no / Introduction: 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. Materials and methods: 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. Results: 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 100mV, is unaffected by cell movement and is identical to the
AP recorded simultaneously with a patch electrode. However, the APD90 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 ± 16.2ms; p< 0.01). Despite this
effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically
or with a patch electrode (91 ± 46 vs. 81 ± 20 nM). Discussion: 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 APD90. 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.
| Identifer | oai:union.ndltd.org:BRADFORD/oai:bradscholars.brad.ac.uk:10454/16510 |
| Date | January 2006 |
| Creators | Hardy, Matthew E., Lawrence, C.L., Standen, N.B., Rodrigo, G.C. |
| Source Sets | Bradford Scholars |
| Language | English |
| Detected Language | English |
| Type | Article, No full-text in the repository |
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