Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC134
Electrical restitution in single cells - effects of acetylcholine and nitric oxide donors
R. R. Patel1,2, K. E. Brack1, G. André Ng1, J. S. Mitcheson2
1. Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom. 2. Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.
Introduction Electrical restitution (RT: the relationship between action potential duration [APD] and diastolic interval [DI]), is a key determinant of ventricular fibrillation initiation. Steep RT slopes confer an increased risk of arrhythmia. We have shown that vagus nerve stimulation (VNS) is protective against VF via a flattening in the RT slope, an effect mediated by nitric oxide (NO). The cellular mechanisms and signaling pathways of this effect are unknown. Our aim was to study RT in single cells and investigate the effect of acetylcholine (ACh) and exogenous NO using the NO donor SNAP. Methods Left ventricular base myocytes were isolated using standard techniques from adult NZW rabbits. Animals were sedated with propofol (10mg/kg IV) and euthanized with an overdose of sodium pentobarbitone (IV). Action potentials were recorded at 33-35oC in current-clamp mode using the whole cell configuration of the patch-clamp technique. RT data were obtained using a single extra-stimulus (S1-S2) protocol. RT curves were plotted and the effect of ACh (1µM) and SNAP (100µM) on maximal RT slope and APD determined. The effect of inhibiting soluble guanylyl cyclase (sGC) with ODQ (100µM) was also investigated. Effective refractory period (ERP), APD90 during constant pacing (APDCP), maximum RT slope and maximal APD90 during RT (APD90MAX) were the electrophysiological parameters measured. Data (mean±SEM) were compared using student’s paired t-test. *P<0.05, **P<0.01, NS P>0.05. Results ACh (n=4-6) and SNAP (n=7-11) had no significant effect on ERP or RT slope. Whilst ACh and SNAP had no effect on APDCP at 2.5 Hz, SNAP significantly prolonged APD90MAX. ODQ not only reversed the effect of SNAP, but caused a profound further shortening at all DIs. Preliminary results (n=2) indicate that application of ODQ alone also causes large decreases in ERP, APDCP and APD90MAX. Mean results are shown in the table below. 1BL vs SNAP, 2BL vs SNAP+ODQ, 3SNAP vs SNAP+ODQ. Conclusion ACh had no significant effects on electrical restitution in single rabbit ventricular cells and maximum RT slope was unaffected by any of the investigated reagents. Although SNAP had no effect on APDCP, it significantly increased APD90MAX, a result similar to that observed in isolated whole heart experiments. This suggests that NO has dynamic, rate dependent effects on cardiac ionic currents. Our results are consistent with this effect of exogenous NO being mediated by a cGMP dependent pathway and also with electrophysiological effects from basal levels of sGC activity under our conditions.
Where applicable, experiments conform with Society ethical requirements