As expectedfor the plasmid with CRT mutation only moderate induction of luciferase activity was seen in the occurrence of E2

Clinically relevant models of heart injuries in mammals involve tissue death, typically due to ischemia. It is conceivable that necrotic tissue represents an obstacle to regeneration that also the zebrafish cannot overcome. To address this, we have established a cryoinjury model of the adult zebrafish heart. We find that zebrafish robustly regenerate ventricular necrotic lesions and that the regenerative response involves early activation of the epicardium and induction of cardiomyocyte proliferation. Thus, our results show that the regenerative abilities of the zebrafish heart are not restricted to damage by tissue removal and that similar cellular mechanisms underlie regeneration after resection and cryoinjury. Our injury model will be of great use for studies of the molecular mechanisms of heart repair. The epicardial epithelium surrounding the myocardium was evident as a single cell layer. The intra-trabecular space was filled with erythrocytes. One day after cryoinjury, the external Tubulin Acetylation Inducer HDAC inhibitor myocardial layer was reduced in width and devoid of cells displaying cardiomyocyte morphology. Likewise, most myocardial cells in the affected trabecular area had lost their typical striated morphology and characteristic nuclei, and displayed vacuolar structures indicative of cell death. Furthermore, erythrocytes were strongly enriched in the lesioned area. The wounded area was found to be infiltrated with leukocytes, most of which displayed the characteristic nuclear morphology of neutrophil granulocytes. This indicates an induction of an inflammatory response to clear cellular debris from the affected area. During the following days the wound area was further remodeled. At 3 dpi the morphology of the ventricular surface had changed to a thickened layer with a loose appearance due to prominent intracellular space and the absence of tightly packed cardiomyocytes. The inner part of the lesion was predominated by erythrocytes. No cardiomyocytes were found in the lesioned area, rather cell debris that was often closely associated with granulocytes. Electron microscopy of peripheral ventricular cell layers in uninjured hearts revealed subepicardial cardiomyocytes with prominent myofilaments and groups of electron dense mitochondria and a single layer of epicardial cells. In contrast, in the lesioned area of cryoinjured hearts at 7 dpi, cellular debris and large tissue gaps could be detected. Furthermore, the lesion contained remnants of cardiomyocytes displaying highly disorganized myofilaments and damaged mitochondria. Electron micrographs also confirmed the presence of heterophil/neutrophil and eosinophil granulocytes in the lesion. Overall, our histological and ultrastructural analyses indicate that cryoinjury resulted in necrotic cell death and loss of cardiomyocytes in the lesioned area, which was accompanied by infiltration of erythrocytes and leukocytes. We describe a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes, epicardial and endocardial cells caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying this regenerative response appear to be similar to the ones utilized during regeneration of ventricular resections. Early after injury, the entire epicardium activates a developmental gene program and becomes proliferative. We have found that epicardium activation in response to ventricular resection and cryoinjury is robustly reported by a wt1b:GFP transgenic zebrafish line, which thus represents a useful tool for future studies of heart regeneration. After both types of injury, the activated epicardial cells cover the lesioned area, presumably by migration. We found that the latter process is completed earlier in cryoinjured hearts than after ventricular resection. Whether this is due to an intrinsic differential response of epicardial cells induced by the type of injury or based on the properties of the lesioned area, eg. a consequence of the properties of the cellular and acellular substrates that the epicardial cells have to migrate on, remains to be tested. We did not detect significant differences in the timing or amplitude of gene expression induction or upregulation of proliferation in epicardial cells in response to the two types of injury, indicating that intrinsic differences in the epicardial response are less likely to be causative for the observed difference in wound coverage. However, after ventricular resection, the wound tissue adheres more strongly to the pericardial sac than after cryoinjury. It is possible that wound coverage by the epicardium is impaired by this adherence. Regeneration of myocardium removed by ventricular resection appears to occur via proliferation of differentiated cardiomyocytes. We likewise find that mature cardiomyocytes, expressing cardiac myosin light chain proliferate in response to cryoinjury and that proliferating cardiomyocytes invade the lesioned area. These data strongly indicate that necrotic lesions are repaired by proliferation of existing mature cardiomyocytes.