S F Glaser(1,2,3), F Cheng(4), A Fischer(1), X Zhang(4), M Muhly-Reinholz(1), A Debes(1), M Klangwart(1), M Ruz Jurado(1,2,3), L Zanders(1,2,3), G Luxan(1,2,3), T Froemel(5), M Yekelchyk(3,6), H Kawase(3,7), T Bozoglu(8,9), N Wettschureck(3,7), T Braun(3,6), C Kupatt(8,9), W Abplanalp(1,2,3), R Brandes(10), A Zeiher(1,2,3), D John(1,2,3), R Wang(4), S Dimmeler(1,2,3)

1. Institute for Cardiovascular Regeneration, Goethe University Frankfurt, Germany; 2. German Centre for Cardiovascular Research (DZHK), partner site Frankfurt Rhine-Main, Berlin, Germany; 3. Cardiopulmonary Institute, Goethe University Frankfurt, Germany; 4. Laboratory for Accelerated Vascular Research, Department of Surgery, Division of Vascular Surgery, University of California, San Francisco, US; 5. Institute for Vascular Signalling, Goethe University Frankfurt, Germany; 6. Department of Cardiac Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; 7. Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; 8. German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance (MHA), Munich, Germany; 9. Klinik und Poliklinik für Innere Medizin I, University Clinic, Technical University of Munich, Munich, Germany; 10. Institute for Cardiovascular Physiology, Goethe University Frankfurt, Germany.

Left ventricular pressure-overload induced by aortic valve stenosis leads to pathological cardiac hypertrophy with LV-remodeling and microvascular dysfunction. Little is known about the role of coronary vasculature in cardiac hypertrophy. Using snRNA-sequencing of hypertrophic human myocardium, we observed a dysregulation of vessel malformation-associated genes in endothelial cells (ECs). Morphologically, coronary angiograms of the sequenced patients demonstrated tortuous coronary vessels. Using lightsheet-microscopy and µCT, similar vessel malformations and vascular convolutes were observed in latex-perfused murine hearts after pressure-induced hypertrophy. Finally, histology revealed dilation and arterialization of microvessels in line with an overall increase in αSMA⁺-cells in whole-heart stainings. Among the regulated malformation genes, the NOTCH family and its downstream targets were profoundly upregulated. Specifically, NOTCH4, a known inducer of developmental vascular malformation, was among the top upregulated genes in ECs. Importantly, ECs with a high NOTCH4 expression demonstrated a particular upregulation of malformation-associated genes, e.g. Vegfa, Efnb2, Dll4 and Hes1. Increased Notch4 expression was validated on protein level in hypertrophic human and mouse tissue specimens. To address the role of Notch4 in vivo, we used an endothelial-specific overexpression mouse model. Notch4 overexpression resulted in profound coronary vascular abnormalities and significant changes in single-cell transcriptomic signatures not only in ECs, in cardiomyocytes, which showed metabolic alterations and, in fibroblasts, which exerted signs if distinct ECM-compositions. A screen for upstream modulators of Notch4 identified hypoxia as inducer, validated by in vitro experiments. In summary, chronic pressure-overload is associated with increased endothelial Notch4 and coronary vascular malformations in both human and murine hearts. Locally inadequate oxygen supply due to microvascular dysfunction in cardiac hypertrophy may contribute to the development of coronary vascular malformations via hypoxia-induced upregulation of endothelial Notch4, which might further propagate remodeling.