Conditional Knockout PDK1 In SHF Affects The Development Of The Outflow Tract And Results In Pulmonary Stenosis

The second heart field progenitors,residing in the splanchnic and pharyngeal mesoderm,migrates to the both poles of heart tube,increases its length for cardiac looping.The anterior portion of this wave of cells,defined as anterior second heart field(AHF)progenitors,are added to the arterial pole at the time of cardiac looping and form all of the mesoderm-derived components of the cardiac OFT and the right ventricle(RV).The septation of OFT to pulmonary artery and aorta follows heart looping at E12.5,which is quite important for construction of systemic pulmonary circulation.Defects in this process cause life-threatening conditions in both syndromic and nonsyndromic CHDs.Mutation of BMP,FGF and Notch signaling in AHFs causes failure of initiation of OFT septation,displaying loss of CNCs in OFT.AHFs initially locate the outer layer of OFT and then invade after sepital bridge formation to accomplish OFT septation.A portion of AHFs expresses smooth muscle actin and contributes to the muscle component of future aorta and pulmonary artery.PDK1 is an essential protein kinase that can activate AKT/PKB and many other AGC kinases such as PKC,S6K,SGK.It is involved in signaling pathways that activate many growth factors and hormones such as insulin signaling.However,the role of PDK1 in cardic development especially in OFT development remains unknown.Here,we employed Mef2c-cre mice to delete PDK1 specifically in SHF cells.The absence of PDK1 in SHF(cKO)resulted decreased lumen size of OFT but not the length at E10.5.Meanwhile the truncal myocardial cuff around the OFT of Pdkl cko mice was much thinner.In contrast,the cushion formation was normal in Pdk1 cko OFT until E11.5.At E12.5,compared to aorta,the stenosis of pulmonary artery showed significantly decreased lumen size,even through both vessels showed thinner myocardial cuff.Histological analysis revealed the number of progenitor cells decreased in arterial pole at E10.5,which could explain the thinner myocardial cuff.In the other side,we used Mef2c-creER to label OFT cells drived from SHF,which could delineate all progeny produced by the OFT cells.Tamoxifen injection performed at different stages,showing that cardiomyocytes labeled in distal OFT at E9.5\E10.5\E11.5 were mostly located at the pulmonary artery.Our results indicate the heterogeneity in the cardiomyocytes of the outflow tract in the mouse embryo,suggest that PDK1 is essential to future sub-pulmonary myocardium,which could be a developmental function of PDK1 that is precisely regulated the sub-pulmonary myocardium during pulmonary artery development.