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Optically recording the cardiac action potential from isolated ventricular myocytes

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posted 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 award


Author affiliation

Cell Physiology and Pharmacology

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD



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