Construction of a Zebrafish Model of Cardiac Hypertrophy Caused by ATIC Gene Deletion and Preliminary Exploration of Aerobic Exercise Improvement

Figures

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  Figure 1. Schematic diagram of atic gene knockout in zebrafish. (A) Schematic diagram of sgRNA targeting for atic gene knockout. The green horizontal line represents the genomic DNA of atic, the green rectangle represents the exon of atic, the red represents the target sequence, and the blue represents PAM. (B) Schematic diagram of the three heritable mutant alleles of atic resulting from gene knockout and their encoded protein sequences. Sequencing peaks of three mutant alleles of (C) atic, where A, G, C, and T are represented by green, black, blue, and red curves, respectively. (D) qRT-PCR to verify the expression level of atic−/−RNA in mutant lines, data are shown as mean ± standard error, **** p < 0.0001, ns: p > 0.05 (n = 3 TU, n = 3 KO, n = 3 EX).

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  Figure 2. (A) H&E staining results of zebrafish adult heart; (B) H&E staining result analysis of zebrafish adult hearts (n = 3); (C) Masson staining results of zebrafish adult hearts; (D) Masson staining results analysis of zebrafish adult hearts (n = 3). * p < 0.05, ** p < 0.01. Scale bars: (A) 100 µm; (C) 200 µm.

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  Figure 3. Cardiac function analysis of adult zebrafish (A–C). Note: Ventricular relaxation is depicted in red and ventricular contraction is depicted in green. This image is used to measure the cardiac function of zebrafish by tracing its ventricular epicardium. Each group consisted of 10 fish (n = 10) and each fish was analyzed in duplicate for three different cardiac cycles. (A–E) TU wild-type, atic−/−, atic + EX−/− adult 3-month-old zebrafish echocardiography result analysis diagram; data are shown as mean ± standard error; note: (D): zebrafish echocardiography epicardial and endocardial ejection fraction result analysis comparison diagram (ns: p > 0.05); (E): zebrafish echocardiogram epicardial result analysis (* p < 0.05, **** p < 0.0001); (F): zebrafish echocardiography endocardial ejection fraction result analysis chart (** p < 0.01, **** p < 0.0001); (G): analysis graph of heart rate results of zebrafish TU wild-type, atic−/−, atic + EX−/− groups (* p < 0.05, *** p < 0.001, ns: p > 0.05); (H): Analysis graph of shortening scores of zebrafish TU wild-type, atic−/−, atic + EX−/− groups (**** p < 0.0001, ns: p > 0.05).

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  Figure 4. Correlation map of samples, volcano map, and heat map of differential gene expression. (A) Sample correlation plot; (B) differential gene summary statistical table; (C) atic−/−KO vs. TU group; (D) atic−/−EX vs. atic−/−KO group; (E) heat map; note: correlation of RNA-seq samples of 3-month-old wild-type zebrafish (TU), atic−/−, atic-EX−/− groups; significantly up-regulated genes are indicated by red dots, significantly down-regulated genes are indicated by blue dots, and non-significantly different genes are indicated by gray dots. The abscissa and ordinate represent the negative correlation coefficient of expression level change. The larger the value, the more obvious the differential expression, and the higher the reliability of screening differential genes.

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  Figure 5. (A) atic−/−VS wild-type TU zebrafish GO (BP) analysis histogram; (B) atic−/−EX vs. atic−/− group GO (BP) analysis histogram; note: green indicates KO enriched GO (BP) pathway in KO vs. TU group, blue indicates TU enriched GO (BP) pathway in KO vs. TU group; (C) atic−/− vs. wild-type TU zebrafish KEGG analysis histogram; (D) atic−/− EX vs. atic−/− group KO KEGG analysis histogram; note: green indicates KO-enriched KEGG pathway in KO vs. TU group and blue indicates TU-enriched KEGG pathway in KO vs. TU group.

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  Figure 6. (A–D) qRT-PCR verification of the expression levels of actc1, myh7, tfgb3, tnnt2, and other genes in the heart after atic gene knockout. The data are displayed as mean ± standard error, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 7. GSEA analysis results chart; (A,B) calcium signaling pathway GSEA analysis diagram, (C–F) qRT-PCR validation pathway of htr2b, agtr1b, fgfr3 gene expression level, *** p < 0.001, **** p < 0.0001, ns: p > 0.05; (G,H) Apelin signaling pathway GSEA analysis diagram, (I–L) qRT-PCR validation pathway of actca, agtr1b, ccn2a, adcy2b gene expression level, *** p < 0.001, **** p < 0.0001, ns: p > 0.05; (M,N): ECM–receptor interaction pathway GSEA analysis diagram, (O–R) qRT-PCR verification pathway of npnta, vwf, itga3a, col6a4a gene expression level, *** p < 0.001, **** p < 0.0001.