The Development Of The Cardiac Conduction System Of Embryonic Heart

Sinoatrial node, atrioventricular node, atrioventricular bundle and bundle branches constitute the central conduction system, while the distal part of bundle branches and purkinje fiber networks constitute the peripheral conduction system. With the rapid progress of developmental biology and molecular genetics, plenty of studies about the development of the cardiac conduction system have been made in different species by using cell lable, in situ hybridization and technology of transgene or gene knock-out. Due to the complexity of the conduction system development and difference in species, there are many debates on origin and mechanism underling the conduction system development. Exploration of the development of conduction system is not only important theoretical research, but also in favor of unraveling the mechanism of congenital heart disease resulting from disturbance of conduction system development. It is fairly significant to prevent and treat such congenital heart disease. Moreover, it may provide theoretical basis to research adult arrhythmia mechanism.Sinoatrial node which is nomal leading pacemaker of the heart generates and propagates electric impulse to working myocardium through cardiac conduction system, which stimulates atrium and ventricle myocardium contraction and completes cardiac ejection. The differentiation and development of conduction system myocardium and working myocardium are regulated by distinct genes which encode distinct protein. To date, since research about the development of conduction system myocardium and working myocardium was mainly converged on the study of transcription level and for the transgene or gene knock-out mice, there are little dates about spatio-temporal expressions of protein encoded by corresponding genes in normal developmental progress of conduction system myocardium and working myocardium.Due to lacking of human embryo materials, there are rare studies about the development of the human embryonic cardiac conduction system which are only limited to morphological description or spatial-temporal expression patterns of non-specific marker protein, such as neural tissue antigen G1N2and HNK-I on older embryos after Carnegie stage14, while the date about the early development of human embryonic cardiac conduction system is still lacking. Studies for the transgene or gene knock-out mice have showed that multiple transcription factors and ion channel protein are involved in regulating the development of cardiac conduction system in mouse embryo heart, but it is still not clear whether these transcription factors and ion channel protein will play the same role in regulating the development of human embryonic cardiac conduction system.In chapter Ⅰ, we observed the spatio-temporal expression patterns of MHC (myosin heavy chain), HCN4(hyperpolarization-activated cyclic nucleotide-gated cation4), Cx43(connexin43) and PODOPLANIN in mouse embryonic hearts from embryonic day (ED)9to ED16to investigate the expression patterns of HCN4, Cx43and PODOPLANIN in the mouse embryonic heart and development of mouse cardiac conduction system. In chapter Ⅱ, the spatio-temporal expression patterns of Cx43in mouse embryonic hearts from ED9to ED17were observed and Cx43protein content in ED11, ED13, ED15, ED16and ED17embryonic hearts were examined to explore the relationship of the spatio-temporal expression pattens of Cx43in the mouse embryonic heart with development of cardiac conduction system myocardium and working myocardium. In chapter Ⅲ, we observed the spatio-temporal expression patterns of MHC, HCN4, DES (desmin), TBX3, Nkx2.5and ISL-1in human embryonic hearts from Carnegie stage9to Carnegie stage16to explore the early development mechanism of the cardiac conduction system of the human embryonic heart.Chapter Ⅰ Expression patterns of HCN4, Cx43and PODOPLANIN in the mouse embryonic heart and development of cardiac conduction systemSerial transverse sections of forty mouse embryonic hearts from embryonic day (ED)9to ED16were stained immunohistochemically or immunofluorescently with antibodies against myosin heavy chain (MHC), hyperpolarization-activated cyclic nucleotide-gated cation4(HCN4), connexin43(Cx43) and PODOPLANIN to investigate the expression patterns of HCN4, Cx43and PODOPLANIN in the mouse embryonic heart and development of mouse cardiac conduction system. The results showed that at ED9, strong positive of HCN4was concentrated in the MHC weak or negative atria and sinus venosus. At ED10, right sinus horn showed strong HCN4expression. From ED11to ED16, with formation and development of sinoatrial node, strong positive of HCN4gradually shifted to sinoatrial node, while the HCN4positive in other parts of the heart gradually decreased. From ED11onward, Cx43negative expression showed site-specific patterns:Cx43negative dorsal wall of atria was seen connecting with the ventral wall of atrioventricular canal along septum primum: Cx43negative staining of sinoatrial node extended along the dorsal right atrial wall and left and right venous valves towards the dorsal wall of atrioventricular canal; With left and right venous valves fusion with base of the septum primum, Cx43negative staining continued with developing atrioventricular node derived from dorsal wall of atrioventricular canal along right venous valves, orifice of coronary sinus and the front of dorsal mesenchymal protrusion. Developing atrioventricular bundle on the top of the interventricular septum was found connecting with atrioventricular node. From ED9to ED10, strong PODOPLANIN expression was detected both in MHC positive myocardium and in MHC negative myocardial precursors of splanchnic mesoderm of dorsal pericardial wall and mesenchyme surrounding sinus venosus. Between ED11and ED13, PODOPLANIN positive mesenchymal cells spreading along outer surface of the heart formed PODOPLANIN positive mesothelial epicardium. After ED14, PODOPLANIN positive of epicardium disappeared, while the expression of PODOPLANIN in most MHC positive myocardium decreased, and strong PODOPLANIN positive was gradually limited to atrial trabeculae, bundle branches and parts of ventricular trabeculae. From the results we conclude that at early stage of mouse heart development, strong HCN4expression is located in sinus venous, then gradually sihfts to sinoatrial node with development, which is consistent with the transfer path of dominant pacemaker found in electrophysilolgical study. Cx43negative myocardium observed at ED11represents the establishment of the prototype of the developing cardiac conduction system. PODOPLANIN promotes myocardial precursor cells differentiating into cardiocytes and is involved in the formation of early myocardium as well as parts of working myocardium and conduction myocardium. PODOPLANIN may promote the migration and different ion of epicardium into myocardium.Chapter II Spatio-temporal expression patterns of Cx43in the mouse embryonic heart and development of cardiac conduction and working myocardiumSerial transverse sections of mouse embryonic hearts from ED9to ED17were stained immunohistochemically or immunofluorescently with antibodies against connexin43(Cx43), myosin heavy chain (MHC) and a-sarcomeric actin(a-SCA); Cx43protein content was examined by western blotting in EDI1. ED13, ED15, ED16and ED17embryonic hearts to explore the relationship of the spatio-temporal expression pattens of Cx43in the mouse embryonic heart with development of cardiac conduction system myocardium and working myocardium. It was observed that during ED9and ED10, weakly expressed Cx43was first detectable only in the ventral wall of left ventricle and primary trabeculae. From ED11onward, Cx43expression in atria and ventricle was seen expanding and increasing gradually. Between ED11and ED16, in atria, expression of the Cx43was sequentially detected in the top, anterior, lateral wall of atria and atrial trabeculae. In ventricle, Cx43expression extended from the apex of the left ventricle towards the trabeculae and compact myocardium of the left and right ventricle leaving the center of sinoatrial node, left and right venous valves, septum primum, dorsal wall of right atrium, atrioventricular canal, atrioventricular node and atrioventricular bundle showing Cx43negative expression that constituted the prototype of embryonic developing conduction system. At EDI7, Cx43negative expression of left and right venous valves, septum primum and dorsal wall of right atrium was turned into Cx43weak positive. Cx43negative expression was found only in sinoatrial node, atrioventricular node and atrio ventricular bundle. Cx43negative atrioventricular bundle on the top of interventricular septum was seen connecting with Cx43positive left and right bundle branches and ventricular trabeculae. Cx43negative myocardium constituted the developing central conduction system of embryonic heart, while Cx43positive myocardium constituted the peripheral conduction system myocardium and working myocardium.Western blotting analysis showed that from EDI1to EDI7, Cx43protein content gradually increased with development. We conclude that expansion of Cx43expression in the artia and ventricule indicates the transformation of primary myocardium towards the working myocardium. Both Cx43negative myocardium and Cx43positive peripheral conduction system myocardium constitute the integral cardiac conduction system. At late stage of heart development, atrial conduction system myocardium is reduced, on the contrary, working myocardium gradually expands. Spatio-temporal expression patterns of Cx43in the mouse embryonic heart are correlated well with the development and conduction function of both working and conduction system myocardium.Chapter EtI The early development of the cardiac conduction system in human embryonic heartSerial transverse sections of27human embryonic hearts from Carnegie stage9to Carnegie stage16(C9-C16,20眑-37postovulatory day) were stained immunohistochemically with antibodies against myosin heavy chain (MHC), hyperpolarization-activated cyclic nucleotide-gated cation4(HCN4), Desmin (DES), TBX3, Nkx2.5and1SL-1to explore early development mechanism of the cardiac conduction system of the human embryonic heart. We found that during C9and CIO, TBX3was expressed broadly in primitive heart. From CM onward, TBX3expression difference was displayed in different parts of primitive myocardium. From CM to C16, strong TBX3expression is gradually limited to developing sinoatrial node, left and right venous valves, atrioventricular canal, atrioventricular node, atrioventricular bundle on the top of interventricular septum and bundle branches. Observed from serial sections, strong TBX3positive of atrioventricular canal was seen extending to developing sinoatrial node through right venous valves, and to atrio ventricular bundle on the top of interventricular septum and bundle branches along right wall of atrioventricular canal, respectively. Strong DES positive was first detected in the right wall of atrioventricular canal in C10embryonic heart. With development, DES positive progressively expanded to sinoatrial node, left and right venous valves, atrioventricular canal, atrioventricular node, atrioventricular bundle and bundle branches, which was the same as the expression patterns of TBX3. Moreover, strong DES positive was detected in dorsal wall of atria, coronary sinus and ventricular trabeculae. Between C10to C13, the development of left sinus horn and right sinus horn at caudal end of venous pole was showed asymmetry. Nkx2.5^MHC expression wasn't detected in the myocardium of right sinus horn which showed ISL-1positive, while the myocardium of left sinus horn obviously expressed Nkx2.5and MHC, but had lost ISL-1expression. At C14, with the right lateral wall of right superior vena cava becoming thickened, the Nkx2.5negative, ISL-1,DES positive sinoatrial node was formed. TBX3positive in sinoatrial node was seen expanding from the myocardium which was adjacent to right venous valve. At C9, weak HCN4positive was detected throughout primitive heart tube. Between C10and C13, HCN4was expressed not only in the outflow tract, atrium, atrioventricular canal and ventricle, but also in the left and right sinus horn. Moreover, HCN4expression gradually increased from arterial pole to venous pole. The HCN4expression of Nkx2.5positive left sinus horn was stonger than that of Nkx2.5negative right sinus horn. During C14and C16, with formation of sinoatrial node and venous valves on the right side of venous pole, strong HCN4positive is gradually limited to sinoatrial node and venous valves, while the expression of HCN4of myocardium in atria and ventricle gradually declined. We suggest that TBX3positive delineates the central conduction system in early human embryonic heart. DES can mark the early human embryonic cardiac conduction system including both the central conduction system and the peripheral conduction system. Expression patterns of HCN4in human embryonic heart provide morphological proof for transfer path of dominant pacemaker found in electrophysilolgical study. The negative expression of Nkx2.5may ensure that the sinoatrial node was normally formed on the right side of venous pole. The regions in the adult heart that are more prone to arrhythmia may have specific embryonic origin.