| Part I: The Isolation, Culture, Identification of Sinoatrial Pacemaker Cells and Ventricular Myocyte of Neonatal rats.Objective: To isolate, culture and identify sinoatrial pacemaker cells and ventricular myocyte of neonatal rats. To record funny current(If) of sinoatrial pacemaker cells and ventricular myocyte of neonatal rats and to analyze its relative pacemaker current gene(Hyperpolarization-activated cyclic nucleotide-gated channel 4, HCN) expression by real-time quantitative polymerase chain reaction(real-time PCR).Methods: sinoatrial pacemaker cells and ventricular myocyte were enzymatically isolated from 1-3 days rat. Light electricity microscopy and Spontaneous action potential(AP) were studied for identification. If current was recorded through whole-cell patch clamp. The expression of HCN gene(m HCN1, m HCN2 and m HCN4) were measured by real-time PCR.Results: The sinoatrial pacemaker cells had higher AP rate than ventricular myocyte(174.4±14.6bpm vs. 69.7±10.1bpm, p<0.05, n=20). The sinoatrial pacemaker cells had higher automaticity than ventricular myocyte. If current were recorded in both sinoatrial pacemaker cells and ventricular myocyte of neonatal rats. The half maximal activation potential of sinoatrial pacemaker cells was-77.74 士 1.79 m V, and the half maximal activation potential of ventricular myocyte was-93.58±0.98 m V. The ratio of m HCN1:m HCN2:m HCN4 for sinoatrial pacemaker cells was(4.16±0.19):(0.03±0.19): 1,and the ratio of m HCN2: m HCN4 for ventricular myocyte was(4.62±0.23):1, m HCN1 was not detected in ventricular myocytes.Conclusion: Both ventricular myocytes and sinoatrial pacemaker cells can express If current. The major HCN subtypes of ventricular myocytes were m HCN2 and m HCN4, and the major HCN subtypes for sinoatrial pacemaker cells were m HCN4 and m HCN1. The different m HCN subtype composition may be one of the reasons for different automaticity.Part II. Lentiviral Transfection of h HCN4 Gene to Create Biological-Pacemaker Cells in VitroObjective: Mesenehymal stem cells transfected with h HCN4 via lentivirus vector to create biological pacemaker cells in vitro.Methods: The shuttle plasmid containing h HCN4 gene(pCDH1-GFP-hHCN4) was built; p CDH1-GFP-h HCN4 was packaged into lentiviral particles; Lentiviral particles containing h HCN4 was transfected into rat MSCs; Puro was used to purify MSCs which stably overexpressed h HCN4; MSCs transfected with lentiviral particles only containing GFP gene was defined as h HCN4-MSCs. h HCN4 expression in MSCs was conducted by RT-PCR, western blot and observation of GFP fluorescence under fluorescence microscope. If current was recorded by Voltage-clamp. h HCN4+MSCs and neonatal rat ventricular myocytes were cocultured to build biological pacemaker model; Where h HCN4-MSCs and myocytes cocultured was defined as h HCN4-MSCs control group; To evaluate automaticity of biological pacemaker, spontaneously beat frequency of myocytes was counted, and spontaneous action potentials of myocytes was record by current patch-clamp.Results: Restriction enzyme digestion and gene sequencing confirmed h HCN4 gene had been successfully constructed into the shuttle plasmid p CDH-GFP-h HCN4. 7 days after transfection, h HCN4 gene expression in MSCs was confirmed by fluorescence microscope, RT-PCR and western blot. High level time- and voltage-dependent inward hyperpolarization current was detected in h HCN4+MSCs which was sensitive to 4mmol/L Cs+, confirming that functional h HCN4 channel was expression in MSCs. Genetically modified MSCs Coculturing with neonatal rat ventricular myocytes(NRVM), spontaneously beating frequency in myocytes coclutured with h HCN4+MSCs was faster than in which myocytes coclutured with HCN4-MSCs(98.1±11.2bpm vs. 70.5±8.2bpm, P<0.05, n=10). Recorded action potential in myocytes demonstrated that myocytes coclutured with h HCN4+MSCs was characterized with a more regular and faster beating frequency. A lower Maximal diastolic potential(MDP)(-84±4m V vs.-76±3m V, P<0.05,n=10) than in which myocytes coclutured with h HCN4-MSCs. Addition of carbenoxolone(200 micro M), a gap junction channel blocker, lowered spontaneous activity of myocytes in biological pacemaker model.Conclusion: Biological pacemakercells were successfully created by h HCN4 transfection to mesenehymal stem cells via lentivirus vector., which can increase the spontaneous beating rate of NRVM, which were cocultured with it.Part III. Co-expression of h HCN4 and h HCN1 to Improve the Function of Biological Pacemaker CellsObjective: To study the efficacy of biological pacemaker cells when co-transfected with h HCN1 and h HCN4.Methods: The lentiviral particles containing hHCN1 and red fluorescent protein(RFP) were constructed with shuttle plasmid p CDH-RFP-h HCN1. h HCN1 was transfect to MSCs with stable expression of h HCN4, screen with puro. MSCs with co-expression of h HCN1 and h HCN4 were built. MSCs only expressing h HCN1 expression was also built. Fluorescence microscope, RT-PCR and western blot were used to identify the expression of h HCN1 and h HCN4. If current was recorded by Voltage-clamp. MSCs and neonatal rat ventricular myocytes were cocultured to build biological pacemaker model. The spontaneous AP were recorded by patch clamp. The maximal diastolic potential(MDP) and velocity of diastolic depolarization(VDD) were recorded.Results: Restriction enzyme digestion and gene sequencing confirmed h HCN1 gene had been successfully constructed into the shuttle plasmid p CDH-RFP-h HCN1. With fluorescence microscope, RT-PCR and western blot, co-expression of h HCN1 and h HCN4 were identified. If current could be recorded in h HCN4+MSCs, h HCN1+ MSCs and h HCN1+4 co-expression MSCs group. Cocultured with NRVM, the spontaneous beating rate of co-expression group(138.1±11.5bpm) was higher than that of h HCN4 group(99.1±8.7bpm) and h HCN1 group(96.3±7.5bpm)(p<0.05, n=10). The MDP of co-expression group(-69.6±3.7) was lower than that of h HCN1 group(-75.9±5.2) and h HCN4 group(-75.2±1.9). The VDD of co-expression group(55.2±8.9 m V/s) was faster than that of h HCN1 group(33.4±2.4 m V/s) and h HCN4 group(33.8±5.3 m V/s).Conclusion: Co-expression of hHCN1 and hHCN4 will elevate the automaticity of biological pacemaker cells based on h HCN1 or h HCN4 modified MSCs.Part IV. Kinetic Properties of Co-expressed h HCN1 and h HCN4 ChannelsObjective: Using whole cell patch clamp to study membrane current in the MSCs transfected with HCN genes and the electrophysiology feature of HCN channels.Methods: Whole cell patch clamp was used to study the eleetrophysiology features of membrane current in the MSCs transfected the HCN channels, such as activation curves of If current, curves of voltage dependent activation kinetics, If current density of each group and the effects of c AMP, Cs+, ivabradine on HCN channels.Results: In whole-cell voltage-dependent mode, inward current could be induced in h HCN1 group, h HCN4 group and h HCN1+4 group. The currents were time dependent without deactivation. The half maximal activation potential(V1/2) of Ih HCN1 was-92.27±0.53 m V,V1/2 of Ih HCN4 was-102.09±0.72 m V, V1/2 of Ih HCN1+4 was-90.88±0.48 m V. Ih HCN4 had more negative V1/2 than Ih HCN1和 Ih HCN1+4. When testing potential was-90 m V,the current density of Ih HCN1 was11.6±6.01 p A/p F, the current density of Ih HCN4 was-20.5±5.46 p A/p F, and the current density of Ih HCN1+4 was-59.9±8.02 p A/p F. Ih HCN1+4 had highest current density in three groups(n=10,p<0.05). When testing potential was-90 m V, the activation time for Ih HCN1 was 150±27.31 ms,for Ih HCN4 was 920±86.9ms,for Ih HCN1+4 was 140±18.02 ms. The activation time for Ih HCN4 was longer than Ih HCN1 and Ih HCN1+4(n=10,p<0.05). 100μmol/L c AMP of bath solution for 10 min did not change If of all three groups. But 100μmol/L c AMP increased Ih HCN1+4 by shifting V1/2 from-92.38±0.57 m V to-83.39±0.20 m V. With testing potential of-90 m V, 100μmol/L c AMP shortened activation time from140±18.02 ms to 84±14.42ms(n=10, p<0.05). The If current was blocked by 4mmol/L Cs+, the IC50 of Cs+ to Ih HCN1+4 was 125.8±14.9μmol/L. The block of 4mmol/L Cs+ was partially released by extracellular washout. The If current was blocked by ivabradine. The IC50 of ivabradine to Ih HCN1+4 was 1.07±0.039μmol/L. The block of 5 μmol/L ivabradine was totally released by extracellular washout.Conclusion: co-expression of h HCN1 and h HCN4 can produce If with higher current density, shorter activation time. It can be activated by intracellular c AMP and can be blocked by Cs+ and ivabradine. Co-expression of h HCN1 and h HCN4 will elevate automaticity of biological pacemaker cells. |
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