Sodium channel trafficking pathways in cardiomyocyte – TRAFFIC

We used different cell models (murine cardiomyocytes and induced pluripotent stem cell (hIPS) derivatives) to invalidate CASK and study the organization of cell contacts (corresponding to disks in vitro), at both molecular and functional levels. Proteomic analysis was also carried out to obtain a comprehensive view of the pathways regulated by CASK. We then tested the benefit of the CASK invalidation strategy in a human in vitro model of arrhythmia characterized by the loss of desmosomes. We used a murine animal model to study the functional effects (pump and electrical function) of the CASK invalidation strategy. To carry out this project, we 1) developed the various plasmid constructs needed to produce the viruses required to infect cardiac cells in vitro and in vivo, 2) developed contact quantification algorithms, 3) developed constructs enabling real-time monitoring of membrane proteins, and finally 4) generated cardiomyocyte lines derived from control and mutation-carrying induced pluripotent stem cells.

Our work has shown that depletion of the CASK protein 1) promotes cardiac function in vivo, and 2) promotes in vitro the organization of disc proteins: electrical junctions and desmosomes. This strategy has proved particularly effective in restoring desmosomal junctions in arrhythmogenic right ventricular cardiomyopathy. This discovery formed the basis of a new project focusing on the CASK depletion strategy in this hereditary pathology, for which the only current therapeutic solution is heart transplantation.

Proof of concept of the efficacy of CASK invalidation to promote desmosome stabilization in arrhythmogenic cardiomyopathy in vitro.
Potential application in gene therapy for arrhythmogenic cardiomyopathy.

Publication 1: Blandin CE, Dilanian G, Fontaine V, Mougenot N, Gravez B, Bobin P, Duboscq-Bidot L, Farhi D, Chardonnet S, Nadaud S, Sanchez-Alonzo JL, Shevchuk A, Gorelik J, Gandjbakhch E, Hatem SN, Villard E, Balse E.
Depleting trafficking regulator CASK promotes intercalated disc organization and ventricular function, BIORXIV/2024/618172
Publication 2: Blandin CE, Gravez BJ, Hatem SN, Balse E.
Remodeling of Ion Channel Trafficking and Cardiac Arrhythmias. Cells 2021. doi: 10.3390/cells10092417.

Context: Localization and ion channel density at the cell surface of cardiomyocytes (CMs) determine the cardiac electrical activity. Disorganization or functional alteration of these ion channels can lead to cardiac arrhythmias, often fatal. The sodium current, carried by NaV1.5 channels, is responsible for the fast depolarization and propagation of the action potential form one CM to another. NaV1.5 channels are heterogeneously distributed in the membrane of CMs with high concentration at the intercalated disk and lower density at the lateral membrane. This is critical for the anisotropic conduction (longitudinal>transversal) of the depolarization wave front in the myocardium. NaV1.5 is positively regulated by various protein partners mainly located at the intercalated disk. In contrast, we have identified the original partner CASK exclusively located at the lateral membrane, which negatively regulates the channel by impairing its anterograde trafficking.

Research hypothesis and Objectives: Considering its specific location at the lateral membrane (notably at the focal adhesion) and its inhibitory function on the channel, CASK could play a central role in the structural and functional organization of CMs and the setting-up of anisotropy. The objective of the project is to understand the molecular basis of the complex interplay between the structural and the electrical organization of CMs.

WP1) To study in real-time the trafficking and targeting of NaV1.5 in the CM (disk vs lateral membrane). We will test the hypothesis of a sorting hub at early stages of trafficking which orientates the targeting of NaV1.5 towards these domains. Partner 2 has developed a retention assay based on a selective hooks (RUSH) system enabling synchronous release of proteins from one donor compartment to one final compartment. Co-trafficking of NaV1.5 and partners will be investigated using multi-color imaging in adult CMs.

WP2) To characterize the role of CASK in the organization of CM microarchitecture and locally regulated exocytosis. The use of truncated forms of CASK developed by Partner 1 and RUSH technology from Partner 2 will allow studying real-time trafficking of the channel in CMs. TIRF microscopy will be used to follow the dynamics of NaV1.5 in the plasma membrane plane. 3D-imaging studies will be conducted to follow the organization of cytoskeleton and focal adhesion in CMs after manipulating CASK expression level. An agnostic approach relying on proteomic analysis will be conducted to unravel new pathways regulated by CASK.

WP3) To study the role of CASK in the setting-up and maintenance of structural polarity and anisotropy of CMs. Two conditions characterized by disorganization/(re)organization of the myocardium will be considered: ontogenic development and ischemic myocardial remodeling. CASK expression will be manipulated in vivo (AAV) in both new-born and adult rats in order to study CASK role on the cardiac function and on the myocardium ex vivo (biochemistry, IHF and electrophysiology). CASK expression level in border and remotes zones of the infarct will be investigated and manipulated (AAV) to determine whether CASK can prevent or worsen the remodeling process.

Originality and relevance related to the state-of-the-art: The mechanisms regulating NaV1.5 trafficking towards specialized membrane domains of the CM are poorly understood. In the context of cardiopathies in which the 3D organization of the tissue is compromised, an altered targeting of ions channels constitutes a major cause of electrical remodeling. Therefore, understanding the spatio-temporal organization of NaV1.5 and its partners in physiological and pathological conditions should open new therapeutic avenues targeting intracellular trafficking.