Lineage Tracing Of Epicardium During Heart Regeneration In Neonatal Mice

Objective:To define the differentiation of activated epicardium during heart regeneration in neonatal mice through lineage tracing.Methods:We use HE staining(n=6)to detect the regeneration after partial resection of left ventricle in neonatal mice.Then,we utilize immunofluorescence(n=6)and RT-PCR(n=4)to confirm the epicardial activation.Finally,the epicardial lineage tracing mice(WtlCreERT2;Rosa26RFP,n=6)and Wtl-expressing mice(Wt1GFPCre,n=6)are performed apical resection to study the differentiation of activated epicardial cells.Results:The results of HE staining(n=6)from 1-day-old wild type mice after apical resection about 10%-15%of left ventricular show that the morphology of heart is normal over 21 days.It is also shown from the spatio-temporal expression of Wtl(n=6)and the expression of Wtl mRNA(n=4)that after 7 days of apex resection in neonatal mice,epicardial activation was highly elevated.The signal of GFP in WtlGFPCre(n=6)co-expressed with the marker of epithelial-mesenchymal transition(EMT),Vimentin.To track the genetic fate mapping of activated epicardial cells,we use Wt1CreERT2;Rosa26RFP(n=6)based lineage tracing system.Immunofluorescence staining show that RFP co-expressed with a-Actinin,a-Smooth Muscle Actinin and Fibroblast specific protein 1,indicating that after activation these cells subsequently go through EMT and differentiate into myocardial cells,smooth muscle cells and fibroblasts.And the cardiomyocytes derived from activated epicardial cells distributed near the incised area,which can complement the loss of myocardial cells and participate in recovery of heart.Adjacent to the resected region,we also observed smooth muscle cells and fibroblasts hierarchized from activated epicardial cells.The newly formed smooth muscle cells are involved in neovascularization,which is a key process in restoring injury.In one way,the fibroblasts will form fibrin blood clots contribute to wound healing.In another way,they will also provide a suitable niche for other cells to undergo proliferation and differentiation by paracrining multiple factors.These findings offer a potential source of cells for the regeneration of cardiac muscle cells and neovascularization,and shed a light on a new avenue for clinical application of heart regeneration.Conclusions:Epicardial cells are highly activated during heart regeneration in neonatal mice,which can differentiate into cardiomyocytes,smooth musle cells and fibroblasts.And our study is aiming to mark the epicardial cells through the fluorescence signal caused by lineage tracing technology instead of nuclear marker.On the one hand,we can directly detect epicardial cells through the fluorescence signal,and then we can utilize these cells to further analyse.On the other hand,we can accurately delineate the epicardial development into other kind of cells,and finally confirmed the process in myocardial regeneration by activating epicardium to restore the heart injury,which is of great significance.There are some limitations in the study,we do not investigate the depth mechanism behind these differentiations from activated epicardial cells.With further study in molecular mechanism,it is possible to use epicardial cells in clinical treatment.