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In vivo relevance of the PY and PDZ-domain binding motifs of the cardiac sodium channel Nav1.5

English title In vivo relevance of the PY and PDZ-domain binding motifs of the cardiac sodium channel Nav1.5
Applicant Abriel Hugues
Number 120707
Funding scheme Project funding (Div. I-III)
Research institution Department for BioMedical Research Medizinische Fakultät Universität Bern
Institution of higher education University of Lausanne - LA
Main discipline Cardiovascular Research
Start/End 01.04.2008 - 31.03.2011
Approved amount 570'000.00
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All Disciplines (2)

Discipline
Cardiovascular Research
Cardiovascular Research

Keywords (5)

Brugada syndrome; sodium channel; ubiquitin; Cardiac arrhythmia; lonq QT syndrome

Lay Summary (English)

Lead
Lay summary
Sudden death caused by disturbances of the cardiac rhythm (arrhythmias) is frequent in industrialized countries. The mechanisms underlying cardiac arrhythmias are complex and dynamic. Thus far, we only partially understand the molecular and cellular mechanisms involved in this type of diseases. The general goal of this project is to obtain detailed information about the regulation of a specific ion channel (a membrane protein mediating the flow of ions across the cell membrane) called Nav1.5. This channel is mainly expressed in the heart and mediates the influx of sodium ions into the cardiac cells, hence playing a key role in the electrical activity of the heart. Many recent studies have demonstrated that malfunction of this channel caused by genetic mutations may lead to a large number of different cardiac diseases. In this project, we postulate that this channel is regulated by two types of proteins interacting directly with it: (1) ubiquitin ligases and (2) anchoring proteins.In order to study the physiological relevance of these interactions, in this project, we plan to generate mouse lines that will be genetically modified. We will alter the domains of the mouse gene coding for Nav1.5 in a way that these proteins will not be able to interact with the channel anymore. Afterwards, we will investigate the consequences of these genetic modifications on the electrical activity of the heart. This will be performed by carrying out biochemistry, cellular and whole-animal experiments. The new knowledge that will be obtained by performing the proposed experiments will allow us to understand much more precisely how Nav1.5 is regulated. This will permit us to propose new models about how this channel is involved in arrhythmic diseases and eventually better prevent sudden cardiac death.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current.
Gillet Ludovic, Rougier Jean-Sébastien, Shy Diana, Sonntag Stephan, Mougenot Nathalie, Essers Maria, Shmerling Doron, Balse Elise, Hatem Stéphane N, Abriel Hugues (2015), Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current., in Heart rhythm, 12(1), 181-92.
PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.
Shy Diana, Gillet Ludovic, Ogrodnik Jakob, Albesa Maxime, Verkerk Arie O, Wolswinkel Rianne, Rougier Jean-Sébastien, Barc Julien, Essers Maria C, Syam Ninda, Marsman Roos F, van Mil Anneke M, Rotman Samuel, Redon Richard, Bezzina Connie R, Remme Carol Ann, Abriel Hugues (2014), PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function., in Circulation, 130(2), 147-60.

Associated projects

Number Title Start Funding scheme
127682 Inherited Channelopathies 01.09.2010 International Exploratory Workshops
135693 In vivo relevance of the PY and PDZ-domain binding motifs of the cardiac sodium channel Nav1.5 01.04.2011 Project funding (Div. I-III)

Abstract

In vivo relevance of the PY and PDZ-domain binding motifs of the cardiac sodium channel Nav1.5Research proposal submitted to the Swiss National Science Foundation, October 2007Applicant: Hugues Abriel; UNI Lausanne, Pharmacology & Toxicology, and CardiologySummary of the Research Plan:Background - Recent genetic findings have unveiled that cardiac ion channel dysfunction plays a central role in the genesis of arrhythmias leading to sudden cardiac death (SCD). Among the different channels expressed in the heart, the voltage-gated sodium channel Nav1.5 is of particular interest as mutations in its gene, SCN5A, lead to many different pathological phenotypes: congenital and acquired long QT syndromes (LQTS), Brugada syndrome, congenital and progressive conduction disorders, sudden infant death syndrome, sinusal dysfunction, atrial fibrillation, and dilated cardiomyopathy. This long and impressive list highlights the important roles played by Nav1.5 in cardiac physiology and suggests that alterations in its function may also participate in arrhythmias linked to acquired cardiac diseases, such as heart failure. The molecular and cellular determinants involved in the regulation of Nav1.5 channel density and localization in cardiac cell membrane are still poorly understood. Our group recently made several important contributions. We showed that Nav1.5 can be ubiquitylated in cardiac tissues and that, by using heterologous expression systems, the ubiquitin ligase Nedd4-2 ubiquitylates and reduces the membrane density of this channel. Nedd4-2 was shown to interact with a PY motif located in the C-terminus of Nav1.5. We also found that Nav1.5 is part of a membrane-associated multiprotein complex comprising syntrophins and dystrophin. Syntrophins were shown to interact with the PDZ-domain binding motif of Nav1.5. We observed a clear reduction of Nav1.5 protein expression and Nav1.5-mediated currents in cardiac cells of dystrophin-deficient mice. Finally, in a series of unpublished experiments, we found that the PDZ-domain binding motif of Nav1.5 also interacts with a MAGUK anchoring protein called SAP-97. SAP-97 has been reported to be enriched at the intercalated disks of cardiac cells, whereas dystrophin, absent from this membrane compartment, is only found in lateral membranes. The proposed experiments are the continuation of a project initiated 5 years ago aimed at deciphering the molecular determinants of Nav1.5 regulation. In order to address the physiological relevance of these previous and preliminary findings, we now plan to tackle this topic by utilizing genetically-modified mouse lines, rather than using heterologous expression systems.Working Hypothesis - At least two pools of Nav1.5 channels, anchored by distinct proteins, are present at the cell membrane of cardiomyocytes: one at the intercalated disks (complexed with SAP-97) and one in lateral membranes (part of the dystrophin complex). Based on our previous findings, we propose that the density of Nav1.5 at the cell membrane of cardiomyocytes also depends on the activity of the ubiquitin ligases Nedd4-2. We postulate that a lack of dystrophin may lead to an increased Nedd4-dependent ubiquitylation of Nav1.5, and that by inhibiting the proteasome with specific drugs we can interfere with this mechanism.Accordingly, the three specific aims of this research proposal are: (1) To show the physiological relevance of the PY motif of Nav1.5 in vivo (2) To demonstrate the role of the interaction between SAP-97 and the PDZ-domain binding motif of Nav1.5 (3) To elucidate the role of the ubiquitin-proteasome system in Nav1.5 down-regulation observed in dystrophin-deficient miceExperimental Design - We plan to study Nav1.5 at the molecular and functional level using cardiomyocytes from genetically-modified mice. The two mouse lines currently available are dystrophin-deficient mdx-5cv mice and transgenic mice expressing a dominant-negative Nedd4-2 (Nedd4-2-DeltaHECT). In addition, two knock-in mouse lines are currently being generated in the laboratory: one harboring a naturally-occurring mutation of the PY motif found in one LQTS patient, Y1977N, and one with a truncation of the PDZ-binding motif (DeltaSIV). In vivo, cellular, and biochemical phenotyping of these mice will be performed using a set of techniques available in our laboratory.Expected Value of the Project - Since Nav1.5 plays a crucial role in cardiac excitability and conduction, we postulate that alterations in its localization and density in cardiac cells may underlie cardiac diseases. The results of this work will elucidate not-yet-described mechanisms to regulate Nav1.5 that may be involved in disorders in which SCD is frequent such as ischemic heart disease or chronic heart failure.
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