Estradiol regulates human QT-interval: acceleration of cardiac repolarization by enhanced KCNH2 membrane trafficking

Abstract

Background: Modulation of cardiac repolarization by sexual hormones is controversial and hormonal effects on ion channels remain largely unknown. In the present translational study, we therefore assessed the relationship between QTc duration and gonadal hormones and studied underlying mechanisms.

Methods and results: We measured hormone levels and QTc intervals in women during clomiphene stimulation for infertility and women before, during, and after pregnancy. Three heterozygous LQT-2 patients (KCNH2-p.Arg752Pro missense mutation) and two unaffected family members additionally were studied during their menstrual cycles. A comprehensive cellular and molecular analysis was done to identify the mechanisms of hormonal QT-interval regulation. High estradiol levels, but neither progesterone nor estradiol/progesterone ratio, inversely correlated with QTc. Consistent with clinical data, in vitro estradiol stimulation (60 pmol/L, 48 h) enhanced IKCNH2. This increase was mediated by estradiol receptor-α-dependent promotion of KCNH2-channel trafficking to the cell membrane. To study the underlying mechanism, we focused on heat-shock proteins. The heat-shock protein-90 (Hsp90) inhibitor geldanamycin abolished estradiol-induced increase in IKCNH2. Geldanamycin had no effect on KCNH2 transcription or translation; nor did it affect expression of estradiol receptors and chaperones. Estradiol enhanced the physical interaction of KCNH2-channel subunits with heat-shock proteins and augmented ion-channel trafficking to the membrane.

Conclusion: Elevated estradiol levels were associated with shorter QTc intervals in healthy women and female LQT-2 patients. Estradiol acts on KCNH2 channels via enhanced estradiol-receptor-α-mediated Hsp90 interaction, augments membrane trafficking and thereby increases repolarizing current. These results provide mechanistic insights into hormonal control of human ventricular repolarization and open novel therapeutic avenues.

Keywords: Gender; Hormones; Ion channels; Long-QT syndrome; Repolarization.

Figure 1

QTc modulation in response to stimulation and pregnancy. (A) Exemplary illustration of in vivo clomiphene effects on hormones and QTc (lead II) in a healthy female. (B) Median values of baseline and clomiphene-stimulated conditions (n = 11). (C) Exemplary illustration of hormone levels and QTc in a healthy female before, during, and after pregnancy. (D) Courses of estradiol and QTc of five females during pregnancies. Of note, QTc shortening coincided with increased estradiol levels. Lines were added for illustration, and symbols represent probands. E, pooled data (pregnant and stimulated; n = 16). Higher estradiol concentrations were associated with QTc shortening (P = 0.000001). E2, estradiol; prog., progesterone; 1^st, 1st trimester; 3^rd, 3rd trimester; pp, postpartum.

Figure 2

Long-term estradiol enhances I_KCNH2 through oestrogen receptor-α-dependent pathway. (A) Representative currents of HEK cells transfected with KCNH2 and oestrogen receptor-α cDNA (protocol in inset). I_KCNH2 increased after estradiol stimulation (60 pmol/L, 48 h). This was abolished by co-incubation with tamoxifen (200 nmol/L). Mean ± SD values for depolarization (‘step’) and repolarization (‘tail’)-induced currents are shown (B, n = 4–6 cells). (C) Acute estradiol exposure did not affect KCNH2 currents in identically transfected cells (mean ± SD, n = 10 each). E2, estradiol; ER, oestrogen receptor; tam, tamoxifen; TP, test potential; pA, pico Ampere; pF, pico Farad.

Figure 3

Estradiol does not affect KCNH2 synthesis but enhances trafficking. Blots are representative of 3–6 experiments with input and pharmacological interventions outlined above each panel, bar graphs at the side represent mean ± SD arbitrary optical densities (OD) for individual lanes. (A) Crude membrane preparations illustrating no change in overall KCNH2 protein expression with ERα co-transfection or estradiol stimulation (60 pmol/L, 48 h). Calnexin expression indicated equal protein loading. (B) Plasma membrane preparations of cells transfected with KCNH2 and ERα showed greater KCNH2 plasma membrane expression with ERα co-transfection and estradiol stimulation (60 pmol/L, 48 h, lane 4, P = 0.047 vs. all others). This effect was abolished by co-incubation with tamoxifen (200 nmol/L). Arrows indicate fully- and core-glycosylated KCNH2 bands. Membrane-marker spectrin was present in the plasma membrane fraction, while calnexin (endoplasmic reticulum marker) was absent. (C) Plasma membrane preparation with KCNH2 and ERβ showed no difference in KCNH2 expression suggesting specificity of the ERα pathway. (D) No effect of estradiol stimulation for plasma membrane preparations from ERα and KCNQ1 co-transfected cells. NT, non-transfected; Q1, KCNQ1; kD, kilo Dalton; AU, arbitrary units. *ANOVA. Other abbreviations are as above.

Figure 4

Enhanced KCNH2 membrane trafficking in response to ERα-mediated estradiol stimulation. (A) (left to right) Cells transfected with KCNH2 (green) alone; with estradiol stimulation (60 pmol/L); KCNH2 and ERα (red); additional estradiol stimulation. Yellow indicates co-localization. Estradiol had no effect on KCNH2 localization without the receptor. Stimulation of co-transfected cells (KCNH2 + ERα) led to nuclear translocation and more prominent KCNH2 expression at the plasma membrane. (B) Analogous experiments with KCNH2 and ERβ. Estradiol led to receptor translocation into the nucleus without effect on KCNH2 membrane localization. (C) Experiments with KCNQ1 and ERα similarly show unchanged KCNQ1 distribution. Images are representative of at least 4–6 experiments. The insets in (A–C) illustrate the optical density along the white bar across the cells. (D) Estradiol stimulation significantly increased the plasma membrane/endoplasmic reticulum ratio in stimulated ERα co-transfected cells. There was no difference for KCNH2 + ERβ or KCNQ1 + ERα (mean ± SD). Bars = 5 µm. End-ret, endoplasmic reticulum. Other abbreviations are as above.

Figure 5

Enhanced chaperone/channel interaction with estradiol stimulation. (A) Estradiol stimulation did not alter mRNA transcription of KCNH2 or ERα. (B) Western blots for various KCNH2 chaperones indicated unchanged expression levels under various transfection conditions (indicated above the blots) with or without estradiol stimulation. The open arrow indicates the specific (62 kDa), the black arrow the unspecific band of STIP-1. (C) mRNA levels of Hsc70 and Hsp90 remained unchanged in various transfection constellations with or without estradiol stimulation. (D) Co-immunoprecipitation of Hsc70 and Hsp90 with KCNH2 illustrated enhanced precipitation of both chaperones with KCNH2 during estradiol stimulation, and bar graphs illustrate mean ± SD data (N = 4). Grp94 did not interact with KCNH2 protein. Hsc70, Heat-shock cognate protein 70kD; Hsp90, Heat-shock protein 90kD; STIP1, Hsc70/Hsp90 organizing protein; DNAJA1, Heat-shock protein 40kD; Grp94, 94kD glucose regulated protein; FKBP38, 38kD FK506-binding protein. *ANOVA. Other abbreviations are as above.

Figure 6

Inhibition of Hsp90 function abrogates enhanced KCNH2 trafficking and ionic current. (A) Plasma membrane preparations of cells transfected with KCNH2 and ERα and exposed to estradiol stimulation (60 pmol/L, 48 h) in the presence or absence of the Hsp90 inhibitor geldanamycin (1 µg/mL). The level of KCNH2 membrane expression was greatly reduced by geldanamycin exposure. (B) KCNH2 currents were similarly reduced with geldanamycin (mean ± SD, n = 10 and 3 cells, respectively). Gelda, geldanamycin. Other abbreviations are as above.

Figure 7

QTc changes during menstrual cycle of female LQT-2 patients. (A) Pedigree of the KCNH2-R752P family. Arrows indicate symptomatic index patients who survived cardiac arrest. Grey are genotype-positives, and hatched lines are unknown genotypes. Asterisks indicate women participating in our study. (B and C) Examples of QTc measurements and respective hormone levels (including estradiol/progesterone ratio) for one genotype-positive (IV-5) and one genotype-negative family member (IV-10) during the menstrual cycle. (D) QTc values from genotype-positive (n = 3) and genotype-negative (n = 2) individuals stratified according to their median of hormone concentrations. High estradiol was associated with abbreviated QTc, whereas no QTc difference was apparent in relation to progesterone, estradiol/progesterone ratio, and serum potassium. Bottom and top of the box are 25th and 75th percentile, the band represents the median, and whiskers illustrate 95% confidence intervals. Broken, horizontal lines beginning at the median of the higher strata have been added for better visual discrimination. Abbreviations are as above.

Figure 8

Enhanced trafficking depends on functional KCNH2 channels. (A) Currents recorded from cells transfected with the trafficking-deficient KCNH2-R752P displayed prominently distorted biophysical behaviour with a rapidly activating and inactivating current that did not give rise to repolarization induced ‘tail’ current. (B) Current–voltage relations for cells expressing KCNH2-R752P alone or in combination with wild-type KCNH2 cDNA. An increase in I_KCNH2 in response to estradiol occurred if wild-type and mutant were co-transfected. (C) Plasma membrane preparations of KCNH2 and KCNH2-R752P indicate almost absent KCNH2-R752P at the plasma membrane and no rescue of mutant protein with estradiol (n = 3). (D) Quantifications of mRNA illustrate unchanged KCNH2-R752P and oestrogen receptor expression. (E) Confocal images confirm lack of enhanced plasma membrane trafficking of KCNH2-R752P with estradiol. Bars = 5 µm. Images are representative of at least 4–6 experiments. (F) Co-immunoprecipitation of KCNH2-R752P with Hsc70 and Hsp90 indicated an unaltered level of interaction. Mean ± SD of n = 12 and 13 cells, respectively. *ANOVA for time series. Abbreviations are as above.