Cardiomyogenic Gene Expression Profile of 5-Azacytidine- Treated Rat Bone Marrow Mesenchymal Stem Cells

Year 2017, Volume: 45 Issue: 3, 343 - 350, 01.09.2017

Handan Sevim Esin Akbay Özer Aylin Gürpınar Serdar Günaydın Mehmet Ali Onur

Abstract

Cell transplantation which aims to introduce healthy myogenic cells into the myocardium of the diseased heart is an important tool in myocardial regeneration therapy. The differentiation potential of bone marrow mesenchymal stem cells BM-MSCs can be key role for this purpose. The present study aimed to evaluate cardiomyocyte gene expression profile of 5-azacytidine treated rat BM-MSCs. 5-Aza treated BM-MSCs started to form cell colonies and especially these colonies stained with desmin and cardiac troponin-T. qRT-PCR results indicate that the expression level of cardiomyocyte specific genes especially α- cardiac actin, Mef-2b, GATA-4 were increased in 5 μM 5-Aza treated BM-MSCs. According ELISA results, nanogram levels of cardiac troponin-I was measured especially 5 μM 5-Aza treated cells.

Keywords

Bone marrow mesenchymal stem cells, 5- Azacytidine, cardiomyocyte differentiation, cardiac troponin

Rat Kemik İliği Mezenşimal Kök Hücrelerinde 5-Azayctidine Uygulaması Sonucu Kardiyomiyojenik Gen İfadesi Profili

Abstract

M iyokardiyal rejenerasyon için parlak bir gelecek sunan hücresel tedavi yöntemleri hastalıklı kalbi miyojenik hücrelerle tedavi etmeyi hedeflemektedir. Kemik iliği mezenkimal kök hücrelerinin Kİ-MKH farklılaşma kapasiteleri nedeniyle miyokardiyal rejenerasyon için anahtar rol oynayabileceği düşünülmektedir. Çalışmamızda in vitro koşullarda Kİ-MKH’den kimyasal indüklemeyle kardiyomiyositlerin gen ifadesi profilinin incelenmesi amaçlanmıştır. 5-Aza uygulanan Kİ-MKH’nin kardiyomiyositlere farklılaşma süresinde, öncelikle hücreler birbirine yaklaşmaya ve hücre kümeleri oluşturmaya başlamış ve özellikle bu hücre kümelerinin desmin ve cTn-T ile immunflorasan boyamalarının pozitif olduğu görülmüştür. RT-PCR sonuçlarına göre ise farklılaşan hücrelerde kardiyak spesifik genlerden özellikle α-kardiak aktin, Mef-2b ve GATA-4 gen ifadesinin artmış olduğu saptanmıştır. ELISA sonuçlarına göre, 5 µM 5-Aza uygulanan hücrelerde nanogram seviyesinde cTn-I ölçülmüştür

Keywords

Kemik iliği mezenkimal kök hücreleri, 5- Azacytidine, hücre farklılaşması, kardiyomiyosit, kardiyak troponin

References

• P.A. McCullough, Coronary artery disease, Clin. J. Am. Soc. Nephrol., 2 (2007) 611-616.
• F.J. Giordano, Oxygen, oxidative stress, hypoxia, and heart failure, J. Clin. Invest., 115 (2005) 500-508.
• R.S. Gardner, T.A. McDonagh, The investigation and treatment of chronic heart failure, Medicine, 34 (2006) 215-219.
• S.H. Choi, S.Y. Jung, S.-M. Kwon, S.H. Baek, Perspectives on stem cell therapy for cardiac regeneration, Circ. J., 76 (2012) 1307-1312.
• C.C. Lee, K.D. Kim, Stem cell microenvironment as a potential therapeutic target, Regen. Med., 7 (2012) 3-5.
• J. Wu, J. Li, N. Zhang, C. Zhang, Stem cell-based therapies in ischemic heart diseases: a focus on aspects of microcirculation and inflammation, Basic Res. Cardiol., 106 (2011) 317-324.
• S. Tomita, R.-K. Li, R.D. Weisel, D.A. Mickle, E.-J. Kim, T. Sakai, Z.-Q. Jia, Autologous transplantation of bone marrow cells improves damaged heart function, Circulation, 100 (1999) 247-256.
• Y. Tomita, S. Makino, D. Hakuno, N. Hattan, K. Kimura, S. Miyoshi, M. Murata, M. Ieda, K. Fukuda, Application of mesenchymal stem cell-derived cardiomyocytes as bio-pacemakers: current status and problems to be solved, Med. Biol. Eng. Comput., 45 (2007) 209-220.
• J.K. Christman, 5-Azacytidine and 5-aza-2’- deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy, Oncogene, 21 (2002) 5483-5495.
• T.C. Doetschman, H. Eistetter, M. Katz, W. Schmidt, R. Kemler, The in vitro development of blastocyst- derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium, Development, 87 (1985) 27-45.
• K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method, Methods, 25 (2001) 402-408.
• J.-Y. Min, Y. Yang, K.L. Converso, L. Liu, Q. Huang, J.P. Morgan, Y.-F. Xiao, Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats, J. J. Appl. Physiol., 92 (2002) 288-296.
• K. Fukuda, Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering, Artif. Organs, 25 (2001) 187-193.
• M.F. Pittenger, B.J. Martin, Mesenchymal stem cells and their potential as cardiac therapeutics, Circ. Res., 95 (2004) 9-20.
• W. Xu, X. Zhang, H. Qian, W. Zhu, X. Sun, J. Hu, H. Zhou, Y. Chen, Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro, Exp. Biol. Med., 229 (2004) 623- 631.
• R. Binato, T. de Souza Fernandez, C. Lazzarotto‐Silva, B. Du Rocher, A. Mencalha, L. Pizzatti, L. Bouzas, E. Abdelhay, Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy, Cell. Prolif., 46 (2013) 10-22.
• M. Ferrari, A. Corradi, M. Lazzaretti, M. De’Cillà, C. Losi, R. Villa, A. Lanfranchi, Adult stem cells: perspectives for therapeutic applications, Vet. Res. Commun., 31 (2007) 1-8.
• V. Santini, H.M. Kantarjian, J.-P. Issa, Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications, Ann. Intern. Med., 134 (2001) 573-586.
• R. Khan, J. Schmidt-Mende, M. Karimi, V. Gogvadze, M. Hassan, T.J. Ekström, B. Zhivotovsky, E. Hellström- Lindberg, Hypomethylation and apoptosis in 5-azacytidine–treated myeloid cells, Exp. Hematol., 36 (2008) 149-157.
• P. Morales‐Ramírez, T. Vallarino‐Kelly, V. Cruz‐Vallejo, Mechanisms of DNA breaks induction in vivo by 5‐ azacytidine: paths of micronucleus induction by azac, J. Appl. Toxicol., 28 (2008) 254-259.
• F. Creusot, G. Acs, J. Christman, Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2’-deoxycytidine, J. Biol. Chem., 257 (1982) 2041-2048.
• S. Makino, K. Fukuda, S. Miyoshi, F. Konishi, H. Kodama, J. Pan, M. Sano, T. Takahashi, S. Hori, H. Abe, Cardiomyocytes can be generated from marrow stromal cells in vitro, J. Clin. Invest., 103 (1999) 697.
• A. Pawlak, R.J. Gil, E. Walczak, P. Seweryniak, Desmin expression in human cardiomyocytes and selected clinical and echocardiographic parameters in patients with chronic heart failure, Kardiol. Pol., 67 (2009) 955-961.
• C.W. Hamm, B.U. Goldmann, C. Heeschen, G. Kreymann, J. Berger, T. Meinertz, Emergency room triage of patients with acute chest pain by means of rapid testing for cardiac troponin T or troponin I, N. Engl. J. Med., 337 (1997) 1648-1653.
• W.S. Shim, S. Jiang, P. Wong, J. Tan, Y.L. Chua, Y.S. Tan, Y.K. Sin, C.H. Lim, T. Chua, M. Teh, Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells, Biochem. Biophys. Res. Commun., 324 (2004) 481-488.
• S. Makino, K. Fukuda, S. Miyoshi, F. Konishi, H. Kodama, J. Pan, M. Sano, T. Takahashi, S. Hori, H. Abe, Cardiomyocytes can be generated from marrow stromal cells in vitro, J. Clin. Invest., 103 (1999) 697- 705.
• D. Orlic, BM stem cells and cardiac repair: where do we stand in 2004, Cytotherapy, 7 (2005) 3-15.
• K. Schwanke, S. Wunderlich, M. Reppel, M.E. Winkler, M. Matzkies, S. Groos, J. Itskovitz‐Eldor, A.R. Simon, J. Hescheler, A. Haverich, Generation and characterization of functional cardiomyocytes from rhesus monkey embryonic stem cells, Stem Cells, 24 (2006) 1423-1432.
• K.R. Boheler, J. Czyz, D. Tweedie, H.-T. Yang, S.V. Anisimov, A.M. Wobus, Differentiation of pluripotent embryonic stem cells into cardiomyocytes, Circ. Res., 91 (2002) 189-201.
• J.D. Molkentin, A.B. Firulli, B.L. Black, J.F. Martin, C.M. Hustad, N. Copeland, N. Jenkins, G. Lyons, E.N. Olson, MEF2B is a potent transactivator expressed in early myogenic lineages, Mol. Cell. Biol., 16 (1996) 3814- 3824.
• B. Ilkovski, S. Clement, C. Sewry, K.N. North, S.T. Cooper, Defining α-skeletal and α-cardiac actin expression in human heart and skeletal muscle explains the absence of cardiac involvement in ACTA1 nemaline myopathy, Neuromuscul. Disord., 15 (2005) 829-835.
• C. Feng, J. Zhu, L. Zhao, T. Lu, W. Zhang, Z. Liu, J. Tian, Suberoylanilide hydroxamic acid promotes cardiomyocyte differentiation of rat mesenchymal stem cells, Exp. Cell. Res., 315 (2009) 3044-3051.
• G. Huang, A. Wessels, B. Smith, K. Linask, J. Ewart, C. Lo, Alteration in connexin 43 gap junction gene dosage impairs conotruncal heart development, Dev. Biol., 198 (1998) 32-44.