Introduction: Aging is a major risk factor for impaired cardiovascular health. The aging heart is characterized by vascular dysfunction, increased hypertrophy, fibrosis and electrophysiological alterations that predispose the elderly to arrhythmic risk. Previously, we described that aging drives left ventricular (LV) denervation leading to reduced heart rate variability and increased susceptibility to ventricular arrhythmia. Our data demonstrated that aging induces vascular senescence in the LV which impairs the neurovascular interaction leading towards a shift of endothelial axon repelling signals with an increase in the repelling factor Sema3a and decline in the neuroprotective factor Vegfb. However, the mechanisms underlying vascular senescence in the aged heart remain elusive.

Methods: Using micro-array, bulk RNA / snRNA-seq and transgenic mouse models as well as miR-mimics in vivo, we aim to elucidate the molecular mechanisms underlying age-related vascular senescence in the heart. Cardiac remodeling and the innervation are assessed by confocal immunofluorescence imaging.

Results: Micro-array data from 3-month-young versus >18-month-old revealed significant repression of the miR-145-5p. TaqMan-PCR on isolated cardiac endothelial cells (n=4, p=0.0037) and RNA-Scope (n=3, p=0.01) revealed the age-related repression of miR-145-5p specifically in cardiac endothelial cells. Interestingly, miR-145 is known to be predominantly expressed by smooth muscle cells but not in endothelial cells. Global and endothelial-specific miR-143/145-cluster-deletion resulted in pronounced senescence exclusively in arteries (n=4, p<00001). MiR143/145-deletion impaired diastolic function and resulted in left atrial enlargement while the LV mass was unchanged. Anti-miR-145 in vitro experiments in endothelial cells resulted in impaired endothelial migration and proliferation and induced endothelial senescence. SnRNA-seq sequencing confirmed the expression of senescence-associated genes in cardiac endothelial cells in endothelial-specific miR143/145-deletion. Ligand-receptor-predictions revealed that endothelial cells increase the expression of axon-repelling genes such as Sema3a and the up-regulation of matrix protein genes such as collagens and laminins. Indeed, endothelial specific miR143/145-deletion mice showed significantly less sympathetic LV innervation and increased vascular fibrosis. To rescue the age-related decline in miR145 in aged mice, we i.v. injected 1000 pmol of miR-145-mimics to 18-month-old mice which resulted in elevated miR145 levels in the heart. Interestingly, already after 3 days LV innervation tended to be increased while vascular senescence was reduced. A long-term experiment including functional assessments is currently ongoing.

Conclusion: Here, we show that miR145 might be an up-stream regulator of endothelial senescence in the aging heart which contributes to LV denervation in the elderly. Rescuing miR145-levels in the aging heart may restore cardiac innervation and thereby improve heart function.