Figure 2.

Figure 2.

BHPF induces cardiac ferroptosis through reduction of YTHDF2-facilitated translation of m⁶A-gch1. A, Changes in the number of zebrafish cardiomyocytes following concentration gradient exposure (0.1–10 μM) to BHPF. The images (left panel) were quantified as a histogram (right panel); n = 6–9 per group. B, Rescue effects of different PCD inhibitors on BHPF-induced reduction of heart rate (bpm) and number of cardiomyocytes. Chloroquine (CQ) is an autophagy inhibitor; Necrostatin-1 (Nec-1) is a necroptosis inhibitor; VX765 (Belnacasan) is a pyroptosis inhibitor; Q-VD-OPh is an apoptosis inhibitor; Ferrostatin-1 (Fer-1) is a ferroptosis inhibitor. All inhibitors were co-treated with BHPF from 4 hpf to 48 hpf; n = 6–13 per group. C, Effects of BHPF exposure on mRNA expression of ferroptosis markers ptgs2a and ptgs2b in FACS sorted zebrafish embryonic cardiomyocytes. D, Pathway enrichment analysis by SMPDB (small molecule pathway database) based on metabolomic profiling. E and F, Effects of exogenous BH2 supplementation on heart rate (bpm refers to beats per minute) (E) and heart activity (F) of BHPF exposed zebrafish; n = 12–17 per group. G, Effects of BHPF exposure on BH2 concentration and GCH1 protein levels. BH2 concentration was detected by LC-MS/MS. H and I, Rescue effects of exogenous gch1 mRNA on heart rate (H) and heart activity (I); n = 10–12 per group. J, Representative images of cardiac sections stained with Hematoxylin and eosin (HE), Sirius red or Wheat germ agglutinin (WGA) in hearts obtained from the offspring of the control and BHPF-exposed mice. Red arrows indicate inflammatory cells and blue arrows indicate collagen fibrils. K, Images of immunofluorescence for YTHDF2 and GCH1 staining, and immunohistochemistry for 4-HNE in the offspring of the control and BHPF-exposed mice with quantification. L, Measurement of gch1 mRNA translation profile in mettl3 morphants by polysome profiling. M, Effects of BHPF exposure on YTHDF2 protein levels. N, Measurement of gch1 mRNA translation profile in ythdf2 morphants by polysome profiling. O, Top panel shows the affinity between YTHDF2 and gch1-flag mRNA (wild type and m⁶A modification sites mutant). Bottom panels show the measurement of gch1 mRNA and protein levels in gch1-flag (WT and MUT) mRNA microinjected control embryos and ythdf2 morphants. P, Representative echocardiograms, summary of heart rate, cardiac ejection fraction (EF) and fractional shortening (FS) in wild-type (ythdf2^+/+) and ythdf2 mutant (ythdf2^−/−) zebrafish; n = 5–7 per group. Q, Images of immunofluorescence for GCH1 staining and immunohistochemistry for 4-HNE in wild-type (ythdf2^+/+) and ythdf2 mutant (ythdf2^−/−) zebrafish with quantification. R, Schematic diagram shows BHPF-mediated downregulation of YTHDF2 stimulating ferroptosis-induced cardiac injury by reduction of YTHDF2-facilitated gch1 translation. Molecular biology experiment materials are zebrafish embryonic cardiomyocytes sorted by FACS (C) or whole embryos (D, G, L–O). Zebrafish embryos were collected at 48 hpf (A–I, L–M) or 10 hpf (O). Scale bar, 50 μm (A–B) or 20 μm (J–K, Q). Data are mean ± s.d. Student's t test, ns represents P > 0.05, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.