Single cell profiling of salamander heart under homeostasis and after cryoinjury.

Heart disease is the leading cause of mortality worldwide due to the limited regenerative capacity of the mammalian heart. Myocardial infarction causes massive cardiomyocyte apoptosis that is replaced by a fibrotic scar. In contrast to mammals, certain vertebrates such as fish and amphibians can regenerate cardiac muscle without scarring. Here, we established a cryoinjury model in the salamander species, Pleurodeles waltl. We performed single-cell RNA sequencing to profile the salamander heart and identified CLDN6 as a specific marker of epicardium, the outermost mesothelial layer enclosing the heart. Notably, lineage tracing experiments showed differentiation of CLDN6+ epicardium-derived cell intermediates into cardiomyocytes in response to injury. Additionally, scRNA-seq experiments suggested a differentiation trajectory that describes epicardial cell to myocyte conversion. Furthermore, ablation of these intermediates with Clostridium perfringens enterotoxin (CPE) blocked cardiac regeneration. Overall design: scRNA-seq of salamander heart tissue - sham, 7 days post cryoinjury (dpci), 14 dpci, 28 dpci.