The present study was approved (approval no. 20160512C10) by the Ethics Committee of Shenzhen Nanshan People's Hospital (Shenzhen, China). A total of 20 male Sprague Dawley (SD) rats (10 weeks old; 320-340 g body weight) were purchased from the Animal Experiment Center of Tongji University (Shanghai, China). Myocardial ischemia was established as previously described (22). Briefly, SD rats were anaesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight) and ventilated with oxygen using a small animal ventilator. After an incision in the left thorax at the level between the fourth and fifth ribs, the heart was exposed, and a 6-0 silk suture slipknot was placed around the proximal left anterior descending coronary artery (LAD) approximately 2 mm below the left auricle. Successful myocardial infarction injury was identified by the blanched appearance of the ligation region and marked arrhythmia. Sham-operated rats underwent the same surgical procedures except for the suture around the LAD which was not ligated. Experimental rats were randomly divided into 2 groups. Experimental rats were subjected to intragastric oral administration (p.o.) Tan-IIA (10 mg/kg; n=10) or PBS (n=10). The treatment continued 10 times twice a day for a total 20-day therapeutic period. All the rats in the study were housed in an environment at 23±1.0°C and 50±5% humidity with 12-h light/dark circadian cycle and ad libitum access to food and water. The mice were caged for 24 h since the last injection and the myocardial tissues were collected after cardiac perfusion. No rats succumbed during the experiments. On day 21, experimental animals were euthanized under intravenous injection of pentobarbital (40 mg/kg), and efforts were made to minimize the suffering of the rats. Cervical dislocation was used as the euthanasia method. The myocardial tissues were used for immunohistochemical analysis.

Experimental rats were anesthetized with isoflurane and following procedures were processed according to a previous study (23). The parameters of left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVES) were evaluated using the Sequoia 512 echocardiography system (Siemens Healthineers) following the manufacturer's instructions. The size of the myocardial infarction was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) and Evan blue double staining assay as previously described (24). The infarct size in tissue sections was assessed by computerized planimetry and quantitated using ImageJ v2.0 (National Institutes of Health).

On day 21, myocardial tissues were obtained from experimental rats, immediately excised and placed in a 4% paraformaldehyde solution overnight at 4°C, followed by dehydration, washing with PBS, and paraffin embedding. Paraffin-embedded myocardial tissues were cut into 4-µm sections, subjected to hydrogen peroxide (3%) for 10 min, and blocked with BSA (5%) for 2 h at 37°C. Myocardial tissue sections were incubated with rabbit anti-rat antibodies Bcl-2 (1:1,000; product code ab182858), Bcl-xL (1:1,000; product code ab32370), caspase-3 (1:1,000; product code ab184787), cytoplasmic cytochrome c (Cyto c; 1:1,000; product code ab133504), Apaf-1 (1:1,000; product code ab234436; all from Abcam) overnight at 4°C. All sections were washed 3 times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2,000; product code ab205718; Abcam) for 1 h at 37°C. The myocardial sections were stained with a 3,3-diaminobenzidine substrate system (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Images were captured using a light microscope (BX51; Olympus Corporation) under a magnification of ×100.

Tan-IIA (purity >99.2%) was purchased from Herbasin (Shenyang) Co., Ltd.; Dasherb Corp. and dissolved in dimethyl sulfoxide (DMSO). Myocardiocytes were isolated from experimental rats with myocardial ischemia as previously described (25). Briefly, after dissection, heart tissues were washed, rinsed with HEPES-buffered saline solution and then incubated at 37°C for 2 h with HEPES-buffered saline solution containing 1.2 mg/ml pancreatin and 0.14 mg/ml collagenase (Gibco; Thermo Fisher Scientific, Inc.). After centrifugation (2,000 × g for 10 min at 4°C), cells were resuspended in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 5% fetal bovine serum (FBS; Sigma-Aldrich; Merck KGaA), 0.1 mM ascorbate, insulin-transferring-sodium selenite media supplement (Sigma-Aldrich; Merck KGaA), 100 µg/ml streptomycin, 100 U/ml penicillin and 0.1 mM bromodeoxyuridine. Cells were then diluted to 1×10⁵ cells/ml and cultured in DMEM/F12 supplemented with 10% FBS. Myocardiocytes were treated with H₂O₂ (1 µM) and/or concentrations of Tan-IIA (0-40 µM) and/or tunicamycin (TM; 1 µM; Sigma-Aldrich; Merck KGaA) for 24, 48 and 72 h at 37°C. PBS buffer containing 10% DMSO (pH 7.2) was used as the control. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

The regulatory effects of Tan-IIA on proliferative ability of myocardiocytes were examined by CCK-8 assay as previously described (26). Briefly, the treated myocardiocytes were seeded into 96-well plates (1×10³ cells/well) and cultured at 37°C in a humidified incubator containing 5% CO₂. Then, 10 µl of CCK-8 reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to the cells followed by incubation at 37°C for 2 h according to the manufacturer's instructions. Cell viability was determined by a microplate reader (Eon BioTech, Pte Ltd.) at 450 nm. Each experiment was repeated for 3 times.

The regulatory effects of caspase-3 overexpression on Tan-IIA-regulated apoptotic factors in myocardiocytes were examined by stable transfection. Expression plasmid pRK5-caspase-3 (casp-3OP) with a Flag tag (cat. no. PPL00180-2a; Public Protein/Plasmid Library) at the C-terminus was constructed by Invitrogen; Thermo Fisher Scientific, Inc. Briefly, myocardiocytes (1×10⁵ cells/ml) were cultured at 37°C in DMEM/F12 (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA) in 6-well plates. After 24 h, myocardiocytes were transfected with plasmid containing either pRK5-caspase-3 (0.5 µg) or pRK5-vector (0.5 µg) by using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) at 37°C for 72 h according to the manufacturer's instructions. After 72 h of transfection, expression of caspase-3 was evaluated using western blot analysis and cells were used for further experiments.

The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (Roche Diagnostics GmbH) was used to detect apoptosis in myocardial tissue and myocardiocytes. Briefly, heart tissues were immediately excised and placed in a 4% paraformaldehyde solution for 12 h at 4°C. This was followed by dehydration, washing with PBS, and paraffin embedding. The paraffin blocks were cut into 4-µm sections and stained with hematoxylin and eosin (H&E; Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at room temperature according to the manufacturer's instructions. For myocardiocytes, the treated cells (1×10⁵ cells) were cultured in 6-well plates for 12 h at 37°C, washed with PBS and fixed with 4% paraformaldehyde for 12 h at 4°C. The cells were washed with PBS and stained using a TUNEL kit for 30 min at 25°C according to the manufacturer's protocols. Finally, the cells were counterstained with 5% DAPI (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature. Images from 6 randomly selected fields of view were captured using a fluorescence microscope at a magnification of ×50 (DMI3000B; Leica Microsystems, Inc.).

The treated myocardiocytes were lysed using RIPA lysis buffer (Sigma-Aldrich; Merck KGaA). The cells were centrifuged at 12,000 × g for 10 min at 4°C. The concentration of protein was determined using a bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). A total of 30 µg of protein/lane was separated using 12% SDS-PAGE, blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) overnight at 4°C, and then transferred to a PVDF (EMD Millipore) membrane. Membranes were incubated with appropriate primary antibodies: Bcl-2 (1:1,000), Bak (1:1,000; product code ab32371; Abcam,), caspase-3 (1:1,000), Cyto c (1:1,000), Apaf-1 (1:1,000), Bim (1:1,000; product code ab32158; Abcam), CHOP (1:1,000; product code ab11419; Abcam) and β-actin (1:1,000; product code ab8227; Abcam) overnight at 4°C, washed with PBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2,000, product code ab7090; Abcam) for 2 h at 25°C. Protein expression was evaluated using enhanced chemiluminescence (product no. CPS1A300; Sigma-Aldrich; Merck KGaA). The expression of protein was quantified using LabWorks™ Image Acquisition (version 4.0; UVP, LLC).

The treated myocardiocytes were cultured in 6-well plates for 12 h at 37°C, washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and then incubated with 0.1% Triton X-100 and 1% BSA for 30 min at room temperature. Myocardiocytes were incubated with primary anti-mouse antibodies against Bim (1:1,000), CHOP (1:1,000), activating transcription factor 4 (ATF4; 1:1,000; product code ab216839; Abcam), inositol-requiring enzyme 1α (IRE1α; 1:1,000; product code ab37073; Abcam), thiobarbituric acid reactive substances (TBARS; 1:1,000; ab118970; Abcam), reactive oxygen species (ROS; 1:1,000; ab186027; Abcam), H₂O₂ (1:1,000; ab138874; Abcam) overnight at 4°C. Myocardiocytes were washed with PBS and incubated with corresponding anti-rabbit secondary antibody (1:2,000, product code ab7090; Abcam) for 2 h at 25°C. Subsequently, myocardiocytes were stained with 5% DAPI for 30 min at room temperature. Images of myocardiocytes were captured using a Zeiss confocal spectral microscope (Carl Zeiss AG) at a magnification of ×100.

All data are expressed as the mean ± SD of triplicate dependent experiments. All data were analyzed using SPSS Statistics 22.0 (IBM Corp.) Paired Student's test was used to assess the significant differences between two groups. One-way variance analysis (ANOVA) followed by Tukey's HSD test were used to assess the significant differences among multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To determine the therapeutic effect of Tan-IIA in myocardial ischemia, a myocardial ischemia-reperfusion rat model was established and received treatment with Tan-IIA, or PBS. As demonstrated in Fig. 1A, Tan-IIA attenuated the myocardial infarction sizes after ischemia-reperfusion compared with the PBS group (P<0.01). In addition, the myocardial functions LVEDD, LVESD, LVEF and LVES were significantly ameliorated during reperfusion in the Tan-IIA group compared with the PBS group (Fig. 1B-E). Tan-IIA treatment exhibited a significant elevation in dp/dt(max) throughout the reperfusion period compared with the PBS (P<0.01; Fig. 1F). These data indicated that Tan-IIA played a protective role in a myocardial ischemia-reperfusion rat model.

The anti-apoptotic role of Tan-IIA in a rat model of myocardial ischemia was next analyzed. A TUNEL assay demonstrated that Tan-IIA decreased apoptosis of myocardial tissue compared with PBS (Fig. 2A). Immunohistochemical analysis revealed that Tan-IIA upregulated the anti-apoptotic protein Bcl-2 and Bcl-xL expression in myocardial tissue compared with PBS (Fig. 2B). The data also demonstrated that Tan-IIA downregulated pro-apoptotic protein caspase-3, Cyto c and Apaf-1 expression in myocardial tissue compared with PBS (Fig. 2C). These data indicated that Tan-IIA could inhibit apoptosis of myocardial tissue in rat model of myocardial ischemia.

To verify the protective effect of Tan-IIA on myocardiocytes, viability of myocardiocytes was analyzed in vitro. As demonstrated in Fig. 3A, 30 µM of Tan-IIA presented the optimal protective effect on viability of myocardiocytes. As demonstrated in Fig. 3B, Tan-IIA (30 µM) increased the viability of myocardiocytes in a time-dependent manner compared with PBS. These data indicated that Tan-IIA could increase the viability of myocardiocytes.

The anti-apoptotic effect of Tan-IIA was analyzed in myocardiocytes in vitro. As revealed in Fig. 4A, Tan-IIA produced a significant reduction of apoptosis of myocardiocytes compared with PBS treatment. The results revealed that expression levels of Bcl-2 and Bcl-xl were upregulated by treatment with Tan-IIA in myocardiocytes compared with PBS treatment (Fig. 4B). Western blot analysis demonstrated that apoptotic factors including cleaved caspase-3 (casp-3), Cyto c and Apaf-1 in the mitochondrial apoptotic pathway were downregulated by Tan-IIA in myocardiocytes compared with treatment by PBS (Fig. 4C). These data indicated that Tan-IIA may inhibit myocardiocyte apoptosis via the mitochondrial apoptotic pathway.

The effect of Tan-IIA on oxidative stress was analyzed in myocardiocytes. As demonstrated in Fig. 5A and B, Tan-IIA treatment upregulated the expression of endoplasmic reticulum stress-related proteins Bim and CHOP in myocardiocytes compared with the control group. However, the oxidative stress stimulator TM abolished the Tan-IIA-increased Bim and CHOP expression in myocardiocytes compared with the control group. The data also demonstrated that myocardial injury-induced apoptosis was inhibited by Tan-IIA pretreatment compared with the control group and this effect was abolished by TM treatment in myocardiocytes (Fig. 5C). These results indicated that Tan-IIA could protect myocardiocytes against apoptosis by modulating oxidative stress.

To identify the possible mechanism of Tan-IIA in myocardiocytes, caspase-3-overexpressed (casp-3OP) myocardiocytes were established. As demonstrated in Fig. 6A, caspase-3 overexpression reversed Tan-IIA-decreased Casp-3, Cyto c, and Apaf-1 in myocardiocytes. The results in Fig. 6B demonstrated that Casp-3 overexpression reversed Tan-IIA-decreased apoptosis of myocardiocytes compared with the control group. These results indicated that Tan-IIA could decrease apoptosis of myocardiocytes via inhibition of the mitochondrial apoptotic signaling pathway.

Previous data has revealed that the intracellular calcium level and oxidative stress are increased during apoptosis of myocardiocytes (21). It has been demonstrated that I/R injury induces oxidative and inflammatory responses, and further ultimately damages cardiac function in patients suffering myocardial ischemia (27). To investigate whether the protective effect of Tan-IIA on myocardiocytes in myocardial ischemia rats is related with intracellular calcium and oxidative stress, the intracellular calcium and oxidative stress levels were analyzed. As demonstrated in Fig. 7A, Tan-IIA decreased the level of intracellular calcium in myocardiocytes compared with the control. The results revealed that production of TBARS, ROS and H₂O₂ was inhibited by Tan-IIA compared with the control groups (Fig. 7B-D). The data also demonstrated that Tan-IIA decreased expression of ATF4 and IRE1α expression in myocardiocytes (Fig. 7E and F). Collectively, these results indicated that Tan-IIA could decrease intracellular calcium and oxidative stress in myocardiocytes.