Passive force of muscle cells is largely developed by the elastic protein titin (1). A single titin polypeptide spans the full distance from the z-disk to M-line regions of the sarcomere (Fig. 1A, top). Importantly, the I-band region of the titin molecule is elastic and extends as the sarcomere is stretched. Support for the proposal that titin develops passive force in response to sarcomere stretch has been obtained by experiments with low doses of ionizing radiation to degrade titin and that showed that this greatly reduced the ability of relaxed skeletal muscle fibers to generate passive force (2). This effect was accompanied by axial misalignment of thick filaments, indicating that titin's force maintains the central position of the thick filaments in the sarcomere, ensuring that during contraction, forces in the two half sarcomeres are balanced.
Single molecule and immunolabeling studies have revealed that titin's extensible region is highly flexible and that in the absence of external force, thermally driven bending movements shorten the segment to a near zero end-to-end length. Sarcomere stretch lowers the conformational entropy of titin and this gives rise to passive force, which pulls z-disks toward each other. The extensible region of titin is held away from the z-disk by titin's inextensible near z-disk region (this region binds strongly to the thin filament and can therefore withstand compressive forces), and when the sarcomere shortens to below the slack length, the thick filament moves into titin's near z-disk region (3). As a result, titin's extensible region is stretched in a direction opposite of that when the slack sarcomere is elongated, generating the so-called restoring force, which pushes the z-disks away from each other toward their slack length position. Thus, the extensible region of titin functions as a bidirectional spring.
Titin's extensible region is in skeletal muscle composed of two spring elements: the tandem Ig segments (tandemly arranged immunoglobulin (Ig)-like domains) and the so-called PEVK segment (rich in proline (P), glutamate (E), valine (V), and lysine (K)) (Fig. 1A, bottom). The unique sequence of the N2B element (N2B-Us) forms a third spring element in cardiac muscle (1). The various spring elements of titin have different mechanical properties and, as a result extend at different sarcomere lengths. Stretch of slack sarcomeres initially gives rise to extension of tandem Ig segments (probably due to straightening of sequences that link Ig domains) followed by extension of PEVK and N2B-Us segments (which is likely due to straightening of random coil sequences). The complex composition of titin's extensible region, with multiple subsegments that extend at different sarcomere lengths, results in unique passive force-extension curves (see also in a later section).
ADJUSTING TITIN-BASED PASSIVE TENSION
The contribution of titin to muscle tension is not fixed but can be adjusted in response to changing conditions. A number of adjustment mechanisms have been discovered that we discuss below in a later section.
Differential Splicing of Titin
Titin is encoded by a single gene that is highly conserved in vertebrates. The human titin gene contains 363 exons within a 280 kb genomic segment (4). Of the 4200 kDa titin polypeptide mass encoded by the gene, 90% is organized into modular repeats. This organization provides extensive opportunity for creating titin isoforms through differential splicing. Comparison of the gene sequence with the transcript variants reveals a prominent exon-shuffling pathway in exons 49-224 that make up titin's molecular spring region. All isoforms identified so far contain a constitutively expressed region that consists of 15 Ig domains near the z-disk (that are part of the so-called proximal tandem Ig segment), 22 Ig domains located close to the A-band (that make up the distal tandem Ig segment), and an 180 residue containing PEVK segment (4). In addition, all skeletal muscle isoforms contain the N2A element, a variable number of additional Ig domains located in the proximal tandem Ig segment, and a variable number of additional PEVK residues (Fig. 1(A) shows two examples of skeletal muscle isoforms). Isoforms that contain a longer spring region reach a given degree of sarcomere stretch with a lower fractional extension of the spring than those that contain a shorter spring region, and thus, skeletal muscles that express large isoforms are more compliant than those that express small isoforms.
As for cardiac titins, two classes of isoforms exist (4): the small 2970 kDa cardiac isoform known as N2B titin (so named because it contains the N2B element) and the large so-called N2BA titin isoform (name reflects presence of both N2B and N2A elements). The N2BA titins contain additional PEVK residues and a variable number of additional Ig domains. Due to their longer extensible I-band region, N2BA titins are less stiff than N2B titans. Because high N2B expression levels are encountered in animals with high heart rates (mouse, rat), it has been suggested (5) that high N2B expression allows rapid early diastolic filling (due to high-restoring forces) and rapid setting of the end diastolic volume (due to the high stiffness at long SLs) when diastolic filling times are short. Large mammals coexpress N2B and N2BA titans, with coexpression occurring at the level of the half sarcomere (1). The coexpression ratio can vary and this allows development of graded passive force levels in between those of N2B-and N2BA-pure myocytes (1). How the precise tissue-specific regulation of splicing is achieved remains to be determined, but it is clear that it is an effective means to vary passive muscle stiffness (Fig. 1B).
High-resolution agarose gel systems can only resolve titin isoforms that differ >50 kDa. To obtain a better resolution, we developed an exon microarray with which the expression levels of all 363 titin exons can be determined simultaneously. When using various methods in conjunction, that is high-resolution protein gels, RT-PCR, and the titin exon array, detailed insights can be obtained into the differential expression of cardiac titin isoforms. We used this approach and analyzed expression of cardiac titin isoforms during fetal and postnatal heart development. We discovered fetal N2BA titin isoforms, characterized by additional spring elements both in the tandem Ig and in PEVK region of the molecule. The fetal N2BA isoform predominates in fetal myocardium and gradually disappears during postnatal development with a time course that varies (from days to weeks) in different species (6). The result is that the myocardium stiffens and this is likely to play an important role in functional transitions in diastolic filling that occur during heart development.
Adjustments in cardiac isoform expression have also been found in patients with heart disease. In patients with end-stage heart failure due to DCM, the expression ratio of large N2BA (compliant) and small N2B (stiff) cardiac titin isoforms was significantly increased (7,8). Mechanical measurements on left ventricular (LV) muscle strips dissected from these hearts revealed that passive muscle stiffness was significantly reduced in patients with high N2BA:N2B expression ratio (8). Clinical correlations support the relevance of these changes for LV function. LV function was assessed by hemodynamics and Doppler echocardiography. A positive correlation was found between the N2BA:N2B titin isoform ratio and clinical parameters of chamber compliance (8). Thus in end-stage failing hearts the more compliant N2BA isoform comprises a greater percentage of titin, this lowers myocardial stiffness, and improves diastolic filling (8).
Passive stiffness can also be adjusted via modulating the mechanical properties of titin's extensible region. Like MyBP-C and TnI, titin has a cardiac-specific element that functions as a protein kinase A (PKA) substrate. In cardiac titin, the N2B unique sequence is a PKA substrate and phosphorylation reduces passive tension of cardiac myocytes (9). Reduced passive tension might be due to a phosphorylation-induced increase in the length of the N2B element, possibly due to destabilizing native structures (9), lowering, at a given sarcomere length, the fractional extension (z/L) of titin's extensible region and, hence, lowering passive force. The effect of the β-adrenergic receptor agonist isoprenaline on diastolic tension has been investigated in intact rat ventricular trabeculae. Isoprenaline phosphorylates titin in situ and was shown to reduce diastolic tension to a degree similar to that found in skinned preparations (10). By studying the effect of phosphorylation on passive tension of myocardium that expresses the two cardiac isoforms at different ratios, it has been shown that the passive tension reduction was largest in muscles that express high levels of N2B titin (10), indicating that the effect of phosphorylation on passive tension generated by N2BA titin is less than N2B titin. This is consistent with the notion that the reduced passive tension is due to reduced fractional extension (end-to-end length divided by the contour length) of titin's extensible region. Because the N2BA isoform has a much longer PEVK segment and contains an additional tandem Ig segment, its contour length is longer than N2B titin. This diminishes the effect of the phosphorylation-induced gain in contour length of the N2B unique sequence on the overall fractional extension of titin's extensible region. Overall, these findings suggest that during β-adrenergic stimulation, PKA-based phosphorylation increases ventricular compliance in a titin-isoform dependent manner.
Calcium also has an effect on passive force by binding to titin and regulating the interaction between titin and the thin filament. Our recent single molecule mechanical experiments show that calcium lowers the bending rigidity (P) of the PEVK segment (11). Importantly, the effect requires the presence of E-rich motifs; E-rich motifs are conserved domains found in the PEVK sequence with 30-50% of their primary structure composed of glutamic acid. Physiological significance is suggested by experiments on skinned mouse soleus muscle fibers (which express 9 E-rich motifs): after extraction of thin filaments, fibers show a calcium-dependent passive force response during stretch (11). As for cardiac muscle, a recent study found no calcium effect in N2B-expressing myocardium, in contrast passive stiffness of N2BA-expressing myocardium was significantly increased by calcium (12). These findings are consistent with the absence (N2B titin) and presence (N2BA titin) of E-rich motifs in these different muscle types and suggest that calcium-sensitive passive stiffness requires the presence of E-rich motifs (11).
In addition, calcium also affects interaction between titin and actin. Yamasaki et al. found that the PEVK region of N2B cardiac titin interacts with F-actin (13). The functional significance of PEVK-actin interaction was investigated using both an in vitro motility technique and cardiac myocyte mechanics. The findings suggest that, as the thin filament slides relative to titin, a dynamic interaction between the PEVK domain and F-actin retards filament sliding and, furthermore, that this interaction contributes to passive myocyte stifness. Although physiological calcium levels alone have no effect on PEVK-actin interaction, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibits PEVK-actin interaction in a calcium-sensitive manner (13). Thus, a dynamic interaction between titin and actin contributes to passive stiffness of the sarcomere and the interaction may vary with the physiological state of the myocardium. To test for actin binding along skeletal muscle PEVK, Nagy et al. (14) expressed contiguous N-terminal (PEVKI), middle (PEVKII), and C-terminal (PEVKIII) PEVK segments of the human soleus muscle isoform and tested their binding affinities for actin. The order of apparent affinity was PEVKII > PEVKI > PEVKIII. The authors propose that the local preponderance of polyE motifs conveys an enhanced local actin-binding property to PEVK. This suggests that titin-actin interaction also enhances passive stiffness in skeletal muscle fibers that express E-rich motifs, a possibility that should be tested in experiments on single muscle fibers.
TITIN AS A MECHANOSENSOR
Titin has a multitude of functions that go beyond a pure mechanical role. In particular, titin may function as a stretch sensor with titin-based stiffness coupled to protein turnover and regulation of gene expression (1). These mechanisms involve titin-binding proteins that have been discovered recently (for details, see (1)). An important protein is MURF-1, which binds directly to the titin domains A168/169, located at the periphery of the M-line (15). MURF-1 is a member of the MURF (muscle specific ring finger protein) family that consists of MURF1-3. MURF-1 is found in both skeletal and cardiac muscles and may play a role in controlling proteasome-dependent degradation of muscle proteins. In skeletal muscle, MURF-1 is one of the few genes that is universally upregulated in all skeletal muscle atrophy models Furthermore, studies of a MURF-1-deficient mouse model demonstrated that in absence of MURF-1, muscles were resistant to atrophy (for review see (1)). Thus important roles in protein turnover and atrophy control are played by titin and its binding proteins.
Titin's mechanical properties can be adjusted by calcium. This mechanism requires E-rich motifs and is most pronounced in skeletal muscles that express large titin isoforms. Tuning of titin can also be achieved by phosphorylation of the N2B element and this involves β-adrenergic stimulation. Finally, the splice patterns of titin are adjustable, prominent examples of when this occurs are during muscle development and disease. Adjustment of titin's elastic properties via these multiple mechanisms gives rise to muscles with distinct passive stiffness and, importantly, is expected to tune titin's sensitivity as a mechanosensor.
Supported by grants HL61497/62881 (HG) and Deutsche Forschungsgemeinschaft La 668/7-1 (SL).
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