Circadian rhythms in the effects of autonomic neurotransmitters on proarrhythmic activity in rat pulmonary vein and left atrial cardiomyocytes

Abstract Introduction Burden of atrial fibrillation (AF) in patients demonstrates a nighttime preponderance. Pulmonary vein (PV) ectopic activity is a major driver of AF. The autonomic nervous system (ANS) provides dense innervation playing a key role in AF onset. We previously demonstrated evidence for a peripheral clock in the rat PV. Delayed after depolarisations (DADs) including sensitivity to ANS neurotransmitters, noradrenaline (NA) and acetylcholine (ACh), was circadian in isolated cardiomyocytes. Underlying mechanisms of pro-arrhythmic activity and impact of the ANS remain to be elucidated. Purpose To examine the circadian variation in pro-arrhythmic activity, and effect of NA and ACh, in the PV and left atrial appendage (LAA) of rat hearts. Methods Hearts were removed under terminal anaesthesia from male Wistar rats maintained in a 24-hr cycle of 12-hr light/dark (lights-on at Zeitgeber time, ZT0; lights-off, ZT12). 4 time points were sampled (ZT 0,6,12,18). Whole-cell current clamp recordings were made from cardiomyocytes isolated from the proximal PV (n=75) and LAA (n=65) of 16 rats and loaded with Cal520-AM (5 μM). Frequency and amplitude of DADs, and diastolic spontaneous Ca release transients (spCaT) were measured and the effect of 1µM NA or 1µM ACh examined. RNA extracted from LAA and proximal PV (3 rats per ZT) was sequenced. The circadian nature of expression of genes involved in adrenergic and cholinergic signalling was assessed, with specific genes of interest confirmed by real-time quantitative polymerase chain reaction. Results Frequency and amplitude of DADs and spCaT were greatest during the lights-on resting phase and overall in PV cardiomyocytes under control conditions. Superfusion with NA increased DAD and spCaT frequency and amplitude in both cell types, with greatest effect during the resting phase and in PV cells. ACh reduced DAD and spCaT frequency and amplitude, with the greatest effect during the resting phase and in LAA cells. DAD amplitude positively correlated with spCaT amplitude. Following NA superfusion, the DAD/spCaT slope was increased markedly, with the effect of NA being greatest during the resting phase and in PV cardiomyocytes. Twelve of 130 genes detected from the KEGG pathway, ‘Adrenergic signaling in cardiomyocytes’ (rno04261) were differentially expressed, including, Adrb1 (β1-adrenoceptor), Adcy5 (adenylyl cyclase 5) and Kcnk2 (β-adrenoceptor-regulated two-pore K+ channel, TREK). Additionally, Chrm2 (M2 receptor) was circadian in the LAA. Conclusions The frequency of spCaTs and DADs was increased by NA and reduced by ACh in a circadian manner. Expression of key genes involved in ANS signalling were circadian. Susceptibility of PV and LAA cardiomyocytes to pro-arrhythmia is highly sensitive to NA and ACh and shows circadian rhythm. Furthering our understanding of underlying mechanisms and the impact of the ANS is important in the development of therapeutic agents for patients with AF.