Abstract Body: Background:
Adult mammalian cardiomyocytes exhibit limited regenerative capacity after injury. In contrast, neonatal mouse hearts retain robust regenerative potential at postnatal day 1 (P1), which is largely lost by postnatal day 7 (P7). However, the cellular niches and microenvironmental mechanisms underlying this transition remain poorly defined.
Aim:
We sought to identify cardiac microenvironments that distinguish regenerative and non-regenerative neonatal hearts after ischemic injury.
Methods:
Spatial transcriptomic profiling was performed on mouse hearts 4 days after left anterior descending (LAD) coronary artery ligation at P1 or P7. Spatial datasets were integrated with single-cell reference data and analyzed using computational approaches, including spatial cell-type mapping and gene ontology pathway analysis. Endocardial endothelial cells (EndoECs) were assessed using inducible lineage tracing (Npr3-CreERT2;Rosa26-tdTomato) with tamoxifen administration at P0-P2 prior to LAD injury, and their functional contribution was evaluated using diphtheria toxin A-mediated genetic ablation.
Results:
Spatial analysis revealed distinct cardiac microenvironments between regenerative and non-regenerative hearts. EndoECs were prominently enriched in the sub-endocardium, vasculature, and injured border zone of regenerative P1 hearts but were largely absent in P7 hearts. Lineage tracing demonstrated that EndoECs labeled at P0-P2 localized adjacent to cardiomyocytes or were incorporated into vascular structures at 4 days post-injury (dpi), with similar localization persisting at 10 dpi and 21 dpi. EndoEC-specific ablation, induced by tamoxifen administration at P0-P2 prior to LAD ligation at P1, significantly impaired cardiac repair, as evidenced by reduced ejection fraction at 21 dpi. Gene ontology analysis of border zone regions revealed divergent biological programs, with P7 hearts enriched for inflammatory and immune pathways, whereas P1 hearts showed enrichment of genes associated with angiogenesis and developmental regulation.
Conclusion:
These findings identify endocardial endothelial niches as key components of regenerative cardiac microenvironments and provide insight into mechanisms that may help reactivate regenerative programs in the adult heart.