Divergent regulation of cardiac KCND3 potassium channel expression by the thyroid hormone receptors α1 and β1

Abstract

The cardiac transient outward current I(to) is regulated by thyroid hormone (T3). However, it remains unclear whether T3 directly modulates underlying gene transcription and which thyroid receptor (TR) isoform might be responsible for gene transactivation. To clarify this situation, we analysed the role of T3 and its receptors alpha1 (TRalpha1) and beta1 (TRbeta1) in regulation of KCNA4, KCND2, KCND3 and KCNIP2 transcription in rat cardiomyocytes. Initial results demonstrated a T3-mediated increase of I(to) current density. T3 stimulation enhanced KCND2 and KCND3 expression and decreased KCNA4 transcription, while KCNIP2 remained unaffected. To dissect the role of TRalpha1 and TRbeta1 in T3-dependent I(to) modulation, TRalpha1 and TRbeta1 were overexpressed in cardiomyocytes by adenovirus-mediated gene transfer. TRalpha1 increased I(to), while TRbeta1 significantly reduced I(to) in size, which was associated with TRalpha1-mediated increase and TRbeta1-mediated reduction of KCND2/3 transcription. To further evaluate a possible direct interaction of TRalpha1 and TRbeta1 with the KCND3 promoter, TR expression vectors were cotransfected with a construct containing 2335 bp of the KCND3 5'-flanking sequence linked to a luciferase reporter into ventricular myocytes. While the TRalpha1 aporeceptor enhanced KCND3 transcription, the TRbeta1 aporeceptor suppressed KCND3 expression, with both effects exhibiting ligand-dependent amplification upon T3 stimulation. Deletion of the KCND3 5'-flanking region localized the suppressible promoter sequence for TRbeta1 to within -293 bp and the activating promoter sequence for TRalpha1 to within -2335 to -1654 bp of the transcription start site. Disruption of putative TR binding sites by mutagenesis abolished the TRalpha1- (G-1651T) and TRbeta1- (G-73T) mediated effects, indicating that TRalpha1 and TRbeta1 response elements map to different regions of the KCND3 promoter. Thus, I(to) is modulated by diverse T3-dependent regulation of underlying gene transcription. TRalpha1 and TRbeta1 exhibit distinct effects on KCND3 transactivation with TRalpha1 enhancing and TRbeta1 suppressing KCND3 transcription.