iPSC-derived cardiomyocytes from patients with myotonic dystrophy type 1 have abnormal ion channel functions and slower conduction velocities.

Cardiac complications such as electrical abnormalities including conduction delays and arrhythmias are the main cause of death in individuals with Myotonic Dystrophy type 1 (DM1). We developed a disease model using iPSC-derived cardiomyocytes (iPSC-CMs) from a healthy individual and two DM1 patients with different CTG repeats lengths and clinical history (DM1-1300 and DM1-300). We confirmed the presence of toxic RNA foci and mis-spliced MBNL1/2 transcripts in DM1 iPSC-CMs. In DM1-1300, we identified a switch in the cardiac sodium channel SCN5A from the adult to the neonatal isoform. The down-regulation of adult SCN5A isoforms is consistent with a shift in the sodium current activation to depolarized potentials observed in DM1-1300. L-type calcium current density was higher in iPSC-CMs from DM1-1300, which is correlated with the overexpression of the CaV1.2 transcript and proteins. Importantly, INa and ICaL dysfunctions resulted in prolonged action potentials duration, slower velocities, and decreased overshoots. Optical mapping analysis revealed a slower conduction velocity in DM1-1300 iPSC-CM monolayers. In conclusion, our data revealed two distinct ions channels perturbations in DM1 iPSC-CM from the patient with cardiac dysfunction, one affecting Na+ channels and one affecting Ca2+ channels. Both have an impact on cardiac APs and ultimately on heart conduction.

A leaky voltage sensor domain of cardiac sodium channels causes arrhythmias associated with dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a structural heart disease that causes dilatation of cardiac chambers and impairs cardiac contractility. The SCN5A gene encodes Nav1.5, the predominant cardiac sodium channel alpha subunit. SCN5A mutations have been identified in patients with arrhythmic disorders associated with DCM. The characterization of Nav1.5 mutations located in the voltage sensor domain (VSD) and associated with DCM revealed divergent biophysical defects that do not fully explain the pathologies observed in these patients. The purpose of this study was to characterize the pathological consequences of a gating pore in the heart arising from the Nav1.5/R219H mutation in a patient with complex cardiac arrhythmias and DCM. We report its properties using cardiomyocytes derived from patient-specific human induced pluripotent stem cells. We showed that this mutation generates a proton leak (called gating pore current). We also described disrupted ionic homeostasis, altered cellular morphology, electrical properties, and contractile function, most probably linked to the proton leak. We thus propose a novel link between SCN5A mutation and the complex pathogenesis of cardiac arrhythmias and DCM. Furthermore, we suggest that leaky channels would constitute a common pathological mechanism underlying several neuronal, neuromuscular, and cardiac pathologies.

Biophysical, Molecular, and Pharmacological Characterization of Voltage-Dependent Sodium Channels From Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: The ability to differentiate patient-specific human induced pluripotent stem cells in cardiac myocytes (hiPSC-CM) offers novel perspectives for cardiovascular research. A number of studies, that reported mainly on current-voltage curves used hiPSC-CM to model voltage-gated Na+ channel (Nav) dysfunction. However, the expression patterns and precise biophysical and pharmacological properties of Nav channels from hiPSC-CM remain unknown. Our objective was to study the characteristics of Nav channels from hiPSC-CM and assess the appropriateness of this novel cell model. METHODS: We generated hiPSC-CM using the recently described monolayer-based differentiation protocol. RESULTS: hiPSC-CM expressed cardiac-specific markers, exhibited spontaneous electrical and contractile activities, and expressed distinct Nav channels subtypes. Electrophysiological, pharmacological, and molecular characterizations revealed that, in addition to the main Nav1.5 channel, the neuronal tetrodotoxin (TTX)-sensitive Nav1.7 channel was also significantly expressed in hiPSC-CM. Most of the Na+ currents were resistant to TTX block. Therapeutic concentrations of lidocaine, a class I antiarrhythmic drug, also inhibited Na+ currents in a use-dependent manner. Nav1.5 and Nav1.7 expression and maturation patterns of hiPSC-CM and native human cardiac tissues appeared to be similar. The 4 Navß regulatory subunits were expressed in hiPSC-CM, with ß3 being the preponderant subtype. CONCLUSIONS: The findings indicated that hiPSC-CM robustly express Nav1.5 channels, which exhibited molecular and pharmacological properties similar to those in native cardiac tissues. Interestingly, neuronal Nav1.7 channels were also expressed in hiPSC-CM and are likely to be responsible for the TTX-sensitive Nav current.

Nav1.5 channels can reach the plasma membrane through distinct N-glycosylation states.

BACKGROUND: Like many voltage-gated sodium channels, the cardiac isoform Nav1.5 is well known as a glycoprotein which necessarily undergoes N-glycosylation processing during its transit to the plasma membrane. In some cardiac disorders, especially the Brugada syndrome (BrS), mutations in Nav1.5 encoding gene lead to intracellular retention and consequently trafficking defect of these proteins. We used two BrS mutants as tools to clarify both Nav1.5 glycosylation states and associated secretory behaviors. METHODS: Patch-clamp recordings and surface biotinylation assays of HEK293T cells expressing wild-type (WT) and/or mutant Nav1.5 proteins were performed to assess the impact of mutant co-expression on the membrane activity and localization of WT channels. Enzymatic deglycosylation assays and brefeldin A (BFA) treatments were also employed to further characterize recombinant and native Nav1.5 maturation. RESULTS: The present data demonstrate that Nav1.5 channels mainly exist as two differentially glycosylated forms. We reveal that dominant negative effects induced by BrS mutants upon WT channel current result from the abnormal surface expression of the fully-glycosylated forms exclusively. Furthermore, we show that core-glycosylated channels can be found at the surface membrane of BFA-treated or untreated cells, but obviously without generating any sodium current. CONCLUSIONS: Our findings provide evidence that native and recombinant Nav1.5 subunits are expressed as two distinct matured forms. Fully-glycosylated state of Nav1.5 seems to determine its functionality whereas core-glycosylated forms might be transported to the plasma membrane through an unconventional Golgi-independent secretory route. GENERAL SIGNIFICANCE: This work highlights that N-linked glycosylation processing would be critical for Nav1.5 membrane trafficking and function.

Unmasked Brugada pattern by ajmaline challenge in patients with myotonic dystrophy type 1.

BACKGROUND: Myotonic dystrophy type 1 (DM1) generates missplicing of the SCN5A gene, encoding the cardiac sodium channel (Nav 1.5). Brugada syndrome, which partly results from Nav 1.5 dysfunction and causes increased VF occurrence, can be unmasked by ajmaline. We aimed to investigate the response to ajmaline challenge in DM1 patients and its potential impact on their sudden cardiac death risk stratification. METHODS: Among 36 adult DM1 patients referred to our institution, electrophysiological study and ajmaline challenge were performed in 12 patients fulfilling the following criteria: (1) PR interval >200 ms or QRS duration >100 ms; (2) absence of complete left bundle branch block; (3) absence of permanent ventricular pacing; (4) absence of implantable cardioverter-defibrillator (ICD); (5) preserved left-ventricular ejection fraction >50%; and (6) absence of severe muscular impairment. Of note, DM1 patients with ajmaline-induced Brugada pattern (BrP) were screened for SCN5A. RESULTS: In all the 12 patients studied, the HV interval was <70 ms. A BrP was unmasked in three patients but none carried an SCN5A mutation. Ajmaline-induced sustained ventricular tachycardia occurred in one patient with BrP, who finally received an ICD. The other patients did not present any cardiac event during the entire follow-up (15 ± 4 months). CONCLUSION: Our study is the first to describe a high prevalence of ajmaline-induced BrP in DM1 patients. The indications, the safety, and the implications of ajmaline challenge in this particular setting need to be determined by larger prospective studies.

Myotonic dystrophy type 1 mimics and exacerbates Brugada phenotype induced by Nav1.5 sodium channel loss-of-function mutation.

BACKGROUND: Myotonic dystrophy type 1 (DM1), a muscular dystrophy due to CTG expansion in the DMPK gene, can cause cardiac conduction disorders and sudden death. These cardiac manifestations are similar to those observed in loss-of-function SCN5A mutations, which are also responsible for Brugada syndrome (BrS). OBJECTIVE: The purpose of this study was to investigate DM1 effects on clinical expression of a loss-of-function SCN5A mutation causing BrS. METHODS: We performed complete clinical evaluation, including ajmaline test, in 1 family combining DM1 and BrS. We screened the known BrS susceptibility genes. We characterized an SCN5A mutation using whole-cell patch-clamp experiments associated with cell surface biotinylation. RESULTS: The proband, a 15-year-old female, was a survivor of out-of-hospital cardiac arrest with ventricular fibrillation. She combined a DMPK CTG expansion from the father's side and an SCN5A mutation (S910L) from the mother's side. S910L is a trafficking defective mutant inducing a dominant negative effect when transfected with wild-type Nav1.5. This loss-of-function SCN5A mutation caused a Brugada phenotype during the mother's ajmaline test. Surprisingly, in the father, a DM1 patient without SCN5A mutation, ajmaline also unmasked a Brugada phenotype. Furthermore, association of both genetic abnormalities in the proband exacerbated the response to ajmaline with a massive conduction defect. CONCLUSION: Our study is the first to describe the deleterious effect of DM1 on clinical expression of a loss-of-function SCN5A mutation and to show a provoked BrS phenotype in a DM1 patient. The modification of the ECG pattern by ajmaline supports the hypothesis of a link between DM1 and Nav1.5 loss of -function.

The ß1-subunit of Na(v)1.5 cardiac sodium channel is required for a dominant negative effect through α-α interaction.

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na(v)1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na(v)1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na(v)1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel ß(1)-subunit was essential for this dominant negative effect. Indeed, the absence of the ß(1)-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the ß(1)-subunit was present. Our findings reveal a new role for ß(1)-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.

A distinct de novo expression of Nav1.5 sodium channels in human atrial fibroblasts differentiated into myofibroblasts.

Fibroblasts play a major role in heart physiology. They are at the origin of the extracellular matrix renewal and production of various paracrine and autocrine factors. In pathological conditions, fibroblasts proliferate, migrate and differentiate into myofibroblasts leading to cardiac fibrosis. This differentiated status is associated with changes in expression profile leading to neo-expression of proteins such as ionic channels. The present study investigates further electrophysiological changes associated with fibroblast differentiation focusing on the activity of voltage-gated sodium channels in human atrial fibroblasts and myofibroblasts. Using the patch clamp technique we show that human atrial myofibroblasts display a fast inward voltage gated sodium current with a density of 13.28 ± 2.88 pA pF(-1) whereas no current was detectable in non-differentiated fibroblasts. Quantitative RT-PCR reveals a large amount of transcripts encoding the Na(v)1.5 α-subunit with a fourfold increased expression level in myofibroblasts when compared to fibroblasts. Accordingly, half of the current was blocked by 1 µm of tetrodotoxin and immunocytochemistry experiments reveal the presence of Na(v)1.5 proteins. Overall, this current exhibits similar biophysical characteristics to sodium currents found in cardiac myocytes except for the window current that is enlarged for potentials between -100 and -20 mV. Since fibrosis is one of the fundamental mechanisms implicated in atrial fibrillation, it is of great interest to investigate how this current could influence myofibroblast properties. Moreover, since several Na(v)1.5 mutations are related to cardiac pathologies, this study offers a new avenue on the fibroblasts involvement of these mutations.