Differences in genes expression associated with rat cardiac fibroblasts differentiation into cardiomyocyte-like cells induced by 5-azacytidine

CHANG Yu-Qiao; JIA Yang-Yang; HAN Bei; GUO Zhi-Kun

doi:10.16098/j.issn.0529-1356.2020.06.009

PDF(6721 KB)

Acta Anatomica Sinica ›› 2020, Vol. 51 ›› Issue (6) : 861-867. DOI: 10.16098/j.issn.0529-1356.2020.06.009

Differences in genes expression associated with rat cardiac fibroblasts differentiation into cardiomyocyte-like cells induced by 5-azacytidine

CHANG Yu-qiao JIA Yang-yang HAN Bei GUO Zhi-kun*

Author information +

History +

Abstract

Objective To explore the differences in genes expression associated with rat cardiac fibroblasts(CFs) differentiation into cardiomyocyte-like cells induced by 5-azacytidine(5-aza) at 5, 7, 14, 21 and 28 days. Methods CFs were isolated from twelve 1-3-day neonatal SD rats by using combined type Ⅰ collagenase and trypsin digestion method . Cell purity was determined by CFs molecular marker discoidin domain receptor 2(DDR2) immunofluorescence assay. Myocardial induction solution containing 15 μmol/L 5-aza was incubated with passage 3 of CFs. The expression differences of related genes including vimentin, DDR2, Nanog, sox2, c-kit, sca-1, Tbx5, CD73, CD34, cMyc, klf4, Gata4, Oct4, Mef2c and cardiac troponin T(cTnT) were detected by Real-time PCR at different induction time. Cell immunofluorescence for cTnT was observed at day 5, 7, 14, 21 and 28 after myocardial induction. Morphological ultrastructure of induced CFs at day 28 was observed by transmission electron microscopy. Results The cell growth rate was slowed down at 3 days after 5-aza induction, and the morphology of some cells were transformed from triangular, spindle shaped to round and rod shaped. Compared with the CFs before induction, induced after 7, 14, 21 and 28 days, DDR2 had stable expression, while vimentin expression decreased at day 14. But Nanog, c-kit, sox2 and Tbx5 declined accompanied by myocardial process, while CD73, Mef2c, CD34, Gata4, Oct4 and cTnT gradually increased, sca-1 rose at 7 days and then falled, cMyc and klf4 increased at 14 days and declined. At day 5, there were a few cTnT positive cells and increased significantly at day 14-21. cTnT positive cells were stable at day 28 and positive cells had no obviously change cultured from 35 days to 40 days. The cytoplasm appeared in a large amount of collagen fiber bundle, arranged regularly and rich mitochondria under transmission electron microscope. It showed end to end connection between induced fibroblasts. There were a large number of myofibril s in the cytoplasm. However, sarcomere was not obvious, and the H band, Z line and M line were not clear. Conclusion CFs can differentiate into cardiomyocyte-like cells but do not form mature cardiomyocytes induced by 5-aza.

Key words

5-azacitidine / Cardiac fibroblast / Cardiomyocyte-like cell / Gene expression / Real-time PCR / Rat

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

CHANG Yu-qiao JIA Yang-yang HAN Bei GUO Zhi-kun. Differences in genes expression associated with rat cardiac fibroblasts differentiation into cardiomyocyte-like cells induced by 5-azacytidine[J]. Acta Anatomica Sinica. 2020, 51(6): 861-867 https://doi.org/10.16098/j.issn.0529-1356.2020.06.009

References

［1］ Díaz-Araya G, Vivar R, Humeres C, et al. Cardiac fibroblasts as sentinel cells in cardiac tissue: receptors, signaling pathways and cellular functions［J］. Pharmacol Res, 2015, 101:30-40.

［2］ Qian Q, Qian H, Zhang X, et al. 5-Azacytidine induces cardiac differentiation of human umbilical cord-derived mesenchymal stem cells by activating extracellular regulated kinase［J］. Stem Cells Dev, 2012, 21(1):67-75.

［3］ Chandrakanthan Ⅴ, Yeola A, Kwan JC, et al. PDGF-AB and 5-azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells［J］. Proc Natl Acad Sci USA, 2016, 113(16):E2306-E2315.

［4］ Luo H, Li Q, Pramanik J, et al. Nanog expression in heart tissures induced by acute myocardial infarction［J］. Histo Histopathol, 2014, 29 (10):1287-1293

［5］ Chang Y, Guo K, Li Q, et al. Multiple directional differentiation difference of neonatal rat fibroblasts from six organs［J］. Cell Physiol Biochem, 2016, 39(1):157-171.

［6］ Chang YQ, Li CX, Jia YY, et al. Multiple phenotypes in cardiac fibroblasts［J］.Acta Anatomica Sinica, 2018, 49(3):317-323. (in Chinese)

常玉巧, 李辞霞, 贾阳阳, 等. 心脏成纤维细胞生物表型多样性［J］. 解剖学报, 2018, 49(3):317-323.

［7］ Darby IA, Laverdet B, Bonté F, et al. Fibroblasts and myofibroblasts in wound healing［J］. Clin Cosmet Investig Dermatol, 2014, 7:301-311.

［8］ Ieda M, Fu JD, Delgado-Olguin P, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors［J］. Cell, 2010, 142(3):375-386.

［9］ Song K, Nam YJ, Luo X, et al. Heart repair by reprogramming non-myocytes with cardiac transcription factors［J］. Nature, 2012, 485(7400):599-604.

［10］ Jayawardena TM, Egemnazarov B, Finch EA, et al. MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes［J］. Circ Res, 2012, 110(11):1465-1473.

［11］ Nam YJ, Song K, Luo X, et al. Reprogramming of human fibroblasts toward a cardiac fate［J］. Proc Natl Acad Sci USA, 2013, 110(14):5588-5593.

［12］ Fu Y, Huang C, Xu X, et al. Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails［J］. Cell Res, 2015, 25(9):1013-1024.

［13］ Deng F, Lei H, Hu Y, et al. Combination of retinoic acid, dimethyl sulfoxide and 5-azacytidine promotes cardiac differentiation of human fetal liver-derived mesenchymal stem cells［J］. Cell Tissue Bank, 2016, 17(1):147-159.

［14］ Qian Q, Qian H, Zhang X, et al. 5-Azacytidine induces cardiac differentiation of human umbilical cord-derived mesenchymal stem cells by activating extracellular regulated kinase［J］. Stem Cells Dev, 2012, 21(1):67-75.

［15］ Qiao C, Xu W, Zhu W, et al. Human mesenchymal stem cells isolated from the umbilical cord［J］. Cell Biol Int, 2008, 32(1):8-15.

［16］ Pennarossa G, Maffei S, Campagnol M, et al. Reprogramming of pig dermal fibroblast into insulin secreting cells by a brief exposure to 5-aza-cytidine［J］. Stem Cell Rev, 2014, 10(1):31-43.

［17］ Tang ChCh, Ma GSh, Huang J, et al. Repair of infracted myocardium with implantation of 5-azacytidine-induced ventricular fibroblasts［J］. Jiangsu Medical Journal, 2009, 35(6):680-682. (in Chinese)

汤成春, 马根山, 黄峻, 等. 5-氮胞苷诱导的心脏成纤维细胞移植修复心肌损伤［J］. 江苏医药, 2009, 35(6):680-682.

［18］ Lavelle D, Saunthararajah Y, Desimone J. DNA methylation and mechanism of action of 5-azacytidine［J］. Blood, 2008, 111(4):2485.

［19］ Silva J, Nichols J, Theunissen TW, et al. Nanog is the gateway to the pluripotent ground state［J］. Cell, 2009, 138(4): 722-737.

［20］ Chambers Ⅰ, Silva J, Colby D, et al. Nanog safeguards pluripotency and mediates germline development［J］. Nature, 2007, 450(7173):123-1234.

［21］ Pierantozzi E, Gava B, Manini Ⅰ, et al. Pluripotency regulators in human mesenchymal stem cells: expression of NANOG but not of OCT-4 and SOX-2［J］. Stem Cells Dev, 2011, 20(5):915-923.

［22］ Synnestvedt K, Furuta GT, Comerford KM, et al. Ecto-5’- nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia［J］.J Clin Invest, 2002, 110(7):993-1002.

［23］ Beavis PA, Stagg J, Darcy PK, et al. CD73: a potent suppressor of antitumor immune responses［J］. Trends Immunol, 2012, 33(5):231-237.

［24］ Wang L, Tang S, Wang Y, et al. Ecto-5’-nucleotidase (CD73) promotes tumor angiogenesis［J］. Clin Exp Metastasis, 2013, 30(5):671-680.

［25］ Li Q, Qi LJ, Guo ZK, et al. CD73+ adipos-derived mesenchymal stem cells possess higher potential to differentiate into cardiomyocytes in vitro［J］. J Mol Histol, 2013, 44(4):411-422.