Trafficking Defects and Gating Abnormalities of a Novel SCN5A Mutation Question Gene-Specific Therapy in Long QT Syndrome Type 3
Rationale: Sodium channel blockers are used as gene-specific treatments in long-QT syndrome type 3, which is caused by mutations in the sodium channel gene (SCN5A). Response to treatment is influenced by biophysical properties of mutations.
Objective: We sought to investigate the unexpected deleterious effect of mexiletine in a mutation combining gain-of- function and trafficking abnormalities.
Methods and Results: A long-QT syndrome type 3 child experienced paradoxical QT prolongation and worsening of arrhythmias after mexiletine treatment. The SCN5A mutation F1473S expressed in HEK293 cells presented a right-ward shift of steady-state inactivation, enlarged window current, and huge sustained sodium current. Unexpectedly, it also reduced the peak sodium current by 80%. Immunostaining showed that mutant Nav1.5 is retained in the cytoplasm. Incubation with 10 μmol/L mexiletine rescued the trafficking defect of F1473S, causing a significant increase in peak current, whereas sustained current was unchanged. Using a Markovian model of the Na channel and a model of human ventricular action potential, we showed that simulated exposure of F1473S to mexiletine paradoxically increased action potential duration, mimicking QT prolongation seen in the index patient on mexiletine treatment.
Conclusions: Sodium channel blockers are largely used to shorten QT intervals in carriers of SCN5A mutations. We provided evidence that these agents may facilitate trafficking of mutant proteins, thus exacerbating QT prolongation. These data suggest that caution should be used when recommending this class of drugs to carriers of mutations with undefined electrophysiological properties.
Long-QT syndrome is an inherited arrhythmogenic disease characterized by QT interval prolongation and susceptibility to ventricular tachyarrhythmias. Long-QT syndrome type 3 (LQT3) is a variant of long-QT syndrome characterized by high lethality,1 marked prolongation of repolarization, poor response to β-blockers,2 and cardiac events occurring preferentially at rest. LQT3 is caused by mutations in the SCN5A gene that encode for the α subunit of the channel that conducts the inward sodium current responsible for fast depolarization and critical for maintenance of intracardiac conduction.3,4 Following the identification of the first SCN5A mutation published in 1995,5 more than 80 SCN5A mutations have been identified in LQT3 patients.
SCN5A mutations associated with LQT3 increase the sodium current by augmenting either the sustained sodium current (Isus) or the window current, thus prolonging cardiac repolarization.6 Based on this evidence, the use of sodium channel blockers to treat LQT3 patients and reduce QT interval has been proposed. Early in vitro studies7 demonstrated that mexiletine is effective in shortening action potential duration (APD) in cardiac myocytes exposed to anthopleurin, a compound that mimics LQT3 cellular phenotype. Furthermore early clinical studies on the use of mexiletine or flecainide were successful in shortening repolarization8,9 and paved the way to the clinical use of sodium channel blockers in LQT3 patients as a gene-specific therapy.
As the experience with the use of these drugs accumulated, the early enthusiasm was dimmed, and it became clear that flecainide might not be always appropriate for LQT3 patients. Indeed, the response of selected mutations could induce an electrocardiographic pattern of coved type ST segment elevation in right precordial leads typical of Brugada syndrome.10,11 Shortly thereafter, it also became clear that sodium channel blockers do not invariably shorten the QT interval, suggesting that SCN5A mutations act through other mechanisms, questioning the “gain-of-function” paradigm that all LQT3 mutations lead to a common channel dysfunction.
This view is supported by the fact that “some” SCN5A mutations cause “overlapping syndromes” in which QT prolongation is associated with “loss-of-function” clinical phenotypes (Brugada syndrome or conduction defects).12,13–14
We recently demonstrated that biophysical diversity of SCN5A mutations is a determinant of response to mexiletine in the clinical setting.15 Here, we bring the characterization of the diversity of SCN5A mutations to the next level by showing that the interplay between protein trafficking and biophysical abnormalities may detrimentally affect the response to sodium channel blockers and represent a hazard rather than a cure.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Genetic analysis was performed by screening of the open reading frame of the SCN5A, KCNH2, KCNQ1, KCNE1, and KCNE2 genes as we previously reported.16
Site-directed mutagenesis and transfection in HEK 293 cells were performed as we previously reported.15 Membrane currents were measured using whole-cell patch clamp procedures (details of procedures are provided in the Online Data Supplement).
Immunofluorescence and Immunoblotting for Quantification of Plasma Membrane Protein Expression
The distribution of Nav1.5 was accessed by polyclonal Nav1.5 in confocal microscopy. The total proteins and plasma membrane fractions were assessed by western blotting using standardized protocols. For a detailed description, see the Online Data Supplement.
Computer simulation was based on the Markovian model.17 For a detailed description see the Online Data Supplement.
Data are presented as means±SE. Statistical comparisons were made using an unpaired 2-tailed t test or ANOVA with the Tukey post hoc test to evaluate the significance of differences between means. P<0.05 was considered statistically significant.
Clinical History of the Carrier of F1473S Mutation in the SCN5A Gene
The mutation we characterize here was identified in a patient born in 2003; no ECG was recorded at birth, and the baby was discharged as a healthy infant. At 12 months of age, he experienced 5 syncopal spells within a few days, was hospitalized and long-QT syndrome was diagnosed. Propranolol (4 mg/kg per day) was initiated, and a blood sample was sent to Maugeri Foundation for genetic screening. We sequenced the opening reading frame of KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 and identified a heterozygous single-nucleotide transition (4418T>C) in the SCN5A gene, leading to a single amino acid replacement at position 1473 (F1473S) (Figure 1A). This mutation was not present in either the parents of the index case or in 300 control DNA samples.
The patient remained asymptomatic for 2 years on β-blockers; however, in 2006, syncope recurred and torsade de pointes was documented; a ventricular-inhibited pacemaker was implanted, and the dose of β-blockers was increased. One year later, the patient experienced another syncopal episode, accompanied by several runs of torsade de pointes. Oral mexiletine was started in an attempt to shorten QT interval and to control arrhythmias; sinus rhythm was regained and QT duration appeared stable at day 1 of administration (Figure 1B, upper trace); unfortunately, the effect of mexiletine (60 mg tid) decayed as the treatment continued. After 10 days, QTc persistently exceeded 650 ms (Figure 1B, lower trace); furthermore, progressive worsening of intraventricular conduction with QRS widening, and occasional pacemaker capture failures (Figure 1C) were observed. At day 11, the patient developed incessant, intractable tachyarrhythmias and died. The clinicians who treated the patient contacted us and were confounded by the worsening clinical status during mexiletine treatment. The inspection of the available ECG showed QTc shortening just at the beginning of therapy followed by prolongation (Figure 1B). We attempted to understand this uncommon and paradoxical response to mexiletine by functional characterization of the mutant.
Functional In Vitro Characterization of F1473S
As shown in Figure 2A, F1473S depolarized the steady-state inactivation (SSI) curve by 18.9 mV and steady-state activation (SSA) curve by 4.4 mV. The SSI pattern of mutant channels was consistent with our previous data15 showing that mexiletine-insensitive mutations are those causing a depolarizing shift of SSI and it also produced a greater overlap of activation and inactivation relations. As a consequence, the window current was markedly enhanced. Recovery from inactivation is shown in Figure 2B.
Besides enlarged window current, F1473S showed a striking increase of Isus. At −10 mV, F1473S channels continued to show 4.46±0.74 pA/pF (5.39±0.2% of the peak current) at 150 ms as compared to 0.64±0.22 pA/pF (0.25±0.05%) in wild-type (WT) (P<0.0001). Overall, F1473S showed a much higher Isus than other mutations we previously characterized (Figure 2C).15
We further characterized the gain-of-function behavior by ramp voltage protocol (from −100 mV to +50 mV). F1473S conducted larger peak currents than WT (F1473S −6.38±0.71 pA/pF, n=9 versus WT −2.28±0.14 pA/pF, n=8; P<0.0001), and the voltage of the peak was shifted by about +20 mV (F1473S −17.1±3.5 mV, n=9 versus WT, −37.8±3.1 mV, n=8; P<0.0001). WT channels conduct current over a narrow range of voltages near −37 mV, whereas F1473S mutant channels conduct current over a broad range of voltages and increase the inward sodium current (Figure 2D).
We concluded that the remarkable increase of both the window current and the Isus account for QT prolongation and the malignant clinical phenotype.
On the other hand, F1473S peak current density was unexpectedly reduced to 20% of WT (Figure 3A), suggesting a trafficking abnormality. This hypothesis was confirmed by confocal microscopy experiments that showed an abnormal protein distribution characterized by clusters/aggregates retained in the cytoplasm (Figure 3B, F1473S). We attempted to colocalize such clusters to determine whether the mutant polypeptides were retained either in the endoplasmic reticulum (ER) (anti-calnexin antibody) or in the Golgi apparatus (anti-Golgin antibody). The F1473S-Nav1.5 clusters did not colocalize with Golgi (Figure 3C, f) and only mildly colocalized with ER (Figure 3C, c), suggesting that most of the abnormal protein is likely to be either free in the cytosol or segregated in yet unidentified subcellular domain(s).
Response of F1473S-Nav1.5 Channel to Exposure to Sodium Channel Blockers
We compared the tonic block of mexiletine in F1473S Nav1.5 with that observed in WT and in mexiletine-sensitive (R1626P) and mexiletine-insensitive mutations (S941N) that we previously characterized.15 Figure 4A shows that tonic block of peak current with 10 μmol/L mexiletine was significantly higher in R1626P compared with WT, S941N, and F1473S; no significant difference was found between F1473S and WT or the insensitive mutation, S941N. Consistent with previous studies showing that sodium channel blockers inhibit preferentially Isus versus peak current,18 tonic block of Isus was proportionally stronger than tonic block of peak current with 10 μmol/L mexiletine (Figure 4A). The tonic block of Isus was similar among the 3 mutants.
An additional well-known mechanism underlying the LQT3 phenotype is the reopening of sodium channels over voltage ranges for which SSI and SSA overlap (ie, the window current). We tested the effect of 10 μmol/L mexiletine on SSI and SSA in the F1473S mutation. SSI shifted toward the negative potential by −1.15 mV, SSA was slightly shifted toward the positive potential by 0.32 mV leaving the overlapping region substantially unchanged (Figure 4B).
Mexiletine Exposure and Trafficking
Previous studies suggested mexiletine rescued impaired trafficking observed in selected loss-of-function SCN5A mutants associated with Brugada syndrome.19–21 We therefore assessed whether the F1473S-Nav 1.5 trafficking defect could be rescued by the drug. F1473S channels incubated with mexiletine (10 μmol/L for 48 hours) did not change their gating properties (data not shown) but showed significantly increased current density. Figure 4C shows that INa peak current density at −5 mV increased by 42% after incubation but recorded in the absence of mexiletine, indicating that mexiletine does restore trafficking of F1473S. Interestingly, however, peak current density recorded in the presence of mexiletine (after incubation) was still increased by 32%, indicating that rescue effect overwhelmed the blocking effect.
Similar approach was used to quantify Isus: (1) incubation with 10 μmol/L mexiletine (48 hours) and recordings in the absence of mexiletine showed 46% increased Isus density. The Isus to peak current ratio remained the same as in untreated cells, implying that Isus is increased by mexiletine incubation as a direct consequence of rescuing of the trafficking. (2) Isus density recorded after incubation and in presence of 10 μmol/L mexiletine remained same as control (untreated cells) indicating that mexiletine treatment did not decrease Isus density because the rescue effect balanced out the block effect (Figure 4D).
Immunostaining with anti-hNav1.5 antibody confirmed that mexiletine incubation alleviates the trafficking impairment caused by F1473S. As shown in Figure 5A and 5⇓B, after mexiletine incubation the F1473S-Nav 1.5 clusters were markedly reduced with a redistribution of the protein in the cytoplasm and in correspondence of the cell membrane (Figure 5B, f versus c). Analysis of the different cell fractions by Western blot showed that incubation with 10 μmol/L mexiletine increased the abundance of Nav-protein on the plasma membrane without affecting the total content of protein in whole cell homogenates (Figure 5C).
Overall, we conclude that exposure to 10 μmol/L mexiletine did not decrease Isus, because the rescued trafficking of F1473S channel offset the blockade of Isus. On the contrary, peak current (and the resulting window current) was significantly increased by mexiletine as the rescuing effect overwhelmed the blocking effect on peak current.
In Silico Evaluation of F1473S-Nav1.5 and Mexiletine Effects on Action Potential
We also investigated the consequences of the biophysical properties of F1473S-Nav1.5 on action potential by using a Markovian Na+ channel model based from Clancy and Rudy22 (see the Online Data Supplement for details) tuned to simulate the mutant current behavior and incorporating it into a comprehensive action potential model of human ventricular myocyte. Figure 6 shows that the effects of F1473S on the voltage-dependence of activation, inactivation and increased window current (Figure 6A) and the time course of recovery from the inactivated state (Figure 6B) are correctly reproduced. Simulation of Isus and response to a ramp test is shown in Online Figure II.
We then simulated the consequence of mexiletine exposure on action potential in heterozygote conditions by introducing into the model the reduction of peak INa, the reduction of the Isus, the SSI shift and the rescuing effect according to the data obtained experimentally (see the Online Data Supplement for details). After mexiletine, APD prolonged (Figure 7A) and the time course of INa current during the plateau phase showed a significant reactivation of the window current that prolonged the action potential. Interestingly at fast heart rates (eg, 120 bpm) adaptation of APD was impaired resulting in failure to capture (Figure 7B).
Mutations associated with LQT3 are characterized by a gain- of-function,4 suggesting that the pharmacological reduction of INa could attenuate the QT prolongation. Because its inception this hypothesis seemed to gather rapid confirmation in experimental,7 as well as in clinical investigations8,9,23 and it supported the view that LQT3 patients would respond to such a “corrective” pharmacological treatment. Thus, the use of sodium channel blockers has been widely supported and it was incorporated into guidelines for clinical practice to reduce the arrhythmic burden with an appropriately cautious class IIb recommendation.24
As more SCN5A mutations were studied over the years, it has become progressively clear that they may combine different electrophysiological properties and induce complex phenotypes.12 Some mutations can bear, at the same time, gain-of-function and loss-of-function properties. We reported13 a single amino acid deletion (lysine at position 1500) resulting in long-QT syndrome, Brugada syndrome, and conduction disease in the same family. This observation was just one of several signals that raised concern that not all gain-of-function mutations should be considered equal and that the use of flecainide or mexiletine might be life saving for some but not for all LQT3 patients.11
In 2007, we attempted to establish whether the clinical response to mexiletine could be predicted on the basis of the biophysical properties detected with heterologous expression of SCN5A mutants. Our data15 suggested that the single most important factor that correlates with a positive response to therapy is represented by a left-ward displacement of SSI. These findings highlighted the concept that knowing the in vitro consequences of mutants could guide clinical management.
The Heterogeneous Biophysical Properties of SCN5A Mutations in LQT3
Here, we characterize a novel mutant that seems to open another chapter, with therapeutic implications, in support of the view that the loss-of-function/gain-of-function paradigm may dangerously oversimplify our clinical approach to SCN5A mutation carriers25 and that in vitro analysis of mutants might become a prerequisite to gene-specific therapy. Indeed, the evidence clearly indicates that in selected SCN5A mutations associated with a clinical phenotype suggestive of gain-of-function, the use of sodium channel blockers may result in harmful rather than curative effects.
The present investigation was prompted by a clinical case in which a patient affected by an early onset LQT3 showed no clinical improvement with mexiletine and possibly presented a worsening of the substrate leading to in-hospital death.
Interestingly, another mutation at the same site, F1473C, was recently reported to be insensitive to mexiletine,26 and the carrier of the mutation continued to experience torsade de pointes after the drug, thus requiring implantable cardioverter defibrillator implantation and left stellate gangliectomy. The biophysical characterization of the F1473C reported by Bankston et al27 is a completely different profile as compared with F1473S. F1473C causes a much smaller Isus (0.6% versus 5.39% in F1473S) and it does not reduce the peak current density. These observations highlight the fact that it is not possible to predict functional properties based on the location of a mutation and that in-depth functional studies are required.
Increased Isus28 and window current29 are 2 known mechanisms for QT prolongation in LQT3. F1473S induces both to a considerable extent. Thus, 2 parallel biophysical abnormalities concur to generate the severe clinical phenotype observed in the proband. It is worth noting that whereas mexiletine has a strong blocking effect on Isus it has only a mild blocking effect on peak current. In particular, we found that a clinically relevant concentration of mexiletine (10 μmol/L) blocked <3% of the peak current, whereas it blocked 28.8% of Isus. As a result, the improved trafficking induced by mexiletine was offset for the Isus but, because of the small peak INa blocking effect, the absolute amount of window current is greatly increased by this drug. When modeled in silico the effect of mexiletine resulted in a further action potential prolongation, which is the likely culprit that tilted the balance toward the onset of life-threatening arrhythmias.
Mexiletine and the Unexplained Mechanisms for Rescuing Trafficking of Nav 1.5 Mutants
Mexiletine trafficking-rescuing activity has been demonstrated for several Nav1.5 mutants19–21,30 but the underlying mechanisms remain elusive. It has been suggested that drugs may act as “chemical chaperones” to promote protein folding and thereby facilitating the exit of mutant ion channel from the ER.31,32 In the case of F1473S, we observed only a small fraction of the mutant protein in the ER, thus suggesting that this mechanism is unlikely to play a major role in improving membrane localization. Ankyrin-G dependent SCN5A trafficking33 is another potential mechanism. In collaboration with Mohler, we demonstrated that a human SCN5A mutation (E1053K) identified in a Brugada patient impairs ankyrin-G binding leading to a trafficking defect.34 E1053 is localized in the predicted 9-aa ankyrin binding motif in the DII–III intracellular loop of Nav1.5 (VPIAVX[E]SD), but we cannot exclude that F1473S, which is in the DIII–DIV intracellular loop, similarly causes an impaired interaction with ankyrin-G.
Another poorly understood aspect of the efficacy of mexiletine in modulating protein localization is related to the concentration at which the effect is observed. Most of the studies used high concentrations of mexiletine, suggesting that a high dosage might be required to improve membrane targeting. Ackerman and colleagues were the first to report rescuing of G1743R30 using clinical concentrations of mexiletine (10 μmol/L), pointing to the evidence that the efficacy is independent of the dosage; our data with 10 μmol/L mexiletine support this view. Recently, it was suggested that only mutations located in extracellular loop region of the SCN5A could be rescued by sodium channel blockers35: the mutant studied here is located intracellularly; thus, it supports the idea that the position of the mutation is unlikely to be the key trafficking controlling factor of the Nav1.5 polypeptide.
The Need to Revisit Recommendations for the Use of Sodium Channel Blockers in LQT3
Improvement of trafficking in defective SCN5A mutations has been considered as a therapeutic strategy that should be applied irrespective of the biophysical profile of mutants; our study provides in vitro and in silico proof of concept that this assumption may be incorrect and potentially harmful. In an interesting editorial, Bezzina et al hypothesized that rescuing mutant channels with abnormal gating behavior could pose a proarrhythmic threat.36 We now provide the first direct evidence of this hypothesis.
Our data establish a link between experimental data and clinical observations raising a warning flag to the “blanket indication” to use mexiletine in LQT3 patients. The improvement of trafficking of a gain-of-function mutant by mexiletine may be arrhythmogenic despite the channel blocking effect that would tend to counteract such a gain-of-function phenotype. The simulation shows that improvement of trafficking increases window current that is only minimally inhibited by mexiletine and thus contributes to APD prolongation.
It took the unfavorable outcome of the severely ill child affected by LQT3 to demonstrate that the loss-of-function/gain-of-function paradigm to classify SCN5A mutants is inadequate and that use of sodium channel blockers in LQT3 patients may be a double-edged sword posing a hazard to patient carriers of a SCN5A mutation with undefined electrophysiological properties.
We acknowledge the presence of limitations in our study. In vitro experiments in heterologous systems are widely used but they do not get rid of factors that may importantly modulate the effect of a mutation in the “real world,” such as temperature37 or genetic background (influence of modulatory polymorphisms).20,38 Furthermore, in vitro experiments do not consider the important contribution to arrhythmogenesis of the signaling pathways, receptors, and autonomic modulation.
Sources of Funding
This work was supported by Telethon grants nos. GGP04066 and GGP06007 and by funds from the Ministero dell’ Università e della Ricerca Scientifica e Tecnologica: FIRB RBNE01XMP4_006, RBLA035A4X_002, PRIN 2006055828_002, Italian Health Care Ricerca finalizzata RF-MAU-2007-641378.
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Novelty and Significance
What Is Known?
Long QT syndrome type 3 (LQT3) is caused by SCN5A mutations characterized by “gain-of-function” of the Nav 1.5 channel.
In clinical practice, sodium channel blockers are used to reduce sodium current and are considered a gene-specific therapy for LQT3.
SCN5A mutations characterized by “loss-of-function” are often caused by trafficking defect and can be rescued by sodium channel blockers.
What New Information Does This Article Contribute?
Sodium channel blockers can prolong QT interval and worsen arrhythmias in carriers of selected SCN5A mutations.
Bench study may be used to personalize therapy for LQT3 patients.
SCN5A mutations that increase the function of the sodium channel cause LQT3, whereas mutations that reduce the function of the channel cause the Brugada syndrome. Based on the assumption that in all LQT3 patients, there is an increase in sodium current, sodium channel blockers have been proposed as an effective therapy for LQT3. In this study, we demonstrate that the F1473S mutation not only increases sustained current and window current but also causes a trafficking defect of the sodium channel, thus combining gain-of-function and loss-of-function changes. The effect of mexiletine on F1473S channels is deleterious, because even though the drug reduces the late component of sodium current, it also increases the number of channels on membrane. The net result is an increase in sodium current. Our findings suggest that the use of sodium channel blockers may be hazardous to some LQT3 patients and suggest that functional characterization of mutants may guide mutation-specific treatment of the disease.
This manuscript was sent to Michael Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received September 12, 2009; resubmission received February 12, 2010; revised resubmission received March 10, 2010; accepted March 11, 2010.