experimental animal model
The study protocol was approved by the local Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System (YUHS-IACUC: 20157) and complies with ARRIVE reporting guidelines. Healthy New Zealand white rabbits were purchased from Dooyeol Biotech (Dooyeol Biotech, Seoul, Korea) and maintained under the same standard laboratory conditions, at room temperature with a 12-h light cycle with free access to diet and water in each cage. All animals were subjected to daily health status monitoring including weight, food intake, and general activity. All protocols followed the guidelines for the care and use of laboratory animals (National Research Council, USA). The main outcome variable was diastolic dysfunction assessed by echocardiography. Based on a previous study Case number estimation yielded a group size n = 10 (8 animals + 2 spare animals). A total of 30 male rabbits (3.0–3.5 kg, 22–24 weeks old) were randomly allocated to three groups: the control group (n = 10), diabetes (n = 10), diabetes + DAPA (dapagliflozin, 1 mg/kg/day/PO for 8 weeks) (n = 10). The diabetic state was induced by intravenous injection of Alloxan monohydrate (ALX, Sigma-Aldrich, St. Louis, MO, USA) at a dose of 150 mg/kg. Rabbits that showed a fasting blood glucose level above 200 mg/dL were diagnosed as diabetic. All rabbits were fed a 1% cholesterol diet (Dooyeol Biotech) for 6 weeks and a normal diet for 2 weeks after that. After follow-up, echocardiography was performed in the prone position after anesthesia with an intramuscular injection of an appropriate mixture of Zoletil and Rompun. After echocardiography, the animals were euthanized to reduce the discomfort experienced by the animals. The experimental protocol is shown in Supplementary File 1: Figure S3 in detail.
Glucose and cholesterol levels were measured in blood samples at baseline, diabetes modelling, and follow-up after 8 weeks using a blood glucose monitoring system (Osang Healthcare, Anyang, Korea) and DRI-CHEM 4000i (Fujifilm, Tokyo, Japan). Blood samples were taken from the veins of rabbits’ ears after they fasted overnight.
All images were acquired using a commercial ultrasound machine (Vivid 7 Dimension; GE Vingmed Ultrasound AS, Horten, Norway) with S10 probe (2.5 MHz). Images were obtained from the three-chamber, four-chamber, and two-chamber apical views; and short-axis views of the mitral valves, papillary muscles, and the apex .
Left atrial end diameter (LVEDD), left atrial end diameter (LVESD), posterior septal wall thickness, LV, and left atrial diameter (LAD) were measured from standard planes. The LV ejection fraction (EF) was calculated using the Teicholz . formula . Pulsed-flow echocardiography was performed via Doppler. The sample volume was set at the level of the coronal tips in the four-chamber apex view. From recording via transmission, early peak (E) and late diastolic filling velocities were obtained. A four-chamber apical view was also used to obtain tissue Doppler imaging of the coronary annulus. Sample volumes were placed on the septal and lateral sides of the coronary annulus. The values of systolic (S′), early (e′), and late (a′) diastolic velocities were obtained. Echocardiography and analysis were performed in blinded conditions.
Ultrastructural analysis using transmission electron microscopy (TEM)
Samples were cut into 1 mm squares and immediately placed in the initial TEM fixation. After pretreatment, samples were combined with a Poly/Bed 812 kit (Polysciences, Warrington, USA) and then placed in resin and polymerized in an electron microscope oven (TD-700, DOSAKA, Kyoto, Japan) at 65 °C for 12 h. . Thin sections (80 nm) were placed on copper grids and double stained with 3% uranyl acetate and 3% lead citrate for 30 min and 7 min, respectively. Stained sections were then imaged using a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) equipped with a Mega-View III CCD camera (soft imaging system, Münster, Germany).
Quantification of interstitial fibrosis and immunofluorescence staining
Cardiac tissues were fixed in 10% normal buffered formalin, embedded in paraffin, 4 μm thick, sectioned on a RM2235 microtome (Leica, Wetzlar, Germany), and then deparaffinized during the dewatering process. Masson Trichrome and Sirius Red were used for collagen staining. Immunohistochemistry (IHC) and immunofluorescence (IF) were used to assess the expression of fibrosis, macrophages or inflammation. Tissue sections were immunoprecipitated at 4 °C overnight with the antibody. IHC was used to detect α-SMA (Abcam, Cambridge, UK, ab-7817), Fibronectin (Abcam, ab-6328), TGF-β1 (Abcam, Wuhan, China, ABP52598), 3-nitrotyrosine (Abcam, Ab- 61392), Receptor advanced glycation end-products (RAGE) (LifeSpan Biosciences, Seattle, USA, LS-C122375), RAM11 (DAKO, CA, USA, M0633), tumor necrosis factor-α (TNF-α) (Abcam) , ab6671), NHE1 (Santa Cruz Biotechnologies, CA, USA, sc-136239), SGLT1 (Millipore, Overijse, Belgium, 07-1417), SGLT2 (Abcam, ab85626), Fis1 (Santa Cruz Biotechnologies, CA, USA, sc -376447), and Mfn1/Mitofusin1 (Santa Cruz Biotechnology, CA, USA, sc-166644). The primary antibody was detected using a peroxidase-based kit (DAKO, Glostrup, Denmark) and visualized with DAB substrate with enhancer (DAKO). Sections were subsequently counterstained with hematoxylin (DAKO). IHC staining was performed as previously described . Digital images of cardiac tissue were scanned with a SCN 400 scanner (Leica, Wetzlar, Germany), and histology was performed using LAS 4.2 software (Leica). Ten random images of 10 heart tissues per group were analyzed in a blind procedure.
If staining of cardiac tissues for detection of SGK1 kinase 1 (ABCAM, ab43606) and epithelial sodium channel (ENaC) (Burebit, Cambridge, UK, orb100662) was performed according to a published protocol. . Sections were washed for 10 min in 1% PBS and then incubated with FITC-conjugated secondary antibody (Santa Cruz Biotechnologies) for 1 h in the dark at room temperature. Sections were washed in PBS for 10 min, mounted with Fluoroshield containing DAPI (ImmunoBioscience, Mukilteo, WA, USA), and stored in the dark at 4 °C. Confocal microscopy was performed using an LSM 700 system (Carl Zeiss, Oberkochen, Germany).
Cell culture and transfection
Cells of the rat H9C2 cardiomyocyte line were cultured in DMEM containing 10% fetal bovine serum (both from Biowest, MO, USA) supplemented with 10% non-essential amino acids, 1% 2-mercaptoethanol, and 10% of penicillin (all from Gibco, Carlsbad, CA, USA). Cells were maintained at 37 °C in humidified air with 5% CO. Before treatment, cells were washed twice with phosphate-buffered saline pH 7.4 (PBS, Gibco). Cells were incubated in 500 μM palmitate (diluted in 5% bovine serum albumin [BSA]) with or without 35 mM glucose (HG) for 24 h and then treated with 0.4 μM dapagliflozin with 10 μg/ml LPS for 24 h (all from Sigma-Aldrich).
siRNA targeting mice were synthesized siSGK1 (5′-AGGAGAACAUCGAGCACAATT-3′) and siControl (5′-UUCUCCGAACGUGUCACGUTT-3′) (Pioneer, Daejeon, Korea). H9C2 cells were then transfected with siRNAs with LipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to a previously described method. .
Reverse transcription (RT)-PCR and real-time PCR
Cardiac tissues and H9C2 cells were isolated from total RNA using a published procedure . cDNA was synthesized using a quantitative reverse transcription kit (QIAGEN, Hilden, Germany), then cDNA was amplified using AccuPower PCR Premix (Bioneer, Daejeon, Korea) and SYBR Green 2X Fast Q-PCR Master Mix kit (SMOBIO, Hsinchu City, Taiwan). Relative mRNA levels were determined by comparison with GAPDH or beta-actin. Rabbit and H9C2 primers used for target genes are shown in Supplementary File 1: Table S1 and S2.
Western blot analysis
Cardiac tissues and H9C2 cells were lysed using RIPA buffer (Biosesang, Seongnam, Korea) containing EDTA-free complete mini-protease inhibitor cocktail (Roche, Basel, Switzerland). Protein samples were resolved by SDS-PAGE and then electrotransferred to a PVDF immunoblot membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% skim milk (Noble Bio, Hwaseong, Korea) in 10% TBS-T for 1 h at room temperature. Membranes were incubated with primary antibodies against Fibronectin (Abcam, ab-6328), TGF-β1 (Abkin, Wuhan, China, ABP52598), SGK1 (Abcam, ab43606), ENaC Gamma (Biorbyt, Cambridge, UK, orb100662), NHE1 ( Santa Cruz Biotechnologies, CA, USA, sc-136239), TNF-α (Abcam, ab6671), IL-6 (Santa Cruz Biotechnologies), NF-kB (p65, Enzo Life Sciences, Farmingdale, NY, USA ), and pNF-kB (p65, Santa Cruz Biotechnologies) at 4 °C overnight and washed with TBS-T. They were incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then subjected to ECL detection (GE Healthcare, Chicago, USA). GAPDH was detected on the same membrane to serve as a loading control. Densitometric analysis was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).
All data are expressed as mean ± SEM. Statistical analyzes were performed using SPSS v26 (SPSS Inc., Chicago, IL, USA) and point graphs were generated using GraphPad Prism 8.4 (GraphPad Inc., San Diego, CA, USA). When our data follow a normal distribution, parametric tests are used, other than that, nonparametric methods are used to compare groups. s– Values less than 0.05 were considered statistically significant.
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