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(Circulation Research. 1997;80:290-294.)
© 1997 American Heart Association, Inc.

Articles

The Giant Protein Titin

Emerging Roles in Physiology and Pathophysiology

Siegfried Labeit, Bernhard Kolmerer, Wolfgang A. Linke

From the European Molecular Biology Laboratory (S.L., B.K.), Heidelberg, and the Institute of Physiology II (W.A.L.), University of Heidelberg (Germany).

Correspondence to the European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany.

	Abstract

Top Abstract Introduction Elasticity of Titin Emerging Roles of Titin... Pathophysiological Aspects References

 
Abstract Titin is a giant protein of vertebrate striated muscles (M_r, 3000 kD). Its molecules are of filamentous shape and span from the Z disk to the M line, thereby forming a third filament system of the sarcomere. This filament system is important for both the structural integrity of the myofibril and the passive tension response of a stretched muscle fiber. The determination of the cDNA sequence of human cardiac titin has shown that the cardiac titin filament is formed by a single, giant, 27 000-residue-long polypeptide chain. The titin strand has a modular structure, and different modular arrangements are expressed in different muscle tissue types by differential splicing. In the A band, the titin modules provide regular arrays of binding sites for other sarcomeric proteins, thereby contributing to a precise assembly of myofibrillar proteins in vivo. In the I band, two specific motif families, tandem-immunoglobulin domains and PEVK-rich sequences, confer extensibility to the titin filament. Expression of muscle tissue–specific length variants of the PEVK region by alternative splicing may explain the differences in the passive tension properties between various striated muscle types. Apart from the titin sequences with apparent functions for muscle structure and elasticity, the titin molecule contains a class of unique sequence insertions. Among these sequences are phosphorylation sites, a serine/threonine kinase domain, and binding sites for muscle-specific calpain proteases. Thus, it is likely that the titin filament also plays a role in myofibrillar signal transduction pathways.

Key Words: titin (connectin) • elasticity • muscle ultrastructure • PEVK region

	Introduction

Top Abstract Introduction Elasticity of Titin Emerging Roles of Titin... Pathophysiological Aspects References

 
Standard protein gels with an acrylamide content ranging from 6% to 15% have commonly been used to detect protein bands in the 10- to 200-kD range. Since this method excludes the detection of larger proteins, it was only two decades ago that researchers discovered a novel gigantic protein with an extremely low apparent mobility by using unconventional 2% gels. The molecular mass of this novel protein, titin¹ (also referred to as connectin² ), was earlier estimated to range between 1.2 and 3.0 MD (for reviews, see References 3³ and 4⁴ ). When the intensity of titin bands on SDS gels is compared with that of other myofibrillar proteins, it appears that titin is, after myosin and actin, the third most abundant muscle protein, making up 10% of the combined muscle protein content. A human adult with 80 kg body weight thus may contain approximately half a kilogram of titin. Considering its abundance, titin's late discovery is quite surprising.

During the 1980s, electron microscopic studies with titin-specific antibodies had demonstrated that titin is an integral part of the myofibril. Single titin molecules were shown to span from the Z disk to the M line, thus forming a third sarcomeric filament system, apart from the thick (mostly myosin) and thin (mostly actin) filaments.⁵ Accordingly, purified native titin molecules visualized by electron microscopy have been found to be >1 µm long.⁶ Furthermore, these molecules appeared as a rod with a beaded substructure,⁶ because their peptide chains are mainly composed of Ig-like and FN3-like repeats,⁷ which fold into globular domains and account for nearly 90% of titin's mass⁸ (Fig 1). In the A band, these repeats are arranged into highly ordered patterns and help organize the thick-filament structure by providing regularly spaced binding sites to other A-band proteins, notably light meromyosin and C protein (for review, see Reference 15¹⁵ ). Because of a tight association with the thick-filament proteins, titin's A-band portion is functionally stiff under physiological conditions. In contrast, titin's I-band section is elastic.^{5 17} When titin filaments are extracted from the sarcomere or degraded by radiation or proteases, the stiffness of the relaxed myofibril decreases (for reviews, see References 3³ and 4⁴ ). More recently, titin has been shown to bear most of the resting tension during physiological extensions in both skeletal and cardiac muscle.^{18 19}

View larger version (0K): [in this window] [in a new window]   Figure 1. Domain architecture and sarcomeric layout of the titin filament. The domain structure of the human soleus titin, as predicted by its 100-kb mRNA, is shown. The 3.7-MD soleus titin peptide contains 297 copies of 100-residue repeats, which are members of the Ig and FN3 superfamilies.8 Each of these domains folds into a 10- to 12-kD small globular subunit, as shown by structural studies.9 Specific for the I-band segment of titin are strings of tandemly repeated Ig domains (tandem-Ig titin) and the "PEVK domain," rich in proline, glutamate, valine, and lysine residues. The tandem-Ig and the PEVK region of titin represent those parts of the titin filament that extend during physiological amounts of stretch.13 14 Specific for the A-band titin are regular patterns of Ig and FN3 domains, referred to as "super repeats."7 These super repeats provide multiple and structurally ordered binding sites for myosin and C protein.7 15 In addition to the Ig/FN3 repeats and the PEVK region of titin, 8% to 10% of titin's mass is formed by unique sequence insertions. Among the encoded peptides are phosphorylation motifs (Pi) and a serine/threonine kinase. The mapped calpain p94-binding sites16 are shown. Arrows above the domain pattern indicate the sites at which muscle type–specific alternative splicing occurs.8

Molecular-level approaches to investigate titin's role in muscle structure and elasticity have become easier after the determination of the cDNA sequence of human skeletal and cardiac titins.⁸ Also, immunoelectron microscopy with epitope-mapped titin-specific antibodies allows us to estimate which segments of the sequence encode Z-disk, I-band, A-band, and M-line^{10 11 12} titin. As suggested earlier from the different mobilities of skeletal and cardiac titins on SDS-gels,^{20 21} it is now clear that titin is expressed in different isoforms in various muscle tissues.⁸ Human cardiac titin, for example, is encoded by a giant 82-kb mRNA, which contains an 81-kb open reading frame coding for a 27 000-residue peptide (M_r, 2993 kD). In contrast, human soleus titin is a significantly larger polypeptide, with a molecular mass of 3700 kD. These differences are the result of a series of alternative splicing events of I-band titin in the different muscle tissues.⁸

	Elasticity of Titin

Top Abstract Introduction Elasticity of Titin Emerging Roles of Titin... Pathophysiological Aspects References

 
When relaxed striated muscle fibers are stretched, a retracting force results, which is referred to as passive or resting tension (stiffness). It has long been known that cardiac muscle is much stiffer than skeletal muscle. Earlier, the high passive stiffness of cardiac muscle tissue had been thought to arise mostly from the low compliance of extracellular structures such as collagen,²² but with the advent of the single myocyte preparation,²³ it became clear that stiff structures must be located, at least partially, within the cell.^{24 25} The stiff extracellular elements, which help prevent overstretch of muscle tissue, may be relevant only at more extreme stresses. Recently, it could also be demonstrated that significant passive force develops upon stretch of relaxed, single, isolated myofibrils, with cardiac specimens being approximately an order of magnitude stiffer than skeletal preparations.¹⁹ These stiffness differences are now known to result from the muscle type–specific expression of different-length variants of titin or, more precisely, from the differential expression of two distinct titin motif families in the I band (Fig 1).^{8 13} One motif type is represented by tandemly arranged Ig domains. The stiffest vertebrate striated muscle, the heart, expresses 37 tandem-Ig domains, whereas the much more compliant soleus muscle expresses 90 tandem-Ig domains. The other differentially expressed I-band titin segment is a distinct motif type termed the PEVK domain, because proline, glutamate, valine, and lysine residues constitute 70% of its sequence.⁸ This PEVK domain is also shortest in the heart and much longer in skeletal muscle: the human cardiac PEVK region comprises 163 residues, whereas the human soleus PEVK region has 2200 residues (Fig 1).⁸

The discovery of differential expression of both the PEVK and the tandem-Ig regions of titin has made it necessary to identify the relative contribution of these two distinct I-band segments to myofibril elasticity. Such identification has recently been attempted by monitoring the position of selected I-band titin antibody epitopes in stretched single myofibrils¹³ or muscle fibers,¹⁴ in addition to measuring the passive tension response of the specimens (Fig 2; compare with Reference 13¹³ ). It was suggested that at slack sarcomere length, I-band titin domains are in the compact state, whereas small stretch may induce straightening of the tandem-Ig regions.^{13 14 27 28} This initial extension is correlated with a small (cardiac muscle) or even negligible (skeletal muscle) passive tension increase only^{20 21 25} and may thus not be brought about by unfolding of tandem-Ig modules, which have been shown to independently fold into thermodynamically stable domains.⁹ At moderate to long stretches, the main extension appears to occur within the PEVK region, at least in skeletal myofibrils,^{13 14} whereas passive tension increases steadily (Fig 2). In cardiac myofibrils, the short PEVK region⁸ may also support passive tension, but only to a limited degree (compare with legend to Fig 2). Once the PEVK segment extensibility has been exhausted, passive tension might be determined mainly by unfolding of Ig domains^{27 28} ; however, since the maximum length of cardiac sarcomeres does not exceed 2.4 µm in vivo, such unfolding is unlikely to occur under physiological conditions.¹³

View larger version (24K): [in this window] [in a new window]   Figure 2. Current model of titin extension with sarcomere stretch. Shown is a major portion of the half-sarcomere, including the I-band portion, which harbors the elastic titin segment. This model of titin arrangement reflects the situation in psoas muscle (adapted from Linke et al13 ) and also considers the recently reported position of the MIR epitope at the very edge of the A band.11 The inset shows a typical passive length-tension curve of single psoas myofibrils,13 with the letters A through D referring to the sarcomere lengths depicted in the main figure. It is thought that at slack length, I-band titin domains are in the compact state (A). During a small stretch, tandem-Ig domains straighten, but the PEVK region extends only little, resulting in very low passive tension (B). With a moderate stretch, Ig domains barely extend further, whereas the PEVK region unravels, which results in a steady passive tension increase (C). In extremely stretched sarcomeres (toward the high end of the physiological sarcomere length range), the PEVK element is maximally unraveled, and the Ig domains become highly strained; passive tension now reaches a maximum before previously bound A-band titin is released into the I band (strain limit).20 It should be pointed out that this model proposed for psoas titin extension may not adequately address the situation in cardiac muscle, where the contribution of the short PEVK segment8 to I-band titin extensibility is very small.13 In cardiac sarcomeres, a significant passive tension increase appears shortly above slack length and seems to be correlated with extension of the tandem-Ig region.25 26 The precise mechanism of titin elasticity remains to be elucidated. Color codes are as follows: blue, actin; green, myosin; yellow, PEVK region of titin; and red, non-PEVK domains. The filled circles represent the I-band tandem-Ig modules. T12, N2-A, MIR, and BD6 are known binding sites of titin antibodies used to measure the extension properties of I-band titin in single isolated myofibrils.13 9D10? indicates the possible epitope position of the 9D10 antibody; the arrow doublets in C and D indicate that the epitope widened at longer sarcomere lengths.

In conclusion, the high extensibility of the PEVK region during physiological amounts of stretch^{13 14} suggests that this domain is able to unravel to an extended polypeptide chain. Therefore, titin's PEVK region and the tandem-Ig domains may constitute a two-spring system acting in series. The tissue-specific expression of both springs in different length variants can now explain why the passive mechanical properties of striated muscles are so diverse. Expression of different tandem-Ig segment lengths in various muscle types could be important for setting the physiological slack sarcomere length, whereas differential splicing of PEVK-rich sequences may control the characteristic stiffness of a relaxed muscle tissue. An important task now is to uncover which tertiary structure allows the PEVK region of titin to undergo the massive and rapidly reversible conformational changes that principally determine myofibrillar passive tension and elasticity.

	Emerging Roles of Titin in Muscle Cell Biology

Top Abstract Introduction Elasticity of Titin Emerging Roles of Titin... Pathophysiological Aspects References

 
At present, it is unknown which cellular signal transduction machinery may control the translation, assembly, and also the disassembly and turnover of the giant titin polypeptide during myogenesis and growth. Titin contains hundreds of binding sites for myosin, C protein, and M-line proteins^{7 15} and probably for a significant number of not-yet-identified Z-disk and I-band proteins. How then does the muscle cell control the translation of the 27 000- to 33 000-residue titin peptide, and how can the titin synthesis be tightly coupled with the assembly of titin ligands during myogenesis? An attractive model would be that titin, myosin, and C-protein mRNAs are colocalized and cotranslationally assembled,²⁹ thereby forcing the nascent peptide chains into the protein meshwork of the paracrystalline order found in vivo. Clearly, a better understanding as to how the titin/thick-filament supramolecular assembly is able to constitute a highly ordered three-dimensional meshwork must now come from a biochemical characterization of expressed titin, myosin, and C-protein fragments and perhaps from studies of titin's mRNA metabolism.

Another mechanism to control titin filament assembly might be implied in some characteristic features of the titin sequence: in addition to the PEVK region and the 244 to 297 copies of Ig and FN3 structural repeats (dependent on muscle type), titin also contains 19 unique sequence insertions, which together constitute 300 kD, or 8% to 10%, of titin's mass.⁸ Two unique sequence insertions, located in the N-terminal and C-terminal titin regions, encode tandemly arranged SP motifs (Fig 1). The serine residues in the SP repeats can be phosphorylated in vitro by muscle extracts,^{30 31} and this could explain why titin becomes rapidly labeled in vivo when [³²P]phosphate is injected into animals.³² Possibly, as-yet-unidentified phosphorylation/ dephosphorylation pathways may thus control titin filament assembly. In the future, it should be interesting to investigate the functional consequences of phosphorylation at titin's Z-disc and M-line ends.

One of the unique sequence insertions of titin located close to the C-terminus encodes a serine/threonine kinase domain (Fig 1).⁷ This domain and the organization of flanking Ig and FN3 repeats are very similar to those of the giant invertebrate proteins, twitchin and projectin.^{33 34} In the kinase domains of both twitchin and titin, calmodulin binding sites have been demonstrated.^{35 36} Recently, the twitchin kinase from the mollusk Aplysia has been shown to be activated by several orders of magnitude through the ubiquitous calcium-regulated cofactor S100.³⁷ Therefore, it appears likely that the twitchin—and perhaps also the titin—filaments represent a novel calcium-sensitive filament system in muscle. Despite the growing insights into the factors that control the activity of the titin/twitchin kinases on artificial substrates, the genuine substrate of these kinases (and thus their physiological role) remains unknown.

To better understand the physiology of titin assembly/disassembly at a molecular level, the discovery of specific binding sites on titin for the calpain protease, p94,¹⁶ might be an important step (compare with Fig 1). In contrast to the ubiquitous calpains expressed in all cell types, p94 is expressed only in muscle tissues. Using p94 as a bait for a yeast two-hybrid screen, two distinct p94-binding loci were identified on the titin filament.¹⁶ The first site is located in the central region of I-band titin. Possibly, cleavage of titin at this site by p94 or p94-regulated proteases may explain why titin easily degrades to the so-called T2 titin (or beta-connectin).³ Then, T2 might be a physiological breakdown product of titin implicated in myofibrillar turnover. Furthermore, the second binding site on titin for p94 is located at the filament's C-terminal end, coinciding with the last unique sequence insertion of titin (Fig 1). Although it is unclear why at least two distinct binding sites for p94 are present in titin, a possibility is that—since the soluble p94 is extremely rapidly degradable and has a half-life of 30 minutes—p94-binding sites in titin may function to sequester the calpain protease to a complexed, stabilized state. Interestingly, the C-terminal p94-binding motif of titin is skipped in some muscle tissues by differential splicing,³⁸ which adds a further level of complexity to the interactions between p94 and titin and raises the possibility of a tissue-specific control of titin stability.

	Pathophysiological Aspects

Top Abstract Introduction Elasticity of Titin Emerging Roles of Titin... Pathophysiological Aspects References

 
Finally, a molecular understanding of the interactions between the titin filament and p94 or other calpain proteases may reward us with a more thorough understanding of muscle degeneration and regeneration, with particular respect to the pathophysiological situation. A careful survey of the proteins present in muscle biopsies from normal and dystrophic patients revealed degradation of titin in DMD and FCMD.³⁹ More recently, mutations in the muscle-specific calpain protease p94 were found to cause LGMD-2A.⁴⁰ Since titin provides specific binding sites for p94,¹⁶ the intriguing possibility is raised that genetically distinct muscular dystrophies, such as FCMD, DMD, and LGMD-2A, share a dysregulation of the p94-titin interaction, which then leads to a pathological fragility of the titin filament system as a common, secondary, disease mechanism.

In summary, titin filaments play an important role in both the physiological and pathophysiological functioning of muscle. Whereas a regular titin structure in the A band appears to be critical for an ordered sarcomere assembly, it is clear that elasticity of titin in the I band determines the passive mechanical properties of the myofibril. In the future, an improved molecular understanding of the elastic properties of I-band titin may evolve from a combined approach, using both biophysical and molecular biological techniques. As for the potential kinase activity of titin and its anticipated role in signal transduction, we still await more detailed exploration. Finally, to further uncover titin's involvement in pathophysiological processes, it will be necessary to study expressed titin modules for possible interactions with other myofibrillar and cytosolic proteins, thereby functionally characterizing those interactions at the molecular level.

	Selected Abbreviations and Acronyms

 

DMD = Duchenne muscular dystrophy FCMD = Fukuyama-type congenital muscular dystrophy FN3 = fibronectin type 3 Ig = immunoglobulin LGMD-2A = limb girdle muscular dystrophy type 2A SP = serine/proline dipeptide

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (La 668/2-3, Li 690/2-1), the European Community, and the "Forschungsfond der Fakultät für Klinische Medizin Mannheim." We thank J.C. Rüegg for continuous support.

Received December 16, 1996; accepted December 17, 1996.

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