Redox status has emerged as critical in modulating stemness and lineage commitment in several precursor cell types. However, a role for redox genes, specifically NADPH oxidases (Nox), in cardiac precursor cells (CPCs) has not been established. We tested whether CPCs marked by type III receptor tyrosine kinase c-kit (c-kit(+)) exhibit a unique NADPH oxidase signature that confers precursor status and whether alterations in this profile are functionally linked to changes in lineage specification. Dihydroethidium (DHE) microfluorography indicated reduced basal reactive oxygen species (ROS) formation within early postnatal c-kit(+) CPCs. Real-time quantitative PCR revealed downregulation of ROS generator Nox2 and its subunit p67(phox) in c-kit(+) CPCs under basal conditions but upregulation of Nox2 and Nox4 over the course of differentiation. Adenoviral silencing of Nox2 and Nox4 increased expression of CPC markers c-kit and Flk-1 and blunted smooth and cardiac muscle differentiation, respectively, while overexpression of Nox2 and Nox4 significantly reduced c-kit expression. These changes were accompanied by altered expression of transcription factors regulating cardiac lineage commitment, Gata6 and Gata4, and cytokine transforming growth factor (TGF)-β1. Similar to other precursor cell types, RT(2)Profiler PCR Arrays revealed that c-kit(+) CPCs also exhibit enhanced antioxidant capacity at the mRNA level. In conclusion, we report that c-kit(+) CPCs demonstrate reduced Nox2 expression and ROS levels and that increases in Nox2 and Nox4 influence their differentiation into mature cells. We speculate that ROS generators Nox2 and Nox4, along with the antioxidant genes identified by PCR Arrays, may be novel targets in CPCs that could prove useful in cell-based therapy of the heart.

Through directed differentiation of embryonic stem cells, it has been demonstrated that mesodermal lineages in the mammalian heart (smooth muscle, endothelial, and cardiac) develop from a common, multipotent cardiovascular precursor (Dev Biol 265:262-275, 2004; Cell 127:1137-1150, 2006; Dev Cell 11:723-732, 2006). Identification of cardiovascular precursor cells at various stages of lineage commitment has been determined by expression of multiple markers, including the stem cell factor receptor c-kit. Utilizing a bacterial artificial chromosome (BAC) transgenic mouse model in which EGFP expression is placed under control of the c-kit promoter (c-kit(BAC)-EGFP), work from our laboratory indicates that c-kit expression identifies a multipotent cardiovascular precursor cell population within the early postnatal heart that can be isolated, expanded, and differentiated in vitro into all three cell lineages that specify the heart (Proc Natl Acad Sci U S A 106:1808-1813, 2009).