Epigenetically modified cardiac mesenchymal stromal cells limit myocardial fibrosis and promote functional recovery in a model of chronic ischemic cardiomyopathy

  • Joseph B. MooreIVEmail author
  • Xian-Liang Tang
  • John Zhao
  • Annalara G. Fischer
  • Wen-Jian Wu
  • Shizuka Uchida
  • Anna M. Gumpert
  • Heather Stowers
  • Marcin Wysoczynski
  • Roberto Bolli
Original Contribution


Preclinical investigations support the concept that donor cells more oriented towards a cardiovascular phenotype favor repair. In light of this philosophy, we previously identified HDAC1 as a mediator of cardiac mesenchymal cell (CMC) cardiomyogenic lineage commitment and paracrine signaling potency in vitro—suggesting HDAC1 as a potential therapeutically exploitable target to enhance CMC cardiac reparative capacity. In the current study, we examined the effects of pharmacologic HDAC1 inhibition, using the benzamide class 1 isoform-selective HDAC inhibitor entinostat (MS-275), on CMC cardiomyogenic lineage commitment and CMC-mediated myocardial repair in vivo. Human CMCs pre-treated with entinostat or DMSO diluent control were delivered intramyocardially in an athymic nude rat model of chronic ischemic cardiomyopathy 30 days after a reperfused myocardial infarction. Indices of cardiac function were assessed by echocardiography and left ventricular (LV) Millar conductance catheterization 35 days after treatment. Compared with naïve CMCs, entinostat-treated CMCs exhibited heightened capacity for myocyte-like differentiation in vitro and superior ability to attenuate LV remodeling and systolic dysfunction in vivo. The improvement in CMC therapeutic efficacy observed with entinostat pre-treatment was not associated with enhanced donor cell engraftment, cardiomyogenesis, or vasculogenesis, but instead with more efficient inhibition of myocardial fibrosis and greater increase in myocyte size. These results suggest that HDAC inhibition enhances the reparative capacity of CMCs, likely via a paracrine mechanism that improves ventricular compliance and contraction and augments myocyte growth and function.


Cardiac mesenchymal cell therapy Histone deacetylase inhibitors Myocardial fibrosis Myocardial infarction Paracrine signaling Cardiomyogenic lineage commitment 



This work was supported by National Institutes of Health grants R01 HL141081 (JBM), P01 HL078825 (RB), and UM1 HL113530 (RB). Additional funding was provided by the University of Louisville’s School of Medicine Basic Grant Program (JBM).

Compliance with ethical standards

Conflict of interest

The authors indicate no potential conflicts of interest.

Supplementary material

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  1. 1.
    Avolio E, Meloni M, Spencer HL, Riu F, Katare R, Mangialardi G, Oikawa A, Rodriguez-Arabaolaza I, Dang ZX, Mitchell K, Reni C, Alvino VV, Rowlinson J, Livi U, Cesselli D, Angelini G, Emanueli C, Beltrami AP, Madeddu P (2015) Combined intramyocardial delivery of human pericytes and cardiac stem cells additively improves the healing of mouse infarcted hearts through stimulation of vascular and muscular repair. Circ Res 116:E81–E94. CrossRefPubMedGoogle Scholar
  2. 2.
    Behfar A, Yamada S, Crespo-Diaz R, Nesbitt JJ, Rowe LA, Perez-Terzic C, Gaussin V, Homsy C, Bartunek J, Terzic A (2010) Guided cardiopoiesis enhances therapeutic benefit of bone marrow human mesenchymal stem cells in chronic myocardial infarction. J Am Coll Cardiol 56:721–734. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Charoenviriyakul C, Takahashi Y, Morishita M, Matsumoto A, Nishikawa M, Takakura Y (2017) Cell type-specific and common characteristics of exosomes derived from mouse cell lines: yield, physicochemical properties, and pharmacokinetics. Eur J Pharm Sci 96:316–322. CrossRefPubMedGoogle Scholar
  4. 4.
    Gnecchi M, Zhang ZP, Ni AG, Dzau VJ (2008) Paracrine Mechanisms in Adult Stem Cell Signaling and Therapy. Circ Res 103:1204–1219. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Granger A, Abdullah I, Huebner F, Stout A, Wang T, Huebner T, Epstein JA, Gruber PJ (2008) Histone deacetylase inhibition reduces myocardial ischemia-reperfusion injury in mice. FASEB J 22:3549–3560. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Guo YR, Wysoczynski M, Nong YB, Tomlin A, Zhu XP, Gumpert AM, Nasr M, Muthusamy S, Li H, Book M, Khan A, Hong KU, Li QH, Bolli R (2017) Repeated doses of cardiac mesenchymal cells are therapeutically superior to a single dose in mice with old myocardial infarction. Basic Res Cardiol 112:18. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hadjal Y, Hadadeh O, Yazidi CEI, Barruet E, Binetruy B (2013) A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells. Cell Death Dis 4:e737. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Halevy O (1993) P53 Gene Is up-Regulated during Skeletal-Muscle Cell-Differentiation. Biochem Bioph Res Co 192:714–719. CrossRefGoogle Scholar
  9. 9.
    Hodgkinson CP, Bareja A, Gomez JA, Dzau VJ (2016) emerging concepts in paracrine mechanisms in regenerative cardiovascular medicine and biology. Circ Res 118:95–107. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hong KU, Guo Y, Li QH, Cao P, Al-Maqtari T, Vajravelu BN, Du J, Book MJ, Zhu X, Nong Y, Bhatnagar A, Bolli R (2014) c-kit + cardiac stem cells alleviate post-myocardial infarction left ventricular dysfunction despite poor engraftment and negligible retention in the recipient heart. PLoS One 9:e96725. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hong KU, Li QH, Guo Y, Patton NS, Moktar A, Bhatnagar A, Bolli R (2013) A highly sensitive and accurate method to quantify absolute numbers of c-kit + cardiac stem cells following transplantation in mice. Basic Res Cardiol 108:346. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D (2010) Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142:375–386. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ahn SY, Chang YS, Sung DK, Yoo HS, Sung SI, Choi SJ, Park WS, J Pharmacol Exp TherJ Am Heart Assoc (2015) Cell type-dependent variation in paracrine potency determines therapeutic efficacy against neonatal hyperoxic lung injury. Cytotherapy 17:1025–1035. CrossRefPubMedGoogle Scholar
  14. 14.
    Kanazawa H, Tseliou E, Dawkins JF, De Couto G, Gallet R, Malliaras K, Yee K, Kreke M, Valle I, Smith RR, Middleton RC, Ho CS, Dharmakumar R, Li D, Makkar RR, Fukuda K, Marban L, Marban E (2016) Durable benefits of cellular postconditioning: long-term effects of allogeneic cardiosphere-derived cells infused after reperfusion in pigs with acute myocardial infarction. J Am Heart Assoc 5:e002796. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lee TM, Lin MS, Chang NC (2007) Inhibition of histone deacetylase on ventricular remodeling in infarcted rats. Am J Physiol Heart Circ Physiol 293:H968–977. CrossRefPubMedGoogle Scholar
  16. 16.
    Litwin SE, Katz SE, Morgan JP, Douglas PS (1994) Serial echocardiographic assessment of left ventricular geometry and function after large myocardial infarction in the rat. Circulation 89:345–354CrossRefGoogle Scholar
  17. 17.
    Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, Sano M, Takahashi T, Hori S, Abe H, Hata J, Umezawa A, Ogawa S (1999) Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 103:697–705. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Martire A, Bedada FB, Uchida S, Poling J, Kruger M, Warnecke H, Richter M, Kubin T, Herold S, Braun T (2016) Mesenchymal stem cells attenuate inflammatory processes in the heart and lung via inhibition of TNF signaling. Basic Res Cardiol 111:54. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Mayourian J, Cashman TJ, Ceholski DK, Johnson BV, Sachs D, Kaji DA, Sahoo S, Hare JM, Hajjar RJ, Sobie EA, Costa KD (2017) Experimental and computational insight into human mesenchymal stem cell paracrine signaling and heterocellular coupling effects on cardiac contractility and arrhythmogenicity. Circ Res 121:411–423. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mayourian J, Ceholski DK, Gorski PA, Mathiyalagan P, Murphy JF, Salazar SI, Stillitano F, Hare JM, Sahoo S, Hajjar RJ, Costa KD (2018) Exosomal microRNA-21-5p mediates mesenchymal stem cell paracrine effects on human cardiac tissue contractility. Circ Res 122:933–944. CrossRefPubMedGoogle Scholar
  21. 21.
    Mazzaro G, Bossi G, Coen S, Sacchi A, Soddu S (1999) The role of wild-type p53 in the differentiation of primary hemopoietic and muscle cells. Oncogene 18:5831–5835. CrossRefPubMedGoogle Scholar
  22. 22.
    Moore JB 4th, Zhao J, Fischer AG, Keith MCL, Hagan D, Wysoczynski M, Bolli R (2017) Histone deacetylase 1 depletion activates human cardiac mesenchymal stromal cell proangiogenic paracrine signaling through a mechanism requiring enhanced basic fibroblast growth factor synthesis and secretion. J Am Heart Assoc 6:e006183. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Moore JB 4th, Zhao J, Keith MC, Amraotkar AR, Wysoczynski M, Hong KU, Bolli R (2016) The Epigenetic regulator HDAC1 modulates transcription of a core cardiogenic program in human cardiac mesenchymal stromal cells through a p53-dependent mechanism. Stem cells 34:2916–2929. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Moscoso I, Centeno A, Lopez E, Rodriguez-Barbosa JI, Santamarina I, Filgueira P, Sanchez MJ, Dominguez-Perles R, Penuelas-Rivas GP, Domenech N (2005) Differentiation “in vitro” of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation. Transpl P 37:481–482. CrossRefGoogle Scholar
  25. 25.
    Oyama T, Nagai T, Wada H, Naito AT, Matsuura K, Iwanaga K, Takahashi T, Goto M, Mikami Y, Yasuda N, Akazawa H, Uezumi A, Takeda S, Komuro I (2007) Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo. J Cell Biol 176:329–341. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25:1463–1465. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Qian Q, Qian H, Zhang X, Zhu W, Yan YM, Ye SQ, Peng XJ, Li W, Xu Z, Sun LY, Xu WR (2012) 5-azacytidine induces cardiac differentiation of human umbilical cord-derived mesenchymal stem cells by activating extracellular regulated kinase. Stem Cells Dev 21:67–75. CrossRefPubMedGoogle Scholar
  28. 28.
    Rossini A, Frati C, Lagrasta C, Graiani G, Scopece A, Cavalli S, Musso E, Baccarin M, Di Segni M, Fagnoni F, Germani A, Quaini E, Mayr M, Xu QB, Barbuti A, DiFrancesco D, Pompilio G, Quaini F, Gaetano C, Capogrossi MC (2011) Human cardiac and bone marrow stromal cells exhibit distinctive properties related to their origin. Cardiovasc Res 89:650–660. CrossRefPubMedGoogle Scholar
  29. 29.
    Ruau D, Ensenat-Waser R, Dinger TC, Vallabhapurapu DS, Rolletschek A, Hacker C, Hieronymus T, Wobus AM, Muller AM, Zenke M (2008) Pluripotency associated genes are reactivated by chromatin-modifying agents in neurosphere cells. Stem cells 26:920–926. CrossRefPubMedGoogle Scholar
  30. 30.
    Sanganalmath SK, Bolli R (2013) Cell therapy for heart failure a comprehensive overview of experimental and clinical studies, current challenges, and future directions. Circ Res 113:810–834. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Segura AM, Frazier OH, Buja LM (2014) Fibrosis and heart failure. Heart Fail Rev 19:173–185. CrossRefPubMedGoogle Scholar
  32. 32.
    Soddu S, Blandino G, Scardigli R, Coen S, Marchetti A, Rizzo MG, Bossi G, Cimino L, Crescenzi M, Sacchi A (1996) Interference with p53 protein inhibits hematopoietic and muscle differentiation. J Cell Biol 134:193–204. CrossRefPubMedGoogle Scholar
  33. 33.
    Stein AB, Tiwari S, Thomas P, Hunt G, Levent C, Stoddard MF, Tang XL, Bolli R, Dawn B (2007) Effects of anesthesia on echocardiographic assessment of left ventricular structure and function in rats. Basic Res Cardiol 102:28–41. CrossRefPubMedGoogle Scholar
  34. 34.
    Subramanian S, Bates SE, Wright JJ, Espinoza-Delgado I, Piekarz RL (2010) Clinical toxicities of histone deacetylase inhibitors. Pharmaceuticals 3:2751–2767. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Suzuki G, Weil BR, Leiker MM, Ribbeck AE, Young RF, Cimato TR, Canty JM (2014) global intracoronary infusion of allogeneic cardiosphere-derived cells improves ventricular function and stimulates endogenous myocyte regeneration throughout the heart in swine with hibernating myocardium. PloS one 9:e113009. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Tang XL, Li Q, Rokosh G, Sanganalmath SK, Chen N, Ou Q, Stowers H, Hunt G, Bolli R (2016) Long-term outcome of administration of c-kit(POS) cardiac progenitor cells after acute myocardial infarction: transplanted cells do not become cardiomyocytes, but structural and functional improvement and proliferation of endogenous cells persist for at least 1 year. Circ Res 118:1091–1105. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Tang XL, Rokosh G, Sanganalmath SK, Tokita Y, Keith MC, Shirk G, Stowers H, Hunt GN, Wu W, Dawn B, Bolli R (2015) Effects of intracoronary infusion of escalating doses of cardiac stem cells in rats with acute myocardial infarction. Circ Heart Fail 8:757–765. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Tang XL, Rokosh G, Sanganalmath SK, Yuan F, Sato H, Mu J, Dai S, Li C, Chen N, Peng Y, Dawn B, Hunt G, Leri A, Kajstura J, Tiwari S, Shirk G, Anversa P, Bolli R (2010) Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction. Circulation 121:293–305. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Weil BR, Suzuki G, Leiker MM, Fallavollita JA, Canty JM Jr (2015) Comparative efficacy of intracoronary allogeneic mesenchymal stem cells and cardiosphere-derived cells in swine with hibernating myocardium. Circ Res 117:634–644. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Wysoczynski M, Dassanayaka S, Zafir A, Ghafghazi S, Long BW, Noble C, DeMartino AM, Brittian KR, Bolli R, Jones SP (2016) A new method to stabilize C-Kit expression in reparative cardiac mesenchymal cells. Front Cell Dev Biol 4:78. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Wysoczynski M, Guo Y, Moore JB 4th, Muthusamy S, Li Q, Nasr M, Li H, Nong Y, Wu W, Tomlin AA, Zhu X, Hunt G, Gumpert AM, Book MJ, Khan A, Tang XL, Bolli R (2017) Myocardial reparative properties of cardiac mesenchymal cells isolated on the basis of adherence. J Am Coll Cardiol 69:1824–1838. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Xie M, Kong YL, Tan W, May H, Battiprolu PK, Pedrozo Z, Wang ZV, Morales C, Luo X, Cho G, Jiang N, Jessen ME, Warner JJ, Lavandero S, Gillette TG, Turer AT, Hill JA (2014) Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy. Circulation 129:1139–U1170. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Yang G, Tian J, Feng C, Zhao LL, Liu Z, Zhu J (2012) Trichostatin a promotes cardiomyocyte differentiation of rat mesenchymal stem cells after 5-azacytidine induction or during coculture with neonatal cardiomyocytes via a mechanism independent of histone deacetylase inhibition. Cell Transpl 21:985–996. CrossRefGoogle Scholar
  44. 44.
    Yang G, Tian J, Feng C, Zhao LL, Liu ZG, Zhu J (2012) Trichostatin A promotes cardiomyocyte differentiation of rat mesenchymal stem cells after 5-azacytidine induction or during coculture with neonatal cardiomyocytes via a mechanism independent of histone deacetylase inhibition. Cell Transpl 21:985–996. CrossRefGoogle Scholar
  45. 45.
    Yoon CH, Koyanagi M, Iekushi K, Seeger F, Urbich C, Zeiher AM, Dimmeler S (2010) Mechanism of improved cardiac function after bone marrow mononuclear cell therapy: role of cardiovascular lineage commitment. Circulation 121:2001–2011. CrossRefPubMedGoogle Scholar
  46. 46.
    Zhang L, Qin X, Zhao Y, Fast L, Zhuang S, Liu P, Cheng G, Zhao TC (2012) Inhibition of histone deacetylases preserves myocardial performance and prevents cardiac remodeling through stimulation of endogenous angiomyogenesis. J Pharmacol Exp Ther 341:285–293. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Zhang LX, Zhao Y, Cheng G, Guo TL, Chin YE, Liu PY, Zhao TC (2010) Targeted deletion of NF-kappaB p50 diminishes the cardioprotection of histone deacetylase inhibition. Am J Physiol Heart Circ Physiol 298:H2154–2163. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Zhao J, Ghafghazi S, Khan AR, Farid TA, Moore JB 4th (2016) Recent developments in stem and progenitor cell therapy for cardiac repair. Circ Res 119:e152–e159. CrossRefPubMedGoogle Scholar
  49. 49.
    Zhao Y, Lu SL, Wu LP, Chai GL, Wang HY, Chen YQ, Sun J, Yu Y, Zhou W, Zheng QH, Wu M, Otterson GA, Zhu WG (2006) Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1). Mol Cell Biol 26:2782–2790. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Joseph B. MooreIV
    • 1
    Email author
  • Xian-Liang Tang
    • 1
  • John Zhao
    • 1
  • Annalara G. Fischer
    • 1
  • Wen-Jian Wu
    • 1
  • Shizuka Uchida
    • 2
  • Anna M. Gumpert
    • 1
  • Heather Stowers
    • 1
  • Marcin Wysoczynski
    • 1
  • Roberto Bolli
    • 1
  1. 1.Institute of Molecular Cardiology, Division of Cardiovascular MedicineUniversity of LouisvilleLouisvilleUSA
  2. 2.Department of Medicine, Cardiovascular Innovation InstituteUniversity of LouisvilleLouisvilleUSA

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