The H9c2 cell line consists of subclonal cells obtained from the heart tissue of embryonic rats. H9c2 cells were provided by the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. H9c2 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 5.5 mM D-glucose, 100 U/ml penicillin, 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 mg/ml streptomycin at 37°C in a humidified 5% CO₂ incubator. Prior to experimentation, H9c2 cells were pretreated with 20 and 50 µM OSS-128167 (Selleck Chemicals) for 1 h, followed by stimulation with high glucose (HG; 33 mM) for various durations (both steps were conducted at 37°C). For analysis of transforming growth factor (TGF)-β, collagen (COL)-1, Bax, Bcl-2 and cleaved-poly ADP-ribose polymerase (PARP) protein, cells were stimulated with HG for 36 h; for RT-qPCR analysis of TGF-β, COL-1, Bax, Bcl-2 and cleaved-PARP mRNA, cells were stimulated with HG for 12 h; for western blotting of tumor necrosis factor (TNF)-α and 3-nitrotyrosine (NT), cells were stimulated with HG for 24 h; for RT-qPCR analysis of TNF-α, cells were stimulated with HG for 8 h; and to detect malondialdehyde (MDA) levels, cells were stimulated with HG for 4 h.

Selective SIRT6 antagonist OSS-128167 was dissolved in dimethyl sulfoxide (DMSO) to obtain 50 mM stock solution, which was diluted in DMSO for use in further experiments. The chemical structure of OSS_128167 is shown in Fig. 1C. DMSO had a final concentration of 0.1% (v/v). All other chemical biological agents were purchased from Sigma-Aldrich; Merck KGaA. Antibodies for SIRT6, TNF-α, COL-1, TGF-β, PARP, 3-NT and GAPDH were purchased from Abcam.

Animals were provided by the Animal Center of Wenzhou Medical University. The present study was approved by the Wenzhou Medical University Animal Policy and Welfare Committee (Wenzhou, China), and experiments were conducted according to the National Institutes of Health (NIH) guidelines regarding the care and use of laboratory animals. A total of 40 male C57BL/6 mice (weight, 22–26 g; age, 10 weeks; Riken BioResource Center of Japan) were housed in an environment with room temperature maintained at 22±2.0°C and humidity at 50±5%. In addition, they were kept under a 12-h light/dark cycle, and were fed with a standard rodent diet and water. After 1 week of acclimation, mice were intraperitoneally injected with 50 mg/kg STZ (Sigma-Aldrich; Merck KGaA) formulated in citrate buffer (100 mM, pH 4.5) for 5 consecutive days. A glucometer was used to detect the levels of fasting blood-glucose (FBG). OSS-128167 intervention was commenced once it was confirmed that type 1 diabetes mellitus (FBG >12 mmol/l) was successfully established. Mice with STZ-induced diabetes (STZ-DM1) were orally administered OSS-128167 (20 or 50 mg/kg) or vehicle (1% CMC-Na) through gavage every other day (n=10/group). As shown in Fig. S1A, the body weight and blood glucose were recorded at certain time points. A total of 16 weeks after administration, the mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight; Ketanest; Pfizer) and xylazine hydrochloride (16 mg/kg body weight; Rompun 2%; Bayer). Blood was collected from the retro-orbital vein after the mice were anesthetized; 500 µl-1 ml blood was collected. Subsequently, the mice were euthanized by cervical dislocation, the thoracic cavity was opened, and the heart was removed after perfusion with normal saline. The heart tissues were embedded in paraffin, and maintained at 65°C for 1.5 h. Paraffin samples were subsequently stored at 4°C, while other heart tissues were placed in liquid nitrogen for snap-freezing before further analysis. The collected blood samples were centrifuged for 5 min (1,500 × g, 4°C) to collect the serum.

Fixed heart sections (5-µm) described above were incubated with 3% H₂O₂ for 30 min and blocked for 30 min with 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA), both at room temperature in PBS. Slides were then incubated overnight with a primary antibody against TNF-α (1:500; Santa Cruz Biotechnology, Inc.; cat. no. sc-52746) at 4°C, followed by incubation with a secondary antibody (1:200; Santa Cruz Biotechnology, Inc.; cat. no. sc-516102) for 1 h and with DAB (A:B, 1:20) for 5 min at room temperature. Hematoxylin was used to stain the cell nuclei for 3 min at room temperature and resin was used to seal the dehydrated sections. A light microscope (magnification, ×400; Nikon Corporation) was used to capture the images.

TUNEL staining was performed using the TUNEL Apoptosis Detection kit (R&D Systems, Inc.), according to the manufacturer's protocol. Heart tissue sections were counterstained with DAPI (Beyotime Institute of Biotechnology) at room temperature for 5 min. Images were captured using the Leica A1 laser confocal microscope (Leica Microsystems GmbH). The number of TUNEL-positive cells was counted using ImageJ 1.52a software (NIH) in three randomly selected fields per sample.

Heart tissues were fixed in 4% paraformaldehyde at room temperature for 24 h, embedded in paraffin and sectioned into 5-µm thick samples. The tissue sections were incubated at 60–70°C for 4–6 h, then deparaffinized in xylene at room temperature for 10 min and another subsequent 15 min to complete deparaffinization. The sections were then rehydrated in a descending series of ethanol, prior to being immersed in PBS trice at room temperature for 5 min each and finally washed in distilled water at room temperature for 20 min for subsequent staining. H&E and Masson's trichrome staining were used to evaluate the intima-media thickness and fibrosis content of the tissues.

For H&E staining (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. G1120), the sections were stained with eosin for 2 min, wash in distilled water for 20 min and stained with hematoxylin for 5 min (all at room temperature). Subsequently, sections were washed in distilled water for 5 min thrice, dehydrated using an ascending ethanol series and the slides were then mounted with neutral gum.

For Masson's trichrome staining (Beijing Solarbio Science & Technology Co., Ltd.; cat. no. G1340), the sections were stained with ponceau staining solution dye for 5 min, washed with distilled water for 1 min twice and incubated with 1% phosphotungstic acid solution for 5 min. Then, the sections were incubated with aniline blue dye solution for 5 min, treated with 1% glacial acetic acid water for 1 min, dehydrated using an ascending ethanol series and then stored at room temperature. The entire staining process was performed at room temperature. A light microscope (magnification, ×400; Nikon Corporation) was used to observe the stained sections.

Dihydroethidium (DHE) staining was used to evaluate superoxide production. Briefly, the isolated mouse heart tissues were embedded in OCT compound (Sakura Finetek USA, Inc.) immediately after the heart tissues were excised from the mice, after which they were split into frozen 5-µm sections. The sections were then incubated in DHE in PBS (10 mmol/l) for 45 min in a dark and moist container at 37°C. DHE was oxidized upon the reaction with superoxide to ethidium bromide, which binds to DNA in the nucleus and fluoresces red (23). DHE dilutions were prepared as 1:5,000 to treat the sections, and images were captured using the Leica A1 laser confocal microscope (Leica Microsystems GmbH).

An MDA kit (Beyotime Institute of Biotechnology; cat. no. S0131S) was used to determine the levels of MDA in cells and tissues, according to the manufacturer's protocol.

PBS and 0.025% trypsin (T6325-25 g; Macklin Inc.) were used to wash and digest H9c2 cells, respectively. The cells then underwent centrifugation at 600 × g for 5 min at 4°C. The supernatant was discarded, and PBS was used to wash the precipitate twice. Lysis buffer was then added at a ratio of 100 µl lysate/2 million cells according to the kit's instructions (cat. no. C1115; Beyotime Institute of Biotechnology). The mixture then underwent centrifugation at 20,000 × g for 15 min at 4°C, after 15 min of lyophilization in an ice bath. Centrifuged supernatant (50 µl) and Ac-DEVD-pNA (10 µl) were mixed in a 96-well plate (without generating air bubbles while mixing). Finally, the 96-well plate was incubated for 90 min at 37°C, after which a spectrophotometer was used to measure the optical density value at an absorbance of 405 nm.

According to a standard protocol, H9c2 cells were homogenized in TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) to extract RNA. The two-step M-MLV Platinum SYBR Green qPCR SuperMix- UDG kit (Thermo Fisher Scientific, Inc.) and Eppendorf Mastercycler eprealplex detection system (Eppendorf) were used to conduct RT and qPCR. Reverse transcription was performed at 37°C for 15 min and 85°C for 5 sec, and then maintained at 4°C. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 3 min; followed by 50 cycles at 95°C for 15 sec and 60°C for 30 sec, and a melting curve stage at 95°C for 15 sec and 60°C for 1 min. The primers were provided by Thermo Fisher Scientific, Inc. and primer sequences are presented in Table SI. The Tm values were normalized to β-actin. The 2^−∆∆Cq method was used to quantify the expression levels (24).

Cells were collected and underwent lysis using a buffer (cat. no. AR0103-100; Boster Biological Technology). Protein was quantified using a BCA assay and protein lysates (40 µg) were then separated by 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes. The membranes then underwent 1 h of blocking in TBS (pH 7.6) containing 5% non-fat milk and 0.05% Tween 20 at room temperature. The membranes were incubated with primary antibodies overnight at 4°C and with the secondary antibodies at room temperature for 2 h. TGF-β (1:500; cat. no. sc-146), B-cell lymphoma-2 (Bcl-2; 1:500; cat. no. sc-7382), NF-κB P65 (1:500; cat. no. sc-7151), cleaved (cle)-PARP (1:500; cat. no. sc-56196) and Bcl-2-associated X (Bax; 1:500; cat. no. sc-7480) antibodies were purchased from Santa Cruz Biotechnology, Inc. SIRT6 (1:1,000; cat. no. ab191385), COL-1 (1:1,000; cat. no. ab34710), TNF-α (1:1,000; cat. no. ab6671), 3-NT (1:1,000; cat. no. ab61392) and GAPDH (1:1,000; cat. no. ab8245) antibodies were provided by Abcam. The primary and secondary antibodies (1:1,000; Cell Signaling Technology, Inc.; cat. nos. 7076 and 7074) were diluted in TBS-0.05% Tween-20 prior to use. The enhanced chemiluminescence kit (Bio-Rad Laboratories, Inc.) was used to detect the specific bands. Densitometric analysis was performed using Image Lab 5.1 software (Bio-Rad Laboratories, Inc.).

Experiments were repeated three times and data were expressed as the mean ± SEM. GraphPad Pro Prism 6.0 (GraphPad Software, Inc.) was used to conduct statistical analyses. Student's t-test was used to analyze the differences between two datasets, while a one-way ANOVA and Bonferroni correction were used to assess multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Type 1 diabetes in male C57BL/6 mice was induced by successively injecting a low dose of STZ intraperitoneally. In the STZ-mediated mouse model of type 1 diabetic cardiomyopathy (STZ-DM1), the protein expression levels of SIRT6 were significantly lower in myocardial tissues of STZ-DM1 mice compared with those in the non-diabetic controls (Fig. 1A and B). Subsequently, the mouse model of type 1 diabetic cardiomyopathy was employed to determine whether OSS-128167 (Fig. 1C) aggravated diabetes-induced cardiac damage. Diabetic mice were treated with OSS-128167 (20 and 50 mg/kg) for 2 months, and the effects of OSS-128167 on diabetic cardiac morphology were examined. As shown using H&E and Masson's trichrome staining, diabetic hearts exhibited an abnormal structure compared with the hearts of normal mice. Compared with the vehicle group, H&E staining showed myocardial fibrosis in the STZ-DM1 group was disordered, while Masson's trichrome staining showed increased myocardial fibrosis in the STZ-DM1 group. Notably, these structural abnormalities were aggravated by treatment of diabetic mice with OSS-128167 (Fig. 1D and E). Finally, TUNEL staining was used to detect myocardial tissue apoptosis. As shown in Fig. 1F, OSS-128167 treatment markedly increased cell apoptosis, as indicated by an increase in the TUNEL-positive staining area. Notably, it was revealed that such adverse effects imposed by OSS-128167 on cardiac morphology were not caused by metabolic changes. OSS-128167 treatment had no impact on FBG and serum insulin levels (Fig. S1B and C). Moreover, compared with that in the vehicle group, heart weight/body weight (HW/BW) was significantly increased in the STZ-DM1 and OSS-128167 treatment groups (Fig. S1D). However, no significant difference in HW/BW was apparent in the OSS-128167 treatment group compared with in the STZ-DM1 group. These results indicated that OSS-128167 exacerbated cardiac structural destruction, and myocardial fibrosis and apoptosis in type 1 diabetic mice.

In order to determine the potential molecular mechanisms underlying the deleterious effect exerted by OSS-128167 in vivo, the expression levels of inflammatory mediators and the levels of oxidative stress in the myocardium were detected in response to OSS-128167. Notably, the mRNA and protein expression levels of the inflammatory factor TNF-α were significantly increased in the STZ-DM1 group compared with those detected in the vehicle group; a further increase in these levels was also detected in the OSS-128167 treatment group (Fig. 2A and C). The biological effects of OSS-128167 on oxidative stress were further detected in the hearts of each group. DHE and MDA kits were used to detect oxidative stress in the myocardium; higher levels of superoxide and MDA were detected in the hearts of mice in the OSS-128167 group compared with those in the STZ-DM1 group (Fig. 2B and D). These results indicated that OSS-128167 aggravated myocardial injury in DCM by exacerbating hyperglycemia-mediated inflammation and oxidative stress.

The results of the present study were verified by observing cultured cardiomyocytes in vitro and the potential molecular mechanisms were explored. Briefly, H9c2 cells were cultured and treated with HG (33 mM) to simulate hyperglycemia. Following treatment, immunoblotting was conducted to assess the expression levels of biomarkers for cell fibrosis (Fig. 3A). OSS-128167 significantly increased the expression levels of pro-fibrotic markers COL-1 and TGF-β compared with in the HG group (Fig. 3A). In addition, OSS-128167 induced a dose-dependent increase in the mRNA expression levels of COL-1 and TGF-β, as well as CTGF (Fig. 3B-D). The present in vivo studies revealed that OSS-128167 increased apoptosis in the heart tissues of mice. Consequently, the effects of OSS-128167 on HG-induced apoptosis in H9c2 cells were examined. Under a HG challenge, the expression levels of the apoptotic biomarker Bax were markedly upregulated by OSS-128167, whereas the opposite was observed for Bcl-2 (Fig. 3E). Treatment with OSS-128167 also significantly reduced the protein expression ratio of Bcl-2/Bax compared with that in the HG group (Fig. 3F). As shown in Fig. 3G, OSS-128167 readily increased the protein expression levels of cle-PARP, which were initially induced by HG. Moreover, caspase-3 activity was increased in the OSS-128167 group compared with that in the HG group (Fig. 3H). These results revealed that OSS-128167 could significantly increase myocardial fibrosis and the expression of apoptotic proteins induced by hyperglycemia in vivo.

The protein and mRNA expression levels of the inflammatory cytokine, TNF-α were measured to evaluate the pro-inflammatory biological function of OSS-128167. OSS-128167 was revealed to significantly aggravate HG-induced increases in TNF-α expression levels (Fig. 4A and B). These findings were in accordance with the results of the in vivo studies and suggested the negative impact of OSS-128167 on cardiomyocytes through the aggravation of inflammation. Oxidative stress also serves as an important pathological mechanism of DCM. HG-stimulated H9c2 cells underwent immunoblotting (Fig. 4C), and the expression levels of 3-NT, a biomarker of the nitrogen free radical species, exhibited markedly increased accumulation in H9c2 cells treated with HG which was more prominent in the OSS-128167-treated groups. Furthermore, MDA levels were measured in H9c2 cells. Polyunsaturated fatty acids produce MDA via lipid peroxidation; in the present study, OSS-128167 aggravated the HG-induced increase in MDA (Fig. 4D). These results demonstrated that OSS-128167 aggravated HG-induced cell inflammatory responses and oxidative stress in H9c2 cells.