Male C57BL/6J mice (8 weeks old) were purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences. The mouse atrial muscle HL-1 cell line was purchased from ATCC. A lentiviral plasmid containing HA-Senp1 was constructed by Suzhou Jima gene Co., Ltd. AS-IV was purchased from Sigma-Aldrich; Merck KGaA. Claycomb Medium (51800C), penicillin/streptomycin (V900929) and glutamine (200 mM, G8540) were from Sigma-Aldrich; Merck KGaA. Fetal bovine serum (10099141C) and trypsin (25200072) were purchased from Gibco; Thermo Fisher Scientific, Inc. Antibodies against cleaved-caspase-3 (cat. no. 9661, 1:1,000), caspase-3 (cat. no. 9662, 1:1,000), BCL2 (cat. no. 15071, 1:1,000), Bax (cat. no. 2774, 1:1,000), Senp1 (cat. no. 11929, 1:1,000) and β-actin (cat. no. 3700, 1:1,000) were purchased from Cell Signaling Technology, Inc. Goat anti-mouse IgG (cat. no. 115-035-003) and goat anti-rabbit IgG (cat. no. 111-035-003) were purchased from Jackson Laboratory. Amplex Red reagent (cat. no. A22177) was purchased from Invitrogen; Thermo Fisher Scientific, Inc. Mitochondrial membrane potential detection kit (kit. no. C2006), lactate dehydrogenase (LDH) cytotoxicity test kit (kit. no. C0016), and reactive oxygen species (ROS) detection kit (kit. no. S0033M) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (kit. no. 11684817910) was purchased from Roche Diagnostics GmbH.

Animal experiments were performed according to the regulations and guidelines approved by the Animal Ethics Committee of The Fifth Central Hospital of Tianjin (approval. no. TJWZX2019018). Animal studies adhered to the Guide for the Care and Use of Laboratory Animals (8th edition, 2011, the Institute for Laboratory Animal Research of the National Research Council in the USA). A total of 24 male C57BL/6J mice (8-week-old; mean weight, 20 g) were purchased from the Animal Center of Nanjing University. Animals were housed in the Experimental Animal Center of The Fifth Central Hospital of Tianjin, and maintained under a controlled temperature (22-24˚C), stable humidity (40-60%) and a 12 h-light/dark cycle with ad libitum access to food and water. To establish the HF model, the mice were anesthetized with 2% isoflurane from Zaozhuang Shuitailan Chemical Co. Ltd., and then transferred to the operating table. Following endotracheal intubation, they were connected to a ventilator, and further anesthetized with 1.5% isoflurane. After the mouse chest was disinfected, a longitudinal incision was made in the second rib to open the chest and expose the thoracic segment of the aorta. Finally, a 7-0 thread was inserted below the brachiocephalic trunk of the aorta and the left common carotid artery. A 26 G fine needle was used to ligate the aortic arch, and the needle was removed following ligation. After 6 weeks, echocardiography was used to determine whether the model was successfully established. The mice were randomly divided into control group, HF group, HF+AS-IV group and AS-IV group. The HF+AS-IV group and AS-IV group were intraperitoneally injected with 40 mg/kg AS-IV every other day. The control group and HF group were given the same dose of solvent (dimethyl sulfoxide, 0.1%). After 4 weeks of drug intervention, the mice underwent cardiac ultrasound examination. At the end of the experiments, the mice were euthanized by CO₂ exposure (CO₂ displacement rate equivalent to 20% of the chamber volume/min, October 2019) and cervical dislocation.

HL-1 cardiomyocytes from mouse heart tissue were purchased from ATCC. The cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum, 100 U penicillin/streptomycin and 2 mM glutamine at 37˚C, 5% CO₂, 95% air and 95% humidity. Following vector or HA-Senp1 plasmid transfection for 24 h, the HF group was treated with isoprenaline (ISO, 20 µmol/l) for 24 h, and the HF+AS-IV group was pretreated with 25, 50, 100 or 200 µmol/l AS-IV for 30 min at 37˚C, and then with 20 µmol/l ISO for 24 h at 37˚C. The mitochondrial membrane potential (Δψm) and ROS were examined after 24 h of treatment.

The mitochondria and cytoplasm were separated by a Tissue Mitochondrial Separation kit (Beyotime Institute of Biotechnology) using differential centrifugation, according to the manufacturer's protocols. Briefly, heart tissue blocks of the same weight and from the same region were washed with precooled PBS solution, cut into small pieces and transferred to a precooled glass homogenizer. Mitochondrial isolation solution containing protease inhibitors was added and the tissue was homogenized in an ice bath ~10 times. The supernatant was extracted following centrifugation at 4˚C for 5 min at 600 x g. Subsequently, the supernatant was discarded following centrifugation at 4˚C for 10 min at 11,000 x g. The remaining precipitate comprised the isolated mitochondria.

Isolated mitochondria or HL-1 cells were incubated with JC-1 (10 µg/ml) for 20 min at 37˚C. Mitochondria were examined by a fluorescence reader and HL-1 cells were examined by a laser scanning confocal microscope (Olympus FV 1200; Olympus Corporation; magnification x60). The excitation and emission wavelengths of the green fluorescence of JC-1 monomer were 488 and 525 nm, respectively, and of the red fluorescence of JC-1 aggregate were 543 and 590 nm, respectively. The change in fluorescence intensity of each experimental group is expressed as polymer/monomer. The temperature was maintained at 37˚C.

Isolated myocardial mitochondria or HL-1 cells were incubated with DCFH-DA (10 µM) for 20 min at 37˚C. Mitochondria were examined by a fluorescence reader and HL-1 cells were examined by a laser scanning confocal microscope (Olympus FV 1200; Olympus Corporation; magnification x100). The excitation and emission wavelengths of DCFH-DA were 488 and 525 nm, respectively. The fluorescence intensity of DCFH-DA is expressed as a percentage of the control group.

The Amplex Red H₂O₂ detection kit was used according to the manufacturer's protocols; H₂O₂ in isolated mitochondria or HL-1 cells was detected by a fluorescence plate reader. The excitation and emission wavelengths of H₂O₂ were 543 and 590 nm, respectively. The change in fluorescence intensity in each experimental group is expressed as a percentage of the control group.

Total protein was extracted from cells or tissues using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 1 mM PMSF and 20 mM N-ethylmaleimide. The protein supernatant was collected following centrifugation at 10,000 x g at 4˚C for 15 min. Protein concentration was measured using a BCA protein assay (Beijing Solarbio Science & Technology Co., Ltd.). Equal amounts of protein lysates (40 µg per lane; 2 µg/µl) were separated on SDS-polyacrylamide gel using 10% gels and were transferred onto PVDF membranes. The membranes were then blocked with 5% skimmed milk for 1 h at room temperature. Next, the membranes were incubated overnight with the aforementioned primary antibodies at 4˚C overnight, and then with secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized by enhanced chemiluminescence (EMD Millipore). Densitometric semi-quantification analysis of the Western blot bands was performed using image analysis software (ImageJv1.48; National Institutes of Health). Protein band intensity was normalized to β-actin and expressed as a percentage of the naive control.

To detect apoptosis, a TUNEL kit (Roche Diagnostics GmbH) was used. Following treatment, HL-1 cells were fixed in 4% paraformaldehyde for 40 min at 25˚C, blocked with 3% H₂O₂ for 10 min at 25˚C, and permeated with 0.1% Triton X-100 for 3 min. Apoptotic cells were labeled with the TUNEL reaction mixture and nuclei were stained with DAPI (1 µg/ml) for 3 min at 25˚C. Cells were washed twice with PBS for 5 min at room temperature and mounted with ProLong Gold Antifade reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Images were randomly obtained using a fluorescence microscope (Olympus ix73; Olympus Corporation; dp73 camera; magnification x10).

Eight weeks after the operation, mice were anesthetized with pentobarbital sodium (30 mg/kg) and placed on a warm pad. Vevo 770^® (Visualsonics Inc.) and a 716 probe were used to dynamically evaluate the cardiac function of mice by echocardiography. Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEED) were obtained by M-type tracing. Ejection fraction (EF) and shortening fraction (FS) were automatically obtained by high-resolution echocardiography (ECG).

Myocardial tissue was fixed with 4% paraformaldehyde for 24 h at 4˚C, dehydrated, paraffin embedded and sectioned (4 µm thick). Pathological sections were stained with hematoxylin for 5 min at 4˚C and eosin for 3 min at 4˚C (Nanjing SenBeiJia Biological Technology Co., Ltd.). H&E staining was observed under a light microscope (Olympus Medical Systems; magnification, x20). The hypertrophy of cardiomyocytes was quantified by counting the cross-sectional area of cardiomyocytes using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

Total RNA was extracted from the HL-1 cells using a TRIzol^® RNA extraction kit (Tiangen Biotech Co., Ltd.). The RNA extracted from HL-1 cells was used to obtain the full-length cDNA of mouse Senp1 by RT-PCR using a RT-PCR kit according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). The full-length Senp1 cDNA (Accessions; NM_001379573.1; Insert size: 2019 bp) was subcloned into mammalian Lentivirus Expression Vector pwpxl (Thermo Fisher Scientific, Inc.) with a HA tag at the C-terminus (HA-SENP1), and the empty plasmid, pwpxl, was used as the negative control group (vector). The empty vector and the vectors encoding the SENP1 (HA-SENP1) were transiently transfected into HL-1 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols (DNA=4 µg, reagent=10 µl). In brief, cells were seeded into a 6-well plate at 37˚C, 5% CO₂ and 95% humidity. A total of 18-24 h after seeding, the density of the HL-1 cells reached 40-50%, prior to being transfected with empty vector or HA-SENP1 using Lipofectamine 2000 transfection reagent (DNA=4 µg, reagent=10 µl). Cells were then inoculated into fresh medium 24 h after the transfection. All subsequent experiments were performed 48 h after transfection.

Cell viability was examined using a Cell Counting Kit-8 (CCK-8; Beijing Solarbio Science & Technology Co., Ltd.) assay. Hl-1 cells were cultured in 96-well plates (2x10³ cells/well) and treated as described in ‘cell culture’. Subsequently, 10 µl CCK-8 solution was added to each well and incubated at 37˚C for 2 h. A microplate reader (VersaMax™ Microplate Reader; Molecular Devices, LLC) was used to analyze the absorbance at 450 nm. To increase the reliability of measures, each experiment was performed three times.

All HL-1 cell culture dishes and mice were randomly assigned to different experimental groups. Data are presented as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, Inc.). The differences between the experimental and control groups were tested using unpaired t-test. For comparing differences among more than two experimental groups, the means were compared using one-way analysis of variance (ANOVA), followed by Tukey's test, or two-way ANOVA, followed by Bonferroni's test. Normal distribution was assessed with the Shapiro-Wilk test. P<0.05 was considered to indicate a statistically significant difference.

Compared with the control group, the heart weight and the heart mass/body weight ratio were increased in the HF group (P<0.05); however, AS-IV reversed this increase induced by pressure overload-driven heart failure (P<0.05; Fig. 1). The ultrasound results demonstrated that, compared with the control group, the EF, FS, LVID;d and LVID;s were increased in the HF group (P<0.05; Fig. 1A and B), suggesting that the heart function of the HF group was impaired. However, AS-IV improved the heart function.

H&E staining demonstrated that, compared with the control group, myocardial cells in the HF group exhibited hypertrophy, disordered arrangement, increased intercellular stroma and inflammatory infiltration. However, AS-IV reversed the morphological changes of the myocardial tissue induced by TAC (Fig. 2). Furthermore, compared with the control group, the ROS levels in myocardial mitochondria of the HF group were increased, while AS-IV decreased the ROS content in HF myocardial cells (Fig. 3A). The Amplex Red results demonstrated that, compared with the control group, the H₂O₂ content in myocardial mitochondria of the HF group was increased, which was abrogated by AS-IV (Fig. 3B). The JC-1 results demonstrated that, compared with the control group, the Δψm of the HF group was decreased, which was also reversed by AS-IV (Fig. 3C).

Western blotting demonstrated that, compared with the control group, the cleaved-caspase-3/caspase-3 ratio was increased, but the Bcl2/Bax ratio was decreased in the HF group, indicating that apoptosis of cardiomyocytes in the HF group was increased. This effect was abolished by AS-IV (Fig. 4A and B). Additionally, compared with the control group, the expression of Senp1 was increased in the HF model, while AS-IV decreased this expression (Fig. 4A and B), indicating that Senp1 may participate in the myocardial protection of AS-IV.

To further investigate the effect of Senp1-overexpression on oxidative stress in HL-1 cells, the present study examined the effect of different concentrations of AS-IV on the survival of HL-1 cells subjected to ISO, which induces myocardial injury. The CCK-8 results demonstrated that HL-1 cells treated with ISO had significantly decreased cell viability. AS-IV at 50 and 100 mmol/l significantly improved the cell viability, but the protective effect of 25 and 200 mmol/l was not notable (Fig. 5A). Next, the HA-Senp1 plasmid was transfected into HL-1 cells. In the subsequent experiments, 50 mmol/l AS-IV was used as the intervention concentration. Western blotting demonstrated that, compared with the vector group, the expression of Senp1 was increased in the HA-Senp1 group (Fig. 5B). Compared with the HF group, AS-IV decreased the increase in intracellular H₂O₂, while the Senp1-overexpression in HL-1 cells inhibited the effect of AS-IV (Fig. 5C). Furthermore, confocal detection of DCF demonstrated that AS-IV decreased the increase in ROS compared with the HF group, while Senp1-overexpression in HL-1 cells abolished the effect of AS-IV (Fig. 6).

Mitochondria are associated with apoptosis, and the decline in Δψm is an important indicator of early apoptosis. Confocal detection of JC-1 demonstrated that, compared with the HF group, AS-IV inhibited the decrease in Δψm, while Senp1-overexpression in HL-1 cells abrogated the effect of AS-IV (Fig. 7). The TUNEL assay also demonstrated that, compared with the HF group, AS-IV decreased the apoptotic rate, while Senp1-overexpression inhibited the protective effect of AS-IV (Fig. 8).