EXPRESSION OF CONNEXIN 40 AND HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATEDCATION CHANNEL 4 IN RAT BONE MARROW MESENCHYMAL STEM CELLS COCULTURED WITHSINOATRIAL NODE TISSUES IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

SONG Bo ^1# , LIAO Bin ¹ , YU Fengxu ¹ , XIA Zengliang. ²

1Department of Cardiothoracic Surgery, the Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan, 646000, P.R.China;;
2West China Molecular GeneticsLaboratory, Sichuan University.;

Corresponding author：

Keywords：

Bone marrow mesenchymal stem cells, Sinoatrial node cells, Coculture Connexin 40, Hyperpolarization-activated cycl ic nucleotide-gated cation channel 4 ,Rat

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

【Abstract】 Objective To investigate the expression of connexin 40 (Cx40) and hyperpolarization-activated cycl ic
nucleotide-gated cation channel 4 (HCN4) in rat bone marrow mesenchymal stem cells (BMSCs) cocultured with the sinoatrial
node (SAN) tissues in vitro, so as to evaluate the possibil ity of BMSCs differentiation into SAN cells. Methods BMSCs were
isolated from Sprague Dawley rats (aged 4-6 weeks, male or female) by the adhesive method and cultured; BMSCs at the 3rd
passage were marked with carboxyfluorescein succinimidyl ester, and then were incubated on 6-well culture plate; cell climing
sl ices were prepared at the same time. SAN tissue was taken and cut into 0.3 cm × 0.3 cm mass, and then placed into 4℃ PBS
solution. The SAN tissue mass was cocultured with marked BMSCs at the 3rd passage for 3 weeks as the experimental group, and
BMSCs at 3rd passage were cultured alone for 1 week as the control group. At 1, 2, and 3 weeks after coculture, the mean integrated
absorbance (MIA) values of Cx40 and HCN4 were measured by Image pro plus 5.0 through the method of immunohistochemistry,
and the mRNA expressions of Cx40 and HCN4 were identified by real-time fluorescent quantitative PCR. Results The
MIA values of Cx40 and HCN4 in the experimental group were higher than that in the control group, showing significant
differences (P lt; 0.01). In the experimental group, the expressions of Cx40 and HCN4 increased gradually with time. The longer
the culture time was, the higher the expressions of Cx40 and HCN4 were, showing significant differences (P lt; 0.05). The mRNA
expressions of Cx40 and HCN4 in the experimental group were significantly higher than those in the control group (P lt; 0.01); in
the experimental group, the mRNA expressions of Cx40 and HCN4 increased gradually with time, showing significant differences
between different time points (P lt; 0.05). Conclusion The expressions of Cx40 and HCN4 increase obviously after coculturing
BMSCs with SAN tissue, indicating that BMSCs could differentiate into SAN cells by coculturing with SAN tissue in vitro.

Citation： SONG Bo,LIAO Bin,YU Fengxu,XIA Zengliang.. EXPRESSION OF CONNEXIN 40 AND HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATEDCATION CHANNEL 4 IN RAT BONE MARROW MESENCHYMAL STEM CELLS COCULTURED WITHSINOATRIAL NODE TISSUES IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(2): 146-151. doi: Copy

1.	Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A, 2003, 100(25): 15235-15240.
2.	Kwong KF, Schuessler RB, Green KG, et al. Differential expression of gap junction proteins in the canine sinus node. Circ Res, 1998, 82(5): 604-612.
3.	李枫. 图像分析中光密度参数物理意义的正确理解和使用. 解剖学杂志, 2009, 32(2): 271-274.
4.	Moreno AP. Connexin phosphorylation as a regulatory event linked to channel gating. Biochim Biophys Acta, 2005, 1711(2): 164-171.
5.	Parthasarathi K, Ichimura H, Monma E, et al. Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries. J Clin Invest, 2006, 116(8): 2193-2200.
6.	Vozzi C, Dupont E, Coppen SR, et al. Chamber-related differences in connexin expression in the human heart. J Mol Cell Cardiol, 1999, 31(5): 991-1003.
7.	Verheule S, van Kempen MJ, Postma S, et al. Gap junctions in the rabbit sinoatrial node. Am J Physiol Heart Circ Physiol, 2001, 280(5): H2103-2115.
8.	Beblo DA, Wang HZ, Beyerr EC, et al. Unique conductance, gating, and selective permeability properties of gap junction channels formed by connexin40. Circ Res, 1995, 77(4): 813-822.
9.	宋慧芳, 夏仲年, 高雨仁, 等. 正常成人窦房结形态学及Cx43、Cx40的表达. 山西医科大学学报, 2008, 39(2): 97-101.
10.	Verheule S, van Batenburg CA, Coenjaerts FE, et al. Cardiac conduction abnormalities in mice lacking gap junction protein Connexin40. J Cardiovasc Electrophyisiol, 1990, 10(10): 1380-1389.
11.	Olgin JE, Verheule S. Transgenic and knockout mouse models of artrial arrhythmias. Cardiovasc Res, 2002, 54(2): 280-286.
12.	Bader A, Schilling T, Teebken OE, et al. Tissue engineering of heart valves-human endothelial cell seeding of detergent acellularized porcine valves. Eur J Cardiothorac Surg, 1998, 14(3): 279-284.
13.	朱妙章, 袁文俊, 吴博威, 等. 心血管生理学与临床. 西安: 高等教育出版社, 2004: 24-25.
14.	富冀枫, 胡优敏, 张照, 等. 心肌缺血时腺苷对绵羊浦肯野纤维If电流的影响. 中国应用生理学杂志, 2000, 16(2): 129-132.
15.	萧永福, Chandler N, Dobrzynski H, et al. Hysteresis in human HCN4 channels: A crucial feature potentially affecting sinoatrial node pacemaking. 生理学报, 2010, 62(1): 1-13.
16.	Yeh YH, Burstein B, Qi XY, et al. Funny current downregulation and sinus node dysfunction associated with atrial tachyarrhythmia: a molecular basis for tachycardia-bradycardia syndrome. Circulation, 2009, 119(12): 1576-1585.
17.	Nadal-Ginard B, Kajstura J, Leri A, et al. Myocyte death, growth, and regeneration in cardiac hypertrophy and failure. Circ Res, 2003, 92(2): 139-150.
18.	Mezey E, Key S, Vogelsang G, et al. Transplanted bone marrow generates new neurons in human brains. Proc Natl Acad Sci U S A, 2003, 100(3): 1364-1369.
19.	Tsuchiya H, Kitoh H, Sugiura F, et al. Chondrogenesis enhanced by overexpression of sox9 gene in mouse bron marrow-derived mesenchymal stem cells. Biochem Biophys Res Commun, 2003, 301(2): 338-343.
20.	杨玉霞, 郑健樑, 张平, 等. 密度梯度离心结合贴壁法培养成年大鼠骨髓间充质干细胞的生物学特性. 中国组织工程研究与临床康复, 2007, 11(3): 583-586.
21.	魏欣, 陈连凤, 刘博江, 等. 大鼠骨髓间充质干细胞体外诱导分化为具有起搏功能的心肌细胞. 基础医学与临床, 2008, 28(10): 1060-1065.
22.	李艳菊, 邓兰, 黄睿, 等. 荧光染料CFSE标记大鼠间充质干细胞的体外研究. 南方医科大学学报, 2009, 29(1): 148-150.

1. Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A, 2003, 100(25): 15235-15240.
2. Kwong KF, Schuessler RB, Green KG, et al. Differential expression of gap junction proteins in the canine sinus node. Circ Res, 1998, 82(5): 604-612.
3. 李枫. 图像分析中光密度参数物理意义的正确理解和使用. 解剖学杂志, 2009, 32(2): 271-274.
4. Moreno AP. Connexin phosphorylation as a regulatory event linked to channel gating. Biochim Biophys Acta, 2005, 1711(2): 164-171.
5. Parthasarathi K, Ichimura H, Monma E, et al. Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries. J Clin Invest, 2006, 116(8): 2193-2200.
6. Vozzi C, Dupont E, Coppen SR, et al. Chamber-related differences in connexin expression in the human heart. J Mol Cell Cardiol, 1999, 31(5): 991-1003.
7. Verheule S, van Kempen MJ, Postma S, et al. Gap junctions in the rabbit sinoatrial node. Am J Physiol Heart Circ Physiol, 2001, 280(5): H2103-2115.
8. Beblo DA, Wang HZ, Beyerr EC, et al. Unique conductance, gating, and selective permeability properties of gap junction channels formed by connexin40. Circ Res, 1995, 77(4): 813-822.
9. 宋慧芳, 夏仲年, 高雨仁, 等. 正常成人窦房结形态学及Cx43、Cx40的表达. 山西医科大学学报, 2008, 39(2): 97-101.
10. Verheule S, van Batenburg CA, Coenjaerts FE, et al. Cardiac conduction abnormalities in mice lacking gap junction protein Connexin40. J Cardiovasc Electrophyisiol, 1990, 10(10): 1380-1389.
11. Olgin JE, Verheule S. Transgenic and knockout mouse models of artrial arrhythmias. Cardiovasc Res, 2002, 54(2): 280-286.
12. Bader A, Schilling T, Teebken OE, et al. Tissue engineering of heart valves-human endothelial cell seeding of detergent acellularized porcine valves. Eur J Cardiothorac Surg, 1998, 14(3): 279-284.
13. 朱妙章, 袁文俊, 吴博威, 等. 心血管生理学与临床. 西安: 高等教育出版社, 2004: 24-25.
14. 富冀枫, 胡优敏, 张照, 等. 心肌缺血时腺苷对绵羊浦肯野纤维If电流的影响. 中国应用生理学杂志, 2000, 16(2): 129-132.
15. 萧永福, Chandler N, Dobrzynski H, et al. Hysteresis in human HCN4 channels: A crucial feature potentially affecting sinoatrial node pacemaking. 生理学报, 2010, 62(1): 1-13.
16. Yeh YH, Burstein B, Qi XY, et al. Funny current downregulation and sinus node dysfunction associated with atrial tachyarrhythmia: a molecular basis for tachycardia-bradycardia syndrome. Circulation, 2009, 119(12): 1576-1585.
17. Nadal-Ginard B, Kajstura J, Leri A, et al. Myocyte death, growth, and regeneration in cardiac hypertrophy and failure. Circ Res, 2003, 92(2): 139-150.
18. Mezey E, Key S, Vogelsang G, et al. Transplanted bone marrow generates new neurons in human brains. Proc Natl Acad Sci U S A, 2003, 100(3): 1364-1369.
19. Tsuchiya H, Kitoh H, Sugiura F, et al. Chondrogenesis enhanced by overexpression of sox9 gene in mouse bron marrow-derived mesenchymal stem cells. Biochem Biophys Res Commun, 2003, 301(2): 338-343.
20. 杨玉霞, 郑健樑, 张平, 等. 密度梯度离心结合贴壁法培养成年大鼠骨髓间充质干细胞的生物学特性. 中国组织工程研究与临床康复, 2007, 11(3): 583-586.
21. 魏欣, 陈连凤, 刘博江, 等. 大鼠骨髓间充质干细胞体外诱导分化为具有起搏功能的心肌细胞. 基础医学与临床, 2008, 28(10): 1060-1065.
22. 李艳菊, 邓兰, 黄睿, 等. 荧光染料CFSE标记大鼠间充质干细胞的体外研究. 南方医科大学学报, 2009, 29(1): 148-150.

Chinese Journal of Reparative and Reconstructive Surgery

EXPRESSION OF CONNEXIN 40 AND HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATEDCATION CHANNEL 4 IN RAT BONE MARROW MESENCHYMAL STEM CELLS COCULTURED WITHSINOATRIAL NODE TISSUES IN VITRO

Abstract Full text Figures/Tables Video References Cited by

Format

Content