Identification of high molecular weight proteins in squid muscle by Western blotting analysis and postmortem rheological changes

The high molecular weight protein connectin (also called titin) in Japanese common squid (Todarodes pacificus) mantle muscle was identified by western blotting analysis with 3B9, the mouse anti-chicken skeletal muscle connectin monoclonal antibody. Similarly to vertebrate samples, there exists connectin in invertebrate squid mantle muscle, and the amino acid sequences are assumed to resemble those present in the A band of vertebrate connectin, judging by the specificity of 3B9. Moreover, the connectin in squid muscle migrated in this study as a closely spaced doublet of alpha and beta (titins 1 and 2). Between 5 and 7 h post-mortem, the SDS PAGE patterns of the squid sample indicated a change of the doublet bands into a single beta-connectin band. Simultaneously, the rheological properties of the squid muscle changed substantially. This degradation of alpha-connectin into beta-connectin in the muscle can explain the critical change that occurs during the post-mortem tenderization of squid muscle.     

Connectin content and its post-mortem changes in fish muscle

Connectin was isolated from fish dorsal myofibrils by an SDS-gel filtration method and estimated to account for approximately 13% of the total myofibrillar proteins. There was no significant difference in the amount of connectin among seven fish species but rabbit skeletal myofibrils contained a slightly higher content (16%) of connectin. The high molecular weight connectins from carp and rabbit both showed a doublet band, consisting of bands 1 and 2, on SDS-polyacrylamide gel electrophoresis using a large-pore gel. However, rabbit band 1 (a component of the connectin doublet) was found to migrate more slowly than carp band 1. During post-mortem ageing of the muscles, it was observed that the band 1 component rapidly disappeared with a concomitant increase in band 2 component and then the band 2 component was transformed slowly into faster migrating components. These results suggest that post-mortem ageing has qualitatively similar effects on the submolecular compositions of carp and rabbit connectins. However, the apparent rate of disappearance of the band 1 component was considerably higher in carp muscle than that in rabbit muscle.     

Native connectin from porcine cardiac muscle

Native connectin was isolated from porcine cardiac muscle using the method developed for the preparation of native connectin from chicken breast muscle (Kimura et al. (1984) J. Biochem. 96, 1947-1950). It was not necessary to keep cardiac muscle at 0 degrees C before preparation: the proteolysis of alpha-connectin to beta-connectin proceeded during the preparation of myofibrils. Cardiac connectin showed almost the same properties as those of skeletal muscle connectin: mobility in SDS gel electrophoresis, filamentous structure under an electron microscope, circular dichroism spectra, UV absorption spectra, and amino acid composition. Porcine cardiac connectin cross-reacted with antiserum against chicken breast muscle connectin as revealed by an immunoblot method. Immunoelectron microscopical observations revealed an abundance of connectin antigenic sites around the A-I junction area of cardiac myofibrils. Cardiac connectin also interacted with myosin and actin filaments at low ionic strengths to form aggregates. The extent of interaction was somewhat weaker in the case of cardiac connectin than skeletal muscle connectin, regardless of the origin of myosin and actin (porcine cardiac and rabbit skeletal muscles). In conclusion, cardiac connectin is very similar, but not identical to skeletal muscle connectin.     

Calcium-induced splitting of connectin filaments into beta-connectin and a 1,200-kDa subfragment.

When rabbit skeletal muscle myofibrils were treated with a solution containing 0.1 mM Ca2+ and 30 micrograms of leupeptin/ml, alpha-connectin, which forms very thin filaments in myofibrils, was split into beta-connectin and a 1,200-kDa subfragment. A part of beta-connectin located near the junction between beta-connectin and the subfragment seems to have an affinity for calcium ions and to be susceptible to the binding of large amounts of calcium ions. The calcium-binding site on beta-connectin is localized near the N2 line in the I band, and the subfragment is localized adjacent to the Z disk. It is possible that connectin filaments change their elasticity during the contraction-relaxation cycle of skeletal muscle at the physiological concentration of calcium ions. Because postmortem skeletal muscles lose their elasticity and become plastic in association with the calcium-specific splitting of connectin filaments, the splitting is considered to be a factor in meat tenderization during postrigor ageing.     

Circular dichroism spectra show abundance of beta-sheet structure in connectin, a muscle elastic protein

Circular dichroism spectra of native connectin from chicken breast muscle strongly suggested the abundant presence of beta-sheet structure, as much as 70% in 0.5 M KCl and 50 mM phosphate buffer, pH 7.5. alpha-Helix was not detected. These results are in contradiction with the conclusion that native connectin from rabbit skeletal muscle consists entirely of random coil [(1984) J. Mol. Biol. 180, 331-356].     

Elastic properties and beta-sheet structure of connectin threads

Connectin is a very long and flexible protein of striated muscle, linking myosin filaments to z discs in a sarcomere. Isolated native connectin in solution frequently forms elastic threads upon concentration of the solution, by side-by-side association of molecules. An X-ray diffraction study was performed to examine the presence of beta-sheet structure in artificially prepared threads. The elastic properties of such threads were measured at various temperatures. Negative temperature dependence of the elastic coefficient suggests that the elasticity of connectin threads is due to deformation of the three-dimensional structure and not to rubber-like behavior.     

Chicken breast muscle connectin: passive tension and I-band region primary structure

We performed cDNA cloning of chicken breast muscle connectin. Together with previous results, our analysis elucidated a 24.2 kb sequence encoding the amino terminus of the protein. This corresponded to the I-band region of the skeletal muscle sarcomere, which is involved in extension and contraction between the Z-line and the A-I junction. There were fewer middle immunoglobulin domains and amino acid residues in the PEVK segment of chicken breast muscle connectin than in human skeletal muscle connectin, but more than in human cardiac muscle connectin. We measured passive tension generation by stretching mechanically skinned myofibril bundles. This revealed that appreciable tension development in chicken breast muscle began at longer sarcomere spacings than in rabbit cardiac muscle, but at shorter spacings than in rabbit psoas and soleus muscles. We suggest that the chicken breast muscle sarcomere remains in a relatively extended state even in unstrained sarcomeres. This would explain why chicken breast muscle does not extend under force to the same degree as rabbit psoas and soleus muscles.     

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.     

Connectin filaments in stretched skinned fibers of frog skeletal muscle

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.     

Profiles of connectin (titin) in atrophied soleus muscle induced by unloading of rats.

Responses of the properties of connectin molecules in the slow-twitch soleus (Sol) and fast-twitch extensor digitorum longus muscles of rats to 3 days of unloading with or without 3-day reloading were investigated. The wet weight (relative to body wt) of Sol, not of extensor digitorum longus, in the unloaded group was significantly less than in the age-matched control (P < 0.05). Immunoelectron microscopic analyses showed that a monoclonal antibody against connectin (SM1) bound to the I-band region close to the edge of the A band at resting length and moved reversibly away from the Z line as the muscle fibers were stretched. In Sol, the displacement of the SM1-bound dense spots in response to stretching decreased after hindlimb suspension. There were no changes in the molecular weights and the percent distributions of alpha- and beta-connectin in both muscles after hindlimb suspension. A significant increment of percent beta-connectin in Sol was observed after 3 days of reloading after hindlimb suspension (P < 0.05). It is suggested that the elasticity of connectin filaments in the I-band region of the atrophied Sol fibers was reduced relative to that of the control fibers. The lack of the elasticity in atrophied muscle fibers may cause a decrease in contractile function.     

Fine structure of the myotendinous junction of lathyritic rat muscle with special reference to connectin, a muscle elastic protein

The fine structure of the myotendinous junction of the skeletal muscle of lathyritic rats caused by β-aminopropionitrile was investigated. In the junction there are finger-like processes of muscle fibers, in which thin filaments were extended from the last Z lines of myofibrils and attached to the sarcolemma of the processes. By the heavy meromyosin decoration technique, these thin filaments were identified as actin filaments. In the lathyritic muscle, the thin filaments were markedly fewer in number and distributed sparsely in the sarcoplasm.The content of connectin, an elastic protein, which is localized in myofibrils and also in sarcolemma was significantly decreased in the lathyritic muscle. A possible relationship between the changes in the fine structure of the myotendinous junction and in the connectin contents is discussed.     

Effects of Corticosterone on Connectin Content and Protein Breakdown in Rat Skeletal Muscle

We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.     

Effects of corticosterone on connectin content and protein breakdown in rat skeletal muscle

We examined the effects of a glucocorticoid, corticosterone, on calpain activity, connectin content and protein breakdown in rat muscle. The results indicated that calpain activity was increased by corticosterone and thus breakdown of connectin was stimulated followed by increased breakdown of skeletal muscle protein.     

Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments

To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.     

Age-related changes in the reducible cross-links on connectin from human skeletal muscle.

After NaB³H₄-reduction of connectin from human skeletal muscle, the changes in the amounts of the reducible cross-links and specific radioactivity of this elastic protein were followed throughout the whole life-span from embryo to old age. The reducible cross-links, aldimine forms of lysinonorleucine and histidino-hydroxymerodesmosine, and unidentified reducible compounds, which were assumed to be cross-linking amino acids, were found to remarkably decrease with age. A progressive decrease in the incorporation of tritium into the reducible compounds was also observed. We conclude that the conversion of the reducible cross-links derived from lysine and hydroxylysine aldehydes to non-reducible compounds is an essential step in the maturation of connectin fibrils, similar to collagen fibrils.     

An historical perspective of the discovery of titin filaments –Part 2

In 2017, a Special Issue of Biophysical Reviews was devoted to “Titin and Its Binding Partners. The issue contained a review: “An historical perspective of the discovery of titin filaments” by dos Remedios and Gilmour that was intended to be a history of the discovery of the giant protein titin, previously named connectin. The review took readers back to the earliest discovery of the so-called third filament component of skeletal and cardiac muscle sarcomeres and ended in 1969. Recently, my colleague Shin’ichi Ishiwata gently reminded me of two papers published in 1990 and 1993 that were unwittingly omitted from the original historical perspective. In the first paper (J Cell Biol 110:53–62, 1990), Funatsu et al. examined the elastic filaments in skeletal muscle using a combination of light and electron microscopy, but they also measured resting as well as passive stiffness mechanical measurements to establish that connectin (titin) is responsible for both stiffness and fiber tension. In the second paper (J Cell Biol 120:711–724, 1993), Funatsu et al. used permeabilised cardiac muscle myocytes (from rabbit papillary muscles) and focussed on filament ultrastructure using either freeze-substitution or deep-etched replica methods to visualise connectin/titin filaments in fibers with and without actin and myosin filaments.     

Analysis of the reducible components of the muscle protein, connectin: absence of lysine-derived cross-links

After exhaustive salt extractions of rabbit and human skeletal muscle, the amino acid compositions of the residual proteins were similar to those reported for connectin. Complete removal of collagen contamination was achieved only after treatment of the connectin preparations with bacterial collagenase. On reduction with KB³H₄, the small amounts of lysine-derived reducible cross-links that were present in the initial connectin preparations were completely absent after treatment with collagenase. In adult human connectin some hexitol-lysine derivatives were present after reduction. These results indicate that, in contrast to previous reports, connectin does not participate in the same lysyl oxidase-mediated cross-linking system that occurs in collagen and elastin.     

Towards a Molecular Understanding of the Elasticity of Titin

Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type.     

Organization of connectin/titin filaments in sarcomeres of differentiating chicken skeletal muscle cells

Very long, elastic connectin/titin molecules position the myosin filaments at the center of a sarcomere by linking them to the Z line. The behavior of the connectin filaments during sarcomere formation in differentiating chicken skeletal muscle cells was observed under a fluorescent microscope using the antibodies to the N terminal (located in the Z line), C terminal (M line), and C zone (myosin filament) regions of connectin and was compared to the incorporation of -actinin and myosin into forming sarcomeres. In early stages of differentiating muscle cells, the N terminal region of connectin was incorporated into a stress fiber-like structure (SFLS) together with -actinin to form dots, whereas the C terminal region was diffusely distributed in the cytoplasm. When both the C and N terminal regions formed striations in young myofibrils, the epitope to the C zone of A-band region, that is the center between the A-I junction and the M-line, initially was diffuse in appearance and later formed definite striations. It appears that it took some time for the N and C terminal regions of connectin to form a regular organization in a sarcomere. Thus the two ends of the connectin filaments were first fixed followed by the specific binding of the middle portion onto the myosin filament during sarcomere formation.     

Connectin, an elastic protein from myofibrils.

The elastic protein isolated from myofibrils of chicken skeletal muscle was compared with extracellular non-collagenous reticulin prepared from chicken liver and skeletal muscle. The amino acid compositions of these proteins were similar except that their contents of Phe, Leu, Cys/2, and Hyp were different. The impregnations of the elastic protein and reticulin with silver were also different. The reticulin was not at all elastic. It also differed from reticulin in solubility and antigenicity. It is proposed to call the intracellular elastic protein connectin.