Abstract 27: Loss of GATA4 in Kit Lineage-derived Endothelium Leads to Vascular Permeability and Increased Heterotypic Fusion

Abstract

The treatment of cardiac ischemic injury using heart-derived c-Kit stem cells showed promising results in clinical trials with scar reduction and improved ejection fraction following coronary infusion. We previously developed a c-Kit lineage tracing mouse model showing that these cells very rarely convert into de novo cardiomyocytes and most lineage-derived cardiomyocytes are due to fusion. To potentially reduce this de novo rate to zero, and unequivocally evaluate the cellular fusion process in vivo , we deleted the essential cardiomyogenic transcription factor Gata4 in c-Kit cells using a Cre-loxP approach (Kit-Gata4 KO). Mice with the tamoxifen inducible Kit-MerCreMer allele crossed into a Gata4 homozygous LoxP targeted background with the Rosa26-eGFP reporter were fed tamoxifen at weaning to delete Gata4 in all c-Kit expressing cells and show them and their progeny as eGFP + . Unexpectedly, we observed a greater than 10-fold increase in Kit lineage-traced cardiomyocytes in Kit-Gata4 KO mice compared to Kit only controls with up to 4 months of tamoxifen treatment (90% v. 81% fusion in Kit-Gata4 KO v. controls). A 6-fold increase in leukocyte (CD45 + ) infiltration was observed suggesting the possibility for greater rates of fusion in the heart. However, bone marrow transplant studies revealed that Kit-Gata4 KO bone marrow does not exhibit increased cardiac infiltration or heterotypic fusion with cardiomyocytes. The reciprocal experiment, donating control bone marrow to recipient mice with Kit-Gata4 KO hearts, showed a 4-fold increase in fusion of control bone marrow showing that increases in fusion was intrinsic to the cells within the heart. Analysis of cardiac endothelial cells in Kit-Gata4 KO mice revealed that total CD31 + cells were increased by 30% with 2 months of tamoxifen treatment with a parallel increase in endothelial progenitor cells (CD133 + ) in the bone marrow. This endothelial phenotype was corroborated using endothelial-specific deletion of Gata4 in a Tie2-CreER mouse which showed a 5-fold increase in both CD31 + cells and vascular permeability after 1 month of tamoxifen. Currently, studies are underway to assess the mechanism of altered endothelial integrity and expansion in the absence of Gata4 in c-kit hemoangioblast progenitors.