title: Inscribing Optical Excitability to Non-Excitable Cardiac Cells: Viral Delivery of Optogenetic Tools in Primary Cardiac Fibroblasts.
abstract: We describe in detail a method to introduce optogenetic actuation tools, a mutant version of channelrhodopsin-2, ChR2(H134R), and archaerhodopsin (ArchT), into primary cardiac fibroblasts (cFB) in vitro by adenoviral infection to yield quick, robust, and consistent expression. Instructions on adjusting infection parameters such as the multiplicity of infection and virus incubation duration are provided to generalize the method for different lab settings or cell types. Specific conditions are discussed to create hybrid co-cultures of the optogenetically modified cFB and non-transformed cardiomyocytes to obtain light-sensitive excitable cardiac syncytium, including stencil-patterned cell growth. We also describe an all-optical framework for the functional testing of responsiveness of these opsins in cFB. The presented methodology provides cell-specific tools for the mechanistic investigation of the functional bioelectric contribution of different non-excitable cells in the heart and their electrical coupling to cardiomyocytes under different conditions.
authors.affiliation: Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY, USA.
authors.forename: Jinzhu
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Biomedical Engineering, Institute for Molecular Cardiology, Stony Brook University, ST-6, Room 120B, Stony Brook, NY, 11794-8661, USA. emilia.entcheva@stonybrook.edu.
authors.forename: Emilia
authors.initials: E
authors.lastname: Entcheva
mesh_terms: D000200:Action Potentials; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics*; D002478:Cells, Cultured; D000075402:Channelrhodopsins; D018920:Coculture Techniques / Q000379:methods; D055724:Electrophysiological Phenomena; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D062308:Optogenetics / Q000379:methods*; D017207:Rats, Sprague-Dawley; D019076:Transgenes; D017494:Proton Pumps
chemical_list: D019843:Archaeal Proteins; D000075402:Channelrhodopsins; C055476:archaerhodopsin protein, Archaea; D017494:Proton Pumps
keywords: ArchT; Cardiac; ChR2; Fibroblasts; Non-excitable cells; Optogenetics