After washing the samples thrice with IGEPAL CA-630 buffer, the beads were collected. Proteins in the beads were then eluted using 1× SDS sample buffer, separated using SDS–PAGE and a nitrocellulose membrane, and incubated overnight with a primary antibody, GST-Tag monoclonal antibody (Proteintech, #66001-2-Ig, 1:500). The membrane was then incubated with a secondary antibody (anti-mouse or anti-rabbit HRP-conjugated antibodies; Cell Signaling) for 2 h, and the signal was developed with SuperSignal West Dura Extended Duration Substrate. Chemiluminescence was detected using iBright Imaging Systems.
In this study, 7-week-old Tg-tfLC3ΔG male mice were used. To promote systemic autophagy, mice were starved for 48 h.55 During the starvation period, the mice had free access to water.
Cell viability assays
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technology, Kumamoto, Japan). In brief, H9c2 cells and NRCMs (1 × 106 cells per 100 μL) were seeded into 96-well dishes. After 24 h, the cells were incubated under hypoxic conditions at 37 °C for 24 h. Next, the cells were inoculated with either WTP.g. or ΔP.g. (1 × 108 CFU·mL–1) and incubated for 1 h. The cell viability assays were performed according to the manufacturer’s instructions in the CCK-8. The experiments were conducted in triplicates.
Detection of GFP-LC3 or mRFP-GFP-LC3
Adenovirus-harboring tandem fluorescent mRFP-GFP-LC3 was generated using a previously described method.55 NRCMs were grown on gelatinized coverslips, and transduction was performed using mRFP-GFP-LC3. After 48 h, the growth medium was replaced by phenol red-free medium. Then, either WTP.g. or ΔP.g. (1 × 108 CFU·mL–1) was added and the samples were incubated for 1 or 4 h. For tissue samples, Tg-tfLC3ΔG mice were anesthetized with MMB (0.1 mL per 10 g body weight), and thoracotomy was performed. The hearts were excised and immediately embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan), which were stored at −80 °C. Next, 5–7 μm-thick samples were sectioned using a cryostat, air dried for 30 min, and stored at 4 °C. The fluorescence of GFP-LC3 or mRFP-GFP-LC3 was observed under a fluorescence microscope.55,56
To detect P.g. in NRCMs, the Click-iTTM Cell Reaction Buffer Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. Briefly, P.g. was cultured with alkyne-conjugated N-acetylmuramic acid and was inoculated in NRCMs. To prepare electron microscopy specimens, P.g. was labeled with azide-conjugated gold nanoparticles. The samples were fixed in a 2.5% glutaraldehyde solution for 1 h, rinsed in 1% bovine serum albumin (BSA), and the Click-iT TM reaction cocktail was added. The samples were incubated at room temperature for 30 min away from light, followed by washing the samples with 1% BSA and fixing them in 0.3% osmium tetroxide (OsO4) dissolved in 0.1 mol·L−1 PBS for 17 min. They were then washed with 0.1 mol·L−1 PBS, dehydrated in ethanol, and stained with 2% uranyl acetate in 70% ethanol for 1 h at 4 °C. Subsequently, the samples were dehydrated in a graded series of ethanol and placed in Epon 812 (TAAB, Aldermaston, United Kingdom) for 48 h. Ultrathin (70–80 nm) sections were placed on copper grids and were observed under a transmission electron microscope (H-7011; Hitachi, Tokyo, Japan).
Confocal fluorescence microscopic analysis
To detect P.g. in the cultured cardiomyocytes, P.g. was labeled with Acetoxymethyl Ester (BCECF-AM), as previously described.57 NRCMs were seeded into a 24-well circular cover-glass plate and grown overnight. Prior to inoculation, P.g. was incubated with BCECF-AM in PBS at 37 °C for 30 min. Then, NRCMs were inoculated with BCECF-labeled P.g. at 37 °C for 30 min in the dark and the cells were washed thrice with PBS. The cardiomyocytes were fixed with 4% formaldehyde in PBS for 10 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100 for 10 min. TRITC-phalloidin (50 μg·mL-1) was placed on coverslips for 45 min. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Cosmo Bio Co., Ltd., Tokyo, Japan), and the coverslips were set on microscope slides. Confocal images were obtained using a confocal fluorescence microscope (TCS SP8; Leica, Wetzlar, Germany).
Obtained data were expressed as mean ± SEM. Kaplan–Meier survival analysis was used, and the survival rates were compared using the log-rank test. Other statistical analyses were done using one-way analysis of variance test followed by Tukey’s post-hoc test for multiple comparisons. A P-value of <0.05 was considered to indicate statistical significance, and GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used for the analyses.