Inhibition of calcium uptake during hypoxia in developing zebrafish is mediated by hypoxia-inducible factor

The present study investigated the potential role of hypoxia-inducible factor (HIF) in calcium homeostasis in developing zebrafish (Danio rerio). It was demonstrated that zebrafish raised in hypoxic water (30 mmHg; control, 155 mmHg P_O ) until 4 days post-fertilization exhibited a substantial reduction in whole-body Ca²⁺ levels and Ca²⁺ uptake. Ca²⁺ uptake in hypoxia-treated fish did not return to pre-hypoxia (control) levels within 2 h of transfer back to normoxic water. Results from real-time PCR showed that hypoxia decreased the whole-body mRNA expression levels of the epithelial Ca²⁺ channel (ecac), but not plasma membrane Ca²⁺-ATPase (pmca2) or Na⁺/Ca²⁺-exchanger (ncx1b). Whole-mount in situ hybridization revealed that the number of ecac-expressing ionocytes was reduced in fish raised in hypoxic water. These findings suggested that hypoxic treatment suppressed the expression of ecac, thereby reducing Ca²⁺ influx. To further evaluate the potential mechanisms for the effects of hypoxia on Ca²⁺ regulation, a functional gene knockdown approach was employed to prevent the expression of HIF-1αb during hypoxic treatment. Consistent with a role for HIF-1αb in regulating Ca²⁺ balance during hypoxia, the results demonstrated that the reduction of Ca²⁺ uptake associated with hypoxic exposure was not observed in fish experiencing HIF-1αb knockdown. Additionally, the effects of hypoxia on reducing the number of ecac-expressing ionocytes was less pronounced in HIF-1αb-deficient fish. Overall, the current study revealed that hypoxic exposure inhibited Ca²⁺ uptake in developing zebrafish, probably owing to HIF-1αb-mediated suppression of ecac expression.