Fig. 2

Heartbeat dependency of miR-143 expression. (A–C) Expression of miR-143 in the ventricle (red arrowheads) and the OFT (blue arrowheads) becomes evident as heartbeat and blood circulation increase. (D) Heartbeat was arrested from 36 hpf by BDM, then restarted by washing out at 94 hpf. Only faint expression of miR-143 was detected at 96 hpf in the OFT (blue arrowhead). (E) Heartbeat was stopped from 36 to 72 hpf. Continuous beating from 72 to 96 hpf allowed weak expression of miR-143 both in the OFT and the ventricle. (F) Duration of heartbeat for 48 h restored the expression of miR-143. (G) Heartbeat was arrested from 36 to 96 hpf. Expression of mir-143 was not detected at 96 hpf. (H) When heartbeat was stopped from 60 to 72 hpf, only faint expression of miR-143 was observed in the OFT at 72 hpf (blue arrowhead). (I) 36 h arrest of heartbeat (from 60 to 96 hpf) abolished expression completely. (J and K) In Tnnt2 morphants, expression of miR-143 was not detected at both 72 (J) and 96 hpf (K). (L) A schematic representation of miR-143 expression. Normally, heartbeat and blood circulation initiate at 24 hpf. Expression of miR-143 commences soon after at 36 hpf. When heartbeat was stopped from 36 hpf, miR-143 was not expressed. When BDM was washed out to initiate heartbeat at 72 hpf, miR-143 was induced. Likewise, when heartbeat was arrested at 60 hpf, miR-143 expression was decreased. (M) Expression of miR-143 was quantified by quantitative RT-PCR in human umbilical vein endothelial cells (HUVECs), to which 2 Pa (20 dyne/cm²) of shear stress was applied. miR-143 was induced rapidly within 1 h. This induction was transient and returned to a basal level within 4 h.