Experimental Animal Models of myocarditis
Male BALB/c mice (6 weeks of age, body weight: 20-25 g) were purchased from Japan Clea, Co. (Tokyo, Japan).
Experimental autoimmune myocarditis was induced in BALB/c mice by subcutaneous injection of the α-Myosin heavy chain (α-MyHC) peptides (150 µg per mouse) emulsified with Complete Freund’s adjuvant on day 0 and 7. On day 0, 500 ng of pertussis toxin were also administrated intraperitoneally at the same time to induce the EAM mice model. EAM mice were assigned to one of the following two groups: EAM mice group treated with linagliptin (3 mg/kg) and untreated controls. Mice were sacrificed on Day 21. For the experiments of Angiotensin II receptor type 1(AT1) blockade, losartan (LOS) (10 mg/kg/day; MERCK & Co., Inc., Kenilworth, NJ, USA) was given to the mice in their drinking water .
A transthoracic echocardiography was performed on animals anesthetized by intraperitoneal administration of 3.6% chloral hydrate (Wako Pure Chemical Industries, Osaka, Japan) in saline (0.1 mL/10 g body weight). For the left ventricular echocardiographic recording, an echocardiographic machine with a 14-MHz transducer (Toshiba, Tokyo, Japan) was used. A two-dimensional targeted M-mode and B-mode echocardiogram was obtained along the short-axis view of the left ventricle at the level of the papillary muscles. Left ventricular end-diastolic (LVDd) and end-systolic (LVDs) dimensions and left ventricular fractional shortening (LVFS = ((LVDd − LVDs) / LVDd)) were calculated from M-mode echocardiograms over 3 consecutive cardiac cycles according to the American Society for Echocardiography leading edge method. The measurements of 3 consecutive cardiac cycles were averaged. Measurements were made offline by two independent investigators.
Hearts were harvested immediately after animals were sacrificed by cutting the abdominal aorta under deep anesthesia on day 21. After measuring the weight (mg), mid-ventricular slices of the heart were stained with hematoxylin and eosin (HE) and Mallory methods. Blue staining of collagen fibers was quantified as a measure of fibrosis using the Image-Pro Express software program. The average infarct size was obtained by calculating the area ratio (fibrosis area/entire area). The area of the myocardium affected by cell infiltration was determined as infiltrated. All data were analyzed in a blind fashion by two independent investigators and averaged.
Immunohistochemistry was performed to evaluate the amount of a thymus-specific isoform of the RAR-related orphan nuclear receptor gamma (RORγt), cathepsin G, and 8-hydroxyguanosine (8-OHdG), a marker of oxidative stress in DNA, in the hearts of mice on day 21. Frozen sections were fixed in acetone at 4oC. The sections were incubated with unlabeled primary antibodies overnight at 4oC and washed in PBS. The antibody-HRP conjugate was detected with a Histofine Simple Stain Kit (Nichirei Corporation, Tokyo, Japan), following the manufacturer’s instructions. RORγt-positive cells were counted and the number was divided by the entire area.
DPP-4 activity assay
The activity of DPP-4 was evaluated in myocardium tissues using a DPP-4 activity assay kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions.
T cell proliferation assay
Spleen cells were isolated from mice with myocarditis on day 18. Cells (5×105/well) were cultured in 96-well plates with 50 μg/mL purified porcine heart myosin (Sigma, St. Louis, MO). Linagliptin was added to each well at various concentrations. Cells were incubated at 37 °C under 5% CO2 for three days. T cell proliferation was assessed by MTT assay with Cell Counting Kit-8 (Dojindo, Tokyo, Japan). Cell proliferation was expressed as the optical density .
Protein extraction from the myocardium
Heart specimens, which were harvested immediately after mice were sacrificed on day 21, were homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% DOC, 0.1% SDS, 1% NP-40). Regarding the samples used for Tandem Mass Tag (TMT) labeling, heart specimens were homogenized in a Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Inc.). Samples were centrifuged, and the supernatant was transferred to new tubes. Protein concentration was determined using a BCA protein assay (Pierce Biotechnology, Rockford, IL). Proteins were stored at −80°C until further analysis.
Human embryonic kidney 293 cells were cultured for 18 to 24 hours to approximately 80% confluence in 10 cm plates and then transfected with 10 ng of plasmid (Flag-DPP-4 or Flag-Empty) using FuGene6 (20 μL) (Roche, Basel, Switzerland), according to the manufacturer’s instructions. After 48 hours of transfection, cells were harvested, and solubilized in a lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM phenylpethyl sulfonyl fluoride, 10 μg/mL aprotinin). Total cell lysates (500 μg) were immunoprecipitated with Flag antibody-conjugated magnetic beads overnight at 4°C. Immunoprecipitates were washed several times with lysis buffer.
Immunoprecipitates with Flag antibody-conjugated magnetic beads (described above) were incubated with heart lysates extracted with a Minute Plasma Membrane Protein Isolation Kit for 6 hours at 4°C. Cells were washed with 500 uL of TBS-T for 10 min at 4oC five consecutive times. They were then added to the elution buffer (0.1 M glycine with HCl) and neutralized with 1 M Tris-HCl [pH 9.0]. TMT labeling was performed using TMT Mass Tagging Kits and Reagents (Thermo Scientific, Rockford, IL, U.S.A.) according to the manufacturer’s instructions. The eluted proteins were adjusted to 100 µL with 100 mM TEAB; additionally, 5 µL of the 200 mM TCEP were added, and samples were incubated at 55°C for 1 hour. Then, 5 µL of 375 mM iodoacetamide were added to the sample and incubated for 30 minutes protected from light at room temperature. Trypsin was added (final concentration: 2.5%) to sample proteins in TEAB and samples were digested overnight at 37°C. As a labeling step, 41 µL of the TMT Label Reagent were added to each 100 µL sample. After incubating the reaction for 1 hour at room temperature, 8 µL of 5% hydroxylamine were added to the sample and incubated for 15 minutes to quench the reaction.
The aliquot of TMT-labeled samples was analyzed using a LTQ Orbitrap Velos hybrid mass spectrometer (Thermo Scientific) equipped with an Easy nLC II nano-LC system (Proxeon, Denmark) and Chip-Mate nano-ESI interface unit (Advion Inc., U.S.A.). The Nano-LC column was a MonoCap C18 Nano-flow 100 µm i.d. × 750 mm (GL Sciences, Japan). Nano-LC settings were configured as follow: Flow rate was 300 nL/min. Water containing 0.1% formic acid was used as eluent A. Acetonitrile/water (7:3, v/v) containing 0.1% formic acid was used as eluent B. The gradient of eluent B was set as follows: 0% – 5 min – 20% – 45 min – 57% – 10 min. The acquired MS and MS/MS spectra were processed with Proteome Discoverer 1.3 Software (Thermo Scientific).
Cathepsin-G activity assay
The activity of cathepsin-G was evaluated in myocardium tissues and mixtures of recombinant mouse DPP-4 (R&D Systems, Minneapolis, MN), recombinant mouse SerpinA3N (R&D Systems), and cathepsin-G (Enzo Biochem, New York, NY) using a cathepsin-G Activity Assay Kit (Abcam, Cambridge, United Kingdom) according to the manufacturer’s instructions. Briefly, the mixture of DPP-4, SerpinA3N, and cathepsin-G or the mixture of SerpinA3N and cathepsin-G was mixed with assay buffer and incubated at 37°C for 10 min. Then, the substrate solution (assay buffer and p-NA) was added to each well. Absorbance was detected at 405 nm using a microplate reader (Bio-Rad, Hercules, CA) at 0 min. A second read was performed after incubating the reaction at 37°C for 60 min, protected from light.
SerpinA3N activity assay
The activity of SerpinA3N was evaluated in mixtures of DPP-4 and SerpinA3N, and extracted proteins from the myocardium according to the manufacturer’s instructions. The activity of SerpinA3N was measured by its ability to inhibit Granzyme B cleavage of tert-butoxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (Boc-AAD-SBzl). Briefly, the mixture of DPP-4, SerpinA3N, and activated recombinant human Granzyme B (R&D Systems) or the mixture of Serpin A3N and Granzyme B was mixed with the assay buffer (50 mM Tris, pH 7.5). Then, the substrate mixture, which consisted of 5,5’-dithio-bis 2-nitrobenzoic acid (DTNB, Sigma-Aldrich, St. Louis, MO), Boc-AAD-SBzl (SM Biochemicals, Anaheim, CA), and assay buffer, was added to each well. The absorbance was detected at 405 nm in kinetic mode for 5 minutes using a microplate reader (Bio-Rad).
Levels of Serpin A3N in homogenates of EAM hearts were determined by an ELISA using an immunoassay kit (Aviva Systems Biology, San Diego, CA, USA). The ELISA was performed according to the manufacturer’s instructions.
Angiotensin II assay
The amount of Angiotensin II was evaluated in myocardium tissues using an Angiotensin II ELISA kit (Sigma) according to the manufacturer’s instructions.
Chymase activity assay
Chymase activity was measured as described  Briefly, tissue extracts were incubated for 30 min at 37ºC with a substrate Suc-Ala-Ala-Pro-Phe-4-methylcoumaryl-7-amide (5 mmol/l; Fuji Film-Wako) in 100 mmol/l Tris-HCl buffer (pH 8.5, 200 mmol/l NaCl). After termination of the enzyme reaction by adding 3% metaphosphoric acid (w/v), the amount of 7-amino-4-methylcoumarin (AMC) was measured by fluorophotometric determination (excitation, 380 nm; emission, 460 nm). One unit of chymase activity was defined as the amount of enzyme that cleaved 1 μmol AMC/min.
Hydrogen peroxide assay
EAM heart tissues (day 21) were homogenized and sonicated in 50 mM potassium phosphate buffer and 0.5% hexadecyltrimethylammonium bromide (HTAB). Samples were centrifuged, and the supernatant was transferred to new tubes. The concentration of hydrogen peroxide was determined using a hydrogen peroxide colorimetric/fluorometric assay kit (BioVision) according to the manufacturer’s instructions.
All data are expressed as mean ± SEM. All statistical analyses were performed using an unpaired Student’s t-test or One-Way ANOVA, followed by a post hoc Bonferroni-Dunn’s comparison test for multiple group comparisons. A value of P<0.05 was considered to be significant.