Divergent regulation of cardiac KCND3 potassium channel expression by the thyroid hormone receptors alpha1 and beta1

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.

Figure 1. KCND3 5′-flanking nucleotide sequence with TRE half-site consensus and half-site consensus sequence for retinoid acid receptor (RAR) or vitamin D3 receptor (VDR) binding

The point mutations G-1651T, G-707T and G-73T were introduced by site-directed mutagenesis to disrupt putative TREs.

Figure 2. Effect of T3 on native I_to and underlying KCNA4, KCND2, KCND3 and KCNIP2 expression

Families of I_to currents (A) and current density–voltage relations (B) demonstrate that T3 treatment significantly increased I_to current density. Values are means ±s.e.m.C, increased KCND2, KCND3 and diminished KCNA4 mRNA levels were found in T3-stimulated cardiomyocytes. In contrast, KCNIP2 transcript was not affected by T3. Gene expression was normalized to the ribosomal 18S signal. M, molecular weight (123 bp DNA ladder). *P < 0.05 versus control.

Figure 3. TRα1 increased native I_to in cardiomyocytes, while overexpression of TRβ1 resulted in a significant reduction of I_to

Representative original current traces recorded in infected myocytes (A) and current density–voltage relations of each group (B) demonstrated that expression of exogenous TRα1 increased I_to current density which was even more pronounced upon T3 treatment. Conversely, overexpression of TRβ1 significantly suppressed I_to1 current size. Control cells were infected with adenoviral vectors expressing EGFP alone. *P < 0.05 versus control, #P < 0.05 compared with control+T3.

Figure 4. RT-PCR revealed abundant KCND2 and KCND3 expression in TRα1-infected cardiac cells, whereas diminished KCND2 and KCND3 levels were detected in TRβ1-expressing myocytes

KCNA4 expression was reduced in T3-treated control cells. However, KCNA4 mRNA was largely unaffected by exogenous TRα1 or TRß1 overexpression. Representative ethidium bromide-stained agarose gels of RT-PCR products (left) and densitometric analysis of band intensities (right) from at least three independent experiments. *P < 0.05 versus control, **P < 0.05 compared with TRα1, #P < 0.05 compared with TRα1+T3.

Figure 5. Enhancement of KCND3 transcription by the TRα1 aporeceptor, while the TRβ1 aporeceptor suppressed KCND3 expression, with both effects exhibiting ligand-dependent amplification

TR expression vectors were cotransfected with pGL3-2335KCND3 into neonate cardiocytes. Results are expressed as fold activation compared to basal luciferase activity in pGL3-2335KCND3-transfected cells and represent means ±s.e.m. of three to nine separate experiments done in triplicate; *P < 0.05 versus control.

Figure 6. TRα1 and TRβ1 response elements map (A–C) and bind (D) to different regions of the KCND3 promoter

A, TR expression vectors were cotransfected with various 5′-fragments of the KCND3 promoter linked to the luciferase reporter into neonate cardiocytes. B, truncation of the KCND3 promoter to −1654 bp, −661 bp and −293 bp of the 5′-flanking sequence eliminated the TRα1-dependent increase in luciferase activity. C, conversely, cotransfection with TRβ1 resulted in an inhibition of luciferase reporter activity with each of the KCND3 promoter fragments tested. Results are expressed as fold activation compared to basal luciferase activity in pGL3-1654KCND3, pGL3-661KCND3, and pGL3-293KCND3 transfected cells, respectively. D, TR expression vectors were cotransfected with constructs carrying mutations of putative TREs of the KCND3 promoter linked to the luciferase reporter into cardiocytes. Mutation of nucleotide G-1651T (pGL3-2335KCND3mut) of the KCND3 promoter eliminated the TRα1-dependent increase in luciferase activity upon T3 stimulation. Mutation of nucleotide G-707T (pGL3-1654KCND3mut) of the 5′-fragment of the KCND3 promoter did not alter the suppressible effect of T3-liganded TRβ1. Conversely, mutation of nucleotide G-72T (pGL3-293KCND3mut) abolished the reduction of luciferase activity by TRβ1. Results are expressed as fold activation following T3 exposure compared to basal luciferase activity, and represent means ±s.e.m. of three to nine separate experiments done in triplicate; *P < 0.05 versus control.