A Novel Mechanism Involving Four-and-a-half LIM Domain Protein-1 and Extracellular Signal-regulated Kinase-2 Regulates Titin Phosphorylation and Mechanics

Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.     

Titin mutations as the molecular basis for dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a heterogeneous cardiac disease characterized by ventricular dilatation and systolic dysfunction. Recent genetic studies have revealed that mutations in genes for cardiac sarcomere components lead to DCM. The cardiac sarcomere consists of thick and thin filaments and a giant protein, titin. Because one of the loci of familial DCM was mapped to the region of the titin gene, we searched for titin mutations in the patients and identified four possible disease-associated mutations. Two mutations, Val54Met and Ala743Val, were found in the Z-line region of titin and decreased binding affinities of titin to Z-line proteins T-cap/telethonin and alpha-actinin, respectively, in yeast two-hybrid assays. The other two mutations were found in the cardiac-specific N2-B region of titin and one of them was a nonsense mutation, Glu4053ter, presumably encoding for a truncated nonfunctional molecule. These observations suggest that titin mutations may cause DCM in a subset of the patients.     

Association of the chaperone alphaB-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.     

Molecular dissection of N2B cardiac titin's extensibility

Titin is a giant filamentous polypeptide of multidomain construction spanning between the Z- and M-lines of the cardiac muscle sarcomere. Extension of the I-band segment of titin gives rise to a force that underlies part of the diastolic force of cardiac muscle. 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.     

Comprehensive analysis of titin protein isoform and alternative splicing in normal and mutant rats

Titin is a giant protein with multiple functions in cardiac and skeletal muscles. Rat cardiac titin undergoes developmental isoform transition from the neonatal 3.7 MDa N2BA isoform to primarily the adult 2.97 MDa N2B isoform. An autosomal dominant mutation dramatically altered this transformation. Titins from eight skeletal muscles: Tibialis Anterior (TA), Longissimus Dorsi (LD) and Gastrocnemius (GA), Extensor Digitorum Longus (ED), Soleus (SO), Psoas (PS), Extensor Oblique (EO), and Diaphram (DI) were characterized in wild type and in homozygous mutant (Hm) rats with a titin splicing defect. Results showed that the developmental reduction in titin size is eliminated in the mutant rat so that the titins in all investigated skeletal muscles remain large in the adult. The alternative splicing of titin mRNA was found repressed by this mutation, a result consistent with the large titin isoform in the mutant. The developmental pattern of titin mRNA alternative splicing differs between heart and skeletal muscles. The retention of intron 49 reveals a possible mechanism for the absence of the N2B unique region in the expressed titin protein of skeletal muscle.     

Alpha B-crystallin mutation in dilated cardiomyopathy

Mutations in genes for sarcomeric proteins such as titin/connectin are known to cause dilated cardiomyopathy (DCM). However, disease-causing mutations can be identified only in a small proportion of the patients even in the familial cases, suggesting that there remains yet unidentified disease-causing gene(s) for DCM. To explore the novel disease gene for DCM, we examined CRYAB encoding alphaB-crystallin for mutation in the patients with DCM, since alphaB-crystallin was recently reported to associate with the heart-specific N2B domain and adjacent I26/I27 domain of titin/connectin, and we previously reported a N2B mutation, Gln4053ter, in DCM. A missense mutation of CRYAB, Arg157His, was found in a familial DCM patient and the mutation affected the evolutionary conserved amino acid residue among alpha-crystallins. Functional analysis revealed that the mutation decreased the binding to titin/connectin heart-specific N2B domain without affecting distribution of the mutant crystallin protein in cardiomyocytes. In contrast, another CRYAB mutation, Arg120Gly, reported in desmin-related myopathy decreased the binding to both N2B and striated muscle-specific I26/27 domains and showed intracellular aggregates of the mutant protein. These observations suggest that the Arg157His mutation may be involved in the pathogenesis of DCM via impaired accommodation to the heart-specific N2B domain of titin/connectin and its disease-causing mechanism is different from the mutation found in desmin-related myopathy.     

The titin N2B and N2A regions: biomechanical and metabolic signaling hubs in cross-striated muscles

Muscle specific signaling has been shown to originate from myofilaments and their associated cellular structures, including the sarcomeres, costameres or the cardiac intercalated disc. Two signaling hubs that play important biomechanical roles for cardiac and/or skeletal muscle physiology are the N2B and N2A regions in the giant protein titin. Prominent proteins associated with these regions in titin are chaperones Hsp90 and αB-crystallin, members of the four-and-a-half LIM (FHL) and muscle ankyrin repeat protein (Ankrd) families, as well as thin filament-associated proteins, such as myopalladin. This review highlights biological roles and properties of the titin N2B and N2A regions in health and disease. Special emphasis is placed on functions of Ankrd and FHL proteins as mechanosensors that modulate muscle-specific signaling and muscle growth. This region of the sarcomere also emerged as a hotspot for the modulation of passive muscle mechanics through altered titin phosphorylation and splicing, as well as tethering mechanisms that link titin to the thin filament system.     

Titin isoform-dependent effect of calcium on passive myocardial tension

We studied the effects of Ca2+ on titin (connectin)-based passive tension in skinned myocardium expressing either predominantly N2B titin (rat right ventricle, RRV) or predominantly N2BA titin (bovine left atrium, BLA). Actomyosin-based tension was abolished to undetectably low levels by selectively removing the thin filaments with a Ca2+-insensitive gelsolin fragment (FX-45). Myocardium was stretched in the presence and absence of Ca2+, and passive tension was measured. Ca2+ significantly increased passive tension during and after stretch in the BLA. The increase was insensitive to the actomyosin inhibitor 2,3-butanedione 2-monoxime, supporting the conclusion that the effect is titin based. Passive tension did not respond to calcium in the RRV, indicating that passive tension developed by N2B titin is calcium insensitive. Western blot analysis and immunofluorescence studies indicated that N2BA titin expresses E-rich PEVK motifs, whereas they are absent from N2B titin, supporting earlier single molecule studies that reported that E-rich motifs are required for calcium sensitivity. We conclude that calcium affects passive myocardial tension in a titin isoform-dependent manner.     

Mechanics on myocardium deficient in the N2B region of titin: the cardiac-unique spring element improves efficiency of the cardiac cycle

Titin (also known as connectin) is an intrasarcomeric muscle protein that functions as a molecular spring and generates passive tension upon muscle stretch. The N2B element is a cardiac-specific spring element within titin's extensible region. Our goal was to study the contribution of the N2B element to the mechanical properties of titin, particularly its hypothesized role in limiting energy loss during repeated stretch (diastole)-shortening (systole) cycles of the heart. We studied energy loss by measuring hysteresis from the area between the stretch and release passive force-sarcomere length curves and used both wild-type (WT) mice and N2B knockout (KO) mice in which the N2B element has been deleted. A range of protocols was used, including those that mimic physiological loading conditions. KO mice showed significant increases in hysteresis. Most prominently, in tissue that had been preconditioned with a physiological stretch-release protocol, hysteresis increased significantly from 320 ± 46 pJ/mm²/sarcomere in WT to 650 ± 94 pJ/mm²/sarcomere in N2B KO myocardium. These results are supported by experiments in which oxidative stress was used to mechanically inactivate portions of the N2B-Us of WT titin through cysteine cross-linking. Studies on muscle from which the thin filaments had been extracted (using the actin severing protein gelsolin) showed that the difference in hysteresis between WT and KO tissue cannot be explained by filament sliding-based viscosity. Instead the results suggest that hysteresis arises from within titin and most likely involves unfolding of immunoglobulin-like domains. These studies support that the mechanical function of the N2B element of titin includes reducing hysteresis and increasing the efficiency of the heart.     

Developmental changes in passive stiffness and myofilament Ca2+ sensitivity due to titin and troponin-I isoform switching are not critically triggered by birth

The giant protein titin, a major contributor to myocardial mechanics, is expressed in two main cardiac isoforms: stiff N2B (3.0 MDa) and more compliant N2BA (>3.2 MDa). Fetal hearts of mice, rats, and pigs express a unique N2BA isoform ( approximately 3.7 MDa) but no N2B. Around birth the fetal N2BA titin is replaced by smaller-size N2BA isoforms and N2B, which predominates in adult hearts, stiffening their sarcomeres. Here we show that perinatal titin-isoform switching and corresponding passive stiffness (STp) changes do not occur in the hearts of guinea pig and sheep. In these species the shift toward "adult" proportions of N2B isoform is almost completed by midgestation. The relative contributions of titin and collagen to STp were estimated in force measurements on skinned cardiac muscle strips by selective titin proteolysis, leaving the collagen matrix unaffected. Titin-based STp contributed between 42% and 58% to total STp in late-fetal and adult sheep/guinea pigs and adult rats. However, only approximately 20% of total STp was titin based in late-fetal rat. Titin-borne passive tension and the proportion of titin-based STp generally scaled with the N2B isoform percentage. The titin isoform transitions were correlated to a switch in troponin-I (TnI) isoform expression. In rats, fetal slow skeletal TnI (ssTnI) was replaced by adult carciac TnI (cTnI) shortly after birth, thereby reducing the Ca2+ sensitivity of force development. In contrast, guinea pig and sheep coexpressed ssTnI and cTnI in fetal hearts, and skinned fibers from guinea pig showed almost no perinatal shift in Ca2+ sensitivity. We conclude that TnI-isoform and titin-isoform switching and corresponding functional changes during heart development are not initiated by birth but are genetically programmed, species-specific regulated events.     

Titin isoforms

The titin isoform composition in skeletal and cardiac muscles of humans and animals has been studied using SDS elecrtophoresis in agarose-strengthened 1.3-2.3% polyacrylamide gels modified by us and immunoblot analysis in order to reveal new titin isoforms with molecular weights of more than 3700 kDa. The experimental data obtained have provided a basis for the suggestion that the titin bands of high molecular weights discovered by us are intact titin isoforms, while the titin bands designated on electrophoregrams as N2A, N2B and N2BA isoforms are their fragments.     

Extensibility of isoforms of cardiac titin: variation in contour length of molecular subsegments provides a basis for cellular passive stiffness diversity

Titin is a giant polypeptide that spans between the Z- and M-lines of the cardiac muscle sarcomere and that develops force when extended. This force arises from titin's extensible I-band region, which consists mainly of three segment types: serially linked immunoglobulin-like domains (Ig segments), interrupted by the PEVK segment, and the N2B unique sequence. Recently it was reported that the myocardium of large mammals co-expresses small (N2B) and large (N2BA) cardiac isoforms and that the passive stiffness of cardiac myocytes varies with the isoform expression ratio. To understand the molecular basis of the differences in passive stiffness we investigated titin's extensibility in bovine atrium, which expresses predominantly N2BA titin, and compared it to that of rat, which expresses predominantly N2B titin. Immunoelectron microscopy was used with antibodies that flank the Ig segments, the PEVK segment, and the unique sequence of the N2B element. The extension of the various segments was then determined as a function of sarcomere length (SL). When slack sarcomeres of bovine atrium were stretched, the PEVK segment extended much more steeply and the unique N2B sequence less steeply than in rat, while the Ig segments behaved similarly in both species. However, the extensions normalized with the segment's contour length (i.e., the fractional extensions) of Ig, PEVK, and unique sequence segments all increase less steeply with SL in cow than in rat. Considering that fractional extension determines the level of entropic force, these differences in fractional extension are expected to result in shallow and steep passive force-SL curves in myocytes that express high levels of N2BA and N2B titin, respectively. Thus, the findings provide a molecular basis for passive stiffness diversity.     

I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure.

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.     

Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.     

On the titin isoforms

By our modified SDS gel electrophoresis and immunoblotting, the isoform composition of titin in skeletal and cardiac muscles of human and animals was studied to reveal new titin forms above 3700 kDa in size. The data obtained suggest that the new large-size titin species are the intact (original) isoforms of this protein, whereas the known N2A, N2B, and N2BA titin bands in electrophoregrams correspond to their fragments.     

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.     

Seasonal changes in the composition of titin isoforms in muscles of hibernating ground squirrels

An electophoretic study of changes in the content of intact titin isoforms, N2B-, N2BA-, N2A-titins and T2 in skeletal and cardiac muscles of ground squirrel (Spermophillus undulatus) is made in different periods: summer activity, autumnal activity, hibernation, arousal, and winter activity. In atria and ventricles of ground squirrels in the period of autumnal activity an increase (by ～1.5 times) in the N2BA to N2B ratio was observed, in comparison with that in cardiac muscle in summer activity. During hibernation, the decrease in the relative content of N2B-, N2BA-titins and T2 in cardiac muscle as well as of N2A-titin and T2 in skeletal muscles was determined against the background of preservation of the relative amount of intact titin isoforms. At waking of ground squirrels and in a short period of winter activity, a rapid restoration of the content of N2B-, N2BA-, N2A-titisns and T2 in muscles was observed. In the myocardium of hibernating, waking ground squirrels and of those during winter activity the increased N2BA to N2B ratio was retained. The changes in the titin content are discussed in the aspect of adaptation of ground squirrels to hibernation.     

Linagliptin prevents left ventricular stiffening by reducing titin cleavage and hypophosphorylation

The metabolic syndrome (MetS) is an escalating problem worldwide, causing left ventricular stiffening, an early characteristic of diastolic dysfunction for which no treatment exists. As diastolic dysfunction and stiffening in MetS patients are associated with increased circulating dipeptidyl peptidase-4 (DPP-4) levels, we investigated whether the clinically approved DPP-4 inhibitor linagliptin reduces left ventricular stiffness in MetS-induced cardiac disease. Sixteen-week-old obese ZSF1 rats, displaying the MetS and left ventricular stiffness, received linagliptin-supplemented or placebo diet for four weeks. Linagliptin significantly reduced obesity, hyperlipidaemia, and hyperglycaemia and improved left ventricular relaxation. This improved relaxation was related to decreased cardiac fibrosis and cardiomyocyte passive stiffness (F_passive). The reduced F_passive was the result of titin isoform switching from the stiff N2B to the more flexible N2BA and increased phosphorylation of total titin and specifically its N2Bus region (S4080 and S3391). Importantly, DPP-4 directly cleaved titin in vitro, resulting in an increased F_passive, which was prevented by simultaneous administration of linagliptin. In conclusion, linagliptin improves left ventricular stiffness in obese ZSF1 rats by preventing direct DPP4-mediated titin cleavage, as well as by modulating both titin isoform levels and phosphorylation. Reducing left ventricular stiffness by administering linagliptin might prevent MetS-induced early diastolic dysfunction in human.     

Molecular mechanics of cardiac titin's PEVK and N2B spring elements.

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.     

Tibial muscular dystrophy is a titinopathy caused by mutations in TTN,the gene encoding the giant skeletal-muscle protein titin

Tibial muscular dystrophy (TMD) is an autosomal dominant late-onset distal myopathy linked to chromosome 2q31. The linked region includes the giant TTN gene, which encodes the central sarcomeric protein, titin. We have previously shown a secondary calpain-3 defect to be associated with TMD, which further underscored that titin is the candidate. We now report the first mutations in TTN to cause a human skeletal-muscle disease, TMD. In Mex6, the last exon of TTN, a unique 11-bp deletion/insertion mutation, changing four amino acid residues, completely cosegregated with all tested 81 Finnish patients with TMD in 12 unrelated families. The mutation was not found in 216 Finnish control samples. In a French family with TMD, a Leu-->Pro mutation at position 293,357 in Mex6 was discovered. Mex6 is adjacent to the known calpain-3 binding site Mex5 of M-line titin. Immunohistochemical analysis using two exon-specific antibodies directed to the M-line region of titin demonstrated the specific loss of carboxy-terminal titin epitopes in the TMD muscle samples that we studied, thus implicating a functional defect of the M-line titin in the genesis of the TMD disease phenotype.