Influence of V54M mutation in giant muscle protein titin: a computational screening and molecular dynamics approach

Recent genetic studies have revealed the impact of mutations in associated genes for cardiac sarcomere components leading to dilated cardiomyopathy (DCM). The cardiac sarcomere is composed of thick and thin filaments and a giant muscle protein known as titin or connectin. Titin interacts with T-cap/telethonin in the Z-line region and plays a vital role in regulating sarcomere assembly. Initially, we screened all the variants associated with giant protein titin and analyzed their impact with the aid of pathogenicity and stability prediction methods. V54M mutation found in the hydrophobic core region of the protein associated with abnormal clinical phenotype leads to DCM was selected for further analysis. To address this issue, we mapped the deleterious mutant V54M, modeled the mutant protein complex, and deciphered the impact of mutation on binding with its partner telethonin in the titin crystal structure of PDB ID: 1YA5 with the aid of docking analysis. Furthermore, two run molecular dynamics simulation was initiated to understand the mechanistic action of V54M mutation in altering the protein structure, dynamics, and stability. According to the results obtained from the repeated 50 ns trajectory files, the overall effect of V54M mutation was destabilizing and transition of bend to coil in the secondary structure was observed. Furthermore, MMPBSA elucidated that V54M found in the Z-line region of titin decreases the binding affinity of titin to Z-line proteins T-cap/telethonin thereby hindering the protein–protein interaction.     

Structural analysis of four and half LIM protein-2 in dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilated ventricle and systolic dysfunction. Most of the DCM patients are sporadic cases, but a certain population of DCM patients can be familial cases caused by mutations in genes for sarcomere/Z-disc components including titin/connectin. However, disease-causing mutations could be identified only in a part of the familial DCM patients, suggesting that there should be other disease causing genes for DCM. To explore a novel disease gene for DCM, we searched for mutations in FHL2, encoding for four and half LIM protein 2 (FHL2) in DCM patients, because FHL2 is known to associate with titin/connectin. A missense mutation, Gly48Ser, was identified in a patient with familial DCM. Functional analysis demonstrated that the FHL2 mutation affected the binding to titin/connectin. Because FHL2 protein is known to tether metabolic enzymes to titin/connectin, these observations suggest that the Gly48Ser mutation may be involved in the pathogenesis of DCM via impaired recruitment of metabolic enzymes to the sarcomere.     

Weakness of a giant: mutations of the sarcomeric protein titin

In 2002, three reports described for the first time mutations in the sarcomeric protein titin associated with dilated cardiomyopathy in humans. Despite different locations (Z-line region, Z-I transitional zone, N2B region, half A band region) all mutations resulted in heart failure. In addition, an N2B mutation was found in zebrafish embryos with ventricular dilatation and cardiac insufficiency. It is concluded that titin mutations have significant functional consequences and need to be studied intensively in the future.     

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction.

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.     

Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells.

Titin (also known as connectin) is a muscle-specific giant protein found inside the sarcomere, spanning from the Z-line to the M-line. The I-band segment of titin is considered to function as a molecular spring that develops tension when sarcomeres are stretched (passive tension). Recent studies on skeletal muscle indicate that it is not the entire I-band segment of titin that behaves as a spring; some sections are inelastic and do not take part in the development of passive tension. To better understand the mechanism of passive tension development in the heart, where passive tension plays an essential role in the pumping function, we investigated titin's elastic segment in cardiac myocytes using structural and mechanical techniques. Single cardiac myocytes were stretched by various amounts and then immunolabeled and processed for electron microscopy in the stretched state. Monoclonal antibodies that recognize different titin epitopes were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. We found that only a small region of the I-band segment of titin is elastic; its contour length is estimated at approximately 75 nm, which is only approximately 40% of the total I-band segment of titin. Passive tension measurements indicated that the fundamental determinant of how much passive tension the heart develops is the strain of titin's elastic segment. Furthermore, we found evidence that in sarcomeres that are slack (length, approximately 1.85 microns) the elastic titin segment is highly folded on top of itself. Based on the data, we propose a two-stage mechanism of passive tension development in the heart, in which, between sarcomere lengths of approximately 1.85 microns and approximately 2.0 microns, titin's elastic segment straightens and, at lengths longer than approximately 2.0 microns, the molecular domains that make up titin's elastic segment unravel. Sarcomere shortening to lengths below slack (approximately 1.85 microns) also results in straightening of the elastic titin segment, giving rise to a force that opposes shortening and that tends to bring sarcomeres back to their slack length.     

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.     

Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy

About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed "typical" HCM (group B). Mutational analysis of the beta-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head-rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially "malignant" defects as for the associated phenotypes, but no specific mutations of HCM/DCM.     

The hydrophilic domain of small ankyrin-1 interacts with the two N-terminal immunoglobulin domains of titin

Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an approximately 17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line.     

家族性扩张型心肌病的分子遗传研究进展

扩张型心肌病是一种以左心室和,或右心室扩大、心肌收缩功能受损为主要特征的心肌疾病,是除冠心病和高血压以外导致心力衰竭的主要病因之一。家族性扩张型心肌病约占扩张型心肌病的35％。目前为止,发现的和扩张型心肌病相关的基因突变主要是心肌蛋白基因突变和细胞骨架蛋白基因突变,此外还有线粒体DNA的突变和能量代谢相关的基因突变。本文对引起家族性扩张型心肌病的分子遗传进展进行了总结。     

Nuclear localization of the titin Z1Z2Zr domain and role in regulating cell proliferation

Titin (also called connectin) is a major protein in sarcomere assembly as well as providing elastic return of the sarcomere postcontraction in cardiac and striated skeletal muscle tissues. In addition, it has been speculated that titin is associated with nuclear functions, including chromosome and spindle formation, and regulation of muscle gene expression. In the present study, a short isoform of titin was detected in a human osteoblastic cell line, MG-63 cells, by both immunostaining and Western blot analysis. Confocal images of titin staining showed both cytoplasmic and nuclear localization in a punctate pattern. Therefore, we hypothesized that human titin may contain a nuclear localization signal (NLS). A functional NLS, 200-PAKKTKT-206, located in a low-complexity, titin-specific region between Z2 and Z repeats, was found by sequentially deleting segments of the NH₂-terminal sequence in conjunction with an enhanced green fluorescent protein reporter system and confirmed by site-directed mutagenesis. Overexpression of titin's amino terminal fragment (Z1Z2Zr) in human osteoblasts (MG-63) increased cell proliferation by activating the Wnt/β-catenin pathway. RT-PCR screens of tissue panels demonstrated that residues 1–206 were ubiquitously expressed at low levels in all tissues and cell types analyzed. Our data implicate a dual role for titin's amino terminal region, i.e., a novel nuclear function promoting cell division in addition to its known structural role in Z-line assembly. Wnt; catenin; osteoblast     

Titin and the sarcomere symmetry paradox

Titin is thought to play a major role in myofibril assembly, elasticity and stability. A single molecule spans half the sarcomere and makes interactions with both a thick filament and the Z-line. In the unit cell structure of each half sarcomere there is one thick filament with 3-fold symmetry and two thin filaments with approximately 2-fold symmetry. The minimum number of titin molecules that could satisfy both these symmetries is 12. We determined the actual number of titin molecules in a unit cell from scanning transmission electron microscopy mass measurements of end-filaments. One of these emerges from each tip of the thick filament and is thought to be the in-register aggregate of the titin molecules associated with the filament. The mass per unit length of the end-filament (17.1 kDa/nm) is consistent with six titin molecules not 12. Thus the number of titin molecules present is insufficient to satisfy both symmetries. We suggest a novel solution to this paradox in which four of the six titin molecules interact with the two thin filaments in the unit cell, while the remaining two interact with the two thin filaments that enter the unit cell from the adjacent sarcomere. This arrangement would augment mechanical stability in the sarcomere.     

Structural analysis of obscurin gene in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiac disease characterized by left ventricular hypertrophy with diastolic dysfunction. Molecular genetic studies have revealed that HCM is caused by mutations in genes for sarcomere/Z-band components including titin/connectin and its associate proteins. However, disease-causing mutations can be found in about half of the patients, suggesting that other disease-causing genes remain to be identified. To explore a novel disease gene, we searched for obscurin gene (OBSCN) mutations in HCM patients, because obscurin interacts with titin/connectin. Two linked variants, Arg4344Gln and Ala4484Thr, were identified in a patient and functional analyses demonstrated that Arg4344Gln affected binding of obscurin to Z9-Z10 domains of titin/connectin, whereas Ala4484Thr did not. Myc-tagged obscurin showed that Arg4344Gln impaired obscurin localization to Z-band. These observations suggest that the obscurin abnormality may be involved in the pathogenesis of HCM.     

Telethonin protein expression in neuromuscular disorders

Telethonin is a 19-kDa sarcomeric protein, localized to the Z-disc of skeletal and cardiac muscles. Mutations in the telethonin gene cause limb-girdle muscular dystrophy type 2G (LGMD2G).We investigated the sarcomeric integrity of muscle fibers in LGMD2G patients, through double immunofluorescence analysis for telethonin with three sarcomeric proteins: titin, alpha-actinin-2, and myotilin and observed the typical cross striation pattern, suggesting that the Z-line of the sarcomere is apparently preserved, despite the absence of telethonin. Ultrastructural analysis confirmed the integrity of the sarcomeric architecture. The possible interaction of telethonin with other proteins responsible for several forms of neuromuscular disorders was also analyzed. Telethonin was clearly present in the rods in nemaline myopathy (NM) muscle fibers, confirming its localization to the Z-line of the sarcomere. Muscle from patients with absent telethonin showed normal expression for the proteins dystrophin, sarcoglycans, dysferlin, and calpain-3. Additionally, telethonin showed normal localization in muscle biopsies from patients with LGMD2A, LGMD2B, sarcoglycanopathies, and Duchenne muscular dystrophy (DMD). Therefore, the primary deficiency of calpain-3, dysferlin, sarcoglycans, and dystrophin do not seem to alter telethonin expression.     

Cardiac sarcomere mechanics in health and disease

The sarcomere is the fundamental structural and functional unit of striated muscle and is directly responsible for most of its mechanical properties. The sarcomere generates active or contractile forces and determines the passive or elastic properties of striated muscle. In the heart, mutations in sarcomeric proteins are responsible for the majority of genetically inherited cardiomyopathies. Here, we review the major determinants of cardiac sarcomere mechanics including the key structural components that contribute to active and passive tension. We dissect the molecular and structural basis of active force generation, including sarcomere composition, structure, activation, and relaxation. We then explore the giant sarcomere-resident protein titin, the major contributor to cardiac passive tension. We discuss sarcomere dynamics exemplified by the regulation of titin-based stiffness and the titin life cycle. Finally, we provide an overview of therapeutic strategies that target the sarcomere to improve cardiac contraction and filling.     

Mutations of presenilin genes in dilated cardiomyopathy and heart failure

Two common disorders of the elderly are heart failure and Alzheimer disease (AD). Heart failure usually results from dilated cardiomyopathy (DCM). DCM of unknown cause in families has recently been shown to result from genetic disease, highlighting newly discovered disease mechanisms. AD is the most frequent neurodegenerative disease of older Americans. Familial AD is caused most commonly by presenilin 1 (PSEN1) or presenilin 2 (PSEN2) mutations, a discovery that has greatly advanced the field. The presenilins are also expressed in the heart and are critical to cardiac development. We hypothesized that mutations in presenilins may also be associated with DCM and that their discovery could provide new insight into the pathogenesis of DCM and heart failure. A total of 315 index patients with DCM were evaluated for sequence variation in PSEN1 and PSEN2. Families positive for mutations underwent additional clinical, genetic, and functional studies. A novel PSEN1 missense mutation (Asp333Gly) was identified in one family, and a single PSEN2 missense mutation (Ser130Leu) was found in two other families. Both mutations segregated with DCM and heart failure. The PSEN1 mutation was associated with complete penetrance and progressive disease that resulted in the necessity of cardiac transplantation or in death. The PSEN2 mutation showed partial penetrance, milder disease, and a more favorable prognosis. Calcium signaling was altered in cultured skin fibroblasts from PSEN1 and PSEN2 mutation carriers. These data indicate that PSEN1 and PSEN2 mutations are associated with DCM and heart failure and implicate novel mechanisms of myocardial disease.     

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.     

Location of the C-terminus of titin at the Z-line region in the sarcomere.

Limited proteolysis of titin with trypsin yielded a number of polypeptides which were electrophoresed and transferred to a nitrocellulose membrane. Proteolytic removal of the C-terminal residues on the nitrocellulose-bound polypeptides was achieved by using carboxypeptidase Y. The species of the polypeptides left after the digestion was quantified by immunoblotting with two distinct monoclonal anti-titin antibodies A2 and A12 of which the epitopes were located at 0.74 micron and 0.69 micron away from the center of an A-band, respectively. Two polypeptides (266 kd and 84 kd) reactive to both antibodies were identified in the control group. Fifteen minutes after the digestion, the immunoreactivities of A2 on 266 kd and 84 kd polypeptides were disappeared, while those of A12 on these polypeptides were not affected. The results indicate that the C-terminal end of titin is located near the Z-line region and the N-terminal end at the M-line region in the sarcomere.     

Structural and functional studies of titin's fn3 modules reveal conserved surface patterns and binding to myosin S1--a possible role in the Frank-Starling mechanism of the heart.

The A-band part of titin, a striated-muscle specific protein spanning from the Z-line to the M-line, mainly consists of a well-ordered super-repeat array of immunoglobulin-like and fibronectin-type III (fn3)-like domains. Since it has been suspected that the fn3 domains might represent titin's binding sites to myosin, we have developed structural models for all of titin's 132 fn3-like domains. A subset of eight experimentally determined fn3 structures from a range of proteins, including titin itself, was used as homology templates. After grouping the models according to their position within the super-repeat segment of the central A-band titin region, we analyzed the models with respect to side-chain conservation. This showed that conserved residues form an extensive surface pattern predominantly at one side of the domains, whereas domains outside the central C-zone super-repeat region show generally less conserved surfaces. Since the conserved surface residues may function as protein-binding sites, we experimentally studied the binding properties of expressed multi-domain fn3 fragments. This revealed that fn3 fragments specifically bind to the sub-fragment 1 of myosin. We also measured the effect of fn3 fragments on the contractile properties of single cardiac myocytes. At sub-maximal Ca(2+) concentrations, fn3 fragments significantly enhance active tension. This effect is most pronounced at short sarcomere length, and as a result the length-dependence of Ca(2+) activation is reduced. A model of how titin's fn3-like domains may influence actomyosin interaction is proposed.     

Structural analysis of the titin gene in hypertrophic cardiomyopathy: identification of a novel disease gene.

Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy accompanied by myofibrillar disarrays. Molecular genetic analyses have revealed that mutations in 8 different genes cause HCM. Mutations in these disease genes, however, could be found in about half of HCM patients, suggesting that there are other unknown disease gene(s). Because the known disease genes encode sarcomeric proteins expressed in the cardiac muscle, we searched for a disease-associated mutation in the titin gene in 82 HCM patients who had no mutation in the known disease genes. A G to T transversion in codon 740, from CGC to CTC, replacing Arginine with Leucine was found in a patient. This mutation was not found in more than 500 normal chromosomes and increased the binding affinity of titin to alpha-actitin in the yeast two-hybrid assay. These observations suggest that the titin mutation may cause HCM in this patient via altered affinity to alpha-actinin.