Role of the hERG-channel in arrhythmia and teratogenicity : studies in animal models and the human embryonic heart

Sammanfattning: Background: Drugs that inhibit cardiac repolarization are associated with potentially life threatening side effects in the form of ventricular arrhythmias in humans. Animal studies show that this mechanism also is relevant for the embryo, and that the circulatory depression results in hypoxia with embryotoxicity in the form of malformations and death as a consequence. This thesis addresses the pharmacological arrhythmogenic effects on the rodent embryonic heart, and highlight the human relevance of this mechanism by characterization of the human embryonic heart. Methods and Results: Pregnant mice were administered the hypoxia probe pimonidazole followed by phenytoin or saline at gestation day (GD) 10 or 15. Phenytoin treatment resulted in dose-dependent embryonic staining for the hypoxia probe at GD 10. At GD 15 staining was not dose-dependent and less pronounced compared to controls. The effect on cardiac rate and rhythm of antiepileptic drugs (AEDs) was studied in cultured GD 10 mouse embryos. Phenytoin, dimethadione, carbamazepine and phenobarbital induced concentration-dependent prolongation of the inter-beat interval (IBI) and irregular arrhythmias. Exposure to combinations of AEDs in therapeutic concentrations resulted in significant increase in IBI compared to single exposure. ECG was obtained before and after drug exposure from GD 11 rat embryos and embryonic cardiomyocytes (ECMs) cultured in multi-electrode array (MEA) culture dishes. In the embryo model phenytoin and the selective IKr blocker E4031 both induced concentration-dependent bradyarrhythmia and QTC prolongation in cultured GD11 rat embryos. At the higher tested concentrations, phenytoin induced cardiac arrest and E4031 induced AV-nodal block. In the ECM model sensitivity to phenytoin and E4031 was similar but other arrhythmias were observed. The distribution of Isl1+ progenitor cells and their proliferative and differentiating capacity in human first trimester embryonic hearts were determined by immunohistochemistry. Isl1+ cells were present in the heart and a few were Ki67+ and troponinT+. Beating clusters of human embryonic cardiomyocytes, called cardiospheres were derived from human embryonic hearts and characterized with immunohistochemistry, electron microscopy and in the MEA system. The spheres were sensitive to adrenergic stimulation with isoprenaline and displayed rate dependency of the action potential in a pacing experiment. Expression and function of the two components of the delayed rectifier potassium current (IK), IKr and IKs, were characterized in cardiac tissue and ECMs from human, rat and rabbit embryonic hearts. Patch clamp and quantitative RT-PCR were used. IKr was expressed and functional in all species. IKs expression was found in human and rat but not in rabbit hearts. Conclusions: Phenytoin induces dose-dependent embryonic hypoxia. The studied AEDs induce concentration-dependent embryonic bradycardia and arrhythmia. For selective IKr blockers and phenytoin, the effects are associated with QT prolongation. This indicates that QT prolongation can be used as a biomarker for embryonic arrhythmogenicity. Rat ECMs display a similar sensitivity as the embryonic heart but respond differently to drug exposure. Isl1+ cells are present in the human embryonic heart and cardiospheres derived from embryonic hearts display rate dependency of the action potential duration and sensitivity to β-adrenergic stimulation. The IKr current is expressed and functional in the human embryonic heart and in species used in teratology testing. The results support that drug-induced embryonic arrhythmia is a cause of embryotoxicity and indicate human relevance of this mechanism.