| Objective:To study properties of ionic currents in undifferentiated neonatal rat mesenchymal stem cells from bone marrow (MSCs) and pacemaker current gene expression of MSCs.To study the electrophysiological properties of the hHCN2 channel in the transfected-hHCN2 cells after Plasmid hHCN2/pcDNA3 was transfected into MSCs. To study the spontenous beat change in neonatal rat ventricular myocytes after ventricular myocytes cocultured with neonatal rat MSCs transfected plasmid hHCN2/pcDNA3.Methods:1. The neonatal rat MSCs were obtained from bone marrow and cultured. Neonatal rat MSCs were characterized by flow cytometry.2. Pacemaker current gene expression of MSCs were studied by real-time quantitative polymerase chain reaction(Real-time PCR).3. The current characters of undifferentiated neonatal rat MSCs were studied through the whole-cell patch clamp.4. The Plasmid hHCN2/pcDNA3 was transfected into the neonatal rat MSCs with Lipofectin. Real-time PCR and Western blot were used to detect the expression of the mRNA and protein of hHCN2 channel.The ionic currents of transfected hHCN2 (IhHCN2) were recorded and the current characters were studied through the whole-cell patch clamp techniques.5. Fresh ventricular myocytes were enzymatically isolated from 1～3 days rat. Cadiomyocytes could be indentified through the methods of light-electricity microscopy, transmission electron microscopy and immunocytochemistry.The coculture of rat cadiomyocytes and MSCs transfected with hHCN2.Observe the spontenous beating rate change of cadiomyocytes after 1,2,3 days of the coculture. Results:1. Neonatal rat MSCs were negative for markers for hematopoietic cells(CD45, 2.0±0.5%) and markers for T cells,B cells , macrophages and thymocytes(CD44, 0.6±0.3 %). Neonatal rat MSCs were dimly positive for CD31(3.9±1.0)%, a marker for endothelial cells,platelets and leucocytes. However, MSCs were positive for CD90 (97.5±1.5%), a marker for stem cell.2. In the result of Real-time PCR,HCN1, HCN2,HCN3 and HCN4 represented (0.23±0.01)%, (83.58±0.04)%,(0.79±0.01)%, (15.44±0.01)% of total HCN mRNA, respectively.3. It was found that multiple ion channels were present in undifferentiated neonatal rat MSCs,including an inward rectifier K+current(IKir),a noise-like Ca2+-activated K+ current(IKCa),a delayed rectifier K+ current (IKDR),a transient outward K+ current(Ito) and a TTX-sensitive Na+ current (INa.TTX);But not including a funny current(If).4. Whole-cell patch clamp recorded ionic currents of transfected hHCN2 and got current density-voltage curves.The threshold for activation of IhHCN2 was≈-75mV.IhHCN2 is blockaded by 4mmol/L CsCl or 100μmol/L ZD7288.The MSCs were highly expressed hHCN2 mRNA in the result of Real-time PCR and were expressed protein level in the Western blot.5. After 2 and 3 days of the coculture of rat cadiomyocytes and MSCs transfected with hHCN2,The spontenous beating rates significantly increased comparing with transfect pcDNA3 control(131±9 versus 73±13bpm,150±15 versus 80±15,n=12,P <0.05).Conclusion:1. In the neonatal rat MSCs, HCN1,HCN2,HCN3 and HCN4 represent (0.08±0.01)%, (77.16±0.03)%,(0.24±0.01)%,(22.53±0.02)% of total HCN mRNA, respectively. HCN2 and HCN4 represent a large amount of total HCN mRNA in the neonatal rat MSCs.2. Five types of functional ion channels are present in undifferentiated neonatal rat MSCs, including Ikir, INa.TTX,Ito,IKDR and IKCa which provides a basis for investigating how these functional ion channel regulate biological and physiological activity of MSCs in the future.3. MSCs was successfully established with transfection of plasmid pcDNA3-hHCN2 by Lipofectin and IhHCN2 was successfully recorded in whole-cell patch clamp,which provide a basis for the study of antiarrythmic gene therapy.4. The coculture of rat ventricular myocytes and MSCs transfected with hHCN2 accelerated the beating rate of ventricular myocytes, which provides a new methods for the biological pacemaker study.
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