The wide type zebrafish (AB strain) was purchased from the Institute of Hydrobiology of the Chinese Academy of Science (Wuhan, China) and transgenic zebrafish (AB cmlc2:GFP) were gifted by Dr. Lou in the Nanjing University Animal Model Institute. These were kept in the Animal Model Center in the Jiangsu Province Key Laboratory of Human Functional Genomics. Recycled water at 28 + 0.5 °C and a 14 h light cycle per day were set to mimic the circadian rhythm. Zebrafish were fed with brine shrimp twice a day. At 17:00, we transferred the male and female adult zebrafish into the mating tank in a ratio of 2:1 and segregated them with a partition. The next morning, we moved the partition at 9:00 and the mating process lasted for 2 h. After spawning, the embryos were collected in clean fish water. The experiment protocols were approved by the Animal Ethics Committee of Nanjing Medical University.
Chemical exposure experiment
We used 0 mM, 0.01 mM, 0.04 mM, 0.16 mM, 0.64 mM, 2.56 mM, and 10.24 mM doses of isoniazid (I3377-50G; Sigma) for the dose-effect assay according to the therapeutic range of 3–5 μg/mL (0.01–0.04 mM) of isoniazid . No precipitation was observed at room temperature 25°C. The embryos were cultured in sterile water composed of 137 mM/L NaCl, 0.54 mM/L KCl, 0.025 mM/L Na2HPO4, 0.044 mM/L KH2PO4, 1.3 mM/L CaCl2,1 mM/L MgSO4, and 0.42 mM/L NaHCO3. N-acetyl-L-cysteine (NAC; A7250-50G; Sigma), a ROS scavenger, was added (1mM) in advance to clean out ROS in the embryos in the NAC group for the rescue experiments. After 4 h post-fertilization (hpf), 80 well-grown embryos in each group were selected under the microscope and kept in one 100 mm plate (CORNING; Corning; United States) for embryo culture. The culture water that contained isoniazid was changed every 12 h and these embryos were kept in the incubator at 28 °C and 5% CO2. We recorded the number and morphology of malformed and dead embryos and removed dead embryos every 12 h after exposure. Each chemical exposure treatment was performed in triplicate.
We observed the morphology of embryos in different stages of development (24 hpf, 48 hpf, and 72 hpf) under white light with a fluorescent microscope (Nikon, Tokyo, Japan). In each stage, the total number of dead, unhatched, and malformed embryos was recorded. The indication of embryo death was defined based on opacity, egg clotting and stopping of heartbeat. We randomly selected 20 larvae and recorded the heart rate in 1 min in each experimental group. A transgenic zebrafish strain (cmlc2:GFP), where a green fluorescent protein gene was inserted under the promoter of a cardiomyocyte-specific gene of cardiac myosin light chain 2 (cmlc2), was applied to observe the change in morphology during heart development. All experiments were performed in triplicate.
After 48 h of chemical exposure, the total RNA of 20 larvae were isolated using Trizol reagent (Thermofisher, Waltham, United State). The total RNA in each group were reverse transcribed to produce cDNA with a TaKaRa RT reagent kit (TaKaRa, Japan). The primers of cardiomyocyte-specific genes were synthesized by Invitrogen (Shanghai, China) and their sequences are listed in Table 1. Real-time PCR was performed in triplicate with a mixture of SYBR green, primers, cDNA, and RNase-free water in an appropriate ratio. A housekeeping gene, 18s ribosomal RNA, was used for normalization.
Acridine orange (AO) staining
Acridine orange, a permeable nucleic acid-selective fluorescent dye (Thermo Fisher Scientific), was used to assess the apoptosis level of cardiomyocytes during the development of zebrafish that were exposed to different doses of isoniazid. Ten randomly selected embryos at 48 hpf were stained with AO for 30 min and washed with phosphate-buffered saline (PBS). A fluorescence microscope (Nikon, Tokyo, Japan) was used for photography.
2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime Biotechnology, China), a permeable and sensitive probe, was used for the detection of ROS in this study. We randomly selected 10 embryos at 48 hpf and stained them for 40 min with 100 µM of DCFH-DA diluted in PBS. After this was washed out with PBS for 10 min, all groups were photographed under a fluorescence microscope under a fluorescein isothiocyanate channel. All steps were finished in the absence of light.
Antioxidant enzyme activity assay
In each group, 20 zebrafish embryos were lysed in radioimmunoprecipitation assay buffer and cocktails. Commercial kits (Beyotime Biotechnology, China) were used to detect the activity of two important antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). We also evaluated the level of malondialdehyde (MDA), another marker for oxidative stress. Finally, the level of ROS was measured using a commercial kit (Beyotime Biotechnology, China). All experiments were performed in triplicate.
In situ hybridization
Embryos for in situ hybridization were exposed to propylenethiourea, a chemical reagent that removes the melanin from larvae. After that, we fixed the embryos in 4% paraformaldehyde and preserved them at 4 °C. This experiment lasted for 3 days. On the first day, we used 25%, 50%, 75%, and 100% ethanol for dehydration and a protease kinase to digest tissues for 10 min. Next, 0.1 mol/L triethanolamine with 25% acetic anhydride was added to acidized the embryos and a nuclear acid probe of cmlc2 for hybridization was added at 60 °C. On the second day, we used 2 × saline sodium citrate (SSC) and 0.2 × SSC buffer to wash out the residual probes. Then, we added an antibody against digoxin overnight at 4 °C. On the last day, we washed out the antibody with 1% maleic acid buffer and dried the embryos with BM purple for 10 min. Finally, we observed the dried embryos on a plate with 5% agar powder with an upright microscope (Olympus, Japan) under white light.
Pericardial area measurement
After we captured the images of embryos under the white light with a microscope (Nikon, Tokyo, Japan) using a 1.5 × lens, the pericardial area was measured using NIH Image J 1.44 software (http://rsb.info.nih.gov/nih-image/). Boundaries and area of pericardial area were traced and calculated by the computer. We conducted the experiment with three replicates (n = 3) and each group contained 50–80 embryos.
Continuous variables are presented as mean and SD, whereas categorical variables were presented as number and percentage. For continuous variables, the difference between all groups was compared with a one-way ANOVA test. For categorical variables, a chi-square test was used. P < 0.05 was a significant statistical difference. An asterisk denotes a statistical significance between two groups (* P < 0.05, ** P < 0.1, *** P < 0.001). All statistical tests were completed in SPSS 22.0.