The Experimental Research On Constructing A Biological Cardiac Pacemaker By Pacemaker Cells Autotransplantation

Biological cardiac pacemaker(BCP) is an evolution direction of cardiac pacing in future.The theoretical basis of BCP is to transfer the pacemaker gene into a non-pacemaker cell so as to obtain an artificial pacemaker cell,which can be transplanted into the heart to pace it and increase heart rate.The success of BCP study will not only avoid the defects of electronic pacemaker,but also be responsible to neural regulation.This study is based on the long-term research of mesenchymal stem cells(MSCs) of our department and combined with the new progress of BCP nowadays.We will explore the following aspects of BCP in this study.1.To clarify the possibility of biopacing heart by cell transplantation,explore the therapy effect for bradyarrhythmias,provide the techniques and test methods for continuing research by employing the sinus node cells(SNCs) to autograft into ventricle myocardium.2.To construct autologous pacemaker cells with physiological pacemaker function.3.To investigate the effect of autologous pacemaker cell transplantation by implanting autologous pacemaker cells into the ventricle myocardium with complete heart block and evaluating this entirely new method of constructing a biological pacemaker.Methods1.The SNCs were isolated by enzyme digestion after sinus node was dissected. The pacemaker current and action potential were recorded by patch clamp study.2.SNCs solution(1ml) was injected back into the marked site beneath the epicardium of fight ventricle.3.The His bundle or AV node was ablated to induce complete atrioventricular block in two weeks after cells were implanted.The effect of cell transplantation was evaluated by clinical electrophysiological study,isolation of the injection region, histology study and immunochemistry methods. 4.hHCN2 gene was introduced into MSCs by constructing Ad.hHCN2.Patch clamp was performed to evaluate the electrophysiological function after the gene was expressed in MSCs,and to optimize the viral transfection methods of constructing pacemaker cells with physiological pacemaker function.5.The animal models with complete atrioventricular block were established.The autologous pacemaker cells were implanted back in the ventricle myocardium to evaluate the pacemaker function by ECG,Holter,histology study and immunochemistry methods.The viability of this biological pacemaker rhythm was also tested with the administration of epinephrine.Results1.The SNCs were kept in good structures and functions after isolation.These cells showed a typical pacemaker current and kept beating in vitro.2.The SNCs can pace the heart effectively after autotransplantation.This rhythm showed a good reaction to isoprenaline.3.It can be testified that the SNCs survived and gap junction formed between SNCs and myocytes in injection region by isolation of the myocardium of injection site.4.The transfection rate reached 70%and the MSCs were kept in good condition after the introduction of Ad.hHCN2.Patch clamp study showed a pacemaker current with hyperpolarization activated,time depended feature.The coculture of Ad.hHCN2-MSCs and neonatal rat myocytes showed the beating rate of myocytes was increased to a higher level,which provided the proof of the pacemaker function of this kind of MSCs in vitro.5.The surface ECG showed that the biological pacemaker rhythm turned out from the injection region since the second week after autotransplantation.Overdriving test showed the excitation of experiment group was higher than control group. Holter results implied that there were no severe arrhythmias in the second week after transplantation.The average heart rate,total beats,biopacing beats and biopacing rate of experiment group were higher than control group.This biological pacemaker rhythm also showed an viability to the administration of epinephrine.Immunochemistry study showed that the MSCs survived in injection region and expressed the hHCN2 channel protein.Conclusion1.The SNCs can be isolated via enzyme digestion in vitro with structure and function in tact.They can pace the heart after being autotransplanted and responsible to neural regulation.2.Ad.hHCN2 can be introduced into MSCs cultured in vitro and increased the beating rate of cocultured neonatal rat myocytes in vitro.Pacemaker current had been recorded from Ad.hHCN2-MSCs by patch clamp study.3.The MSCs transfected by Ad.hHCN2 can pace the ventricle in vivo and increase the heart rate effectively as a biological pacemaker after being autotransplanted. This biopacing rhythm has a normal viability to the neural regulation.4.Although the ventricle rate after complete heart block could be improved by autologous biological cardiac pacemaker,the rate is still lower than normal sinus rate.For further exploration,modification and optimization should be implemented to fully simulate the sinus rate.