Seamless Genetic Recording of Transiently Activated Mesenchymal Gene Expression in Endothelial Cells During Cardiac Fibrosis

Background: Cardiac fibrosis is a lethal outcome of excessive formation of myofibroblasts that are scar-forming cells accumulated after heart injury. It has been reported that cardiac endothelial cells (ECs) contribute to a substantial portion of myofibroblasts through endothelial to mesenchymal transition (EndoMT). Recent lineage tracing studies demonstrate that myofibroblasts are derived from the expansion of resident fibroblasts rather than from the transdifferentiation of ECs. However, it remains unknown whether ECs can transdifferentiate into myofibroblasts reversibly or EndoMT genes were just transiently activated in ECs during cardiac fibrosis. Methods: By using the dual recombination technology based on Cre-loxP and Dre-rox, we generated a genetic lineage tracing system for tracking EndoMT in cardiac ECs. We used it to examine if there is transiently activated mesenchymal gene expression in ECs during cardiac fibrosis. Activation of the broadly used marker gene in myofibroblasts, αSMA (α-smooth muscle actin), and the transcription factor that induces epithelial to mesenchymal transition, Zeb1 (zinc finger E-box–binding homeobox 1), was examined. Results: The genetic system enables continuous tracing of transcriptional activity of targeted genes in vivo. Our genetic fate mapping results revealed that a subset of cardiac ECs transiently expressed αSMA and Zeb1 during embryonic valve formation and transdifferentiated into mesenchymal cells through EndoMT. Nonetheless, they did not contribute to myofibroblasts, nor transiently expressed αSMA or Zeb1 after heart injury. Instead, expression of αSMA was activated in resident fibroblasts during cardiac fibrosis. Conclusions: Mesenchymal gene expression is activated in cardiac ECs through EndoMT in the developing heart, but ECs do not transdifferentiate into myofibroblasts, nor transiently express some known mesenchymal genes during homeostasis and fibrosis in the adult heart. Resident fibroblasts that are converted to myofibroblasts by activating mesenchymal gene expression are the major contributors to cardiac fibrosis.