Over the past century, life expectancy has remarkably prolonged. However, longevity is a double-edged sword. Despite the fortune of a prolonged life span, ageing is the major risk factor for developing severe diseases, such as cardiovascular disease, which is the primary cause of death globally. The ageing heart goes through various pathological abnormalities at the cellular and molecular level. These lead to several cardiovascular illnesses including changes in heart rate variability. We previously demonstrated that ageing is associated with left ventricular denervation. Nervous fibres co-align with blood vessels that increase the expression of neuro-repelling factors whereas decreased expression of neuro-protective factors results in left ventricular denervation in the aging heart.
We found that nervous fibres, particularly sympathetic, but not for sensory fibres co-align with lymphatic vessels in the heart. Beginning at 16 months of age, we observed a concurrent decline in the lymphatic vasculature and innervation, with impaired co-alignment of sympathetic fibres and lymphatic large pre-collector tubes in the aging murine heart. This suggested an interdependency of the cardiac nervous and lymphatic system. Indeed, disrupting the cardiac lymphatic system in 3-month-old (young) mice, by AAV9-mediated expression of a soluble form of VEGF-receptor 3 (VEGFR3) or genetic deletion of VEGFR3 in lymphatic vessels, led to a decline in sympathetic innervation (p=0.0002, -0.40-fold ± 0.08), whereas sensory innervation was not affected. Of note, impairing cardiac sympathetic innervation pharmacologically using 6-OHDA or surgically by ganglionectomy did not affect cardiac lymphatic density.
To rescue cardiac innervation in aged mice, we overexpressed AAV9 encoding the lymphatic growth factor VEGF-C in aged mice. Vegfc induction rescued cardiac lymphatic density and sympathetic innervation. Again, sensory fibres were not changed. Telemetric ECG tracing demonstrated that heart rate variability was improved within 4 weeks after AAV9-VEGF-C injection, suggesting a recovery of the sympathetic tone.
To identify the molecular mechanism underlying lymphatic-neuro interactions, we performed snRNA-seq of hearts from aged mice with and without AAV9-mediated Vegfc induction. GO term assessment of transcripts upregulated in the lymphatic cluster revealed genes connected to axon guidance. These included transcripts encoding matrix proteins, such as collagen-4a-2 (Col4a2), which were decreased in endothelial cells from aged versus young hearts. To study the effect of lymphatic Col4a2 on sympathetic neurons, we cultured isolated primary sympathetic neurons from mice on top of human lymphatic endothelial cells with and without siRNA-mediated COL4A2-silencing and found that repressing COL4A2 in lymphatic endothelial cells results in impaired axonal outgrowth.
Our results indicate that cardiac lymphatic vessels provide a scaffold for sympathetic nerve fibres and suggest that age-related remodelling of lymphatic vessels in the heart impairs cardiac innervation, which can contribute to cardiac arrhythmias.
