Changing the epigenetic landscape contributes to regenerative reprogramming of stromal cells following cardiac injury in zebrafish

R. Mausam (Giessen)¹, M. Bentsen (Bad Nauheim)², S. Allanki (Bad Nauheim)³, N. Ahmed (Gießen)⁴, D. Stainier (Bad Nauheim)⁵, M. Looso (Bad Nauheim)⁶, S. T. Sossalla (Gießen)⁷, S. Reischauer (Gießen)^8
1Justus-Liebig-University Giessen, Medical Clinic I Cardiology and Angiology Giessen, Deutschland; ²Bioinformatics Core Unit (BCU), Max Planck Institute for Heart and Lung Research Bad Nauheim, Deutschland; ³Max Planck Institute for Heart and Lung Research department of developmental genetics Bad Nauheim, Deutschland; ⁴medical clinic and campus Kerckhoff, Justus Liebig University experimental cardiology Gießen, Deutschland; ⁵Max Planck Institute for Heart and Lung Research Developmental Genetics Bad Nauheim, Deutschland; ⁶Max Planck Institute for Heart and Lung Research Bioinformatics and Deep Sequencing Platform Bad Nauheim, Deutschland; ⁷Universitätsklinikum Gießen und Marburg GmbH Medizinische Klinik I - Kardiologie und Angiologie Gießen, Deutschland; ⁸Justus Liebig university Giessen Internal medicine I - Department of Cardiology Gießen, Deutschland

Background: Epigenetic modifications are known to play a critical role in controlling gene expression and cell differentiation in development and disease. Cardiac injury in humans elicits a complex multicellular response resulting in permanent fibrotic scarring. Conversely, species like zebrafish can regenerate damaged tissue, including the heart, without permanent scar formation. Elucidating the epigenetic mechanisms underlying scar-free heart regeneration in organisms capable of robust cardiac renewal, such as zebrafish, presents a promising avenue for developing regenerative strategies for the human heart. The present study was undertaken in order to gain a deeper comprehension of scar free heart regeneration in zebrafish hearts.

Methods: Utilizing the cryoinjury heart regeneration model in zebrafish, we isolated cardiac stromal cells from regenerative and fibrotic conditions at various stages to assess their chromatin landscape. A comparative analysis of ATACseq data in single nucleus resolution obtained from regenerating hearts from wild-type animals as well as scar-inducing, non-regenerative interleukin-11 receptor (il11ra)-deficient animals allowed us to identify genomic elements regulated during regeneration and fibrosis in a cell type specific manner.

Results: We identified cell type-specific il11ra-dependent and -independent chromatin remodeling of regulatory elements at early and late timepoints post-injury. Our analysis revealed a regeneration-specific regulation of elements in proximity to il11a, aldh1a2, and lepb at 96 hours post-injury (hpci) that are reported to be regulators of cardiac regeneration in zebrafish. We also found elements with higher accessibility in fibrotic il11ra mutants, including the genes acvr1l and sox9b, which are linked to fibrosis in mammals. Moreover, motif enrichment analysis revealed enrichment for Stat transcription factor binding sites that act downstream of il11ra in regenerating stromal cells when compared to our fibrotic model, which shows an enrichment of egr1, a known key regulator of fibrosis in mammals.

Conclusions: Overall, our data suggests that injury-induced and interleukin-dependent chromatin reconfiguration play a central role in governing regenerative reprogramming of stromal cells during regeneration in zebrafish. These findings underscore the critical role played by endocardial cells (EndoCs) and epicardial-derived cells (EPDCs) in the cardiac regeneration process in zebrafish that contribute predominantly to fibrotic remodeling in mammals.