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. 2002 Jun 1;541(Pt 2):335–342. doi: 10.1113/jphysiol.2001.014381

Cardiac titin: an adjustable multi-functional spring

Henk Granzier *, Siegfried Labeit †
PMCID: PMC2290327  PMID: 12042342

Abstract

The giant elastic protein titin contains a molecular spring segment that underlies the majority of myocardial passive stiffness. The mechanical characteristics of this spring may be tuned to match changing mechanical demands placed on muscle, using mechanisms that operate on different time scales and that include post-transcriptional and post-translational processes. Recent work also suggests that titin performs roles that go beyond passive stiffness generation. In contracting myocardium, titin may modulate actomyosin interaction by a titin-based alteration of the distance between myosin heads and actin. Furthermore, novel ligands have been identified that link titin to membrane channels, protein turnover and gene expression. This review highlights that titin is a versatile and adjustable spring with a range of important functions in passive and contracting myocardium.

Titin is the third myofilament type of vertebrate striated muscle, with a single molecule spanning the half-sarcomere, from the Z-line to the M-line (for recent reviews with original citations, see Wang, 1996; Labeit et al. 1997; Maruyama, 1997; Gregorio et al. 1999; Trinick & Tskhovrebova, 1999; Linke, 2000). Passive tension is generated by titin's extensible region, found in the I-band of the sarcomere and comprising serially linked and mechanically distinct spring elements. Two elements are found in skeletal muscles: the tandem Ig and PEVK elements that predominantly extend in short and long sarcomeres, respectively (Linke & Granzier, 1998; Trombitas et al. 1998). In cardiac titin, a third spring is formed by the N2B element whose extension dominates at the upper physiological sarcomere lengths (Helmes et al. 1999; Linke et al. 1999; Trombitas et al. 1999). This three-element spring gives rise to a unique force-extension curve that underlies the majority of the passive force of the myocardium (Fig. 1 and Wu et al. 2000).

Figure 1. Cardiac titin's extensible I-band region is a three-element molecular spring.

Figure 1

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A, in slack sarcomeres the extensible region is in a contracted state. Upon sarcomere stretch, initially extension of tandem Ig segments dominates (spring element 1; red) followed by dominating PEVK (yellow) and N2B unique sequence (blue) extension (elements 2 and 3). When sarcomeres shorten below slack, springs extend in the opposite direction of that during stretch, giving rise to restoring force. The inextensible region of titin in and near the Z-line is indicated in grey. Note that the schematic is not to scale. B, titin-based passive tension contributes more than half to total passive tension of cardiac muscle at a wide range of sarcomere lengths. (Non-titin-based passive tension is largely derived from collagen. For details see Wu et al. (2000).)

Considering that heart rates and ventricular pressures encountered in different species vary greatly, variations in cardiac titin's elastic properties may be anticipated. Titin's stiffness may also respond to exercise and mechanical stress. Indeed, a variety of mechanisms have emerged recently which modulate titin's elastic properties, and we discuss these mechanisms below (section 1). Evidence is also mounting that titin performs roles that go beyond passive force generation; we review work that indicates that titin may affect active force development (section 2) and that titin interacts with signalling molecules (section 3).

(1A) Modulation of titin's elasticity by differential splicing of spring elements

Titin is encoded by a single gene located in human and mouse on chromosome 2 (Fig. 2). The recent genomic analysis of human titin revealed 363 exons that code for a total of 38,138 amino acid residues (mol. mass 4200 kDa; Bang et al. 2001). This includes 108 exons that code for conserved ∼28-residue PEVK-repeats, possibly corresponding to structural spring units (Freiburg et al. 2000; Greaser, 2001). Interestingly, two titin exons, M10 and novex-III, function as alternative C-termini (Bang et al. 2001), giving rise to truncated (Z1-Z2 to novex-III; mol. mass ∼600 kDa), and full-length titins (Z1 to M10; mol. mass > 2970 kDa). The truncated novex-3 titin isoform can integrate into the Z-line lattice but is too short to reach the A-band, and is expressed in both skeletal and cardiac muscles (Bang et al. 2001). Co-expression of truncated and full-length titins may adjust the titin filament system to both 3- and 2-fold symmetries of thick and thin filaments, respectively (Bang et al. 2001).

Figure 2. Exon-intron structure of human titin gene.

Figure 2

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Note novel exons I-III. Binding sites of known titin ligands are indicated. Top left: example of a signalling complex in which titin may contribute to regulation of ion channel activity. (For details see text and Bang et al. (2001).)

Full-length titins, spanning from Z1 to M10, have highly variable I-band segments, generated by differential splicing (Fig. 3). In the heart, titin transcripts are processed by distinct splice routes, giving rise to so-called N2B titins (containing the N2B sequence) and N2BA titins (containing N2B and N2A sequences). N2BA titins have a longer PEVK segment and more Ig domains than N2B titins (Fig. 3). The expression level of N2B and N2BA cardiac titins varies from predominantly N2B (small rodents) to predominantly N2BA (bovine atrium), with many large mammals (including human) co-expressing both isoforms at intermediate levels (Cazorla et al. 2000). Passive stiffness of cardiac myocytes is much higher in N2B-expressing myocytes than in N2BA myocytes (Fig. 4). The higher passive stiffness is likely to result from the shorter I-band segment of N2B titin which, at a given sarcomere length, results in an extensible segment with a fractional extension (end-to-end length divided by the maximal length) that is higher than in N2BA titin (Trombitas et al. 2000).

Figure 3. Splice isoform diversity in titin's I-band regions.

Figure 3

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Skeletal muscles express N2A-based titins that vary in size in different muscle types (two of many possible splice pathways are shown). Heart muscles express large N2BA titins and small N2B titins. All striated muscles express novex-3 titin. (Based on Freiburg et al. (2000) and Bang et al. (2001).)

Figure 4. Co-expression of cardiac isoforms and passive tension modulation.

Figure 4

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Micrographs: sarcomeres of cells labelled with N2BC antibody that binds C-terminal of the N2B unique sequence. To the left of each micrograph an electrophoresis result is shown. N2B-expressing cells (top) have a single N2BC epitope near the A-band (epitope b) and develop high passive tensions (left), whereas N2BA-expressing cells (bottom) have a single N2BC epitope near the Z-line (epitope a) and develop low passive tensions. The two I-band epitopes in the middle micrograph suggest co-expression of N2B and N2BA isoforms at the level of the half-sarcomere. These cells develop intermediate force levels. (Based on Trombitas et al. (2001).)

The high titin-based passive stiffness provided by N2B titin may allow rapid and stable determination of the end-diastolic volume at the high beat frequencies encountered in small mammals (where N2B titin dominates). Titin is also expected to play a role in centring the A-band within the sarcomere (Horowits & Podolsky, 1987) and the high N2B-based passive stiffness may rapidly reset the A-band location during each diastole. It is also worthwhile to consider titin's contribution to restoring forces (Helmes et al. 1996). The segment of titin near the Z-line binds the thin filament and can withstand compressive forces (Fig. 1A and Trombitas & Granzier, 1997). When sarcomeres shorten to below the slack length, the thick filament moves into the stiff region of titin near the Z-line, and titin's extensible region extends in a direction that is opposite of that during stretch (Granzier et al. 2000). This gives rise to so-called restoring forces (pushing Z-lines away from each other) and these forces are expected to be highest in N2B-expressing myocytes. Considering that titin's restoring force may contribute to the early diastolic suction force that aids ventricular filling, expressing high levels of N2B titin may be relevant for achieving rapid diastolic filling.

(1B) Co-expression of cardiac titin isoforms

Ventricular myocardium of large mammals co-expresses titin isoforms at the level of the half-sarcomere, and co-expression results in passive tension levels intermediate between that of N2B and N2BA pure myocytes (Fig. 4). Such intermediate tensions, in theory, could be achieved by varying the number of titin molecules per thick filament. However, this would also influence functions performed by titin's inextensible regions, such as thick-filament length control and construction and maintenance of Z-lines and M-lines (Gregorio et al. 1999). When passive force levels are tuned via variation in the isoform expression ratio, the inextensible regions are not impacted because these regions are the same in different isoforms (Labeit & Kolmerer, 1995). Thus co-expressing isoforms at various ratios is an effective means for tuning passive properties, and any force level intermediate between that of isoform-pure cells may be obtained (see shaded area of passive tension curve in Fig. 4). Studies on both canine and human myocardium (Bell et al. 2000; Wu et al. 2002) suggest that this mechanism may be utilized to adjust diastolic stiffness during heart disease.

(1C) Post-translational modulation of titin's spring properties

It was recently shown (Yamasaki et al. 2002) that the N2B unique sequence of cardiac titin can be phosphorylated by protein kinase A (PKA). Thus as with the well-characterized myofibrillar PKA substrates MyBP-C and TnI, titin contains a PKA-responsive domain expressed only in cardiac muscle. Interestingly, PKA-based phosphorylation of titin results in a reduction of passive tension of cardiac myocytes (Strang et al. 1994; Yamasaki et al. 2002). This reduction may be explained by assuming that phosphorylation destabilizes native structures within the N2B element, causing it to extend and lower its fractional extension. Considering that the activation of PKA via β-adrenergic stimulation constitutes a major regulatory pathway in the heart, the PKA-responsive element of cardiac titins may allow modulation of diastolic function in vivo.

(1D) Titin-actin interaction modulates titin's stiffness

Earlier studies have suggested that in cardiac muscle, interactions between titin and actin occur (reviewed in Yamasaki et al. 2001). In vitro binding studies revealed that the PEVK element of N2B titin binds F-actin at physiological ionic strengths and that as F-actin slides relative to titin in the in vitro motility assay, a dynamic interaction between the PEVK domain and F-actin retards filament sliding (Kulke et al. 2001; Yamasaki et al. 2001). Mechanical experiments suggest that a similar interaction makes a significant contribution to passive stiffness of the sarcomere (Yamasaki et al. 2001). Although physiological levels of calcium alone have no effect, S100A1, a calcium-binding protein found at high concentrations in myocardium, inhibits PEVK-actin interaction in a calcium-sensitive manner. Thus a dynamic interaction between titin and actin contributes to passive stiffness of the sarcomere and the interaction varies with the physiological state of the myocardium.

(2) Effect of titin's passive force on actomyosin interaction

Recent evidence suggests that titin may play a role in regulating active force. Cazorla et al. (2001) reported that titin-based passive force enhances length-dependent activation of cardiac myocytes. Length-dependent activation was studied by measuring the active force-pCa (-log[Ca2+]) relationship at sarcomere lengths (SL) of 2.0 and 2.3 μm, determining at each length the pCa value that gives a force of 50 % of maximal (pCa50) and calculating the difference in pCa50 at 2.3 and 2.0 μm (ΔpCa50). Increasing sarcomere length was found to result in an increase in calcium sensitivity, as reflected by the positive ΔpCa50 values (Fig. 5A). Interestingly, when passive tension at 2.3 μm SL was varied by adjusting the characteristics of the stretch imposed on the passive cell prior to activation, calcium sensitivity varied as well (Fig. 5A). For maximal calcium sensitivity of skinned cardiac myocytes, a high level of titin-based passive tension appears to be required.

Figure 5. Effect of passive tension on active force development of mouse cardiac myocytes.

Figure 5

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A, force-pCa relationship at 2.0 μm SL (▪) and 2.3 μm with high (∼9 mN mm−2; •) and low passive tension (∼2 mN mm−2; ○). Curves at long length are shifted leftwards and shift is largest at high passive tension. Inset: effect of a range of passive tensions on ΔpCa50. B, X-ray patterns from mouse skinned muscle in relaxing solution (SL: 2.0 μm) showing equatorial 1,0 reflections. Following titin degradation (bottom) the 1,0 spacing (S) is reduced, indicating filament lattice expansion. C, model of titin-based modulation of myofilament lattice spacing and head mobility. Titin's force (F) has a longitudinal and radial component, the latter of which compresses the myofilament lattice. At long SL, titin's longitudinal force slightly increases thick filament strain (vertical left line) and this is postulated to increase myosin head mobility (note that heads are further from the thick filament backbone than at short SL) and probability of actomyosin interaction. See text for details. (Based on Cazorla et al. (2001).)

To probe the mechanism for this effect, small-angle X-ray diffraction studies were performed (Cazorla et al. 2001). Degradation of titin was found to significantly increase lattice spacing (Fig. 5B). Inspection of titin's layout in the sarcomere (Fig. 5C) reveals a possible mechanism for how titin may affect lattice spacing. The segment of titin near the Z-line binds strongly to the thin filament (Trombitas & Granzier, 1997) and in the A-band titin attaches to the thick filament (Trombitas et al. 1995). Thus the elastic region of titin runs obliquely to the thin and thick filaments and titin is expected to develop a longitudinal force (FL) and radial (Fr) force, the latter of which compresses the lattice.

Interfilament lattice spacing is widely held as a determinant of the probability of actomyosin interaction at a given calcium concentration (McDonald & Moss, 1995;Fuchs & Wang, 1996), and the effect of titin on lattice spacing may thus explain the effect of titin's passive tension on the length dependence of calcium sensitivity. However, considering the recent work that showed that lattice spacing and calcium sensitivity are not well correlated (Konhilas et al. 2002), it is also worth considering that titin influences active force via an earlier proposed mechanism (Granzier & Wang, 1993) in which the likelihood of cross-bridge interaction is enhanced by passive force-induced thick filament strain (Fig. 5C, bottom). This mechanism also provides an explanation for the reported length-dependent effect of titin on maximal active tension in rat cardiac trabeculae (Fukuda et al. 2001). Thus the Frank-Starling mechanism of the heart (Allen & Kentish, 1985) may in part be due to an effect of titin-based force on the length dependence of maximal active tension and calcium sensitivity.

Recent work (Muhle-Goll et al. 2001) suggests that fibronectin (fn)-like domains from the A-band region of titin may play a role in calcium sensitivity. Recombinant fragments containing fn domains bind in vitro weakly to myosin S1 and when added to skinned myocytes they reduce the length dependence of activation (primarily due to an increase in calcium sensitivity at short length). It was postulated that in endogenous titin, fn domains weakly interact with myosin heads and that this keeps the average position of the heads close to the thick filament backbone, away from their actin binding site. Such an interaction would reduce the likelihood of actomyosin interaction, especially at short sarcomere length where myofilament lattice spacing is largest, and where the crossbridge requires optimal mobility to reach its binding site on actin. The exogenous fragments may compete with native titin for myosin binding sites and abolish the inhibitory effect of native titin on actomyosin interaction at short sarcomere length.

In summary, several recent studies indicate that titin is not just a passive spring, but that titin also influences active force development. We propose that the underlying mechanisms involve titin-based effects on lattice spacing and/or head mobility that modulate the distance between the myosin head and its actin binding site.

(3) Association of titin with signalling molecules

During the last few years a diverse family of titin binding proteins has been identified, which suggests the participation of the titin filament system in the regulation of ion channels, protein turnover and gene expression (see Fig. 2). At the edge of the Z-line, titin's N-terminal domains Z1-Z2 interact with the 19 kDa protein Tcap, which in turn associates with the minK β-subunit of the stretch-regulated IKs potassium channel (Furukawa et al. 2001). Thus titin's passive force might be transmitted to ion channels and may possibly influence their activity. In the I-band, the novex-3 titin isoform (Fig. 2 and Fig. 3) interacts with obscurin (Bang et al. 2001), a ∼720 kDa protein containing several types of signalling domains (Young et al. 2001). Because the novex-3 titin/obscurin complex extends in stretched sarcomeres (Bang et al. 2001), the complex may have signalling properties that respond to sarcomere stretch and may be involved in stretch-initiated sarcomeric restructuring that occurs during muscle adaptation and disease.

The calpain-regulated protease p94/CAPN3 binds near titin's C-terminus and within the N2A element of skeletal muscle and N2BA cardiac titins (Sorimachi et al. 1995 and Fig. 2), suggesting a role for titin in protein turnover. Near the C-terminus, titin also contains a serine/threonine kinase domain (Labeit & Kolmerer, 1995). It has been speculated that the titin kinase phosphorylates the Z-line-associated protein telethonin/Tcap during myofibrillogenesis (Mayans et al. 1998), and that it may play additional roles in adult tissues (Centner et al. 2001). In proximity to the titin kinase domain, the muscle-specific RING finger protein MURF-1 associates with titin (Centner et al. 2001). The interaction of titin and MURF-1 is essential for integrity of the M-line lattice (McElhinny et al. 2002). Interestingly, MURF-1 also interacts with the transcriptional co-factor GMEB1. Therefore, the interaction of MURF-1 with the titin kinase region could be involved in the regulation of muscle gene expression (McElhinny et al. 2002). Thus titin binds in the sarcomere to a host of ligands, and titin's functional significance is likely to go far beyond that of generating passive stiffness.

Conclusion

Titin's elastic properties may be adjusted using mechanisms that operate at different time scales (section 1). Calcium/ S100 may adjust titin's elasticity within single contractile cycles. Phosphorylation of cardiac titin's N2B spring elements involves signalling pathways that require minutes or longer to become mechanically relevant. Finally, the splice patterns of titin in the heart are adjustable, and this requires days/weeks. The adjustment of titin's elastic properties may also influence titin-based modulation of active force (section 2). Recent evidence suggests that titin's passive force modulates actomyosin interaction, possibly via influencing the distance between the myosin head and its binding site on actin. Finally, ligands link titin to membrane channel activity, protein turnover and gene expression (section 3) and modification of titin's spring properties may affect these processes. For example, titin is linked to the stretch-regulated potassium channel IKs and changes in titin's elasticity may affect the channel. Thus titin is a complex and adjustable molecular spring that not only underlies passive stiffness of muscle, but has a plethora of additional properties that place this intriguing molecule at the centre of a wide range of important physiological processes.

Acknowledgments

We thank NIH (HL61497 and HL62881) and the Deutsche Forschungsgemeinschaft (La 668/5-2+6-2) for financial support. Fig. 1 is reproduced with permission of Academic Press, Figs 2, 3 and 5A with permission of Lippincot, Williams and Wilkins Publisher, and Fig. 4 with permission of the American Physiological Society.

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