Introduction: With increasing life expectancy, age-related diseases emerged as a leading cause of mortality. The aging myocardium is characterized by structural/functional alterations, which predispose the elderly to arrhythmias. The autonomic nervous system (ANS) and neurovascular interactions recently gained increasing attention, as previous findings demonstrate that endothelial senescence induced denervation of the left ventricle (LV) in aging. However, the effects of aging on atrial innervation and neurovascular cross-talks remains unexplored. Since alterations of the ANS increases the risk of atrial fibrillation (AFib), which is highly prevalent in the elderly, we explored the innervation and the neuro-vascular cross-talk in the aging atria.
Aims/Objectives: In this project we aim to study age-related changes in atrial innervation and possible impact on arrhythmias.
Methods: To characterize the ageing murine atria, we used immunofluorescence-based stainings and senolytic experiments. Further, we included single-nuclei RNA sequencing and in vivo electrical pacing experiments. Burst stimulation protocol for atria was used to provoke atrial arrhythmias.
Results: To investigate atrial innervation in aged hearts, we stained pan-neuronal marker beta-3-tubulin in LAs of 3-months-old and 22-months-old mice, observing a 1.6±0.1-fold increase (p=0.0141 (unpaired, parametric t-test), 3m n=8, 22m n=6)) in aged mice, while innervation decreased in the LV. This increase in LA innervation was primarily attributed to enhanced sympathetic innervation, with a 1.6±0.1-fold increase (p=0.0003 (unpaired, parametric t-test), 3m n=13, 22m n=8)) as determined by tyrosine-hydroxylase staining.
Since we previously found that vascular senescence controls denervation of the LV, we investigated cellular senescence patterns between the LV and the LA. Acid β-galactosidase staining showed a 3.4±0.1-fold increase (p=0.001 (unpaired, parametric t-test), 3m n=11, 22m n=8) in LA senescence with age. However, double staining with cell type specific markers revealed no increase in endothelial cell senescence but a tendency for increased senescence in CD45-positive immune cells (2.7±0.1-fold increase, p=0.0964 (unpaired, parametric t-test), n=8) and CD68-positive macrophages (2.9±0.1-fold increase, p=0.0585 (unpaired, parametric t-test), n=8).
Consistent with these findings, treating mice with the endothelial cell specific senolytic drug fisetin did not affect LA senescence or hyperinnervation in aged LAs after 2 months of treatment but reduced cellular senescence in the LV. These data indicate that LA hyperinnervation in aging is associated with immune cell but not endothelial cell senescence.
To investigate mechanisms underlying LA hyperinnervation, we performed single-nuclei RNA sequencing. First analyses demonstrate that macrophages show an increased neuroinflammation signature in aged murine LAs. Cell types like endocardial endothelial cells on the other hand showed a more pro-axon guidance phenotype with declined axon-repelling factors such as SEMA3A, while protective factors such as VEGFB were increased.
Preliminary results of electrical pacing experiments in young and 16-months-old mice revealed that aged mice have a higher susceptibility for induced AFib compared to young counterparts. This suggests that LA hyperinnervation might be associated with AFib. Histological analysis of LA samples from human patients (n=3) confirmed high innervation levels in AFib.
Conclusions: In conclusion, we demonstrate hyperinnervation in aged LAs. Future research will focus on the relationship between AFib and LA hyperinnervation while unraveling the molecular mechanisms driving these age-related changes.