The ion channel basis of pharmacological effects of amiodarone on myocardial electrophysiological properties, a comprehensive review.

Amiodarone is a benzofuran-based class III antiarrhythmic agent frequently used for the treatment of atrial and ventricular arrhythmias. The primary target of class III antiarrhythmic drugs is the cardiac human ether-a-go-go-related gene (hERG) encoded channel, KCNH2, commonly known as HERG, that conducts the rapidly activating delayed rectifier potassium current (IKr). Like other class III antiarrhythmic drugs, amiodarone exerts its physiologic effects mainly through IKr blockade, delaying the repolarization phase of the action potential and extending the effective refractory period. However, while many class III antiarrhythmics, including sotalol and dofetilide, can cause long QT syndrome (LQTS) that can progress to torsade de pointes, amiodarone displays less risk of inducing this fatal arrhythmia. This review article discusses the arrhythmogenesis in LQTS from the aspects of the development of early afterdepolarizations (EADs) associated with Ca2+ current, transmural dispersion of repolarization (TDR), as well as reverse use dependence associated with class III antiarrhythmic drugs to highlight electropharmacological effects of amiodarone on the myocardium.

Effect of oxygen delivery during cardiopulmonary bypass on postoperative neurological outcomes in patients undergoing cardiac surgery: A scoping review of the literature.

BACKGROUND: Reduced oxygen delivery (DO2) during cardiopulmonary bypass (CPB) was proposed as a risk factor for the development of postoperative neurological complications (PONCs), including cerebrovascular accidents (CVA), delirium, and postoperative cognitive dysfunction (POCD). We aimed to review the current evidence on the association between intraoperative DO2 and the incidence of PONCs. METHODS: MEDLINE, Embase, the Cochrane Library, and Web of Science were electronically searched to identify comparative studies from inception until July 2023 that reported the association between intraoperative DO2 levels and the incidence of PONCs (as defined by the scales and diagnostic tools utilized by the studies' authors) in adults patients undergoing cardiac surgery using CPB. RESULTS: Of the 2513 papers identified, 10 studies, including 21,875 participants, were included. Of these, three studies reported on delirium, two on POCD, and five on CVA. Eight studies reported reduced intraoperative DO2 in patients who developed delirium and CVA. There was a lack of consensus on the cut-off of DO2 levels or the correlation between the period below these threshold values and the development of PONC. CONCLUSIONS: Limited data suggest that maintaining intraoperative DO2 above the critical threshold levels and ensuring adequate intraoperative cerebral perfusion may play a role in minimizing the incidence of neurological events in adult patients undergoing cardiac surgery on cardiopulmonary bypass.

Animal models of Lyme carditis. Understanding how to study a complex disease.

Lyme carditis, a well-established manifestation of Lyme disease, has been studied in animal models to improve understanding of its pathogenesis. This review synthesizes existing literature on these models and associated disease mechanisms. Searches in MEDLINE, Embase, BIOSIS, and Web of Science yielded 53 articles (47 mice models and 6 other animal models). Key findings include: 1) Onset of carditis correlates with spirochete localization in the heart; 2) Carditis occurs within 10 days of infection, progressing to peak inflammation within 30 days; 3) Infiltrates were predominantly composed of Mac-1+ macrophages and were associated with increases in TNF-α, IL-1 and IL-12 cytokines; 4) Resolution of inflammation was primarily mediated by lymphocytes; 5) Immune system is a double-edged sword: it can play a role in the progression and severity of carditis, but can also have a protective effect. Animal models offer valuable insights into the evolution and pathophysiologic mechanisms of Lyme carditis.

Differential Effects of Remdesivir and Lumacaftor on Homomeric and Heteromeric hERG Channels.

The human ether-a-go-go-related gene (hERG) encodes for the pore-forming subunit of the channel that conducts the rapidly activating delayed K+ current (IKr) in the heart. The hERG channel is important for cardiac repolarization, and reduction of its expression in the plasma membrane due to mutations causes long QT syndrome type 2 (LQT2). As such, promoting hERG membrane expression is a strategy to rescue mutant channel function. In the present study, we applied patch clamp, western blots, immunocytochemistry, and quantitative reverse transcription polymerase chain reaction techniques to investigate the rescue effects of two drugs, remdesivir and lumacaftor, on trafficking-defective mutant hERG channels. As our group has recently reported that the antiviral drug remdesivir increases wild-type (WT) hERG current and surface expression, we studied the effects of remdesivir on trafficking-defective LQT2-causing hERG mutants G601S and R582C expressed in HEK293 cells. We also investigated the effects of lumacaftor, a drug used to treat cystic fibrosis, that promotes CFTR protein trafficking and has been shown to rescue membrane expression of some hERG mutations. Our results show that neither remdesivir nor lumacaftor rescued the current or cell-surface expression of homomeric mutants G601S and R582C. However, remdesivir decreased while lumacaftor increased the current and cell-surface expression of heteromeric channels formed by WT hERG and mutant G601S or R582C hERG. We concluded that drugs can differentially affect homomeric WT and heteromeric WT+G601S (or WT+R582C) hERG channels. These findings extend our understanding of drug-channel interaction and may have clinical implications for patients with hERG mutations. SIGNIFICANCE STATEMENT: Various naturally occurring mutations in a cardiac potassium channel called hERG can impair channel function by decreasing cell-surface channel expression, resulting in cardiac electrical disturbances and even sudden cardiac death. Promotion of cell-surface expression of mutant hERG channels represents a strategy to rescue channel function. This work demonstrates that drugs such as remdesivir and lumacaftor can differently affect homomeric and heteromeric mutant hERG channels, which have biological and clinical implications.

Opioids-induced inhibition of HERG ion channels and sudden cardiac death, a systematic review of current literature.

BACKGROUND: It is estimated that over 60 million individuals regularly use opioids globally, with opioid use disorder increasing substantially in the past decade. Several reports have linked sudden cardiac death, QTc prolongation, and other adverse cardiovascular outcomes with opioid use through their inhibitory effect on the human ether-a-go-go-related gene (HERG) ion channel. Therefore, understanding this underlying mechanism may be critical for risk prevention and management in prescribing opioids and treating patients with opioid dependency. AIM: The present systematic review summarizes the current literature on the impact of opioids-induced inhibition of HERG channel function and its relationship with sudden cardiac death, QTc prolongation, and other cardiovascular adverse effects. METHODS: A systematic review was conducted of the databases PubMed, EMBASE, Cochrane, and ClinicalTrials.gov of primary studies that reported the effects of opioids on HERG channel function and associated cardiovascular outcomes. RESULTS: The search identified 1,546 studies, of which 12 were finally included for data extraction. Based on the current literature, methadone, oliceridine, l-α-acetylmethadol (LAAM), and fentanyl were found to inhibit the HERG channel function and were associated with QTc prolongation. However, other opioids such as morphine, codeine, tramadol, and buprenorphine were not associated with inhibition of HERG channels or QTc prolongation. Additional cardiac outcomes associated with opioid related HERG channels dysfunction included sudden cardiac death and Torsade de Pointes. CONCLUSION: Our findings suggest that certain opioid consumption may result in the inhibition of HERG channels, subsequently prolonging the QTc interval and increasing patient susceptibility to sudden cardiac death.

The role of pericardial fluid biomarkers in predicting post-operative atrial fibrillation, a comprehensive review of current literature.

Post-operative atrial fibrillation (POAF) is a common complication of cardiac surgery which is associated with longer hospital stay, diminished quality of life, and increased mortality. Yet, the pathophysiology of POAF is poorly understood and it is unclear which patients are at highest risk. Pericardial fluid (PCF) analysis is emerging as an important tool for the early detection of biochemical and molecular changes in the cardiac tissue. With the epicardium acting as a semi-permeable membrane, the composition of PCF reflects the activity of the cardiac interstitium. Emerging research on PCF composition has identified promising biomarkers which may help stratify the risk for developing POAF. These include inflammatory molecules, such as interleukin-6, mitochondrial deoxyribonucleic acid, and myeloperoxidase, as well as natriuretic peptides. Additionally, PCF appears to be superior to serum analysis in detecting changes in these molecules during the early postoperative period after cardiac surgery. The aim of the present narrative review is to summarize the current literature on the temporal changes in the levels of potential biomarkers in PCF after cardiac surgery and their association with the development of new-onset postoperative atrial fibrillation.

Effectiveness of intra-operative topical amiodarone for prevention of postcardiac surgery new-onset atrial fibrillation: A review of current evidence.

BACKGROUND: Postoperative atrial fibrillation (POAF) is one of the most common complications following cardiac surgery and is associated with increased morbidity. Intraoperative topical amiodarone application on epicardial tissue has been shown to reduce systemic concentrations while maintaining therapeutic myocardial concentrations, thereby, lowering the risk of extracardiac adverse effects associated with oral and intravenous amiodarone therapy. However, the efficacy and safety of topical amiodarone in preventing POAF is unclear. OBJECTIVES: This study summarizes the clinical studies to-date that have investigated the efficacy and safety of topical amiodarone administration in preventing POAF following cardiac surgery. METHODS: A database search was conducted using Medline, Embase, and Cochrane Library to identify relevant studies. Abstracts were screened and data were extracted from relevant full-text articles that met the inclusion and exclusion criteria. RESULTS: The search returned four studies with variable findings on the effect of topical amiodarone therapy on the incidence of POAF, cardiac effects, extracardiac effects, and hospital length of stay. CONCLUSION: Prophylactic topical application of amiodarone may be effective and safe for preventing post-operative new-onset atrial fibrillation. Further investigation is required to evaluate the efficacy and safety of topical amiodadrone therapy before it can be widely integrated into current practice.

Kv1.5 channels are regulated by PKC-mediated endocytic degradation.

The voltage-gated potassium channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA, 10 nM) diminished Kv1.5 current (IKv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 channel function under PKC activation conditions.

COVID-19 Drugs Chloroquine and Hydroxychloroquine, but Not Azithromycin and Remdesivir, Block hERG Potassium Channels.

Drug-induced long QT syndrome (LQTS) is an established cardiac side effect of a wide range of medications and represents a significant concern for drug safety. The rapidly and slowly activating delayed rectifier K+ currents, mediated by channels encoded by the human ether-a-go-go-related gene (hERG) and KCNQ1 + KCNE1, respectively, are two main currents responsible for ventricular repolarization. The common cause for drugs to induce LQTS is through impairing the hERG channel. For the recent emergence of COVID-19, caused by severe acute respiratory syndrome coronavirus 2, several drugs have been investigated as potential therapies; however, there are concerns about their QT prolongation risk. Here, we studied the effects of chloroquine, hydroxychloroquine, azithromycin, and remdesivir on hERG channels. Our results showed that although chloroquine acutely blocked hERG current (IhERG), with an IC50 of 3.0 µM, hydroxychloroquine acutely blocked IhERG 8-fold less potently, with an IC50 of 23.4 µM. Azithromycin and remdesivir did not acutely affect IhERG When these drugs were added at 10 µM to the cell culture medium for 24 hours, remdesivir increased IhERG by 2-fold, which was associated with an increased mature hERG channel expression. In addition, these four drugs did not acutely or chronically affect KCNQ1 + KCNE1 channels. Our data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns. SIGNIFICANCE STATEMENT: This work demonstrates that, among off-label potential COVID-19 treatment drugs chloroquine, hydroxychloroquine, azithromycin, and remdesivir, the former two drugs block hERG potassium channels, whereas the latter two drugs do not. All four drugs do not affect KCNQ1 + KCNE1. As hERG and KCNQ1 + KCNE1 are two main K+ channels responsible for ventricular repolarization, and most drugs that induce long QT syndrome (LQTS) do so by impairing hERG channels, these data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns.

Fentanyl-Induced Block of hERG Channels Is Exacerbated by Hypoxia, Hypokalemia, Alkalosis, and the Presence of hERG1b.

Human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium current (IKr) important for repolarization of cardiac action potentials. Drug-induced disruption of hERG channel function is a main cause of acquired long QT syndrome, which can lead to ventricular arrhythmias and sudden death. Illicit fentanyl use is associated with sudden death. We have demonstrated that fentanyl blocks hERG current (IhERG) at concentrations that overlap with the upper range of postmortem blood concentrations in fentanyl-related deaths. Since fentanyl can cause respiratory depression and electrolyte imbalances, in the present study we investigated whether certain pathologic circumstances exacerbate fentanyl-induced block of IhERG Our results show that chronic hypoxia or hypokalemia additively reduced IhERG with fentanyl. As well, high pH potentiated the fentanyl-mediated block of hERG channels, with an IC50 at pH 8.4 being 7-fold lower than that at pH 7.4. Furthermore, although the full-length hERG variant, hERG1a, has been widely used to study hERG channels, coexpression with the short variant, hERG1b (which does not produce current when expressed alone), produces functional hERG1a/1b channels, which gate more closely resembling native IKr Our results showed that fentanyl blocked hERG1a/1b channels with a 3-fold greater potency than hERG1a channels. Thus, in addition to a greater susceptibility due to the presence of hERG1b in the human heart, hERG channel block by fentanyl can be exacerbated by certain conditions, such as hypoxia, hypokalemia, or alkalosis, which may increase the risk of fentanyl-induced ventricular arrhythmias and sudden death. SIGNIFICANCE STATEMENT: This work demonstrates that heterologously expressed human ether a-go-go-related gene (hERG) 1a/1b channels, which more closely resemble rapidly activating delayed rectifier potassium current in the human heart, are blocked by fentanyl with a 3-fold greater potency than the previously studied hERG1a expressed alone. Additionally, chronic hypoxia, hypokalemia, and alkalosis can increase the block of hERG current by fentanyl, potentially increasing the risk of cardiac arrhythmias and sudden death.

Mechanical stretch increases Kv1.5 current through an interaction between the S1-S2 linker and N-terminus of the channel.

The voltage-gated potassium channel Kv1.5 plays important roles in atrial repolarization and regulation of vascular tone. In the present study, we investigated the effects of mechanical stretch on Kv1.5 channels. We induced mechanical stretch by centrifuging or culturing Kv1.5-expressing HEK 293 cells and neonatal rat ventricular myocytes in low osmolarity (LO) medium and then recorded Kv1.5 current (IKv1.5) in a normal, isotonic solution. We observed that mechanical stretch increased IKv1.5, and this increase required the intact, long, proline-rich extracellular S1-S2 linker of the Kv1.5 channel. The low osmolarity-induced IKv1.5 increase also required an intact intracellular N terminus, which contains the binding motif for endogenous Src tyrosine kinase that constitutively inhibits IKv1.5 Disrupting the Src-binding motif of Kv1.5 through N-terminal truncation or mutagenesis abolished the mechanical stretch-mediated increase in IKv1.5 Our results further showed that the extracellular S1-S2 linker of Kv1.5 communicates with the intracellular N terminus. Although the S1-S2 linker of WT Kv1.5 could be cleaved by extracellularly applied proteinase K (PK), an N-terminal truncation up to amino acid residue 209 altered the conformation of the S1-S2 linker and made it no longer susceptible to proteinase K-mediated cleavage. In summary, the findings of our study indicate that the S1-S2 linker of Kv1.5 represents a mechanosensor that regulates the activity of this channel. By targeting the S1-S2 linker, mechanical stretch may induce a change in the N-terminal conformation of Kv1.5 that relieves Src-mediated tonic channel inhibition and results in an increase in IKv1.5.

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.

Blockade of the Human Ether A-Go-Go-Related Gene (hERG) Potassium Channel by Fentanyl.

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr). Drug-mediated or medical condition-mediated disruption of hERG function is the primary cause of acquired long-QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. Fentanyl abuse poses a serious health concern, with abuse and death rates rising over recent years. As fentanyl has a propensity to cause sudden death, we investigated its effects on the hERG channel. The effects of norfentanyl, the main metabolite, and naloxone, an antidote used in fentanyl overdose, were also examined. Currents of hERG channels stably expressed in HEK293 cells were recorded using the whole-cell voltage-clamp method. When hERG tail currents were analyzed upon -50 mV repolarization after a 50 mV depolarization, fentanyl and naloxone blocked hERG current (IhERG) with IC50 values of 0.9 and 74.3 µM, respectively, whereas norfentanyl did not block. However, fentanyl-mediated block of IhERG was voltage dependent. When a voltage protocol that mimics a human ventricular action potential (AP) was used, fentanyl blocked IhERG with an IC50 of 0.3 µM. Furthermore, fentanyl (0.5 µM) prolonged AP duration and blocked IKr in ventricular myocytes isolated from neonatal rats. The concentrations of fentanyl used in this study were higher than seen with clinical use but overlap with postmortem overdose concentrations. Although mechanisms of fentanyl-related sudden death need further investigation, blockade of hERG channels may contribute to the death of individuals with high-concentration overdose or compromised cardiac repolarization.

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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 N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1-S2 linkage.

The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel (i.e. Frag(304-613)) to form a functional channel independently of the S1-S2 linkage.

Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility.

The human ether-a-go-go related gene (hERG)-encoded channel hERG undergoes N-linked glycosylation at position 598, which is located in the unusually long S5-pore linker of the channel. In other work we have demonstrated that hERG is uniquely susceptible to proteolytic cleavage at the S5-pore linker by proteinase K (PK) and calpain (CAPN). The scorpion toxin BeKm-1, which binds to the S5-pore linker of hERG, protects hERG from such cleavage. In the present study, our data revealed that, compared with normal glycosylated hERG channels, nonglycosylated hERG channels were significantly more susceptible to cleavage by extracellular PK. Furthermore, the protective effect of BeKm-1 on hERG from PK-cleavage was lost when glycosylation of hERG was inhibited. The inactivation-deficient mutant hERG channels S620T and S631A were resistant to PK cleavage, and inhibition of glycosylation rendered both mutants susceptible to PK cleavage. Compared with normal glycosylated channels, nonglycosylated hERG channels were also more susceptible to cleavage mediated by CAPN, which was present in the medium of human embryonic kidney cells under normal culture conditions. Inhibition of CAPN resulted in an increase of nonglycosylated hERG current. In summary, our results revealed that N-linked glycosylation protects hERG against protease-mediated degradation and thus contributes to hERG channel stability on the plasma membrane.- Lamothe, S. M., Hulbert, M., Guo, J., Li, W., Yang, T., Zhang, S. Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility. FASEB J. 32, 1933-1943 (2018). www.fasebj.org.

Hypoxia reduces mature hERG channels through calpain up-regulation.

Human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium current (IKr) potassium channel, which is important for cardiac repolarization. Impairment of hERG function is the primary cause of acquired long QT syndrome, which predisposes individuals to cardiac arrhythmias and sudden death. Patients with hypoxia due to conditions such as cardiac ischemia or obstructive sleep apnea display increased incidence of cardiac arrhythmias and sudden death. We sought to understand the mechanisms that underlie hypoxia-associated cardiac arrhythmias. Using cell biology and electrophysiologic techniques, we found that hypoxic culture of hERG-expressing human embryonic kidney (HEK) cells and neonatal rat cardiomyocytes reduced hERG current/IKr and mature ERG channel expression with a concomitant increase in calpain expression. Calpain was actively released into the extracellular milieu and degraded cell-surface hERG. In contrast to hERG, the ether-a-go-go (EAG) channel was not reduced by hypoxic culture. By making chimeric channels between hERG and EAG, we identified that hypoxia-induced calpain degraded hERG by targeting its extracellular S5-pore linker. The scorpion toxin BeKm-1, which is known to selectively bind to the S5-pore linker of hERG, prevented hypoxia-induced hERG reduction. Our data provide novel information about hypoxia-mediated hERG dysfunction and may have biological and clinical implications in hypoxia-associated diseases.-Lamothe, S. M., Song, W., Guo, J., Li, W., Yang, T., Baranchuk, A., Graham, C. H., Zhang, S. Hypoxia reduces mature hERG channels through calpain up-regulation.

ß-Arrestin-Mediated Regulation of the Human Ether-a-go-go-Related Gene Potassium Channel.

The rapidly activating delayed rectifier K+ channel (IKr) is encoded by the human ether-a-go-go-related gene (hERG), which is important for the repolarization of the cardiac action potential. Mutations in hERG or drugs can impair the function or decrease the expression level of hERG channels, leading to long QT syndrome. Thus, it is important to understand hERG channel trafficking and its regulation. For this purpose, G protein-coupled receptors (GPCRs), which regulate a vast array of cellular processes, represent a useful route. The development of designer GPCRs known as designer receptors exclusively activated by designer drugs (DREADDs) has made it possible to dissect specific GPCR signaling pathways in various cellular systems. In the present study, by expressing an arrestin-biased M3 muscarinic receptor-based DREADD (M3D-arr) in stable hERG-expressing human embryonic kidney (HEK) cells, we demonstrate that ß-arrestin signaling plays a role in hERG regulation. By exclusively activating M3D-arr using the otherwise inert compound, clozapine-N-oxide, we found that M3D-arr activation increased mature hERG expression and current. Within this paradigm, M3D-arr recruited ß-arrestin-1 to the plasma membrane, and promoted phosphoinositide 3-kinase-dependent activation of protein kinase B (Akt). The activated Akt acted through phosphatidylinositol 3-phosphate 5-kinase and Rab11 to facilitate hERG recycling to the plasma membrane. Potential ß-arrestin signaling-mediated increases in hERG and IKr were also observed in hERG-HEK cells as well as in neonatal rat ventricular myocytes treated with the muscarinic agonist carbachol. These findings provide novel insight into hERG trafficking and regulation.

Clemizole hydrochloride blocks cardiac potassium currents stably expressed in HEK 293 cells.

BACKGROUND AND PURPOSE: Clemizole, a histamine H1 receptor antagonist has a potential therapeutic effect on hepatitis C infection and also potently inhibits TRPC5 ion channels. The aim of the present study was to investigate whether clemizole blocks cardiac K+ currents and thus affects cardiac repolarization. EXPERIMENTAL APPROACH: Whole-cell patch techniques was used to examine the effects of clemizole on hERG channel current, IKs and Kv 1.5 channel current in HEK 293 cell expression systems as well as on ventricular action potentials of guinea pig hearts. Isolated hearts from guinea pigs were used to determine the effect on the ECG. KEY RESULTS: Clemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (IC50 : 0.07 µM). The S631A, S636A, Y652A and F656V hERG mutant channels reduced the inhibitory effect of clemizole (IC50 : 0.82, 0.89, 1.49 and 2.98 µM, respectively), suggesting that clemizole is a pore blocker of hERG channels. Clemizole also moderately decreased IKs and human Kv 1.5 channel current. Moreover, clemizole increased the duration of the ventricular action potential in guinea pig hearts and the QTc interval in isolated perfused hearts from guinea pigs, in a concentration-dependent manner (0.1-1.0 µM). CONCLUSION AND IMPLICATIONS: Our results provide the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac IKs , delays cardiac repolarization and thereby prolongs QT interval. Thus, caution should be taken when clemizole is used as a TRPC5 channel blocker or for treating hepatitis C infection.

The Human Ether-a-go-go-related Gene (hERG) Potassium Channel Represents an Unusual Target for Protease-mediated Damage.

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation, up-regulation, and secretion of various proteolytic enzymes. Here, using whole-cell patch clamp and Western blotting analysis, we demonstrate that the hERG/IKr channel was selectively cleaved by the serine protease, proteinase K (PK). Using molecular biology techniques including making a chimeric channel between protease-sensitive hERG and insensitive human ether-a-go-go (hEAG), as well as application of the scorpion toxin BeKm-1, we identified that the S5-pore linker of hERG is the target domain for proteinase K cleavage. To investigate the physiological relevance of the unique susceptibility of hERG to proteases, we show that cardiac ischemia in a rabbit model was associated with a reduction in mature ERG expression and an increase in the expression of several proteases, including calpain. Using cell biology approaches, we found that calpain-1 was actively released into the extracellular milieu and cleaved hERG at the S5-pore linker. Using protease cleavage-predicting software and site-directed mutagenesis, we identified that calpain-1 cleaves hERG at position Gly-603 in the S5-pore linker of hERG. Clarification of protease-mediated damage of hERG extends our understanding of hERG regulation. Damage of hERG mediated by proteases such as calpain may contribute to ischemia-associated QT prolongation and sudden cardiac death.