Abstract Body (Do not enter title and authors here): Aging is a major risk factor for cardiovascular diseases contributing to the progressive deterioration of heart function. Since the vascular niche was shown to maintain cardiac homeostasis, we explored the epigenomic and transcriptional circuits driving endothelial cell (EC) impairment with aging. Analysis of chromatin accessibility by snATAC-seq and gene expression by RNA-seq of hearts from 18- to 20-month-old mice revealed a significant reduction in chromatin accessibility and expression of the zinc finger transcription factor ZBTB16, which was also confirmed in aged human hearts. Haploinsufficiency of Zbtb16 reduced diastolic function at 3 months of age and was associated with typical hallmarks of cardiac aging, such as hypertrophy, fibrosis, decreased innervation, elevated perivascular senescence, and lower capillary density. In vitro studies confirmed the crucial role of ZBTB16 in endothelial cells. Silencing of ZBTB16 impaired EC proliferation, migration, and network formation while inducing senescence. RNA-seq from ZBTB16-silenced EC and snRNA-seq of hearts from Zbtb16+/- mice further showed an induction of secreted senescence-associated secreted proteins and several growth factors. To assess a potential effect of ZBTB16 on the paracrine communication, we performed co-culture experiments with fibroblasts, cardiomyocytes and nerves. Indeed, supernatants of ZBTB16-silenced ECs induced activation of cardiac fibroblasts, while ZBTB16 overexpression in long-term cultured ECs prevented paracrine fibroblast activation. Supernatants of ZBTB16 silenced ECs additionally induced cardiomyocyte hypertrophy and reduced neuronal sprouting. In silico ligand-receptor analysis additionally confirmed the augmented interaction of EC and fibroblasts in vivo and showed and increased PDGFB signaling between the two cell types. Neutralizing antibodies against PDGFB attenuated the effects of conditioned ZBTB16-repressed EC medium on fibroblasts. Using chromatin-immunoprecipitation-sequencing, we further identified ZBTB16 targets, which include the nuclear receptor-interacting protein 1, which mediates PDGFB expression and paracrine fibroblast activation.
In conclusion, ZBTB16 is a master regulator of cardiac aging. Its reduction induces a pro-inflammatory and pro-fibrotic endothelial cell secretome, which affects fibroblast, cardiomyocytes and nerves in vitro. These findings highlight the role of ZBTB16 in maintaining vascular niche functions during aging.