Titin Research Roundup

Titin is a high-molecular weight protein (3.0-3.8 MDa) in the sarcomeres of vertebrate striated muscles. A plethora of titin isoforms is expressed from the copy of a single titin gene (on chromosome 2 in mouse and humans). A current view of the arrangement of titin in the sarcomere and titin's domain architecture is shown in Fig 1. Titin's role in muscle structure and function is multifaceted. The polypeptide (1) provides a structural framework through association with other proteins of the sarcomere, (2) keeps the thick filament centered in the sarcomere during activation, (3) functions as a molecular spring in muscle sarcomeres, and (4) is involved in myocyte signaling, associates with signaling molecules and probably has a central place in the stress-sensing machinery of the muscle cell. Currently more than 15 sarcomeric and non-sarcomeric proteins are known to interact with titin. Elucidation of titin's primary structure revealed that the polypeptide contains, near its COOH-terminal end (M-line), a kinase domain (Fig. 1), which may be active during muscle development. Titin also contains a number of phosphorylation sites, in the M-line, Z-disk and I-band, which may be important for the function of titin in signal transduction.

Figure 1:

Three-filament model of the sarcomere and domain architecture of titin isoforms according to Freiburg et al. (2000). Shown are the structures of human titin isoforms. The I-band and A-band sections of the molecule are indicated, which represent titin's elastic and stiff regions, respectively. A-band titin consists of two motif types, immunoglobulin-like (Ig) modules and fibronectin type-3-like (FN3) domains; both types are arranged in super-repeat patterns. Also shown is the position of the titin kinase domain. M-line titin and Z-disk titin contain no fibronectin modules but are composed of Ig domains interspersed with unique sequences. The main section of I-band titin is made up of two structurally distinct regions, stretches of tandem-Ig modules that adopt a stable beta-sheet fold (Politou et al., 1995), separated by sequences with poorly defined secondary structure, such as the PEVK domain and the cardiac-specific "N2B-unique sequence". The proximal Ig-segment and the PEVK domain are subject to differential splicing. Cardiac muscle co-expresses N2B and N2BA isoforms, skeletal muscle has N2A isoform only.

Elastic region of titin. A particularly important property of titin filaments is their ability to act as molecular springs providing relaxed myofibrils with elasticity. Although single titin polypeptides span the entire half of a sarcomere, only a molecular segment confined to the I-band is functionally elastic (Fig. 1). This segment consists of two main, structurally distinct, regions. One region is a unique sequence rich in proline (P), glutamate (E), valine (V) and lysine (K) residues, termed the PEVK segment, which has no obvious secondary or tertiary structure. The PEVK domain is flanked on either side by a region made up of serially linked immunoglobulin-like (Ig) domains--modules that fold into seven-stranded beta-barrels. Both regions are expressed in muscle-type specific length isoforms. Skeletal-muscle sarcomeres contain a so-called N2-A isoform, whose length varies in different tissue types. Cardiac cells co-express an N2-B isoform and N2-BA isoforms, the latter of which contain structural elements from both the N2-A and N2-B isoforms and vary in length in their proximal Ig segments and PEVK-domain. Cardiac titin also contains a unique region termed "N2-B" (Fig. 1). We demonstrated that the Ig-domain regions confer extensibility to the sarcomere mainly at low stretch, the PEVK domain predominantly at higher stretch. In cardiac muscle also the N2-B unique sequence extens at high stretch forces. The elastic region of titin interacts with several other proteins, e.g., actin and the chaperone alpha-B-crystallin, but other proteins important for myocyte signaling and turnover as well. Weak interactions of titin's PEVK-domain with the thin filament give rise to a viscous force component during quick length changes of muscle.

Molecular basis of titin elasticity. State-of-the-art techniques have been used to explore titin elasticity at the single molecule level. Laser trapping and atomic force microscopy (AFM) studies on native or engineered titin molecules or titin domains have revealed a nonlinear force response upon stretching, which was explained in terms of the entropic-chain characteristics of the polypeptide. An entropic chain undergoes thermally-induced bending movements that tend to shorten its end-to-end length (Fig. 2). Stretching such a chain reduces its conformational entropy and thus, requires an external force. In the mechanical studies on isolated proteins, titin's force-extension relation could be well fitted with the wormlike chain (WLC) model of entropic elasticity. A WLC is a deformable rod whose bending rigidity is characterized by its persistence length, a distance within which the orientations of the chain are correlated. Rigid polymers have a long persistence length (relative to their contour length) and their straightening requires little external force, while the opposite is true for more flexible polymers. In titin, the Ig-domain regions represent WLC elements with relatively long persistence length. In addition, these regions appear to be capable of adjusting their contour lengths by unfolding a few of their Ig modules. Ig-domain unfolding has been readily shown in AFM stretch experiments on single recombinant polyproteins containing only a small number of Ig modules. The PEVK domain and the N2-B unique sequence are entropic springs with a relatively short persistence length, which explains why these regions extend after the Ig-domain segments. Currently it is debated whether titin's unique sequences also have an "enthalpic" component of elasticity, brought about e.g. by electrostatic or hydrophobic interactions.

Figure 2 : Entropic spring undergoing thermodynamic fluctuations.

Titin and active force generation. The titin filament could play some active role in contraction. The elastic section of titin exerts a retractive force, which tends to shorten the sarcomere (in cardiac muscle at end diastole, this force can be appreciable); the force is effectively contractile. It has been proposed that titin could stiffen upon activation--already before sarcomeric stiffness increases when myosin cross-bridges attach strongly to actin--perhaps due to the rise in intracellular [Ca2+]. Titin's "contractile" force would thus increase. Indeed, calcium binds to titin at the PEVK domain. Thus, the possibility exists that Ca2+ affects titin stiffness. Recently, we found that the PEVK domain of cardiac titin interacts with actin filaments in a Ca2+-regulated manner, thereby modulating thin-thick filament interaction. It is likely that titin plays other important roles for active contractile functions in cardiac muscle, e.g., through an effect on the length-dependent contractility of the heart (the so-called Frank-Starling mechanism).

Cardiac titin and human heart disease. Human-heart sarcomeres coexpress a stiffer N2B titin and more compliant N2BA titin isoforms. The ratio between N2BA and N2B titin is about 30:70 in normal human heart, but can increase to approximately 50:50 in failing hearts from coronary artery disease (CAD) patients. The titin-isoform shift leads to decreased titin-derived stiffness of the cardiomyofibrils, although whole hearts usually are globally stiffened due to increased fibrosis. Thus, titin can be modified in response to chronic human heart disease. Higher (sometimes perhaps lower) than normal or unchanged N2BA:N2B titin ratios may be found under different disease conditions.

Cardiac titin in heart development. Before birth the hearts of rats, mice, pigs, and other species, express very long, compliant, embryonic/fetal titin isoforms of the N2BA-type, which can be as large as 3.7 MDa. The postnatal heart contains much shorter, stiffer, titin isoforms, including the N2B-isoform.

Further reading (related original publications from our laboratory)

  1. Association of the chaperone alpha-B-crystallin with titin in heart muscle
    Bullard B., C. Ferguson, A. Minajeva, M.C. Leake, M. Gautel, D. Labeit, L. Ding, S. Labeit, J. Horwitz, K. Leonard & W.A. Linke , J. Biol. Chem. (2004) , 279 , 7917-7924

  2. Interaction between PEVK-titin and actin filaments: origin of a viscous force component in cardiac myofibrils
    Kulke M., S. Fujita-Becker, E. Rostkova, C. Neagoe, D. Labeit, D.J. Manstein, M. Gautel & W.A. Linke , Circ. Res. (2001) , 89 , 874-881

  3. Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle
    Kulke M., C. Neagoe, B. Kolmerer, A. Minajeva, H. Hinssen, B. Bullard & W.A. Linke , J. Cell Biol. (2001) , 154 , 1045-1057

  4. Reverse engineering of the giant muscle protein titin
    Li H., W.A. Linke, A.F. Oberhauser, M. Carrion-Vazquez, J.B. Kerkvliet, H. Lu, P.E. Marszalek & J.M. Fernandez , Nature (2002) , 418 , 998-1002

  5. Towards a molecular understanding of the elasticity of titin
    Linke W.A., M. Ivemeyer, N. Olivieri, B. Kolmerer, J.C. Rüegg & S. Labeit , J. Mol. Biol. (1996) , 261 , 62-71

  6. Nature of PEVK-titin elasticity in skeletal muscle
    Linke W.A., M. Ivemeyer, P. Mundel, M.R. Stockmeier & B. Kolmerer , Proc. Natl. Acad. Sci. USA (1998) , 95 , 8052-8057

  7. Passive stiffness changes caused by upregulation of compliant titin isoforms in human dilated cardiomyopathy hearts
    Makarenko, I., C.A. Opitz, M.C. Leake, C. Neagoe, M. Kulke, J.K. Gwathmey, F. del Monte, R.J. Hajjar & W.A. Linke , Circ. Res. (2004) , 95 , 708-716

  8. Titin-based contribution to shortening velocity of rabbit skeletal myofibrils
    Minajeva, A., C. Neagoe, M. Kulke & W.A. Linke , J. Physiol. (2002) , 540 , 177-188

  9. Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils
    Minajeva A., M. Kulke, J.M. Fernandez & W.A. Linke , Biophys. J. (2001) , 80 , 1442-1451

  10. Titin isoform switch in ischemic human heart disease
    Neagoe C., M. Kulke, F. del Monte, J.K. Gwathmey, P.P. de Tombe, R.J. Hajjar & W.A. Linke , Circulation (2002) , 106 , 1333-1341

  11. Damped elastic recoil of the titin spring in myofibrils of human myocardium
    Opitz, C.A., M. Kulke, M. C. Leake, C. Neagoe, H. Hinssen, R.J. Hajjar & W.A. Linke , Proc. Natl. Acad. Sci. USA (2003) , 100 , 12688-12693

  12. Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart
    Opitz, C.A., M.C. Leake, I. Makarenko, V. Benes & W.A. Linke , Circ. Res. (2004) , 94 , 967-975

  13. Isoform diversity of giant proteins in relation to passive and active contractile properties of rabbit skeletal muscles.
    Prado, L., I. Makarenko, C. Andresen, M. Krüger, C.A. Opitz & W.A. Linke , J. Gen. Physiol. (2005) , 126 , 461-480

Review Articles

  1. The giant protein titin: Emerging roles in physiology and pathophysiology
    Labeit, S., B. Kolmerer & W.A. Linke , Circ. Res. (1997) , 80 , 290-294

  2. A spring tale: New facts on titin elasticity
    Linke, W.A. & H. Granzier , Biophys. J. (1998) , 75 , 2613-2614

  3. Gigantic variety: Expression patterns of titin isoforms in striated muscles and consequences for myofibrillar passive stiffness
    Neagoe, C., C.A. Opitz, I. Makarenko & W.A. Linke , J. Muscle Res. Cell Motil. (2003) , 24 , 175-189