Jean-Sébastien Rougier

Cardiac Voltage-Gated Sodium Channel Nav1.5 Is Regulated by Nedd4-2 Mediated Ubiquitination

Nav1.5, the cardiac isoform of the voltage-gated Na+ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Nav1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Nav1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Nav1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Nav1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2–dependent ubiquitination of Nav1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Nav1.5 and Nedd4-2. Nav1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Nav1.5. Consistent with the functional findings, localization at the cell periphery of Nav1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Nav1.5, suggesting that Nedd4-2 is a specific regulator of Nav1.5. These results demonstrate that Nav1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Nav1.5 by decreasing the channel density at the cell surface.

Identification of a novel loss-of-function calcium channel gene mutation in short QT syndrome (SQTS6)

AIMSnShort QT syndrome (SQTS) is a genetically determined ion-channel disorder, which may cause malignant tachyarrhythmias and sudden cardiac death. Thus far, mutations in five different genes encoding potassium and calcium channel subunits have been reported. We present, for the first time, a novel loss-of-function mutation coding for an L-type calcium channel subunit.nnnMETHODS AND RESULTSnThe electrocardiogram of the affected member of a single family revealed a QT interval of 317 ms (QTc 329 ms) with tall, narrow, and symmetrical T-waves. Invasive electrophysiological testing showed short ventricular refractory periods and increased vulnerability to induce ventricular fibrillation. DNA screening of the patient identified no mutation in previously known SQTS genes; however, a new variant at a heterozygous state was identified in the CACNA2D1 gene (nucleotide c.2264G > C; amino acid p.Ser755Thr), coding for the Ca(v)α(2)δ-1 subunit of the L-type calcium channel. The pathogenic role of the p.Ser755Thr variant of the CACNA2D1 gene was analysed by using co-expression of the two other L-type calcium channel subunits, Ca(v)1.2α1 and Ca(v)β(2b), in HEK-293 cells. Barium currents (I(Ba)) were recorded in these cells under voltage-clamp conditions using the whole-cell configuration. Co-expression of the p.Ser755Thr Ca(v)α(2)δ-1 subunit strongly reduced the I(Ba) by more than 70% when compared with the co-expression of the wild-type (WT) variant. Protein expression of the three subunits was verified by performing western blots of total lysates and cell membrane fractions of HEK-293 cells. The p.Ser755Thr variant of the Ca(v)α(2)δ-1 subunit was expressed at a similar level compared with the WT subunit in both fractions. Since the mutant Ca(v)α(2)δ-1 subunit did not modify the expression of the pore-forming subunit of the L-type calcium channel, Ca(v)1.2α1, it suggests that single channel biophysical properties of the L-type channel are altered by this variant.nnnCONCLUSIONnIn the present study, we report the first pathogenic mutation in the CACNA2D1 gene in humans, which causes a new variant of SQTS. It remains to be determined whether mutations in this gene lead to other manifestations of the J-wave syndrome.

PDZ Domain–Binding Motif Regulates Cardiomyocyte Compartment-Specific NaV1.5 Channel Expression and Function

Background— Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain–binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results— To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (&Dgr;SIV). &Dgr;SIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of &Dgr;SIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and &Dgr;SIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued &Dgr;SIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. Conclusions— Our results demonstrate the in vivo significance of the PDZ domain–binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Variable Na(v)1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-) mouse model.

Background Loss-of-function mutations in SCN5A, the gene encoding Nav1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a +/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. Methodology/Principal Findings Based on ECG, 10-week-old Scn5a +/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a +/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a +/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a +/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a +/− mice had similar Nav1.5 mRNA but higher Nav1.5 protein expression, and moderately larger INa current than severely affected Scn5a +/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a +/− mice than in mildly affected ones. Conclusions Scn5a +/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a +/− mice, phenotype severity correlates with wild-type Nav1.5 protein expression.

TRPM4 channels in the cardiovascular system: Physiology, pathophysiology, and pharmacology

The transient receptor potential channel (TRP) family comprises at least 28 genes in the human genome. These channels are widely expressed in many different tissues, including those of the cardiovascular system. The transient receptor potential channel melastatin 4 (TRPM4) is a Ca(2+)-activated non-specific cationic channel, which is impermeable to Ca(2+). TRPM4 is expressed in many cells of the cardiovascular system, such as cardiac cells of the conduction pathway and arterial and venous smooth muscle cells. This review article summarizes the recently described roles of TRPM4 in normal physiology and in various disease states. Genetic variants in the human gene TRPM4 have been linked to several cardiac conduction disorders. TRPM4 has also been proposed to play a crucial role in secondary hemorrhage following spinal cord injuries. Spontaneously hypertensive rats with cardiac hypertrophy were shown to over-express the cardiac TRPM4 channel. Recent studies suggest that TRPM4 plays an important role in cardiovascular physiology and disease, even if most of the molecular and cellular mechanisms have yet to be elucidated. We conclude this review article with a brief overview of the compounds that have been shown to either inhibit or activate TRPM4 under experimental conditions. Based on recent findings, the TRPM4 channel can be proposed as a future target for the pharmacological treatment of cardiovascular disorders, such as hypertension and cardiac arrhythmias.

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) controls the sorting of newly synthesized Ca(V)1.2 calcium channels.

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Cav calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Cav1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Cav whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Cav pore-forming α1 subunit nor the associated Cavβ and Cavα2δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Cav channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Cav currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Cav channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Cavβ subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Cavβ at the endoplasmic reticulum/Golgi level to prevent the delivery of Cav channels at the plasma membrane.

Molecular characterization of two founder mutations causing long QT syndrome and identification of compound heterozygous patients

Background. Mutations of at least six different genes have been found to cause long QT syndrome (LQTS), an inherited arrhythmic disorder characterized by a prolonged QT interval on the electrocardiogram (ECG), ventricular arrhythmias and risk of sudden death. Aim. The aims were to define the yet undetermined phenotypic characteristics of two founder mutations and to study clinical features in compound heterozygotes identified during the course of the study. Methods. To maximize identification of the compound heterozygotes, we used an extended group of LQTS patients comprising 700 documented or suspected cases. Functional studies were carried out upon transient expression in COS‐7 or HEK293 cells. Results. The KCNQ1 IVS7‐2A>G (KCNQ1‐FinB) mutation associated with a mean QTc interval of 464u2005ms and a complete loss‐of‐channel function. The HERG R176W (HERG‐FinB) mutation caused a reduction in current density as well as slight acceleration of the deactivation kinetics in vitro, and its carriers had a mean QTc of 448u2005ms. The HERG R176W mutation was also present in 3 (0.9%) out of 317 blood donors. A total of six compound heterozygotes were identified who had the HERG R176W mutation in combination with a previously reported LQTS mutation (KCNQ1 G589D or IVS7‐2A>G). When present simultaneously with an apparent LQTS‐causing mutation, the HERG R176W mutation may exert an additional in vivo phenotypic effect. Conclusions. The HERG R176W mutation represents a population‐prevalent mutation predisposing to LQTS. Compound heterozygosity for mutant LQTS genes may modify the clinical picture in LQTS.

Modulation of Nav1.5 Channel Function by an Alternatively Spliced Sequence in the DII/DIII Linker Region

In the present study, we identified a novel splice variant of the human cardiac Na+ channel Nav1.5 (Nav1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Nav1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Nav1.5d was not detected in the mouse heart, indicating that alternative splicing of Nav1.5 is species-dependent. In HEK293 cells, splice variant Nav1.5d generated voltage-dependent Na+ currents that were markedly reduced compared with wild-type Nav1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Nav1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Nav1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Nav1.5.

Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current

BACKGROUNDnMembrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization.nnnOBJECTIVEnThe purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes.nnnMETHODSnSAP97(fl/fl) mice were generated by inserting loxP sequences flanking exons 1-3 of the SAP97 gene. SAP97(fl/fl) mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97(fl/fl) mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase-polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice.nnnRESULTSnAction potential duration was greatly prolonged in αMHC-Cre/SAP97(fl/fl) cardiomyocytes compared to SAP97(fl/fl) controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97(fl/fl) mice.nnnCONCLUSIONnThese data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.

Regulation of the Cardiac Sodium Channel Nav1.5 by Utrophin in Dystrophin Deficient Mice

AIMSnDuchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we investigated mice deficient in both dystrophin and utrophin [double knockout (DKO)] to assess the role of utrophin in the regulation of the cardiac sodium channel (Na(v)1.5) in mdx mice.nnnMETHODS AND RESULTSnCo-immunoprecipitation studies in HEK293 cells showed that utrophin interacts with Na(v)1.5 via syntrophin proteins, an interaction abolished by deletion of the PDZ (PSD-95, Dlg, and Zona occludens) domain-binding motif of Na(v)1.5. We also provide evidence for such interaction in mouse heart using Na(v)1.5 C-terminus fusion proteins. In hearts of DKO mice, Na(v)1.5 protein levels were decreased by 25 ± 8%, together with a 42 ± 12% reduction of syntrophins compared with mdx, where utrophin was up-regulated by 52 ± 9% compared with C57BL/10 control mice. Sodium current was found to be reduced by 41 ± 5% in DKO cardiomyocytes compared with mdx, representing a loss of 63 ± 3% when compared with C57BL/10 wild-type control mice. Decreased Na(v)1.5 protein and current in DKO were reflected in a significant slowing of 27 ± 6% of maximal upstroke velocity of the cardiac action potential compared with mdx.nnnCONCLUSIONnUtrophin plays a central role in the regulation of Na(v)1.5 in mdx mice. These findings provide support for therapeutic strategies aimed at overexpressing utrophin in the hopes of reducing cardiac pathology in DMD patients.