Titin Fragments Research Articles

P.3.12 Characterization of CAPN3-dependent proteolysis of C-terminal titin

The titinopathies tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the C-terminus of titin, residing in the sarcomeric M-band. Mutations identified so far affect the last Ig domain M10 or the preceding is7 region. The FINmaj mutation, underlying TMD/LGMD2J in patients of Finnish origin, causes the exchange of four amino acids in M10 and presumably leads to domain unfolding. The other known mutations cause missense changes of single amino acids or truncation of the ultimate C-terminus. Loss of C-terminal titin epitopes in IF microscopy and reduced amount of C-terminal titin fragments in western blotting suggest that increased or abnormal proteolytic turnover of mutant titin may contribute to the pathomechanism. The muscle-specific protease calpain 3 (CAPN3) binds M-band titin at the is7 region; the interaction is thought to regulate the autolytic activation of CAPN3. LGMD2J patients and FINmaj knock-in mice show secondary CAPN3 deficiency, reflecting loss of the binding site and consequent dysregulation of CAPN3 activity. To elucidate the proteolytic events involved in the pathogenesis of M-band titinopathies, we investigated the cleavage of C-terminal titin by CAPN3. Cleavage fragments generated by CAPN3 were identified by coexpressing various wild-type and mutant titin constructs with active or inactive CAPN3 in cell culture, and comparing the resulting fragment patterns. Active CAPN3 produced several titin fragments, which were characterized by western blotting, protein sequencing and mass spectrometry for identification of cleavage sites. Furthermore, targeted mutagenesis of predicted CAPN3 recognition sites was utilized for understanding the sequence determinants of CAPN3 cleavage and studying the order of cleavage events.

Abstract 18454: Calcium Calmodulin Dependent Protein Kinase II delta Modulates Cardiomyocytes Passive Stiffness by Phosphorylating Titin Spring Elements

Background: High diastolic stiffness of the failing heart can result from altered cardiomyocyte mechanical properties. Cardiomyocyte passive force (F passive ) is attributed mainly to the mechanical properties of titin, particularly isoform composition and phosphorylation. Titin can be phosphorylated by protein kinase(PK)A or PKG at the N2-Bus, which decreases F passive . However, PKCα phosphorylates titin's PEVK-region, which increases F passive . Whether other kinases relevant for cardiomyocyte function phosphorylate the titin springs has been unknown. Aim: To investigate whether Ca 2+ /calmodulin dependent protein kinase IIδ (CaMKIIδ) phosphorylates cardiac titin and affects cardiomyocyte F passive . Methods: Cardiac titin phosphorylation was assessed in CaMKIIδ/γ double knockout (DKO; n=8) and transgenic CaMKIIδC overexpressing mice (TG; n=6). A SILAC-DKO mouse was generated for quantitative proteomics in vivo . Cardiac titin separated by titin gel electrophoresis was also studied for phosphorylation sites using MudPIT mass spectrometry. Recombinant wildtype (WT) and mutant fragments of human cardiac titin were expressed and phosphorylated in vitro by CaMKIIδ. F passive of permeabilized isolated WT, DKO and TG mouse cardiomyocytes was recorded between 1.8 and 2.4 µm sarcomere length before and after CaMKIIδ administration. Results: Total titin phosphorylation was reduced in DKO and elevated in TG compared to WT hearts. CaMKIIδ treatment of cardiac tissue ex vivo increased titin phosphorylation. A CaMKII-dependent phosphosite was identified within the N2-Bus (S4062) by MudPIT and two phosphosites within the PEVK-region ( T12007 and S12022 ) by SILAC. Western blotting using phosphospecific antibodies against these sites in cardiac tissues or recombinant WT/mutant titin constructs confirmed that CaMKIIδ indeed phosphorylates these 3 sites. F passive was elevated in DKO and lowered in TG, compared to WT cardiomyocytes. Administering CaMKIIδ ex vivo lowered F passive in DKO but did not affect TG cardiomyocytes. Conclusion: CaMKIIδ phosphorylates the titin springs , thereby lowering F passive . Reducing titin-based stiffness through CaMKIIδ-dependent phosphorylation of titin N2-Bus and PEVK may benefit diastolic function.

G.P.33 Protein turnover mechanisms in M-band titinopathies

The titinopathies tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J) are caused by mutations in the C-terminus of titin, residing in the sarcomeric M-band. Mutations identified so far affect the last Ig domain M10 or the preceding is 7 region. The FINmaj mutation, underlying TMD/LGMD2J in patients of Finnish origin, causes the exchange of four amino acids in M10 and presumably leads to domain unfolding. The other known mutations cause missense changes of single amino acids or truncation of the ultimate C-terminus. Loss of C-terminal titin epitopes in IF microscopy and reduced amount of C-terminal titin fragments in western blotting suggest that increased or abnormal proteolytic turnover of mutant titin may contribute to the pathomechanism. However, the detailed molecular mechanisms responsible for titin proteolysis and downstream processing of the fragments remain unknown. We recently identified mutations in the co-chaperone DNAJB6 as the molecular cause of LGMD1D, a dominant form of limb-girdle muscular dystrophy with myofibrillar and rimmed vacuolar pathology. Our findings also suggested that DNAJB6 is associated with CASA (chaperone-assisted selective authophagy), a protein turnover pathway important for Z-disk maintenance. Interestingly, DNAJB6 and the CASA proteins BAG3 and HSPB8 also show occasional localization to the M-band, suggesting that the CASA pathway might have additional functions there. To assess whether CASA could be involved in mediating the normal and pathological turnover of M-band titin, we have characterized the expression and localization of these proteins in muscles of titinopathy patients and the FINmaj knock-in mouse. We have also studied the effects of overexpression and knockdown of CASA components on the behaviour of C-terminal titin fragments. Our preliminary results suggest there might be cross-talk between regulatory maintenance functions of the Z-disk and the M-band of the sarcomere.

Clinical evaluation of a matrix metalloproteinase-12 cleaved fragment of titin as a cardiovascular serological biomarker.

BackgroundTitin is a muscle-specific protein found in cardiac and skeletal muscles which is responsible for restoring passive tension. Levels and functioning of titin have been shown to be affected by cardiac damage. Due to the inherent difficulty of measuring titin levels in vivo in a clinical setting, we aimed to develop an assay that could reliably measure fragments of degraded titin in serum and potentially be used in the assessment of cardiac muscle damage.MethodsA competitive ELISA was developed to specifically measure levels of the titin sequence 12670’ NVTVEARLIK 12679’, derived by the degradation of titin by matrix metalloproteinase (MMP)-12. Serum samples from 90 individuals were divided into 3 equally sized groups. One group had been diagnosed with acute myocardial infarction (AMI) while the remaining two were asymptomatic individuals either with CT-scan signs of coronary calcium (CT-plusCa) or without coronary calcium (CT-noCa).ResultsMean geometric levels of the titin fragment in the CT-noCa group were 506.5 ng/ml (±43.88). The CT-plusCa group showed 50.6% higher levels of the marker [763 ng/ml (±90.14)] (P < 0.05). AMI patients showed 56.3% higher levels [792 ng/ml (±149)] (P < 0.05).ConclusionsThe titin-12670 fragment is present in both individuals with undiagnosed and diagnosed CVD. The statistically significant increase in level of the marker in the AMI group is indicative that this neoepitope biomarker may be a useful serological marker in AMI.

Are titin properties reflected in single myofibrils?

Titin is a structural protein in muscle that spans the half sarcomere from Z-band to M-line. Although there are selected studies on titin's mechanical properties from tests on isolated molecules or titin fragments, little is known about its behavior within the structural confines of a sarcomere. Here, we tested the hypothesis that titin properties might be reflected well in single myofibrils. Single myofibrils from rabbit psoas were prepared for measurement of passive stretch-shortening cycles at lengths where passive titin forces occur. Three repeat stretch-shortening cycles with magnitudes between 1.0 and 3.0μm/sarcomere were performed at a speed of 0.1μm/s·sarcomere and repeated after a ten minute rest at zero force. These tests were performed in a relaxation solution (passive) and an activation solution (active) where cross-bridge attachment was inhibited with 2,3 butanedionemonoxime. Myofibrils behaved viscoelastically producing an increased efficiency with repeat stretch-shortening cycles, but a decreased efficiency with increasing stretch magnitudes. Furthermore, we observed a first distinct inflection point in the force-elongation curve at an average sarcomere length of 3.5μm that was associated with an average force of 68±5nN/mm. This inflection point was thought to reflect the onset of Ig domain unfolding and was missing after a ten minute rest at zero force, suggesting a lack of spontaneous Ig domain refolding. These passive myofibrillar properties observed here are consistent with those observed in isolated titin molecules, suggesting that the mechanics of titin are well preserved in isolated myofibrils, and thus, can be studied readily in myofibrils, rather than in the extremely difficult and labile single titin preparations.

Small Heat Shock Proteins Associate under Stress Conditions with Elastic Titin Filaments and Provide Protection from Aggregation

The I-band portion of the giant muscle-protein titin is elastic but detailed knowledge of titin arrangement and interactions in this region is lacking. We determined whether or not select domains from this region can associate with one another, as observed for titin's “distal” immunoglobulin domain-like (Ig-) region. Using sedimentation-velocity centrifugation and size-exclusion chromatography of purified human titin fragments, we detected a monomeric state of the titin N2B-domain or the N2B-unique sequence contained therein. The constitutively expressed PEVK-domain and the Ig-only fragment I9-I12 also behaved as monomers in-vitro, whereas the N2A-segment showed both dimeric and (to a lesser degree) monomeric behavior. Yeast-2-hybrid and GST-pulldown interaction tests demonstrated that both the N2B and the N2A domain, but not the PEVK-fragment, bind to the small heat-shock proteins (sHSPs), alphaB-crystallin and HSP27. These interactions were confirmed on isolated human cardiac or rabbit psoas myofibrils incubated ex-vivo with either sHSP type. Stretching myofibrils to promote titin-domain unfolding increased the propensity of sHSPs to bind I-band titin. Protein unfolding in-vitro by 8M urea caused aggregation of the N2A-segment under acidic conditions (pH6.7), but not at pH7.2. Importantly, alphaB-crystallin prevented the aggregation of the N2A-segment at pH6.7 partially (molar ratio of N2A:alphaB-crystallin, 1:5) or fully (ratio, 1:10). In cultured neonatal rat cardiomyocytes, both sHSPs translocated from the cytosol to the sarcomeric Z-disk/I-band region on inhibition of the proteasome. In adult rat cardiomyocytes both sHSPs associated with I-band titin already under normal culture conditions, suggesting an age-related increase in chaperoning activity. We conclude that titin filaments may run through the elastic segment mainly as monomers; sHSPs associate with elastic titin domains under various stress conditions; and sHSPs are able to protect I-band titin regions from aggregation under adverse intracellular circumstances.

Assembly of a protein “brush” by end-grafting titin fragments to liposomes

Titin is a giant filamentous protein of striated muscle composed of >300 immunoglobulin-like domains linked in tandem. Here, we demonstrate that a six-immunoglobulin fragment of titin carrying a poly-histidine tag forms a protein "brush" on liposomes containing metallochelating lipids. These specimens might allow the direct visualization by cryo-EM of frozen hydrated and unstained titin chains with preserved architectural features.

Transcriptional and Posttranslational Modifications of Titin

See related article, pages 87–94 Myocardial diastolic stiffness has been variably attributed to extracellular matrix composition, cytoskeletal properties of cardiomyocytes, or residual diastolic crossbridge cycling because of incomplete relaxation or cytosolic calcium removal.1 Extracellular matrix and cardiomyocyte cytoskeleton are presumed to mediate chronic rises in myocardial diastolic stiffness, as occur during aging, pressure overload or heart failure, whereas residual diastolic crossbridge cycling accounts for acute changes, as observed during ischemia, exercise, or pharmacological interventions. The elegant study by Kruger et al, published in this issue of Circulation Research , challenges this conceptual framework.2 The study demonstrates that protein kinase (PK)G is capable of phosphorylating the giant cytoskeletal protein titin, as previously reported for PKA3,4 and that phosphorylation by PKG or PKA of a serine residue within the N2B fragment of titin leads to an acute fall in cardiomyofibrillar stiffness. An acute effect produced by a cytoskeletal protein invalidates the concept of distinct mediators for chronic or acute changes in myocardial diastolic stiffness. From these and other recent observations it becomes evident that the cytoskeletal protein titin can alter myocardial diastolic stiffness, both acutely and chronically, through multiple mechanisms such as isoform shifts, phosphorylation by PKG or PKA, and titin–actin interaction at the Z-disc (Figure). Figure. Titin alters cardiomyocyte stiffness through isoform shifts, phosphorylation, and titin–actin interaction. A, Sarcomeric structure with detailed view of I-band region of N2B titin isoform showing tandem immunoglobulin (Ig), N2B, and elastic PEVK segments. B through D, Shift from N2B to N2BA titin isoform (B) and phosphorylation by PKG or PKA at S469 (C) reduce stiffness of the elastic PEVK segment, whereas …

Calpain 1 Binding Capacities of the N1-Line Region of Titin Are Significantly Enhanced by Physiological Concentrations of Calcium

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.

Ultralow-resolutionab initiophasing of filamentous proteins: crystals from a six-Ig fragment of titin as a case study

Low-resolution diffraction data (resolution below 12 angstroms) from crystals of a filamentous six-Ig fragment of titin, I65-I70, were used in ab initio phasing with the aim of calculating its lattice packing and molecular envelope. Filamentous molecules, characterized by marked anisometry and idiosyncratic crystal lattices, have not been addressed before using this methodology. In this study, low-resolution phasing (19-122 angstroms) successfully identified the region of the unit cell occupied by the molecule. Phase extension to a higher resolution (12 angstroms) yielded regions of high density that corresponded either to the positions of individual Ig domains or to zones of dense intermolecular contacts, hindering the identification of individual domains and the interpretation of electron-density maps in terms of a molecular model. This problem resulted from the acutely uneven packing of the molecules in the crystal and it was further accentuated by the presence of partially disordered regions in the molecule. Addition of low-resolution reflections with phases computed ab initio to those obtained experimentally using MIRAS improved the initial electron-density maps of the atomic model, demonstrating the generic utility of low-resolution phases for the structure-elucidation process, even when individual molecules cannot be resolved in the lattice.

Titin expression in human articular cartilage and cultured chondrocytes : A novel component in articular cartilage biomechanical sensing?

In striated muscle tissues, the giant protein titin acts as a biomechanically active filament system, coupling stress/strain to gene expression. The objective of the study is to show the existence of titin fragments in human articular cartilage, as in diarthodial joints, chondrocytes are also known to sense and respond to stretching. We have surveyed human cultured cartilage collected from adults with osteoarthritis (OA), without OA and from infants with a set of titin antibodies and primer pairs. Three different antibodies were used for immunolabelling, reacting with titin's N-terminal Z1–Z2 domains, its Novex III exon, and with its PEVK region. An antibody directed to a titin ligand was included, since in cardiac muscle, this has been shown to participate in the transmission of stretch dependent titin-based signals. Our results indicate that although at low levels, titin is expressed in cartilage. Primer pairs detected titin transcripts in cartilage, and consistent with this, antibodies directed to titin's Z-disc region and to its elastic region stained cartilage. Moreover, we also could detect transcription of the titin ligand CARP. Components of the stretch dependent signal machinery in muscle are also expressed in cartilage. Further studies are warranted to address if common stress/strain dependent signalling are conserved in muscle and cartilage tissues.

The Ig Doublet Z1Z2: A Model System for the Hybrid Analysis of Conformational Dynamics in Ig Tandems from Titin

Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.

Protein kinase-A phosphorylates titin in human heart muscle and reduces myofibrillar passive tension

Protein kinase-A (PKA) is activated during beta-adrenergic stimulation of the heart and is known to phosphorylate several sarcomeric proteins including the giant polypeptide titin. A PKA phosphorylation site on titin is located within the N2B-unique sequence, which is present in the elastic segment of the two major isoforms of cardiac titin, N2B and N2BA, but not in the skeletal-muscle isoforms of the N2A-type. In bovine and rat cardiomyocytes, PKA-mediated phosphorylation decreases passive tension (PT), an effect ascribed to titin phosphorylation. Whether titin is phosphorylated by PKA upon beta-adrenergic stimulation in human heart has not been shown to date. Here we report that PKA induces phosphorylation of N2B and N2BA titin isoforms, as well as a characteristic proteolytic fragment of titin, T2, in human donor hearts. The PKA-induced phosphorylation signals were stronger when myofilaments were first de-phosphorylated by protein phosphatase-1, suggesting inherent phosphorylation of titin in human heart. Titin phosphorylation was associated with a reduction in PT of skinned human cardiac strips; the relative decrease was higher at shorter than at longer physiological sarcomere lengths. The PKA-dependent PT drop was substantially larger when fibers were pre-treated with protein phosphatase-1, indicating that inherent phosphorylation of titin is important for the basal myocardial PT level. Mechanical measurements on isolated myofibrils from rat heart confirmed the PKA effect on passive stiffness and also showed a more pronounced effect in the presence of reducing agent, DTT. In contrast, PKA did not alter the PT of single skinned rat diaphragm muscle fibers; however, the kinase was still able to phosphorylate the skeletal N2A-titin isoform, which lacks the N2B-unique sequence. Thus, an additional phosphorylation site in titin may exist outside the cardiac N2B-unique sequence. We conclude that PKA mediates phosphorylation of titin in normal human myocardium. Titin phosphorylation lowers titin-based passive stiffness in heart but not in skeletal muscle.

Nuclear Titin interacts with A- and B-type lamins in vitro and in vivo

Lamins form structural filaments in the nucleus. Mutations in A-type lamins cause muscular dystrophy, cardiomyopathy and other diseases, including progeroid syndromes. To identify new binding partners for lamin A, we carried out a two-hybrid screen with a human skeletal-muscle cDNA library, using the Ig-fold domain of lamin A as bait. The C-terminal region of titin was recovered twice. Previous investigators showed that nuclear isoforms of titin are essential for chromosome condensation during mitosis. Our titin fragment, which includes two regions unique to titin (M-is6 and M-is7), bound directly to both A- and B-type lamins in vitro. Titin binding to disease-causing lamin A mutants R527P and R482Q was reduced 50%. Studies in living cells suggested lamin-titin interactions were physiologically relevant. In Caenorhabditis elegans embryos, two independent C. elegans (Ce)-titin antibodies colocalized with Ce-lamin at the nuclear envelope. In lamin-downregulated [lmn-1(RNAi)] embryos, Ce-titin was undetectable at the nuclear envelope suggesting its localization or stability requires Ce-lamin. In human cells (HeLa), antibodies against the titin-specific domain M-is6 gave both diffuse and punctate intranuclear staining by indirect immunofluorescence, and recognized at least three bands larger than 1 MDa in immunoblots of isolated HeLa nuclei. In HeLa cells that transiently overexpressed a lamin-binding fragment of titin, nuclei became grossly misshapen and herniated at sites lacking lamin B. We conclude that the C-terminus of nuclear titin binds lamins in vivo and might contribute to nuclear organization during interphase.

Calpain 1–titin interactions concentrate calpain 1 in the Z‐band edges and in the N2‐line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.

Persistence Length of Titin from Rabbit Skeletal Muscles Measured with Scattering and Microrheology Techniques

The persistence length of titin from rabbit skeletal muscles was measured using a combination of static and dynamic light scattering, and neutron small angle scattering. Values of persistence length in the range 9-16 nm were found for titin-II, which corresponds to mainly physiologically inelastic A-band part of the protein, and for a proteolytic fragment with 100-nm contour length from the physiologically elastic I-band part. The ratio of the hydrodynamic radius to the static radius of gyration indicates that the proteins obey Gaussian statistics typical of a flexible polymer in a -solvent. Furthermore, measurements of the flexibility as a function of temperature demonstrate that titin-II and the I-band titin fragment experience a similar denaturation process; unfolding begins at 318 K and proceeds in two stages: an initial gradual 50% change in persistence length is followed by a sharp unwinding transition at 338 K. Complementary microrheology (video particle tracking) measurements indicate that the viscoelasticity in dilute solution behaves according to the Flory/Fox model, providing a value of the radius of gyration for titin-II (63 +/- 1 nm) in agreement with static light scattering and small angle neutron scattering results.

Regulation of the actin–myosin interaction by titin

Titin is known to interact with actin thin filaments within the I-band region of striated muscle sarcomeres. In this study, we have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure the effect of this interaction on the functional properties of the actin-myosin complex. MALDI-TOF MS revealed that T800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) domain. In the presence of tropomyosin-troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)-coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and with an inhibition of HMM binding to actin N-terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca(2+)-controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin-HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the actin-myosin complex.

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.

Association of the Chaperone αB-crystallin with Titin in Heart Muscle

alphaB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. alphaB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of alphaB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. alphaB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, alphaB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between alphaB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by alphaB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with alphaB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.

Myofibrillar tightly bound calcium in skeletal muscle fibers: a possible role of this cation in titin strands aggregation

In muscle cells, part of the calcium is tightly bound to the N1- and N2-line of the sarcomere but its physiological significance was unknown. In the present work we reported the ability of a recombinant titin fragment spanning titin domains Z9 to I1 to tightly bind calcium ions with a K d of 0.049±0.004 nM. We further showed that calcium induced a spontaneous aggregation of the titin fragment and that the major aggregate is a tetramer. The implication of these findings on the organization of the six titin strands that emanate from the end of the thick filament within the I-band is discussed.