Decline of the epigenetically regulated transcription factor ZBTB16 in the aged heart results in impaired endothelial niche functions and leads to cardiac dysfunction

Kathrin Stilz (Frankfurt am Main)1, L. Tombor (Frankfurt am Main)1, L. Kettenhausen (Frankfurt am Main)1, A. Fischer (Frankfurt am Main)1, D. John (Frankfurt am Main)1, K. Schmitz (Frankfurt am Main)1, M. Shumliakivska (Frankfurt am Main)1, M. Yekelchyk (Bad Nauheim)2, T. Braun (Bad Nauheim)2, A. M. Zeiher (Frankfurt am Main)1, J. Wagner (Frankfurt am Main)1, S. Dimmeler (Frankfurt am Main)1

1Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; 2Max-Planck-Institut für Herz- und Lungenforschung Bad Nauheim, Deutschland


Extended lifespan is accompanied by cellular aging, a major risk factor for cardiovascular diseases. Chronic injury and aging impair endothelial cell (EC) functions and induce distinct transcriptomic signatures. These genetic landscapes are orchestrated by altered expression and activity of transcription factors and epigenetic regulatory pathways, which may contribute to disease-associated dysfunction, DNA damage responses, cellular senescence and a pro-inflammatory senescence-associated secretory phenotype (SASP). However, little is known about the mechanisms underlying these alterations.

This study aims to identify epigenetically controlled transcriptional pathways contributing to age-related impairment of EC function. We analyzed cardiac EC single-nucleus ATAC-sequencing and respective RNA-sequencing of 3- and 20-month-old C57Bl/6J mice.

Analysis of the data revealed a significant reduction in chromatin accessibility and a down-regulation of mRNA expression of the transcription factor Zbtb16 in cardiac endothelial cells of aged mice. A similar reduction of ZBTB16 expression was observed in the EC cluster of single nuclei RNA sequencing data of aged (> 55 y) versus young human hearts. Zbtb16 encodes for a zinc finger transcription factor (also known as PLZF), which plays a role in embryonic development and hematopoietic cell differentiation. However, its molecular functions in endothelial cells in aging and disease are unknown. Genetic deletion of Zbtb16 resulted in a diminished birth rate of Zbtb16-/- mice, whereas Zbtb16+/- mice were viable and showed a markedly reduced diastolic function, cardiac hypertrophy and increased diffuse fibrosis at 3 months of age. Zbtb16+/- mice further demonstrated increased senescence in the perivascular area, a decline in capillary and nerve density and reduced ex vivo aortic ring sprouting. In vitro studies confirmed the crucial function of ZBTB16 as cell proliferation, migration and network formation were impaired in ZBTB16-silenced cultured ECs. Interestingly, secreted factors that are part of the senescence-associated secretome (e.g. SERPINEs, IL1A, IL6, TGFB2, GDF15, CCL2) were upregulated. Functionally, conditioned media of ZBTB16-depleted ECs activated primary cardiac fibroblasts, increased rat cardiomyocyte hypertrophy and accelerated beating. Moreover, mouse neurons treated with the respective supernatant degenerated. Consistent with the outcomes of the knockdown experiments, ZBTB16 overexpression in long-term passaged ECs rescued cellular senescence, as assessed by beta-galactosidase staining. This overexpression also enhanced network formation and prevented the paracrine activation of fibroblasts.

Here, we identified ZBTB16 as a master regulator of endothelial dysfunction in aging and diastolic heart failure. Heterozygote deletion of Zbtb16 recapitulated all major hallmarks of cardiac aging and induced premature senescence in mice. Reduction of ZBTB16 in EC induced paracrine activation of fibroblasts and cardiomyocytes, while overexpression of ZBTB16 in long-term passaged ECs rescued cellular senescence. These findings suggest that ZBTB16 plays a pivotal role in safeguarding vascular niche functions in aging.
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