DMEM-F12 medium, phosphate buffered solution (PBS) fetal bovine serum (FBS), DCFH-DA probe and mitochondria-targeted MitoSOX probe (M36008) were purchased from Invitrogen. Hcy, ATX, MTT, propidium iodide (PI) were obtained from Sigma. TUNEL-DAPI kit, Mito-SOX probe and BCA assay kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Primary antibodies, including cleaved PARP (#94885), active-caspase-3 (#9664), active-caspase-7 (#8438), active-caspase-9 (#9507), Bax (#2772), Bad (#9292), Bcl-xL (#2764), Ser428-ATR (#2853), Ser15-p53 (#9284), total-p53 (#2524), and Ser139-histone (#9718) were all obtained from Cell Signaling Technology (Beverly, USA). Bcl-2 (#14-6992-82), CD-34 (#MA1-10202) and Ser-1981-ATM (#14-9046-82) were bought from Invitrogen (Carlsbad, USA). All solvents used were of high performance liquid chromatography (HPLC) grade.

H9c2 rat myocardial cells were obtained from ATCC company (USA). Cells were cultured with DMEM-F12 medium containing 10% FBS at 37° and 5% CO₂ in a incubator. Cell viability with Hcy or/and ATX was detected by MTT assay. Briefly, H9c2 cells (8 × 10³ cells/well) seeded in 96-well plate were treated with Hcy (1–16 mM) for 72 h, or cells were treated with 8 mM Hcy for 6, 12, 24, 48, and 72 h. For protective treatment, cells were pre-treated with 0.5–8 μM ATX for 6 h and co-incubated with Hcy for 72 h. After treatment, 20 μl of MTT solution was added and incubated for another 5 h. Then the medium was removed and 150 μl of dimethyl sulfoxide (DMSO) was added. The cell viability was measured by detecting the absorbance at 570 nm. H9c2 cells morphology was observed by phase microscope. All data and images were obtained from three independent trials and were conducted in accordance with the relevant guidelines and regulations of Taishan Medical University.

Cell apoptosis and cell cycle distribution in H9c2 cells were analyzed by FCM. Briefly, cells were pre-treated with or without 4 μM ATX for 6 and co-treated with 8 mM Hcy for 72 h. After treatment, cells were washed, collected and incubated with PI buffer at 37°C for 5 h in darkness. Then the stained cells were monitored by FCM. Cell cycle (G0/G1, S, and G2/M phases) was analyzed by Modfit software. The hypodiploid DNA content (Sub-G1 peak) was employed to quantify the apoptotic cell death. About 10⁴ cells/sample were recorded. All data and images were obtained from three independent trials and were conducted in accordance with the relevant guidelines and regulations of Taishan Medical University.

Cells apoptosis in H9c2 cells were also detected by TUNEL-DAPI staining. Briefly, Treated H9c2 cells seeded on a cover glass were fixed with 4% formaldehyde, and permeabilized with 0.1% Triton X-100. Then cells were incubated with TUNEL reaction solution for 1 h in darkness at 37°C. After incubation, cells were stained with 10 μg/ml DAPI for 10 min, and washed with PBS for three times. The cells apoptosis was detected by an inverted fluorescence microscope. The TUNEL-positive cells represent the apoptotic cells. All data and images were obtained from three independent trials and were conducted in accordance with the relevant guidelines and regulations of Taishan Medical University.

Mitochondrial function was evaluated by the mitochondrial membrane potential (Δψ_m) and mitochondrial morphology, which were examined by JC-1 and Mito-Tracker probes, respectively. Briefly, cells seeded in 6-cm plate were treated with ATX or/and Hcy. Cells after treatment were washed and incubated with 10 μM JC-1 or Mito-Tracker for 15 min in darkness. Then cells were washed and imaged under an inverted fluorescence microscope. The green fluorescence intensity after JC-1 staining was quantified by ImagePlus Software. All data and images were obtained from three independent trials and were conducted in accordance with the relevant guidelines and regulations of Taishan Medical University.

The intracellular ROS and superoxide anion were measured by DCFH-DA and Mito-SOX probes in live cells, respectively. Briefly, treated cells cultured on glass were washed and incubated with 10 μM DCFH-DA or 0.5 μM Mito-SOX. After reaction, cells were washed and observed by an inverted fluorescence microscope for detection of ROS (green fluorescence) and superoxide anion (red fluorescence). The images showed here were obtained from three independent experiments. All data and images were obtained from three independent trials and were conducted in accordance with the relevant guidelines and regulations of Taishan Medical University.

Protein expression was examined by western blotting method. Briefly, cells after treatment were washed, collected and lysed by RIPA lysis buffer. The intracellular total protein was extracted by centrifugation at 11,000 g for 10 min at 4°C. Total protein after quantification was boiled and loaded (40 μg/lane) for separation by SDS-PAGE. Then protein was transferred onto a nitrocellulose membrane at 100 V for 1.5 h. Membrane subsequently was blocked, incubated with the primary antibody, and second antibody, respectively. Then the membrane was washed and the protein bands were visualized on an X-ray film using an enhanced ECL chemiluminescence system. The proteins expression was quantified by Quantity-One Software, and the proteins expression rate was labeled under the bands. All data and images were obtained from three independent trials and were conducted in accordance with the relevant guidelines and regulations of Taishan Medical University.

The therapeutic effect of ATX against Hcy-induced cardiotoxicity was also evaluated in vivo in mouse. Briefly, 40 mice were divided into four groups (10 mice/group), and administrated with 5 mg/kg/day ATX or/and 300 mg/kg/day Hcy for 4 weeks. The control group was given equal normal saline. After administration, mouse was given euthanasia and the body weight and heart weight were measured. The contents of reduced glutathione (GSH-Rs) and malondialdehyde (MDA) in heart tissue were examined by ELISA kits according to the manufacture's instruction. Part of heart tissue was cut into 4-μM section for immunohistochemical assay (IHC). All the animal experiments were conducted in accordance with the relevant guidelines and regulations of Taishan Medical University.

All data and images were done from three independent experiments at least. The statistical analysis was carried out by SPSS013.0 software. The significance between two groups was analyzed by two-tailed Student's test. The difference among three or more groups was analyzed by multiple comparisons. Bars with “^*” or “^**” represent the P < 0.05 or P < 0.01, respectively. Bars with different characters indicates the statistical different at P < 0.05 level.

The cytotoxicity of Hcy and possible protective effect of ATX on H9c2 cells were firstly examined by MTT assay. As shown in Figure 1 and Supplementary Figure 1, Hcy treatment alone significantly inhibited H9c2 cells viability in a dose- and time-dependent manner (Figures 1A,B). For instance, treatment of cells with 8 mM Hcy for 72 h significantly decreased the cell viability to 52.6%. However, pre-treatment of cells with ATX (1, 2 and 4 μM) for 6 h effectively blocked Hcy-induced cytotoxicity in H9c2 cells from 52.6% (Hcy) to 87.9, 95.4, and 99.8%, respectively (Figure 1D). ATX alone showed no obvious cytotoxicity toward H9c2 cells (Figure 1C). The improvement of cell morphology further confirmed this protective potential (Figure 1E). These results suggested that ATX has the potential to alleviate Hcy-induced cytotoxicity.

We next evaluated Hcy-induced apoptosis and the possible protective mechanism of ATX on H9c2 cells by FCM. As shown in Figure 2A, Hcy alone caused apparent apoptosis, as reflected by the increase of Sub-G1 peak. However, the cell apoptosis in Hcy-treated cells was significantly attenuated by ATX pre-treatment from 52.6 to 4.1%. This protection of ATX on H9c2 cells was evaluated by TUNEL-DAPI co-staining. As shown in Figure 2B, Hcy treatment alone resulted in significant apoptosis, as convinced by the increase of TUNEL-positive cells. However, pre-treatment with ATX effectively prevented H9c2 cells from HCY-induced apoptosis. Moreover, the PARP cleavage and caspase activation were also conducted to investigate the underlying mechanism. As shown in Figure 2C, incubation of cells with Hcy alone induced dramatically poly-ADP-ribose polymerase (PARP) cleavage and the activation of caspase-3, -7, and -9. The activation of caspase-7 and caspase-9 indicated that Hcy activated the mitochondria-mediated apoptosis. The slight activation of capase-8 indicated that Hcy also triggered death receptor-mediated apoptosis, but not dominant (Supplementary Figure 2). As expected, ATX pre-treatment markedly inhibited Hcy-induced PARP cleavage and caspase activation. Taken together, these results above indicated that ATX suppressed the mitochondria-mediated apoptosis in Hcy-treated H9c2 cells.

To elucidate the role of mitochondria in Hcy-induced apoptosis of H9c2 cells, the Δψ_m and mitochondrial morphology were detected by JC-1 and Mito-tracker probes, respectively. As shown in Figure 3A, Hcy treatment induced significant loss of Δψ_m, as reflected by the fluorescent shift from red to green. ATX pre-treatment completely improved the Δψ_m in Hcy-treated cells. The statistical result further confirmed this protective effect (Supplementary Figure 3). Moreover, Hcy treatment also caused obvious mitochondrial fragmentation, as demonstrated by the mitochondrial morphological changes from protonema to punctiform. Interestingly, ATX pre-treatment completely blocked Hcy-induced mitochondrial fragmentation.

Bcl-2 family plays important role in regulating mitochondrial permeability and inducing apoptosis. Therefore, a time-course effect of Hcy on Bcl-2 family members was investigated. As show in Figure 3B, Hcy treatment caused continuous down-regulation of Bcl-2 at 12 h. Bad expression in Hcy-treated H9c2 cells showed significant up-regulation at 12 h. However, the expression level of pro-apoptosis (Bax and Bad) and pro-survival (Blc-2 and Bcl-XL) in Hcy-treated cells were effectively normalized by ATX pre-treatment (Figure 3C). Taken together, these results revealed that ATX blocked Hcy-induced mitochondrial dysfunction by regulating Bcl-2 family proteins.

Accumulated evidences have confirmed that Hcy could trigger oxidative damage through inducing ROS accumulation. Therefore, the oxidative status in Hcy-treated H9c2 cells was investigated. Primarily, the intracellular superoxide anion and ROS were detected by Mito-SOX (a mitochondria-targeted red probe) and DCFH-DA (green), respectively. As shown in Figures 4A,B, Hcy treatment caused significant accumulation of superoxide anion and ROS, as demonstrated by the enhanced red fluorescence and green fluorescence in H9c2 cells, respectively. Secondly, Hcy-induced DNA damage was also detected. As shown in Figure 4C, cells treated with 8 mM Hcy showed significant phosphorylation activation of ataxia telangiectasia mutated (ATM) (Ser1981), and Rad3-related (ATR) (Ser428), p53 (Ser15), and histone (Ser139) in a time-dependent manner, indicating that Hcy induced oxidative damage in H9c2 cells. However, pre-treatment with ATX inhibited Hcy-induced generation of ROS and superoxide anion. The statistical results further confirmed ATX's anti-oxidative effect (Supplementary Figure 4). ROS inhibition by ATX eventually attenuated Hcy-induced oxidative damage (Figure 4D). Taken together, these results above demonstrated that ATX attenuated Hcy-induced oxidative damage through inhibition ROS overproduction.

To validate the in vivo protective effect and possible mechanism of ATX against Hcy-induced cardiotoxicity, mouse administrated with ATX or/and Hcy were employed. The results showed that Hcy treatment in vivo caused slight decrease in mice heart weight, but not affect the mice body weight (Figures 5A,B). Hcy treatment also decreased the content of reduced-glutathione (GSH-Rs) (Figure 5C) and increased the content of MDA (Figure 5D), indicating the induction of oxidative damage by Hcy. IHC analysis revealed that Hcy treatment in vivo induced significant myocardial apoptosis (active-caspase-3 staining) and inhibited angiogenesis (CD-34 staining). Importantly, ATX treatment in vivo effectively inhibited the oxidative damage and myocardial apoptosis, and improved the angiogenesis in Hcy-treated mouse (Figure 5E), which consisted with the in vitro mechanism. These results suggested that ATX inhibited Hcy-induced cardiotoxicity in vivo.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.