Extraction and Replacement of the Tropomyosin–Troponin Complex in Isolated Myofibrils

  • Beatrice Scellini
  • Nicoletta Piroddi
  • Corrado Poggesi
  • Chiara Tesi
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 682)

Abstract

Tropomyosin (Tm) is an essential component in the regulation of striated muscle contraction. Questions about Tm functional role have been difficult to study because sarcomere Tm content is not as easily manipulated as Troponin (Tn). Here we describe the method we recently developed to replace Tm-Tn of skeletal and cardiac myofibrils from animals and humans to generate an experimental model of homogeneous Tm composition and giving the possibility to measure a wide range of mechanical parameters of contraction (e.g. maximal force and kinetics of force generation). The success of the exchange was determined by SDS–PAGE and by mechanical measurements of calcium dependent force activation on the reconstituted myofibrils. In skeletal and cardiac myofibrils, the percentage of Tm replacement was higher than 90%. Maximal isometric tension was 30–35% lower in the reconstituted myofibrils than in control myofibrils but the rate of force activation (kACT) and that of force redevelopment (kTR) were not significantly changed. Preliminary results show the effectiveness of Tm replacement in human cardiac myofibrils. This approach can be used to test the functional impact of Tm mutations responsible for human cardiomyopathies.

Keywords

Regulatory proteins Tropomyosin Troponin Myofibrils Cardiac Skeletal 

Notes

Acknowledgement

This work was supported by 7th Framework Programs of the European Union (STREP Project “BIG-HEART”, grant agreement 241577) and by Telethon-Italy (GGP07133). Authors gratefully acknowledge Dr. Earl Homsher for helpful discussion and technical advice.

References

  1. Brandt PW, Diamond MS, Schachat FH (1984) The thin filament of vertebrate skeletal muscle co-operatively activates as a unit. J Mol Biol 180:379–84PubMedCrossRefGoogle Scholar
  2. Brenner B (1988) Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc Natl Acad Sci U S A 85:3265–3269PubMedCrossRefGoogle Scholar
  3. Brenner B, Kraft T, Yu L, Chalovich JM (1999) Thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle. Biophys J 77:2677–2691PubMedCrossRefGoogle Scholar
  4. Chang AN, Harada K, Ackerman MJ, Potter JD (2005) Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in alpha-tropomyosin. J Biol Chem 280:34343–3439PubMedCrossRefGoogle Scholar
  5. Clemmens EW, Entezari M, Martyn DA, Regnier M (2005) Different effects of cardiac versus skeletal muscle regulatory proteins on in vitro measures of actin filament speed and force. J Physiol 566:737–46PubMedCrossRefGoogle Scholar
  6. Colomo F, Piroddi N, Poggesi C, te Kronnie G, Tesi C (1997) Active and passive forces of isolated myofibrils from cardiac and fast skeletal muscle of the frog. J Physiol 500:535–548PubMedGoogle Scholar
  7. Cummins P, Perry SV (1973) The subunits and biological activity of polymorphic forms of tropomyosin. Biochem J 133:765–777PubMedGoogle Scholar
  8. de Tombe PP, Belus A, Piroddi N, Scellini B, Walker JS, Martin AF, Tesi C, Poggesi C. (2007) Myofilament calcium sensitivity does not affect cross-bridge activation-relaxation kinetics. Am J Physiol Regul Integr Comp Physiol 292:R1129–1136PubMedCrossRefGoogle Scholar
  9. Fujita H, Yasuda K, Niitsu S, Funatsu T, Ishiwata S (1996) Structural and functional reconstitution of thin filaments in the contractile apparatus of cardiac muscle. Biophys J 71:2307–2318PubMedCrossRefGoogle Scholar
  10. Fujita H, Sasaki D, Ishiwata S, Kawai M (2002) Elementary steps of the cross-bridge cycle in bovine myocardium with and without regulatory proteins. Biophys J 82:915–28PubMedCrossRefGoogle Scholar
  11. Gordon AM, Homsher E, Regnier M (2000) Regulation of contraction in striated muscle. Physiol Rev 80:853–924PubMedGoogle Scholar
  12. Gunning P, O’Neill G, Hardeman E (2008) Tropomyosin-based regulation of the actin cytoskeleton in time and space. Physiol Rev 88: 1–835PubMedCrossRefGoogle Scholar
  13. Jagatheesan G, Rajan S, Schulz EM, Ahmed RP, Petrashevskaya N, Schwartz A, Boivin GP, Arteaga GM, Wang T, Wang YG, Ashraf M, Liggett SB, Lorenz J, Solaro RJ, Wieczorek DF (2009) An internal domain of {beta}-tropomyosin increases myofilament Ca2+ sensitivity. Am J Physiol Heart Circ Physiol 297:H181–190PubMedCrossRefGoogle Scholar
  14. Kominz DR, Yoshioka K (1969) The influence of native tropomyosin on the ATP threshold for turbidity development of actomyosin and myofibril suspensions. Arch Biochem Biophys 129:609–614PubMedCrossRefGoogle Scholar
  15. Kreutziger KL, Piroddi N, Scellini B, Tesi C, Poggesi C, Regnier M (2008) Thin filament Ca2+ binding properties and regulatory unit interactions alter kinetics of tension development and relaxation in rabbit skeletal muscle. J Physiol 586:3683–700PubMedCrossRefGoogle Scholar
  16. Kruger M, Zittrich S, Redwood C, Blaudeck N, James J, Robbins J, Pfitzer G, Stehle R (2005) Functional consequences of hypertrophic and dilated cardiomyopathy-causing mutations in alpha-tropomyosin. J Physiol 564:347–357PubMedCrossRefGoogle Scholar
  17. Moss RL, Allen JD, Greaser ML (1986) Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament. J Gen Physiol 87:761–74Google Scholar
  18. Muthuchamy M, Pieples K, Rethinasamy P, Hoit B, Grupp IL, Boivin GP, Wolska B, Evans C, Solaro RJ, Wieczorek DF (1999) Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction. Circ Res 85:47–56PubMedCrossRefGoogle Scholar
  19. Narolska NA, Piroddi N, Belus A, Boontje NM, Scellini B, Deppermann S, Zaremba R, Musters RJ, dos Remedios C, Jaquet K, Foster DB, Murphy AM, van Eyk JE, Tesi C, Poggesi C, van der Velden J, Stienen GJ (2006) Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction-relaxation cycle in isolated cardiac myofibrils. Circ Res 99:1012–1020PubMedCrossRefGoogle Scholar
  20. Ochala J, Li M, Ohlsson M, Oldfors A, Larsson L (2008) Defective regulation of contractile function in muscle fibres carrying an E41K beta-tropomyosin mutation. J Physiol 586:2993–3004PubMedCrossRefGoogle Scholar
  21. Perry SV, Corsi A (1958) Extraction of proteins other than myosin from the isolated rabbit myofibril. Biochem J. 68:5–12PubMedGoogle Scholar
  22. Pieples K, Arteaga G, Solaro RJ, Grupp I, Lorenz JN, Boivin GP, Jagatheesan G, Labitzke E, DeTombe PP, Konhilas JP, Irving TC, Wieczorek DF (2002) Tropomyosin 3 expression leads to hypercontractility and attenuates myofilament length-dependent Ca(2+) activation. Am J Physiol Heart Circ Physiol 283:H1344–1353PubMedGoogle Scholar
  23. Piroddi N, Tesi C, Pellegrino MA, Tobacman LS, Homsher E, Poggesi C (2003) Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils. J Physiol 552:917–931PubMedCrossRefGoogle Scholar
  24. Piroddi N, Belus A, Eiras S, Tesi C, van der Velden J, Poggesi C, Stienen GJ (2006) No direct effect of creatine phosphate on the cross-bridge cycle in cardiac myofibrils. Pflugers Arch 452:3–6PubMedCrossRefGoogle Scholar
  25. Piroddi N, Belus A, Scellini B, Tesi C, Giunti G, Cerbai E, Mugelli A, Poggesi C (2007) Tension generation and relaxation in single myofibrils from human atrial and ventricular myocardium. Pflügers Arch 454:63–73PubMedCrossRefGoogle Scholar
  26. Salviati G, Betto R, Danieli Betto D (1982) Polymorphism of myofibrillar proteins of rabbit skeletal-muscle fibres. An electrophoretic study of single fibres. Biochem J. 207:261–272Google Scholar
  27. She M, Trimble D, Yu L, Chalovich JM (2000) Factors contributing to troponin exchange in myofibrils and in solution. J Mus Res Cell Mot 21:737–745CrossRefGoogle Scholar
  28. Siththanandan VB, Tobacman LS, Van Gorder N, Homsher E (2009) Mechanical and kinetic effects of shortened tropomyosin reconstituted into myofibrils. Pflugers Arch 458:761–776PubMedCrossRefGoogle Scholar
  29. Tesi C, Colomo F, Nencini S, Piroddi N, Poggesi C (1999) Modulation by substrate concentration of maximal shortening velocity and isometric force in single myofibrils from frog and rabbit fast skeletal muscle. J Physiol 516:847–853PubMedCrossRefGoogle Scholar
  30. Tesi C, Colomo F, Nencini S, Piroddi N, Poggesi C (2000) The effect of inorganic phosphate on force generation in single myofibrils from rabbit skeletal muscle. Biophys J 78:3081–3092PubMedCrossRefGoogle Scholar
  31. Tesi C, Colomo F, Piroddi N, Poggesi C (2002a) Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles. J Physiol. 541:187–99PubMedCrossRefGoogle Scholar
  32. Tesi C, Piroddi N, Colomo F, Poggesi C (2002b) Relaxation kinetics following sudden Ca(2+) reduction in single myofibrils from skeletal muscle. Biophys J 83:2142–2151PubMedCrossRefGoogle Scholar
  33. Wolska BM, Keller RS, Evans CC, Palmiter KA, Phillips RM, Muthuchamy M, Oehlenschlager J, Wieczorek DF, de Tombe PP, Solaro RJ (1999) Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta-tropomyosin. Circ Res 1999 84:745–51PubMedCrossRefGoogle Scholar
  34. Yang Z, Yamazaki M, Shen QW, Swartz DR (2009) Differences between cardiac and skeletal troponin interaction with the thin filament probed by troponin exchange in skeletal myofibrils. Biophys J 97:183–94PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Beatrice Scellini
  • Nicoletta Piroddi
  • Corrado Poggesi
  • Chiara Tesi
    • 1
  1. 1.Dipartimento di Scienze FisiologicheUniversità di FirenzeFirenzeItaly

Personalised recommendations