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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.