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Ascorbic acid and salvianolic acid B enhance the valproic acid and 5-azacytidinemediated cardiac differentiation of mesenchymal stem cells

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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.

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Data availability

I declare that I have no other data to share. All the information is included in the manuscript.

References

  1. 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

    Article  CAS  Google Scholar 

  2. 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

    Article  CAS  Google Scholar 

  3. Fuchs FD, Whelton PK (2020) High blood pressure and cardiovascular disease. Hypertension 75(2):285–292. https://doi.org/10.1161/HYPERTENSIONAHA.119.14240

    Article  CAS  PubMed  Google Scholar 

  4. 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

    Article  Google Scholar 

  5. 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

    Article  CAS  PubMed  Google Scholar 

  6. 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

    Article  Google Scholar 

  7. 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

    Article  CAS  Google Scholar 

  8. 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

    Article  CAS  Google Scholar 

  9. 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

    Article  PubMed  PubMed Central  Google Scholar 

  10. 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

    Article  PubMed  PubMed Central  Google Scholar 

  11. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 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

    Article  CAS  Google Scholar 

  13. 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

  14. 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

    Article  PubMed  Google Scholar 

  15. 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

  16. 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

    Article  CAS  PubMed  Google Scholar 

  17. 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

    Article  CAS  PubMed  Google Scholar 

  18. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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

    Article  CAS  PubMed  Google Scholar 

  20. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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

    Article  PubMed  Google Scholar 

  22. 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

    Article  CAS  PubMed  Google Scholar 

  23. 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

    Article  CAS  PubMed  Google Scholar 

  24. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 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

    Article  CAS  PubMed  Google Scholar 

  27. 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

    Article  CAS  PubMed  Google Scholar 

  28. 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

    Article  CAS  PubMed  Google Scholar 

  29. 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

    Article  PubMed  PubMed Central  Google Scholar 

  30. 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

    Article  PubMed  PubMed Central  Google Scholar 

  31. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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

    Article  PubMed  Google Scholar 

  33. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 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

    Article  PubMed  PubMed Central  Google Scholar 

  35. 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

    Article  PubMed  PubMed Central  Google Scholar 

  36. 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

    Article  CAS  PubMed  Google Scholar 

  37. 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

    Article  CAS  PubMed  Google Scholar 

  38. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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).

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Authors and Affiliations

  1. Dr. Zafar H. Zaidi Center for Proteomics, University of Karachi, Karachi, 75270, Pakistan

    Nukhba Akbar, Hira Anum, Syeda Saima Razzaq & Kanwal Haneef

  2. Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan

    Asmat Salim

  3. Department of Molecular Medicine, Ziauddin University, Karachi, Pakistan

    Shumaila Usman

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  1. Nukhba Akbar
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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.

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Correspondence to Kanwal Haneef.

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The current research project was approved by the institutional bioethical committee of University of Karachi (Protocol #: IBC KU-270/2022).

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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

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