Hyperpolarization-activated Cyclic Nucleotide-gated Channel HCN4 Gene Lentiviral Transfection To Create Biological-Pacemaker Cells In Vitro

Background:The implantation of electronic devices has become the preferred treatment for symptomatic bradyarrhythmias with excellent success and minimal morbidity. The shortcomings of electronic pacemakers include limited battery life, need for lead implantation into heart, which in most cases can no longer be removed, and lack of response to autonomic and physiologic demands on the heart. Furthermore, cardiac pacing in the pediatric age still needs improvements, as the devices cannot follow the somatic growth of the little patients and have to be changed over the years. Nonetheless, the ideal therapy for these disorders may be the development of a biological solution allowing reconstitution of the physiological electrical activity of the cardiac conduction system with the same plasticity and adaptability to the human body and to the physiology of the cardiovascular system. At present, molecular approaches to the development of a biological pacemaker are a conceptually attractive alternate treatment modality for bradyarrhythmias. In this field, both gene and stem cell therapies represent new and promising strategies for the development of a biological pacemaker. This paper will focus on hyperpolarization-activated cyclic nucleotide-gated channel HCN4 gene lentiviral transfection to create biological-pacemaker cells in vitro.Partâ… Culture of Mesenchymal Stem Cells from Porcine Bone Marrow and Transformation to Myogenic Cell Induced by 5-azaSection ACulture of Mesenchymal Stem Cells from Swine Bone MarrowObjective: By establishing a method of the culture of swine mesenchymal stem cells (MSCs) from bone marrow, a new cell source for the cardiomyocytes induced by 5-aza. Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer.Results: The adherent, fibroblast-like cells were confluent in single layer after plating for 10～12 days. The cell cycle analysis showed that 73% of MSCs was in G₀/G₁ phase.Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherent ability. It is suggesting that MSCs may be a new cell source for the cardiomyocytes induced by 5-aza.Section BMesenchymal Stem Cells Transformed to Myogenic Cell Induced by 5-azacytidine inVitroObjective: By establishing a method for the culture of porcine mesenchymal stem cells(MSCs) from bone marrow and the transformation to myogenic cells in vitro, a new cell source to create biological-pacemaker cells in vitro.Methods: MSCs were isolated from bone marrow and purified by centrifuge. The proliferation and growth characteristics were observed in primary and passage culture. After being co-cultured with 5-azacytidine (5-aza) for 24h, the cultured cells were evaluated by immunohistochemical stains.Results: After being co-cultured with 5-aza, some of MSCs became spindle-like and were found to be stained positively with desmin, MHC, cTnI, and Cx-43.Conclusion: Porcine MSCs can be isolated from postnatal bone marrow through their adherentability. Myogenic cells can be generated from MSCs in vitro. It is suggesting that MSCs may be a new cell source to create biological-pacemaker cells .Partâ…¡Construct the Shuttle Plasmid pCDHl-GFP-HCN4 and Pack theRecombined Lentivirus Section AConstruct the Shuttle Plasmid pCDH1-GFP-HCN4Objective: HCN4 gene fragment to be unloaded from plasmid pCDNA3-hHCN4 generously provided by Dr Stieber, and to be cloned into the shuttle plasmid pCDHl-GFP-HCN4. HCN4 gene fragment to be unloaded from plasmid pCDNA3-hHCN4 generously provided by Dr Stieber, and to be cloned into the shuttle plasmid pCDH1-GFP-HCN4.Methods: HCN4 gene fragment was unloaded from plasmid pCDNA3-hHCN4 and cloned into plasmid Puc19 by Enzymes Eco RI and Xbaâ… ; then the target gene was cutted and cloned into plasmid pcDNA3.1 (A) by Enzymes Eco RI and Hindâ…¢. In the end, HCN4 gene fragment was cutted into plasmid pCDH1-MCS1 -EF1-copGFP by Enzymes Xbaâ… . Double Enzymes Xbaâ… and EcoR I verified approach and analysising the sequence were used to confirm the positive plasmid. The positive plasmid was then transfected into 293 cells and the MCSc.Results: HCN4 gene fragment was cloned into the plasmid pCDH1-MCS1 -EF1-copGFP confirmed by Enzymes Xbaâ… and EcoRI and the sequence in the positive plasmid was confirmed by comparison with the published gene bank; GFP was observed in the transfected 293 cells and the MSCs under the fluorescent microscope.Conclusion: HCN4 gene fragment was successfully unloaded from plasmid pCDNA3-hHCN4 cloned into the shuttle plasmid pCDHl-GFP-HCN4 which would be constructed lentivirus in the next step.Section BPack the Recombined LentivirusObjective: Using the pPACKH1-Lentivector Packaging Kit to construct HCN4 recombined lentivirus.Methods: HCN4 recombined lentivirus was constructed according to operating manual of Lentivector Expression System. pPACKHl- Lentivector Packaging Kit with three plasmids was used to transfect MSCs and 293 T cells. After 48 hours culturing, GFP (green) was detected by the fluorescence microscope.Results: The recombined lentivirus was used to transfect MSCs and 293 T cells and GFP was observed in the transfected 293 cells and the MSCs under the fluorescent microscope. The efficiency of infection was above 90 percents.Conclusion:The recombined lentivirus was successfully constructed and had high efficiency of infection.Partâ…¢Using an HIV-Based Lentiviral Vector to Transfect Mesenchymal Stem Cells to Create Biological-Pacemaker Cells in VitroObjective: Using HCN4 recombined lentivirus to transfect Mesenchymal stem cells to make them Stably overexpressing HCN4 to create biological-pacemaker cells in vitro.Methods: MSCs were transfected by HCN4 recombined lentivirus according to operating manual of Lentivector Expression System. After two weeks' culturing, GFP (green) was detected by the fluorescence microscope. Protein was extracted for Western blot analysis and real time RT-PCR was carried out using superscript one step RT-PCR with Promega Taq kits and Sybergreen. GAPDH was used as a housekeeping gene. Whole-cell patch clamp to study membrane currents in control MSCs and those transfected with hHCN4 and the state of transfected MSCs long-term cultured were observed.Results: After 48 hours culturing, GFP (green) was detected in transfected MSCs by the fluorescence microscope. Protein expression by hHCN4 was confirmed by comparison with the published one according Western blot analysis. The expression intensity of HCN4 in MSCs remained strong 2 weeks later with detected by real time RT-PCR. Whole-cell patch clamp recorded an inward current in response to hyperpolarization which was time-dependent and voltage-dependent. Spontaneous beating in cell group was found in the microscope and the beating rate was 15±lbpm after the transfected MSCs were cultered for 2 months.Conclusion: Biological-pacemaker cells were successfully created by HCN4 recombined lentivirus to transfect Mesenchymal stem cells, which were stably overexpressed HCN4 in vitro. Partâ…£Electrophysiological characterization of the expressed hHCN4 channel inthe MSCs transfected with lentivirisObjective: Whole-cell patch clamp to study membrane currents in the MSCs transfected with hHCN4 and the electrophysiology feature of hHCN4 channel.Methods: Whole-cell patch clamp was studied the electrophysiology feature of membrane currents in the MSCs transfected with hHCN, such as activation curves of hHCN4 current, curve of voltage dependence of activation kinetics and effect of extracellular K⁺,Na⁺,TTX,TEA,cAMP,ISO,Ach,Cs⁺,ZD7288 and amiodarone on the HCN4 currents.Results:1.In whole-cell voltage-clamp mode, hyperpolarizing voltage steps negative to -80 mV induced inward currents in hHCN4 expressing cells. It began to slowly active in voltage and time dependent manner without deactivation.The membrane potential of threshold voltage (V_th) and half-maximal activation (V_1/2) obtained by fits to Boltzmann equations was-83±8mV and-108±2mV (n=10) for the hHCN4 current. The hHCN4 activation time constants ranged from 0.66±0.1s (n = 10) at -140 mV to 3.1±0.7s (n = 10) at -110 mV. The reversal potential was -21.2±2.3mV(n=10). The relative permeability ratio for Na⁺ versus K⁺ (P_Na/P_K), as determined by the Goldmann-Hodgkin-Katz equation, was 0.23.2.At -140mV, I_hHCN4 current density decrease from -300±25pA/pF^-1 to -87±10 pA/pF^-1 with reducing the extracellular Na⁺ concentration from 110 mmol/Lto 50 mmol/L when the extracellular K⁺ concentration was 30 mmol/L and decrease from -302±30pA/pF^-1 to -244±19 pA/pF^-1 with reducing the extracellular K⁺ concentration from 30 mmol/Lto 5 mmol/L when the extracellular Na⁺ concentration was 110 mmol/L.3. Neither current was sensitive to TTX nor TEA, two classic potassium channel blockers.4. In whole-cell mode measurements, I_hHCN4 did not change in the presence of 1 mM cAMP in the bath solution for 10 minutes. But 1 mmol/L cAMP increased the hHCN4 current by shifting the activation curves 17 mV in the positive direction when the cell exposed to 1 mM cAMP intracellular solution. The V_1/2 values for the hHCN4 current were -109±2mV in the absence and -92±2mV in the presence of 1 mM cAMP in the intracellular solution for 10 minutes (n=10, p<0.05).5.Application of 1μmol/L ISO to the extracellular solution resulted in lightly accelerated I_hHCN4·And I_hHCN4 did not change in the presence of 1 umol/L Ach in the bath solution for 10 minutes.6. The I_hHCN4 was blocked by low concentrations of extracellular Cs⁺. The IC50 was 128.8±28.1μmol/L and the block was released by (extracellular) washout. The I_hHCN4 was blocked by low concentrations of extracellular ZD7288 but the block could not be released by (extracellular) washout.7. Amiodarone decreased the peak current of I_hHCN4 and could block the current. The IC50 was 1.34±0.33μmol/L and the block could not be released by (extracellular) washout. The hHCN4 activation time constants prolonged at every clamp voltage.Conclusion:The current of expressed hHCN4 channel in the MSCs transfected with lentivirus has the electrophysiological characterization of native If, that is, activation upon membrane hyperpolarization, slow activation in voltage and time dependent manner without deactivation, combine-conduction of Na⁺ and K⁺, enhancement by cAMP and blockage by Cs⁺,ZD7288 and amiodarone. This experment provides theory ground for biological-pacemaker study in treatment of arrhythmia.