2016KhuwailehRAAPhD.pdf (6.22 MB)
Electrical restitution and action potential repolarisation studies in acutely isolated cardiac ventricular myocytes
thesisposted on 2016-12-05, 12:09 authored by Rawan Abdulla Aied Khuwaileh
Cardiac death is a major clinical problem that most commonly results from ventricular arrhythmias. Electrical restitution, which is the relationship between action potential duration (APD) and diastolic interval (DI), is a determinant of the risk of ventricular fibrillation (VF), a leading cause of sudden cardiac death. The maximum slope of the electrical restitution curve is an indicator of the risk of VF. Steep maximum slopes indicate an increased risk of arrhythmia. The aim of the project was to study electrical restitution in single cells and investigate the effect modulators of the nitric oxide (NO) pathways have on action potentials. Experiments were done on acutely isolated left ventricular guinea-pig myocytes at 37°C using the perforated patch clamp technique. The diurnal variation in electrical restitution was also studied by isolating myocytes during the active or resting periods for guinea-pigs. The active period myocytes were more prone to arrhythmias than the resting period cells as the maximum slope of the electrical restitution curve was steeper in the active period myocytes under basal conditions (P≤0.01). Inhibition of NO synthases (NOS) caused substantial shortening of APD₉₀ at high stimulation frequencies (4 and 5 Hz) by 35 ms, 39 ms respectively, in active compared to resting period cells but only following β-adrenergic receptor (β-AR) stimulation with isoprenaline (ISO). These effects are consistent with NO inhibiting effects of ISO on cardiac repolarisation (a protective effect) seen on whole heart studies. While comparing the response to ISO of the active versus resting period myocytes at each stimulation frequency, showed that there was a statistically significant shortening at 1 Hz by 18 ms (P≤0.001) and at 2 Hz by 19 ms (P≤0.05); with the resting period myocytes being more responsive. Applying NO signalling pathway modulators failed to modulate the electrical restitution curve. However, during constant pacing (2 Hz) experiments, NO signalling pathway modulators changed action potential (AP) duration in the presence of ISO. Enhancing the effect of cGMP-dependent pathway by adding the soluble guanylyl cyclase activatore BAY 60-2770 prolonged APD₉₀ by 20.5 ms (P≤0.05), while enhancing S-nitrosylation by inhibiting S-nitrosoglutathione reductase with N6022 shortened APD₉₀ by 49.2 ms which was statistically significant (P≤0.05). In conclusion, NO modulate cardiac repolarization of single cells in the presence of β-AR stimulation through a complex interplay of both cGMP-dependent and nitrosylation dependent mechanisms. However, NO modulation did not affect the electrical restitution slope in single myocytes. The effect of the bradycardiogenic drug ivabradine on AP repolarization was also studied ivabradine had variable effect on AP repolarization. However, the main finding was shortening of APD90 which suggests it affects predominantly currents other than IKᵣ.
Supervisor(s)Mitcheson, John; Davies, Noel
Date of award2016-12-01
Author affiliationDepartment of Cell Physiology and Pharmacology
Awarding institutionUniversity of Leicester