EXPERIMENTAL RESEARCH OF RECOMBINANT LENTIVIRUS MEDIATED HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CATION CHANNEL 4 GENE TRANSFECTING BONE MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YU Fengxu , FANG Yibing , LIU Xuan , LAI Qiancheng , JIA Jianbo , LIAO Bin.

Department of Cardiothoracic Surgery, the Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan, 646000, P.R.China. Corresponding author: LIAO Bin, E-mail: liaobindr@tom.com;

Corresponding author：

Keywords：

Hyperpolarization-activated cyclic nucleotide-gated cation channel 4; Bone marrow mesenchymal stem cells; Lentivirus transfection; Pacemaker current

DOI：

10.7507/1002-1892.20130331

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Objective To investigate the feasibility of recombinant lentivirus (LVs) mediated hyperpolarization- activated cyclic nucleotide-gated cation channel 4 (HCN4) gene transfecting rat bone mesenchymal stem cells (BMSCs) so as to construct the biological pacemaker cells. Methods Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein (EGFP) as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid (experimental group) and LVs-EGFP null virus liquid (control group). Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group. Results After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group (33.75 ± 0.41) was significantly higher than that of the control group (23.39 ± 0.33) (t=17.524, P=0.013). In the experimental group, the pacemaker current was recorded, and it could be blocked by CsCl, in accordance with the characteristics of pacemaker current. Conclusion The recombinant LVs mediated HCN4 gene is successfully transfected into rat BMSCs, and the expression of HCN4 protein and the pacemaker current can be detected.

Citation： YU Fengxu,FANG Yibing,LIU Xuan,LAI Qiancheng,JIA Jianbo,LIAO Bin.. EXPERIMENTAL RESEARCH OF RECOMBINANT LENTIVIRUS MEDIATED HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CATION CHANNEL 4 GENE TRANSFECTING BONE MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(12): 1512-1516. doi: 10.7507/1002-1892.20130331 Copy

1.	Senaratne J, Irwin ME, Senaratne MP. Pacemaker longevity: are we getting what we are promised? Pacing Clin Electrophysiol, 2006, 29(10): 1044-1054.
2.	Hauser RG, Hayes DL, Kallinen LM, et al. Clinical experience with pacemaker pulse generators and transvenous leads: an 8-year prospective multicenter study. Heart Rhythm, 2007, 4(2): 154-160.
3.	Yu FX, Ke JJ, Fu Y, et al. Protective effects of adenosine in rabbit sinoatrial node ischemia-reperfusion model in vivo: control of arrhythmia by hyperpolarization activated cyclic nucleotide-gated (HCN) 4 channels. Mol Biol Rep, 2011, 38(3): 1723-1731.
4.	Meng F, Wang K, Aoyama T, et al. Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology, 2012, 143(3): 765-776. e1-3.
5.	邓松柏, 佘强. 窦房结自动除极相关离子通道的研究进展. 心血管病学进展, 2008, 29(5): 736-739.
6.	Verkerk AO, van Ginneken AC, Wilders R. Pacemaker activity of the human sinoatrial node: role of the hyperpolarization-activated current, I(f). Int J Cardiol, 2009, 132(3): 318-336.
7.	Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker. Circulation, 2009, 119(12): 1562-1575.
8.	Moosmang S, Biel M, Hofmann F, et al. Diferential distribution of four hyperpolarzation activated cation channels in mouse brainl. Biol Chem, 1999, 380(7-8): 975-980.
9.	Rosen MR, Robinson RB, Brink PR, et al. The road to biological pacing. Nat Rev Cardiol, 2011, 8(11): 656-666.
10.	Roubille F, Tardif JC. New therapeutic targets in cardiology: heart failure and arrhythmia: HCN channels. Circulation, 2013, 127(19): 1986-1996.
11.	Später D, Abramczuk MK, Buac K, et al. A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat Cell Biol, 2013, 15(9): 1098-1106.
12.	Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node insights into the function of the cardiac pacemaker. Circulation, 2009, 119(12): 1562-1575.
13.	Choi KH, Park JK, Kim HS, et al. Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin. PLoS One, 2013, 8(8): e72184.
14.	Tiscornia G, Singer O, Verma IM, et al. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc, 2006, 1(1): 234-240.
15.	Lim TT, Geisen C, Hesse M, et al. Lentiviral vector mediated thymidine kinase expression in pluripotent stem cells enables removal of tumorigenic cells. PLoS One, 2013, 8(7): e70543.
16.	贾建博, 方易冰, 卞铁荣, 等. 脂质体介导hHCN2基因转染HEK293细胞的效率研究. 泸州医学院学报, 2012, 35(3): 248-250.
17.	Nong Y, Zhang C, Wei L, et al. In situ investigation of allografted mouse HCN4 gene transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices. Cytotherapy, 2013, 15(8): 905-919.
18.	El Khoury N, Mathieu S, Marger L, et al. Upregulation of the hyperpolarization activated current increases pacemaker activity of the sinoatrial node and heart rate during pregnancy in mice. Circulation, 2013, 127(20): 2009-2020.
19.	Bosman A, Sartiani L, Spinelli V, et al. Molecular and functional evidence of HCN4 and caveolin-3 interaction during cardiomyocyte differentiation from human embryonic stem cells. Stem Cells Dev, 2013, 22(11): 1717-1727.
20.	Scavone A, Capilupo D, Mazzocchi N, et al. Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells. Circ Res, 2013, 113(4): 389-398.
21.	Lu W, Yaoming N, Boli R, et al. mHCN4 genetically modified canine mesenchymal stem cells provide biological pacemaking function in complete dogs with atrioventricular block. Pacing Clin Electrophysiol, 2013, 36(9): 1138-1149.
22.	Müller M, Stockmann M, Malan D, et al. Ca2+ activated K channels-new tools to induce cardiac commitment from pluripotent stem cells in mice and men. Stem Cell Rev, 2012, 8(3): 720-740.

1. Senaratne J, Irwin ME, Senaratne MP. Pacemaker longevity: are we getting what we are promised? Pacing Clin Electrophysiol, 2006, 29(10): 1044-1054.
2. Hauser RG, Hayes DL, Kallinen LM, et al. Clinical experience with pacemaker pulse generators and transvenous leads: an 8-year prospective multicenter study. Heart Rhythm, 2007, 4(2): 154-160.
3. Yu FX, Ke JJ, Fu Y, et al. Protective effects of adenosine in rabbit sinoatrial node ischemia-reperfusion model in vivo: control of arrhythmia by hyperpolarization activated cyclic nucleotide-gated (HCN) 4 channels. Mol Biol Rep, 2011, 38(3): 1723-1731.
4. Meng F, Wang K, Aoyama T, et al. Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology, 2012, 143(3): 765-776. e1-3.
5. 邓松柏, 佘强. 窦房结自动除极相关离子通道的研究进展. 心血管病学进展, 2008, 29(5): 736-739.
6. Verkerk AO, van Ginneken AC, Wilders R. Pacemaker activity of the human sinoatrial node: role of the hyperpolarization-activated current, I(f). Int J Cardiol, 2009, 132(3): 318-336.
7. Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker. Circulation, 2009, 119(12): 1562-1575.
8. Moosmang S, Biel M, Hofmann F, et al. Diferential distribution of four hyperpolarzation activated cation channels in mouse brainl. Biol Chem, 1999, 380(7-8): 975-980.
9. Rosen MR, Robinson RB, Brink PR, et al. The road to biological pacing. Nat Rev Cardiol, 2011, 8(11): 656-666.
10. Roubille F, Tardif JC. New therapeutic targets in cardiology: heart failure and arrhythmia: HCN channels. Circulation, 2013, 127(19): 1986-1996.
11. Später D, Abramczuk MK, Buac K, et al. A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat Cell Biol, 2013, 15(9): 1098-1106.
12. Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node insights into the function of the cardiac pacemaker. Circulation, 2009, 119(12): 1562-1575.
13. Choi KH, Park JK, Kim HS, et al. Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin. PLoS One, 2013, 8(8): e72184.
14. Tiscornia G, Singer O, Verma IM, et al. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc, 2006, 1(1): 234-240.
15. Lim TT, Geisen C, Hesse M, et al. Lentiviral vector mediated thymidine kinase expression in pluripotent stem cells enables removal of tumorigenic cells. PLoS One, 2013, 8(7): e70543.
16. 贾建博, 方易冰, 卞铁荣, 等. 脂质体介导hHCN2基因转染HEK293细胞的效率研究. 泸州医学院学报, 2012, 35(3): 248-250.
17. Nong Y, Zhang C, Wei L, et al. In situ investigation of allografted mouse HCN4 gene transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices. Cytotherapy, 2013, 15(8): 905-919.
18. El Khoury N, Mathieu S, Marger L, et al. Upregulation of the hyperpolarization activated current increases pacemaker activity of the sinoatrial node and heart rate during pregnancy in mice. Circulation, 2013, 127(20): 2009-2020.
19. Bosman A, Sartiani L, Spinelli V, et al. Molecular and functional evidence of HCN4 and caveolin-3 interaction during cardiomyocyte differentiation from human embryonic stem cells. Stem Cells Dev, 2013, 22(11): 1717-1727.
20. Scavone A, Capilupo D, Mazzocchi N, et al. Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells. Circ Res, 2013, 113(4): 389-398.
21. Lu W, Yaoming N, Boli R, et al. mHCN4 genetically modified canine mesenchymal stem cells provide biological pacemaking function in complete dogs with atrioventricular block. Pacing Clin Electrophysiol, 2013, 36(9): 1138-1149.
22. Müller M, Stockmann M, Malan D, et al. Ca2+ activated K channels-new tools to induce cardiac commitment from pluripotent stem cells in mice and men. Stem Cell Rev, 2012, 8(3): 720-740.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL RESEARCH OF RECOMBINANT LENTIVIRUS MEDIATED HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED CATION CHANNEL 4 GENE TRANSFECTING BONE MESENCHYMAL STEM CELLS

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