Introduction: Aging is a major risk factor for impaired cardiovascular function. The aging heart is characterized by vascular dysfunction, increased hypertrophy, fibrosis and electrophysiological alterations. Studies show impaired endothelial cell function associates with senescence in aging. Since vessels are aligned with nerves, and this interplay is critical for tissue homeostasis, we investigated whether an impairment of the neuro-vascular interface may contribute to age-associated pathologies in the heart.
Methods and Results: To investigate the innervation of the aging mouse heart, we histologically assessed the cardiac autonomic nervous system in 12-week and 20-month old mice using the pan-neuronal marker Tuj1 and the sympathetic marker tyrosine hydroxylase. Interestingly, sympathetic innervation of the left ventricle, especially around arteries, was reduced by 0.66±0.07-fold with aging (n=9; p<0.05) indicating that aging reduces innervation of the left ventricle. Similar findings were observed in premature aging models of TERT-deficient and endothelial-transgenic progeria mice. Electrophysiological studies confirmed an increased incidence of ventricular arrhythmias and reduced heart rate variability in aged mice.
In order to determine the potential contribution of the neurovascular cellular cross-talk to the reduced innervation, we analyzed gene expression of isolated endothelial cells in the aging mouse heart by bulk RNA sequencing. Aging significantly induced the expression of genes associated with GO-terms related to neuronal cell death and axon injury. Sema3a, a known axon repelling factor, was significantly increased by 1.5±0.2-fold in endothelial cells of aged mice hearts, a finding which was confirmed by qPCR and by histology at protein level (p<0.05). Next we determined the mechanism of Sema3a de-repression. We show that miR-145 acts as an up-stream regulator of Sema3a expression. miR-145-/- mice showed increased Sema3a expression and reduced cardiac innervation.
We observed an increase in acidic β-galactosidase activity as a marker for cellular senescence in the aging heart, particularly in the proximate arterial space. In addition, Sema3a expression was significantly increased in senescent endothelial cells in vitro, thus suggesting a putative role for senescence to impair cardiac innervation. To test whether eliminating senescent cells may restore left ventricular axon density, we treated 18-month old mice with a combination of the two senolytic drugs dasatinib and quercetin, which are known to deplete senescent cells in vivo. After 2 months of senolytic treatment, cellular senescence was reduced by 0.4±0.1-fold (p<0.05) in the aging heart in parallel with reduced expression of endothelial Sema3a. Importantly, senolytic treatment restored ventricular innervation (by 1.8±0.4-fold vs. old-placebo mice), augmented heart rate variability, and normalized electrophysiological abnormalities (all p<0.05).
Conclusion: In conclusion, we show that aging augments axon repelling signals in endothelial cells and reduces left ventricular innervation. The depletion of senescent cells prevented age-induced impairment of innervation and normalized electrophysiological abnormalities suggesting a critical role of senescence-associated axon repulsion in the aging heart.