(A) The cGMP level was dramatically increased in the nitrite-exposed embryos at 24, 36, 48 and 76 hpf, respectively. **: P<0.01. The values of cGMP amount in all the control embryos were normalized to 1.0, respectively. The value of cGMP amount in the nitrite-exposed embryos was the fold of the control embryos at the same developmental stage. (B) A scatter plot showing the increased EIs in the nitrite-exposed embryos were significantly reduced by microinjecting ODQ (sGC inhibitor) into nitrite-exposed embryos. Different treatments of embryos were shown in X-axis. EI (shown in Y-axis) of each embryos was shown in the plot. Average ± standard errors of EIs were shown in lines. (C–E) Defective histological structures of heart caused by excessive nitrite were partially rescued by microinjection of ODQ into nitrite-exposed embryos. Embryos were observed at 108 hpf. Compared to control embryos with normal pericardial membrane, myocardium and both superior and inferior valve leaflets (C), 6/8 embryos exposed to the nitrite showed thinner myocardium and defective formation of either superior or inferior leaflets (D). ODQ microinjection resulted in 3/6 of the embryos developed both superior and inferior leaflets (E). (F–T) Microinjection of ODQ partially rescued the diminished expressions of valve progenitor makers in zebrafish embryos at 48 hpf. nppa is not expressed in the AVC (rectangular box) of control embryos (F) but was ectopically expressed in the AVC of nitrite-exposed embryos (K). Microinjection of ODQ into nitrite-exposed embryos prevented 7 of 15 embryos from expressing nppa in AVC (P). Compared to control embryos, nitrite exposure significantly decreased or abolished expressions of bmp4 (G, L), vcana (H, M), notch1b (I, N) and has2 (J, O) in AVC. However, microinjection of ODQ into nitrite-exposed embryos resumed expressions of bmp4 (Q), vcana (R), notch1b (S) and has2 (T) in about half embryos. Red star (*): pericardial membrane; Black arrow: position of superior valve leaflet; Black arrowhead: position of inferior valve leaflet; A: atria; V: ventricle.
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