An experimental study regarding the effectiveness of phosphodiesterase type 5 inhibition to prevent atrial fibrillation

Abstract

Introduction Atrial fibrillation (AF) is a common arrhythmia in need of new treatment options. Diastolic leak of calcium within atrial myocytes is believed to be a fundamental driving force in the underlying pathophysiology, where it is responsible for increased triggered activity and abnormal remodelling of atrial electrophysiology and structure. Whilst calcium cycling within the myocyte is modulated by numerous pathways, one mechanism which may be clinical interest is the cyclic nucleotide secondary messenger system, which is under strict regulation by phosphodiesterases (PDEs). Inhibition of PDE5 has been identified as a novel target for preventing AF as it has been hypothesised it will reduce sarcoplasmic reticulum (SR) calcium content, thereby decreasing diastolic leak and the downstream pathological atria remodelling. The aims of this thesis were: to determine if PDE5 inhibition reduces the incidence of AF in an ovine model by reducing diastolic calcium leak and its associated effects of the cellular mechanisms of arrhythmia and atrial remodelling. Methods Inhibition of PDE5 was assessed both acutely and chronically in wild-type adult Welsh mountain sheep. Acute in vitro effects of 50nM Tadalafil were studied in single atria myocytes isolated from healthy sheep using current clamp experiments to measure SR content, the calcium transient and the action potential duration (APD); and voltage clamp experiments to measure ICaL and intracellular calcium buffering. Acute in vivo effects were studied on healthy sheep under general anaesthesia by utilising an electrophysiology study to measure the vulnerability to induced AF, the atrial effective refractory period (AERP), the rate threshold for atrial alternans and action potential restitution curve; both before and after 10mg intravenous Sildenafil. The chronic effects of PDE5 inhibition were studied in a intermittently tachypaced model of AF, allocated to taking either 20mg Tadalafil daily or placebo. Assessment of the burden of AF (duration of induced AF and number of inductions lasting 30 seconds), the dominant frequency of AF, P wave duration and left atrial diameter was performed over an eight week period. This was followed by euthanasia and isolation of single atrial myocytes to undergo current clamp electrophysiology for SR calcium content, the calcium transient, APD and calcium waves. Results Acute PDE5 inhibition in vitro reduced sarcoplasmic reticulum calcium content by ~40% (p=0.002), at least in part due to a ~35% reduction calcium influx through the L type calcium channel (p=0.01) as no difference was observed in intracellular calcium buffering or sarcoplasmic reticulum calcium ATPase function. The amplitude of the calcium transient was reduced by ~20% (p=0.01), however no difference was observed in the APD at 90% repolarisation. Myocytes were unable to consistently generate triggered activity to assess if the reduction in SR content resulted in a meaningful reduction in diastolic leak. Acute PDE5 inhibition in vivo reduced the vulnerability to AF, manifesting as a ~50% reduction in episodes >5 seconds (p=0.001) and ~35% reduction in median duration of AF (p=0.013). The primary anti-arrhythmic mechanism identified was a median 15msec prolongation in the AERP (p=0.016). An anti-arrhythmic shift in the restitution curve was identified however this did not translate to a difference in the rate threshold for atrial action potential alternans. The methodology for recording atrial alternans in vivo was investigated further as it was believed to be confounded by the timing of ventricular contraction. An alternating 5msec delay in atrioventricular (AV) conduction was found to mimic atrial electrical alternans due to the induction of ventricular mechanical alternans. As a result, a modified protocol based dual chamber pacing with a fixed AV delay was proposed for future accurate assessment of atrial alternans. The effects of chronic PDE5 inhibition in a tachypaced ovine mode

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andy Trafford (Supervisor) & Katharine Dibb (Supervisor)