Behaviour of connectin (titin) and nebulin in skinned muscle fibres released after extreme stretch as revealed by immunoelectron microscopy

Summary Stretching of skinned fibres of frog skeletal muscle beyond the overlap of the thin and thick filaments followed by release to resting length results in disorganization of the thin filaments at the A-I junction of a sarcomere (Higuchiet al. (1988) J. Muscl. Res. Cell. Motility 9, 491–8). Immunoelectron microscopic observations showed that the binding sites of antibodies against connectin (titin) returned to the original position after extreme stretch and release but those of anti-nebulin antibodies were largely disorganized. The binding sites of anti-connectin antibodies moved within an I band with the change in sarcomere length, but those of anti-nebulin antibodies did not. Nebulin remained in the I band at extreme stretch. Thus connectin filaments appear to be responsible for maintaining mechanical continuity of a sarcomere and appear to behave independently of thin filaments. It is suggested that nebulin is localized in the I band but not in the A band and is associated with thin filaments but not with the elastic structure of myofibrils.     

Calcium binding to an elastic portion of connectin/titin filaments

-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the -connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on -connectin. Purified -connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 10⁷ M^–1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 M. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of -connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of -connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl₂ and 70 M leupeptin to split connectin filaments into -connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while - and -connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 m at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N₂ line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction–relaxation cycle of skeletal muscle.     

Effects of eccentric exercise on joint stiffness and muscle connectin (titin) isoform in the rat hindlimb

We investigated the effects of repeated eccentric exercise for rat medial gastrocnemius muscle on ankle joint stiffness and muscle connectin (titin) isoform composition (longer form, alpha-connectin; shorter form, beta-connectin). Male Wistar rats were trained on a custom-made, isokinetic dynamometer (eccentric-exercise group, n = 6; sham-operated group, n = 6). The exercise session consisted of 20 eccentric contractions elicited by submaximal electric stimulations under anesthesia. The contracting muscle was forcibly lengthened by an isokinetic dorsi-flexion of the ankle joint (velocity, 30 degrees/s; range of motion, 45 degrees). Rats in the eccentric-exercise group were trained every two days for 20 days (10 sessions in total). The static passive resistive torque (PRT) of 45 degrees at the ankle joint was used as a measure of the joint stiffness, and was determined before and after the experimental period. After 10 sessions of eccentric exercise, the wet weight of medial gastrocnemius muscle significantly increased (P < 0.05), whereas the static PRT significantly decreased (P < 0.05) in the eccentric-exercise group, when compared to the sham-operated group. Myosin-ATPase staining showed a decrease in the number of type IIb/IId fibers (P < 0.001) and an increase in the number of type IIa fibers (P < 0.05). However, no significant difference was seen in the connectin (titin) isoform composition between the eccentric-exercise group and the sham-operated group, suggesting that the reduction in PRT was not due to change in resting mechanical properties of muscle fibers.     

Plasticity of cardiac titin/connectin in heart development

Many sarcomeric proteins in the myocardium alter their isoform pattern during perinatal development to adjust to the intensified pump function of the postnatal heart. These changes also involve the giant protein titin/connectin. Here we show by low-percentage polyacrylamide-gel electrophoresis that developmentally regulated switching of cardiac titin/connectin size occurs in the hearts of mouse, rat, pig, and chicken. Mammalian hearts express, well before birth, large foetal (∼3.7 MDa) N2BA-titin/connectin isoform but no N2B-isoform (3.0 MDa). During perinatal heart development the 3.7-MDa N2BA-isoform is replaced by a mix of smaller isoforms. At birth a plethora of intermediate-size N2BA-isoforms appears together with the N2B-isoform. In postnatal heart development the larger-size N2BA-isoforms disappear and smaller-size N2BA-isoforms are upregulated, whereas the proportion of N2B-titin/connectin increases to species-specific adult levels. The time courses of isoform switching are faster in small than in large mammals. Titin/connectin isoform switching also takes place in developing chicken hearts, but the largest embryonic isoform found here was less than 3.4 MDa. At hatching, various smaller-size isoforms appeared and within a week the adult expression pattern was established representing a major 3.0-MDa isoform and a minor 3.15-MDa isoform. The ratio between the two adult isoforms differed between the left ventricle and the right atrium. The perinatal changes toward smaller cardiac titin/connectin isoforms in mammals and chicken greatly increase the myofibrillar passive tension of postnatal hearts. Plasticity of titin/connectin at approximately the time of birth thus affects myocardial mechanics but could also be an important factor in developmentally regulated assembly and signalling processes.Proceedings of the International Symposium on Muscle Elastic Proteins:Koscak Maruyama Memorial Meeting, Chiba, Japan, November 2004     

Projectin is an invertebrate connectin (titin): Isolation from crayfish claw muscle and localization in crayfish claw muscle and insect flight muscle

Summary A filamentous protein was isolated from crayfish claw muscle. This protein had physiochemical properties very similar to vertebrate skeletal muscle connectin (titin), although its apparent molecular mass ( 1200 kDa) was considerably lower than that of connectin ( 3000 kDa). Polyclonal as well as monoclonal antibodies against chicken skeletal muscle connectin reacted with the 1200 kDa protein from crayfish claw muscle. Conversely, polyclonal antibodies against crayfish 1200 kDa protein crossreacted with chicken connectin. Circular dichroic spectra indicated the abundance of-sheet structure ( 60 %). Low-angle shadowed images showed filamentous structures (0.2 0.5m) by electron microscopy. Proteolysis of the 1200 kDa protein by -chymotrypsin or V8 protease rapidly resulted in formation of 1000 kDa or 1100 and 800 kDa peptides. The amino acid composition was very similar to those of vertebrate connectins and of honeybee flight muscle projectin. Based on the molecular weight and amino acid composition, the 1200 kDa protein is regarded to be crayfish projectin.Immunofluorescence and immunoelectron microscopy revealed that crayfish projectin was localized in the A/I junction area and A-band except for its centre region in crayfish claw muscles. Polyclonal antibodies against crayfish claw muscle projectin reacted with 1200 kDa projectin of honeybee and beetle flight muscle. A monoclonal antibody against chicken skeletal muscle connectin also reacted with honeybee and beetle projectin. Immunoelectron microscopic observations revealed that anti-crayfish projectin antibodies bound the connecting filaments linking the Z-line and the thick filaments up to the M-line of honeybee muscle sarcomere. Anti-crayfish projectin antibodies bound the I-band region near the Z-line of beetle flight muscle.It is concluded that the 1200 kDa projectin from crayfish claw muscle is an invertebrate connectin (titin). Recent work with locust flight muscle mini-titin (Nave & Weber, 1990) is in good agreement with the present study, except that the isolated minititin estimated as 600 kDa appears to be a proteolytic product ( 1100 kDa) of the parent molecule ( 1200 kDa).     

Functional analysis of titin/connectin N2-B mutations found in cardiomyopathy

Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of “idiopathic” cardiomyopathy. Recent molecular genetic analyses have now revealed that “idiopathic” cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.     

Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments

Summary The localization and elasticity of connectin (titin) filaments in skinned fibres of frog skeletal muscle were examined for changes in the localization of connectin and in resting tension during partial depolymerization of thick filaments with a relaxing solution containing increased KCl concentrations. Immunoelectron microscopic studies revealed that deposites of antibodies against connectin at a sarcomere length of 3.0 m remained at about 0.8 m from the M-line, until the thick filament was depolymerized to the length of approximately 0.4 m. On further depolymerization, the bound antibodies were found to move towards the Z-line and, on complete depolymerization, were observed to be within 0.3 m of the Z-line; a marked decrease in resting tension accompanied this further depolymerization. These results suggest that connectin filament starts from the Z-line, extends to the M-line, and contributes to resting tension. After partial depolymerization of thick filaments, the distances between the anti-connectin deposits and the Z-line and between anti-connectin deposits and the M-line increased with sarcomere length, suggesting that connectin filaments are elastic along their entire length.     

Restoring force development by titin/connectin and assessment of Ig domain unfolding

Titin/connectin is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is composed of serially-linked immunoglobulin (Ig)-like domains and several unique sequence elements. Here we address the role of titin/connectin in sarcomeres shortened to below the slack length (length attained by an un-activated cell in absence of external forces). Such shortened cells develop so-called restoring forces that re-extend the cells upon relaxation. The experiments that we present are based on a high throughput method with a rapid solution switching system which allows unattached single cardiac myocytes to be activated (resulting in shortening below the slack length) and then to be rapidly relaxed while their maximal re-lengthening velocity is measured at the sarcomere level (dSL/dt _max), with high-resolution imaging techniques. Experiments were carried out on myocytes that express different isoforms of titin/connectin. We measured the relation between dSL/dt _max and the minimal SL during contraction (SL_min) and determined the slope of this relation as a measure of ‘restoring stiffness.’ We found that the restoring stiffness correlates with the isoform expression profile with myocytes that express high levels of the stiff isoform (N2B) having the highest restoring stiffness. These results support the notion that titin/connectin is a bi-directional spring that develops passive force when stretched above the slack length and restoring force when shortened to below this length. We also discuss in detail the mechanisms that underlie titin/connectin’s restoring force development and focus on whether or not unfolding of Ig domains plays a role.     

Myofibrillogenesis in primary tissue cultures of adult human skeletal muscle: expression of desmin,titin, and nebulin

To investigate the in vitro development of myofibrils in skeletal muscle cells derived from adult human muscle biopsies, immunohistochemical analysis was performed using monoclonal antibodies against desmin, titin, and nebulin. Diffuse desmin reactivity was detected 48 h after plating in about 60% of all mononucleated cells. This supports the use of desmin as a marker for undifferentiated rhabdomyosarcomas in man. Titin was visible from day 4 onwards, while nebulin was not found in mononucleated cells. After 1 week polynucleated myotubes appeared, and grew up to 30 days. Desmin was distributed diffusely throughout the cytoplasm until day 21, when the pattern became patchy. Titin began to be organized in a predominantly longitudinal orientation at day 15, while nebulin, which appeared for the first time in fusing myoblasts on the fifth to the seventh day, was almost immediately organized in a dotted longitudinal pattern, which became a Z line connected striation in matured myotubes.Abbreviations FCS fetal calf serum - mAb monoclonal antibody - TBS Tris-buffered saline Correspondence to: T. Behr     

Assembly of the cardiac I-band region of titin/connectin: expression of the cardiac-specific regions and their structural relation to the elastic segments

Summary The giant molecule titin (also called connectin) provides an elastic connection in the I-band between the Z-disk and A-band of striated muscle. This region is assembled in a tissue-specific way by extensive differential splicing events. We have raised monoclonal antibodies against the two N2-line isoforms of titin and demonstrate that both forms of cardiac I-band titin are constitutively co-expressed in atrial and ventricular muscle. In developing mouse embryos, the expression of the cardiac N2-B isoform remains strictly cardiac-specific and is linked to the expression of the ubiquitous N2-A isoform. The mechanical function of the cardiac N2-line region was investigated ultrastructurally. Immunoelectron microscopy reveals that the N2-B region separates two mechanically distinct sections of titin with a hyperextensible segment spanning the distance to the Z-disk. The formation of a plateau in the extension of cardiac titin rules out that Ig-domains can be unfolded as a mechanism of elasticity.     

Developmental changes in rat cardiac titin/connectin: transitions in normal animals and in mutants with a delayed pattern of isoform transition

Rat cardiac titin undergoes developmental changes in isoform expression during the period from late embryonic through the first 20–25 days of life. At least five size classes of titin isoforms have been identified using SDS agarose gel electrophoresis. The longest normal isoform is expressed in the embryonic stages, and it is progressively replaced with increasingly smaller versions. The isoform switching is consistent with changes in resting tension from lower values in one-day neonates to higher levels in adult myocytes. Considerable micro-heterogeneity in alternative splicing patterns also was found, particularly in the N2BA PEVK region of human, rat, and dog ventricle. A rat mutation has been identified in which the embryonic-neonatal titin isoform transitions are markedly delayed. These mutant animals may prove useful for examining the role of titin in stretch-activated signal transduction and in the Frank–Starling relationship.     

The muscular organization of the stomach of Capybara (Hydrochoerus hydrochaeris): an architectural view

Twenty-two stomachs from adult capybaras were used in this study, and an acid digestion mesoscopic technique was pursued using different concentrations of nitric acid to observe the muscular organization of the stomach. The capybara's stomach possessed a muscular coat composed of four layers or strata: external longitudinal, external oblique, circular and internal oblique. Also, the cardiac and pyloric sphincter muscles were comprised of three or two different layers, respectively. Furthermore, the internal oblique fibres were observed extending from the cardiac portion of the stomach to the smaller curvature, where they participated in the formation of the Ansa cardiaca together with the external longitudinal fibres. This muscular architectural arrangement was compared to that in small rodents (rat, hamster, guinea pig), as well as in rabbits and pigs. In conclusion, the stomach of the capybara has a very particular, complex and defined muscular organization that differs from that in other rodents, or domestic animals, in particular, pigs.     

The relaxation of sarcomeres in ischemic injury of myocardium

Summary Polarization-microscopic and micrometric investigations of the myocardium during infarction at autopsy and in experimental ischemic conditions, has shown that one of the earliest and most typical morphological signs of ischemic myocardium is sarcomere relaxation. The latter is expressed by the loss of contractility of muscle cells in life time and in strongly frozen corpses, and due to the effects of fixatives. Increase of the percentage of relaxed sarcomeres parallels the duration of experimental ischemia and the time after myocardial infarction. This allows the indirect calculation of irreversible cell change.     

The organization and hypothalamic projections of the tuberomammillary nucleus in the rat: an immunohistochemical study of adenosine deaminase-positive neurons and fibers

The intense immunohistochemical reaction for the enzyme adenosine deaminase displayed by neurons in the tuberomammillary nucleus in the rat was used to study the distribution and morphology of cells comprising this nucleus, their fiber fields within the posterior hypothalamus and their projection pathways from the hypothalamus. Neurons immunoreactive for adenosine deaminase were found along ventricular and basal aspects of the hypothalamus from the level of the dorsomedial nucleus to the caudal pole of the mammillary body. Approximately 4500 neurons were seen on each side of the brain. Positive neurons showed a complex distribution, largely avoiding nuclear boundaries within the posterior basal hypothalamus and mammillary body. This distribution is mapped in detail and a nomenclature based on topography is introduced so that different regions of the cell distribution may be discussed more easily. Reactive neurons showed a Golgi-like staining which allowed careful study of their morphology. In general, neurons were large, with major axes of from 22 to 30 micron, and bipolar in shape. A second, smaller cell type, 14-16 micron in diameter was also seen and, although often less intensely stained, it was considered a constituent of tuberomammillary nucleus of the hypothalamus as well. Stained dendritic arbours extended considerable distances from the parent cell bodies and branched regularly. Dendrites showed very sparse spines and had an apparently scalloped surface. Features suggestive of varicose segments of dendrites were also noted. The long, smooth dendrites of positive neurons were often seen to aggregate into bundles which avoided nuclear boundaries and tended to collect adjacent to basal and ventricular surfaces of the posterior hypothalamus. Varicose fibers immunoreactive for adenosine deaminase formed a dense network within the hypothalamus. These fibers were considered to derive from the positive neurons in the tuberomammillary nucleus and were similar to adenosine deaminase-immunoreactive fibers seen throughout much of the rest of the brain. The density of this type of positive fiber was, however, much greater within the hypothalamus. The region of the posterior basal hypothalamus also contained relatively sparse populations of adenosine deaminase-positive fibers, apparently distinct from this network. These consisted of a field of fine fibers in the median division of the medial mammillary nucleus and a few large varicosities in the dorsolateral part of the median eminence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spatiotemporal organization of brain dynamics and intelligence: an EEG study in adolescents.

This study investigated relationships between global dynamics of brain electric activity and intelligence. EEG was recorded monopolarly from 10 symmetric leads (10-20 system) in 37 (17 males) healthy subjects (mean age 13.7 years) at rest and during performance of two visually presented cognitive tasks, verbal (semantic grouping) and spatial (mental rotation). On another occasion, the subjects were administered the Intelligence Structure Test (IST). Both total IST score and some individual subtests of specific abilities showed significant positive correlations with EEG coherence in the theta band and significant negative relationships with EEG dimension, a measure of complexity and unpredictability of neural oscillatory dynamics underlying the EEG time series. Furthermore, EEG coherence and dimensional complexity were inversely related. Taken together, these EEG metrics accounted for over 30% of the variability of the total IST score in this sample. No significant effects of the task type (spatial vs. verbal) or specific abilities were observed. Long-distance theta coherence between frontal and parieto-occipital areas showed the most consistent relationship with cognitive abilities. The results suggest that order to chaos ratio in task-related brain dynamics may be one of the biological factors underlying individual differences in cognitive abilities in adolescents.     

The significance of the supratrochlear aperture (STA) in elbow range of motion: an anatomical study

Assessment of the range of motion at a joint is among the methods employed by orthopedic surgeons and physiotherapists to determine courses of therapy and joint recovery. Females tend to have a greater range of motion at the elbow joint than males. In the present case–control study, the elbow extension angle was compared between males and females with and without the supratrochlear aperture. A total of 453 dry humeri and their corresponding ulnae were included in the study, and elbow extension angle was measured using a goniometer. The average extension angle in this sample was 173°, and it was significantly greater when the STA was present (\(\bar{X}\) = 175.4°) than when it was absent (\(\bar{X}\) = 171°). It was greater in females (\(\bar{X}\) = 174.5°) than in males (\(\bar{X}\) = 171.3°) irrespective of STA status, and was greater on the left in both sexes. Hyperextension characterized 13 % of the sample, whereas the majority (76 %) showed hypoextension and only a few (11 %) exhibited normal extension. Trochlear notch depth and olecranon–coronoid distance would found to be useful for predicting the presence of the supratrochlear aperture, while the transverse and vertical diameters of the supratrochlear aperture were found to be the most useful parameters when predicting the degree of extension. The functional benefits of hyperextension at the elbow joint are not fully understood. However, these results are important to orthopedic surgeons and physiotherapists as they permit a greater understanding of normal elbow range of motion in the South African population.     

The giant titin: how to evaluate its role in cardiomyopathies

Journal of Muscle Research and Cell Motility - Titin, the largest protein known, has attracted a lot of interest in the cardiovascular field in recent years, since the discovery that truncating...     

The urokinase plasminogen activator (u-PA) and its inhibitor (PAI-1) in embryo-fetal bone formation in the human: an immunohistochemical study

The role of urokinase plasminogen activator and plasminogen activator inhibitor-1 in human embryofetal bone formation between the 9th and the 20th week of gestation has been studied immunohistochemically. While mature osteocytes of the secondary spongiosa and resting chondrocytes of the bone epiphyses were negative for both antigens in each developmental stage, metabolically active parts of the osseocartilaginous system showed a strong immunoreactivity. Until the end of the 10th week of gestation urokinase plasminogen activator and plasminogen activator inhibitor-1 could not be demonstrated in the shaft of the preexisting cartilaginous models of bones, which correlates with the morphological developmental stage of the embryos. Later, osteoblasts and chondrocytes in the areas of enchondral ossification, and the perivascular chondrocytes of the epiphyseal secondary ossification centres, showed similarly high concentrations of urokinase plasminogen activator and plasminogen activator inhibitor-1. Moreover, the individual ossification stages of the different bones in embryo-fetal development could be demonstrated immunohistochemically. While humeri and femora showed diaphyseal immunoreactivities at an early stage, positive reactions in the phalanges were found only much later. Thus, the enzymes of the fibrinolytic system studied are clearly involved in the desmal and enchondral ossification process in the osseocartilaginous compartment.     

The genome organization of the broad bean necrosis virus (BBNV)

Summary.  The genome of the broad bean necrosis virus Oita-isolate (BBNV-O) [RNA1 (6.0 kb), RNA2 (2.8 kb) and RNA3 (2.4 kb)] was cloned and sequenced. Computer analysis indicates that methyltransferase, helicase and RNA-dependent RNA polymerase (RdRp) motifs are present in RNA1. The viral capsid protein (CP) cistron is located at the 5′ terminal end of RNA2 and the Mr of CP (20 K) is close to that determined by SDS-PAGE analysis. An ochre codon (UAA) in the CP cistron is thought to be partially suppressed to produce a large readthrough protein. RNA3 possesses typical motifs of triple gene block proteins, which are also reported in several other plant viruses. The furovirus genome organization and phylogenetic analysis using RdRp and CP amino acid sequences suggest that BBNV is closely related to potato mop-top virus (PMTV), but is relatively distantly related to other furoviruses. The data also suggest that the genus Furovirus should be separated into several genera: the prototypical genus Furovirus, which excludes the following viruses: the PMTV group including BBNV; the beet necro- tic yellow vein virus (BNYVV) group; and the peanut clump virus (PCV) group. Received November 14, 1997 Accepted Febuary 12,1998