Loss of cpt1b function inhibits cardiomyocyte proliferation in zebrafish embryos. (A) A schematic representation of the design for generating the cpt1b knockout zebrafish. (B) A representative agarose gel electrophoresis image for the genotyping of cpt1b^+/+, cpt1b^+/−, and cpt1b^−/− zebrafish. (C) Genomic sequences of wild-type (WT) and cpt1b knockout zebrafish around cpt1b guide RNA target sites. The guide RNA target sequences are denoted by red letters, with the protospacer adjacent motif (PAM) sequences underscored, and the coding sequences are represented by capital letters. (D) Whole-mount in situ hybridization analysis of cpt1b expression in cpt1b^+/+ and cpt1^−/− zebrafish embryos at 4 dpf. The black triangle indicates muscle, the yellow triangle indicates liver, and the red triangle indicates heart. Scale bar: 500 μm. (E) Quantitative RT-PCR analysis of cpt1b mRNA expression in 4 dpf cpt1b^+/+, cpt1b^+/−, and cpt1b^−/− embryos. (F) Quantitative RT-PCR analysis of cpt1b mRNA expression in the hearts of adult cpt1b^+/+, cpt1b^+/−, and cpt1b^−/− zebrafish. ** p < 0.01, *** p < 0.001. (G) Representative maximum intensity projection confocal images of dissected hearts from 4 dpf Tg(pcna^mGFP;myl7:H2A-mCherry;cpt1b^+/+) and Tg(pcna^mGFP;myl7:H2A-mCherry;cpt1b^−/−) zebrafish. Enlarged single optical section images of the boxed regions are shown on the right. White triangles indicate proliferating cardiomyocytes. Scale bar: 50 µm. (H) Quantitative analysis of proliferating cardiomyocytes in G. n = 9–20. ** p < 0.01. (I) Quantification of ventricular areas of adult cpt1b^+/+ and cpt1b^−/− zebrafish. n = 9–10. *** p < 0.001.

Overexpression of cpt1b promotes cardiomyocyte proliferation in zebrafish embryos. (A) A schematic representation of the Tg(myl7:cpt1b-TBFP) construct. (B) A stereo fluorescence image of a 3 dpf wild-type sibling and Tg(myl7:cpt1b-TBFP) embryos. The triangles indicate the embryonic hearts. Scale bar: 200 µm. (C) Whole-mount in situ hybridization analysis of cpt1b expression with WT and cpt1b^OE zebrafish embryos at 3 dpf. The red boxes demarcate enlarged ventral views of the hearts (red triangles). Scale bar: 500 µm. (D) In situ hybridization analysis of cpt1b expression in cryosections of adult WT and cpt1b^OE zebrafish hearts. The red boxes show enlarged views of the denoted regions. Scale bar: 50 µm. (E) Quantitative RT-PCR analysis of cpt1b expression in the hearts of adult WT and cpt1b^OE zebrafish. *** p < 0.001. (F) Representative maximum intensity projection confocal images of dissected hearts from 3 dpf Tg(pcna^mGFP;myl7:H2A-mCherry) and Tg(pcna^mGFP;myl7:H2A-mCherry;myl7:cpt1b-TBFP) embryos. Enlarged single optical section images of the boxed regions are shown on the right. White triangles indicate proliferating cardiomyocytes. Scale bar: 50 µm. (G) Quantitative analysis of proliferating cardiomyocytes in F. * p < 0.05. n = 14–17. (H) Quantification of ventricular areas of adult wild-type and cpt1b^OE zebrafish. n = 9–10. ** p < 0.01.

Transcriptomic analysis reveals the potential contribution of Glul in cpt1b-regulated cardiomyocyte proliferation in zebrafish. (A) Nine-quadrant plot illustrating the log2(fold change) of recovered genes in the cpt1b^−/− vs. cpt1b^+/+ analysis and cpt1b^OE vs. WT analysis. Red dots denote genes that were significantly upregulated in cpt1b^−/− hearts and concomitantly downregulated in cpt1b^OE hearts, blue dots represent genes that were downregulated in cpt1b^−/− hearts and upregulated in cpt1b^OE hearts, and pink dots highlight the two glutamine synthetase genes. (B) Gene Ontology enrichment analysis of 154 differentially expressed genes revealed significant enrichment of biological processes. The y-axis represents the enriched biological processes, and the x-axis represents the negative logarithm of the p-value. (C) Quantitative RT-PCR analysis of glula and glulb expression in cpt1b^+/+ and cpt1b^−/− hearts. (D) Quantitative RT-PCR analysis of glula and glulb expression in WT and cpt1b^OE hearts. (E) Quantitative analysis of Pcna-positive cardiomyocytes in 4 dpf WT and cpt1b^OE zebrafish embryos treated with either vehicle (Ctrl) or 250 µM MSO. n = 10–13. ** p < 0.01, *** p < 0.001, and ns indicates p > 0.05.