Optical Action Potential Screening on Adult Ventricular Myocytes as an Alternative QT-screen

Abstract

Background/Aims: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. Methods: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. Results: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. Conclusion: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

“…When using gene transfer with adenoviruses, protein expression of the genetically encoded fusion proteins or biosensors reached optically detectable levels within 24 hours. 30,31 Adenovirus-mediated expression is rather stable during the time course of 1 week, and we did not observe adverse effects caused by the viral transduction or induced gene expression during that period. 32,33 Therefore, adenovirus-mediated gene transfer seems to be the most suitable method for the expression of proteins in cultured adult cardiomyocytes.…”

“…Such ratios are not independent of uneven bleaching and contraction artifacts, which in beating myocytes are particularly advantageous properties of ratiometric measurements. 31 Quantitative FRET signals rely on 2 major determinants: (1) the FRET efficiency of the sensor itself, which is determined by the distance between the donor and the acceptor and the angle of their dipoles to one another and (2) the number of molecules that interact. For FRET-based genetically encoded Ca 2+ sensors, a particular molecule is expected to be in 1 of 2 conformations, either Ca 2+ free or Ca 2+ occupied.…”

“…120,121 A robust isolation procedure 122 and greatly improved cell culture 23 enable the use of adult cardiomyocytes, which is a prerequisite that seems to be compulsory, particularly in consideration of the different gene expression patterns of embryonic, neonatal, and adult cardiac myocytes. 31,123 The major limitation is the requirement for electrodes in multiwell plates, which can be expensive; current designs are limited to 24-well plates. 84,121 However, the counterparts of GECI and genetically encoded optical manipulators, such as channel rhodopsins, have been successfully used in the heart.…”

Multiparameter In Vitro Assessment of Compound Effects on Cardiomyocyte Physiology Using iPSC Cells

“…The calcium spark is similar to the AP but more physiologically like a true cardio contraction. There are other dyes and dye systems that can be employed to monitor cardiomyocyte contractions including Di-8-ANEPPS, 40 the Mermaid fluorescence protein FRET system, 41 and DiBAC4(3). 42 The first two dye systems use ratio metric readouts, making the instrumentation and analysis required more complicated.…”

Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

“…25 In this case, PWF might also be used to reconstruct the time course of individual APs from optical recordings using fluorescent potential sensors. 32,33 Therefore, the PWF of APs may not only give results similar to those of classic electrophysiological experiments but also might add the ability to spatially resolve optical AP recordings, possibly yielding new insight into the distribution and propagation of APs.…”