Deciphering the Epigenetic Regulation of Cardiac Fibrosis: Insights from Single-Cell Multi-Omics

Adel Elsherbiny (Heidelberg)1, P. Laurette (Heidelberg)1, L. M. Weiß (Frankfurt)2, G. Güven (Heidelberg)1, N. Díaz i Pedrosa (Heidelberg)1, F. Rezende (Frankfurt am Main)3, R. Gilsbach (Heidelberg)1

1Universitätsklinikum Heidelberg Innere Medizin VIII, Institut für Experimentelle Kardiologie Heidelberg, Deutschland; 2Goethe-Universität Institut für Kardiovaskuläre Physiologie Frankfurt, Deutschland; 3Universitätsklinikum Frankfurt Institut für Kardiovaskuläre Physiologie Frankfurt am Main, Deutschland


Background: In heart diseases, fibrosis preserves the heart's structural integrity by replacing dead cells and forming scar tissue. However, this excess production of collagen-rich scar tissue impairs heart function by enhancing muscle's passive stiffness, interfering with electrical conduction and promoting cardiac arrhythmia. Thus, understanding and steering the regulatory processes controlling the progression of cardiac fibrosis provides a basis for innovative anti-fibrotic treatment.


Methods and Results: To decode cardiac fibrosis, we combined single-nucleus Multiome (snRNA-seq and snATAC-seq from the same nuclei) with transgenic lineage tracing of fibroblasts. Lineage traced FB isolated by FACS from AngII/PE and control mic (> 20.000 fibroblast nuclei). We combined snMultiome with deep HiC analysis (around 2 billion reads). The snMultiome analysis revealed ten FB clusters, of which three were specifically detected after AngII/PE stimulation and marked with canonical FB activation markers (e.g., Postn). Overall, we observed a relatively homogeneous activation of cardiac fibroblasts using AngII/PE. Pseudobulk chromatin accessibility analysis and transcription factor footprinting showed that distal regulatory elements increased the binding of RUNX1 and SMAD family members. HiC analysis shows that these distal regulatory elements showed differential promoter interactions during FB activation. For example, synergistic epigenetic and spatial genome organization changes were evident at the Postn and Runx1 locus.


Conclusions: Integrating single-cell Multiome data with higher-order chromatin structure and transcription factor footprinting revealed that fibroblast activation is tightly linked to epigenetic changes, including chromatin rewiring of promoter interactions with distal regulatory elements. These distal regulatory are marked by specific sets of transcription factors representing promising therapeutic targets.

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