Biology subjects

Doisne, N.

Publications and source records attributed to Doisne, N..

4 recordsLinked to original sources

bioRxiv2024

Gene Therapy with the N-terminal Fragment of Nav1.5 for Cardiac Channelopathies: A Novel Transcomplementation Mechanism Potentiating the Cardiac Sodium Current

BACKGROUNDCardiac channelopathies, caused by mutations in ion-channel genes, can lead to sudden cardiac death (SCD) via ventricular arrhythmias. Brugada syndrome (BrS) is a rare inherited channelopathy characterized by a unique ECG pattern and a high incidence of ventricular fibrillation leading to SCD in the absence of structural heart defects. The main gene responsible for 20-25% of BrS cases is SCN5A, encoding the cardiac sodium channel -subunit Nav1.5, which carries the sodium current (INa) responsible for the rapid depolarization phase of the action potential (AP). While current treatments do not target the genetic cause of channelopathies, this study explores the therapeutic potential of overexpressing the N-terminal region of Nav1.5 (Nter) to restore electrical activity by rescuing INa, in the context of SCN5A deficiency. METHODSWe overexpressed the Nter peptide using viral vectors in Scn5a+/- mice, in CRISPR/Cas9 edited-SCN5A+/- cardiomyocytes derived from induced-pluripotent stem cells (iPSC-CMs) and in BrS patient iPSC-CMs. We assessed molecular and functional effects of Nter overexpression in vitro and in vivo by measuring Nav1.5 subcellular expression and electrophysiological activity and by recording ECGs and arrhythmias. RESULTSWhereas Scn5a+/- mice showed an impaired INa associated with a slowed-cardiac conduction characteristic of the BrS phenotype, cardiac-specific overexpression of Nter corrected AP parameters by restoring INa density in Scn5a+/- mouse cardiomyocytes. This increase in INa density was caused by a translocation of Nav1.5 to the cell membrane in Nter-overexpressing mice. Most importantly, Nter overexpression normalized atrioventricular and ventricular conduction and protected Scn5a+/- mice from arrhythmias triggered by programmed electrical stimulation. Similarly, Nter overexpression in SCN5A+/- human iPSC-CMs led to a 2-fold increase in Nav1.5 cell-surface expression, resulting in normalization of INa and AP parameters and abolition of early after depolarizations observed during spontaneous AP recordings. Similar results were obtained in iPSC-CMs derived from a BrS patient, confirming the potential of this therapy in human models. CONCLUSIONSThis study identified a novel therapeutic peptide effective in restoring cardiac excitability in animal and cellular models of BrS, paving the way for future development of therapies for life-threatening arrhythmias in patients with SCN5A deficiency.

physiology↗

bioRxiv2020

HIGH FAT DIET STIMULATES BETA-OXIDATION, ALTERS ELECTRICAL PROPERTIES AND INDUCES ADIPOGENICITY OF ATRIA IN OBESE MICE

Metabolic disorders such as obesity are risk factors of atrial fibrillation, not only by sharing comorbidities but likely through their direct impact on atria, notably its adipogenicity. Here, we submitted mice that lack cardiac adipose tissue to a high fat diet and first studied the atrial metabolomic and lipidomic phenotypes using liquid chromatography-mass spectrometry. We found an increased consumption of free fatty acid by the beta-oxidation and an accumulation of long-chain lipids in atria of obese mice. Free fatty acid was the main substrate of mitochondrial respiration studied in the saponin-permeabilized atrial muscle. Conducted action potential recorded in atrial trabeculae was short, and ATP-sensitive potassium current was increased in perforated patch-clamp atrial myocytes of obese mice. There was histological and phenotypical evidence for an accumulation of adipose tissue in obese mice atria. Thus, an obesogenic diet transforms the energy metabolism, causes fat accumulation and induces electrical remodeling of atria myocardium. HIGHLIGHTS- Untargeted metabolomic and lipidomic analysis revealed that a high fat diet induces profound transformation of atrial energy metabolism with beta-oxidation activation and long-chain lipid accumulation. - Mitochondria respiration studied in atrial myocardial trabecula preferentially used Palmitoyl-CoA as energy substrate in obese mice. - Atria of obese mice become vulnerable to atrial fibrillation and show short action potential due to the activation of K-ATP dependent potassium current. - Adipocytes and fat molecular markers were detected in atria of obese mice together with an inflammatory profile consistence with a myocardial accumulation of fat.

physiology↗

bioRxiv2019

Distinct CASK domains control cardiac sodium channel membrane expression and focal adhesion anchoring

Membrane-associated guanylate kinase (MAGUK) proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein CASK (Calcium/CAlmodulin-dependent Serine protein Kinase) negatively regulates the main cardiac sodium channel, NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex, and like syntrophin, binds to the C-terminal domain of the channel. Here we show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. Thus, the multi-modular structure of CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocyte.\n\nSUMMARYSequential functional domain deletion approach identifies three critical domains of CASK in cardiomyocytes. CASK binds the cell adhesion dystrophin-glycoprotein complex through HOOK domain and inhibits NaV1.5 channel membrane expression by impeding trafficking through L27B and GUK domains.

physiology↗

bioRxiv2019

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusturization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule-dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane.

cell biology↗