Pathogenesis of Myocardial Injury and Cell Death in Myocarditis: Its Relation to the Fas/Fas Ligand Pathway

  • Michiaki Hiroe
  • Tetsuya Toyozaki
Conference paper


The Fas/APO-1(CD95) cell surface receptor binds to the Fas ligand, leading to apoptotic cell death under a variety of pathological conditions. In viral myocarditis, a perforin-based mechanism involving killer cells is known to play a role in necrotic myocardial injury. However, little is known about apoptotic cell death in myocarditis. Serum levels of the soluble form of Fas and Fas ligand were much higher in patients with myocarditis than in other cardiac patients. There was a positive correlation between sIL-2R and sFas levels in patients with myocarditis. In the myocarditis group, sFasligand levels increased with the severity of heart failure, as defined by New York Heart Association functional class. Cardiac tissues from rats with autoimmune myocarditis showed in situ DNA fragments by DNA laddering formation and TUNEL assay, the biological hallmark of apoptosis, in some cardiomyocytes and infiltrating lymphocytes. The expression of Fas ligand was up-regulated in some cardiomyocytes and lymphocytes. Fas ligand mRNA and protein were expressed in some CD4+ T cells. Coexpression of Fas with the Fas ligand was observed in some infiltrating lymphocytes. Furthermore, when cultured neonatal cardiomyocytes were exposed to the soluble form of Fas ligand in the presence of actinomycin D, the cells underwent apoptotic cell death, as demonstrated by electron microscopy, flow cytometry, and the TUNEL assay. These results suggest that some portion of the cardiomyocytes and infiltrating lymphocytes are subjected to apoptotic cell death, possibly due to T cell-mediated cytotoxicity, which is modulated by the Fas/Fas ligand pathway in myocarditis.

Key words

Apoptosis Myocarditis Fas/Fas ligand pathway TUNEL assay Electron microscopy Flow cytometry 


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  1. 1.
    Sekiguchi M, Hiroe M, Take M, Hirosawa K (1980) Clinical and histopathological profile of the sarcoidosis of heart and acute idiopathic myocarditis. Concepts through a study employing endomyocardial biopsy II. Myocarditis. Jpn Circ J 44: 264–273PubMedCrossRefGoogle Scholar
  2. 2.
    Hiroe M, Sekiguchi M, Take M, Matsuda M, Hirosawa K (1985) Hemodynamic studies and response to a combined therapy of nitroglycerin ointment and dopamine in patients with acute myocarditis. Heart Vessels Suppl 1: 180–186PubMedCrossRefGoogle Scholar
  3. 3.
    Hiroe M, Sekiguchi M, Take M, Kusakabe K, Shigeta A, Hirosawa K (1985) Long follow-up study in patients with prior myocarditis by radionuclide methods. Heart Vessels Suppl 1: 199–203PubMedCrossRefGoogle Scholar
  4. 4.
    Dec GW, Alacios IF, Fallon JT, Artez HT, Mills J, Lee DC, Johnson RA (1985) Active myocarditis in the spectrum of acute dilated cardiomyopathies: clinical features, histologic correlates, and clinical outcome. N Engl J Med 312: 885–890PubMedCrossRefGoogle Scholar
  5. 5.
    Fenoglio JJ, Ursell PC, Kellogg CF, Drusin RE, Weiss MB (1983) Diagnosis and classification of myocarditis by endomyocardial biopsy. N Engl J Med 308: 112–118CrossRefGoogle Scholar
  6. 6.
    Aretz JT (1987) Myocarditis: the Dallas criteria. Hum Pathol 18: 619–635PubMedCrossRefGoogle Scholar
  7. 7.
    Lieberman EB, Hutchins GM, Herskowitz A, Rose NR, Baughman KL (1991) Clinico-pathologic description of myocarditis. J Am Coll Cardiol 18: 1617–1626PubMedCrossRefGoogle Scholar
  8. 8.
    Woodruff JF (1980) Viral myocarditis: a review. Am J Pathol 101: 427–484Google Scholar
  9. 9.
    Bowles NE, Richardson Pi Olsen EGJ,Archard LC (1986) Detection of Coxsackie-Bvirus specific RNA sequence in myocardial biopsy sample from patients with myocarditis and dilated cardiomyopathy. Lancet 1: 1120–1123PubMedCrossRefGoogle Scholar
  10. 10.
    Jin O, Sole M, Butany J (1990) Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and cardiomyopathy using polymerase chain reaction. Circulation 82: 8–16PubMedCrossRefGoogle Scholar
  11. 11.
    Martin AB, Webber S, Fricker J, Jaffe R, Demmler G, Kearney D, Zhang Y-H, Bodurtha J, Gelb B, Ni J, Bricker T, Towbin JA (1994) Acute myocarditis: rapid diagnosis by PCR in children. Circulation 90: 330–339PubMedGoogle Scholar
  12. 12.
    Berke G (1995) The CTL’s kiss of death. Cell 81: 9–12PubMedCrossRefGoogle Scholar
  13. 13.
    Liu CC, Young LHY, Young JDE (1996) Lymphocyte-mediated cytolysis and disease. N Engl J Med 335: 1651–1659PubMedCrossRefGoogle Scholar
  14. 14.
    Kâgi D, Vignaux F, Ledeman B, Burki K, Depraetere V, Nagata S, Hengarttner H. Golstein P (1994) Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265: 528–530PubMedCrossRefGoogle Scholar
  15. 15.
    Young JD, Hengartner H, Podack ER, Cohn ZA (1986) Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity. Cell 44: 849–859PubMedCrossRefGoogle Scholar
  16. 16.
    Shi L, Kam C-M, Powers JC, Aebersold R, Greenberg AH (1992) Purification of three cytotoxic lymphocytes granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J Exp Med 176: 1521–1529PubMedCrossRefGoogle Scholar
  17. 17.
    Arase H, Arase N, Saito S (1995) Fas-mediated cytotoxicity by freshly isolated natural killer cells. J Exp Med 181: 1235–1238PubMedCrossRefGoogle Scholar
  18. 18.
    Nagata S (1997) Apoptosis by death factor. Cell 88: 355–365PubMedCrossRefGoogle Scholar
  19. 19.
    Krammer PH, Dhein J, Walczak H, Behrmann I, Mariani S, Matiba B, Fath M, Daniel PT, Knipping E, Westendorp MO, Stricker K, Baumler C, Hellbardt S, Germer M, Peter ME, Debatin K-M (1994) The role of APO-1 mediated apoptosis in the immune system. Immunol Rev 142: 175–191PubMedCrossRefGoogle Scholar
  20. 20.
    Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239–257PubMedCrossRefGoogle Scholar
  21. 21.
    Li Y, Sharov VG, Jiang N, Zaloga C, Sabbah HN, Chopp M (1995) Ultrastructual and light microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat. Am J Pathol 146: 1045–1051PubMedGoogle Scholar
  22. 22.
    Majino G, Joris I (1995) Apoptosis, oncosis, and necrosis: an overview of cell death. Am J Pathol 146: 3–15Google Scholar
  23. 23.
    Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous nuclease activation. Nature 284: 555–556PubMedCrossRefGoogle Scholar
  24. 24.
    Wyllie AH, Morris RG, Smith AL, Dunlop D (1984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macro-molecular synthesis. J Pathol 142: 67–77PubMedCrossRefGoogle Scholar
  25. 25.
    Raff MC (1992) Social control on cell survival and cell death. Nature 356: 397–400PubMedCrossRefGoogle Scholar
  26. 26.
    Thompson CB (1995) Apoptosis in the pathogenesis and treatment of diseases. Science 267: 1456–1462PubMedCrossRefGoogle Scholar
  27. 27.
    Feuerstein G, Rufflo RR Jr, Yue TL (1997) Apoptosis and congestive heart failure. Trends Cardiovasc Med 7: 249–255PubMedCrossRefGoogle Scholar
  28. 28.
    MacLellan WR, Schneider MD (1997) Death design: programmed cell death in cardiovascular biology and disease. Circ Res 81: 137–144PubMedGoogle Scholar
  29. 29.
    Haunstetter A, Izumo S (1998) Apoptosis: basic mechanisms and implications for cardiovascular diseases. Circ Res 82: 1111–1126PubMedGoogle Scholar
  30. 30.
    Anversa P, Leri A, Beltrami CA, Guerra S, Kajstura J (1998) Myocyte death and growth in the failing heart. Lab Invest 78: 767–786PubMedGoogle Scholar
  31. 31.
    Sabbah H, Sharov VG (1998) Apoptosis in heart failure. Prog Cardiovasc Dis 40: 549–562PubMedCrossRefGoogle Scholar
  32. 32.
    Kajstura J, Mansukhan IM, Cheng W, Reis Anversa P (1995) Programmed cell death and the expression of the protooncogene Bel-2 in myocyte during postnatal maturation of the heart. 219: 110–121Google Scholar
  33. 33.
    Takeda K, Yu ZX, Nishikawa T, Tanaka M, Hosoda S, Ferrans VJ, Kasajima T (1996) Apoptosis and DNA fragmentation in the bulbus cordis of the developing rat heart. J Mol Cell Cardiol 28: 209–215PubMedCrossRefGoogle Scholar
  34. 34.
    Narula J, Haider N, Virmani R, DiSalvo TG, Kolodgie FD, Hajjar RJ, Schmidt U, Semigran MJ, Dec GW, Khaw B-A (1996) Apoptosis in myocytes in end-stage heart failure. N Engl J Med 335: 1182–1189PubMedCrossRefGoogle Scholar
  35. 35.
    Olivetti JT, Abbi R, Quaini F, Kajstura J, Cheng W, Wuaini E, Loreto CD, Beltrrrami CA, Krajewski S, Reed JC, Anversa P (1997) Apoptosis in failing human heart. N Engl J Med 336: 1131–1141PubMedCrossRefGoogle Scholar
  36. 36.
    Liu Y, Cigola E, Cheng E, Kajstura J, Olivetti G, Hintze TH, Anversa P (1995) Myocyte nuclear mitotic division and programmed cell death characterize the cardiac myopathy induced by rapid right ventricular pacing in dogs. Lab Invest 73: 771–787PubMedGoogle Scholar
  37. 37.
    Sharov VG, Sabbah HN, Shimoyama H, Goussev AV, Lesch M, Goldstein S (1996) Evidence of cardiocyte apoptosis in myocardium of dogs with chronic heart failure. Am J Pathol 148: 141–149PubMedGoogle Scholar
  38. 38.
    Bing OHL (1994) Hypothesis: apoptosis may be a mechanism for the transition to heart failure with chronic pressure overload. J Mol Cell Cardiol 26: 943–948PubMedCrossRefGoogle Scholar
  39. 39.
    Itoh G, Tamura J, Suzuki M, Suzuki Y, Ikeda H, Koike M, Nomura M, Jie T, Ito K (1995) DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis. Am J Pathol 146: 1325–1331PubMedGoogle Scholar
  40. 40.
    Saraste AK, Pulkki K, Henrikesen M, Parvinen M, Voipio-Pulkki L-M (1997) Apoptosis in human acute myocardial infarction. Circulation 95: 320–323PubMedGoogle Scholar
  41. 41.
    Gottlieb RA, Burleson KO. Kloner RA, Bablor BM, Engler RL (1994) Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94: 1621–1628PubMedCrossRefGoogle Scholar
  42. 42.
    Fliss H, Gattinger D (1996) Apoptosis in ischemic and reperfused rat myocardium. Circ Res 79: 949–956PubMedGoogle Scholar
  43. 43.
    Kajstura J, Cheng W, Reiss K, Clark WA, Sonnenblick EH, Krajewski S, Reed JC, Olivetti G, Anversa P (1996) Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 74: 86–107PubMedGoogle Scholar
  44. 44.
    Fliss H, Gattinger D (1996) Apoptosis in ischemia and reperfused rat myocardium. Circ Res 79: 949–956PubMedGoogle Scholar
  45. 45.
    Mallat Z, Tedgui A, Fontaliran F, Frank R, Durigon M, Fontaine G (1996) Evidence of apoptosis in arrhythmogenic right ventricular dysplasia. N Engl J Med 335: 1190–1196PubMedCrossRefGoogle Scholar
  46. 46.
    Valente M, Calabrese F, Thiene G. Angnelini A, Basso C, Nava A, Rossi L (1998) In vivo evidence of apoptosis in arrhythmogenic right ventricular cardio-myopathy. Am J Pathol 132: 479–484Google Scholar
  47. 47.
    James TN, Nichols MM, Sapire DW, DiPatre PL, Lopez SM (1996) Complete heart block and fatal right ventricular failure in an infant. Circulation 93: 1588–1600PubMedGoogle Scholar
  48. 48.
    Diez J, Panizo A, Hernandez M, Vega F. Sola I, Fortufio MA, Pardo J (1997) Cardiomyocyte apoptosis and cardiac angiotensin-converting enzyme in spontaneously hypertensive rats. Hypertension 30: 1029–1034PubMedGoogle Scholar
  49. 49.
    Frusutaci A, Chimenti C, Setoguchi M, Guerra S, Corsello S, Crea F, Len A, Kajstura J, Anversa P, Maseri A (1999) Cell death in acromegalic cardiomyopathy. Circulation 99: 1426–1434Google Scholar
  50. 50.
    Tanaka M, Ito H, Adachi S, Akimoto H, Nishikawa T, Kasajima T, Marumo H, Hiroe M (1994) Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res 75: 426–433PubMedGoogle Scholar
  51. 51.
    Laderoute KR, Webster KA (1997) Hypoxia/reoxygenation stimulate Jun kinase activity through redox signaling in cardiac myocytes. Circ Res 80: 336–344PubMedGoogle Scholar
  52. 52.
    Cheng W, Li B, Kajstura J, Li P, Wolin MS, Sonnenblick EH, Hintze TH, Olivetti G, Anversa P (1995) Stretch-induced programmed myocyte cell death. J Clin Invest 96: 2247–2259PubMedCrossRefGoogle Scholar
  53. 53.
    Krown KA, Page MT, Nguyen C, Zechner D, Gutierrez V, Comstock KL, Glembotski CC, Quintana PJE, Sabbadini RA (1996) Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death. J Clin Invest 98: 2854–2856PubMedCrossRefGoogle Scholar
  54. 54.
    Kajstura J, Cigola E, Malhotra A, Cheng W, Meggs LG, Anversa P (1997) Angiotensin II induces apoptosis of adult ventricular myocytes in vitro. J Mol Cell Cardiol 29: 859–870PubMedCrossRefGoogle Scholar
  55. 55.
    Wu CF, Bishopric NH, Pratt RE (1997) Atrial natriuretic peptide induces apoptosis in neonatal rat cardiac myocytes. J Biol Chem 272: 14860–14866PubMedCrossRefGoogle Scholar
  56. 56.
    Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Tanaka M, Shiojima I, Hiroi Y, Yazaki Y (1997) Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest 100: 1813–1821PubMedCrossRefGoogle Scholar
  57. 57.
    Shimojo T, Hiore M, Ishiyama S, Ito H, Nishikawa T, Marumo F (1999) Nitric oxide induces apoptotic death of cardiomyocytes via a cyclic-GMP-dependent pathway. Exp Cell Res 247: 38–47PubMedCrossRefGoogle Scholar
  58. 58.
    Watanabe FR, Brannan CI, Itoh N, Yonehara S, Copeland NG, Jenkins NA, Nagata S (1992) The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J Immunol 148: 1274–1279Google Scholar
  59. 59.
    Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, Nagata S (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66: 233–243PubMedCrossRefGoogle Scholar
  60. 60.
    Yonehara S, Ishi A, Yonehara M (1989) A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 169: 1747–1756PubMedCrossRefGoogle Scholar
  61. 61.
    Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, Itoh N, Suda T, Nagata S (1993) Lethal effect of the anti-Fas antibody in mice. Nature 364: 306–309CrossRefGoogle Scholar
  62. 62.
    Suda T, Nagata S (1994) Purification and characterization of the Fas-ligand that induces apoptosis. J Exp Med 179: 873–879PubMedCrossRefGoogle Scholar
  63. 63.
    Dhein J, Walczak H, Bâumler C, Debatin KM, Krammer PH (1995) Autocrine T-cell suicide mediated by Apo-1/Fas/CD95. Nature 373: 438–441PubMedCrossRefGoogle Scholar
  64. 64.
    Ju S-T, Panka DJ, Cui H, Ettinger R, El-Khatib M, Sherr DH, Stanger BZ, MarshakRothstein A (1995) Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature 373: 444–448PubMedCrossRefGoogle Scholar
  65. 65.
    Cheng J, Zhou T, Liu C, Shapiro JP, Brauer MJ, Kiefer MC, Barr PJ, Mountz JD (1994) Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science 263: 1759–1762PubMedCrossRefGoogle Scholar
  66. 66.
    Liu C, Cheng J, Mountz JD (1995) Differential expression of human Fas mRNA species upon peripheral blood mononuclear cell activation. Biochem J 310: 957–963PubMedGoogle Scholar
  67. 67.
    Papoff G, Cascino I, Eramo A, Starace G, Lynch DH, Ruberti G (1996) An N-terminal domain shared by Fas/Apo-1(CD95) soluble variants prevents cell death in vitro. J Immunol 156: 4622–4630PubMedGoogle Scholar
  68. 68.
    Nishigaki K, Minatoguchi S, Seishima M, Asano K, Noda T, Yasuda N, Sano H, Kumada H, Takemura M, Noma A, Tanaka T, Watanabe S, Fujiwara H (1997) Plasma Fas ligand, an inducer of apoptosis, and plasma soluble Fas, an inhibitor of apoptosis, in patients with chronic congestive heart failure. J Am Coll Cardiol 29: 1214–1220PubMedCrossRefGoogle Scholar
  69. 69.
    Okuyama M, Yamaguchi S, Nozaki N, Yamaoka M, Shirakabe M, Tomoike H (1997) Serum levels of soluble form of Fas molecule in patients with congestive heart failure. Am J Cardiol 79: 1698–1701PubMedCrossRefGoogle Scholar
  70. 70.
    Toyozaki T, Hiroe M, Saito T, Iijima Y. Takano H, Hiroshima K, Kohno H, Ishiyama S, Marumo F, Masuda Y, Ohwada H (1998) Levels of soluble Fas in patients with myocarditis, heart failure of unknown origin, and in healthy volunteers. Am J Cardiol 81: 798–800Google Scholar
  71. 71.
    Kayagaki N, Kawasaki A, Ebata T. Ohmoto H, Ikeda S, Inoue S, Yoshini K, Nozawa K, Okuyama K, Yagita H (1995) Metalloproteinase-mediated release of human Fas ligand. J Exp Med 182: 1777–1783Google Scholar
  72. 72.
    Tanaka M, Suda T, Haze K, Nakamura N, Sato K, Kimura F, Motoyoshi K, Mizuki M, Tagawa S, Ohga S, Hatake K, Drummoud AH, Nagata S (1996) Fas ligand in human serum. Nature Med 2: 317–322PubMedCrossRefGoogle Scholar
  73. 73.
    Nozawa K, Kayagaki N,Tokano Y, Yagita H, Okumura K, Hashimoto H (1997) Soluble Fas (APO-1, CD95) and soluble Fas ligand in rheumatic diseases. Arthritis Rheum 40: 1126–1129Google Scholar
  74. 74.
    Tanaka M, Suda T, Takahashi T, Nagata S (1995) Expression of the functional soluble form of human Fas ligand in activated lymphocytes. EMBO J 14: 1129–1135PubMedGoogle Scholar
  75. 75.
    Toyozaki T, Hiroe M, Tanaka M, Nagata S, Ohwada H, Marumo F (1998) Levels of soluble Fas ligand in myocarditis. Am J Cardiol 82: 246–248PubMedCrossRefGoogle Scholar
  76. 76.
    Neu N, Rose NR, Beisel KW, Herskowitz A, Gurri-Glass G, Craig SW (1987) Cardiac myosin induces myocarditis in genetically predisposed mice. J Immunol 139: 3630–3636PubMedGoogle Scholar
  77. 77.
    Smith SC, Allen PM (1991) Myosin-induced acute myocarditis is a T cell-mediated disease. J Immunol 147: 2141–2147PubMedGoogle Scholar
  78. 78.
    Kodama M, Matsumoto Y, Fujiwara M (1992) In vivo lymphocyte-mediated myocardial injuries demonstrated by adoptive transfer of experimental autoimmune myocarditis. Circulation 85: 1918–1926PubMedGoogle Scholar
  79. 79.
    Ishiyama S, Hiroe M, Nishikawa T, Abe S, Shimojo T, Ito H, Ozasa S, Yamakawa K, Matsuzaki M, Mohammed MU, Nakazawa H, Kasajima T, Marumo F (1997) Nitric oxide contributes to the progression of myocardial damage in experimental autoimmune myocarditis in rats. Circulation 95: 489–496PubMedGoogle Scholar
  80. 80.
    Davidoff R, Palacios I, Southern J, Fallon JT, Newell J, Dec GW (1991) Giant cell versus lymphocytic myocarditis: a comparison of their clinical features and long-term outcomes. Circulation 83: 953–961PubMedGoogle Scholar
  81. 81.
    Ishiyama S, Hiroe M, Nishikawa T, Shimojo T, Abe S, Fujisaki H, Ito H, Yamakawa K, Kobayashi N, Kasajima T, Marumo F (1998) The Fas/Fas ligand system is involved in the pathogenesis of autoimmune myocarditis in rats. J Immunol 161: 4695–4701PubMedGoogle Scholar

Copyright information

© Springer-Verlag Tokyo 2000

Authors and Affiliations

  • Michiaki Hiroe
    • 1
  • Tetsuya Toyozaki
    • 2
  1. 1.Division of Cardiology, Second Department of Internal MedicineTokyo Medical and Dental UniversityBunkyo-ku, TokyoJapan
  2. 2.Division of Pathology, Institute of Pulmonary Cancer ResearchChiba University School of MedicineChibaJapan

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