Towards Cardiac Safety Screens by Single Cell Imaging Procedures

Abstract

To address cardiac safety screens in vitro, neonatal cells may not represent a good model due to non-adult gene expression patterns. Therefore we explored a novel concept of high content screens utilising isolated adult cardiomyocytes. This approach based on a long term culture procedure developed and established for these cells enabling culturing of cardiomyocytes for one week without major dedifferentiation. Such an approach is suitable as a safety screen since experiments can be performed in acute and chronic stimulation conditions. In our hands fluorescence microscopy appears to be an ideal tool to perform such screens. An essential prerequisite for long term fluorescence recoding, ideally even using an identical cell population, was the application of genetically encoded biosensors. They represent an almost ideal sensor-system since transfection is easy, characterised by high (almost 100%) transfection rates, fast expression (<24h) and high biocompatibility. In addition appropriate transfection systems are available for both in vitro (Adenovirus) and in vivo (Lentivirus) situations. Here, we demonstrate the result of a long term expression of various calcium sensors while imaging individual cells for several hours. We compared results obtained in cardiac myocytes expressing various such sensors with respect to their biophysical properties and putative changes of calcium handling and biocompatibility.The combination of various imaging techniques, such as fast video microscopy, total internal reflection fluorescence microscopy, fluorescence lifetime imaging and fluorescence redistribution after photobleach will allow flexible and complex screening protocols. Furthermore, online image processing algorithms will allow intelligent alterations of screening processes that depend on the cellular response. We demonstrate how all these component add up to use single cell models of adult cardiac myocytes for high-content safety screenings.This work is supported by the Federal Ministry for Education and Research (BMBF) in the framework “Biophotonics III”.