Spontaneous Ca²⁺ signalling in pulmonary vein sleeve cells increases during ageing

Atrial fibrillation (AF) is the most common form of a sustained cardiac arrhythmia, with age being a significant risk factor. Substantial evidence indicates that cardiomyocytes located in the pulmonary veins (pulmonary vein sleeve cells; PVCs) cause AF by generating ectopic electrical activity. Electrical ablation, to isolate PVCs from their left atrial junctions, is a major treatment for AF. In small rodents, the sleeve of PVC cardiomyocytes extends along the pulmonary veins deep inside the lungs. Since ageing is a major risk factor for AF development, we investigated changes in the morphology of PVCs, and their spontaneous and electrically-paced Ca2+ signals in aged mice. Lung slices were prepared, as previously described, from mice between 3 and 24 months of age. Briefly, lungs were injected with 1.8% agarose and subsequently sliced into 180 µm thick sections. Lung slices were either used for live-cell Ca2+ imaging studies, or were fixed in 4% paraformaldehyde for immunofluorescence staining or electron microscopic (EM) studies. For Ca2+ imaging, the slices were loaded with 5 µM fluo-4 AM, and line scans were collected on a Zeiss LSM 510 confocal microscope. Immunofluorescence staining was performed as previously described (5), using an antibody against type 2 ryanodine receptor (RyR2). For EM studies, the slices were post-fixed in 1% osmium tetroxide, dehydrated through a graded series of acetone concentrations before flat-embedding in Epon resin. Ultrathin sections ~ 70 nm were cut and counter-stained with 4% uranyl acetate and Reynolds' lead citrate before imaging in a JEM 1400 transmission EM. PVCs from lung slices of all ages showed spontaneous Ca2+ transients, with a higher spontaneous activity in slices from 24 month compared to 3 month mice (1.0 ± 0.19 vs. 0.71 ± 0.11 Hz, n.s., unpaired t-test, n = 3 and 4 slices, respectively). Electrical pacing with all frequencies was more successful in slices from 3 month mice (full pacing with 1 Hz in 40.3 ± 9.9% of the slices, 3 Hz 37.4 ± 9.4 %, and 5 Hz 32.6 ± 10.1%, 4 slices) compared to slices from 24 month mice (12.5 % full pacing at all frequencies, 3 slices). However, the differences are not statistically significant (unpaired t-test). Immunofluorescence staining showed a striated expression of RyR2 in lung slices from all age groups, with no difference in the distance between the striations (1.9 ± 0.03 vs. 1.9 ± 0.06 µm, respectively; unpaired t-test). EM images confirmed the presence of striations in PVCs. The data presented here indicate that PVCs become increasingly spontaneously active and more difficult to electrically pace as they age, but do not have gross morphological changes. The reason underlying the enhanced spontaneous Ca2+release is unknown. However, it is likely that an increased rate of spontaneous Ca2+ signalling within the PVCs will contribute to the genesis of AF