Department of Biomedical Engineering, Duke University, 101 Science Drive, Room 1427, Fitzpatrick CIEMAS, Durham, North Carolina 27708, USA.
Nat Commun. 2016 Oct 18;7:13132. doi: 10.1038/ncomms13132.
The ability to directly enhance electrical excitability of human cells is hampered by the lack of methods to efficiently overexpress large mammalian voltage-gated sodium channels (VGSC). Here we describe the use of small prokaryotic sodium channels (BacNa) to create de novo excitable human tissues and augment impaired action potential conduction in vitro. Lentiviral co-expression of specific BacNa orthologues, an inward-rectifying potassium channel, and connexin-43 in primary human fibroblasts from the heart, skin or brain yields actively conducting cells with customizable electrophysiological phenotypes. Engineered fibroblasts ('E-Fibs') retain stable functional properties following extensive subculture or differentiation into myofibroblasts and rescue conduction slowing in an in vitro model of cardiac interstitial fibrosis. Co-expression of engineered BacNa with endogenous mammalian VGSCs enhances action potential conduction and prevents conduction failure during depolarization by elevated extracellular K, decoupling or ischaemia. These studies establish the utility of engineered BacNa channels for induction, control and recovery of mammalian tissue excitability.
直接增强人类细胞的电兴奋性受到缺乏有效过表达大型哺乳动物电压门控钠离子通道 (VGSC) 方法的限制。在这里，我们描述了使用小型原核钠离子通道 (BacNa) 来创建新的兴奋性人类组织并增强体外受损动作电位传导的方法。慢病毒共表达特定的 BacNa 同源物、内向整流钾通道和连接蛋白 43 可在心脏、皮肤或大脑的原代人成纤维细胞中产生具有可定制电生理表型的主动传导细胞。工程化成纤维细胞（“E-Fibs”）在大量传代或分化为肌成纤维细胞后保留稳定的功能特性，并在心脏间质纤维化的体外模型中挽救传导减慢。与内源性哺乳动物 VGSCs 共表达工程化的 BacNa 可增强动作电位传导，并在升高的细胞外 K、去耦或缺血时防止去极化引起的传导失败。这些研究确立了工程化 BacNa 通道在诱导、控制和恢复哺乳动物组织兴奋性方面的实用性。
