Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC126
A comparison between standard and dynamic electrical restitution in single cells
K. E. Brack1, R. R. Patel2, G. Ng1, J. S. Mitcheson2
1. Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom. 2. Cell physiology and pharmacology, University of Leicester, Leicester, United Kingdom.
Electrical restitution (RT - the relationship between action potential duration [APD] and diastolic interval [DI]), is a key determinant of ventricular fibrillation (VF) initiation. Whilst restitution is usually studied in whole hearts, its investigation in single cells is relatively unexplored. There are two main methods to measure electrical restitution - standard (STRT) and dynamic restitution (DYRT)1. Our aim is to compare STRT and DYRT in single cells. Adult NZW rabbits (n=3) were anaesthetised with propofol (10mg/kg, IV) and euthanized with an overdose of pentobarbitone (IV). Hearts were isolated and standard digestion techniques used to obtain myocytes from left ventricular base. Action potentials were recorded (33-35oC) in current-clamp mode using the whole cell configuration of the patch-clamp technique. STRT was obtained using a single extra-stimulus protocol, where cells were paced at 400ms cycle length (CL, 20 beats [S1]) followed by an ‘S2’ stimulus at programmed intervals down to effective refractory period (ERP - the longest CL that failed to capture). DYRT data was obtained by constant pacing for 100 beats from 350ms to ERP. RT curves were plotted and the slopes analysed. Maximal slope, DI@slope = 1, DI range for slope>1 (DI range), APDmax, slope at DI of 30ms, 50ms and 90ms (sRT30, sRT50, sRT90 respectively). For additional analysis of DYRT, APD alternans developed (APD difference >5ms) and were quantified, including the CL at which alternans occurred; maximum amplitude of alternans, the CL range that alternans occurred and sum of all alternans amplitudes (alternans index)). Data (mean±SEM) were analysed using paired students t-test, P<0.05 considered significant. Most analysed parameters were comparable (Table) between STRT and DYRT (n=5), but there was a trend towards a smaller ERP in DYRT. There were differences in the RT slopes (Figure) with sRT50 and sRT90 greater in DYRT compared with STRT. This is the first detailed study of restitution parameters in isolated ventricular myoyctes. Parameters from STRT and DYRT are comparable to those from whole hearts. Noticeable differences exist with STRT curves following a mono-exponential curve, whereas dynamic RT is more linear. The key outcome is that there is comparable and quantifiable data so that single cells can be used to determine the cellular mechanisms behind arrhythmias generation.
Where applicable, experiments conform with Society ethical requirements