Introduction.

Episodes of atrial fibrillation (AF) in patients tend to occur at night. Cardiomyocytes in the pulmonary vein (PV) sleeves are a major source of the ectopic activity driving AF. The pulmonary vein sleeves are highly innervated and it is known that the autonomic nervous system plays a role in the onset of AF. In addition, spontaneous diastolic Ca²⁺ release has been suggested to play a role in the generation of delayed afterdepolarisations (DADs), triggered activity and abnormal automaticity in PV cardiomyocytes.

Aims/objectives.

This study addresses the hypothesis that 24-hr variation in Ca²⁺ release plays a role in circadian rhythms in proarrhythmic activity of PV cardiomyocytes.

Methods.

Animal procedures were approved by the Animal Welfare and Ethics Review Board of the University of Bristol and conducted in accordance with UK law. Male Wistar rats were maintained in a 24-hr cycle of 12-hr light/dark (lights-on at Zeitgeber time, ZT=0; lights-off, ZT12). Hearts were removed under terminal general anaesthesia at 4 times (ZT, Zeitgeber Times) through the 24-hour cycle (ZT 0, 6, 12, 18). Whole-cell current clamp recordings were made from cardiomyocytes isolated from the proximal PV (n=75 cells) and the left atrial appendage (LAA, n=65 cells) of 16 rats and loaded with Cal520-AM (5 μM). The frequency and amplitude of DADs, and diastolic spontaneous Ca release transients (spCaT) were measured and the effects of the cardiac sympathetic neurotransmitter, noradrenaline (NA, 1 µM), examined. DAD and spCaT amplitude and frequency were plotted against ZT of recording and fitted with a sine wave. Fitted curves were compared using an extra-sum-of-squares F-test (P<0.05).

Results and Discussion.

Under control conditions, DADs and spCaT could be recorded in both PV and LAA cardiomyocytes, with the frequencies and amplitudes being greatest during the resting phase (ZT0-12) compared to the active phase (ZT12-24) in both cell types. However, DAD/spCaT frequencies and amplitudes were generally greater in PV than in LAA cardiomyocytes in both rest and active phases. Superfusion with NA increased DAD and spCaT frequency and amplitude in both cell types with the greatest effects during the resting phase and in PV cardiomyocytes. DAD amplitude was closely correlated with spCaT amplitude in both cell types, with the slope of the relation being greater in PV than LAA cardiomyocytes and being increased markedly by NA. There was a ZT-dependence to the slope of the DAD amplitude/spCaT relation under control conditions and in the presence of NA.

Conclusions.

Taken together, these data suggest a role for noradrenergic control of diastolic spontaneous Ca²⁺ release in the circadian rhythms in proarrhythmic activity of PV cardiomyocytes.