Abstract Body: Cardiac voltage-gated sodium channels (Na_V1.5) are critical for the initiation of action potentials within the heart. Alterations in Na_V1.5 function significantly contribute to diverse inherited and acquired arrhythmias, such as Long QT syndrome (LQT), Brugada syndrome (BrS), mixed-syndrome, and heart failure. These pathologies are closely associated with two distinct modifications in Na_V1.5 function: (1) a gain-of-function phenotype, characterized by incomplete inactivation of Na_V1.5 leading to a sustained Na⁺ current (I_NaL), and (2) a loss-of-function phenotype resulting in diminished peak Na⁺ current (I_NaP). Despite the broad understanding of Na_V1.5 implication in cardiac pathophysiology, the development of effective therapeutic strategies to reverse these dysfunctions remains challenging.
Here, we introduce a novel bifunctional actuator that inhibits I_NaL and upregulates I_NaP, as a common molecular strategy to reverse pathophysiological changes in Nav-linked cardiac arrhythmias. To do so, we use a genetically encoded approach by engineering a selective nanobody (nb82) targeting Na_V1.5, linked to (1) a peptide inhibitor targeting I_NaL (FixR), and (2) the catalytic domain of a deubiquitinase (DUB) to enhance Na_V1.5 membrane expression. Changes in I_NaP and I_NaL were assessed using whole-cell and multichannel electrophysiology techniques in both wild-type and channelopathic mutant channels. Our results demonstrate that NbFixR-DUB effectively reduces I_NaL while concurrently increasing I_NaP for multiple channelopathic mutations. Overall, this approach holds considerable promise for rectifying both loss-of-function and gain-of-function phenotypes in Nav channelopathies.