Endothelial inactivation of Sox9 protects from aging-induced cardiac fibrosis and heart failure

F. A. Trogisch (Mannheim)¹, M. Fuhrmann (Mannheim)¹, N. Weinzierl (Mannheim)¹, E. Hofmann (Mannheim)¹, J. U. G. Wagner (Frankfurt am Main)², R. Kist (Newcastle)³, S. Dimmeler (Frankfurt am Main)⁴, J. Heineke (Mannheim)^5
1Medizinische Fakultät Mannheim der Universität Heidelberg Abteilung für kardiovaskuläre Physiologie Mannheim, Deutschland; ²Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration Frankfurt am Main, Deutschland; ³Newcastle University School of Dental Sciences Newcastle, Großbritannien; ⁴Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; ⁵Medizinische Fakultät Mannheim der Universität Heidelberg Kardiovaskuläre Physiologie Mannheim, Deutschland

Introduction
Previously, we reported that the transcription factor Sox9 serves as master regulator of fibrotic remodeling in endothelial cells (ECs) in preclinical models of heart failure with preserved or reduced ejection fraction. One important adverse effect of Sox9 overexpression in ECs was to reduce vascular and nerve density. In addition, it was recently shown that the neurovascular interface is an important modulator of cardiac impairment in aged hearts. Whether endothelial Sox9 orchestrates cardiac failure during aging, has so far not been investigated.

Objective
Aim of the study was to investigate the impact of endothelial Sox9 on the development of fibrotic cardiac disease in aged mice.

Methods & Results
Cell type-specific RNA sequencing revealed induction of Sox9 in several cardiac cell types during aging in mice (up to 24 months vs. 3 months of age). We validated global cardiac and especially endothelial induction of Sox9 utilizing immunofluorescence. In order to investigate the role of endothelial Sox9 during aging, we specifically inactivated Sox9 in endothelial cells by using Sox9^fl/fl and Cdh5-CreERT2 mice injected with Tamoxifen at the age of 2 months (Sox9^EC-KO). We monitored the mice up to 24 months of age. While Sox9^fl/fl control mice developed diastolic cardiac dysfunction at the age of approximately 18 months, Sox9^EC-KO mice were protected as shown by normalized global longitudinal strain and LV end-diastolic pressure. Additionally, aged Sox9^EC-KO mice developed less fibrosis and displayed a more physiologic shape of cardiomyocyte hypertrophy, as shown by uniform increase of cross-sectional area and length. Interestingly, while Sox9^fl/fl control mice suffered from reduction of both capillary and nerve density in the myocardium, aged Sox9^EC-KO mice preserved both throughout the study, as shown by isolectin B4 and tubulin beta 3 staining.

Conclusions
Inactivation of Sox9 in endothelial cells protects from aging-induced cardiac fibrosis and failure while preserving myocardial capillary and nerve density. Hence, Sox9 in ECs might serve as therapeutic target in aging-related heart failure.