Zebrafish husbandry and reproduction
Both the Tg:tnnt2a+/- mutant strain and wild-type AB strain were purchased from Fudan University. Zebrafish were reared under standard temperature (28°C) and 14h light:10h dark cycle photoperiod conditions, and were fed with fresh paramecia 3 times a day. Fertilized embryos were collected promptly and placed in an egg-water incubator at 28°C. Embryos at 24h post-fertilization(hpf) were fed with egg water containing 0.2 mM N-phenylthiourea (Sigma-Aldrich, St. Louis, MO, USA) to suppress skin pigment production, which was beneficial during subsequent visual observations were performed in microscopy imaging experiments. All animal experiments were performed according to European guidelines and in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
DCM model in zebrafish
We have reported previously that tnnt2a mutant zebrafish was generated by knocking out the related gene using CRISPR/Cas9 technology[11]. Moreover, mature F0 zebrafish were crossed with wild-type AB to produce offspring containing heterozygous mutants that were in crossed to obtain homozygous mutants. Genotyping was performed via PCR-sequencing to indicate that tnnt2a was deleted as reported.
tnnt2a Genotyping Primer Forward: 5’-gtaagcgcatggagaaggac-3’,
tnnt2a Genotyping Primer Reversed: 5’-gcgacatcacagagccaaat-3’.
DCM zebrafish models were divided into two groups: One group named model 1 was tnnt2a-/- generated by crossing with tnnt2a+/-, which is similar to the clinical phenotypes of patients with dilated cardiomyopathy, such as cardiac arrest,atrial and ventricular enlargement,and also obvious influence on activity (Figure 1).
Tol2 transposon technology was used to insert the target gene tnnt2a into the zebrafish genome, and the Tet-On doxycycline (DOX) system induced the expression of tnnt2a[20]. Treatment with DOX produced higher levels of tnnt2a mRNA that were comparable to wild-type hearts. In addition, Tg:tnnt2a-/- transgenic zebrafish had persistently enlarged atria and ventricles, persistent abdominal edema, and typically died at 6-7 day-post-fertilization(dpf). Therefore, our study used the Tg:tnnt2a-/- transgenic zebrafish model with the heart recovered to beat after the addition of DOX as model 2 (Figure 2).
Drugs and Collocation
APS(LOT:C18M6Y1)was purchased from Shanghai Yuanye Biotechnology (Shanghai, China).Doxycycline(DOX), N-phenylthiourea(PTU), Trizol were purchased from Sigma-Aldrich (St. Louis, MO, United States).HE staining kit, One Step TdT-Mediated dUTP Nick End Labeling(TUNEL)Apoptosis Assay Kit, and 4,6diamidino-2-phenylindole (DAPI) staining solution was purchased from Beyotime Biotechnology (Shanghai, China). APS stock solution was prepared with dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml, stored at -20 °C, and diluted to 15 μg/ml when used.
Assessment Effects of APS on DCM Zebrafish
Homozygous zebrafish(tnnt2a-/-) with DCM phenotype were picked under the microscope after 30 hpf. Zebrafish were placed in a 12-well plate with 10 well-grown zebrafish embryos per well, 30 per group. Preliminary experimental results show that the safe concentration of APS is 15μg/ml. The zebrafish has severe heart malformation with obvious phenotype from 3dpf,both model 1 and model 2 were treated with APS for 48h. The embryos receiving egg medium only served as control. Untreated zebrafish were used to confirm that the vehicle solvent did not cause any side effects on zebrafish. The medium of all treatment groups was refreshed every 24h. Each experiment was repeated at least three times.
Morphological observation of zebrafish
At 48 hpf and 72 hpf, zebrafish were visually tracked from microplates and observed for viability, heart rate, and cardiac dilatation under a fluorescence microscope(Olympus IX81 Motorized Inverted Microscope, Japan)equipped with a digital camera(DP controller, Soft Imaging System, Olympus).
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)
Zebrafish embryos from each group were collected and the hearts were dissected under a microscope. Under light-proof conditions, add 30~40μl TUNEL and DAPI to the zebrafish heart, incubate at 37°for 12h, and the incubation time can be appropriately extended. Then washed with PBST and sealed with small coverslips after gradient addition of 20%, 50%, and 70% glycerol. Analyze the samples under the fluorescence microscope and observe the red fluorescence. Laser confocal scanning of DAPI- stained blue channel and TUNEL-stained red channel.
RNA-seq and Bioinformatics Analysis
The zebrafish transcriptome was sequenced, and the bioinformatics data were analyzed according to the methods of our group's previous studies[21]. Double-terminal sequencing was performed on the Illumina Hi-2000 genome analysis platform. The terminal RNA sequences were paired and the data processed using the fastq format of the Illumina genome analyzer. Cuffdiff software was used for differential gene analysis. Differentially expressed genes were subjected to GO functional annotation and analysis and KEGG Pathway significant enrichment analysis in the DAVID database.
qRT-PCR
Total RNA was extracted from zebrafish embryos using a TRIzol kit. Next,approximately 1μg total RNA was reverse transcribed to synthesiize the cDNA.qRT-PCR was performed using SYBR Green detection reagents in a StepOne Plus PCR machine.The relative mRNA levels were calculated by the 2−ΔΔCt method and displayed as fold changes relative to the control group, and β-actin was used as an endogenous control gene to normalize gene expression.The zebrafish β-actin primers were 5’-TTGACAACGGCTCCGGTATG-3’(F) and 5’-TCCCATGCCAACCATCACTC-3’(R);bcl-2 primers were 5’-TGTGCGTGGAAAGCGTCAA-3’(F) and5’-GTCTCTCTGCTGACCGTACAT-3’(R);bax primers were 5’-CGGAGATGAGCTGGATGGAA-3’(F) and5’-CAAGGCGACAGGCAAAGTAG-3’(R);caspase-3 primers were 5’-CCCAGTGGAGGCAGATTTCC-3’(F) and5’-GAGCATTGAGACGATGCAGG-3’(R);caspase-9 primers were 5’-TAAATGACCGCAAGGGCTCC-3’(F) and5’-GCCTCAGTGCCATGTGAAAG-3’(R).Each sample was tested in triplicates.
Statistical Analysis
All results are expressed as means ± standard deviation (SD). The survival rate was compared by the Cox Mantel test, One way ANOVA was used for comparison between multiple groups, and the LSD-t test was used for comparison between two groups by using GraphPad Prism 6.0 software. Values of p < 0.05 were considered to indicate statistical significance.