This dataset contains the data used to generate the central figure in the associated publication. BACKGROUND:
Previously, we have shown that NavSheP D60A, an engineered prokaryotic voltage-gated sodium channels (BacNav), can augment electrical excitability and reduce arrhythmia incidence in cardiomyocyte cultures and infarcted primate hearts. While NavSheP is a promising candidate for BacNav gene therapy, its activation and inactivation kinetics are slower than those of Nav1.5 channels. The discovery of novel BacNav orthologs with faster gating kinetics would more efficiently augment peak INa and rescue impaired conduction in diseased myocardium. More generally, different therapeutic applications may require engineered channels with different biophysical properties, prompting us to expand the pool of functional BacNav channels.
METHODS:
Fifty novel BacNav orthologs were identified from a comprehensive search through Uniprot bacteria database. These orthologs were human-codon optimized, synthesized, and lentivirally expressed in 293KC cells (a monoclonal HEK293 line stably expressing Kir2.1 and connexin-43), which were subsequently characterized using whole-cell patch clamp recording and optical mapping. Top BacNav candidate was further validated in engineered 3D neonatal rat cardiac tissues (“cardiobundles”) and a Scn5a+/- mouse model.
RESULTS:
Twenty-nine functional BacNav were identified and characterized by whole-cell patch clamp recordings. Among these, NavRhi exhibited the most favorable electrophysiological properties, demonstrating significantly higher conduction velocity (CV) and maximum capture rate (MCR) than NavSheP in transduced 293KC cells. Furthermore, NavRhi expression significantly improved cardiobundle CV by 15.4% compared to control tissues (44.9±1.4 cm/s vs. 51.8±1.9 cm/s). In the proof-of-concept in vivo study, AAV-mediated NavRhi expression in Scn5a+/- mice rescued the conduction slowing in right ventricles to levels approaching WT littermates.
CONCLUSIONS:
In this study, we for the first time exploited rich bacterial diversity to screen, identify, and validate novel BacNav gene therapy candidates. Specifically, we identified a BacNav ortholog NavRhi that effectively improved cardiac AP conduction in engineered cardiac tissues in vitro and murine Scn5a+/- model of reduced excitability ex vivo.
