Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

• Published in: Journal of pharmacological and toxicological methods 2006-09, Vol.54 (2), p.173-182
• Main Authors: Hardy, M.E.L., Lawrence, C.L., Standen, N.B., Rodrigo, G.C.
• Format: Article
• Language: English
• Subjects: Action potential; Action Potentials - drug effects; Animals; Arrhythmia; Calibration; Cardiac muscle; Coloring Agents; Drug Evaluation, Preclinical; Guinea Pigs; Guinea-pig myocyte; In Vitro Techniques; Male; Membrane Potentials - drug effects; Microscopy, Confocal; Myocytes, Cardiac - drug effects; Potentiometry; Pyridinium Compounds - pharmacology; Radiometry; Voltage-sensitive dyes
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2006.02.013

Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD 90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6 ± 13.5 vs. 296.2 ± 16.2 ms; p < 0.01). Despite this effect, the apparent IC 50 for cisapride, which prolongs the AP by blocking I Kr, was not significantly different whether determined optically or with a patch electrode (91 ± 46 vs. 81 ± 20 nM). These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD 90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.