Abstract 14943: High-Throughput Contactless Optogenetic Assay for Cellular Coupling: Illustration by Chr2-Light-Sensitized Cardiac Fibroblasts and Cardiomyocytes
Cellular coupling is an important determinant of cardiac function. The most direct way to quantify coupling, the dual patch clamp, lacks scalability and applicability in situ; other approaches are indirect and low-throughput, using fluorescent molecule diffusion to infer coupling strength. We demonstrate experimentally and computationally a conceptually new contactless assay for electrical coupling based on selective illumination of inexcitable cells expressing ChR2 (light-sensitive ion channel) and optical sensing of the response of coupled excitable cells, e.g. cardiomyocytes, that are light-insensitive. Cell-cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-excited cell(s). In vitro, we transduced primary cardiac fibroblasts (FB) with ChR2 using Ad-viral delivery, patterned them in a focal zone, and probed their coupling to a monolayer of neonatal rat cardiomyocytes (CM) by all-optical interrogation of excitability, Fig A-B. Cx43-mediated coupling was confirmed between the two cell types, Fig C. We found that the irradiance needed to excite at long pulses (90ms) was sensitive to different levels of coupling obtained by manipulating gap junction conductance with antagonists (heptanol) or agonists (4-phenylbutyrate, 4PB), Fig D. Computationally, we confirm that irradiance at long-duration pulses is a suitable quantitative measure of coupling; sensitivity was improved when light-sensitized cells had a more hyperpolarized resting potential (vs. normal, -50mV for FB), Fig E. The proposed assay is applicable for probing electrical coupling between multiple cell types with high specificity and spatial resolution; it could also be used in a sub-threshold regime if subtle membrane potential changes can be optically captured. Model-facilitated calibration will permit the quantification of coupling in situ and/or in high-throughput settings.
- © 2013 by American Heart Association, Inc.