Molecular and Cellular Biochemistry

, Volume 313, Issue 1–2, pp 167–178 | Cite as

Lipopolysaccharide induces cellular hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 myocardiac cells

  • Chung-Jung Liu
  • Yi-Chang Cheng
  • Kung-Wei Lee
  • Hsi-Hsien Hsu
  • Chun-Hsien Chu
  • Fuu-Jen  Tsai
  • Chang-Hai Tsai
  • Chia-Yih Chu
  • Jer-Yuh Liu
  • Wei-Wen Kuo
  • Chih-Yang Huang


Evidences suggest that lipopolysaccharide (LPS) participates in the inflammatory response in the cardiovascular system; however, it is unknown if LPS is sufficient to cause the cardiac hypertrophy. In the present study, we treated H9c2 myocardiac cells with LPS to explore whether LPS causes cardiac hypertrophy, and to identify the precise molecular and cellular mechanisms behind hypertrophic responses. Here we show that LPS challenge induces pathological hypertrophic responses such as the increase in cell size, the reorganization of actin filaments, and the upregulation of hypertrophy markers including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in H9c2 cells. LPS treatment significantly promotes the activation of GATA-4 and the nuclear translocation of NFAT-3, which act as transcription factors mediating the development of cardiac hypertrophy. After administration of inhibitors including U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor), FK506 (calcineurin inhibitor), and QNZ (NFκB inhibitor), LPS-induced hypertrophic characteristic features, such as increases in cell size, actin fibers, and levels of ANP and BNP, and the nuclear localization of NFAT-3 are markedly inhibited only by calcineurin inhibitors, CsA and FK506. Collectively, these results suggest that LPS leads to myocardiac hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 cells. Our findings further provide a link between the LPS-induced inflammatory response and the calcineurin/NFAT-3 signaling pathway that mediates the development of cardiac hypertrophy.


Lipopolysaccharide Myocardiac cell Hypertrophy Calcineurin NFAT-3 GATA-4 





Toll-like receptor


Extracellular signal regulated kinase

p38 MAPK

p38 Mitogen-activated protein kinase


c-Jun N-terminal kinase


Nuclear factor of activated T-cells


Atrial natriuretic peptide


B-type natriuretic peptide


Cyclosporine A


Dulbecco’s modified Eagle’s medium


Glyceraldehyde-3-phosphate dehydrogenase


Reverse transcription


Phosphate-buffered saline


6-amino-4-(4-phenoxyphenylethylamino) quinazoline


  1. 1.
    Lorell BH, Carabello BA (2000) Left ventricular hypertrophy: pathogenesis, detection, and prognosis. Circulation 102:470–479PubMedGoogle Scholar
  2. 2.
    Olson EN, Schneider MD (2003) Sizing up the heart: development redux in disease. Genes Dev 17:1937–1956PubMedCrossRefGoogle Scholar
  3. 3.
    Molkentin JD, Dorn IG 2nd (2001) Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu Rev Physiol 63:391–426PubMedCrossRefGoogle Scholar
  4. 4.
    Sugden PH, Clerk A (1998) Cellular mechanisms of cardiac hypertrophy. J Mol Med 76:725–746PubMedCrossRefGoogle Scholar
  5. 5.
    Wang Y, Su B, Sah VP et al (1998) Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a specific activator for c-Jun NH2-terminal kinase in ventricular muscle cells. J Biol Chem 273:5423–5426PubMedCrossRefGoogle Scholar
  6. 6.
    Towbin JA, Bowles NE (2002) The failing heart. Nature 415:227–233PubMedCrossRefGoogle Scholar
  7. 7.
    Hunter JJ, Chien KR (1999) Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 341:1276–1283PubMedCrossRefGoogle Scholar
  8. 8.
    Mann DL (1999) Mechanisms and models in heart failure: a combinatorial approach. Circulation 100:999–1008PubMedGoogle Scholar
  9. 9.
    Levy D, Garrison RJ, Savage DD et al (1990) Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med 322:1561–1566PubMedGoogle Scholar
  10. 10.
    Drazner MH, Rame JE, Marino EK et al (2004) Increased left ventricular mass is a risk factor for the development of a depressed left ventricular ejection fraction within five years: the Cardiovascular Health Study. J Am Coll Cardiol 43:2207–2215PubMedCrossRefGoogle Scholar
  11. 11.
    Frey N, Olson EN (2003) Cardiac hypertrophy: the good, the bad, and the ugly. Annu Rev Physiol 65:45–79PubMedCrossRefGoogle Scholar
  12. 12.
    Sugden PH, Clerk A (1998) “Stress-responsive” mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res 83:345–352PubMedGoogle Scholar
  13. 13.
    Abe J, Baines CP, Berk BC (2000) Role of mitogen-activated protein kinases in ischemia and reperfusion injury: the good and the bad. Circ Res 86:607–609PubMedGoogle Scholar
  14. 14.
    Glennon PE, Kaddoura S, Sale EM et al (1996) Depletion of mitogen-activated protein kinase using an antisense oligodeoxynucleotide approach downregulates the phenylephrine-induced hypertrophic response in rat cardiac myocytes. Circ Res 78:954–961PubMedGoogle Scholar
  15. 15.
    Bueno OF, van Rooij E, Molkentin JD et al (2002) Calcineurin and hypertrophic heart disease: novel insights and remaining questions. Cardiovasc Res 53:806–821PubMedCrossRefGoogle Scholar
  16. 16.
    Yue TL, Gu JL, Wang C et al (2000) Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy. J Biol Chem 275:37895–37901PubMedCrossRefGoogle Scholar
  17. 17.
    Takeishi Y, Huang Q, Abe J et al (2001) Src and multiple MAP kinase activation in cardiac hypertrophy and congestive heart failure under chronic pressure-overload:comparison with acute mechanical stretch. J Mol Cell Cardiol 33:1637–1648PubMedCrossRefGoogle Scholar
  18. 18.
    Clerk A, Michael A, Sugden PH (1998) Stimulation of the p38 mitogen-activated protein kinase pathway in neonatal rat ventricular myocytes by the G protein-coupled receptor agonists, endothelin-1 and phenylephrine: a role in cardiac myocyte hypertrophy? J Cell Biol 142:523–535PubMedCrossRefGoogle Scholar
  19. 19.
    Behr TM, Berova M, Doe CP et al (2003) p38 mitogen-activated protein kinase inhibitors for the treatment of chronic cardiovascular disease. Curr Opin Investig Drugs 4:1059–1064PubMedGoogle Scholar
  20. 20.
    Komuro I, Kudo S, Yamazaki T et al (1996) Mechanical stretch activates the stress-activated protein kinases in cardiac myocytes. FASEB J 10:631–636PubMedGoogle Scholar
  21. 21.
    Choukroun G, Hajjar R, Kyriakis JM et al (1998) Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy. J Clin Invest 102:1311–1320PubMedCrossRefGoogle Scholar
  22. 22.
    Ramirez MT, Zhao XL, Schulman H et al (1997) The nuclear deltaB isoform of Ca2+/calmodulin-dependent protein kinase II regulates atrial natriuretic factor gene expression in ventricular myocytes. J Biol Chem 272:31203–31208PubMedCrossRefGoogle Scholar
  23. 23.
    Yano M, Kim S, Izumi Y et al (1998) Differential activation of cardiac c-jun amino-terminal kinase and extracellular signal-regulated kinase in angiotensin II-mediated hypertension. Circ Res 83:752–760PubMedGoogle Scholar
  24. 24.
    Choukroun G, Hajjar R, Fry S et al (1999) Regulation of cardiac hypertrophy in vivo by the stress-activated protein kinases/c-Jun NH(2)-terminal kinases. J Clin Invest 104:391–398PubMedCrossRefGoogle Scholar
  25. 25.
    Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ et al (1996) Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion. Circ Res 79:162–173PubMedGoogle Scholar
  26. 26.
    Molkentin JD, Lu JR, Antos CL et al (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93:215–228PubMedCrossRefGoogle Scholar
  27. 27.
    Molkentin JD, Olson EN (1997) GATA4: a novel transcriptional regulator of cardiac hypertrophy? Circulation 96:3833–3835PubMedGoogle Scholar
  28. 28.
    Yang RB, Mark MR, Gray A et al (1998) Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature 395:284–288PubMedCrossRefGoogle Scholar
  29. 29.
    Stoll LL, Denning GM, Weintraub NL (2004) Potential role of endotoxin as a proinflammatory mediator of atherosclerosis. Arterioscler Thromb Vasc Biol 24:2227–2236PubMedCrossRefGoogle Scholar
  30. 30.
    Niebauer J, Volk HD, Kemp M (1999) Endotoxin and immune activation in chronic heart failure: a prospective cohort study. Lancet 353:1838–1842PubMedCrossRefGoogle Scholar
  31. 31.
    Jardin F, Brun-Ney D, Auvert B et al (1990) Sepsis-related cardiogenic shock. Crit Care Med 18:1055–1060PubMedCrossRefGoogle Scholar
  32. 32.
    Packer M (1995) Is tumor necrosis factor an important neurohormonal mechanism in chronic heart failure? Circulation 92:1379–1382PubMedGoogle Scholar
  33. 33.
    Boyd JH, Mathur S, Wang Y, Bateman RM et al (2006) Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response. Cardiovasc Res 72:384–393PubMedCrossRefGoogle Scholar
  34. 34.
    Yasuda S, Lew WY (1997) Lipopolysaccharide depresses cardiac contractility and beta-adrenergic contractile response by decreasing myofilament response to Ca2+ in cardiac myocytes. Circ Res 81:1011–1020PubMedGoogle Scholar
  35. 35.
    Clerk A, Cullingford TE, Fuller SJ et al (2007) Signaling pathways mediating cardiac myocyte gene expression in physiological and stress responses. J Cell Physiol 212:311–322PubMedCrossRefGoogle Scholar
  36. 36.
    Bartunek J, Weinberg EO, Tajima M et al (2000) Chronic N(G)-nitro-l-arginine methyl ester-induced hypertension: novel molecular adaptation to systolic load in absence of hypertrophy. Circulation 101:423–429PubMedGoogle Scholar
  37. 37.
    Wagner DR, Combes A, McTiernan C et al (1998) Adenosine inhibits lipopolysaccharide-induced cardiac expression of tumor necrosis factor-alpha. Circ Res 82:47–56PubMedGoogle Scholar
  38. 38.
    Levine B, Kalman J, Mayer L et al (1990) Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 323:236–241PubMedGoogle Scholar
  39. 39.
    Blum A, Sclarovsky S, Rehavia E et al (1994) Levels of T-lymphocyte subpopulations, interleukin-1 beta, and soluble interleukin-2 receptor in acute myocardial infarction. Am Heart J 127:1226–1230PubMedCrossRefGoogle Scholar
  40. 40.
    Anker SD, von Haehling S (2004) Inflammatory mediators in chronic heart failure: an overview. Heart 90:464–470PubMedCrossRefGoogle Scholar
  41. 41.
    Rauchhaus M, Doehner W, Francis DP et al (2000) Plasma cytokine parameters and mortality in patients with chronic heart failure. Circulation 102:3060–3067PubMedGoogle Scholar
  42. 42.
    Kumar A, Haery C, Parrillo JE (2000) Myocardial dysfunction in septic shock. Crit Care Clin 16:251–287PubMedCrossRefGoogle Scholar
  43. 43.
    Kumar A, Brar R, Wang P et al (1999) Role of nitric oxide and cGMP in human septic serum-induced depression of cardiac myocyte contractility. Am J Physiol 276:R265–276PubMedGoogle Scholar
  44. 44.
    Medzhitov R, Preston-Hurlburt P, Janeway CA Jr (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394–397PubMedCrossRefGoogle Scholar
  45. 45.
    Methe H, Kim JO, Kofler S, Weis M, Nabauer M, Koglin J (2005) Expansion of circulating Toll-like receptor 4-positive monocytes in patients with acute coronary syndrome. Circulation 111:2654–2661PubMedCrossRefGoogle Scholar
  46. 46.
    Frantz S, Kobzik L, Kim YD et al (1999) Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium. J Clin Invest 104:271–280PubMedCrossRefGoogle Scholar
  47. 47.
    Baumgarten G, Knuefermann P, Nozaki N et al (2001) In vivo expression of proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4. J Infect Dis 183:1617–1624PubMedCrossRefGoogle Scholar
  48. 48.
    Li C, Ha T, Kelley J et al (2004) Modulating toll-like receptor mediated signaling by (1 → 3)-beta-d-glucan rapidly induces cardioprotection. Cardiovasc Res 61:538–547PubMedCrossRefGoogle Scholar
  49. 49.
    Ha T, Li Y, Hua F et al (2005) Reduced cardiac hypertrophy in toll-like receptor 4-deficient mice following pressure overload. Cardiovasc Res 68:224–234PubMedCrossRefGoogle Scholar
  50. 50.
    Karin M, Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol 18:621–663PubMedCrossRefGoogle Scholar
  51. 51.
    Dash R, Schmidt AG, Pathak A et al (2003) Differential regulation of p38 mitogen-activated protein kinase mediates gender-dependent catecholamine-induced hypertrophy. Cardiovasc Res 57:704–714PubMedCrossRefGoogle Scholar
  52. 52.
    Cook SA, Novikov MS, Ahn Y et al (2003) A20 is dynamically regulated in the heart and inhibits the hypertrophic response. Circulation 108:664–667PubMedCrossRefGoogle Scholar
  53. 53.
    Li Y, Ha T, Gao X et al (2004) NF-kappaB activation is required for the development of cardiac hypertrophy in vivo. Am J Physiol Heart Circ Physiol 287:H1712–1720PubMedCrossRefGoogle Scholar
  54. 54.
    Thompson M, Kliewer A, Maass D et al (2000) Increased cardiomyocyte intracellular calcium during endotoxin-induced cardiac dysfunction in guinea pigs. Pediatr Res 47:669–676PubMedCrossRefGoogle Scholar
  55. 55.
    Okamura H, Aramburu J, Garcia-Rodriguez C et al (2000) Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol Cell 6:539–550PubMedCrossRefGoogle Scholar
  56. 56.
    Lim HW, Molkentin JD (1999) Calcineurin and human heart failure. Nat Med 5:246–247PubMedCrossRefGoogle Scholar
  57. 57.
    Bueno OF, Wilkins BJ, Tymitz KM et al (2002) Impaired cardiac hypertrophic response in calcineurin abeta-deficient mice. Proc Natl Acad Sci USA 99:4586–4591PubMedCrossRefGoogle Scholar
  58. 58.
    Haq S, Choukroun G, Lim H et al (2001) Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation 103:670–677PubMedGoogle Scholar
  59. 59.
    Laverriere AC, MacNeill C, Mueller C et al (1994) GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut. J Biol Chem 269:23177–23184PubMedGoogle Scholar
  60. 60.
    Liang Q, De Windt LJ, Witt SA et al (2001) The transcription factors GATA4 and GATA6 regulate cardiomyocyte hypertrophy in vitro and in vivo. J Biol Chem 276:30245–30253PubMedCrossRefGoogle Scholar
  61. 61.
    Diedrichs H, Chi M, Boelck B, Mehlhorn U et al (2004) Increased regulatory activity of the calcineurin/NFAT pathway in human heart failure. Eur J Heart Fail 6:3–9PubMedCrossRefGoogle Scholar
  62. 62.
    Yang TT, Xiong Q, Enslen H et al (2002) Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases. Mol Cell Biol 22:3892–3904PubMedCrossRefGoogle Scholar
  63. 63.
    Morimoto T, Hasegawa K, Wada H et al (2001) Calcineurin-GATA4 pathway is involved in beta-adrenergic agonist-responsive endothelin-1 transcription in cardiac myocytes. J Biol Chem 276:34983–34989PubMedCrossRefGoogle Scholar
  64. 64.
    Crabtree GR (1999) Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96:611–614PubMedCrossRefGoogle Scholar
  65. 65.
    McCaffrey PG, Luo C, Kerppola TK et al (1993) Isolation of the cyclosporin-sensitive T cell transcription factor NFATp. Science 262:750–754PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Chung-Jung Liu
    • 1
  • Yi-Chang Cheng
    • 2
  • Kung-Wei Lee
    • 3
  • Hsi-Hsien Hsu
    • 4
  • Chun-Hsien Chu
    • 1
  • Fuu-Jen  Tsai
    • 5
  • Chang-Hai Tsai
    • 6
  • Chia-Yih Chu
    • 7
  • Jer-Yuh Liu
    • 1
  • Wei-Wen Kuo
    • 8
  • Chih-Yang Huang
    • 9
    • 10
    • 11
  1. 1.Institute of Biochemistry and BiotechnologyChung Shan Medical UniversityTaichungTaiwan
  2. 2.Emergency Department China Medical University HospitalTaichungTaiwan
  3. 3.Department of Internal Medicine, Division of CardiologyChina Medical University HospitalTaichungTaiwan
  4. 4.Division of Colorectal SurgeryMackay Memorial HospitalTaipeiTaiwan
  5. 5.Department of Pediatrics, Medical Research and Medical GeneticsChina Medical UniversityTaichungTaiwan
  6. 6.Department of Healthcare AdministrationAsia UniversityTaichungTaiwan
  7. 7.School of Applied ChemistryChung Shan Medical UniversityTaichungTaiwan
  8. 8.Department of Biological Science and TechnologyChina Medical UniversityTaichungTaiwan
  9. 9.Graduate Institute of Chinese Medical ScienceChina Medical UniversityTaichungTaiwan
  10. 10.Institute of Basic Medical ScienceChina Medical UniversityTaichungTaiwan
  11. 11.Department of Health and Nutrition BiotechnologyAsia UniversityTaichungTaiwan

Personalised recommendations