hERG Protein Expression Research Articles

Deacetylation mechanism and potential reversal strategy of long QT syndrome on hERG K+ channel under hypoxia

Clinically, hypoxia is a major risk factor for long QT syndrome (LQTS), which is associated with many diseases, such as myocardial ischemia. LQTS can be caused by the deficiency of hERG, a potassium ion channel that plays a key role in cardiac repolarization. Modifications such as acetylation of histones or non-histone proteins can affect the protein expression. In the present study, we explored the mechanism underlying hypoxia-induced LQTS and a potential reversal strategy. Experiments were performed under hypoxia to determine transcriptional and post-transcriptional expression changes. We used real-time PCR, chromatin immunoprecipitation assay, and western blotting to determine the histones acetylation in the hERG gene and the mechanism. Molecular docking, western blotting, IP, and patch -clamp assay were performed to determine the acetylation and ubiquitination levels of hERG protein and the mechanism. hERG mRNA and protein expression were found to decrease under hypoxia. The histone deacetylation level increased under hypoxia at both H3K27 and H4 of the hERG gene. HDAC1 and HDAC2 are the key enzymes for the mechanism. HDAC6 directly interacts with hERG. The acetylation level of hERG decreased and the ubiquitination level of hERG increased under hypoxia. The inhibitors of HDAC1, HDAC2, and HDAC6 could reverse the reduction of hERG mRNA and hERG protein expression under hypoxia. In conclusion, deacetylation of hERG gene-associated histones and hERG protein might be the mechanisms for LQTS in patients with hypoxia, and the inhibition of HDAC1, HDAC2, and HDAC6 might be a promising reversal strategy for reducing hERG expression under different pathological conditions.

Downregulation of hERG channel expression by tyrosine kinase inhibitors nilotinib and vandetanib predominantly contributes to arrhythmogenesis

Cardiotoxicity by tyrosine kinase inhibitors remains an important concern. Nilotinib and vandetanib clinically carry high proarrhythmic risk and the exact mechanism underlying arrhythmogenesis is not fully understood. In this study, we investigated the effects of nilotinib and vandetanib on the abundance of human ether-á-go-go-related gene (hERG) K+ channel and assessed the potential role of acute hERG blockage versus chronic effects in arrhythmogenesis. We found that both nilotinib and vandetanib prolonged the field potential duration reflecting the repolarisation process and induced cellrythmias of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in a time-and concentration-dependent manner after, after chronic exposure. Patch-clamp recordings revealed significant reductions of hERG current densities by nilotinib or vandetanib after chronic incubation with hERG-HEK293 cells in addition to the acute inhibition. Western blot analysis showed that nilotinib and vandetanib decreased mature hERG protein (155-kDa) expression, in a greater extent than that of the immature form (135-kDa). A serum and glucocorticoid kinase 1 (SGK1) activator, C4-ceramide, prevented the nilotinib-and vandetanib-induced hERG protein downregulation and thus the incidence of cellrrhythmias. Taken together, our data demonstrated that the downregulation of hERG channel abundance on the cellular membrane predominantly contributed to the proarrhythmic effect of nilotinib and vandetanib.

Pharmacological corrections of the mutant hERG channels by posaconazole.

Properties of mutant human ether-à-go-go-related gene (hERG) channels can be modified by some antibiotics. However, the pharmacological effects of posaconazole on cardiomyocyte hERG channels remain unclear. Whole-cell patch clamping, western blotting and laser confocal scanning microscopy were used to evaluate the effects of posaconazole on wild-type (WT)-, A561V- and L539fs/47-hERG channels expressed in human embryonic kidney (HEK) 293 cells. In electrophysiological experiments, HEK 293 cells were transiently co-transfected with equal amounts of WT-hERG, WT+A561V-hERG and WT+L539fs/47-hERG plasmids to mimic a heterozygous genotype. Posaconazole (30μM) increased tail currents in cells expressing WT-hERG, WT+A561V-hERG and WT+L539fs/47-hERG by 82.65%, 147.72% and 134.73%, respectively, compared to controls. Posaconazole increased hERG protein expression in cells expressing WT-hERG, WT+A561V-hERG and WT+L539fs/47-hERG compared to controls condition as well as their trafficking to the cell membrane. To our knowledge, this is the first study to show that antifungal agent posaconazole rescues the mutant A561V-hERG and L539fs/47-hERG channels by altering the gating kinetics, enhancing the expression and trafficking of hERG channels. The results demonstrate that posaconazole could be a promising candidate for the prevention and treatment of long QT syndrome and other arrhythmia-related diseases.

Hypoxia induced hERG trafficking defect linked to cell cycle arrest in SH-SY5Y cells.

The alpha subunit of the voltage gated human ether-a-go-go-related (hERG) potassium channel regulates cell excitability in a broad range of cell lines. HERG channels are also expressed in a variety of cancer cells and control cell proliferation and apoptosis. Hypoxia, a common feature of tumors, alters gating properties of hERG currents in SH-SY5Y neuroblastoma cells. In the present study, we examined the molecular mechanisms and physiological significance underlying hypoxia-altered hERG currents in SH-SY5Y neuroblastoma cells. Hypoxia reduced the surface expression of 150kDa form and increased 125kDa form of hERG protein expression in the endoplasmic reticulum (ER). The changes in protein expression were associated with ~50% decrease in hERG potassium conductance. ER retention of hERG 125kDa form by CH was due to defective trafficking and was rescued by exposing cells to hypoxia at low temperatures or treatment with E-4031, a hERG channel blocker. Prolonged association of hERG with molecular chaperone Hsp90 resulting in complex oligomeric insoluble aggregates contributed to ER accumulation and trafficking defect. Hypoxia increased reactive oxygen species (ROS) levels and manganese (111) tetrakis (1methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant prevented hypoxia-induced degradation of 150kDa and accumulation of 125kDa forms. Impaired trafficking of hERG by hypoxia was associated with reduced cell proliferation and this effect was prevented by antioxidant treatment. These results demonstrate that hypoxia through increased oxidative stress impairs hERG trafficking, leading to decreased K+ currents resulting in cell cycle arrest in SH-SY5Y cells.

Intracellular Mechanism of Rosuvastatin-Induced Decrease in Mature hERG Protein Expression on Membrane.

The hERG potassium channel (IKr) encoded by human ether-a-go-go-related gene plays an important role in cardiac repolarization. Decreased IKr may lead to long QT syndrome, which subsequently causes torsade de pointes and sudden cardiac death. Previous studies have shown that statins inhibit IKr and are more potent in inhibiting hERG currents when combined with other drugs. Since chemical structure of rosuvastatin is similar to that of several IKr blockers (ibutilide and E-4031), the present study aimed to reveal the mechanism that underlies rosuvastatin-induced hERG current reduction and to evaluate the possibility of cardiac toxicity. The results showed that rosuvastatin reduced hERG currents by accelerating the inactivation and prolonged action potential duration (APD) in hiPSC-CMs. Meanwhile, it was observed that rosuvastatin reduced the expression of the mature hERG. Transcription factor Sp1 was involved in hERG protein downregulation induced by rosuvastatin, and the result was verified by Sp1 siRNA and Sp1 agonist epicatechin. These results indicated that rosuvastatin could potentially inhibit transcription and reduce hERG mRNA expression. The interaction between hERG and heat shock protein was evaluated to study the mechanism of trafficking inhibition through co-immunoprecipitation. We found that rosuvastatin reduces the interaction of heat shock protein 70 (Hsp70) with the hERG protein, thereby affecting the folding of the hERG channel. Additionally, rosuvastatin significantly activates ATF6, which plays a key role in the activation of the unfolded protein response (UPR) pathway. Increased expression of the molecular chaperone calnexin and calreticulin, which are activated by ATF6 to help channel folding, further confirmed UPR activation. Meanwhile, the degradation of the hERG channel was mediated by lysosomes and proteasomes. In conclusion, Rosuvastatin reduced the expression of hERG plasma membrane by two pathways, the first is to disrupt the transport of immature hERG channels to the membrane, and the second is to increase the degradation of mature hERG channels. In addition, Rosuvastatin potently blocked hERG current, delayed cardiac repolarization, and thereby prolonged APDs and QTc intervals. Therefore, caution should be taken when rosuvastatin is used in the treatment of hyperlipidemia, especially when combined with drugs that can prolong the QT interval.

Anti-Ro52 antibody acts on the S5-pore linker of hERG to chronically reduce channel expression.

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (IKr). Malfunction of hERG/IKr is the primary cause of acquired long QT syndrome (LQTS), an electrical disorder of the heart that can cause arrhythmias and sudden death. Patients with autoimmune diseases display a high incidence of LQTS. While dysfunction of hERG channels induced by autoantibodies such as anti-Ro52 may play a role in this pathology, the underlying mechanisms are not well understood. Here, we investigated the acute and chronic effects of anti-Ro52 antibody on hERG channels stably expressed in human embryonic kidney (hERG-HEK) 293 cells as well as IKr in neonatal rat ventricular myocytes. Using whole-cell patch clamp, western blot analyses, and immunocytochemistry, we found that a 12-h treatment of hERG-HEK cells with patients' sera containing anti-Ro52 autoantibody decreased the hERG current (IhERG) by 32% compared to cells treated with autoantibody-negative patients' sera. Commercial anti-Ro52 antibody at 100 µg/mL did not acutely block IhERG. Instead, a 12-h treatment with anti-Ro52 antibody at a concentration of 4 µg/mL significantly reduced mature hERG protein expression and IhERG. Specifically, anti-Ro52 antibody did not acutely block hERG current but chronically facilitated hERG endocytic degradation. The extracellular S5-pore linker of hERG, which forms the turret of the channel on the outside of the cell, is the target region for anti-Ro52-mediated hERG reduction since its replacement with the analogous region of EAG abolished the anti-Ro52 effect. In neonatal rat ventricular myocytes, 100 µg/mL anti-Ro52 antibody did not acutely block IKr, but a 12-h treatment of cells with 4 µg/mL anti-Ro52 antibody selectively reduced IKr and prolonged the action potential duration. Our results indicate that anti-Ro52 antibody acts on the hERG S5-pore linker to chronically decrease hERG expression and current. These findings provide novel insights into hERG regulation and anti-Ro52 antibody-associated LQTS.

The role and mechanism of chaperones Calnexin/Calreticulin in which ALLN selectively rescues the trafficking defective of HERG-A561V mutation.

Long QT (LQT) type 2 (LQT2) is caused by HERG mutation. L539fs/47 encodes a truncated protein, and its mechanisms in HERG mutation are unknown. HERG mutation plasmids were overexpressed in HEK293T cells, respectively, followed by analyzing lysates with Western blot. Transfected HEK293T cells were treated with or without N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) and Propranolol (Prop) at 24 or 48 h. HERG-WT, HERG-A561V, WT/A561V, HERG-L539fs/47, WT/L539fs/47, and Calnexin (CNX)/Calreticulin (CRT) protein expression and their interactions were detected by Western blot and immunoprecipitation. Each group with HERG currents (Ikr) were detected by Patch-clamp technique. Treated with ALLN, the expression of mature HERG protein and the CNX/CRT protein increased. The interaction of HERG-A561V and WT/A561V protein with the chaperone CNX/CRT increased significantly. The maximum peak currents and tail currents density increased by 70% and 73%, respectively, while maximal peak currents density (24%) and tail currents density (19%) were slight increased in WT-HERG cells. Treated with Prop, the expression and interaction of mature HERG and chaperones CNX/CRT had no difference in each group. The maximal currents and tail currents density were slight increased. CNX/CRT might play a crucial role in the trafficking-deficient process and degradation of HERG-A561V mutant protein, however they had no effect on L539fs/47 HERG due to protein transport deletion. ALLN can restore HERG-A561V mutant protein trafficking process and rescue the dominant negative suppression of WT-HERG.

MiRNAs Regulate hERG.

The human ether-a-go-go-related gene (hERG) is the major molecular component of the rapidly activating delayed rectifier K+ current (Ikr ). Impairment of hERG function is believed to be a mechanism causing long-QT syndromes (LQTS). Growing evidences have shown that microRNAs (miRNAs) are involved in functional modulation of the hERG pathway. The purpose of this study was to screen and validate miRNAs that regulate the hERG pathway. The miRNAs identified in this study will provide new tools to assess the mechanism of LQTS. Six miRNAs were selected by algorithm predictions based on potential interaction with hERG. The effects of each miRNA on hERG were assessed by use of the Dual-Luciferase Reporter assay system, qRT-PCR, Western blotting, and confocal fluorescence microscopy. Furthermore, whole-cell patch clamp technique was used to validate the effect of miR-103a-1 on the electrophysiological characteristic of the Ikr of the hERG protein channel. miR-134, miR-103a-1, miR-143, and miR-3619 significantly downregulated luciferase activity (P < 0.05) in a reporter test system. These 4 miRNAs significantly suppressed expression of hERG mRNA and protein in U2OS cells (P < 0.05).Corresponding AMOs rescued expression of hERG mRNA and protein. Confocal microscopy showed that all 4 miRNAs reduced the expression of both immature and mature hERG protein. miR-103a-1 decreased the maximum current and tail current amplitudes of hERG channel. Expression and functions of hERG are regulated by specific miRNAs.

Fluconazole-induced long QT syndrome via impaired human ether-a-go-go-related gene (hERG) protein trafficking in rabbits.

hERG protein trafficking deficiency has long been known in drug-induced long QT syndrome (LQTS). However, validated evidence from in vivo data kept scanty. Our goal was to investigate the proarrhythmic action of fluconazole and its underlying mechanism in an animal model. Twenty female Japanese long-eared white rabbits were randomly distributed into a control group and a fluconazole group for a chronic 2-week treatment. The control group was treated with 0.5% sodium carboxymethylcellulose (CMCNa), and the fluconazole group was treated with fluconazole. Electrocardiograms (ECGs) were recorded during the experimental period. Isolated arterially perfused left ventricular wedge preparations from the rabbits were made 2 weeks after treatment, and the arrhythmia events, the transmural ECG, and action potential from both the endocardium and epicardium were recorded. The changes in hERG protein expression were measured by western blot. The fluconazole group showed a longer QT interval 1 week after treatment (P < 0.05) and a higher arrhythmia occurrence 2 weeks after treatment (P < 0.05) than the control group. The fluconazole group also showed a longer transmural dispersion of repolarization and a higher occurrence of life-threatening torsades de pointes in arterially perfused left ventricular preparations. Furthermore, western blot analysis showed that the density of mature hERG protein was lower in the fluconazole group than that in the control group. Fluconazole can prolong the QT interval and possess proarrhythmic activity due to its inhibition of hERG protein trafficking in our experimental model. These findings may impact the clinical potential of fluconazole in humans.

A Molecular Substrate for Long QT in HIV Patients: Tat Protein Reduces IKR in Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes

Compared to the general population, individuals with HIV have a 4.5 fold higher risk of sudden cardiac death, and up to 20% of them present with a long QT syndrome (LQTS). Notably, torsades de pointes arrhythmias have been described even in the absence of drug therapy, suggesting a rather direct implication of HIV in LQTS. The HIV-encoded Tat protein appears as a potential candidate for inducing ventricular arrhythmias, since it was shown to prolong the action potential (AP) of guinea-pig ventricular cardiomyocytes by reducing IKr. Tat protein serves as a transactivator of transcription required for HIV replication. Tat is released from infected cells and is found circulating in the blood of HIV-infected patients.The aim of this study was to evaluate the human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CMs) as a model to study the cellular mechanisms involved in the Tat-dependent alteration of cardiac electrical activity in human.Our data show that Tat incubation reduces IKr in hiPS-CMs while it does not alter IhERG in transfected COS-7 and HEK293 cellular models. hERG protein expression was reduced only in Tat-incubated hiPS-CMs; such reduction likely contributes to IKr reduction. Ventricular AP durations (APD70 and APD90) were significantly increased in hiPS-CMs incubated with Tat compared to buffer incubation. In addition, most Tat-treated cells showed a higher APD90 dispersion which resulted from AP duration and amplitude alternans. Alternans were exacerbated at faster stimulation rates.This work highlights that Tat-treated hiPS-CMs recapitulate alterations of the cardiomyocyte electrical activity, consistent with the arrhythmias observed in HIV patients. hiPS-CMs represent a relevant model for further investigations of the cellular mechanisms involved in AIDS, and, more generally, in cardiac non-genetic diseases.

Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia.

Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+) current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein degradation by IH.

Reduced expression of voltage-gated Kv11.1 (hERG) K(+) channels in aganglionic colon in Hirschsprung's disease.

The pathophysiology of Hirschsprung's disease (HSCR) is not entirely understood. There is no clear explanation for the occurrence of the spastic or tonically contracted aganglionic segment of bowel. Kv11.1 (hERG) channels play a critical role in the regulation of the resting membrane potential as well as affecting either the force or frequency of contraction of smooth muscles. We designed this study to investigate the expression and distribution of hERG channels in the normal colon and the colon of patients with HSCR. We investigated hERG protein expression in both the ganglionic and aganglionic regions of HSCR patients (n=10) versus normal control colon (n=10). Protein distribution was assessed using immunofluorescence and confocal microscopy. Gene and protein expressions were quantified using real-time polymerase chain reaction, western blot analysis and densitometry. Confocal microscopy of the normal colon revealed strong hERG channel expression in interstitial cells of Cajal, platelet-derived growth factor-alpha receptor- (PDGFRα(+)) positive cells and enteric neurons. hERG expression was markedly decreased in aganglionic bowel, whereas colonic hERG gene expression levels were significantly decreased in aganglionic compared to ganglionic bowel and controls (p<0.05). Western blotting revealed decreased colonic hERG protein expression in aganglionic HSCR specimens compared to controls. We demonstrate, for the first time, the expression and distribution of hERG channels in the human colon. The decreased expression of hERG in the aganglionic colon may be responsible for the increased tone in the aganglionic narrow spastic segment of bowel.

Mechanisms underlying probucol-induced hERG-channel deficiency.

The hERG gene encodes the pore-forming α-subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Reduction of IhERG due to genetic mutations or drug interferences causes long QT syndrome, leading to life-threatening cardiac arrhythmias (torsades de pointes) or sudden death. Probucol is a cholesterol-lowering drug that could reduce hERG current by decreasing plasma membrane hERG protein expression and eventually cause long QT syndrome. Here, we investigated the mechanisms of probucol effects on IhERG and hERG-channel expression. Our data demonstrated that probucol reduces SGK1 expression, known as SGK isoform, in a concentration-dependent manner, resulting in downregulation of phosphorylated E3 ubiquitin ligase Nedd4-2 expression, but not the total level of Nedd4-2. As a result, the hERG protein reduces, due to the enhanced ubiquitination level. On the contrary, carbachol could enhance the phosphorylation level of Nedd4-2 as an alternative to SGK1, and thus rescue the ubiquitin-mediated degradation of hERG channels caused by probucol. These discoveries provide a novel mechanism of probucol-induced hERG-channel deficiency, and imply that carbachol or its analog may serve as potential therapeutic compounds for the handling of probucol cardiotoxicity.

The protease inhibitor atazanavir blocks hERG K(+) channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane.

Atazanavir (ATV) is a HIV-1 protease inhibitor for the treatment of AIDS patients, which is recently reported to provoke excessive prolongation of the QT interval and torsades de pointes (TdP). In order to elucidate its arrhythmogenic mechanisms, we investigated the effects of ATV on the hERG K(+) channels expressed in HEK293 cells. hERG K(+) currents were detected using whole-cell patch clamp recording in HEK293 cells transfected with EGFP-hERG plasmids. The expression of hERG protein was measured with Western blotting. Two mutants (Y652A and F656C) were constructed in the S6 domain within the inner helices of hERG K(+) channels that were responsible for binding of various drugs. The trafficking of hERG protein was studied with confocal microscopy. Application of ATV (0.01-30 μmol/L) concentration-dependently decreased hERG K(+) currents with an IC50 of 5.7±1.8 μmol/L. ATV (10 μmol/L) did not affect the activation and steady-state inactivation of hERG K(+) currents. Compared with the wild type hERG K(+) channels, both Y652A and F656C mutants significantly reduced the inhibition of ATV on hERG K(+) currents. Overnight treatment with ATV (0.1-30 μmol/L) concentration-dependently reduced the amount of fully glycosylated 155 kDa hERG protein without significantly affecting the core-glycosylated 135 kDa hERG protein in the cells expressing the WT-hERG protein. Confocal microscopy studies confirmed that overnight treatment with ATV obstructed the trafficking of hERG protein to the cell membrane. ATV directly blocks hERG K(+) channels via binding to the residues Y652 and F656 in the S6 domain, and indirectly obstructs the transport of the hERG protein to the cell membrane.

Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins

We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

GW24-e1194 The proarrhythmic action of Fluconazole and the underlying mechanism

ObjectivesTo investigate the proarrhythmic action of fluconazole and the underlying mechanism.MethodsTwenty Japanese long-eared white rabbits were randomly distributed into Normal group and Fluconazole group, the Normal group were treated with...

Intermittent Hypoxia‐induced hERG degradation involves ROS Activated Calpains

Human ether‐go‐go‐related gene (hERG) protein, the alpha subunit of voltage gated potassium channel, mediates a variety of cellular functions including the cell excitability. Intermittent hypoxia (IH) is a hallmark feature of recurrent apnea. The present study examined the effects of IH on hERG protein expression in neuroblastoma (SH‐SY5Y) cells. IH decreased hERG current density by ~60%. The decreased current density was associated with stimulus‐dependent down‐regulation of hERG protein, which could not be rescued with proteasomal and lysosomal inhibitors. IH increased calpain activity and ALLM a calpain protease inhibitor prevented IH‐evoked degradation of hERG protein. Further analysis showed that IH‐induced calpain activation involves elevation of [Ca2+]I by reactive oxygen species. Similar downregulation of hERG was also observed in adrenal chromaffin cells from IH exposed neonatal rats, and anti‐oxidant treatment abolished this effect. The IH evoked hERG degradation might contribute to increased cell excitability and augmented catecholamine release from neonatal adrenal chrmoaffin cells.

Arsenic trioxide-induced hERG K+ channel deficiency can be rescued by matrine and oxymatrine through up-regulating transcription factor Sp1 expression

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating, delayed rectifier potassium channel (IKr) important for cardiac repolarization. Dysfunction of the hERG channel can cause Long QT Syndrome (LQTS). A wide variety of structurally diverse therapeutic compounds reduce the hERG current by acute direct inhibition of the hERG current or/and selective disruption of hERG protein expression. Arsenic trioxide (As2O3), which is used to treat acute promyelocytic leukemia, can cause LQTS type 2 (LQT2) by reducing the hERG current through the diversion of hERG trafficking to the cytoplasmic membrane. This cardiotoxicity limits its clinical applications. Our aim was to develop cardioprotective agents to decrease As2O3-induced cardiotoxicity. We reported that superfusion of hERG-expressing HEK293 (hERG-HEK) cells with matrine (1, 10μM) increased the hERG current by promoting hERG channel activation. Long-term treatment with 1μM matrine or oxymatrine increased expression of the hERG protein and rescued the hERG surface expression disrupted by As2O3. In addition, Matrine and oxymatrine significantly shortened action potential duration prolonged by As2O3 in guinea pig ventricular myocytes. These results were ascribed to the up-regulation of hERG at both mRNA and protein levels via an increase in the expression of transcription factor Sp1, an established transactivator of the hERG gene. Therefore, matrine and oxymatrine may have the potential to cure LQT2 as a potassium channel activator by promoting hERG channel activation and increasing hERG channel expression.

Sphingosine 1-phosphate and human ether-a′-go-go-related gene potassium channels modulate migration in human anaplastic thyroid cancer cells

Anaplastic thyroid cancer (ATC) is the most aggressive form of human thyroid cancer, lacking any effective treatment. Sphingosine 1-phosphate (S1P) receptors and human ether-a'-go-go-related gene (HERG (KCNH2)) potassium channels are important modulators of cell migration. In this study, we have shown that the S1P(1-3) receptors are expressed in C643 and THJ-16T human ATC cell lines, both at mRNA and protein level. S1P inhibited migration of these cells and of follicular FTC-133 thyroid cancer cells. Using the S1P(1,3) inhibitor VPC-23019, the S1P(2) inhibitor JTE-013, and the S1P(2) receptor siRNA, we showed that the effect was mediated through S1P(2). Treatment of the cells with the Rho inhibitor C3 transferase abolished the effect of S1P on migration. S1P attenuated Rac activity, and inhibiting Rac decreased migration. Sphingosine kinase inhibitor enhanced basal migration of cells, and addition of exogenous S1P inhibited migration. C643 cells expressed a nonconducting HERG protein, and S1P decreased HERG protein expression. The HERG blocker E-4031 decreased migration. Interestingly, downregulating HERG protein with siRNA decreased the basal migration. In experiments using HEK cells overexpressing HERG, we showed that S1P decreased channel protein expression and current and that S1P attenuated migration of the cells. We conclude that S1P attenuates migration of C643 ATC cells by activating S1P(2) and the Rho pathway. The attenuated migration is also, in part, dependent on a S1P-induced decrease of HERG protein.

HIV Tat protein inhibits hERG K + channels: A potential mechanism of HIV infection induced LQTs

HIV-infected patients have a high prevalence of long QT syndrome (LQTs). hERG K + channel encoded by human ether-a-go-go related gene contributes to IKr K + currents responsible for the repolarization of cardiomyocytes. Inhibition of hERG K + channels leads to LQTs. HIV Tat protein, the virus transactivator protein, plays a pivotal role in AIDS. The aim of the present study is to examine the effects of HIV Tat protein on hERG K + channels stably expressed in HEK293 cells. The hERG K + currents were recorded by whole-cell patch-clamp technique and the hERG channel expression was measured by real-time PCR and Western blot techniques. HIV Tat protein at 200 ng/ml concentration showed no acute effect on hERG currents, but HIV Tat protein (200 ng/ml) incubation for 24 h significantly inhibited hERG currents. In HIV Tat incubated cells, the inactivation and the recovery time from inactivation of hERG channels were significantly changed. HIV Tat protein incubation (200 ng/ml) for 24 h had no effect on the hERG mRNA expression, but dose-dependently inhibited hERG protein expression. The MTT assay showed that HIV Tat protein at 50 ng/ml and 200 ng/ml had no effect on the cell viability. HIV Tat protein increased reactive oxygen species (ROS) generation and the inhibition of hERG channel protein expression by HIV Tat protein was prevented by antioxidant tempol. HIV Tat protein in vivo treatment reduced IKr currents and prolonged action potential duration of guinea pig cardiomyocytes. We conclude that HIV Tat protein inhibits hERG K + currents through the inhibition of hERG protein expression, which might be the potential mechanism of HIV infection induced LQTs.