For Western blotting and immunohistochemistry, anti-OGT antibody (sc-32921), anti-BNP antibody (sc-67455), and anti-NFATc3 antibody (sc-8405) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-O-GlcNAc antibody (MA1-072) was purchased from Affinity Thermo Scientific (Waltham, MA, USA). Anti-ANP (ab-91250) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-phospho-GSK-3β, anti-GSK-3β, anti-phospho-NF-κB, anti-NF-κB, anti-phospho-Smad3, anti-Smad3, and anti-NFAT antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Collagen III antibody (BS1531) was purchased from Bioworld Technology (Bloomington, MN, USA). For cell culture and in vivo experiments, GSK-3β inhibitor, TDZD-8, was purchased from Tokyo Chemical Industry (Tokyo, Japan). Thiamet G (TMG) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Angiotensin II (Ang II) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

All animal experiments were conducted under the guidelines for the care and use of animals approved by Osaka Medical and Pharmaceutical University (protocol #2020-087). We used WT and Ogt-Tg mice (C57BL/6J, male) that expresses Ogt under the control of the CAG promoter (31). The mice (10-12 week-old) were anesthetized with 2,2,2-tribromoethanol. TAC surgery was performed under a dissecting microscope, with a small animal respirator, at a rate of 110 cycles/min. Aortic constriction was performed by tying a 7-0 silk string ligature around a 26-gauge needle, and then removing the needle. Since it was difficult for Ogt-Tg mice to survive for 4 weeks under the pressure overload by normal TAC surgery, we reduced the severity of the TAC surgery in this study. A sham surgery was performed following the same surgical procedure without tying the silk suture for the control group. Echocardiography (Nemio30; Toshiba Medical Systems, Japan) was performed without anesthetics before surgery, and 4 weeks after surgery, with the following parameters: LV ejection fraction (EF), LV fractional shortening (FS), end-diastolic LV internal dimension (LVIDd), end-systolic LV internal dimention (LVIDs), and then the heart was excised from the mice after being euthanized for Western blot and histological analyses.

Hearts excised from mice 4 weeks after the TAC or sham surgery were homogenized in lysis buffer (50 mM HEPES (pH 7.4), 5 mM sodium pyrophosphate, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride) containing a proteasome inhibitor cocktail (WAKO Pure Chemical Industries, Osaka, Japan). Homogenates were centrifuged at 4°C for 10 min at 10,000 rpm. Protein concentration was measured by the bicinchoninic acid assay method according to the manufacturer’s instructions. Supernatants were mixed with sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min. The boiled samples were cooled at room temperature (22–28°C) and subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred to a PVDF membrane (MERK Millipore, Burlington, MA, USA). The membrane was incubated in tris-buffered saline (TBST) containing 5% skim milk at room temperature for 1 h. Subsequently, the membrane was incubated with primary antibody in TBST containing 5% skim milk at 4°C overnight. The membrane was washed in TBST for 10 min three times and then incubated with secondary antibody in TBST containing 5% skim milk for 1 h. The membrane was washed in TBST for 10 min three times and detection was performed using Luminata Crescendo Western HRP (MERK Millipore) and Fusion FX7 (Vilber-Luormat, Germany).

Co-immunoprecipitation was performed using Sure Beads Protein G Magnetic Beads (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The pull-downed eluates were used for Western blot analysis with the antibodies of interest.

For histological analysis, hearts were arrested in diastole, fixed with 4% paraformaldehyde, embedded in paraffin. Paraffin-embedded sections were stained with Masson’s trichrome for the detection of collagen fibers.

One week after the TAC or sham surgery, mice were injected intraperitoneally daily with a GSK-3β inhibitor, TDZD-8 (10 mg/kg/day, i.p.), dissolved in dimethylsulfoxide (DMSO): phosphate-buffered saline (PBS); 1:10 for 3 weeks. When daily intraperitoneal injection ended, echocardiography was performed, then the hearts were excised from the mice for Western blot analysis, Masson’s trichrome staining, and immunofluorescence staining.

Rat cardiomyoblast cells (H9c2 cells) were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. After 48 h incubation with serum-free medium, the cells were treated with PBS (solvent control), 100 nM Ang II, or Ang II plus 20 μM TDZD-8 for 48 h. The cells were further treated with or without 5 μM TMG for 2 h.

The paraffin-embedded LV sections and H9c2 cells were fixed with 4% paraformaldehyde for 10 min, followed by blocking and permeabilization with 10% bovine serum albumin and 0.1% Triton-X100 for 15 min. The LV sections and H9c2 cells were then incubated with primary antibody against NFATc3 (1:200) at room temperature overnight. After washing three times with PBS, they were incubated with secondary Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:200) at room temperature for 1 h. After washing 3 times with PBS, they were mounted using Vectashield mounting medium (Vector Laboratories) with 4’ 6-diamidino-2-phenylindole (DAPI) and observed under a confocal laser microscope (SP8, Leica, Germany).

Differences between more than two groups were analyzed using a two-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test. The significant differences between two groups were evaluated by the F-test followed by the Student’s t-test.

To investigate the effect of OGT overexpression on pressure overload-induced cardiac hypertrophy or failure, TAC or sham surgery was performed in WT and Ogt-Tg mice. After TAC4W, O-GlcNAcylation was significantly increased in the hearts of Ogt-Tg mice, although the increase was not significant in those of WT mice, probably due to the less severity of the TAC surgery than normal ( Figures 1A, C ). There were no differences in the expression level of OGT in WT and Ogt-Tg mice after TAC4W ( Figures 1A, B ). To clarify the discrepancy between the rate of changes of O-GlcNAcylation and OGT expression levels in Ogt-Tg mice after TAC4W, we examined the expression level of glutamine-fructose-6-phosphate transaminase (GFAT) that generates UDP-N-acetylglucosamine (UDP-GlcNAc), the substrate for O-GlcNAcylation; the expression level of GFAT1 increased drastically in Ogt-Tg mice after TAC4W ( Figures 1A, D ), whereas GFAT2 expression was not significantly changed ( Figures 1A, E ). These data suggest that increased O-GlcNAcylation in the hearts of Ogt-Tg mice after TAC4W may be due to increased expression level of GFAT1, not GFAT2.

Heart weight/body weight ratio was significantly increased in Ogt-Tg mice after TAC4W compared to sham group, whereas the increase was not significant in WT mice ( Figures 2A, B ). The echocardiography showed that EF and FS were significantly decreased and LVIDd was significantly increased in WT and Ogt-Tg mice after TAC4W. LVIDs was significantly increased in Ogt-Tg mice after TAC4W, but not in WT mice. The decrease rates in EF and FS and increase rates in LVIDd and LVIDs in Ogt-Tg mice after TAC4W were significantly higher compared to those in WT mice ( Figures 2C–F ). Myocardial ANP, a biochemical marker for left ventricular dysfunction, was significantly increased in Ogt-Tg mice after TAC4W ( Figures 3A, B ).

To investigate the molecular mechanisms underlying TAC-induced cardiac dysfunction in Ogt-Tg mice, we examined the GSK-3β and NF-κB signaling pathways that are involved in cardiac hypertrophy (21) (32). We found that the phosphorylation of GSK-3β, which reflects inactivation of the pathway, in the heart tissues of Ogt-Tg mice was significantly lower than those of WT mice after TAC4W ( Figures 3A, C ). In contrast, the phosphorylation of NF-κB, which reflects its activation of the pathway, was not significantly changed ( Figures 3A, D ). Collectively, the GSK-3β signaling pathway, but not NF-κB, may be involved in cardiac hypertrophy after TAC4W that induces cardiac dysfunction.

Four weeks after the TAC (TAC4W) or sham surgery in WT and Ogt-Tg mice, morphological changes in the hearts from both mice groups was evaluated. The macroscopic images of Masson’s trichrome staining showed that the myocardium in Ogt-Tg mice did not show hypertrophy, whereas the LV dimension was enlarged compared to WT mice ( Figures 4A, B ). We found that TDZD-8 improved the TAC-induced hypertrophy in Ogt-Tg mice, indicating that the GSK-3β signaling pathway was involved in this morphology ( Figure 4B ). Moreover, increased LV wall thickness observed in WT mice after TAC4W was restored with TDZD-8 treatment ( Figures 4A, E ). In microscopic images, cardiac fibrosis was significantly increased in Ogt-Tg mice after TAC4W compared to sham mice, and TDZD-8 reduced the cardiac fibrosis in Ogt-Tg mice ( Figures 4D, F ), whereas it was not significantly changed in WT mice after TAC4W compared to sham mice, probably due to the less severity of the TAC surgery than normal ( Figures 4C, F ).

The echocardiography showed that EF and FS were decreased and LVIDd and LVIDs were increased in WT and Ogt-Tg mice after TAC4W, and the changes of cardiac parameters were all restored by the treatment of TDZD-8 ( Figures 5A–D ).

Since cardiac fibrosis was significantly increased in Ogt-Tg mice after TAC4W and the increase was restored with the treatment of TDZD-8 ( Figure 4 ), we examined the effect of the TAC surgery and TDZD-8 treatment on the Smad signaling pathway related to fibrosis and the expression of collagen III that is the main constituent of the interstitial matrix to form fibrosis. The result showed that the phosphorylation level of Smad2 and expression level of Collagen III were both increased in Ogt-Tg mice after TAC4W, and the increase was restored with the treatment of TDZD-8 ( Figures 6A, F, G ). The TAC surgery significantly reduced GSK-3β phosphorylation in the hearts of Ogt-Tg mice, and the reduced phosphorylation was restored by the treatment of TDZD-8 ( Figures 6A, E ). Given that total O-GlcNAcylation level was increased in the Ogt-Tg mice and the level was synergistically increased after TAC4W ( Figures 1A, C ), we examined whether O-GlcNAcylation was involved in the reduction of the GSK-3β signaling pathway. Co-immunoprecipitation study showed that the levels of O-GlcNAcylated GSK-3β in the heart tissues of Ogt-Tg mice was significantly increased after TAC4W, which was restored by the treatment of TDZD-8 ( Figures 6H, I ). O-GlcNAcylation generally competes with phosphorylation of target proteins (11, 33); therefore, these results imply that increased O-GlcNAcylation of GSK-3β in Ogt-Tg mice after TAC4W may result in the reduction of phosphorylated GSK-3β and the restoration of the GSK-3β phosphorylation via reduced GSK-3β O-GlcNAcylation by the treatment of TDZD-8. O-GlcNAcylation ( Figures 6A, C ) and myocardial ANP ( Figures 6A, D ) were significantly increased in Ogt-Tg mice after TAC4W, and the increase was restored with the treatment of TDZD-8. There were no differences in the expression level of OGT in WT and Ogt-Tg mice after TAC4W with or without the treatment of TDZD-8 ( Figures 6A, B ).

NFAT is a master transcription factor that regulates genes involved in cardiac hypertrophy. Phosphorylated NFAT by GSK-3β is retained in the cytoplasm and cannot induce cardiac hypertrophy, indicating that the phosphorylation state of GSK-3β is important for the NFAT signaling pathway (16). To examine whether TMG treatment, which increases O-GlcNAcylation, affects subcellular localization of NFAT, we employed an Ang II-induced cardiomyocyte hypertrophic model with H9c2 cells. As shown in Figures 7A–D , nuclear translocation of NFAT was observed with Ang II stimulation in dimethyl sulfoxide (DMSO) (solvent control)-treated H9c2 cells, whereas it was not observed in TMG-treated H9c2 cells. The translocation was accelerated by the treatment of TDZD-8 in DMSO-treated H9c2 cells ( Figures 7A, C ), whereas it was not changed by the treatment of TDZD-8 in TMG-treated H9c2 cells ( Figures 7B, D ), which is consistent with the data showing that the treatment of TDZD-8 restored cardiac function and cardiac hypertrophy in Ogt-Tg mice after TAC4W ( Figures 4 , 5 ). NFAT is one of the major promotors of cardiac hypertrophy; therefore, the cell size was measured in Ang II-stimulated H9c2 cells in the presence or absence of TMG or TDZD-8. The cell size in H9c2 cells was increased with Ang II stimulation regardless of TDZD-8 addition, whereas TMG treatment minimized the effect, which was associated with nuclear translocation of NFAT ( Figures 7E, F ). Collectively, the activation of GSK-3β by O-GlcNAcylation prevents pressure overload and Ang II-induced cardiac hypertrophy by inhibiting the NFAT signaling pathway.