SETD4 Controlled Quiescent C-Kit~+ Cells Contribute To Cardiac Neovascularization Of Capillaries Beyond Activation

Vessels in the adult exist in a highly organized and stable state,angiogenesis only occurs in response to a physiological stimulus,such as hypoxia for blood vessels.In the ischemic heart,limited expansion capacity of myocardial vascular bed cannot satisfy demand for oxygen supply and myocardium eventually undergoes irreversible damage.Endogenous c-Kit⁺cells are convinced to predominantly contribute to cardiac endothelial cells according to recent reports,which attract great attention in the potential of treatments for cardiac ischemic injury.In adult hearts,endogenous c-Kit⁺cells exist in either an active or quiescent state.And quiescent cells in other tissues are known to contribute to the long-term maintenance of a cell pool,preserve proliferation capacity and upon activation facilitate tissue homeostasis and regeneration in response to tissue injury.However,the regulatory determinant of controlling c-Kit⁺cells quiescence and the function of quiescent c-Kit⁺cells in cardiac homeostasis and response to injury remains unknown.In this study,we presented the existence of a quiescent c-Kit⁺cell population in the adult mouse heart by analyzing the cell proliferation markers Ki67 and H3p S10,as well as using Ed U incorporation and Brd U retention experiments.In our previous study,we found an evolutionarily conserved mechanism of cell quiescence epigenetically regulated by SETD4.By analyzing the molecular characteristics of quiescent c-Kit⁺cells,we clarified that these population of quiescent c-Kit⁺cells were catalyzed by SETD4 to form H4K20me3 promoting the formation of heterochromatin.On this basis,in order to investigate the function of SETD4-regulated quiescent c-Kit⁺cells in mouse hearts,we constructed SETD4 conditional knockout mice and achieved SETD4conditional knockout in the c-Kit⁺cells of adult and newborn mouse hearts.The results showed that the SETD4 gene knockout could lead to the production of cardiac endothelial cells and promote the production of microvessels both in the adult and newborn mouse hearts.However,the endothelial cells in large size coronary vessels are rarely derived from c-Kit⁺cells.Meanwhile,we found that the activation of quiescent c-Kit⁺cells via knockout of SETD4 was achieved by activating the PI3K-Akt-m TOR signaling pathway.In addition,we performed SETD4 gene conditional knockout in myocardial infarction(MI)model mice,the results showed that Setd4 knock-out lead to generating more microvascular cells in MI-injured hearts,which would attenuate cardiomyocyte apoptosis by providing additional oxygen and improved cardiac function.In order to investigate the biological functions of SETD4⁺cells during the stage of embryo development,we constructed Setd4-Cre;Rosa26^{Td Tomato} mice.We found that Setd4⁺cells appeared early in the embryonic day 6.5 and contributed to each cardiac lineage including c-Kit⁺cells.The quiescent Setd4⁺c-Kit⁺cells may represent a developmental remnant that has persisted in the heart throughout embryonic and adult period.In conclusion,our study identified a population of quiescent c-Kit⁺cells regulated by SETD4 protein,and revealed that these endogenous quiescent c-Kit⁺cells were able to improve cardiac function in myocardial infarction injured mice via neovascularization of capillaries beyond activation.