The Journal of international medical research · 2013
Genetically-engineered mesenchymal stem cells transfected with human HCN1 gene to create cardiac pacemaker cells.
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Zhou Ya-Feng, Yang Xiang-Jun, Li Hong-Xia, Han Lian-Huan, Jiang Wen-Ping
Аннотация
OBJECTIVE: To test the proof-of-principle that genetically-engineered mesenchymal stem cells (MSCs) transfected with the human hyperpolarization-activated cyclic nucleotide-gated channel 1 (hHCN1) gene can be modified to become cardiac pacemaker cells.
METHODS: MSCs were transfected with the hHCN1 gene using lentiviral-based transfection. The expressed pacemaker current (I(f)) in hHCN1-transfected MSCs was recorded using whole-cell patch-clamp analysis. The effect of the hHCN1-transfected MSCs on cardiomyocyte excitability was determined by coculturing the MSCs with neonatal rabbit ventricular myocytes (NRVM). The spontaneous action potentials of the NRVM were recorded by whole-cell current-clamp analysis.
RESULTS: A high level time- and voltage-dependent inward hyperpolarization current that was inhibited by 4 mM caesium chloride was detected in hHCN1-transfected MSCs, suggesting that the HCN1 proteins acted as I(f) channels in MSCs. The mean ± SE beating frequency in NRVMs cocultured with control MSCs transfected with the pcDNA3 plasmid control was 82 ± 8 beats/min (n = 5) compared with 129 ± 11 beats/min (n = 5) in NRVMs cocultured with hHCN1-transfected MSCs.
CONCLUSIONS: Genetically-engineered MSCs transfected with the hHCN1 gene can be modified to become cardiac pacemaker cells.
METHODS: MSCs were transfected with the hHCN1 gene using lentiviral-based transfection. The expressed pacemaker current (I(f)) in hHCN1-transfected MSCs was recorded using whole-cell patch-clamp analysis. The effect of the hHCN1-transfected MSCs on cardiomyocyte excitability was determined by coculturing the MSCs with neonatal rabbit ventricular myocytes (NRVM). The spontaneous action potentials of the NRVM were recorded by whole-cell current-clamp analysis.
RESULTS: A high level time- and voltage-dependent inward hyperpolarization current that was inhibited by 4 mM caesium chloride was detected in hHCN1-transfected MSCs, suggesting that the HCN1 proteins acted as I(f) channels in MSCs. The mean ± SE beating frequency in NRVMs cocultured with control MSCs transfected with the pcDNA3 plasmid control was 82 ± 8 beats/min (n = 5) compared with 129 ± 11 beats/min (n = 5) in NRVMs cocultured with hHCN1-transfected MSCs.
CONCLUSIONS: Genetically-engineered MSCs transfected with the hHCN1 gene can be modified to become cardiac pacemaker cells.
Предметные рубрики MeSH
Action PotentialsAnimalsAnimals, NewbornCell EngineeringCesiumChloridesCoculture TechniquesGene ExpressionGenetic VectorsHeart VentriclesHumansHyperpolarization-Activated Cyclic Nucleotide-Gated ChannelsLentivirusMesenchymal Stem CellsMyocytes, CardiacPatch-Clamp TechniquesPlasmidsPotassium ChannelsRabbitsTransfection