Background:

Following Myocardial Infarction (MI), the myocardium heals through a process of scarring in which healthy heart muscle is replaced by non-contractile fibrous tissue causing high morbidity due to arrhythmia, ventricular remodelling, and heart failure¹. Remarkably, rather than forming a permanent fibrous scar, neonatal mice (P1) regenerate functional cardiomyocytes, and a provisional collagen ‘patch’ at the site of injury is actively resorbed². By post-natal day 7 (P7) this property is lost, and mice form permanent scars, as in humans. The understanding of important cell types and key processes involved in cardiac regeneration in neonatal mice up to P7 is limited.

Methods:

To identify the important cell types and candidates associated with neonatal heart regeneration, we performed single-cell RNA sequencing (scRNA-seq) experiments on regenerative (P1) and non-regenerative (P7) hearts in healthy, 1- and 7-days post induction of MI in mice (n=3 for each condition). We hypothesised that changes in chromatin accessibility during development is associated with the differential gene expression in the hearts of regenerative P1 and non-regenerative P7 mice. We performed single-cell ATAC-seq on P1 and P7 hearts (n=6 for each condition) to delineate the changes in chromatin accessibility in single-cell populations in hearts. The scRNA-seq and scATAC-seq data were generated using 10x genomics platform and analysed using R-based packages Seurat and Signac, respectively.

Result:

The study reveals dysregulation of functionally important genes implicated in the processes of ECM remodelling and angiogenesis in Endothelial cells. The most significant differentially regulated genes, upregulated in the regenerative groups was Timp4, a matrix metalloproteinase inhibitor that regulates matrix remodelling. Independent works have established that Timp4 plays a protective role in MI and mice lacking Timp4 have compromised ECM, increased accumulation of neutrophils and increased post-MI mortality³. Several animal and cell culture studies in the recent years have established the importance of ECM remodelling in promoting cardiac regeneration⁴. We also observed an upregulation of Plvap, an EC-specific gene associated with angiogenesis and is known to be upregulated in the ECs in the border zone in infarcted hearts⁵. We performed single-cell ATAC-seq to map the differences in chromatin landscape between regenerative and non-regenerative hearts and observed differential chromatin accessibility associated with the Timp4 and Plvap genes in the regenerative mice compared to the non-regenerative mice.

Conclusion:

Our preliminary results based on scRNA-seq and scATAC-seq from P1 and P7 hearts point towards an important role of Endothelial cells in the P1 hearts. We have identified upregulation of Timp4 in regenerative hearts, and propose that Endothelial Cells in the hearts of regenerative neonatal (P1) mice regulate ECM remodelling and promote angiogenesis, both the processes being necessary (but not sufficient) in promoting regeneration of cardiomyocytes after MI.