The Development Of The Outflow Tract, Sinus Venosus And Cardiac Conduction System Of Embryonic Heart

After complex septation and remodeling,the outflow tract of embryonic heart developed intrapericardial asending aorta and pulmonary trunk,outlets of ventricles and semilunar valves. Abnormality in outflow tract development and septation resulted in congenital heart defects, such as persistent truncus arteriosus,ventricular septal defects,double outlets of right ventricle, transition of great arteries and tetralogy of Fallot,etc.Due to effects of transgene and gene knock-out on embryonic development and differences in species,there were many debates on mechanism underling the outflow tract development.The outflow tract of human embryonic heart completed septation and remodeling before the eighth week.The investigation of the outflow tract development of human embryonic heart was mainly converged on the older embryos,lack of studies of the very younger embryos.Exploration of the early development of outflow tract was not only important to theoretical research,but also in favor of unraveling the mechanism of congenital heart defects resulting from disturbance of outflow tract development and septation.It was reported that myocardium of sinus venosus derived from mesenchyme in the posterior heart-forming field,controlled by distinct gene program.It was even argued that sinus venosus had never been appeared in mammals.There was no date about the early development of sinus venosus of human embryonic heart since studies were almost manipulated on old embryos after C14(Carnegie stage 14),when the sinus venosus had incorporated into right atrium.Mutation in DES gene caused DES myopathy,often accompanied with conduction blocks,arrhythmias and sudden death.The cause of DES myopathy was still unclear so far.In chapter I,we observed the spacio-temporal expression patterns ofα-SMA(α-smooth muscle actin),α-SCA(α-sarcomeric actin),myosin heavy chain(MHC) and active Caspase-3 (CAS-3) in cardiac outflow tract and pharyngeal mesoderm of human embryos from C10 to C16 (22±1～37 postovulatory day) and morphologic charicteristic of heart and pharyngeal mesoderm at each stage with HE staining to explore the mechanism underling the elongation of outflow tract,the development of the endocardial cushions and the division of the aortic sac.In chapterⅡ, the spacio-temporal expression patterns ofα-SMA,α-SCA and desmin(DES) in human embryonic heart through C10 to C16 were observed to investigate the early development of sinus venosus and cardiac conduction system.In chapterⅢ,the spacio-temporal expression patterns ofα-SMA,α-SCA,GATA-4,MHC,CAS-3 and PCNA(proliferating cell nuclear antigen) in mouse embryonic heart from E9(embryonic day 9) to E12 were detected to explore the shortening mechanism of outflow tract during its remodeling.ChapterⅠThe development of the outflow tract in the early human embryonic heartSerial sections of twenty-nine human embryonic hearts from Carnegie stage 10 to Carnegie stage 16(C10～C16) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA(α-sarcomeric actin),MHC(myosin heavy chain) and CAS-3 (active Caspase-3) to investigate the relationship of splanchnic epithelium lining the dorsal wall of the pericardial cavity,the prepharyngeal mesenchyme and the aortic sac with the embryogenesis of the outflow tract myocardium.HE staining was carried out to observe morphologic charicteristic of heart and pharyngeal mesoderm at each stage.We found that with the caudal translocation of the aortic sac and outflow tract relative to the pharyngeal arches during C10 to C15 and the dorsal expansion of the pericardial cavity on both lateral sides of the outflow tract,the aortic sac originally embedded in the prepharyngeal mesenchyme gradually protruded into the pericardial cavity.The progressive differentiation of the pericardial splanchnic epithelium covering the mesenchymal wall of the aortic sac intoα-SCA and MHC positive cardiomyocytes resulted in the elongation of the myocardial outflow tract.The ability of the dorsal pericardial splanchnic epithelium to differentiate into cardiomyocyte was terminated at C16.From C10 to C12,splanchnic pericardium distal to the outflow tract and dorsal pericardium were stratified epithelium,when extending into parietal mesoderm pericardium gradually transformed into simple squamous epithelium.During the following three stages, dorsal pericardium gradually changed into simple epithelium.During C10 to C13,expression ofα-SMA andα-SCA reached splanchnic pericardium and pharyngeal mesenchymal cells distal to the outflow tract,which caused the progressively elongation of the outflow tract.From C14 to C15,the outflow tract gradually elongated.Over these stages,splanchnic pericardial cells distal to the outflow tract proliferated and progressively migrated into the outflow tract, expresssingα-SMA andα-SCA when closed to cardiomyocytes.The prepharyngeal mesenchyme migrated to the dorsal and ventral walls of the arterial pole of the outflow tract was seen being apoptosed at C15,the outflow tract cardiomyocytes were detected to proliferate, migrate into and replace the apoptosised outflow tract mesenchymal masses.α-SMA positive neural crest cells began to appear in the endocardium of the outflow tract at C12 and gradually aggregated to form two opposite spiral ridges during the following stages.During C15 and C16, α-SMA positive neural crest cells in the posterior wall of the aortic sac proliferated and grew into the aortic sac to form the aorto-pulmonary septum that divided the aortic sac into the intrapericardial ascending aorta and pulmonary trunk.We suggest that the splanchnic mesodermal epithelium of the pericardial cavity is the secondary heart field of the human embryonic heart,the continuous differentiation of which into cardiomyocytes brings about the increase in the length of the myocardial outflow tract.CAS-3 positive staining suggests that not all of the mesenchymal cells migrated to the arterial pole of the outflow tract can differentiate into cardiomyocytes.Myocardium of distal outflow tract can induce cells derived from the secondary heart field(or the anterior heart field) to differentiate into cardiomyocytes. Appearance ofα-SMA positive neural crest cells in the endocardium of the outflow tract and aorto-pulmonary septum at different developmental stages indicates that migration of the neural crest cells towards the outflow tract ridges and the aorto-pulmonary septum is along the different routes.ChapterⅡThe early development of the sinus venosus and the cardiac conduction system in human embryonic heartSerial transverse sections of 29 human embryonic hearts from Carnegie stage 10 to Carnegie stage 16(C10-C16) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA(α-sarcomeric actin) and DES(desmin).We found that during C12 and C13,the sinus venosus formed by confluence of systematic veins at the caudal end of the pericardial cavity could be recognized in the mesenchyme of primitive transverse septum. The mesenchymal cells of the sinus venosus gradually differentiated intoα-SCA positive cardiocyocytes.At C14,the sinus venosus was within the pericardial cavity due to expansion of the pericardial cavity and incorporated into the right atrium.Differentiation of DES positive conductive cardiomyocyte was initiated in the right wall of atrio-ventricular canal of C10 embryonic heart and with the development,extended towards the myocardium of the interventricular sulcus to form His bundle,left and right bundle branches as well as the ventricular trabecular myocardium.In the atium,the strong expression of DES was first detected in the dorsal wall of C11 atrium.At C13,unique myocardial band showingα-SCA,α-SMA and DES expression in the left dorsal wall of the sinus venosus were found to be continuous with the basal wall of left atium and the dorsal wall of the atrio-ventricular canal,this band might be related to the development of conduction system from sinoatrial node to atrio-ventricular canal. During C14 to C16,primary conduction pathway of atria with strong DES expression was formed that extended from sinoatrial node along venous valves,DES positive myocardium in the dorsal and ventral walls of the atria to the right atrio-ventricular canal,respectively.We suggest that the mesenchyme of the primitive transverse septum is heart forming field of human embryos responsible for formation of sinus venosus myocardium,and cardiomyocytes differentiated from mesenchymal cells in the primitive transverse septum progressively add to the venous pole of the heart tube to form myocardial sinus venosus.The differentiation of CCS of the early human embryo initiates in the atrio-ventricular canal and develops gradually towards the arterial and venous poles of the heart tube.By C16,DES positive embryonic CCS can be clearly recognized morphologically.ChapterⅢCardiac outflow tract in the early development incorporating into right ventricle in mouse embryoSections of embryonic mouse heart from E9(embryonic day9)-E12(harvested respectively at eight,twelve,sixteen,twenty o'clock at E12) were stained with mouse monoclonal antibody respectively againstα-SCA(α-sarcomeric actin),α-SMA(α-smooth muscle actin),GATA-4, MHC(myosin heavy chain),PCNA(proliferating cell nuclear antigen) and rabbit polyclonal antibody against active CAS-3(Caspase-3).We found that the length of outflow tract from embryonic mouse heart became shortened during eight to twenty o'clock at E12.Before and during its shortening,no CAS-3 positive cell was detected in the whole outflow tract.Through E11 to 8 o'clock at E12,with the translocation of the aortic sac into the pericardial cavity,the expression ofα-SCA,α-SMA,GATA-4 and MHC at the distal part of the outflow tract retracted in the pericardial cavity towards the ventricle.GATA-4 staining suggested at the distal border of the outflow tract no cardiomyocytes transdifferentiated into cell components of the great arteries, and mesenchymal cells derived from the secondary heart field continued differentiating into cardiomyocytes to add to the arterial pole of the heart,which still resulted in the elongation of the outflow tract.Through E11 to E12,α-SMA positive cardiac neural crest-derived cells migrated into the distal outflow tract,α-SCA,α-SMA,GATA-4,MHC and PCNA expression showed that in the proximal outflow tract,cardiomyocytes proliferated into trabecula invading into adjacent ridges,resulting in the myocardilization of it and the outflow tract incorporating into the right ventricle.At E12,α-SCA andα-SMA weak positive mesenchymal cell confluents continuous with the myocardium of the outflow tract were detected.With higher magnification, mesenchymal cell processes were observed to extend towards myocardiocytes,some of which even contacted with myocardiocyte.Independent cells withα-SCA,α-SMA,GATA-4 coexisting were observed in the outflow tract ridges during E12.These results suggested that mesenchymal cells in the outflow tract ridges transdifferentiated into cardiomyocytes.We suggest that just before the fusion of outflow tract ridges into the outflow tract septum,their proximity musculizes through myocardilization and mesenchymal cell transdifferention into cardiomyocytes,with the result of the outflow tract being absorbed into the right ventricle,the shortening of the outflow tract and retraction of the distal end of the outflow tract toward the ventricle.In the proximal outflow tract ridges,mesenchymal cell transdifferention into cardiomyocytes is induced by contact with cardiomyocytes and substance released from cardiomyocytes.Neither apoptosis of cardiomyocytes nor transdifferention of cardiomyocytes into cell components of the great arteries at the distal border of the outflow tract is the mechanism underlying the outflow tract shortening during remodeling.