Peptide-Based Targeting of the L-Type Calcium Channel Corrects the Loss-of-Function Phenotype of Two Novel Mutations of the CACNA1 Gene Associated With Brugada Syndrome.

Vittoria Di Mauro,Silvia G Priori,Francesco Lodola,Carlo Napolitano,Paola Ceriotti,Daniele Catalucci,Nicolò Salvarani,Jessica Modica,Marie-Louise Bang,Andrea Mazzanti

doi:10.3389/fphys.2020.616819

Abstract

Brugada syndrome (BrS) is an inherited arrhythmogenic disease that may lead to sudden cardiac death in young adults with structurally normal hearts. No pharmacological therapy is available for BrS patients. This situation highlights the urgent need to overcome current difficulties by developing novel groundbreaking curative strategies. BrS has been associated with mutations in 18 different genes of which loss-of-function (LoF) CACNA1C mutations constitute the second most common cause. Here we tested the hypothesis that BrS associated with mutations in the CACNA1C gene encoding the L-type calcium channel (LTCC) pore-forming unit (Cavα1.2) is functionally reverted by administration of a mimetic peptide (MP), which through binding to the LTCC chaperone beta subunit (Cavβ2) restores the physiological life cycle of aberrant LTCCs. Two novel Cavα1.2 mutations associated with BrS were identified in young individuals. Transient transfection in heterologous and cardiac cells showed LoF phenotypes with reduced Ca2+ current (ICa). In HEK293 cells overexpressing the two novel Cavα1.2 mutations, Western blot analysis and cell surface biotinylation assays revealed reduced Cavα1.2 protein levels at the plasma membrane for both mutants. Nano-BRET, Nano-Luciferase assays, and confocal microscopy analyses showed (i) reduced affinity of Cavα1.2 for its Cavβ2 chaperone, (ii) shortened Cavα1.2 half-life in the membrane, and (iii) impaired subcellular localization. Treatment of Cavα1.2 mutant-transfected cells with a cell permeant MP restored channel trafficking and physiologic channel half-life, thereby resulting in ICa similar to wild type. These results represent the first step towards the development of a gene-specific treatment for BrS due to defective trafficking of mutant LTCC.

Highlights

Brugada syndrome (BrS) is an inherited arrhythmogenic disorder causing sudden death in young individuals (Napolitano et al, 2012) and has been associated with mutations in 18 different genes
The phenotypic consequences of mutations that are incompletely known and only in part experimentally reproduced in pharmacological models of L-type calcium channel (LTCC) LoF (Fish and Antzelevitch, 2004) are: (1) ST segment elevation due to a transmural voltage gradient, which is accentuated in the right ventricular outflow tract epicardium that shows a prominent transient outward potassium current
The Cavα1.2 T320M mutation was identified in a 33-yearold asymptomatic male patient, who was admitted at the emergency room for abdominal pain

Summary

Introduction

Brugada syndrome (BrS) is an inherited arrhythmogenic disorder causing sudden death in young individuals (Napolitano et al, 2012) and has been associated with mutations in 18 different genes. In the precordial unipolar electrocardiographic leads V1 and V2 covering this region the typical Type I BrS pattern is detected; (2) QT shortening due to the reduction of the inward Ca2+ current (ICa) in the ventricular myocardium (Burashnikov et al, 2010; Napolitano and Antzelevitch, 2011); (3) ST elevation in inferior or lateral leads, called early repolarization (ERP; Burashnikov et al, 2010). All these conditions predispose to an increased risk of sudden death. The coexistence of ST segment elevation and abbreviated repolarization (short QT interval) often coexist in association with CACNA1C mutations and lead to an overlapping syndrome combining phenotypes of BrS and Short QT syndromes

Methods

Results

Conclusion