Construction Of Pacemaker Cells By Transducing Rat Mesenchymal Stem Cells With The Mouse HCN4 Gene And Cx45 Gene

Objective: To construct biological pacemaker cells by transducing rat mesenchymal stem cells(r MSC) with two mouse genes, hyperpolarization-activated cyclic nucleotide-gated channel 4(m HCN4) and connexin 45(m Cx45).Methods 1. Query the gene sequence of m HCN4 and m Cx45 from Gene Bank, chemically synthesize the c DNAs and amplify by PCR, then homologously recombine the c DNAs into the Eco RⅠ sites of the p Lenti-CMV-EGFP/RFP vectors to produce the plasmids of p Lenti-CMV-m HCN4-RFP and p Lenti-CMV-m Cx45-EGFP. Introduc the four plasmid system into 293 T cells to package lentivirus, and the titer of the lentivirus was determinated by the real-time PCR assay.2. The mesenchymal stem cells were isolated from SD rats by flushing the bone marrow cavity. The neonatal rat ventricular myocytes were isolated by trypsin digestion, and the fibroblasts were removed by differential adhension. The beating rates of the NRVM were observed daily.3. The experimental group r MSC were transfected by the lentivirus carrying m HCN4-RFP, while the control group r MSC were transfected by the lentivirus only carrying EGFP. Fluorescence expression was observed after 72 hours by fluorescence microscopy, and the transfection efficiency of the lentivirus was detected by flow cytometry. The funny current(If) of the positive transfected cells were recorded by the voltage-clamp. The experimental group(m HCN4-r MSC) and the control group(null-r MSC) were co-cultured with NRVM, and the beating rates of the NRVM in each group(n=6) were observed every two days.4. The mHCN4-rMSCs were transfected by the lentivirus carrying mCx45-EGFP. The r MSCs co-expressing m HCN4 and m Cx45 were defined as the experimental group, while the control group was m HCN4-r MSC. Fluorescence expression was observed after 72 hours by fluorescence microscopy, and the transfection efficiency of the lentivirus was detected by flow cytometry. The experimental group(m HCN4-m Cx45-r MSC) and the control group(m HCN4-r MSC) were co-cultured with NRVM, and the beating rates of the NRVM in each group(n=6) were observed every two days.Results1. The c DNA of m HCN4(m Cx45) was successfully cloned into the plasmid of p Lenti-CMV-RFP(p Lenti-CMV-EGFP). The lentivirus carrying m HCN4(m Cx45) was successfully packaged by four plasmid system. The titers of lentivirus liquid were 2.2x108TU/ml(m HCN4) and 1.93x108TU/ml(m Cx45). The titer of the control lentivirus only carrying EGFP was known, 1.89x109TU/ml.2. The third passage of the cultured rat marrow mesenchymal stem cells was highly homogeneous and the purity was up to 90%. The living rate of neonatal rat ventricular myocytes was about 80% after digestion. After culturing 3 days, about 80% of the myocytes beated spontaneously at the rate of about 60 beats/min. After culturing 10 days, the beating rate started to slow down, and the myocytes almost stopped beating at the 14 th day.3. After 72 hours of lentivirus transfection, flaky red fluorescence was observed by fluorescence microscopy from the experimental group(m HCN4-RFP), while flaky green fluorescence was observed from the control group(null-EGFP). By flow cytometry fluorescence detection, the efficiencies of lentivirus transfection were(57.1±2.3)% in experimental group and(62.3±2.1)% in control group. A high level voltage- and time-dependent hyperpolarization-activated inward funny current(If) was recorded by voltage clamp from the experimental group, and the hyperpolarization potential for If channel activating was about-80 m V. The control group did not recorded any current. After co-culturing 4 days, the beating rate(76±4 beats/min) of NRVM in experimental group was significantly higher than the control group(62±2 beats/min).4. After 72 hours of lentivirus transfection, flaky red fluorescence and punctate green fluorescence were observed by fluorescence microscopy from the experimental group(m HCN4-RFP+m Cx45-EGFP), while the control group(m HCN4-RFP) only expressed flaky red fluorescence. By flow cytometry fluorescence detection, the efficiencies of lentivirus transfection were(58.4±1.1)% in experimental group and(57.1±2.3)% in control group. After co-culturing 4 days, the beating rate(93±4 beats/min) of NRVM in experimental group was significantly higher than the control group(76±4 beats/min).Conclusion: The rMSC transduced with the mHCN4 gene can function as a biological pacemaker cell. The m Cx45 gene co-expressing in m HCN4-r MSC can improve the pacemaking function.