Deleting titin's I-band/A-band junction reveals critical roles for titin in biomechanical sensing and cardiac function.
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ABSTRACT:
SUBMITTER: Granzier HL
PROVIDER: S-EPMC4210014 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Titin is a giant elastic protein that spans the half-sarcomere from Z-disk to M-band. It acts as a molecular spring and mechanosensor and has been linked to striated muscle disease. The pathways that govern titin-dependent cardiac growth and contribute to disease are diverse and difficult to dissect. METHODS:To study titin deficiency versus dysfunction, the authors generated and compared striated muscle specific knockouts (KOs) with progressive postnatal loss of the complete titin protein by removing exon 2 (E2-KO) or an M-band truncation that eliminates proper sarcomeric integration, but retains all other functional domains (M-band exon 1/2 [M1/2]-KO). The authors evaluated cardiac function, cardiomyocyte mechanics, and the molecular basis of the phenotype. RESULTS:Skeletal muscle atrophy with reduced strength, severe sarcomere disassembly, and lethality from 2 weeks of age were shared between the models. Cardiac phenotypes differed considerably: loss of titin leads to dilated cardiomyopathy with combined systolic and diastolic dysfunction-the absence of M-band titin to cardiac atrophy and preserved function. The elastic properties of M1/2-KO cardiomyocytes are maintained, while passive stiffness is reduced in the E2-KO. In both KOs, we find an increased stress response and increased expression of proteins linked to titin-based mechanotransduction (CryAB, ANKRD1, muscle LIM protein, FHLs, p42, Camk2d, p62, and Nbr1). Among them, FHL2 and the M-band signaling proteins p62 and Nbr1 are exclusively upregulated in the E2-KO, suggesting a role in the differential pathology of titin truncation versus deficiency of the full-length protein. The differential stress response is consistent with truncated titin contributing to the mechanical properties in M1/2-KOs, while low titin levels in E2-KOs lead to reduced titin-based stiffness and increased strain on the remaining titin molecules. CONCLUSIONS:Progressive depletion of titin leads to sarcomere disassembly and atrophy in striated muscle. In the complete knockout, remaining titin molecules experience increased strain, resulting in mechanically induced trophic signaling and eventually dilated cardiomyopathy. The truncated titin in M1/2-KO helps maintain the passive properties and thus reduces mechanically induced signaling. Together, these findings contribute to the molecular understanding of why titin mutations differentially affect cardiac growth and have implications for genotype-phenotype relations that support a personalized medicine approach to the diverse titinopathies.
| S-EPMC6453709 | biostudies-literature
Project description:AimsHeart failure (HF) patients commonly experience symptoms primarily during elevated heart rates, as a result of physical activities or stress. A main determinant of diastolic passive tension, the elastic sarcomeric protein titin, has been shown to be associated with HF, with unresolved involvement regarding its role at different heart rates. To determine whether titin is playing a role in the heart rate (frequency-) dependent acceleration of relaxation (FDAR). W, we studied the FDAR responses in live human left ventricular cardiomyocytes and the corresponding titin-based passive tension (TPT) from failing and non-failing human hearts.Methods and resultsUsing atomic force, we developed a novel single-molecule force spectroscopy approach to detect TPT based on the frequency-modulated cardiac cycle. Mean TPT reduced upon an increased heart rate in non-failing human hearts, while this reduction was significantly blunted in failing human hearts. These mechanical changes in the titin distal Ig domain significantly correlated with the frequency-dependent relaxation kinetics of human cardiomyocytes obtained from the corresponding hearts. Furthermore, the data suggested that the higher the TPT, the faster the cardiomyocytes relaxed, but the lower the potential of myocytes to speed up relaxation at a higher heart rate. Such poorer FDAR response was also associated with a lesser reduction or a bigger increase in TPT upon elevated heart rate.ConclusionsOur study established a novel approach in detecting dynamic heart rate relevant tension changes physiologically on native titin domains. Using this approach, the data suggested that the regulation of kinetic reserve in cardiac relaxation and its pathological changes were associated with the intensity and dynamic changes of passive tension by titin.
| S-EPMC6918063 | biostudies-literature
Project description:The thick filament-associated A-band region of titin is a highly repetitive component of the titin chain with important scaffolding properties that support thick filament assembly. It also has a demonstrated link to human disease. Despite its functional significance, it remains a largely uncharacterized part of the titin protein. Here, we have performed an analysis of sequence and structure conservation of A-band titin, with emphasis on poly-FnIII tandem components. Specifically, we have applied multi-dimensional sequence pairwise similarity analysis to FnIII domains and complemented this with the crystallographic elucidation of the 3D-structure of the FnIII-triplet A84-A86 from the fourth long super-repeat in the C-zone (C4). Structural models serve here as templates to map sequence conservation onto super-repeat C4, which we show is a prototypical representative of titin's C-zone. This templating identifies positionally conserved residue clusters in C super-repeats with the potential of mediating interactions to thick-filament components. Conservation localizes to two super-repeat positions: Ig domains in position 1 and FnIII domains in position 7. The analysis also allows conclusions to be drawn on the conserved architecture of titin's A-band, as well as revisiting and expanding the evolutionary model of titin's A-band.
| S-EPMC10665226 | biostudies-literature
Project description:In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.
| S-EPMC2150553 | biostudies-literature
Project description:Titin is largely comprised of serially-linked immunoglobulin (Ig) and fibronectin type-III (Fn3) domains. Many of these domains are arranged in an 11 domain super-repeat pattern that is repeated 11 times, forming the so-named titin C-zone in the A-band region of the sarcomere. Each super-repeat is thought to provide binding sites for thick filament proteins, such as cMyBP-C (cardiac myosin-binding protein C). However, it remains to be established which of titin's 11 C-zone super-repeats anchor cMyBP-C as titin contains 11 super-repeats and cMyBP-C is found in 9 stripes only. To study the layout of titin's C-zone in relation to MyBP-C, immunolabeling studies were performed on mouse skinned myocardium with antibodies to titin and cMyBP-C, using both immuno-electron microscopy and super-resolution optical microscopy. Results indicate that cMyBP-C locates near the interface between titin's C-zone super-repeats. Studies on a mouse model in which two of titin's C-zone repeats have been genetically deleted support that the first Ig domain of a super-repeat is important for anchoring cMyBP-C but also Fn3 domains located at the end of the preceding repeat. Furthermore, not all super-repeat interfaces are equal as the interface between super-repeat 1 and 2 (close to titin's D-zone) does not contain cMyBP-C. Finally, titin's C-zone does not extend all the way to the bare zone but instead terminates at the level of the second myosin crown. This study enhances insights in the molecular layout of the C-zone of titin, its relation to cMyBP-C, and its possible roles in cardiomyopathies.
| S-EPMC6639027 | biostudies-literature
Project description: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.
| S-EPMC1300589 | biostudies-other
Project description:The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219-225 deleted, the TtnΔ219-225 model (MGI: TtnTM 2.1Mgot ). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219-225 muscles: a larger isoform that represents the TtnΔ219-225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219-225 soleus muscle (WT 240 ± 9; TtnΔ219-225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219-225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219-225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin's PEVK exons 219-225 have high passive tension, due in part to alterations elsewhere in splicing of titin's spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin's C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.
| S-EPMC7274174 | biostudies-literature
Project description: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.
| S-EPMC4001778 | biostudies-literature
Project description:Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction, and muscle stiffness. TTN splicing generates variable protein sizes with different functions. Therefore, understanding TTN splicing is important to develop a novel treatment for TTN-based diseases. The I-band TTN splicing regulated by RNA binding motif 20 (RBM20) has been extensively studied. However, the Z- and M-band splicing and regulation remain poorly understood. Herein, we aimed to define the Z- and M-band splicing in striated muscles and determined whether RBM20 regulates the Z- and M-band splicing. We discovered four new Z-band TTN splicing variants, and one of them dominates in mouse, rat, sheep, and human hearts. But only one form can be detected in frog and chicken hearts. In skeletal muscles, three new Z repeats (Zr) were detected, and Zr4 to 6 exclusion dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z-band. In the M-band, two new variants were discovered with alternative 3' splice site in exon363 (Mex5) and alternative 5' splice site in intron 362. However, only the sheep heart expresses two new variants rather than other species. Skeletal muscles express three M-band variants with altered ratios of Mex5 inclusion to Mex5 exclusion. Finally, we revealed that RBM20 does not regulate the Z- and M-band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z- and M-band splicing and provided the first evidence of the role of RBM20 in the Z- and M-band TTN splicing.
| S-EPMC6218289 | biostudies-literature
Project description:Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.
| S-EPMC5043576 | biostudies-literature