Alexandra Freiburg

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.

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. Titins 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.

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 titins 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.

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 titins MIR, followed by reactivity along the titin molecule in the course of the disease.

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.

Myasthenia Gravis Patients have a Cellular Immune Response against Titin

Myasthenia gravis (MG) is caused by antibodies against the acetylcholine receptor (AChR). However, some MG patients have antibodies against non-AChR epitopes of skeletal muscle including titin. In this study, we tested peripheral blood lymphocytes from 11 MG patients and 13 blood donors in a lymphocyte transformation test using the antigen MGT-30, a titin epitope that represents the main immunogenic region. Stimulation index (SI) was defined as counts per minute (cpm) in stimulated culture minus background divided by cpm in unstimulated culture minus background. MGT-30 caused a significant stimulation of T-cells from titin-antibody positive MG patients; SI=1.84±0.77, compared with titin-antibody negative patients; SI=0.67±0.28 (p=0.01), and blood-donors; SI=0.65±0.21 (p=0.0005). SI after PHA stimulation was similar in MG patients with and without titin-antibodies and blood-donors. After MGT-30 stimulation, IL-4 concentrations in the supernatant were 84–150pg/mL in all 4 titin-positive patients examined. IL-4 levels were below the detection limit for the ELISA used (60pm/ml) in the cultures from the 8 blood-donors and the 1 titin-negative MG patient that were tested. Thus, MG patients with anti-titin antibodies also have a T-cell mediated immune reactivity against titin.