Pflügers Archiv

, Volume 449, Issue 5, pp 449–457 | Cite as

Titin-based modulation of active tension and interfilament lattice spacing in skinned rat cardiac muscle

  • Norio Fukuda
  • Yiming Wu
  • Gerrie Farman
  • Thomas C. Irving
  • Henk Granzier
Cardiovascular System


The effect of titin-based passive tension on Ca2+ sensitivity of active tension and interfilament lattice spacing was studied in skinned rat ventricular trabeculae by measuring the sarcomere length (SL)-dependent change in Ca2+ sensitivity and performing small angle X-ray diffraction studies. To vary passive tension, preparations were treated with trypsin at a low concentration (0.31 μg/ml) for a short period (13 min) at 20°C, that resulted in ~40% degradation of the I-band region of titin, with a minimal effect on A-band titin. We found that the effect of trypsin on titin-based passive tension was significantly more pronounced immediately after stretch than at steady state, 30 min after stretch (i.e., trypsin has a greater effect on viscosity than on elasticity of passive cardiac muscle). Ca2+ sensitivity was decreased by trypsin treatment at SL 2.25 μm, but not at SL 1.9 μm, resulting in marked attenuation of the SL-dependent increase in Ca2+ sensitivity. The SL-dependent change in Ca2+ sensitivity was significantly correlated with titin-based passive tension. Small-angle X-ray diffraction experiments revealed that the lattice spacing expands after trypsin treatment, especially at SL 2.25 μm, providing an inverse linear relationship between the lattice spacing and Ca2+ sensitivity. These results support the view that titin-based passive tension promotes actomyosin interaction and that the mechanism includes interfilament lattice spacing modulation.


Calcium sensitivity Myocardium Sarcomere length X-ray diffraction 



This work is supported by a National Institutes of Health (NIH) Grant HL62881 (to H.G.). N.F. is a recipient of a grant from the Uehara Memorial Foundation (Tokyo, Japan). We would like to thank J. Henry, G.A. Kumar, and A. Joshi for help with X-ray data analysis. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Energy Research, under Contract No. W-31-109-ENG-38. BioCAT is an NIH-supported Research Center (RR08630).


  1. 1.
    Allen DG, Kentish JC (1985) The cellular basis for length-tension relation in cardiac muscle. J Mol Cell Cardiol 17: 821–840PubMedGoogle Scholar
  2. 2.
    Astier C, Labbe J, Roustan C, Benyamin Y (1993) Effects of different enzymic treatments on the release of titin fragments from rabbit skeletal myofibrils. Biochem J 290:731–734PubMedGoogle Scholar
  3. 3.
    Bang ML, Centner T, Fornoff F, Geach AJ, Gotthardt M, McNabb M, Witt CC, Labeit D, Gregorio CC, Granzier H, Labeit S (2001) The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circ Res 89:1065–1072PubMedGoogle Scholar
  4. 4.
    Cazorla O, Vassort G, Garnier D, Le Guennec JY (1999) Length modulation of active force in rat cardiac myocytes: is titin the sensor? J Mol Cell Cardiol 31: 1215–1227CrossRefPubMedGoogle Scholar
  5. 5.
    Cazorla O, Freiburg A, Helmes M, Centner T, McNabb M, Wu Y, Trombitas K, Labeit S, Granzier H (2000) Differential expression of cardiac titin isoforms and modulation of cellular stiffness. Circ Res 86:59–67PubMedGoogle Scholar
  6. 6.
    Cazorla O, Wu Y, Irving TC, Granzier H (2001) Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes. Circ Res 88: 1028–1035PubMedGoogle Scholar
  7. 7.
    Dobesh DP, Konhilas JP, de Tombe PP (2002) Cooperative activation in cardiac muscle: impact of sarcomere length. Am J Physiol Heart 282:H1055–H1062Google Scholar
  8. 8.
    Fitzsimons DP, Moss RL (1998) Strong binding of myosin modulates length-dependent Ca2+ activation of rat ventricular myocytes. Circ Res 83: 602–607PubMedGoogle Scholar
  9. 9.
    Fuchs F, Wang YP (1996) Sarcomere length versus interfilament lspacing as determinants of cardiac myofilament Ca2+ sensitivity and Ca2+ binding. J Mol Cell Cardiol 28:1375–1383CrossRefPubMedGoogle Scholar
  10. 10.
    Fukuda N, Granzier H (2004) Role of the giant elastic protein titin in the Frank-Starling mechanism of the heart. Curr Vasc Pharmacol 2:135–139PubMedGoogle Scholar
  11. 11.
    Fukuda N, Kajiwara H, Ishiwata S, Kurihara S (2000) Effects of MgADP on length dependence of tension generation in skinned rat cardiac muscle. Circ Res 86:e1–e6PubMedGoogle Scholar
  12. 12.
    Fukuda N, Sasaki D, Ishiwata S, Kurihara S (2001) Length dependence of tension generation in rat skinned cardiac muscle: role of titin in the Frank-Starling mechanism of the heart. Circulation 104:1639–1645PubMedGoogle Scholar
  13. 13.
    Fukuda N, Wu Y, Irving TC, Granzier H (2003) Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle. J Physiol (Lond) 553:147–154Google Scholar
  14. 14.
    Gordon AM, Huxley AF, Julian FJ (1966) The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J Physiol (Lond) 184:170–192Google Scholar
  15. 15.
    Granzier H, Irving TC (1995) Passive tension in cardiac muscle: contribution of collagen, titin, microtubles, and intermediate filaments. Biophys J 68:1027–1044PubMedGoogle Scholar
  16. 16.
    Granzier HL, Wang K (1993) Gel electrophoresis of giant proteins: solubilization and silver-staining of titin and nebulin from single muscle fiber segments. Electrophoresis 14:56–64PubMedGoogle Scholar
  17. 17.
    Haase H, Pagel I, Khalina Y, Zacharzowsky U, Person V, Lutsch G, Petzhold D, Kott M, Schaper J, Morano I (2004) The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction. FASEB J 18:839–841PubMedGoogle Scholar
  18. 18.
    Helmes M, Trombitas K, Granzier H (1996) Titin develops restoring force in rat cardiac myocytes. Circ Res 79: 619–626PubMedGoogle Scholar
  19. 19.
    Helmes M, Lim CC, Liao R, Bharti A, Cui L, Sawyer DB (2003) Titin determines the Frank-Starling relation in early diastole. J Gen Physiol 121:97–110CrossRefPubMedGoogle Scholar
  20. 20.
    Irving TC, Konhilas J, Perry D, Fischetti R, de Tombe PP (2000) Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium. Am J Physiol Heart 279:H2568–H2573Google Scholar
  21. 21.
    Kentish JC (1986) The effects of inorganic phosphate and creatine phosphate on force production in skinned muscles from rat ventricle. J Physiol (Lond) 370:585–604Google Scholar
  22. 22.
    Kentish JC, ter Keurs HEDJ, Ricciardi L, Bucx JJJ, Noble MIM (1986) Comparison between the sarcomere length-force relations of intact and skinned trabeculae from rat right ventricle. Influcence of calcium concentrations on these relations. Circ Res 58:755–768PubMedGoogle Scholar
  23. 23.
    Konhilas JP, Irving TC, de Tombe PP (2002) Myofilament calcium sensitivity in skinned rat cardiac trabeculae: role of interfilament spacing. Circ Res 90:59–65CrossRefPubMedGoogle Scholar
  24. 24.
    Konhilas JP, Irving TC, de Tombe PP (2002) Length-dependent activation in three striated muscle types of the rat. J Physiol (Lond) 544:225–236Google Scholar
  25. 25.
    Konhilas JP, Irving TC, Wolska BM, Jweied EE, Martin AF, Solaro RJ, de Tombe PP (2003) Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing. J Physiol (Lond) 547:951–961Google Scholar
  26. 26.
    Kulke M, Fujita-Becker S, Rostkova E, Neagoe C, Labeit D, Manstein DJ, Gautel M, Linke WA (2001) Interaction between PEVK-titin and actin filaments: origin of a viscous force component in cardiac myofibrils. Circ Res 89:874–881PubMedGoogle Scholar
  27. 27.
    Labeit S, Gautel M, Lakey A, Trinick J (1992) Towards a molecular understanding of titin. EMBO J 11:1711–1716PubMedGoogle Scholar
  28. 28.
    Lakatta EG (1987) Starling’s law of the heart is explained by an intimate interaction of muscle length and myofilament calcium activation. J Am Col Cardiol 10:1157–1164Google Scholar
  29. 29.
    McDonald KS, Moss RL (1995) Osmotic compression of single cardiac myocytes eliminates the reduction in Ca2+ sensitivity of tension at short sarcomere length. Circ Res 77:199–205PubMedGoogle Scholar
  30. 30.
    Mio Y, Fukuda N, Kusakari Y, Tanifuji Y, Kurihara S (2002) Bupivacaine attenuates contractility by decreasing sensitivity of myofilaments to Ca2+ in rat ventricular muscle. Anesthesiology 97:1168–1177CrossRefPubMedGoogle Scholar
  31. 31.
    Moss RL, Fitzsimons DP (2002) Frank-Starling relationship. Long on importance, short on mechanism. Circ Res 90:11–13CrossRefPubMedGoogle Scholar
  32. 32.
    Rice JJ, de Tombe PP (2004) Approaches to modeling crossbridges and calcium-dependent activation in cardiac muscle. Prog Biophys Mol Biol 85:179–195CrossRefPubMedGoogle Scholar
  33. 33.
    Trombitas K, Granzier H (1997) Actin removal from cardiac myocytes show that near Z-line titin attaches to actin while under tension. Am J Physiol 273:C662–C670PubMedGoogle Scholar
  34. 34.
    Trombitas K, Jin JP, Granzier H (1995) The mechanically active domain of titin in cardiac muscle. Circ Res 77:856–861PubMedGoogle Scholar
  35. 35.
    Wu Y, Cazolra O, Labeit D, Labeit S, Granzier H (2000) Changes in titin and collagen underlie diastolic stiffness diversity of cardiac muscle. J Mol Cell Cardiol 32:2151–2162CrossRefPubMedGoogle Scholar
  36. 36.
    Yamasaki R, Berri M, Wu Y, Trombitas K, McNabb M, Kellermayer MS, Witt C, Labeit D, Labeit S, Greaser M, Granzier H (2001) Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1. Biophys J 81:2297–2313PubMedGoogle Scholar
  37. 37.
    Yamasaki R, Wu Y, McNabb M, Greaser M, Labeit S, Granzier H (2002) Protein kinase A phosphorylates titin’s cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes. Circ Res 90:1181–1188CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag  2004

Authors and Affiliations

  • Norio Fukuda
    • 1
  • Yiming Wu
    • 1
  • Gerrie Farman
    • 2
  • Thomas C. Irving
    • 2
  • Henk Granzier
    • 1
  1. 1.Department of Veterinary and Comparative Anatomy, Pharmacology and PhysiologyWashington State UniversityPullmanUSA
  2. 2.Department of Biological, Chemical and Physical SciencesIllinois Institute of TechnologyChicagoUSA

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