Abstract 17526: Engineering Prokaryotic Sodium Channels for Generation and Control of Mammalian Tissue Excitability
Introduction: Treatment of various excitability tissue disorders is often hampered by the inability to stably overexpress large mammalian voltage-gated sodium channels (VGSC) via viral delivery methods. Here we propose that the small gene size of prokaryotic sodium channels (BacNav) could be utilized to generate and directly enhance mammalian tissue excitability.
Methods: We created a mutant library of BacNav with varying biophysical characteristics and performed computational simulation to predict their effects on electrical properties of engineered excitable cells. We then transduced multicistronic lentiviruses expressing select BacNav mutants, Kir2.1, and Cx43 channels in human dermal and ventricular fibroblasts to generate electrically excitable cells capable of action potential conduction. Structural and functional characterization of engineered fibroblasts (E-Fibs) were conducted via immunostaining, cell coupling analyses, patch clamp, and optical mapping.
Results: We efficiently generated excitable cells exhibiting wide ranges of action potential durations (75 to 582 ms), maximum upstrokes (19 to 170 V/s), and conduction velocities (3 to 21 cm/s). E-Fibs maintained stable electrophysiological phenotypes despite significant cell expansion or conversion into myofibroblasts. In an in vitro model of interstitial fibrosis, E-Fibs successfully recovered impaired cardiac conduction (Fig. 1a-c). Furthermore, expression of select BacNav mutant in mammalian excitable tissue rescued conduction failure under high extracellular potassium condition where endogenous Nav1.5 channels were largely inactivated (Fig. 1d-f).
Conclusions: We developed a robust computational and experimental platform for engineering, screening and utilization of BacNav channels to create de novo excitable human tissues or recover impaired tissue excitability. These results could pave ways for new cell and gene therapies for arrhythmogenic heart diseases.
Author Disclosures: H. Nguyen: None. N. Bursac: None.
- © 2016 by American Heart Association, Inc.