U495618.pdf (23.36 MB)
Optically recording the cardiac action potential from isolated ventricular myocytes
thesisposted on 2014-12-15, 10:35 authored by Matthew E. L. Hardy
The application of potentiometric dyes to isolated ventricular myocytes may provide the opportunity to record changes in drug-induced action potential (AP) morphology without the use of more difficult electrophysiological techniques.;Conditions were optimised for recording cardiac APs from isolated guinea pig ventricular myocytes stimulated at 1Hz using the ratiometric fluorescence emission of the dyes, di-4-ANEPPS and di-8-ANEPPS. Using di-8-ANEPPS, APs of steady duration were recorded for up to 28 min, when exposed to excitation light for 30 s in every 3 min, and using di-4-ANEPPS up to 24 min when exposed for 5 s in every 4 min. Using voltage-clamp protocols simultaneously with fluorescent recordings demonstrated a linear relationship between membrane potential and the fluorescence emission of both di-4-ANEPPS and di-8-ANEPPS.;Changes in action potential duration in response to increasing concentrations of cisapride were measured using a patch electrode or the emission of di-8-ANEPPS. Values for IC50 apparent for action potential prolongation were similar between the two assays. However, cells loaded with dye had an increased basal APD90 and a decreased sensitivity compared to patch electrode recordings, suggesting additional actions of the dye.;Screening a number of structurally similar dyes (di-4-ANEPPS, di-8-ANEPPS, di-12-ANEPPS, di-3-ANEPPDHQ and di-4-ANEPPDHQ) or demonstrated a variety of different pharmacological effects.;A double-blinded validation using the fluorescence emission of di-4-ANEPPS (loaded in guinea pig myocytes) was compared to results from standard proarrhythmia screening techniques: sharp electrode recordings from canine Purkinje fibres and M cells. The data suggest that guinea pig myocytes respond to drug-induced changes in AP morphology in a more similar manner to canine M cells from Purkinje fibres and show that di-4-ANEPPS can be used to monitor changes in AP duration and triangulation in isolated ventricular cells.;This method provides a higher throughput method for safety-pharmacology screens than standard microelectrode techniques, whilst still providing an indication of the effects of test compounds in native tissue.
Date of award2007-01-01
Author affiliationCell Physiology and Pharmacology
Awarding institutionUniversity of Leicester