Transcription factor 21 regulates cardiac myofibroblast formation and fibrosis [RNA-Seq]

Transcription factor 21 (TCF21) is a basic helix-loop-helix protein required for developmental specification of cardiac fibroblasts from epicardial progenitor cells that normally surround and invade the heart. In the adult heart, TCF21 is expressed in tissue resident fibroblasts but is downregulated in response to injury or stimuli leading to myofibroblast differentiation. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Here, bulk RNA sequencing was used to determine the effect of loss of TCF21 and enforced TCF21 expression in adult cardiac fibroblasts. Overall design: RNA-sequencing was performed on cardiac fibroblasts isolated by flow cytometry from adult genetically engineered mouse models with loss or gain of function of TCF21 in the healthy heart or after an injury. Tcf21fl/fl mice were crossed with Tcf21-MerCreMer (Tcf21MCM) mice to permit ablation of Tcf21 upon exposure to tamoxifen. Tcf21MCM mice were used as controls. Col1a1-TCF21 mice were crossed with platelet-derived growth factor receptor, alpha polypeptide (Pdgfra) gene (Pdgfra-CreERT2) mice to permit overexpression of TCF21 upon exposure to tamoxifen. Pdgfra-CreERT2 mice were used as controls. All mice contained a Rosa26-loxP-STOP-loxP-enhanced green fluorescent protein for lineage tracing and sorting fibroblasts. To study the baseline effects of loss of Tcf21 in cardiac fibroblasts, tamoxifen was administered to adult mice and fibroblasts were isolated two weeks later by flow cytometry for RNA-sequencing. To study the effects of loss and gain of function of Tcf21 in the presence of an injury, tamoxifen was administered to mice to induce genetic recombination. Mice were then exposed to a continuous infusion of the fibrotic agonists, angiotensin II and phenylephrine for one week before isolation and sorting of fibroblasts by flow cytometry for RNA-sequencing.