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

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.

The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4.

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).

The Nox family of NADPH oxidases: friend or foe of the vascular system?

NADPH (nicotinamide adenine dinucleotide phosphate) oxidases are important sources of reactive oxygen species (ROS). In the vascular system, ROS can have both beneficial and detrimental effects. Under physiologic conditions, ROS are involved in signaling pathways that regulate vascular tone as well as cellular processes like proliferation, migration and differentiation. However, high doses of ROS, which are produced after induction or activation of NADPH oxidases in response to cardiovascular risk factors and inflammation, contribute to the development of endothelial dysfunction and vascular disease. In vascular cells, the NADPH oxidase isoforms Nox1, Nox2, Nox4, and Nox5 are expressed, which differ in their activity, response to stimuli, and the type of ROS released. This review focuses on the specific role of different NADPH oxidase isoforms in vascular physiology and their potential contributions to vascular diseases.

Elevated heart rate triggers action potential alternans and sudden death. translational study of a homozygous KCNH2 mutation.

BACKGROUND: Long QT syndrome (LQTS) leads to arrhythmic events and increased risk for sudden cardiac death (SCD). Homozygous KCNH2 mutations underlying LQTS-2 have previously been termed "human HERG knockout" and typically express severe phenotypes. We studied genotype-phenotype correlations of an LQTS type 2 mutation identified in the homozygous index patient from a consanguineous Turkish family after his brother died suddenly during febrile illness. METHODS AND RESULTS: Clinical work-up, DNA sequencing, mutagenesis, cell culture, patch-clamp, in silico mathematical modelling, protein biochemistry, confocal microscopy were performed. Genetic analysis revealed a homozygous C-terminal KCNH2 mutation (p.R835Q) in the index patient (QTc ∼506 ms with notched T waves). Parents were I° cousins - both heterozygous for the mutation and clinically unremarkable (QTc ∼447 ms, father and ∼396 ms, mother). Heterologous expression of KCNH2-R835Q showed mildly reduced current amplitudes. Biophysical properties of ionic currents were also only nominally changed with slight acceleration of deactivation and more negative V50 in R835Q-currents. Protein biochemistry and confocal microscopy revealed similar expression patterns and trafficking of WT and R835Q, even at elevated temperature. In silico analysis demonstrated mildly prolonged ventricular action potential duration (APD) compared to WT at a cycle length of 1000 ms. At a cycle length of 350 ms M-cell APD remained stable in WT, but displayed APD alternans in R835Q. CONCLUSION: Kv11.1 channels affected by the C-terminal R835Q mutation display mildly modified biophysical properties, but leads to M-cell APD alternans with elevated heart rate and could precipitate SCD under specific clinical circumstances associated with high heart rates.

N-terminal arginines modulate plasma-membrane localization of Kv7.1/KCNE1 channel complexes.

BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks)) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 ß-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks) and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for I(Ks) modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks) resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA') were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks). Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.

Hepatocyte growth factor induces a proangiogenic phenotype and mobilizes endothelial progenitor cells by activating Nox2.

Hepatocyte growth factor (HGF) by stimulating the receptor tyrosine kinase c-Met induces angiogenesis and tissue regeneration. HGF has been shown to antagonize the angiotensin II-induced senescence of endothelial progenitor cells (EPCs), which is mediated by NADPH oxidase-dependent reactive oxygen species (ROS) formation. As growth factors, however, usually require ROS for their signaling, we hypothesized that the proangiogenic effects of HGF require NADPH oxidases and focused on the homolog Nox2, which is most abundantly expressed in EPCs and endothelial cells. Indeed, HGF increased the H(2)O(2) formation in EPCs and human umbilical vein endothelial cells (HUVECs), and this effect was not observed in Nox2-deficient cells. HGF induced the mobilization of EPCs and vascular outgrowth from aortic explants in wild-type (WT) but not Nox2(y/-) mice. HGF also stimulated migration and tube formation in HUVECs, and antisense oligonucleotides against Nox2 prevented this effect. To identify the signal transduction underlying these effects, we focused on the kinases Jak2 and Jnk. In HUVECs, HGF increased the phosphorylation of these in a Nox2-dependent manner as demonstrated by antisense oligonucleotides. Also, the HGF-induced Jak2-dependent activation of a STAT3 reporter construct was attenuated after downregulation of Nox2. Accordingly, the HGF-stimulated tube formation of HUVEC was blocked by inhibitors of Jak2 and Jnk. In vivo treatment with the Jnk inhibitor SP600125 blocked the HGF-induced mobilization of EPCs. Ex vivo, SP600125 blocked HGF-induced migration and tube formation. We conclude that HGF-induced mobilization of EPCs and the proangiogenic effects of the growth factor require a Nox2-dependent ROS-mediated activation of Jak2 and Jnk.