Quantitative analysis of trafficking defects induced by heterozygous expression of hERG voltage sensor domain variants

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1

Defective assembly and trafficking of mutant HERG channels with C-terminal truncations in long QT syndrome

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

Long QT syndrome (LQTS) type 3 although less common than the first two forms, differs in that arrhythmic events are less likely triggered by adrenergic stimuli and are more often lethal. Effective pharmacological treatment is challenged by interindividual differences, mutation dependence, and adverse effects, translating into an increased use of invasive measures (implantable cardioverter-defibrillator, sympathetic denervation) in patients with LQTS type 3. Previous studies have demonstrated the therapeutic potential of polyclonal KCNQ1 antibody for LQTS type 2. Here, we sought to identify a monoclonal KCNQ1 antibody that preserves the electrophysiological properties of the polyclonal form. Using hybridoma technology, murine monoclonal antibodies were generated, and patch clamp studies were performed for functional characterization. We identified a monoclonal KCNQ1 antibody able to normalize cardiac action potential duration and to suppress arrhythmias in a pharmacological model of LQTS type 3 using human-induced pluripotent stem cell-derived cardiomyocytes.NEW & NOTEWORTHY Long QT syndrome is a leading cause of sudden cardiac death in the young. Recent research has highlighted KCNQ1 antibody therapy as a new treatment modality for long QT syndrome type 2. Here, we developed a monoclonal KCNQ1 antibody that similarly restores cardiac repolarization. Moreover, the identified monoclonal KCNQ1 antibody suppresses arrhythmias in a cellular model of long QT syndrome type 3, holding promise as a first-in-class antiarrhythmic immunotherapy.

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.

Mutations in human ether-a-go-go-related gene 1 (hERG) are linked to long QT syndrome type 2 (LQT2). hERG encodes the pore-forming alpha-subunits that coassemble to form rapidly activating delayed rectifier K(+) current in the heart. LQT2-linked missense mutations have been extensively studied in noncardiac heterologous expression systems, where biogenic (protein trafficking) and biophysical (gating and permeation) abnormalities have been postulated to underlie the loss-of-function phenotype associated with LQT2 channels. Little is known about the properties of LQT2-linked hERG channel proteins in native cardiomyocyte systems. In this study, we expressed wild-type (WT) hERG and three LQT2-linked mutations in neonatal mouse cardiomyocytes and studied their electrophysiological and biochemical properties. Compared with WT hERG channels, the LQT2 missense mutations G601S and N470D hERG exhibited altered protein trafficking and underwent pharmacological correction, and N470D hERG channels gated at more negative voltages. The DeltaY475 hERG deletion mutation trafficked similar to WT hERG channels, gated at more negative voltages, and had rapid deactivation kinetics, and these properties were confirmed in both neonatal mouse cardiomyocyte and human embryonic kidney (HEK)-293 cell expression systems. Differences between the cardiomyocytes and HEK-293 cell expression systems were that hERG current densities were reduced 10-fold and deactivation kinetics were accelerated 1.5- to 2-fold in neonatal mouse cardiomyocytes. An important finding of this work is that pharmacological correction of trafficking-deficient LQT2 mutations, as a potential innovative approach to therapy, is possible in native cardiac tissue.

Ventricular dysfunction in a family with long QT syndrome type 3

Mutations in the human ether-a-gogo-related gene (HERG) K(+) channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduce I(Kr) currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K(+) channels without block.

Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD90) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (r= -0.8; P<0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (P=0.01). Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

<i>Circulation</i> Editors’ Picks

Mexiletine shortened QTc interval in an age-dependent manner but suppressed ventricular arrhythmias at all age in children with long QT syndrome type 8

1 Long QT syndrome Type 2 (LQT2) is a genetic disorder caused by missense mutations in the KCNH2 gene that encodes the potassium channel K V 11.1. Previous studies have shown that most K V 11.1 missense mutations with loss-of-function phenotypes result from impaired trafficking from the endoplasmic reticulum to the plasma membrane. To investigate the molecular basis of these defects, we used molecular dynamics simulations to analyze two sets of disease-associated missense mutations: those that suppress and those that maintain normal channel trafficking. We focused initially on the conformational and dynamics differences between wild-type and several mutants of K V 11.1 via molecular dynamics simulations when two K + were placed in the selectivity filter (SF). Our study reveals that missense mutations in the S4 helix allosterically disrupt the selectivity filter, a critical determinant for proper channel trafficking. Trafficking-competent variants largely retained a wild-type selectivity filter structure, whereas trafficking-deficient mutants exhibited pronounced structural perturbations in this region. These findings suggest that certain LQT2-associated missense mutations in KCNH2 impair channel trafficking by compromising the structural integrity of the selectivity filter. We additionally found that second-site variants Y652C in the drug binding vestibule can correct structural defects associated with some mistrafficking variants.

Deciphering HERG mutation in long QT syndrome type 2 using antisense oligonucleotide–mediated techniques: Lessons from cystic fibrosis

The long QT syndrome type 2 (LQT2) is inheritable life threatening arrhythmic disorder and one of the most common genetic variants in long QT syndrome. There are some indications for treatment of the patients with LQT2 but it is impossible to completely prevent fatal arrhythmia. To develop novel therapy for the patients with LQT2, it has been desired to generate diseasespecific and patient-specific disease model. Human induced pluripotent stem (iPS) cells are somatic cell-derived pluripotent stem cells with infinite proliferation ability and multipotency. Patient-specific iPS cells can be derived from patient somatic cells, have all genomic information encoded in patient's genome including mutation and all SNPs, and can be ideal disease models of the patients. To generate disease model for LQT2 by iPS cells, we should firstly generate iPS cells from the patient with LQT2 and confirm the genomic mutation in iPS cells. In this study, we showed the successful generation of iPS cells from a patient with KCNH2 G603D mutation. The patient specific iPS cells properly expressed stem cell markers, such as NANOG and OCT3/4. We also confirmed that the KCNH2 G603D (G1808A) mutation was taken over in patient specific iPS cells. These patient-specific iPS cells may contribute to the future analysis for disease pathogenesis and drug innovation.