Titin's I-band Research Articles

Study of the expression of Cronos titin in TTN-truncation cardiomyopathy and heart development

Cronos is a recently discovered isoform of the giant protein titin driven by an alternative internal promoter. Cronos spans the half-A-band of a sarcomere and binds to myosin filaments, but its role in healthy or diseased hearts is largely unknown. Upregulation of Cronos has been suggested as a possible compensatory mechanism in titin-truncation cardiomyopathy, to help explain why truncations in titin's I-band region are less frequent and often cause a milder disease phenotype than truncations in the A-band region.

Unexpected Low Mechanical Stability of Titin I27 Domain at Physiologically Relevant Temperature.

The extensively studied immunoglobulin (Ig) domain I27 of the giant force-bearing protein titin has provided a basis for our current understanding of the structural stability, dynamics, and function of the numerous mechanically stretched Ig domains in the force-bearing I-band of titin. The current consensus is that titin I27 has a high mechanical stability characterized by very low unfolding rate (<10-3 s-1) in physiological force range and high unfolding forces (>100 pN) at typical physiological force loading rates from experiments at typical laboratory temperatures. Here, we report that when the temperature is increased from 23 to 37 °C, the unfolding rate of I27 drastically increases by ∼100-fold at the physiological level of forces, indicating a low mechanical stability of I27 at physiological conditions. The result provides new insights into the structural states and the associated functions of I27 and other similar titin I-band Ig domains.

Modulation of Titin-Based Stiffness in Hypertrophic Cardiomyopathy via Protein Kinase D

The giant protein titin performs structure-preserving functions in the sarcomere and is important for the passive stiffness (Fpassive) of cardiomyocytes. Protein kinase D (PKD) enzymes play crucial roles in regulating myocardial contraction, hypertrophy, and remodeling. PKD phosphorylates myofilament proteins, but it is not known whether the giant protein titin is also a PKD substrate. Here, we aimed to determine whether PKD phosphorylates titin and thereby modulates cardiomyocyte Fpassive in normal and failing myocardium. The phosphorylation of titin was assessed in cardiomyocyte-specific PKD knock-out mice (cKO) and human hearts using immunoblotting with a phosphoserine/threonine and a phosphosite-specific titin antibody. PKD-dependent site-specific titin phosphorylation in vivo was quantified by mass spectrometry using stable isotope labeling by amino acids in cell culture (SILAC) of SILAC-labeled mouse heart protein lysates that were mixed with lysates isolated from hearts of either wild-type control (WT) or cKO mice. Fpassive of single permeabilized cardiomyocytes was recorded before and after PKD and HSP27 administration. All-titin phosphorylation was reduced in cKO compared to WT hearts. Multiple conserved PKD-dependent phosphosites were identified within the Z-disk, A-band and M-band regions of titin by quantitative mass spectrometry, and many PKD-dependent phosphosites detected in the elastic titin I-band region were significantly decreased in cKO. Analysis of titin site-specific phosphorylation showed unaltered or upregulated phosphorylation in cKO compared to matched WT hearts. Fpassive was elevated in cKO compared to WT cardiomyocytes and PKD administration lowered Fpassive of WT and cKO cardiomyocytes. Cardiomyocytes from hypertrophic cardiomyopathy (HCM) patients showed higher Fpassive compared to control hearts and significantly lower Fpassive after PKD treatment. In addition, we found higher phosphorylation at CaMKII-dependent titin sites in HCM compared to control hearts. Expression and phosphorylation of HSP27, a substrate of PKD, were elevated in HCM hearts, which was associated with increased PKD expression and phosphorylation. The relocalization of HSP27 in HCM away from the sarcomeric Z-disk and I-band suggested that HSP27 failed to exert its protective action on titin extensibility. This protection could, however, be restored by administration of HSP27, which significantly reduced Fpassive in HCM cardiomyocytes. These findings establish a previously unknown role for PKDin regulating diastolic passive properties of healthy and diseased hearts.

Calcium Dependent Interaction Between N2A-Halo and F-Actin: A Single Molecule Study

In the last two decades, interactions between titin's I-band region and actin have been widely investigated for skeletal and cardiac isoforms. Previous studies suggested a calcium-dependent interaction between titin and actin. However, previous in vitro motility studies did not quantify the strength of interaction between titin's N2A region and actin in the presence of calcium. Here, we used AFM single molecule force spectroscopy (SMFS) to probe the interaction strength between a recombinant N2A-HALO construct and actin filaments (F-actin), in the presence (pCa = 4.0) and absence of calcium. The N2A-Halo construct consisted of the N2A region of titin (Ig80-IS-Ig81-Ig82-Ig83, where IS is the insertion sequence) with a C-terminal Halo-tag. The construct was immobilized on an AFM cantilever tip via its N-terminal cysteines. The interaction strength was determined by probing the molecule during multiple approach-retraction cycles at physiological pulling rates (300-1000 nm/sec) over various locations on a substrate functionalized with F-actin stabilized on a lipid bilayer with ATP. Preliminary data show that both the yield and strength of N2A-Halo interactions with F-actin nearly doubled in the presence of Ca2+, with multiple peaks in the rupture force distribution at 50 ± 25 pN, 75 ± 10 pN, and 100 ± 25 pN. The force extension curves also showed different rupture lengths, indicating multiple actin binding sites. No significant interactions occurred between N2A constructs and the charged lipid bi-layer surface in the absence of F-actin. These experiments show that calcium increases the interaction probability and binding strength between N2A-Halo and F-actin and that the binding strength between N2A titin and F-actin is nearly 4 times greater than previously reported for PEVK titin - F-actin interactions. These results support a physiological role for titin in active muscle contraction and force enhancement.

A Multi-Tool Mouse Model to Study the Elasticity of Native Titin

The elasticity of muscle is principally determined by the I-band region of titin, consisting of 40-100 immunoglobulin-like (Ig) domains and two unstructured segments. Traditional force spectroscopy of titin can study only eight tandem Ig domains at a time, and the dominant entropic effect of the massive native titin polypeptide is lost. Here, we have developed a knock-in mouse model to study titin in its entirety. We genetically encoded a HaloTag enzyme, flanked with two protease sites, in the constitutively expressed region of the I-band. This multi-tool mouse model permits: covalent protein immobilization for force spectroscopy, fluorescent labeling for sarcomere length measurements, and titin specific proteolysis for muscle mechanics experiments. The HaloTag, engineered close to the I-A junction of titin, allows titin immobilization on glass coverslips functionalized with chloroalkane chemistry. Using T12 antibody coated paramagnetic beads, the N-terminus of titin can be picked up and stretched with magnetic tweezers. For the first time, this has enabled the study of the mechanical properties of the entire intact titin I-band. For different muscle groups, the results show unfolding and refolding of individual Ig domains at forces in the physiological range, below 10 pN. Moreover, accumulated step size histograms show two populations in the unfolding and refolding transitions, suggesting the presence of reduced and oxidized disulfide bonds in several Ig domains. Additionally, fluorescent labeling of the HaloTag with Alexa488 conjugated chloroalkane permits accurate sarcomere length measurements of intact myofibrils. We propose muscle mechanics experiments in which stretched myofibrils are treated with TEV protease to cleave titin at the I-A junction so that titin's contribution to passive elasticity, and perhaps active contraction, can be measured. This versatile mouse model allows for a diverse set of biophysical experiments to elucidate the role of the giant protein titin in muscle function.

Exploration of pathomechanisms triggered by a single-nucleotide polymorphism in titin's I-band: the cardiomyopathy-linked mutation T2580I.

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.

The Structure of Misfolded Amyloidogenic Dimers: Computational Analysis of Force Spectroscopy Data

Progress in understanding the molecular mechanism of self-assembly of amyloidogenic proteins and peptides requires knowledge about their structure in misfolded states. Structural studies of amyloid aggregates formed during the early aggregation stage are very limited. Atomic force microscopy (AFM) spectroscopy is widely used to analyze misfolded proteins and peptides, but the structural characterization of transiently formed misfolded dimers is limited by the lack of computational approaches that allow direct comparison with AFM experiments. Steered molecular dynamics (SMD) simulation is capable of modeling force spectroscopy experiments, but the modeling requires pulling rates 107 times higher than those used in AFM experiments. In this study, we describe a computational all-atom Monte Carlo pulling (MCP) approach that enables us to model results at pulling rates comparable to those used in AFM pulling experiments. We tested the approach by modeling pulling experimental data for I91 from titin I-band (PDB ID: 1TIT) and ubiquitin (PDB ID: 1UBQ). We then used MCP to analyze AFM spectroscopy experiments that probed the interaction of the peptides [Q6C] Sup35 (6–13) and [H13C] Aβ (13–23). A comparison of experimental results with the computational data for the Sup35 dimer with out-of-register and in-register arrangements of β-sheets suggests that Sup35 monomers adopt an out-of-register arrangement in the dimer. A similar analysis performed for Aβ peptide demonstrates that the out-of-register antiparallel β-sheet arrangement of monomers also occurs in this peptide. Although the rupture of hydrogen bonds is the major contributor to dimer dissociation, the aromatic-aromatic interaction also contributes to the dimer rupture process.

Abstract 308: Targeted Inhibition of Cardiac Ankyrin Repeat Protein Disrupts Sarcomeric ERK-GATA4 Signaling at the Titin I-band and Abrogates Agonist-Induced Cardiac Hypertrophy

Hypertrophic cardiomyocyte growth occurs in response to various stress stimuli including biomechanical stress and neurohormonal factors. Accumulating evidence suggest that sarcomere signaling complexes play a pivotal role in the cardiomyocyte hypertrophic response by transmitting signals to the nucleus to induce gene expression. Cardiac ankyrin repeat protein (CARP, Ankrd1) is a transcriptional regulatory protein that also associates with the titin I-band spring domain, however the exact role of CARP in the heart remains to be elucidated. We report that CARP directly interacts with mitogen activated protein kinase ERK1/2 and cardiac transcription factor GATA4. Phenylephrine (PE) stimulation in cardiomyocytes induced ERK1/2 and GATA4 to transiently co-localize with sarcomeric CARP, followed by translocation of CARP and GATA4 to the nucleus. Four-and-a-half-LIM (FHL) domains proteins are part of a sarcomeric ERK2 sensory complex and knockdown of CARP by small interfering RNA (siRNA) resulted in disruption of FHL1 and FHL2. Moreover, loss of CARP attenuated PE-induced phosphorylation of ERK1/2 and GATA4, decreased GATA4 DNA binding, and prevented PE-induced cardiomyocyte growth. Mice lacking CARP have decreased FHL1 levels, and PE stimulation in wild-type mice resulted in elevated GATA4 phosphorylation and a hypertrophic response, which were completely abrogated in CARP-KO mice. We demonstrate that CARP plays an important role in PE-induced hypertrophic signaling by recruiting ERK2 and GATA4 into a titin I-band macro-molecular complex to induce GATA4 activation, followed by translocation of CARP and GATA4 to the nucleus to enhance GATA4 DNA binding and hypertrophic gene expression. Loss of CARP destabilizes FHL1 and FHL2, resulting in disruption of the PE-induced sarcomeric complex and abrogation of the cardiomyocyte hypertrophic response. These data reveal a novel role for sarcomeric titin I-band as a transcription factor activation hub that induces downstream nuclear signaling in response to agonist-induced hypertrophic stimuli.

Computational Analysis of the Single Molecule AFM Force Spectroscopy Data

AFM force spectroscopy is a widely applied to various molecular systems providing an important information on the strength of intramolecular and intermolecular interactions. However, a structural characterization of the systems is limited by the lack of theoretical approaches capable of direct comparison with experiment. Steered Molecular Dynamic (SMD) simulation is widely used for modeling of the AFM pulling experiments, but it is typically performed at pulling rates 5 nm/ns which are 107 times higher than those for typical experimental conditions. Here we describe the computational approach utilizing Monte Carlo Pulling (MPC) simulation enabling us to model AFM pulling experiments at conditions corresponding to experimental ones. Tested by the comparison with experimental data for titin I-band(PDB ID: 1TIT), the approach was applied to modeling of the AFM force spectroscopy experiments on probing of interaction of misfolded peptides CGNNQQNY and Aβ14-23 within dimers. The structures of the CGNNQQNY dimers were obtained from the Replica Exchanged MD simulations. The results for antiparallel out-of-register β sheet of CGNNQQNY produce the force ∼100 pN which is in line with the experimental data. At the same time, pulling of antiparallel in-register β sheet provides much higher rupture force, suggesting that CGNNQQNY monomers adopt the out-of-register arrangement in the dimer. Similar analysis applied to Aβ14-23 peptide demonstrated that the out-of-register antiparallel β-sheet arrangement of monomers is realized for this peptide as well. The elevated rupture forces for Aβ14-23 compared to CGNNQQNY dimers is explained by elevated contribution of hydrophobic, salt bridge and aromatic interactions in Aβ peptide.

Single Molecule Analysis of PKC Phosphorylation of Titin's PEVK Domain

Titin's I-band region contains three spring-like domains that are primarily responsible for the development of passive tension in cardiac muscle. PKC phosphorylation targets one of these I-band domains_the PEVK region, which is rich in proline (P), glutamate (E), valine (V), and lysine (K). It has been shown that two serine residues within the PEVK are targeted by PKC phosphorylation, S26 and S170. We investigate the effects of PKC phosphorylation of these two residues on the single molecule level using force-extension curves generated by atomic force microscopy (AFM). We constructed four recombinant proteins: two PEVK single mutants (S26A and S170A), a PEVK double mutant (S26AS170A), and a wild-type PEVK segment. All constructs are flanked by immunoglobulin-like domains, Ig27 and Ig84, and the unfolding of these domains generates a single molecular “fingerprint”. The force-extension curve leading up to the first unfolding peak describes the force-extension relationship of the PEVK. Preliminary data suggests that mutating either serine residue alters PEVK resistance to extension, which is quantified by the molecule's persistence length (PL). Wild-type PEVK underwent a large decrease in its PL after phosphorylation by PKC (by ∼50%), and both single mutants have PLs similar to that of phosphorylated wild-type PEVK. Furthermore, phosphorylation of both single mutants resulted in a small PL decrease. Phosphorylation decreased PL for the S26A mutation by 16% (from 0.55 ± 0.02 to 0.46 ± 0.02 (mean ± SE)), and the serine-170 mutation PL by 11% (from 0.53 ± 0.04 to 0.47 ± 0.03). The double mutant was not affected by PKC (from 0.51 ± 0.04 to 0.51 ± 0.03). We conclude that both serines play a structural role in determining the relationship between longitudinal force and PEVK extension, and that this role is modulated through phosphorylation by PKC.

Titin: Physiological Function and Role in Cardiomyopathy and Failure

Titin is a giant protein that constitutes the third myofilament of the sarcomere. Single titin molecules anchor in the Z-disk and extend all the way to the M-line region of the sarcomere. Successive titin molecules are arranged head-to-head and tail-to-tail, providing a continuous filament along the full length of the myofibril. The majority of titin's I-band region is extensible and functions as a molecular spring that when extended develops passive force. We will discuss mechanisms for adjusting titin-based force, including alternative splicing and posttranslational modifications. Multiple biological functions can be assigned to different regions of the titin molecule. In addition to titin's role in determining passive muscle stiffness, recent evidence suggests a role in protein metabolism, compartmentalization of metabolic enzymes, binding of chaperones, and positioning of the membrane systems of the T-tubules and sarcoplasmic reticulum. We will also discuss titin-based force adjustments that occur in various muscle diseases and several disease-causing titin mutations that have been discovered. We will focus on the role of titin in heart failure patients that was recently investigated in patients with end-stage heart failure due to non-ischemic dilated cardiomyopathy. In end-stage failing hearts, compliant titin isoforms comprise a greater percentage of titin and changes in titin isoform expression in heart failure patients with DCM significantly impact diastolic filling by lowering myocardial stiffness.

The Elasticity of Single Titin Molecules Using a Two-Bead Optical Tweezers Assay

Titin is responsible for the passive elasticity of the muscle sarcomere. The mechanical properties of skeletal and cardiac muscle titin were characterized in single molecules using a novel dual optical tweezers assay. Antibody pairs were attached to beads and used to select the whole molecule, I-band, A-band, a tandem-immunoglobulin (Ig) segment, and the PEVK region. A construct from the PEVK region expressing >25% of the full-length skeletal muscle isoform was chemically conjugated to beads and similarly characterized. By elucidating the elasticity of the different regions, we showed directly for the first time, to our knowledge, that two entropic components act in series in the skeletal muscle titin I-band (confirming previous speculations), one associated with tandem-immunoglobulin domains and the other with the PEVK region, with persistence lengths of 2.9 nm and 0.76 nm, respectively (150 mM ionic strength, 22°C). Novel findings were: the persistence length of the PEVK component rose (0.4–2.7 nm) with an increase in ionic strength (15–300 mM) and fell (3.0–0.3 nm) with a temperature increase (10–60°C); stress-relaxation in 10–12-nm steps was observed in the PEVK construct and hysteresis in the native PEVK region. The region may not be a pure random coil, as previously thought, but contains structured elements, possibly with hydrophobic interactions.

Evidence for a direct but sequential binding of titin to tropomyosin and actin filaments

Titin is a giant molecule that spans half a sarcomere, establishing several specific bindings with both structural and contractile myofibrillar elements. It has been demonstrated that this giant protein plays a major role in striated muscle cell passive tension and contractile filament alignment. The in vitro interaction of titin with a new partner (tropomyosin) reported here is reinforced by our recent in vitro motility study using reconstituted Ca-regulated thin filaments, myosin and a native 800-kDa titin fragment. In the presence of the tropomyosin–troponin complex, the actin filament movement onto coated S1 is improved by the titin fragment. Here, we found that two purified native titin fragments of 150 and 800 kDa, covering respectively the N1-line and the N2-line/PEVK region in the I-band and known to contain actin-binding sites, directly bind tropomyosin in the absence of actin. We have also shown that binding of the 800-kDa fragment with filamentous actin inhibited the subsequent interaction of tropomyosin with actin, as judged by cosedimentation. However, this was not the case if the complex of actin and tropomyosin was formed before the addition of the 800-kDa fragment. We thus conclude that a sequential arrangement of contacts exists between parts of the titin I-band region, tropomyosin and actin in the thin filament.

Steered Molecular Dynamics Studies of Titin I1 Domain Unfolding

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging β-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B β-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A′- and G-strands. The disulfide bond also limits the maximum extension of I1 to ∼220 Å. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.

Titin; a multidomain protein that behaves as the sum of its parts

Titin is a giant, multidomain muscle protein forming a major component of the sarcomere in vertebrate striated muscle. As for many other multidomain proteins, the properties of titin are often studied by characterisation of the constituent domains in isolation. This raises the question of to what extent the properties of the isolated domains are representative of the domains in the wild-type protein. We address this question for the I-band region of titin, which is of particular biological interest due to its role in muscle elasticity, by determining the properties of five immunoglobulin domains from the I-band in three different contexts; firstly as isolated domains with the boundaries defined conservatively, secondly, with a two amino acid extension at both the N and C terminus and thirdly as part of multidomain constructs. We show that adjacent domains in the titin I-band have very different kinetic properties which, in general, undergo only a small change in the presence of neighbouring domains and conclude that, provided that care is taken in the choice of domain boundaries, the properties of the titin I-band are essentially “the sum of its parts”. From this and other work we propose that variation in kinetic properties between adjacent domains may be a general property of the I-band thereby preventing misfolding events on muscle relaxation.

The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct (≈15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.