Gata4-Dependent Differentiation of c-Kit+-Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart

Background: Although c-Kit(+) adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation, we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells. Methods:Kit allele-dependent lineage tracing and fusion analysis were performed in mice following simultaneous Gata4 and Gata6 cell type-specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit(+) cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2(CreERT2) transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results: Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2(CreERT2) transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart, and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion because of defective endothelial cell differentiation in the absence of Gata4. Conclusions: Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit(+) cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit(+) endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.