hERG Protein Research Articles

Investigating the cardiotoxicity of N-n-butyl haloperidol iodide: Inhibition mechanisms on hERG channels

The human Ether-à-go-go-Related Gene (hERG) encodes a protein responsible for forming the alpha subunit of the IKr channel, which plays a crucial role in cardiac repolarization. The proper functioning of hERG channels is paramount in maintaining a normal cardiac rhythm. Inhibition of these channels can result in the prolongation of the QT interval and potentially life-threatening arrhythmias. Cardiotoxicity is a primary concern in the field of drug development. N-n-Butyl haloperidol iodide (F2), a derivative of haloperidol, has been investigated for its therapeutic potential. However, the impact of this compound on cardiac toxicity, specifically on hERG channels, remains uncertain. This study employs computational and experimental methodologies to examine the inhibitory mechanisms of F2 on hERG channels. Molecular docking and molecular dynamics simulations commonly used techniques in computational biology to predict protein-ligand complexes' binding interactions and stability. In the context of the F2-hERG complex, these methods can provide valuable insights into the potential binding modes and strength of interaction between F2 and the hERG protein. On the other hand, electrophysiological assays are experimental techniques used to characterize the extent and nature of hERG channel inhibition caused by various compounds. By measuring the electrical activity of the hERG channel in response to different stimuli, these assays can provide important information about the functional effects of ligand binding to the channel. The study's key findings indicate that F2 interacts with the hERG channel by forming hydrogen bonding, π-cation interactions, and hydrophobic forces. This interaction leads to the inhibition of hERG currents in a concentration-dependent manner, with an IC50 of 3.75 μM. The results presented in this study demonstrate the potential cardiotoxicity of F2 and underscore the significance of considering hERG channel interactions during its clinical development. This study aims to provide comprehensive insights into the interaction between F2 and hERG, which will may guid us in the safe use of F2 and in the development of new derivatives with high efficiency while low toxicity.

In silico exploration of anti-prostate cancer compounds from differential expressed genes

Prostate cancer (PCa) is a complex and biologically diverse disease with no curative treatment options at present. This study aims to utilize computational methods to explore potential anti-PCa compounds based on differentially expressed genes (DEGs), with the goal of identifying novel therapeutic indications or repurposing existing drugs. The methods employed in this study include DEGs-to-drug prediction, pharmacokinetics prediction, target prediction, network analysis, and molecular docking. The findings revealed a total of 79 upregulated DEGs and 110 downregulated DEGs in PCa, which were used to identify drug compounds capable of reversing the dysregulated conditions (dexverapamil, emetine, parthenolide, dobutamine, terfenadine, pimozide, mefloquine, ellipticine, and trifluoperazine) at a threshold probability of 20% on several molecular targets, such as serotonin receptors 2a/2b/2c, HERG protein, adrenergic receptors alpha-1a/2a, dopamine D3 receptor, inducible nitric oxide synthase (iNOS), epidermal growth factor receptor erbB1 (EGFR), tyrosine-protein kinases, and C-C chemokine receptor type 5 (CCR5). Molecular docking analysis revealed that terfenadine binding to inducible nitric oxide synthase (-7.833 kcal.mol−1) and pimozide binding to HERG (-7.636 kcal.mol−1). Overall, binding energy ΔGbind (Total) at 0 ns was lower than that of 100 ns for both the Terfenadine-iNOS complex (-101.707 to -103.302 kcal.mol−1) and Ellipticine-TOPIIα complex (-42.229 to -58.780 kcal.mol−1). In conclusion, this study provides insight on molecular targets that could possibly contribute to the molecular mechanisms underlying PCa. Further preclinical and clinical studies are required to validate the therapeutic effectiveness of these identified drugs in PCa disease.

A novel mutation in hERG gene associated with azithromycin-induced acquired long QT syndrome.

Mutations in human ether-à-go-go-related gene (hERG) potassium channels are closely associated with long QT syndrome (LQTS). Previous studies have demonstrated that macrolide antibiotics increase the risk of cardiovascular diseases. To date, the mechanisms underlying acquired LQTS remain elusive. A novel hERG mutation I1025N was identified in an azithromycin-treated patient with acquired long QT syndrome via Sanger sequencing. The mutant I1025N plasmid was transfected into HEK-293 cells, which were subsequently incubated with azithromycin. The effect of azithromycin and mutant I1025N on the hERG channel was evaluated via western blot, immunofluorescence, and electrophysiology techniques. The protein expression of the mature hERG protein was down-regulated, whereas that of the immature hERG protein was up-regulated in mutant I1025N HEK-293 cells. Azithromycin administration resulted in a negative effect on the maturation of the hERG protein. Additionally, the I1025N mutation exerted an inhibitory effect on hERG channel current. Moreover, azithromycin inhibited hERG channel current in a concentration-dependent manner. The I1025N mutation and azithromycin synergistically decreased hERG channel expression and hERG current. However, the I1025N mutation and azithromycin did not alter channel gating dynamics. These findings suggest that hERG gene mutations might be involved in the genetic susceptibility mechanism underlying acquired LQTS induced by azithromycin.

Transfer RNA-mediated restoration of potassium current and electrical correction in premature termination long-QT syndrome hERG mutants

Disease-causing premature termination codons (PTCs) individually disrupt the functional expression of hundreds of genes and represent a pernicious clinical challenge. In the heart, loss-of-function mutations in the hERG potassium channel account for approximately 30% of long-QT syndrome arrhythmia, a lethal cardiac disorder with limited treatment options. Premature termination of ribosomal translation produces a truncated and, for potassium channels, a potentially dominant-negative protein that impairs the functional assembly of the wild-type homotetrameric hERG channel complex. We used high-throughput flow cytometry and patch-clamp electrophysiology to assess the trafficking and voltage-dependent activity of hERG channels carrying patient PTC variants that have been corrected by anticodon engineered tRNA. Adenoviral-mediated expression of mutant hERG channels in cultured adult guinea pig cardiomyocytes prolonged action potential durations, and this deleterious effect was corrected upon adenoviral delivery of a human ArgUGA tRNA to restore full-length hERG protein. The results demonstrate mutation-specific, context-agnostic PTC correction and elevate the therapeutic potential of this approach for rare genetic diseases caused by stop codons.

1656-P: Glucagon-Like peptide-1 (GLP-1) Secretion from Intestinal L-Cells Is Mediated by hERG Potassium Channels

The hERG protein is a member of voltage-gated potassium (Kv) channels. Previous reports showed that patients with long QT syndrome due to mutations in hERG channel had higher responsive glucagon-like peptide-1 (GLP-1) secretion. However, the role of hERG in GLP-1 secretion remains uncertain. Here we report that hERG is co-expressed in GLP-1-expressing L cells in rodent intestinal epithelium (Fig. 1). In a murine L-cell model, GLUTag cell line, downregulation of hERG significantly prolonged action potential duration, increased intracellular calcium concentration ([Ca2+]i), and promoted GLP-1 secretion (Fig. 2) after stimulation of nutrients. These results reveal a direct role of hERG in GLP-1 secretion in murine L-cells, suggesting intestinal hERG is a potential target for the treatment of diabetes. Disclosure J.Yang: None. C.Liu: None. H.Wang: None. Y.Yuan: None. Funding National Natural Science Foundation of China (82170809, 81930019)

Can We Quickly Learn to "Translate" Bioactive Molecules with Transformer Models?

Meaningful exploration of the chemical space of druglike molecules in drug design is a highly challenging task due to a combinatorial explosion of possible modifications of molecules. In this work, we address this problem with transformer models, a type of machine learning (ML) model originally developed for machine translation. By training transformer models on pairs of similar bioactive molecules from the public ChEMBL data set, we enable them to learn medicinal-chemistry-meaningful, context-dependent transformations of molecules, including those absent from the training set. By retrospective analysis on the performance of transformer models on ChEMBL subsets of ligands binding to COX2, DRD2, or HERG protein targets, we demonstrate that the models can generate structures identical or highly similar to most active ligands, despite the models having not seen any ligands active against the corresponding protein target during training. Our work demonstrates that human experts working on hit expansion in drug design can easily and quickly employ transformer models, originally developed to translate texts from one natural language to another, to "translate" from known molecules active against a given protein target to novel molecules active against the same target.

Structure optimization and discovery of novel compound for the treatment of insertion mutations within exon 20 of EGFR and HER2

In previous decades, patients with the most active EGFR mutations in non-small cell lung cancer (NSCLC) have significantly benefited from EGFR tyrosine kinase inhibitors (TKIs). However, a minority with EGFR and HER2 exon 20 mutations are inherently resistant to treatment. Several molecular TKIs (such as TAK788 and Poziotinib) were recently discovered and demonstrated as effective inhibitors against the most prevalent HER2 or EGFR exon 20 mutations. However, low clinical efficiency and uncertain adverse reaction indicated that the development of effective therapies is still demanded. In the present work, we designed several hybrid compounds learning from 3D modeling of kinase structure. One lead compound (compound 56) was found to be the most potent compound with IC50 value of 0.027 nM against EGFR D770-N771 ins NPG and reduced binding affinity with hERG protein. In vitro and in vivo biological results suggested that compound 56 demonstrated good oral bioavailability, and it was significantly capable of inhibiting the growth of tumor cells with a variety of HER2 exon 20 mutations and EGFR mutants with negligible toxic effects. It was identified that compound 56 might be considered a potential drug candidate for NSCLC target therapy.

Verification of Nanomaterial-Induced Size-Dependent Human Ether-à-Go-Go-Related Gene Potassium Channel Blockage Using Three-Dimensional Bioengineered Functional Cardiac Tissue Constructs

Three-dimensional (3D) bioprinted functional cardiac tissue constructs are proposed for the first time as a screening platform to evaluate the nanomaterial-induced hERG potassium channel blockage based on the in situ measurement of K+ permeation and hERG protein immunofluorescence. Multifunctional anticancer spherical gold nanoparticles whose surface was bound with the ligand of thiol-terminated polyethylene glycol (PEG) conjugated to indomethacin (IMC-PEG-SH@AuNPs) were synthesized with mean diameters of 19, 33, and 64 nm. Triphenylphosphine-stabilized gold nanoparticles (F-108@TPP-AuNPs) were fabricated via temperature- and reaction time-controlled synthesis. Both IMC-PEG-SH@AuNPs and F-108@TPP-AuNPs showed a dose-dependent decrease in K+ permeation because of their binding to hERG proteins. The degree of the hERG potassium channel blockage was dependent on the surface ligand conjugated to the AuNPs. The size-dependent effect of the nanomaterials on the hERG K+ channel block was quantitatively evaluated using the three different sizes of IMC-PEG-SH@AuNPs. The effect of the K+ channel block was remarkably proportional to the size of IMC-PEG-SH@AuNPs. This is the first report demonstrating the size-dependent effect of an hERG K+ channel block induced by nanomaterials. In addition, small molecules showed a greater decrease in K+ permeation and hERG immunofluorescence compared with IMC-PEG-SH@AuNPs because of steric limitations in the binding of surface ligands to hERG proteins.

Hydroxychloroquine Attenuates hERG Channel by Promoting the Membrane Channel Degradation: Computational Simulation and Experimental Evidence for QT-Interval Prolongation with Hydroxychloroquine Treatment.

The coronavirus disease 2019 (COVID-19) pandemic has led to millions of confirmed cases and deaths worldwide and has no approved therapy. Currently, more than 700 drugs are tested in the COVID-19 clinical trials, and full evaluation of their cardiotoxicity risks is in high demand. We mainly focused on hydroxychloroquine (HCQ), one of the most concerned drugs for COVID-19 therapy, and investigated the effects and underlying mechanisms of HCQ on hERG channel via molecular docking simulations. We further applied the HEK293 cell line stably expressing hERG-wild-type channel (hERG-HEK) and HEK293 cells transiently expressing hERG-p.Y652A or hERG-p.F656A mutants to validate our predictions. Western blot analysis was used to determine the hERG channel, and the whole-cell patch clamp was utilized to record hERG current (IhERG). HCQ reduced the mature hERG protein in a time- and concentration-dependent manner. Correspondingly, chronic and acute treatment of HCQ decreased the hERG current. Treatment with brefeldin A (BFA) and HCQ combination reduced hERG protein to a greater extent than BFA alone. Moreover, disruption of the typical hERG binding site (hERG-p.Y652A or hERG-p.F656A) rescued HCQ-mediated hERG protein and IhERG reduction. HCQ can reduce the mature hERG channel expression and IhERG via enhancing channel degradation. The QT prolongation effect of HCQ is mediated by typical hERG binding sites involving residues Tyr652 and Phe656.

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.

Differential Regulation of Human Ether-à-Go-Go-Related Gene (hERG) Current and Expression by Activation of Protein Kinase C.

The human ether-à-go-go-related gene (hERG) encodes the channel that conducts the rapidly activating delayed rectifier potassium current (IKr) in the heart. Reduction in IKr causes long QT syndrome, which can lead to fatal arrhythmias triggered by stress. One potential link between stress and hERG function is protein kinase C (PKC) activation; however, seemingly conflicting results regarding PKC regulation of hERG have been reported. We investigated the effects of PKC activation using phorbol 12-myristate 13-acetate (PMA) on hERG channels expressed in human embryonic kidney cell line 293 (HEK293) cells and IKr in isolated neonatal rat ventricular myocytes. Acute activation of PKC by PMA (30 nM, 30 minutes) reduced both hERG current (IhERG) and IKr Chronic activation of PKC by PMA (30 nM, 16 hours) increased IKr in cardiomyocytes and the expression level of hERG proteins; however, chronic (30 nM, 16 hours) PMA treatment decreased IhERG, which became larger than untreated control IhERG after PMA removal for 4 hours. Deletion of amino acid residues 2-354 (Δ2-354 hERG) or 1-136 of the N terminus (ΔN 136 hERG) abolished acute PMA (30 nM, 30 minutes)-mediated IhERG reduction. In contrast to wild-type hERG channels, chronic activation of PKC by PMA (30 nM, 16 hours) increased both Δ2-354 hERG and ΔN136 hERG expression levels and currents. The increase in hERG protein was associated with PKC-induced phosphorylation (inhibition) of Nedd4-2, an E3 ubiquitin ligase that mediates hERG degradation. We conclude that PKC regulates hERG in a balanced manner, increasing expression through inhibiting Nedd4-2 while decreasing current through targeting a site(s) within the N terminus.

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.

Human ether‑à‑go‑go‑related gene mutation L539fs/47‑hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway.

Congenital long QT syndrome (LQTS) is a cardiac channelopathy that often results in fatal arrhythmias. LQTS mutations not only lead to abnormal myocardial electrical activities but are associated with heart contraction abnormalities, cardiomyopathy and congenital heart defects. In vivo and in vitro studies have found that LQTS mutations are associated with cardiomyocyte apoptosis, cardiac developmental disorders and even embryonic mortality. Cardiac delayed rectifier potassium channel dysfunction due to the human ether-à-go-go-related gene (hERG) mutation causes congenital LQTS type 2. The majority of LQTS 2 mutations are characterized by mutant protein accumulation in the endoplasmic reticulum (ER). Unfolded or misfolded protein retention in the ER causes an unfolded protein reaction, which is characteristic of ER stress (ERS). Therefore, the present study hypothesized that LQTS mutations can cause cardiac structural abnormalities via ERS-mediated cardiomyocyte apoptosis. To test this hypothesis, 293 cells were transiently transfected with an L539fs/47-hERG plasmid to generate an LQTS 2 model. L539fs/47-hERG is an LQTS 2 mutation, which consists of a 19-bp deletion at 1619-1637 and a point mutation at 1692. Using confocal laser scanning microscopy analysis, it was verified that the L539fs/47-hERG protein was retained in the ER. Hoechst 33342 apoptosis staining indicated that apoptosis was increased in the L539fs/47-hERG-transfected cells, and this be reversed by treatment with 4-phenyl butyric acid. Western blot analysis revealed increased expression levels of the ERS chaperone glucose regulated protein 78 and pro-apoptotic ERS-induced factors, including protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation-initiation factor-2α and C/EBP homologous protein, in the L539fs/47-hERG-transfected cells. The B-cell lymphoma (Bcl-2)-associated X protein/Bcl-2 ratio and caspase-12 were also increased in the mutated cells. These results demonstrate that L539fs/47-hERG induces cell apoptosis and the potential molecular mechanism involves the activation of ERS and ERS-mediated cell apoptosis.

Trafficking of the human ether-a-go-go-related gene (hERG) potassium channel is regulated by the ubiquitin ligase rififylin (RFFL)

The QT interval is an important diagnostic feature on surface electrocardiograms because it reflects the duration of the ventricular action potential. A previous genome-wide association study has reported a significant linkage between a single-nucleotide polymorphism ∼11.7 kb downstream of the gene encoding the RING finger ubiquitin ligase rififylin (RFFL) and variability in the QT interval. This, along with results in animal studies, suggests that RFFL may have effects on cardiac repolarization. Here, we sought to determine the role of RFFL in cardiac electrophysiology. Adult rabbit cardiomyocytes with adenovirus-expressed RFFL exhibited reduced rapid delayed rectifier current (IKr). Neonatal rabbit cardiomyocytes transduced with RFFL-expressing adenovirus exhibited reduced total expression of the potassium channel ether-a-go-go-related gene (rbERG). Using transfections of 293A cells and Western blotting experiments, we observed that RFFL and the core-glycosylated form of the human ether-a-go-go-related gene (hERG) potassium channel interact. Furthermore, RFFL overexpression led to increased polyubiquitination and proteasomal degradation of hERG protein and to an almost complete disappearance of IKr, which depended on the intact RING domain of RFFL. Blocking the ER-associated degradation (ERAD) pathway with a dominant-negative form of the ERAD core component, valosin-containing protein (VCP), in 293A cells partially abolished RFFL-mediated hERG degradation. We further substantiated the link between RFFL and ERAD by showing an interaction between RFFL and VCP in vitro We conclude that RFFL is an important regulator of voltage-gated hERG potassium channel activity and therefore cardiac repolarization and that this ubiquitination-mediated regulation requires parts of the ERAD pathway.

The visualization of hERG channels in living cells via a fluorescent probe regulated by the synergy between solvatochromism and molecular rotation based on simple targeting of the group 4-benzylaniline.

A highly sensitive fluorescent probe CBH based on solvatochromism and molecular rotation was designed and developed for imaging of hERG channels by employing a novel targeting group 4-benzylaniline. More importantly, CBH has the potential for the quantitative analysis of the hERG channels expressed in tumor cells.

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.

Electrophysiological evaluation of pentamidine and 17-AAG in human stem cell-derived cardiomyocytes for safety assessment

Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs’ actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. Cav1.2 or Nav1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.