Cell and Tissue Research

, Volume 264, Issue 3, pp 577–587 | Cite as

Myofibrillar and cytoskeletal assembly in neonatal rat cardiac myocytes cultured on laminin and collagen

  • Lula L. Hilenski
  • Louis Terracio
  • Thomas K. Borg


Neonatal rat cardiomyocytes were cultured on extracellular matrix components laminin and collagens I+III to examine effects of extracellular matrix on the assembly of cytoskeletal proteins during myofibrillogenesis. Myofibril assembly was visualized by immunofluorescence of marker proteins for myofibrils (f-actin for I bands and α-actinin for Z bands), focal adhesions (vinculin), and transmembrane extracellular matrix receptors (β1 integrin) as cells spread for various times in culture. By 4 h in culture, f-actin appeared organized into nonstriated stress-fiber-like structures while α-actinin, vinculin and β1 integrin were localized in small streaks and beads. Subsequently, striated patterns were observed sequentially in the intracellular cytoskeletal components α-actinin, vinculin, f-actin, and then in the transmembrane β1 integrin receptor. These data support an earlier model for sarcomerogenesis in which stress-fiber-like structures serve as initial scaffolds upon which α-actinin and then vinculin-containing costameres are assembled. This sequential and temporal assembly was the same on both laminin and collagens I+III. A quantitative difference, however, was apparent on the 2 matrices. There was an increased appearance on collagens I+III of rosettes (also called podosomes or cortical actin-containing bodies in other cells) which consisted of an f-actin core surrounded by α-actinin, vinculin and β1 integrin rims. The increased incidence of rosettes in neonatal myocytes on collagens I+III suggests that these cytoskeletal complexes are involved in recognition and interaction with extracellular matrix components.

Key words

Myofibrils Cytoskeleton Extracellular matrix Laminin Collage Actin filaments Vinculin-Rat 


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  1. Abercrombie M, Heaysman JEM, Pegrum SM (1971) The locomotion of fibroblasts in culture. IV. Electron microscopy of the leading lamella. Exp Cell Res 67:359–367Google Scholar
  2. Atherton BT, Meyer DM, Simpson DG (1986) Assembly and remodelling of myofibrils and intercalated discs in cultured neonatal rat heart cells. J Cell Sci 86:233–248Google Scholar
  3. Blikstad I, Carlsson L (1982) On the dynamics of the microfilament system in HeLa cells. J Cell Biol 93:122–128Google Scholar
  4. Blobel G, Dobberstein B (1975) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol 67:835–851Google Scholar
  5. Borg TK, Terracio L (1988) Cellular adhesion to artificial substrates and long term culture of adult cardiac myocytes: In: Clark WA, Decker RS, Borg TK (eds) Biology of isolated adult cardiac myocytes. Elsevier, New York, pp 14–24Google Scholar
  6. Borg TK, Terracio L (1989) Interaction of the extracellular matrix with cardiac myocytes during development and disease. In: Kinne R, Stolte H (eds) Issues in biomedicine: cardiac myocyteconnective tissue interactions in health and disease. Karger, Basel, pp 113–129Google Scholar
  7. Borg TK, Rubin K, Lundgren E, Borg K, Öbrink B (1984) Recognition of extracellular matrix components by neonatal and adult cardiac myocytes. Dev Biol 104:86–96Google Scholar
  8. Borg TK, Xuehui M, Hilenski L, Vinson N, Terracio L (1990) The role of the extracellular matrix on myofibrillogenesis in vitro. In: Clark EB, Takao A (eds) Developmental cardiology: morphogenesis and function. Futura, Mt Kisco, NY, pp 175–190Google Scholar
  9. Brugge JS, Erikson RL (1977) Identification of transformationspecific antigen induced by an avian sarcoma virus. Nature 269:346–348Google Scholar
  10. Buck CA, Horwitz AF (1987) Cell surface receptors for extracellular matrix molecules. Ann Rev Cell Biol 3:179–205Google Scholar
  11. Burnette WN (1981) Western blotting:electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203Google Scholar
  12. Burridge K (1986) Substrate adhesions in normal and transformed fibroblasts:organization and regulation of cytoskeletal, membrane and extracellular matrix components at focal contacts. Cancer Rev 4:18–78Google Scholar
  13. Carley WW, Barak LS, Webb WW (1981) F-actin aggregates in transformed cells. J Cell Biol 90:797–802Google Scholar
  14. Caufield JB, Borg TK (1979) The collagen network of the heart. Lab Invest 40:364–372Google Scholar
  15. Chen W-T (1989) Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. J Exp Zool 251:167–185Google Scholar
  16. Chen W-T, Singer SJ (1982) Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts. J Cell Biol 95:205–222Google Scholar
  17. Chen W-T, Olden K, Bernard BA, Chu F-F (1984) Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites. J Cell Biol 98:1546–1555Google Scholar
  18. Chen W-T, Hasegawa E, Hasegawa T, Weinstock C, Yamada KM (1985a) Development of cell surface linkage complexes in cultured fibroblasts. J Cell Biol 100:1103–1114Google Scholar
  19. Chen W-T, Chen J-M, Parsons SJ, Parsons JT (1985b) Local degradation of fibronectin at sites of expression of the transforming gene product pp60src. Nature 316:156–158Google Scholar
  20. Colley NJ, Tokuyasu KT, Singer SJ (1990) The early expression of myofibrillar proteins in round postmitotic myoblasts of embryonic skeletal muscle. J Cell Sci 95:11–22Google Scholar
  21. Croop J, Holtzer H (1975) Response of myogenic and fibrogenic cells to cytochalasin B and to colcemid. 1. Light microscope observations. J Cell Biol 65:271–285Google Scholar
  22. Damsky CH, Knudsen KA, Bradley D, Buck CA, Horwitz AF (1985) Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture. J Cell Biol 100:1528–1539Google Scholar
  23. David-Pfeuty T, Singer SJ (1980) Altered distribution of the cytoskeletal proteins vinculin and α-actinin in cultured fibroblasts transformed by Rous sarcoma virus. Proc Natl Acad Sci USA 77:6687–6691Google Scholar
  24. Dejana E, Colella S, Conforti G, Abbadini M, Gaboli M, Marchisio PC (1988) Fibronectin and vitronectin regulate the organization of their respective arg-gly-asp adhesion receptors in cultured human endothelial cells. J Cell Biol 107:1215–1223Google Scholar
  25. Denning GM, Kim IS, Fulton AB (1988) Shedding of cytoplasmic actins by developing muscle cells. J Cell Sci 89:273–282Google Scholar
  26. Dlugosz AA, Antin PB, Nachmias VT, Holtzer H (1984) The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes. J Cell Biol 99:2268–2278Google Scholar
  27. Duband J-L, Nuckolls GH, Ishihara A, Hasegawa T, Yamada KM, Thiery JP, Jacobson K (1988) Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells. J Cell Biol 107:1385–1396Google Scholar
  28. Fath KR, Edgell C-JS, Burridge K (1989) The distribution of distinct integrins in focal contacts is determined by the substratum composition. J Cell Sci 92:67–75Google Scholar
  29. Fischman DA (1986) Myofibrillogenesis and the morphogenesis of skeletal muscle. In: Engel AG, Banker BQ (eds) Myology: basic and clinical. McGraw-Hill, New York, pp 5–37Google Scholar
  30. Fürst DO, Osborn M, Weber K (1989) Myogenesis in the mouse embryo: differential onset of expression of myogenic protein and the involvement of titin in myofibril assembly. J Cell Biol 109:517–527Google Scholar
  31. Geiger B, Avnur Z, Kreis TE, Schlessinger J (1984) The dynamics of cytoskeletal organization in areas of cell contact. Cell Muscle Motil 5:195–234Google Scholar
  32. Gullberg D, Terracio L, Borg TK, Rubin K (1989) Identification of integrin-like matrix receptors with affinity for interstitial collagens. J Biol Chem 264:12686–12694Google Scholar
  33. Handel SE, Wang S-M, Greaser ML, Schultz E, Bulinksi JC, Lessard JL (1989) Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the α- and gamma-isoforms of actin. Dev Biol 132:35–44Google Scholar
  34. Hilenski LL, Terracio L, Sawyer R, Borg TK (1989) Effects of extracellular matrix on cytoskeletal and myofibrillar organization in vitro. Scanning Microsc 3:535–548Google Scholar
  35. Horwitz A, Duggan K, Buck C, Berkerle MC, Burridge K (1986) Interaction of plasma membrane fibronectin receptor with talin: a transmembrane linkage. Nature 320:531–533Google Scholar
  36. Hynes RO (1987) Integrins: a family of cell surface receptors. Cell 48:549–554Google Scholar
  37. Isobe Y, Warner FD, Lemanski LF (1988) Three-dimensional immunogold localization of α-actinin within the cytoskeletal networks of cultured cardiac muscle and nonmuscle cells. Proc Natl Acad Sci USA 85:6758–6762Google Scholar
  38. Kaufmann R, Frösch D, Westphal D, Weber L, Klein CE (1989) Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures. J Cell Biol 109:1807–1815Google Scholar
  39. Lampugnani MG, Dejana E, Abbadini M, Marchisio PC (1988) Organization of vitronectin and fibronectin receptors in the endothelial cell membrane. In: Rousset BAF (ed) Colloque INSERM, vol 171 Libbey, London, pp 133–138Google Scholar
  40. Legato MJ (1972) Ultrastructural characteristics of the rat ventricular cell grown in tissue culture, with special reference to sarcomerogenesis. J Mol Cell Cardiol 4:299–317Google Scholar
  41. Levinson AD, Oppermann H, Levintow L, Varmus HE, Bishop JM (1978) Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein. Cell 15:561–572Google Scholar
  42. Lin Z, Eshleman J, Grund C, Fischman DA, Masaki T, Franke WW, Holtzer H (1989a) Differential response of myofibrillar and cytoskeletal proteins in cells treated with phorbol myristate acetate. J Cell Biol 108:1079–1091Google Scholar
  43. Lin Z, Holtzer S, Schultheiss T, Murray J, Masaki T, Fischman DA, Holtzer H (1989b) Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes. J Cell Biol 108:2355–2367Google Scholar
  44. Marchisio PC, Cirillo D, Teti A, Zambonin-Zallone A, Tarone G (1987) Rous sarcoma virus-transformed fibroblasts and cells of monocytic origin display a peculiar dot-like organization of cytoskeletal proteins involved in microfilament-membrane interactions. Exp Cell Res 169:202–214Google Scholar
  45. Marchisio PC, Bergui L, Corbascio GC, Cremona O, D'Urso N, Schena M, Tesio L, Caligaris-Cappio F (1988) Vinculin, talin, and integrins are localized in specific adhesion sites of malignant B lymphocytes. Blood 72:830–833Google Scholar
  46. Markwald RR (1973) Distribution and relationship of precursor Z material to organizing myofibrillar bundles in embryonic rat and hamster ventricular myocytes. J Mol Cell Cardiol 5:341–350Google Scholar
  47. Otey CA, Pavalko FM, Burridge K (1989) Integrin-binding proteins from chicken embryo fibroblasts. J Cell Biol 109:190aGoogle Scholar
  48. Pardo JV, Siliciano JD, Craig SW (1983) A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements (“costameres”) mark sites of attachment between myofibrils and sarcolemma. Proc Natl Acad Sci USA 80:1008–1012Google Scholar
  49. Peng HB, Wolosewick JJ, Cheng P-C (1981) The development of myofibrils in cultured muscle cells: a whole-mount and thinsection electron microscope study. Dev Biol 88:121–136Google Scholar
  50. Ruoslahti E, Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins. Science 238:491–497Google Scholar
  51. Schultheiss T, Lin Z, Lu M-H, Murray J, Fischman DA, Weber K, Masaki T, Imamura M, Holtzer H (1990) Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils. J Biol 110:1159–1172Google Scholar
  52. Sefton BM, Beemon K, Hunter T (1978) Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo. J Virol 28:957–971Google Scholar
  53. Shimada Y, Fischman DA (1975) Cardiac cell aggregation by scanning electron microscopy. In: Lieberman M, Sano T (eds) Developmental and physiological correlates of cardiac muscle. Raven Press, New York, pp 81–101Google Scholar
  54. Shimada Y, Komiyama M, Terai M, Maruyama K (1990) Early phases of myofibril assembly in embryonic chick cardiac myocytes in vitro. In: Clark EB, Takao A (eds) Developmental cardiology: morphogenesis and function. Futura, Mt Kisco, New York, pp 63–77Google Scholar
  55. Siddiqui MAQ, Kumar CC (1987) Molecular genetics and control of contractile proteins. In: Spry CJF (ed) Immunology and molecular biology of cardiovascular diseases. MTP Press, Lancaster, pp 3–20Google Scholar
  56. Singer II (1979) The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell 16:675–685Google Scholar
  57. Singer II, Scott S, Kawka DW, Kazazis DM, Gailit J, Ruoslahti E (1988) Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation. J Cell Biol 106:2171–2182Google Scholar
  58. Terai M, Komiyama M, Shimada Y (1989) Myofibril assembly is linked with vinculin, α-actinin and cell substrate contacts in embryonic cardiac myocytes in vitro. Cell Motil Cytoskeleton 12:185–194Google Scholar
  59. Terracio L, Gullberg D, Rubin K, Craig S, Borg TK (1989) Expression of collagen adhesion proteins and their association with the cytoskeleton in cardiac myocytes. Anat Rec 223:62–71Google Scholar
  60. Terracio L, Simpson DG, Hilenski L, Carver W, Decker RS, Vinson N, Borg TK (1990) Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy. J Cell Physiol 145:78–87Google Scholar
  61. Timpl R, Rohde H, Robey PG, Rennard SI, Foidart J-M, Martin GR (1979) Laminin — a glycoprotein from basement membranes. J Biol Chem 254:9933–9937Google Scholar
  62. Tokuyasu KT, Maher PA (1987a) Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. I. Presence of immunofluorescent titin spots in premyofibril stages. J Cell Biol 105:2781–2793Google Scholar
  63. Tokuyasu KT, Maher PA (1987b) Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. II. Generation of α-actinin dots within titin spots at the time of the first myofibril formation. J Cell Biol 105:2795–2801Google Scholar
  64. Turner CE, Glenney JR Jr, Burridge K (1990) Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol 111:1059–1068Google Scholar
  65. Volk T, Fessler LI, Fessler JH (1990) A role for integrin in the formation of sarcomeric cytoarchitecture. Cell 63:525–536Google Scholar
  66. Wang K (1985) Sarcomere-associated cytoskeletal lattices in striated muscle: review and hypothesis. In: Shay JW (ed) Cell and muscle motility, vol 6. Plenum Press, New York, pp 315–369Google Scholar
  67. Wang K, McClure J, Tu A (1979) Titin: major myofibrillar components of striated muscle. Proc Natl Acad Sci USA 76:3698–3702Google Scholar
  68. Wang S-M, Greaser ML, Schultz E, Bulinski JC, Lin JJ-C, Lessard JL (1988) Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin. J Cell Biol 107:1075–1083Google Scholar
  69. Yin HL, Stossel TP (1979) Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature 281:583–586Google Scholar
  70. Ylänne J, Virtanen I (1989) The Mr 140000 fibronectin receptor complex in normal and virus-transformed human fibroblasts and in fibrosarcoma cells: identical localization and function. Int J Cancer 43:1126–1136Google Scholar
  71. Zambonin-Zallone A, Teti A, Grano M, Rubinacci A, Abbadini M, Gaboli M, Marchisio PC (1989) Immunocytochemical distribution of extracellular matrix receptors in human osteoclasts: a β1 integrin is colocalized with vinculin and talin in the podosomes of osteoclastoma giant cells. Exp Cell Res 182:645–652Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Lula L. Hilenski
    • 1
  • Louis Terracio
    • 2
  • Thomas K. Borg
    • 1
  1. 1.Department of PathologyUniversity of South CarolinaColumbiaUSA
  2. 2.Department of Anatomy, Cell Biology and NeurosciencesUniversity of South CarolinaColumbiaUSA

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