Tandem Ig Segment Research Articles

Stability of tandem Ig domain unfolding within proximal Ig segment of titin.

Titin is a giant filamentous protein contributing to the passive elasticity of muscle and is composed of approximately 300 serially linked immunoglobulin-like domains. The tandem domains of titin's proximal immunoglobulin (Ig) segment have been shown to separate at relatively low forces and thus contribute to the protein's elasticity. While titin's elastic behavior is attributed to extension of the proximal Ig segment and PEVK region beyond passive slack length, the potential unfolding of individual Ig domains has been posited to contribute to even greater extensibility. Computational modeling and mechanical stability testing have demonstrated Ig domain unfolding under physiologically relevant force, with neighboring domains exhibiting variable degrees of stability. Thermal and chemical stability experiments were conducted on domains I65-I70 of the proximal tandem Ig segment to evaluate the stability of adjacent domains. Testing revealed variable free energies of unfolding, with I65 demonstrating the lowest thermal and chemical stability out of all domains of interest, and I67 demonstrating the highest. The quantification of each domain's relative stability helps elucidate the role of the proximal Ig segment in titin's elasticity and can be used to assess the unfolding potential of other domains along the protein's extensible regions.

Removal of immunoglobulin-like domains from titin’s spring segment alters titin splicing in mouse skeletal muscle and causes myopathy

Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin's stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin's stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin's constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin's stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.

Myocardial Titin and Collagen in Cardiac Diastolic Dysfunction

High myocardial diastolic stiffness has usually been attributed to excessive myocardial collagen deposition. Over the last decennium, stiff cardiomyocytes were also identified as important contributors to high myocardial diastolic stiffness, especially in heart failure (HF) with preserved ejection fraction (HFPEF).1–3 Cardiomyocyte stiffness relates to elasticity of the giant cytoskeletal protein titin, which spans the sarcomere from the Z disk to the M line and functions as a bidirectional spring responsible for early diastolic recoil and late diastolic distensibility of cardiomyocytes.4 In HFPEF patients and in HFPEF animal models,5 the observed increase in cardiomyocyte stiffness was always accompanied by increased deposition of collagen; therefore, it remained unclear whether impaired elasticity of titin could be solely responsible for high myocardial diastolic stiffness and HFPEF. In this issue of Circulation , however, Chung et al6 provide compelling evidence for titin being the sole perpetrator in the diastolic left ventricular (LV) dysfunction of an HFPEF mouse model. They generated mice with a deletion of nine immunoglobulin (Ig)-like domains from the proximal tandem Ig segment of the titin spring region (IG KO). This deletion extended the remaining titin spring segments and increased overall titin stiffness. Despite unaltered myocardial collagen content or composition, the IG KO mice developed HFPEF, evident from a reduced exercise tolerance, an enlarged left atrium, and a steeper LV end-diastolic pressure-volume relationship. The elegant study by Chung et al therefore clearly establishes myocardial titin to be able to sufficiently compromise diastolic LV function to induce HFPEF. Article see p 19 Titin, with a molecular mass of up to 3800 kDa, spans half-sarcomeres from the Z disk to the M band and contains a molecular spring segment, the I-band region, that supports early diastolic recoil and late diastolic resistance to stretch (Figure 1A). The I-band region has …

Shortening of the Elastic Tandem Immunoglobulin Segment of Titin Leads to Diastolic Dysfunction

Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established. We generated a mouse model in which 9 Ig-like domains (Ig3-Ig11) were deleted from the proximal tandem Ig segment of the spring region of titin (IG KO). Exon microarray analysis revealed no adaptations in titin splicing, whereas novel phospho-specific antibodies did not detect changes in titin phosphorylation. Passive myocyte stiffness was increased in the IG KO, and immunoelectron microscopy revealed increased extension of the remaining titin spring segments as the sole likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels, with no consistent changes in extracellular matrix composition or extracellular matrix-based passive stiffness, supporting a titin-based mechanism for in vivo diastolic dysfunction. Additionally, IG KO mice have a reduced exercise tolerance, a phenotype often associated with diastolic dysfunction. Increased titin-based passive stiffness is sufficient to cause diastolic dysfunction with exercise intolerance.

Shortening of Titin's Elastic Tandem Ig Segment Leads to Cardiac Hypertrophy and Diastolic Dysfunction

Diastolic dysfunction is a poorly understood but clinically pervasive syndrome. Titin is the main determinant of cellular passive stiffness. However, the physiological role that titin's tandem Ig segment plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction needs to be established by performing studies at a wide range of organizational levels (skinned cells to the in vivo LV chamber). We produced a novel KO model by deleting nine immunoglobulin(Ig)-like domains from the proximal tandem Ig segment (Ig 3-11) of titin's spring region (IG KO). Exon microarray analysis revealed no adaptations in titin splicing, while novel phospho-specific antibodies did not detect changes in titin phosphorylation. Passive myocyte stiffness was increased in the KO and immunoelectron microscopy revealed increased extension of the remaining titin spring segments as the likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels; myocardial stiffness studies did not detect changes in extracellular matrix based passive stiffness, supporting a titin-based mechanism. IG KO mice have a reduced exercise tolerance and develop LV hypertrophy that is associated with a marked increase in expression of hypertrophy-associated four and a half LIM proteins (FHL). These findings suggest that titin-based stiffness plays a role in diastolic dysfunction and hypertrophic signaling and that increased titin-based passive stiffness is sufficient to cause diastolic dysfunction with exercise intolerance and LV hypertrophy.

Effect of Shortening Titin's Proximal IG Segment on Passive Cellular Mechanics

Titin is widely considered the main contributor to passive stiffness of cardiac myocytes. To test this we studied a novel mouse model, in which a large fraction of titin's proximal tandem Ig segment is deleted (we refer to the model as IG KO; for details on model, see Chung et al poster). The proximal tandem Ig segment extends greatly under physiological conditions and shortening the tandem Ig segment is predicted to increase titin-based passive tension. We first studied passive tension in skinned cardiac myocytes by stretching cells in relaxing solution with a velocity of 1 base-length/s (∼in vivo speed during diastole), and determining stiffness from the slope of the passive stress - SL relation in the physiological SL range of 1.95-2.10 μm. Cellular stiffness was significantly increased in the IG KO by ∼50%. Using a sinusoidal analysis, the elastic moduli at physiological SLs were found to be increased in the IG KO but the viscous moduli were not different. This shows that the tandem Ig segment is a pure elastic spring. Currently we are investigating intact myocytes using a carbon-fiber based system for attaching cells and measuring force. We conclude that theproximal Ig segment of titin's I band region is a pure elastic spring and that shortening of this spring element increases titin-based cellular stiffness in the physiological SL range. Increased titin-based stiffness is found in patients with heart failure with preserved ejection fraction (HFpEF) and the IGKO is well-suited to study the effect of increased titin-based stiffness on heart function (see poster by Chung et al).

Single Molecule Force Spectroscopy of the Cardiac Titin N2B Element: EFFECTS OF THE MOLECULAR CHAPERONE αB-CRYSTALLIN WITH DISEASE-CAUSING MUTATIONS

The small heat shock protein alphaB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of alphaB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type alphaB-crystallin on the single molecule force-extension curve was determined as well as that of mutant alphaB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type alphaB-crystallin decreased the persistence length of the N2B-Us (from approximately 0.7 to approximately 0.2 nm) but did not alter its contour length. alphaB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 +/- 3 piconewtons); the rate constant of unfolding at zero force was estimated to be approximately 17-fold lower in the presence of alphaB-crystallin (1.4 x 10(-4) s(-1) versus 2.4 x 10(-3) s(-1)). We also found that alphaB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 +/- 6 piconewtons. The effects of alphaB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that alphaB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing alphaB-crystallin mutations suggests that the interaction between alphaB-crystallin and titin is important for normal heart function.

Evidence for the Oligomeric State of ‘Elastic’ Titin in Muscle Sarcomeres

The giant protein titin has important roles in muscle sarcomere integrity, elasticity and contractile activity. The key role in elasticity was highlighted in recent years by single-molecule mechanical studies, which showed a direct relationship between the non-uniform structure of titin and the hierarchical mechanism of its force-extension behavior. Further advances in understanding mechanisms controlling sarcomere structure and elasticity require detailed knowledge of titin arrangement and interactions in situ. Here we present data on the structure and self-interactive properties of an ∼ 290 kDa ( ∼ 100 nm long) tryptic fragment from the I-band part of titin that is extensible in situ. The fragment includes the conserved ‘distal’ tandem Ig segment of the molecule and forms side-by-side oligomers with distinctive 4 nm cross-striations. Comparisons between these oligomers and the end filaments seen at the tips of native thick filaments indicate identical structure. This shows that end-filaments are formed by the elastic parts of six titin molecules connecting each end of the thick filament to the Z-line. Self-association of elastic titin into stiff end-filaments adds a further hierarchical level in the mechanism of titin extensibility in muscle cells. Self-association of this part of titin may be required to prevent interference of the individual flexible molecules with myosin cross-bridges interacting with actin.

Targeted deletion of titin N2B region leads to diastolic dysfunction and cardiac atrophy

Titin is a giant protein that is in charge of the assembly and passive mechanical properties of the sarcomere. Cardiac titin contains a unique N2B region, which has been proposed to modulate elasticity of the titin filament and to be important for hypertrophy signaling and the ischemic stress response through its binding proteins FHL2 and alphaB-crystallin, respectively. To study the role of the titin N2B region in systole and diastole of the heart, we generated a knockout (KO) mouse deleting only the N2B exon 49 and leaving the remainder of the titin gene intact. The resulting mice survived to adulthood and were fertile. Although KO hearts were small, they produced normal ejection volumes because of an increased ejection fraction. FHL2 protein levels were significantly reduced in the KO mice, a finding consistent with the reduced size of KO hearts. Ultrastructural analysis revealed an increased extension of the remaining spring elements of titin (tandem Ig segments and the PEVK region), which, together with the reduced sarcomere length and increased passive tension derived from skinned cardiomyocyte experiments, translates to diastolic dysfunction as documented by echocardiography. We conclude from our work that the titin N2B region is dispensable for cardiac development and systolic properties but is important to integrate trophic and elastic functions of the heart. The N2B-KO mouse is the first titin-based model of diastolic dysfunction and, considering the high prevalence of diastolic heart failure, it could provide future mechanistic insights into the disease process.

Adaptations in titin's spring elements in normal and cardiomyopathic hearts.

The giant elastic protein titin contains an extensible segment that underlies the majority of physiological passive muscle stiffness. The extensible segment comprises mechanically distinct and serially-linked spring elements: the tandem Ig segments, the PEVK and the cardiac-specific N2B unique sequence. Under physiological conditions the tandem Ig segments are likely to largely consist of folded Ig domains whereas the N2B unique sequence and PEVK are largely unfolded and behave as wormlike chains with different persistence lengths. The mechanical characteristics of titin's extensible region may be tuned to match changing mechanical demands placed on muscle, using mechanisms that operate at different time scales and that include post-transcriptional and post-translational processes.

Molecular Mechanics of Cardiac Titin's PEVK and N2B Spring Elements

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.

Different Molecular Mechanics Displayed by Titin's Constitutively and Differentially Expressed Tandem Ig Segments

Titinis a giant elastic protein responsible for passive force generated by the stretched striated-muscle sarcomere. Passive force develops in titin's extensible region which consists of the PEVK segment in series with tandemly arranged immunoglobulin (Ig)-like domains. Here we studied the mechanics of tandem Ig segments from the differentially spliced (I65–70) and constitutive (I91–98) regions by using an atomic force microscope specialized for stretching single molecules. The mechanical stability of I65–70 domains was found to be different from that of I91–98 domains. In the range of stretch rates studied (0.05–1.00 μm/s) lower average domain unfolding forces for I65–70 were associated with a weaker stretch-rate dependence of the unfolding force, suggesting that the differences in the mechanical stabilities of the segments derive from differences in the zero force unfolding rate (K0u) and the characteristic distance (location of the barrier) along the unfolding reaction coordinate (ΔXu). No effect of calcium was found on unfolding forces and persistence length of unfolded domains. To explore the structural basis of the differences in mechanical stabilities of the two fragment types, we compared the amino acid sequence of I65–70 domains with that of I91–98 domains and by using homology modeling analyzed how sequence variations may affect folding free energies. Simulations suggest that differences in domain stability are unlikely to be caused by variation in the number of hydrogen bonds between the force-bearing β-strands at the domain's N- and C-termini. Rather, they may be due to differences in hydrophobic contacts and strand orientations.

Titin–Actin Interaction in Mouse Myocardium: Passive Tension Modulation and Its Regulation by Calcium/S100A1

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK–actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK–actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK–actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin’s extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1–PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.

Molecular Dissection of N2B Cardiac Titin’s Extensibility

Titin is a giant filamentous polypeptide of multidomain construction spanning between the Z- and M-lines of the cardiac muscle sarcomere. Extension of the I-band segment of titin gives rise to a force that underlies part of the diastolic force of cardiac muscle. Titin’s force arises from its extensible I-band region, which consists of two main segment types: serially linked immunoglobulin-like domains (tandem Ig segments) interrupted with a proline (P)-, glutamate (E)-, valine (V)-, and lysine (K)-rich segment called PEVK segment. In addition to these segments, the extensible region of cardiac titin also contains a unique 572-residue sequence that is part of the cardiac-specific N2B element. In this work, immunoelectron microscopy was used to study the molecular origin of the in vivo extensibility of the I-band region of cardiac titin. The extensibility of the tandem Ig segments, the PEVK segment, and that of the unique N2B sequence were studied, using novel antibodies against Ig domains that flank these segments. Results show that only the tandem Igs extend at sarcomere lengths (SLs) below ∼2.0 μm, and that, at longer SLs, the PEVK and the unique sequence extend as well. At the longest SLs that may be reached under physiological conditions (∼2.3 μm), the PEVK segment length is ∼50 nm whereas the unique N2B sequence is ∼80 nm long. Thus, the unique sequence provides additional extensibility to cardiac titins and this may eliminate the necessity for unfolding of Ig domains under physiological conditions. In summary, this work provides direct evidence that the three main molecular subdomains of N2B titin are all extensible and that their contribution to extensibility decreases in the order of tandem Igs, unique N2B sequence, and PEVK segment.

Mechanically driven contour-length adjustment in rat cardiac titin's unique N2B sequence: titin is an adjustable spring.

The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force-sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline [P], glutamate [E], valine [V], and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above approximately 2.2 microm SL titin's elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin's extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable.