Abstract
Background
Cardiovascular diseases remain a major cause of death globally. Cardiac cells once damaged, cannot resume the normal functioning of the heart. Bone marrow derived mesenchymal stem cells (BM-MSCs) have shown the potential to differentiate into cardiac cells. Epigenetic modifications determine cell identity during embryo development via regulation of tissue specific gene expression. The major epigenetic mechanisms that control cell fate and biological functions are DNA methylation and histone modifications. However, epigenetic modifiers alone are not sufficient to generate mature cardiac cells. Various small molecules such as ascorbic acid (AA) and salvianolic acid B (SA) are known for their cardiomyogenic potential. Therefore, this study is aimed to examine the synergistic effects of epigenetic modifiers, valproic acid (VPA) and 5-azacytidine (5-aza) with cardiomyogenic molecules, AA and SA in the cardiac differentiation of MSCs.
Methods and results
BM-MSCs were isolated, propagated, characterized, and then treated with an optimized dose of VPA or 5-aza for 24 h. MSCs were maintained in a medium containing AA and SA for 21 days. All groups were assessed for the expression of cardiac genes and proteins through q-PCR and immunocytochemistry, respectively. Results show that epigenetic modifiers VPA or 5-aza in combination with AA and SA significantly upregulate the expression of cardiac genes MEF2C, Nkx2.5, cMHC, Tbx20, and GATA-4. In addition, VPA or 5-aza pretreatment along with AA and SA enhanced the expression of the cardiac proteins connexin-43, GATA-4, cTnI, and Nkx2.5.
Conclusion
These findings suggest that epigenetic modifiers valproic acid and 5-azacytidine in combination with ascorbic acid and salvianolic acid B promote cardiac differentiation of MSCs. This pretreatment strategy can be exploited for designing future stem cell based therapeutic strategies for cardiovascular diseases.
Similar content being viewed by others
Data availability
I declare that I have no other data to share. All the information is included in the manuscript.
References
Haneef K, Habib R, Naeem N, Salim A (2021) Stem cell factor gene overexpression enhances the Fusion potential of rat bone marrow mesenchymal stem cells with cardiomyocytes. Pak J Zool 53(6):2305. https://doi.org/10.17582/journal.pjz/20200823170859
Karimian M, Nouri N, Ghasemi LV, Mohammadi AH, Behjati M (2023) Administration of stem cells against cardiovascular diseases with a focus on molecular mechanisms: current knowledge and prospects. Tissue Cell Apr 81:102030
Fuchs FD, Whelton PK (2020) High blood pressure and cardiovascular disease. Hypertension 75(2):285–292. https://doi.org/10.1161/HYPERTENSIONAHA.119.14240
Guo Y, Yu Y, Hu S, Chen Y, Shen Z (2020) The therapeutic potential of mesenchymal stem cells for cardiovascular diseases. Cell Death Dis 11(5):1–10. https://doi.org/10.1038/s41419-020-2542-9
Demurtas J, Fanelli GN, Romano SL, Solari M, Yang L, Soysal P, Veronese N (2021) Stem cells for treatment of cardiovascular diseases: an umbrella review of randomized controlled trials. Ageing Res Rev 67:101257. https://doi.org/10.1016/j.arr.2021.101257
Dias IE, Pinto PO, Barros LC, Viegas CA, Dias IR, Carvalho PP (2019) Mesenchymal stem cells therapy in companion animals: useful for immune-mediated diseases? BMC Vet Res 15(1):1–14. https://doi.org/10.1186/s12917-019-2087-2
Guo X, Bai Y, Zhang L, Zhang B, Zagidullin N, Carvalho K, Cai B (2018) Cardiomyocyte differentiation of mesenchymal stem cells from bone marrow: new regulators and its implications. Stem Cell Res Ther 9(1):1–12. https://doi.org/10.1186/s13287-018-0773-9
Szaraz P, Gratch YS, Iqbal F, Librach CL (2017) In vitro differentiation of human mesenchymal stem cells into functional cardiomyocyte-like cells. J Vis Exp 126:e55757. https://doi.org/10.3791/55757
Basu A, Tiwari VK (2021) Epigenetic reprogramming of cell identity: lessons from development for regenerative medicine. Clin Epigenetics 13(1):144. https://doi.org/10.1186/s13148-021-01131-4
Handy DE, Castro R, Loscalzo J (2011) Epigenetic modifications: basic mechanisms and role in cardiovascular disease. Circulation 123(19):2145–2156. https://doi.org/10.1161/CIRCULATIONAHA.110.956839
Aguirre-Vázquez A, Salazar-Olivo LA, Flores-Ponce X, Arriaga-Guerrero AL, Garza-Rodríguez D, Camacho-Moll ME, Velasco I, Castorena-Torres F, Dadheech N, de Bermúdez M (2021) 5-Aza-2’-Deoxycytidine and valproic acid in combination with CHIR99021 and A83-01 induce pluripotency genes expression in human adult somatic cells. Molecules 26(7):1909. https://doi.org/10.3390/molecules26071909
Naeem N, Haneef K, Kabir N, Iqbal H, Jamall S, Salim A (2013) DNA methylation inhibitors, 5-azacytidine and zebularine potentiate the transdifferentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes. Cardio vasc Ther 31(4):201–209. https://doi.org/10.1111/j.1755-5922.2012.00320.x
Rashid S, Salim A, Qazi REM, Malick TS, Haneef K (2022) Sodium Butyrate induces hepatic differentiation of mesenchymal stem cells in 3D collagen scaffolds. Appl Biochem Biotechnol 1–12. https://doi.org/10.1007/s12010-022-03941-5
Najafipour H, Bagheri-Hosseinabadi Z, Eslaminejad T, Mollaei HR (2019) The effect of sodium valproate on differentiation of human adipose-derived stem cells into cardiomyocyte-like cells in two-dimensional culture and fibrin scaffold conditions. Cell Tissue Res 378(1):127–141. https://doi.org/10.1007/s00441-019-03027-5
Vukićević V, Qin N, Balyura M, Eisenhofer G, Wong ML, Licinio J, Ehrhart-Bornstein M (2015) Valproic acid enhances neuronal differentiation of sympathoadrenal progenitor cells. Mol Psychiatry 20(8). 941szxdfsf2t354m5j55ydsrgi0-950
Rashid S, Qazi REM, Malick TS, Salim A, Khan I, IlyasA, Haneef K (2021) Effect of valproic acid on the hepatic differentiation of mesenchymal stem cells in 2D and 3D microenvironments. Mol Cell Biochem 476(2):909–919. https://doi.org/10.1007/s11010-020-03955-9
Okubo T, Fujimoto S, Hayashi D, Suzuki T, Sakaue M, Miyazaki Y, Takizawa T (2019) Valproic acid promotes mature neuronal differentiation of adipose tissue-derived stem cells through iNOS–NO–sGC signaling pathway. Nitric Oxide 93:1–5. https://doi.org/10.1016/j.niox.2019.08.008
KalantarMotamedi Y, Peymani M, Baharvand H, Nasr-Esfahani MH, Bender A (2016) Systematic selection of small molecules to promote differentiation of embryonic stem cells and experimental validation for generating cardiomyocytes. Cell Death Discov 2:16007. https://doi.org/10.1038/cddiscovery.2016.7
Minami I, Yamada K, Otsuji TG, Yamamoto T, Shen Y, Otsuka S, Kadota S, Morone N, Barve M, Asai Y, Tenkova-Heuser T, Heuser JE, Uesugi M, Aiba K, Nakatsuji N (2012) A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. Cell Rep 2(5):1448–1460. https://doi.org/10.1016/j.celrep.2012.09.015
Wang C, Luo H, Xu Y, Tao L, Chang C, Shen X (2018) Salvianolic acid B-Alleviated angiotensin II induces Cardiac Fibrosis by suppressing NF-κB pathway in Vitro. Med Sci Monit 24:7654–7664. https://doi.org/10.12659/MSM.908936
Lv Y, Gao CW, Liu B, Wang HY, Wang HP (2017) BMP-2 combined with salvianolic acid B promotes cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells. Kaohsiung J Med Sci 33(10):477–485. https://doi.org/10.1016/j.kjms.2017.06.006
Cao N, Liu Z, Chen Z, Wang J, Chen T, Zhao X, Ma Y, Qin L, Kang J, Wei B, Wang L, Jin Y, Yang HT (2012) Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells. Cell Res 22(1):219–236. https://doi.org/10.1038/cr.2011.195
Chan SSK, Chen JH, Hwang SM, Wang IJ, Li HJ, Lee RT, Hsieh PC (2009) Salvianolic acid B–vitamin C synergy in cardiac differentiation from embryonic stem cells. Biochem Biophys Res Commun 387(4):723–728
Haneef K, Lila N, Benadda S, Legrand F, Carpentier A, Chachques JC (2012) Development of bioartificial myocardium by electrostimulation of 3D collagen scaffolds seeded with stem cells. Heart Int 7(2):e14. https://doi.org/10.4081/hi.2012.e14
Kong Y, Xu R, Darabi MA, Zhong W, Luo G, Xing MM, Wu J (2016) Fast and safe fabrication of a free-standing chitosan/alginate nanomembrane to promote stem cell delivery and wound healing. Int J Nanomedicine 11:2543. https://doi.org/10.2147%2FIJN.S102861
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, ProckopDj, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317. https://doi.org/10.1080/14653240600855905
Haneef K, Ali A, Khan I, Naeem N, Jamall S, Salim A (2018) Role of interleukin-7 in fusion of rat bone marrow mesenchymal stem cells with cardiomyocytes in vitro and improvement of cardiac function in vivo. Cardiovasc Ther 36(6):e12479. https://doi.org/10.1111/1755-5922.12479
Ali SR, Ahmad W, Naeem N, Salim A, Khan I (2020) Small molecule 2′-deoxycytidine differentiates human umbilical cord-derived MSCs into cardiac progenitors in vitro and their in vivo xeno-transplantation improves cardiac function. Mol Cell Biochem 470(1):99–113. https://doi.org/10.1007/s11010-020-03750-6
Markmee R, Aungsuchawan S, Pothacharoen P, Tancharoen W, Narakornsak S, Laowanitwattana T, Pangjaidee N (2019) Effect of ascorbic acid on differentiation of human amniotic fluid mesenchymal stem cells into cardiomyocyte-like cells. Heliyon 5(7):e02018. https://doi.org/10.1016/j.heliyon.2019.e02018
Razzaq SS, Khan I, Naeem N, Salim A, Begum S, Haneef K (2022) Overexpression of GATA binding protein 4 and myocyte enhancer factor 2 C induces differentiation of mesenchymal stem cells into cardiac-like cells. World J Stem Cells 14(9):700–713. https://doi.org/10.4252/wjsc.v14.i9.700
Yilbas AE, Hamilton A, Wang Y, Mach H, Lacroix N, Davis DR, Li Q (2014) Activation of GATA4 gene expression at the early stage of cardiac specification. Front Chem 2:12. https://doi.org/10.3389/fchem.2014.00012
NakaoK,Minobe W, Roden R, Bristow MR, Leinwand LA (1997) Myosin heavy chain gene expression in human heart failure. J Clin Invest 100(9):2362–2370. https://doi.org/10.1172/JCI119776
Ang YS, Rivas RN, Ribeiro AJ, Srivas R, Rivera J, Stone NR, Srivastava D (2016) Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis. Cell 167(7):1734–1749. https://doi.org/10.1016/j.cell.2016.11.033
Rodríguez-Sinovas A, Sánchez JA, Valls-Lacalle L, Consegal M, Ferreira-González I (2021) Connexins in the heart: regulation, function and involvement in Cardiac Disease. Int J Mol Sci 22(9):4413. https://doi.org/10.3390/ijms2209441
Vaez SA, Ebrahimi-Barough S, Soleimani M, Kolivand S, Farzamfar S, AhmadiTafti SH, Azami M, Noorbakhsh F, Ai J (2018) The cardiac niche role in cardiomyocyte differentiation of rat bone marrow-derived stromal cells: comparison between static and microfluidic cell culture methods. EXCLI J 17:762–774. https://doi.org/10.17179/excli2018-153
Phiel CJ, Zhang F, Huang EY, Guenther MG, Lazar MA, Klein PS (2001) Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem 276(39):36734–36741. https://doi.org/10.1074/jbc.M101287200
Mirza A, Khan I, Qazi RE, Salim A, Husain M, Herzig JW (2022) Role of Wnt/βcatenin pathway in cardiac lineage commitment of human umbilical cord mesenchymal stem cells by zebularine and 2’-deoxycytidine. Tissue Cell 77:101850. https://doi.org/10.1016/j.tice.2022.10185
Yu Z, Zou Y, Fan J, Li C, Ma L (2016) Notch1 is associated with the differentiation of human bone marrow derived mesenchymal stem cells to cardiomyocytes. Mol Med Rep 14(6):5065–5071. https://doi.org/10.3892/mmr.2016.5862
Acknowledgements
This study was financially supported by Higher Education, Commission, Pakistan Scholarship for Ph.D. studies to Akbar N (No. 121-FBS3-003).
Funding
This study was financially supported by Higher Education, Commission, Pakistan. Scholarship for Ph.D. studies to Akbar N (No. 121-FBS3-003).
Author information
Authors and Affiliations
Contributions
NK performed the experiments, analyzed the data, and wrote part of the manuscript; HA assisted in cell culture and wrote part of the manuscript; SSR assisted in immunocytochemistry; AS assisted in in vitro studies and edited the manuscript; SU assisted in q-PCR; KH conceived the idea, designed the experiments, and finalized the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
All the authors declare that they have no conflict of interest.
Ethical approval
The current research project was approved by the institutional bioethical committee of University of Karachi (Protocol #: IBC KU-270/2022).
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Akbar, N., Anum, H., Razzaq, S.S. et al. Ascorbic acid and salvianolic acid B enhance the valproic acid and 5-azacytidinemediated cardiac differentiation of mesenchymal stem cells. Mol Biol Rep 50, 7371–7380 (2023). https://doi.org/10.1007/s11033-023-08634-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-023-08634-8