Titin Research Articles

Crystallographic Structures of Titin Immunoglobulin-Like I21 Domains Involved in Dilated Cardiomyopathy

The protein titin is the main responsible for the passive elasticity of the sarcomere, determining the stiffness and contractile function of cardiomyocytes. Titin is also a key factor in the etiology of heart disease, given that truncating mutations in the titin gene are the most common cause of dilated cardiomyopathy (DCM). However, missense mutations have commonly been classified as variants of uncertain significance because of their high frequency in the general population and the absence of functional annotation. We have recently identified a missense mutation targeting a conserved cysteine in the I21 domain of titin as a cause of DCM in humans. Here, we present the structural characterization of the wild type domain, that follows a fold characteristic for the intermediate set (type I) of the immunoglobulin superfamily: 2 antiparallel 4-strand-β-sheets packed in a β-sandwich shape. Interestingly, we observed that the I21 titin domain contains a second cysteine located 5Å away from the cysteine position mutated in dilated cardiomyopathy patients. We also report the structural and biophysical characterization of the mutated domain. The mutated domain presents unaltered overall structure, although its thermal stability is severely compromised, showing a decrease of 17ºC in its melting temperature when compared to the wild type domain. Our results suggest that titin domain destabilization by missense variants can lead to DCM, even if the structure of the native state is preserved. Future research will investigate the origin of the thermal destabilization in the mutated domain, and the potential influence of intramolecular disulfide bonds.

Graded Titin Cleavage Uncovers the Protein's Role for Sarcomere Structure and Force Generation of Contracting Muscle

The giant muscle protein titin is a major contributor to passive force; however, its role during active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of the titin springs to quantify how titin-based forces define myocyte ultrastructure and mechanics. We conduct a series of passive and active mechanical tests on permeabilized psoas fiber bundles from wildtype, heterozygote, and homozygote TC mice. We show that under mechanical strain, as titin cleavage doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly non-linear, while passive and active forces are progressively reduced. Furthermore, interactions of elastic titin with sarcomeric actin filaments are revealed, as cleaved I-band titins only partially recoil to the Z-disk, and instead stick to the thin filaments. Strikingly, when titin-cleaved fibers contract, myosin-containing A-bands quickly stream to the point that they split and adjacent thick filaments move in opposite directions. Cleavage of I-band titin also destabilizes the myosin filaments during contraction, which leads to a shedding of individual myosin rods from the D-zone of thick filaments. Taken together, these results establish intact titin filaments as critical force-transmission networks, buffering the forces between myosin filaments during contraction, while also stabilizing the myosin rods within the thick filament. Based on our calculations, a structural change in titin that increases stiffness compared to passive muscle is critical for titin to perform these buffering tasks, unveiling its fundamental role as an activation-dependent spring in contracting muscle.

Unusual Mobility of the Elastic Protein Titin in Thirteen-Lined Ground Squirrel Hearts

Small mammalian hibernators, such as thirteen-lined ground squirrels, undergo rapid, 100-fold reductions in heart rate, leading to a substantial prolongation of the cardiac cycle when entering torpor. Initial studies using high Urea solubilization buffers and Vertical Agarose Electrophoresis suggest no seasonally dependent change in the ratio of the longer, compliant N2BA to the shorter, stiff N2B cardiac titin isoform (N2BA:N2B). However, an unusually large protein was observed. Previously, this band was observed in preparations using urea-free solubilization buffers and agarose-acrylamide gel systems.

Mechanisms of TTNtv-Related Dilated Cardiomyopathy: Insights from Zebrafish Models.

Dilated cardiomyopathy (DCM) is a common heart muscle disorder characterized by ventricular dilation and contractile dysfunction that is associated with significant morbidity and mortality. New insights into disease mechanisms and strategies for treatment and prevention are urgently needed. Truncating variants in the TTN gene, which encodes the giant sarcomeric protein titin (TTNtv), are the most common genetic cause of DCM, but exactly how TTNtv promote cardiomyocyte dysfunction is not known. Although rodent models have been widely used to investigate titin biology, they have had limited utility for TTNtv-related DCM. In recent years, zebrafish (Danio rerio) have emerged as a powerful alternative model system for studying titin function in the healthy and diseased heart. Optically transparent embryonic zebrafish models have demonstrated key roles of titin in sarcomere assembly and cardiac development. The increasing availability of sophisticated imaging tools for assessment of heart function in adult zebrafish has revolutionized the field and opened new opportunities for modelling human genetic disorders. Genetically modified zebrafish that carry a human A-band TTNtv have now been generated and shown to spontaneously develop DCM with age. This zebrafish model will be a valuable resource for elucidating the phenotype modifying effects of genetic and environmental factors, and for exploring new drug therapies.

Impact of prolonged sepsis on neural and muscular components of muscle contractions in a mouse model.

BackgroundProlonged critically ill patients frequently develop debilitating muscle weakness that can affect both peripheral nerves and skeletal muscle. In‐depth knowledge on the temporal contribution of neural and muscular components to muscle weakness is currently incomplete.MethodsWe used a fluid‐resuscitated, antibiotic‐treated, parenterally fed murine model of prolonged (5 days) sepsis‐induced muscle weakness (caecal ligation and puncture; n = 148). Electromyography (EMG) measurements were performed in two nerve–muscle complexes, combined with histological analysis of neuromuscular junction denervation, axonal degeneration, and demyelination. In situ muscle force measurements distinguished neural from muscular contribution to reduced muscle force generation. In myofibres, imaging and biomechanics were combined to evaluate myofibrillar contractile calcium sensitivity, sarcomere organization, and fibre structural properties. Myosin and actin protein content and titin gene expression were measured on the whole muscle.ResultsFive days of sepsis resulted in increased EMG latency (P = 0.006) and decreased EMG amplitude (P < 0.0001) in the dorsal caudal tail nerve–tail complex, whereas only EMG amplitude was affected in the sciatic nerve–gastrocnemius muscle complex (P < 0.0001). Myelin sheath abnormalities (P = 0.2), axonal degeneration (number of axons; P = 0.4), and neuromuscular junction denervation (P = 0.09) were largely absent in response to sepsis, but signs of axonal swelling [higher axon area (P < 0.0001) and g‐ratio (P = 0.03)] were observed. A reduction in maximal muscle force was present after indirect nerve stimulation (P = 0.007) and after direct muscle stimulation (P = 0.03). The degree of force reduction was similar with both stimulations (P = 0.2), identifying skeletal muscle, but not peripheral nerves, as the main contributor to muscle weakness. Myofibrillar calcium sensitivity of the contractile apparatus was unaffected by sepsis (P ≥ 0.6), whereas septic myofibres displayed disorganized sarcomeres (P < 0.0001) and altered myofibre axial elasticity (P < 0.0001). Septic myofibres suffered from increased rupturing in a passive stretching protocol (25% more than control myofibres; P = 0.04), which was associated with impaired myofibre active force generation (P = 0.04), linking altered myofibre integrity to function. Sepsis also caused a reduction in muscle titin gene expression (P = 0.04) and myosin and actin protein content (P = 0.05), but not the myosin‐to‐actin ratio (P = 0.7).ConclusionsProlonged sepsis‐induced muscle weakness may predominantly be related to a disruption in myofibrillar cytoarchitectural structure, rather than to neural abnormalities.

(Epi)genetic variants of the sarcomere-desmosome are associated with premature utero-contraction in spontaneous preterm labor

Spontaneous preterm birth is a syndrome with clinical and genetic heterogeneity. Few studies have focused on the genetic and epigenetic defects and pathogenic mechanisms associated with premature uterine contraction in spontaneous preterm birth. The objective of this study was to investigate the (epi)genetic variations associated with premature uterine contraction of spontaneous preterm birth. A systems biology approach with an integrated multiomic study was employed. Biobanked pregnancy tissues selected from a pregnancy cohort were subjected to genomic, transcriptomic, methylomic, and proteomic studies, with a focus on genetic loci/genes related to uterine muscle contraction, specifically, genes associated with sarcomeres and desmosomes. Thirteen single nucleotide variations and pathogenic variants were identified in the sarcomere gene, TTN, which encodes the protein Titin, from 146 women with spontaneous preterm labor. Differential expression profiles of five long non-coding RNAs were identified from loci that overlap with four sarcomeric genes. Longitudinally, the long non-coding RNA of gene TPM3 that encodes the protein tropomysin 3 was found to significantly regulate the mRNA of TPM3 in the placenta, compared to maternal blood. The majority of genome methylation profiles related to premature uterine contraction were also identified in the CpG promoters of sarcomeric genes/loci. Differential expression profiles of mRNAs associated with premature uterine contraction showed 22 genes associated with sarcomeres and three with desmosomes. The results demonstrated that premature uterine contraction was associated mainly with pathogenic variants of the TTN gene and with transcriptomic variations of sarcomeric premature uterine contraction genes. This association is likely regulated by epigenetic factors, including methylation and long non-coding RNAs.

Making sense of missense variants in TTN-related congenital myopathies

Mutations in the sarcomeric protein titin, encoded by TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.

Molecular Mechanisms of Muscle Tone Impairment under Conditions of Real and Simulated Space Flight.

Kozlovskaya et al. [1] and Grigoriev et al. [2] showed that enormous loss of muscle stiffness (atonia) develops in humans under true (space flight) and simulated microgravity conditions as early as after the first days of exposure. This phenomenon is attributed to the inactivation of slow motor units and called reflectory atonia. However, a lot of evidence indicating that even isolated muscle or a single fiber possesses substantial stiffness was published at the end of the 20th century. This intrinsic stiffness is determined by the active component, i.e. the ability to form actin-myosin cross-bridges during muscle stretch and contraction, as well as by cytoskeletal and extracellular matrix proteins, capable of resisting muscle stretch. The main facts on intrinsic muscle stiffness under conditions of gravitational unloading are considered in this review. The data obtained in studies of humans under dry immersion and rodent hindlimb suspension is analyzed. The results and hypotheses regarding reduced probability of cross-bridge formation in an atrophying muscle due to increased interfilament spacing are described. The evidence of cytoskeletal protein (titin, nebulin, etc.) degradation during gravitational unloading is also discussed. The possible mechanisms underlying structural changes in skeletal muscle collagen and its role in reducing intrinsic muscle stiffness are presented. The molecular mechanisms of changes in intrinsic stiffness during space flight and simulated microgravity are reviewed.

Digenic Inheritance Involving a Muscle Specific Protein Kinase and the Giant Titin Protein Causes a Skeletal Muscle Myopathy

Digenic Inheritance Involving a Muscle Specific Protein Kinase and the Giant Titin Protein Causes a Skeletal Muscle Myopathy

TTN GENE’S VARIANTS AS POTENTIAL MARKERS ASSOCIATED WITH MUSCLE TISSUE’S DISFUNCTIONS AND PHYSICAL PERFORMANCE

A titin, encoded by a TTN gene, is a third most abundant sarcomere component. Although, this myofilament plays a wide range of key roles in muscle tissue such as structural, developmental, mechanical, and regulatory functions, it is a usually missed aspect of the muscle properties formation. At first, the TTN gene variants was described in development of skeletal and cardiac muscle diseases. Recently, the gene is also considered a very promising genetic marker for sport performance which may underlie differences in the potential to be an elite athlete. The aim of the present study is to provide the comprehensive update of the titin protein and the TTN gene variants role in formation of skeletal and cardiac muscle properties. We review function and structure of the protein, the gene, and the isoforms, as well as molecular mechanisms, disease-causing mutations, associated phenotypes, and their implications for human health, physical performance, adaptive changes of muscles in response to training, and injury risk.

Investigation of the properties of the Cr,Mo-(Cr,Mo,Zr,Nb)N-(Cr,Mo,Zr,Nb,Al)N multilayer composite multicomponent coating with nanostructured wear-resistant layer

The article discusses the properties of the Cr,Mo-(Cr,Mo,Zr,Nb)N-(Cr,Mo,Zr,Nb,Al)N multilayer composite multicomponent coating with a nanostructured wear-resistant layer. The structure and phase composition of the coating were investigated. The comparative cutting tests were carried out on carbide cutting tools with the coating under study and carbide cutting tools with the Ti–TiN-(Ti,Al,Cr)N commercial coating. Particular attention was paid to the study of the wear and fracture patterns typical for the coating and the diffusion processes on the “coating–material being machined” interface. The studies found the difference in the content of Fe in the coating nanolayers. In particular, the high content of Fe was detected in the nanolayers with the increased content of Cr–Mo, while the nanolayers with the increased content of Al demonstrated the low content of Fe. The depth of the Fe diffusion into the coating is 2–3 times lower compared to the Fe diffusion into a carbide substrate. The influence of the nanolayer structure of the coating on the crack propagation was considered. The studies revealed signs of plastic deformation in the surface layers of the coating. The investigation was also focused on the structure of a build-up of the material being machined with a layer structure demonstrating different levels of the oxidation inits layers.

The Interplay Between Titin, Polygenic Risk, and Modifiable Cardiovascular Risk Factors in Atrial Fibrillation

BackgroundCommon and rare variants, including those in the gene for the cardiac structural protein titin (TTN), have been implicated in the risk of developing atrial fibrillation (AF). However, the effect of genetic variants on risk of AF compared with established modifiable risk factors is unclear. The objective of this study was to evaluate the risk of AF and associated cardiovascular complications in TTN variant carriers and examine interactions between TTN variants or common variants and modifiable AF risk factors. MethodsWe used whole exome sequencing data of 49,881 individuals and genotyping data of 408,572 individuals from the UK Biobank to examine the associations of TTN variants, polygenic risk, and 4 risk factors (hypertension, diabetes, obesity, and smoking) with AF. Adjusted hazard ratios (aHRs) were calculated with the use of Cox proportional hazards models. ResultsTTN variant carrier status was associated with a higher risk of AF (aHR 2.10, 95% CI 1.59-2.79; P = 2.54 × 10−7) and higher risk of dilated cardiomyopathy in AF patients (aHR 10.39, 95% CI 5.31-20.33; P = 8.37 × 10−12). We identified additive effects between TTN variants and polygenic risk with hypertension, diabetes, obesity, and smoking on the risk of AF. ConclusionsGenetic and modifiable cardiovascular risk factors contribute to the probability of developing AF. Our findings highlight the potential utility of incorporating data from targeted sequencing or genotyping of common variants to further inform AF risk stratification and aggressive management of modifiable cardiovascular risk factors.

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle.

The giant muscle protein titin is a major contributor to passive force; however, its role in active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of elastic titin to quantify how titin-based forces define myocyte ultrastructure and mechanics. We show that under mechanical strain, as TC doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly out of register while passive and active forces are reduced. Interactions of elastic titin with sarcomeric actin filaments are revealed. Strikingly, when titin-cleaved muscles contract, myosin-containing A-bands become split and adjacent myosin filaments move in opposite directions while also shedding myosins. This establishes intact titin filaments as critical force-transmission networks, buffering the forces observed by myosin filaments during contraction. To perform this function, elastic titin must change stiffness or extensible length, unveiling its fundamental role as an activation-dependent spring in contracting muscle.

Investigation of the Influence of Microdroplets on the Coatings Nanolayer Structure

The paper presents the results of studies focused on the specific features typical for the formation of the shape and structure of microdroplets embedded into the structure of the Ti–TiN–(Ti,Cr,Mo,Al)N and Ti–TiN–(Ti,Al,Nb,Zr)N coatings during their deposition. Three main microdroplet shapes—a sphere, a tear, and a lens—have been considered. The specific features typical for the formation of secondary layered structures on the surface of some microdroplets have also been examined. As a result of the conducted investigations, with the use of scanning and transmission electron microscopy, the influence of microdroplets on the distortion of the nanolayer structure of the coatings was studied. A hypothesis has been proposed concerning a relationship between the microdroplet shape and the presence or absence of secondary structures and the microdroplet sizes and weight, as well as the conditions in the unit chamber during the movement of a microdroplet from a cathode to the deposition surface. Based on the study focused on the shape of the microdroplet core and the specific features typical for the formation of the secondary structure around it, a hypothesis has been proposed, according to which, for some microdroplets, it takes much more time than previously assumed for the movement from a cathode to the deposition surface.

Myopathy or Myasthenia Positive for Antibodies Against MuSK, Lrp4, and Titin.

Myopathy or Myasthenia Positive for Antibodies Against MuSK, Lrp4, and Titin.

The Poly-E motif in Titin's PEVK region undergoes pH dependent conformational changes

The muscle protein titin plays a crucial role in passive elasticity and the disordered PEVK region within titin is central to that function. The PEVK region is so named due to its high proline, glutamate, valine and lysine content and the high charge density in this region results in a lack of organized structure within this domain. The PEVK region is highly extensible but the molecular interactions that contribute to the elastic nature of the PEVK still remain poorly described. The PEVK region is formed by two unique sequence motifs. The PPAK motif is a 26 to 28 amino acid sequence that contains a mixture of charged and hydrophobic residues and is the primary building block for the PEVK region. Poly-E sequence motifs vary in length and contain clusters of 3–4 glutamic acids distributed throughout the motif. In this study, we derived two 28-residue peptides from the human titin protein sequence and measured their structural characteristics over a range of pHs. Our results demonstrate that the poly-E peptide undergoes a shift from a more rigid and elongated state to a more collapsed state as pH decreases with the midpoint of this transition being at pH ~5.5. Interestingly, a similar conformational shift is not observed in the PPAK peptide. These results suggest that the poly-E motif might provide a nucleating site for the PEVK when the muscle is not in an extended state.

Evaluation of cardiac titin expression after long-term exercise in a rat model

Abstract Introduction The main role of the giant elastic protein titin is to provide passive stiffness to striated (i.e. cardiac and skeletal) muscles. The adult cardiac muscle contains two titin isoforms: the more compliant N2BA and the stiffer N2B isoforms. Titin reduces passive stiffness in cardiac muscle by increased expression of the more compliant N2BA isoform (elevated N2BA:N2B ratio). Moreover, decreased passive stiffness is associated with increased exercise tolerance. Long-term exercise induces physiological adaptation of the heart, termed athlete's heart. Currently, there is limited data of titin's role in the athlete's heart. Aims Our aim is to evaluate exercise-induced morphological and functional changes of the heart. Furthermore, to determine the N2BA:N2B ratio in the rat model of athlete's heart. Methods Rats were divided into exercised (n=12) and control (n=12) groups. Athlete's heart was induced by a 12-week-long swim training (200min/day). The control group swam 5 min/day. Following the training period cardiac changes were assessed by echocardiography. In vivo cardiac function was examined by left ventricular (LV) pressure-volume (P-V) analysis. Titin isoform expressions were detected by sodium-dodecyl-sulfate (SDS)-agarose gel electrophoresis. Results Echocardiography and post-mortem measured cardiomyocyte diameters confirmed athlete's heart and LV hypertrophy in exercised rats. P-V analysis showed improved contractility, active relaxation and mechanoenergetics in the exercised group. The N2BA:N2B titin ratio was significantly increased in exercised rats compared to controls (0.28 vs. 0.21, p&amp;lt;0.05). Conclusions Our results confirm the morphological and functional changes of the athlete's heart. The increased ratio of N2BA:N2B titin corresponds to a more compliant heart in the exercised rats. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Research, Development and Innovation Office of Hungary (K 120277) and the ÚNKP-19-3-I New National Excellence Program of The Ministry For Innovation and Technology.

Hyperbolic rules of the cooperative organization of eukaryotic and prokaryotic genomes

The author's method of oligomer sums for analysis of oligomer compositions of eukaryotic and prokaryotic genomes is described. The use of this method revealed the existence of general rules for the cooperative oligomeric organization of a wide list of genomes. These rules are called hyperbolic because they are associated with hyperbolic sequences including the harmonic progression 1, 1/2, 1/3, .., 1/n. These rules are demonstrated by examples of quantitative analysis of many genomes from the human genome to the genomes of archaea and bacteria. The hyperbolic (harmonic) rules, speaking about the existence of algebraic invariants in full genomic sequences, are considered as candidates for the role of universal rules for the cooperative organization of genomes. The results concerns additionally the problem of the origin of life. The described phenomenological results were obtained as consequences of the previously published author's quantum-information model of long DNA sequences. The oligomer sums method was also applied to the analysis of long genes and viruses including the COVID-19 virus; this revealed, in characteristics of many of them, the phenomenon of such rhythmically repeating deviations from model hyperbolic sequences, which are associated with DNA triplets. In addition, an application of the oligomer sums method is shown to the analysis of amino acid sequences in long proteins like the protein Titin. The topics of the algebraic harmony in living bodies and of the quantum-information approach in biology are discussed.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx)

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Maintenance of sarcomeric integrity in adult muscle cells crucially depends on Z-disc anchored titin

The giant protein titin is thought to be required for sarcomeric integrity in mature myocytes, but direct evidence for this hypothesis is limited. Here, we describe a mouse model in which Z-disc-anchored TTN is depleted in adult skeletal muscles. Inactivation of TTN causes sarcomere disassembly and Z-disc deformations, force impairment, myocyte de-stiffening, upregulation of TTN-binding mechanosensitive proteins and activation of protein quality-control pathways, concomitant with preferential loss of thick-filament proteins. Interestingly, expression of the myosin-bound Cronos-isoform of TTN, generated from an alternative promoter not affected by the targeting strategy, does not prevent deterioration of sarcomere formation and maintenance. Finally, we demonstrate that loss of Z-disc-anchored TTN recapitulates muscle remodeling in critical illness ‘myosinopathy’ patients, characterized by TTN-depletion and loss of thick filaments. We conclude that full-length TTN is required to integrate Z-disc and A-band proteins into the mature sarcomere, a function that is lost when TTN expression is pathologically lowered.