With increasing life expectancy, age-related diseases have become a leading cause of mortality. The aging heart undergoes structural and functional changes that predispose to arrhythmias. While endothelial cell (EC) senescence causes denervation of the left ventricle (LV), the impact of aging on atrial innervation remains unclear. Given that autonomic nervous system alterations increase the risk of atrial fibrillation (AF), we investigated innervation patterns and neurovascular cross-talk in the aging atria.
To understand the impact of aging on cardiac innervation, we assessed nerve density using the pan-neuronal marker Tuj1. We observed major regional differences showing a 1.6±0.1-fold increase (p=0.01) in the left atrium (LA), while LV innervation decreased in 22-month-old mice compared to young controls. Increased LA innervation was attributed to enhanced sympathetic innervation, as demonstrated by tyrosine hydroxylase staining (1.6±0.1-fold increase; p=0.0003).
To unravel mechanisms underlying LA hyperinnervation, we performed single-nuclei RNA sequencing of young and old mouse LAs. Since ECs play a crucial role in LV denervation, we specifically looked into differentially expressed genes of ECs. GO term analysis revealed significant downregulation of axon-repelling factors in endocardial but not in vascular ECs. Flrt2, Slit3, and Rtn4 were among the top regulated factors with Flrt2 being the highest expressed. Culturing primary sympathetic mouse neurons with and without recombinant FLRT2 confirmed the axon-repelling property of FLRT2. Deleting Flrt2 in ECs of 3-month-old mice led to a 1.3±0.1-fold increase (p=0.07) in LA Innervation. We thereby propose a novel endocardial-axon-axis being an important factor for increased LA innervation in aging.
Having demonstrated that an endocardial-axon-axis increased innervation in the aging LA, we further question whether the hyperinnervation may influence cardiomyocyte functions. Indeed, ligand-receptor analysis predicted an increased interaction between LA neurons and cardiomyocytes. RNA sequencing of young and old mouse stellate ganglia, the primary source of cardiac sympathetic innervation, confirmed an age-dependent upregulation of gene networks associated with muscle contraction. Based on this prediction, we hypothesize a change in paracrine activity of sympathetic neurons with age, impacting cardiomyocyte contractile properties.
Treating iPSC-derived atrial cardiomyocytes with supernatant of isolated neurons from old mice led to higher beating frequency compared to treatment with young conditioned medium. As this cross-talk might contribute to AF, we studied hearts from young and old mice for AF susceptibility in vivo. Electrical pacing revealed that AF burden correlates with LA hyperinnervation in aged mice, which have a higher susceptibility (p=0.07) for induced AF than young mice, suggesting that LA hyperinnervation might contribute to AF. Clinical relevance was provided by histological analysis of human LA samples confirmed 1.3±0.1-fold (p=0.09) higher innervation levels in AF patients compared to non-AF patients and underscores the potential role of hyperinnervation in AF.
In conclusion, we propose a novel endocardial-axon-axis driving hyperinnervation in the aging LA, which may contribute to AF. Targeting EC-derived neuronal guidance cues may represent a new therapeutic approach to prevent age-related AF.
