Teratogenicity by the hERG potassium channel blocking drug almokalant: use of hypoxia marker gives evidence for a hypoxia-related mechanism mediated via embryonic arrhythmia

The rapid component of the delayed rectifying potassium ion current (IKr), plays an important role in cardiac repolarization. In rats, potent IKr channel blocking drugs cause similar stage-specific malformations (such as orofacial clefts and digital reductions) on gestational days (GDs) 10-14 as after periods of embryonic oxygen deprivation (hypoxia). The idea of a hypoxia-related teratogenic mechanism is supported by studies using rat embryos cultured in vitro. These studies show that the embryonic heart reacts with concentration-dependent bradycardia, arrhythmia, and cardiac arrest when exposed to IKr blockers on GDs 10-14. The main purpose of this study was to investigate whether previously shown teratogenic doses on GD 11 and 13 of the selective IKr blocker almokalant (ALM) induce hypoxia in rat embryos in vivo by using the hypoxia marker pimonidazole hydrochloride (PIM). Rats were orally dosed with almokalant or tap water on GD 11 (150 micromol/kg), 13 (50 micromol/kg), or 16 (800 micromol/kg), followed by PIM intravenously 30 min later. Two hours after the PIM dose, the embryonic heart activity was videotaped and analysed, and the embryos were fixed, sectioned, and immunostained. Computer-assisted image analysis showed a two- and threefold increase in hypoxia staining in embryos exposed to teratogenic doses of ALM on GDs 11 and 13. Embryonic arrhythmia was observed in almokalant groups on these GDs, but not in controls. In contrast, dosing on GD 16, with a much higher dose (800 micromol/kg), caused neither hypoxia nor any effects on heart rhythm. The results support the IKr-related arrhythmia-hypoxia hypothesis, by showing that the potent IKr-blocking drug, almokalant, (1) causes severe embryonic hypoxia and arrhythmia at stages (GDs 11 and 13) when developmental toxicity could be induced and IKr is functional and (2) does not cause hypoxia or affect heart rhythm at a developmental stage when IKr is suppressed (GD 16) and potent IKr blockers do not induce developmental toxicity.