The Role Of SEMA6D In Cardiac Development And Homeostasis

Congenital heart diseases remain the leading cause of infant morbidity and mortality in developed countries,affecting¹%of newborns,and they are also risk factors for devastating diseases in the cardiovascular and other systems,such as heart failure,arrhythmia,sudden death,lung dysfunction and Alzheimer's disease.Understanding the molecular and genetic mechanisms that control normal cardiogenesis would provide crucial clues for the development of novel clinical strategies fighting against congenital heart diseases and is thus highly significant for both basic and clinical research.Semaphorins are a family of secreted and membranebound molecules that play critical functions in diverse biological processes.SEMA1A/Fasciclin IV is the founding member of the semaphorin family and was first reported in 1992 as a regulator of axon branching in the growth cone of grasshopper embryos.To date,more than 20 semaphorin members have been discovered in viruses,invertebrates,and vertebrates.In addition to acting as guidance cues for axon growth in the nervous system,semaphorins have been implicated in many other systems,such as immune responses,tumor angiogenesis,bone development and homeostasis,and cardiovascular development.Semaphorins are categorized into 8 classes.Classes 1and 2 are found in invertebrates,classes 3 to 7 are present in vertebrates,and class V semaphorins are found only in viruses.The heart is the first functional organ formed in mammals.Initially,the heart develops from clusters of progenitor cells,which coalesce to form the cardiac crescent and the heart tube.Subsequently,the heart tube undergoes elongation,looping,septation,remodeling,and maturation to form the final 4-chambered organ.Multiple cell types,including myocardial,endocardial,epicardial,and neural crest cells?NCCs?act coordinately during complicated cardiogenesis in vertebrates.Numerous signaling pathways and downstream transcription factors are required for normal cardiogenesis and blood vessel development.In the past 2decades,accumulated studies have indicated that impaired semaphorin signaling results in various cardiovascular disorders during development and in multiple disease states.SEMA6D is a member of the Semaphorin signaling family and act both locally through cell-cell interaction and at a long distance through secretion of its ectodomain.We previously showed that endocardial expression of SEMA6D is essential for the initiation of mesenchyme formation in the atrioventricular canal region of mouse embryonic hearts.In this study,we focused on its role in cardiomyocytes.A previous chicken study suggested that SEMA6D regulates myocardial wall compaction;however,the function of Sema6D in mammalian cardiomyocytes has not been reported in the literature.In our current research we applied a conditional gene inactivation approach to delete Sema6D specifically in developing cardiomyocytes at different stages,including embryo,newborn and adult,to detect the role of SEMA6D in cardiac development and homeostasis.Methods:1.Detecting the effect of conditional knockout of Sema6D on cardiac morphogenesis during embryonic period.We crossed cTnt-Cre;Sema6D^loxp/+male mice with Sema6D^loxp/loxpoxp/loxp female mice to acquire mutant(cTnt-Cre;Sema6D^loxp/loxp)and control(Sema6D^loxp/+or Sema6D^loxp/loxp)animals.A mutant embryo and its litter-mate control embryo at E10.5 were sagittally sectioned and immunostained to investigate the expression of Sema6D in the heart.Mutant and control hearts were HE stained at E18.5.P1 and P6and heart weights were recorded.Mutant and control hearts were immunostained at E16.5,E17.5 and P1 to detect cell proliferation.A mutant and a control heart at E17.5 were immunostained to detect apoptotic cardiomyocytes.2.To examine the effect of exogenous SEMA6D on cardiomyocyte proliferation.We cultured cardiomyocytes isolated from E16.5 hearts,and treated them with conditional medium containing the ectodomain of SEMA6D and examined their cell proliferation through EDU labeling.3.To investigate the expression of MYCN,Cyclin D1,Cyclin D2 and ID2 in hearts at E17.5.To detect the expression of mature sarcomeric isoforms, including ACTC1,TNNI3 and?-MHC,and the expression of their corresponding embryonic isoforms,including ACTA1,ACTA2,TNNI1 and ?-MHC in hearts at P1.Mutant and control heart proteins were extracted at E17.5 and P1,and the expression levels of different proteins were detected by Western blotting.4.Detecting embryonic heart deletion of Sema6D in heart function in adult mice.To examined heart function in 3-month old male and female control and mutant mice by echocardiography.Recording mouse heart weight and tibia length.5.To detect the potency of rAAV?Cre?virus using R26R and mT/mG genotype mice.We injected subcutaneously with rAAV?Vector?or rAAV?Cre?virus at P1mice,and performed X-gal staining and immunofluorescence staining at P7 mice.6.To investigate the effect of conditional knockout of Sema6D on the body weight and cardiac function of Sema6D ^loxp/loxpoxp/loxp male mice after neonatal birth;to investigate whether SEMA6D play a protective role in different doses of DOX-induced myocardial injury model of Sema6D ^loxp/loxpoxp/loxp male mice.P1 mice were injected subcutaneously with rAAV?Vector?or rAAV?Cre?virus,and body weights were recorded every 3 days,and cardiac function was examined by echocardiography at P28.P1 mice were injected subcutaneously with rAAV?Vector?or rAAV?Cre?virus,then the mice were injected with low dose DOX?5mg/kg?,high dose DOX?10 mg/kg?or Vehicle according to the body weight of the mice at different times?P14,P21,P28,P35?.1 week later,HE staining of cross sections and echocardiography were performed after the last intraperitoneal injection,and the heart weight and length of the tibia were recorded.7.To detect TAM-induced?MHC-MerCreMer recombinase efficacy in adult mice with R26R and mT/mG genotypes mice.12-week-old?MHC-MerCreMer;Sema6D ^loxp/+;R26R^+/-genotype mice were injected intraperitoneally with tamoxifen for 5 days,and X-gal staining was performed 3 weeks later.12-week-old?MHC-MerCreMer;Sema6D ^loxp/+;mTmG^+/-genotype mice were injected intraperitoneally with tamoxifen for 5 days,and heart sections were immunostained 3 weeks later.8.To investigate the effect of tamoxifen on the cardiac function of adult mice after conditional knockout of Sema6D.12-week-old Sema6D ^loxp/loxpoxp/loxp and?MHC-MerCreMer;Sema6D ^loxp/loxpoxp/loxp genotypes adult male mice were injected intraperitoneally with tamoxifen or corn oil for 5 consecutive days.After 3 weeks,the heart cross sections were performed HE staining,Massson trichrome staining and echocardiography.The mouse heart weight and tibia length were recorded.Results:1.Our immunostaining analysis showed that expression of Sema6D was efficiently inactivated in the myocardium of mutant embryos at E10.5.At P1,mutant hearts displayed the hypoplastic myocardial wall defect in both ventricles.The wall of the left ventricle?LV?of the mutant hearts was significantly thinner than that the control one at P6.Cardiomyocyte proliferation was significantly reduced in mutant hearts after birth at E17.5 and P1.2.Exogenous SEMA6D significantly increased cell proliferation on cultured fetal cardiomyocytes.3.We found that expression of mature sarcomeric isoforms were all increased,whereas expression of their corresponding embryonic isoforms were all decreased in mutant hearts at P1.4.The heart weight and weight-to-tibia length ratio were significantly reduced in both male and female mutant mice?3-months old?.The ejection fraction and stroke volume were both significantly reduced in male mutant mice.Activities of female mutant hearts are not significantly reduced compared to control female mice.5.Our X-gal staining of R26R mice at P7 indicated that most myocardia were blue.GFP were activated in most cardiomyocytes of mT/mG mice.6.There were no significant differences in body weight and cardiac function between the newborn Sema6D ^loxp/loxpoxp/loxp male mice after conditional knockout of Sema6D.7.Our X-gal staining of R26R mice demonstrated that myocardia turned blue,and the effect of TAM?80mg/kg?injection was the best.GFP were activated in most cardiomyocytes of mT/mG mice after injection of TAM?80mg/kg?.8.The heart weight and heart weight/tibia length ratio of?MHC-MerCreMer;Sema6D ^loxp/loxpoxp/loxp group were significantly higher than other groups,and cardiac function was significantly lower than other groups.Massson trichrome staining showed that myocardial collagen fibers were increased in?MHC-MerCreMer;Sema6D ^loxp/loxpoxp/loxp group compared to other groups.Conclusion:Embryonic heart deletion of Sema6D results in hypoplastic myocardial wall in fetal and neonatal hearts.SEMA6D acts through MYCN to promote cardiomyocyte proliferation in embryonic hearts.Embryonic heart deletion of Sema6D leads to reduced heart function in adult mice in a sex-dependent manner.There are no significant differences in body weight and cardiac function between the newborn Sema6D ^loxp/loxpoxp/loxp male mice after conditional knockout of Sema6D.SEMA6D does not play a protective role in different doses of DOX-induced myocardial injury male mice model.Conditional knockout of Sema6D with tamoxifen leads to myocardial collagen fibrosis and decreased cardiac function in adult male mice.