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.

Tissue-specific expression and alpha-actinin binding properties of the Z-disc titin: implications for the nature of vertebrate Z-discs.

Titins are giant filamentous proteins which connect Z-discs and M-lines in the sarcomeres of vertebrate striated muscles. Comparison of the N-terminal region of titin (Z-disc region) from different skeletal and cardiac muscles reveals a 900-residue segment which is expressed in different length variants, dependent on tissue type. When searching for ligands of this differentially expressed domain by a yeast-two hybrid approach, we detected binding to alpha-actinin. The isolated alpha-actinin cDNAs were derived from the C-terminal region of the alpha-actinin isoform (alpha-actinin-2) encoded by the ACTN2 gene. Therefore, the two antiparallel subunits of an alpha-actinin-2 homodimer will attach to actin at their respective C termini, whereas they will bind to the Z-disc titin at their N termini. This may thus explain how alpha-actinins can cross-link antiparallel titin and thin filaments from opposing sarcomeres. The alpha-actinin-2 binding site of the Z-disc titin is located within a sequence of 45-residue repeats, referred to as Z-repeat region. Both the N-terminal and C-terminal Z-repeats have alpha-actinin binding properties and are expressed in all striated muscles. By contrast, the more central Z-repeats are expressed in slow and fast skeletal muscles, as well as embryonic and adult cardiac muscles, in different copy numbers. Such alternative splicing of the Z-disc titin appears to be important for the tissue and fibre type diversity of the Z-disc lattice.

Paraneoplastic myasthenia gravis: detection of anti-MGT30 (titin) antibodies predicts thymic epithelial tumor.

It has been suggested that antibodies against non-acetylcholine receptor proteins of striated muscle are markers of the presence of a thymic epithelial tumor in patients with myasthenia gravis (MG). These antibodies may be measured using an immunofluorescence assay against striated muscle (anti-STR) or an ELISA with a recombinant 30-kd titin fragment (anti-MGT30). To directly compare anti-STR with anti-MGT30, we examined the sera of 276 consecutive patients with known or suspected MG. Definite diagnoses and thymic histology, if available, were correlated with the antibody assays. Of the 276 patients, 164 had MG. Thymic histology was obtained in 44 patients: 18 had lymphofollicular hyperplasia, 13 thymic epithelial tumors, 8 atrophy, and 5 were normal. When compared with anti-STR, anti-MGT30 showed a sensitivity of 69% (STR 77%), specificity of 100% (STR 56%, p = 0.026), negative predictive value of 82% (STR 77%), and positive predictive value of 100% (STR 56%, p = 0.003) for the identification of a thymic epithelial tumor versus thymic hyperplasia. We conclude that the anti-MGT30 ELISA is better than the anti-STR immunofluorescence assay for the diagnosis of paraneoplastic MG.

Striational autoantibodies in myasthenia gravis patients recognize I-band titin epitopes.

Myasthenia gravis (MG) patients develop autoantibodies primarily against the acetylcholine receptor in the motor endplate, but also against intracellular striated muscle proteins, notably titin, the giant elastic protein of the myofibrillar cytoskeleton. Titin antibodies have previously been shown to be directed against a single epitope on the molecule, located at the A-band/I-band junction and referred to as the main immunogenic region (MIR) of titin. By using immunofluorescence microscopy on stretched single myofibrils, we now report that approximately 40% of the sera from 18 MG/thymoma patients and 8 late-onset MG patients with thymus atrophy contain antibodies that bind to a more central I-band titin region. This region consists of homologous immunoglobulin domains and is known to be differentially spliced dependent on muscle type. All patients with I-band titin antibodies also had antibodies against the MIR. Although a statistically significant correlation between the occurrence of I-band titin antibodies and MG severity was not apparent, the results could hint at an initial immunoreactivity to titin's MIR, followed by reactivity along the titin molecule in the course of the disease.

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.

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.

A molecular map of the interactions between titin and myosin-binding protein C. Implications for sarcomeric assembly in familial hypertrophic cardiomyopathy.

The thick filaments of vertebrate striated muscles contain with myosin a number of accessory proteins of the intracellular immunoglobulin superfamily, which are localized in a distinct pattern of stripes 43 nm apart. The specific localization of these proteins is believed to be due partly to their interaction with the giant muscle protein titin (also called connectin), which spans the entire sarcomere and may act as a molecular ruler. We have used recombinant fragments of titin covering the thick filament region to investigate their interaction with myosin-binding protein C (MyBP-C) from skeletal and cardiac muscle. The interaction of titin and MYBP-C is directed by a subset of titin immunoglobulin domains that are specific for the C-region of the thick filament, supporting the ruler hypothesis for the myosin-binding proteins. The interaction of recombinant titin with overlapping fragments of human cardiac MyBP-C maps the titin-binding site within the C-terminal region, which is deleted in patients suffering from the chromosome-11-associated form of familial hypertrophic cardiomyopathy. This disorder is therefore likely to be the result of thick-filament misassembly by abolishing the ternary interaction of titin, myosin and MyBP-C.

Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction?

Cardiac myosin binding protein-C (cardiac MyBP-C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP-dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP-C now reveals regulatory motifs specific for this isoform. Site-directed mutagenesis identifies a LAGGGRRIS loop in the N-terminal region of cardiac MyBP-C as the key substrate site for phosphorylation by both PKA and a calmodulin-dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform-specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP-C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP-C in cardiac disease. The gene coding for cardiac MyBP-C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP-C a candidate gene for chromosome 11-associated FHC.

Titin antibody positive myasthenia gravis patients have a cellular immune response against the main immunogenic region of titin.

Some myasthenia gravis (MG) patients have antibodies against non-acetylcholine receptor (AChR) epitopes of skeletal muscle including titin. Peripheral blood lymphocytes from 11 MG patients and 13 blood-donors were tested for lymphocyte proliferation after stimulation with the titin peptide MGT-30, which represents the main immunogenic region. Four out of seven titin antibody positive patients had significant stimulation defined as a stimulation index (SI) above 2. Neither of the four titin antibody negative patients nor the 13 blood-donors had SI above 2 (p = 0.001). Mean SI was significantly higher for T-cells from titin antibody positive MG patients, SI = 2.2 ± 0.8, compared to titin antibody negative patients, SI = 0.9 ± 0.2 (p = 0.01), and blood-donors, SI = 0.8 ±0.3 (p > 0.0005). After MGT-30 stimulation, IL-4 was detected in the blood lymphocyte culture supernatant from four of the five MG patients examined, but from none of the eight blood-donors. Thus, MG patients with anti-titin antibodies have a T-cell mediated immune reaction against titin.

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.

Cell-mediated immune response against titin in myasthenia gravis: evidence for the involvement of Th1 and Th2 cells.

Myasthenia gravis (MG) patients may have circulating autoantibodies against titin. In this study, we have stimulated T cells from MG patients with a recombinant polypeptide containing the main immunogenic region of titin, MGT-30 (myasthenia gravis titin-30 kDa). In an ELISpot assay, MGT-30 reactive interferon (IFN)-gamma secreting cells (Th1 cells) were detected in six of 10 titin antibody positive MG patients. Such cells were not detected in any of the five titin antibody negative MG patients or in the seven blood donors. In three patients, the stimulated number of cells decreased when total remission of MG symptoms was achieved after thymectomy or following a period of intensive immunosuppressive medication. We detected MGT-30 interleukin (IL)-4 secreting cells (Th2 cells) in two of five titin antibody positive MG patients, but not in the two titin antibody negative patients or the five blood donors examined. We conclude that titin antibody positive MG patients have a combined Th1/Th2 cell mediated immunity against the muscle protein titin.

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.

Titin transcripts in thymomas.

More than 90% of myasthenia gravis (MG) patients with a thymoma have antibodies against titin. We have identified titin mRNA transcripts in thymomas by RT-PCR and Southern blotting. The transcripts cover the main immunogenic region (MIR) and a central I-band epitope reactive with some MG patients' antibodies. The presence of the central I-band epitope was confirmed by immunohistochemistry as a titin antibody reactive with this part of titin, stained thymoma epithelial cells and a thymoma extract in Western blots. Our findings suggest that the initiation of paraneoplastic titin reactivity is correlated with the expression of titin sequences within the thymoma.