Identifying downstream targets of ankrd1a in heart regeneration after cryoinjury in adult zebrafish

In contrast to humans, zebrafish have a remarkable ability to regenerate injured heart through a complex and highly orchestrated process involving all cardiac structures. During regeneration, the major source of new myocardial cells are resident cardiomyocytes, which dedifferentiate and reinitiate proliferation, invading the area of injury to replace the lost myocardium. The response of the myocardium and the coronary vasculature is preceded by activation of epi- and endocardium, which form active scaffolds to provide mechanical and paracrine support to guide regeneration.Zebrafish ankrd1a was found activated in differentiated and proliferating cardiomyocytes restricted to the border zone, as well as in protrusions of cardiomyocytes that invade the scar. The TgBAC(ankrd1a:EGFP) transgene was not detected in epicardial or endocardial cells of regenerating zebrafish heart. Transcriptomic analysis of cryoinjured ventricles showed that lack of functional ankrd1a at 7 dpci leads to change in expression of genes involved in antigen processing and presentation of peptide or polysaccharide antigen via MHC class II, cellular extravasation, cell-cell adhesion and heart valve development. Overall design: The ankrd1abns502 mutant zebrafish line was generated by CRISPR/Cas9 genome editing. Heart cryoinjury was performed after the incisions made in the body wall and pericardial sac and the exposed ventricles were placed in contact with the cryoprobe previously cooled in liquid nitrogen. After 7 days of recovery, RNA was isolated from the heart ventricle of cryoinjured or sham operated fish. Four experimental groups (cryoinjured wt, sham wt, cryoinjured mutant, and sham mutant) were subjected to RNA-seq. Each experimental group comprised four samples and one sample contained RNA isolated from ventricles of three fish.