The Expression Pattern And The Construction Of Desmin And LaminA Of Intermediate Filament Protein In The Conduction System Of Early Human Embryonic Hearts

Background:The efficient contraction of the heart depends on the coordination of the working myocardium of chambers with the special myocardium of the cardiac conduction system.The sinus node(SAN),the atrioventricular node(AVN),the His bundle and the proximal part of the bundle branches constitute the central conduction system(CCS),while the distal part of the bundle branches and the peripheral ventricular conduction networks are included in the peripheral conduction system(PCS).Since embryonic heart development is a complex process and acquisition of human embryonic specimens is challenging,the mechanism by which the embryonic conduction system develops remains unclear.Recent advances in techniques of molecular biology provide some insight into the mechanism underlying the development of the conduction system in chicken,rodents and zebrafish embryonic heart.It has been demonstrated that the development and differentiation of the conduction system are regulated by complicated network of transcription factors,including T-box transcription factors Tbx3.Tbx3 functions as a repressor to inhibit the expressions of chamber specific proteins in the central conduction system,including atrial natriuretic factor(Nppa),and fast conduction gap junction proteins connexin43(CX43)and connexin40(CX40),to delineate the region of the central conduction system.Studies on transgenic animals have shown that the ectopic expression of Tbx3 can result in the dysfunction of the conduction system or fatal arrhythmia.Desmin is a principal muscle-specific intermediate filament protein,which is an important component of cytoskeleton.It connects adjacent myofibrils laterally at the Z-band level,links myofibrils to the sarcolemma and nuclear LaminA,and participates in the assembly of intercalated disk in cardiac muscle.In the adult,desmin and LaminA establish entire cell framework structure throughout the nucleus and cytoplasm by connecting to the nucleoskeleton and cytoskeleton complex(LINC),which penetrates throughout the inner and the outer nuclear membranes.Patients of desminopathies due to DES gene mutation produce arrhythmogenesis such as severe atrioventricular block,even complete heart block with requirement of insertion of a permanent pacemaker at young ages.Mutation in LMNA gene results in the disease usually with conduction disease or arrhythmia as the first symptoms.It is suggested that desmin and LaminA are closely related to the development of conduction system in human embryonic heart.However,to our knowledge,there are few reports about their expression patterns in early human embryonic hearts.In present study,the morphologic evolution process of Carnegie10-20 human embryonic hearts was systematically observed,and the expression patterns of intermediate filament proteins desmin and LaminA in the cardiac conduction system and their relationship with the expression patterns of different transcription factors such as Tbx3,Isl-1 and Nkx2.5 were expounded.Compared with model animals,our data provide a direct morphological basis for understanding the arrhythmogenesis caused by mutations in human DES and LMNA genes.Part ? The spatio-temporal expression pattern and significance of desmin and LaminA in developing conduction system of early human embryonic hearts Objective:To clarify the spatio-temporal expression patterns of the intermediate filament protein desmin and LaminA in the conduction system of early human embryonic hearts and its function in the development of the conduction system of human hearts,providing morphological basis for the study on the pathogenesis of conduction dysfunction caused by mutations of human DES and LMNA genes.Methods:Serial paraffin sections of human embryos from Carnegie stage 10 to 20(3-8 weeks after fertilization)were stained with H.E.,immunohistochemistry and double immunofluorescence staining methods.In this study,monoclonal antibodies against desmin,myosin heavy chain(MHC)and ?-smooth muscle actin(?-SMA)and polyclonal antibodies against Tbx3,LaminA and CX43 were used.Three-dimensional reconstruction from the stained serial sections of some embryos were performed.Results:Our experiment revealed that the expression of Tbx3 was limited to the central conduction system(CCS)only after stage 11,desmin was coexpressed with Tbx3 in the myocardium of atrioventricular canal,although it was stronger and earlier than that of Tbx3.And before stage 11,Tbx3 was expressed in the myocardium throughout the heart except the sinus venosus.The weak LaminA was first detected in trabeculation surrounding the cavity of the ventricles,where the Purkinje fibers of peripheral conduction system(PCS)started to develop.At stage 12,the myocardial band positive for both Tbx3 and desmin was seen running from the ventral wall of AVC through the roof of the common atrium to meet with dorsal wall of the right atrium,where the septum spurium was developing.Furthermore,the expression of desmin and Tbx3 extending from the dorsal wall of AVC along the wall of the left atrium to the left pulmonary ridge was also detected.At stage 13,the thickened area of the dorsal wall of AVC was considered as the primordium of the atrioventricular node,with sparse cardiomyocytes and weak eosinophilic cytoplasm.The expression intensity of Tbx3,desmin,LaminA and Nkx2.5 is stronger than that in atrial or ventricular cardiomyocytes,but the expression of ?-SMA is absent.At stage 14,the His bundle formed and connected with the right dorsal wall of the atrioventricular canal,and both expressed desmin and Tbx3.The expression of desmin extended from the His bundle of the central conduction system to the distal bundle branches and Purkinje fibers of the peripheral conduction system.At stage 15 and 17,the sinus node and the atrioventricular node could be clearly recognized morphologically,respectively.The right venous valve extended caudally to connect with the atrioventricular node,while caudal extension of the left venous valve was seen to insert into the dorsal mesenchymal protrusion.The two conduction bands positive for Tbx3 and desmin was observed between the sinus node and the atrioventricular node in C17-C20 human embryos.The atrioventricular node was found connecting to the His bundle,which is connected to the left and right bundle branches and Purkinje fibers.Thus,the morphologically recognizable conduction pathway from the sinus node to the ventricular Purkinje fibers was established.The expression of Tbx3 was confined in the myocardium of the central conduction tissue,while desmin was expressed in the central conduction system during early development of human embryonic heart,with earlier than that of Tbx3.At stage 20,the expression of desmin was recognized extending all the way down to the peripheral conduction system and the working myocardium in addition to the compact myocardium.Positive expression of LaminA was significantly later than that of desmin,and the positive expression area extended gradually upward from Purkinje fiber to the myocardium of the central conduction system.The stronger expression of LaminA was not obtained in the sinus node until stage 20.Conclusion:1.The expression of desmin is relatively colocalized with that of Tbx3 in the central conduction system of early human embryos.As an important cytoskeletal structure,desmin is indispensable in the initial framework of the central conduction system.Furthermore,Tbx3 promotes the differentiation of primitive cardiomyocytes into slow-conduction cardiomyocytes of the central conduction system,ensuring that the morphogenesis is adapted to the function in the developing heart.2.The development of cardiac conduction system is a complex dynamic process,and the combined application of desmin and Tbx3 as marker proteins can well demonstrate the complete cardiac conduction system from the sinus node to the Purkinje fiber network in the early human embryonic heart.3.The expression of LaminA is later than that of desmin and expanding from the peripheral conduction system to the central conduction system,suggesting that the coupling of LaminA with desmin does not occur until stage 20 in human embryonic heart.4.Desmin is also involved in the development and maturation of the peripheral conduction system and working myocardium of atrium in human embryos.Part ? The early development of the sinus venosus and the sinus node in human embryonic heartsObjective:To investigate the origin and morphogenesis of the sinus node in human embryonic hearts,and to explore the relationship between the expression of Tbx3 and Isl1 and the development and differentiation of the sinus node,providing the morphological data for the etiology of human congenital dysfunction of the sinus node.Methods: After fixation with MAW solution,the collected human embryos(3-8 weeks after fertilization)were made into 7?m-thick paraffin-embed continuous sections.Then the sections were stained with ?-smooth muscle actin(?-SMA),myosin heavy chain(MHC)and desmin monoclonal antibodies and Nkx2.5,Isl-1 and Tbx3 rabbit polyclonal antibody,respectively.Results: At stage 10,a discrete symmetrical sinus venosus already could be identified at the end of the common atrium,and the absence of Nkx2.5 expression allowed us to identify the sinus venosus at the molecular level.The Isl-1 positive cells were seen extending from mesenchyme in the ventral of the foregut to the dorsal wall of the sinus venosus.At stage 11,the presence of ?-SMA in the wall of sinus venosus indicated the onset of myocardialization of mesenchymal cells.At stage 12,the junction of Nkx2.5-negative cranial part of the sinus venosus and Nkx2.5-positive caudal component of the right atrial appendage folded into a two layers right venous valve.The right venous valve continued toward the cranial part of the right atrium and fused gradually with the septum spurium.The left venous valve could not be recognized at this stage.At stage 13,the right and tiny left venous valves were observed in the dorsal-lateral wall at the caudal end of the right atrium and merged with septum spurium that acquired Nkx2.5 expression and lost Isl-1 expression.In contrast to adjacent atrial myocardium,the part between the two valves showed a positive expression of Isl1 and ?-SMA,and a negative expression of Nkx2.5.At stage 14,the length of the left and right valves increased greatly,the expression of Nkx2.5 decreased with the caudal approach,the opposite was true for the expression of Isl-1.Instead of taking up into the right atrium,the part of Isl-1 positive cells in the sinus venosus participates to form the right horn of the sinus venosus and the coronary sinus,with the expression pattern of ?-SMA(+),desmin(+)and Tbx3(-).The right horn of the sinus venosus was significantly thicker than that of stage 13 and located between the right superior caval vein and the right atrium.At stage 15,the sinus node was located in the dorsal and cranial position of the right atrium.The head of the sinus node wrapped the junction of right atrium with right superior caval vein,and the tail extended along the right venous valve(terminal crest).As a result,the anatomic position of the sinus node similar to that of the adult and its comma or U-shaped morphology could be identified.Nevertheless,the expression of Tbx3 was only detected in a small region proximal to the right atrium from the venous valve,with obviously lagging behind that of desmin and Isl-1.We noted that the area that acquired Tbx3 expression became negative for ?-SMA and loosely organized.After stage 16,the expression of Tbx3 extended further along the right venous valve to the distal part of the head of the sinus node.Until stage 20,the head of the sinus node still exhibited the strictly complementary expression of Tbx3 and ?-SMA.Isl-1,a marker of cardiac progenitor cells in the second region,remained strongly positive at the head of the sinus node,though the muscle specific protein MHC was already expressed.Conclusion: 1.The mesenchymal wall of the sinus venosus first developed as early as at stage 10 and began myocardialization at stage 11 in human embryos.And the myocardial cells that accomplished myocardialization evolved gradually into the striated muscle cells from the head end to the tail end of the venous sinus at stage 12.2.We demonstrated that the special area in the wall of right atrium that was positive for Isl-1 and negative for Nkx2.5 represented the primordium of the tail of the sinus node at stage 13,which was confined by the right and left valves and remained traceable in stage 14 embryos.At this stage,the caudal entrance of the thickened wall in the right horn that was directly connected with right superior caval vein was recognized as the primordium of the head of the sinus node.At stage 15,the head of the sinus node was located in the dorsal and cranial lateral of the right atrium due to the convergence of the chambers of the heart that resulted in the shift of the sinus venosus from the caudal end to the cranial end.3.From stage 15,the expression of Tbx3 had expanded along the right venous valve to the area of the head of the sinus venosus proximal to the right atrium,and the expression of Tbx3 and ?-SMA showed a strict complementary pattern,implying that the cells in the sinus node began to differentiate and mature from the tail to the head.4.As with mouse embryos,both the sinus venosus and the sinus node have a special Nkx2.5 negative and Isl-1 positive expression pattern,and the transcription factors Nkx2.5 and Isl-1 are ideal markers for them in human embryos.