No / Evaluation of drug candidates in in-vitro assays of action potential duration (APD) is one component of preclinical safety assessment. Current assays are limited by technically-demanding, time-consuming electrophysiological methods. This study aimed to assess whether a voltage-sensitive dye-based assay could be used instead. Methods Optical APs were recorded using di-4-ANEPPS in electrically field stimulated Beagle left ventricular midmyocardial myocytes (LVMMs). Pharmacological properties of di-4-ANEPPS on the main cardiac ion channels that shape the ventricular AP were investigated using IonWorks™ and conventional electrophysiology. Effects of 9 reference drugs (dofetilide, E4031, d-sotalol, ATXII, cisapride, terfenadine, alfuzosin, diltiazem and pinacidil) with known APD-modulating effects were assessed on optically measured APD at 1 Hz. Results Under optimum conditions, 0.1 μM di-4-ANEPPS could be used to monitor APs paced at 1 Hz during nine, 5 s exposures without altering APD. di-4-ANEPPS had no effect on either hIERG, hINa, hIKs and hIto currents in transfected CHO cells (up to 10 µM) or ICa,L current in LVMMs (at 16 µM). di-4-ANEPPS had no effect on APs recorded with microelectrodes at 1 or 0.5 Hz over a period of 30 min di-4-ANEPPS displayed the sensitivity to record changes in optically measured APD in response to altered pacing frequencies and sequential vehicle additions did not affect the optically measured APD. APD data obtained with 9 reference drugs were as expected except (i) d-sotalol-induced increases in duration were smaller than those caused by other IKr blockers and (ii) increases in APD were not detected using low concentrations of terfenadine. Discussion Early in drug discovery, the di-4-ANEPPS-based method can reliably be used to assess drug effects on APD as part of a cardiac risk assessment strategy.

Cardiovascular complications associated with elevated levels of glucose in the blood (Hyperglycemia, HG) is a growing health concern. HG is known to be associated with a variety of cardiovascular morbidities including higher incidence of electrical disturbances. Although effects of chronic HG have been widely investigated, electrophysiological effects of acute hyperglycemia are relatively less known. Further, hyperglycemic effects on adrenergic response is not widely investigated. We used excised ventricular tissues from mice to record trans-membrane potentials during a variety of pacing protocols to investigate cellular/tissue level electrophysiological effects of acute hyperglycemia and adrenergic stimulation (1µM Isoproterenol, a β-adrenergic agonist). A custom program was used to compute action potential durations (APD), maximal rates of depolarization (dv/dtmax), and action potential amplitudes (APA) from the recorded trans-membrane potentials. From these computed measures, electrical restitution and alternans threshold were quantified. Restitution was quantified using the Standard Protocol (SP; basic cycle length BCL= 200ms), Dynamic Protocol (DP; 200-40ms or until blockade) and a novel diastolic interval (DI) control protocol with Sinusoidal Changes in DI. Results from 6 mice show that acute hyperglycemia causes prolongation of the APD. Effects of adrenergic stimulation during acute hyperglycemia were partially blunted compared with non-hyperglycemic state, i.e. hyperglycemia minimized the decrease in APD that was produced by adrenergic stimulation. Similar, but less consistent (across animals) effects were seen in other electrophysiological parameters such as alternans threshold. These results show that acute hyperglycemia may itself alter cellular level electrophysiology of myocytes and importantly, modify adrenergic response. These results suggest that in addition to long term re-modeling that occurs in diabetes, acute changes in glucose levels also affect electrical function and further may contribute to systemically observed changes in diabetes by blunting adrenergic response. Therefore, further investigation into the electrophysiological effects of acute changes in glucose levels are warranted.

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