Hierarchical extensibility in the PEVK domain of skeletal-muscle titin.

Titin is the main determinant of passive muscle force. Physiological extension of titin derives largely from its PEVK (Pro-Glu-Val-Lys) domain, which has a different length in different muscle types. Here we characterized the elasticity of the full-length, human soleus PEVK domain by mechanically manipulating its contiguous, recombinant subdomain segments: an N-terminal (PEVKI), a middle (PEVKII), and a C-terminal (PEVKIII) one third. Measurement of the apparent persistence lengths revealed a hierarchical arrangement according to local flexibility: the N-terminal PEVKI is the most rigid and the C-terminal PEVKIII is the most flexible segment within the domain. Immunoelectron microscopy supported the hierarchical extensibility within the PEVK domain. The effective persistence lengths decreased as a function of ionic strength, as predicted by the Odijk-Skolnick-Fixman model of polyelectrolyte chains. The ionic strength dependence of persistence length was similar in all segments, indicating that the residual differences in the elasticity of the segments derive from nonelectrostatic mechanisms.

Molecular basis of passive stress relaxation in human soleus fibers: assessment of the role of immunoglobulin-like domain unfolding.

Titin (also known as connectin) is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is constructed of the PEVK domain and tandem-immunoglobulin segments comprising serially linked immunoglobulin (Ig)-like domains. It is unresolved whether under physiological conditions Ig domains remain folded and act as "spacers" that set the sarcomere length at which the PEVK extends or whether they contribute to titin's extensibility by unfolding. Here we focused on whether Ig unfolding plays a prominent role in stress relaxation (decay of force at constant length after stretch) using mechanical and immunolabeling studies on relaxed human soleus muscle fibers and Monte Carlo simulations. Simulation experiments using Ig-domain unfolding parameters obtained in earlier single-molecule atomic force microscopy experiments recover the phenomenology of stress relaxation and predict large-scale unfolding in titin during an extended period (> approximately 20 min) of relaxation. By contrast, immunolabeling experiments failed to demonstrate large-scale unfolding. Thus, under physiological conditions in relaxed human soleus fibers, Ig domains are more stable than predicted by atomic force microscopy experiments. Ig-domain unfolding did not become more pronounced after gelsolin treatment, suggesting that the thin filament is unlikely to significantly contribute to the mechanical stability of the domains. We conclude that in human soleus fibers, Ig unfolding cannot solely explain stress relaxation.

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.

Cardiac titin isoforms are coexpressed in the half-sarcomere and extend independently.

Titin, the third myofilament type of cardiac muscle, contains a molecular spring segment that gives rise to passive forces in stretched myocardium and to restoring forces in shortened myocardium. We studied cardiac titin isoforms (N2B and N2BA) that contain length variants of the molecular spring segment. We investigated how coexpression of isoforms takes place at the level of the half-sarcomere, and whether coexpression affects the extensibility of the isoforms. Immunoelectron microscopy was used to study local coexpression of isoforms in a range of species. It was found that the cardiac sarcomere of large mammals coexpresses N2B and N2BA titin isoforms at the level of the half-sarcomere, and that when coexpressed, the isoforms act independently of one another. Coexpressing isoforms at varying ratios results in modulation of the passive mechanical behavior of the sarcomere without impacting other functions of titin and allows for adjustment of the diastolic properties of the myocardium.

Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain.

The giant myofibrillar protein titin contains within its C-terminal region a serine-threonine kinase of unknown function. We have identified a novel muscle specific RING finger protein, referred to as MURF-1, that binds in vitro to the titin repeats A168/A169 adjacent to the titin kinase domain. In myofibrils, MURF-1 is present within the periphery of the M-line lattice in close proximity to titin's catalytic kinase domain, within the Z-line lattice, and also in soluble form within the cytoplasm. Yeast two-hybrid screens with MURF-1 as a bait identified two other highly homologous MURF proteins, MURF-2 and MURF-3. MURF-1,2,3 proteins are encoded by distinct genes, share highly conserved N-terminal RING domains and in vitro form dimers/heterodimers by shared coiled-coil motifs. Of the MURF family, only MURF-1 interacts with titin repeats A168/A169, whereas MURF-3 has been reported to affect microtubule stability. Association of MURF-1 with M-line titin may potentially modulate titin's kinase activity similar to other known kinase-associated proteins, whereas differential expression and heterodimerization of MURF1, 2 and 3 may link together titin kinase and microtubule-dependent signal pathways in striated muscles.

Extensibility of isoforms of cardiac titin: variation in contour length of molecular subsegments provides a basis for cellular passive stiffness diversity.

Titin is a giant polypeptide that spans between the Z- and M-lines of the cardiac muscle sarcomere and that develops force when extended. This force arises from titin's extensible I-band region, which consists mainly of three segment types: serially linked immunoglobulin-like domains (Ig segments), interrupted by the PEVK segment, and the N2B unique sequence. Recently it was reported that the myocardium of large mammals co-expresses small (N2B) and large (N2BA) cardiac isoforms and that the passive stiffness of cardiac myocytes varies with the isoform expression ratio. To understand the molecular basis of the differences in passive stiffness we investigated titin's extensibility in bovine atrium, which expresses predominantly N2BA titin, and compared it to that of rat, which expresses predominantly N2B titin. Immunoelectron microscopy was used with antibodies that flank the Ig segments, the PEVK segment, and the unique sequence of the N2B element. The extension of the various segments was then determined as a function of sarcomere length (SL). When slack sarcomeres of bovine atrium were stretched, the PEVK segment extended much more steeply and the unique N2B sequence less steeply than in rat, while the Ig segments behaved similarly in both species. However, the extensions normalized with the segment's contour length (i.e., the fractional extensions) of Ig, PEVK, and unique sequence segments all increase less steeply with SL in cow than in rat. Considering that fractional extension determines the level of entropic force, these differences in fractional extension are expected to result in shallow and steep passive force-SL curves in myocytes that express high levels of N2BA and N2B titin, respectively. Thus, the findings provide a molecular basis for passive stiffness diversity.

Connecting filaments: a historical prospective.

This short review covers the development of the extensible filament research from the very beginning until the most recent results. This work emphasizes the milestones of discovery, which led us from initial observations that were solely ultrastructural to the molecular understanding of the extensible process of these filaments.

Molecular tools for the study of titin's differential expression.

Although vertebrate genomes appear to contain only one titin gene, a large variety of quite distinct titin isoforms are expressed in striated muscle tissues. The isoforms appear to be generated by a series of complex, not yet fully characterized differential splicing mechanisms. Here, we provide an overview of the titin-specific antibodies that have been raised by our laboratory to study individual differentially expressed isoforms of titin. The staining patterns obtained in different tissues will contribute to the identification of both the particular titin isoforms that are expressed in the different tissues, as well as their intracellular distributions. In addition, antibodies to titin that are available are rapidly allowing for the refinement of our knowledge of titin's elastic spring properties. Knowledge of the nature and structure of vertebrate titins that may also be expressed in nonmuscle tissues may be broadened using these antibodies.

Mechanical properties of titin isoforms.

Titin is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the sarcomere. As a result of differential splicing, length variants of titin are expressed in different skeletal and cardiac muscles. Here we first briefly review some of our previous work that has revealed that titin develops force in sarcomeres either stretched beyond their slack length (passive force) or shortened to below the slack length (restoring force) and that titin's force underlies a large fraction of the diastolic force of cardiac muscle. Next we present our mechanical and immunoelectron microscopical (IEM) studies of skeletal and cardiac muscles that express titin isoforms. The previously deduced molecular properties of titin were used to model titin's extensible region in the sarcomere as serially linked WLCs: rigid segments (containing folded Ig/Fn domains) and more flexible segments (PEVK segment). The model was tested on skeletal muscle fibers that express titin isoforms with tandem Ig and PEVK length variants. The model adequately predicts titin's behavior along a wide sarcomere length range in skeletal muscle, but at long sarcome lengths (SLs), predicted forces are much higher than those determined experimentally. IEM reveals that this may result from Ig domain unfolding. Experiments were also performed on cardiac myocytes from mouse and cow that express predominantly a small cardiac titin isoform (N2B titin) or a large isoform (N2BA titin), respectively. The passive tension-SL relation of myocytes was found to increase more steeply with SL in mouse than in cow. IEM revealed an additional source of extensibility within both of these cardiac titins: the unique N2B sequence (absent in skeletal muscle). Furthermore, the PEVK segment of the N2BA isoform extended to a maximal length of approximately 200 nm, as opposed to approximately 60 nm for the N2B isoform. We propose that, along the physiological SL range, the long PEVK segment found in N2BA titins results in a low PEVK fractional extension and that this underlies the lower passive tensions of N2BA-expressing cow myocytes.

From connecting filaments to co-expression of titin isoforms.

The molecular basis of elasticity in insect flight muscle has been analyzed using both the mechanism of extensibility of titin filaments (Trombitás et al., J. Cell Biol. 1998;140:853-859), and the sequence of projectin (Daley et al., J. Mol. Biol. 1998;279:201-210). Since a PEVK-like domain is not found in the projectin sequence, it is suggested that the sarcomere elongation causes the slightly "contracted" projectin extensible region to straighten without requiring Ig/Fn domain unfolding. Thus, the extensible region of the projectin may be viewed as a single entropic spring. The serially linked entropic spring model developed for skeletal muscle titin was applied to titin in the heart. The discovery of unique N2B sequence extension in physiological sarcomere length range (Helmes et al., Circ. Res. 1999;84:1339-1352) suggests that cardiac titin can be characterized as a serially linked three-spring system. Two different cardiac titin isoform (N2BA and N2B) co-exist in the heart. These isoforms can be differentiated by immunoelectron microscopy using antibody against sequences C-terminal of the unique N2B sequence, which is present in both isoforms. Immunolabeling experiments show that the two different isoform are co-expressed within the same sarcomere.

Series of exon-skipping events in the elastic spring region of titin as the structural basis for myofibrillar elastic diversity.

Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.

Differential expression of cardiac titin isoforms and modulation of cellular stiffness.

Extension of the I-band segment of titin gives rise to part of the diastolic force of cardiac muscle. Previous studies of human cardiac titin transcripts suggested a series of differential splicing events in the I-band segment of titin leading to the so-called N2A and N2B isoform transcripts. Here we investigated titin expression at the protein level in a wide range of mammalian species. Results indicate that the myocardium coexpresses 2 distinct titin isoforms: a smaller isoform containing the N2B element only (N2B titin) and a larger isoform with both the N2B and N2A elements (N2BA titin). The expression ratio of large N2BA to small N2B titin isoforms was found to vary greatly in different species; eg, in the left ventricle the ratio is approximately 0.05 in mouse and approximately 1.5 in pig. Differences in the expression ratio were also found between atria and ventricles and between different layers of the ventricular wall. Immunofluorescence experiments with isoform-specific antibodies suggest that coexpression of these isoforms takes place at the single-myocyte level. The diastolic properties of single cardiac myocytes isolated from various species expressing high levels of the small (rat and mouse) or large (pig) titin isoform were studied. On average, pig myocytes are significantly less stiff than mouse and rat myocytes. Gel analysis indicates that this result cannot be explained by varying amounts of titin in mouse and pig myocardium. Rather, low stiffness of pig myocytes can be explained by its high expression level of the large isoform: the longer extensible region of this isoform results in a lower fractional extension for a given sarcomere length and hence a lower force. Implications of our findings to cardiac function are discussed.

Adaptation of a super-sensitive epitope detection technique for the immunoelectron microscopy of titin filaments in vertebrate striated muscle.

A super-sensitive epitope-detection technique based on gold-silver intensification was adapted for pre-embedding immunolabelling of titin filaments in vertebrate striated muscle. Indirect immunoelectron microscopy of titin filaments was performed with monoclonal titin antibodies as primary antibodies and Fab anti-mouse IgG conjugated with 1.4 nm gold particles as secondary antibodies. The secondary antibodies penetrated easily into the tissue owing to their reduced size and the very small gold particles. After the labelling procedure, the tissue was fixed in glutaraldehyde. Since the gold particles were not visible by conventional transmission electron microscopy, they were intensified with a silver developing system. Although the particle size varied nonlinearly with the developing time, very fine grain size was achievable. The technique provided super-sensitive detection with excellent contrast and demonstrated epitopes with both strong and weak affinities.

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 approximately 2.0 microm, 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 ( approximately 2.3 microm), the PEVK segment length is approximately 50 nm whereas the unique N2B sequence is approximately 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.

Muscle carnitine acetyltransferase and carnitine deficiency in a case of mitochondrial encephalomyopathy.

Profound decrease of the carnitine acetyltransferase activity (0.08 U/g wet weight; 1.67% of control) and carnitine deficiency (total carnitine was 230 nmol/g wet weight in the patient vs 2730 in the controls) was detected in the skeletal muscle of a female paediatric patient. She died of her illness, which included cerebellar symptoms and slight muscle spasticity affecting mainly the lower extremities, at 1 year of age. Histological examination of the autopsy specimens revealed a selective Purkinje cell degeneration in the cerebellum: the cells had abnormal position, were shrunken and decreased in number, and displayed abnormal dendritic trees and fragmented, disorganized axons. Electron microscopy revealed mitochondrial abnormalities in skeletal and cardiac muscle and also in the Purkinje cells. Deletions of the mitochondrial DNA were detected in the muscle in heteroplasmic form (up to 7%). Mainly the ND4-ND4L region was affected, as evidenced by the PCR; however, other regions of the mitochondrial genome also showed deletions of varying size and extent, suggesting multiple deletions of the mitochondrial DNA.

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.

Role of reactive oxygen species and poly-ADP-ribose polymerase in the development of AZT-induced cardiomyopathy in rat.

The short term cardiac side-effects of AZT (3'-azido-3'-deoxythymidine, zidovudine) was studied in rats to understand the biochemical events contributing to the development of AZT-induced cardiomyopathy. Developing rats were treated with AZT (50 mg/kg/day) for 2 wk and the structural and functional changes were monitored in the cardiac muscle. AZT treatment provoked a surprisingly fast appearance of cardiac malfunctions in developing animals characterized by prolonged RR, PR and QT intervals and J point depression. Electron microscopy showed abnormal mitochondrial structure but the cardiomyocyte had normal myofibers. The AZT treatment of rats significantly increased ROS and peroxynitrite formation in heart tissues as determined by the oxidation of nonfluorescent dihydrorhodamine123 and dichlorodihydro-fluorescein diacetate (H2DCFDA) to fluorescent dyes, and induced single-strand DNA breaks. Lipid peroxidation and oxidation of cellular proteins determined from protein carbonyl content were increased as a consequence of AZT treatment. Activation of the nuclear poly-ADP-ribose polymerase and the accelerated NAD+ catabolism were also observed in AZT-treated animals. Western blot analysis showed that mono-ADP-ribosylation of glucose regulated protein (GRP78/BIP) was enhanced by AZT treatment, that process inactivates GRP78. In this way moderate decrease in the activity of respiratory complexes was detected in the heart of AZT-treated animals indicating a damaged mitochondrial energy production. There was a significant decrease in creatine phosphate concentration resulting in a decrease in creatine phosphate/creatine ratio from 2.08 to 0.58. ATP level remained close to normal but the total extractable ADP increased with 45%. The calculated free ATP/ADP ratio decreased from 340 to 94 in the heart of AZT-treated rats as a consequence of increased free ADP concentration. It was assumed that the increased free ADP in AZT-treated cardiomyocyte may help cells to compensate the defective ATP production in damaged mitochondria by activating the ATP synthesis in undamaged mitochondria. Southern blot analysis did not show decreased quantity of mtDNA deriving from AZT-treated rat hearts indicating that under our experimental conditions AZT-induced heart abnormalities are not the direct consequence of the mtDNA depletion. These data show that ROS-mediated oxidative damages, activated ADP-ribosylation reactions and accelerated NAD+ catabolism play basic roles in the development of AZT-induced cardiomyopathy in our animal model and indicated that these ROS-mediated processes can be important factors in the development of myopathy and cardiomyopathy in zidovudine-treated AIDS patients.

The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

PEVK extension of human soleus muscle titin revealed by immunolabeling with the anti-titin antibody 9D10.

Titin is a giant protein that spans half of the striated muscle sarcomere. The I-band portion of titin extends as the sarcomere is stretched, developing what is known as passive force. This portion of the molecule is composed mainly of tandem immunoglobulin (Ig) segments, consisting of serially linked Ig-like domains, and a recently discovered unique sequence termed the PEVK segment. The tandem Ig and PEVK segments have been suggested to extend sequentially when sarcomeres are stretched, with PEVK extension dominating at moderate to high degrees of sarcomere stretch (M. Gautel and D. Goulding, 1996, FEBS Lett. 385, 11-14; W. Linke et al., 1996, J. Mol. Biol. 261, 62-71; K. Trombitás et al., 1998). Previously we observed that the anti-titin antibody 9D10 labels a region in the I-band that increases in width as sarcomeres are stretched. Here we tested whether 9D10 labels the PEVK segment. The 9D10-labeled region of human soleus fibers was followed by immunoelectron microscopy as sarcomeres were stretched. It was found that 9D10 labeled a region in the I-band that was approximately 100 nm wide at a sarcomere length of 2.4 micrometer and approximately 550 nm wide at a sarcomere length of 4.0 micrometer. Results were compared with those obtained with sequence-specific antibodies that were used to mark the boundaries of the PEVK segment. Findings indicate that 9D10 labels the PEVK segment from close to its N-terminal end to its C-terminal end. Consistent with this conclusion are the results on cardiac myocytes that express a much shorter PEVK segment than skeletal muscle and where 9D10 labels a region that is much less wide than in skeletal muscle. The anti-titin antibody 9D10 is a useful tool for investigating the extensible behavior of the PEVK segment in both skeletal and cardiac muscles.