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.     

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.     

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.     

Proteolytic fragments of connectin cause aggregation of myosin filaments but not of actin filaments

Proteolytic fragments of 400 kD isolated from chymotrypsin-treated connectin, a muscle elastic protein, still retained the ability to cause aggregation of myosin filaments but lost the actin-bundling action. Tryptic digests of connectin showed similar effects. However, when connectin was hydrolyzed by pepsin to peptides smaller than approximately 40 kD, no such action was seen for both myosin and actin filaments. It is suggested that the actin bundling action of connectin filaments is due to topological restrictions. A modified reproducible procedure for the preparation of native connectin from chicken breast muscle is described in detail.     

Interactions of muscle beta-connectin with myosin, actin, and actomyosin at low ionic strengths

The interaction of the muscle elastic protein connectin with myosin and actin filaments was investigated by turbidimetry, viscosity, flow birefringence measurements, and electron microscopic observations. In KCl concentrations lower than 0.15 M at pH 7.0 at 25 degrees C, both myosin and actin filaments were aggregated by connectin. Myosin filaments were entangled with each other in the presence of connectin. Actin filaments were assembled into bundles under the influence of connectin just as under that of alpha-actinin. The physiological significance of the interactions of connectin with myosin and actin filaments is discussed in relation to the localization of connectin in myofibrils. The Mg2+-activated ATPase activity of actomyosin was appreciably enhanced by connectin in the presence of KCl concentrations lower than 0.1 M. The extent of activation by connectin was smaller than by alpha-actinin. The enhancement of the ATPase activity may be due to acceleration of the onset of superprecipitation of actomyosin.     

Cross-linking of connectin, an elastic protein in muscle

Following reduction with NaB³H₄, connectin, an elastic protein prepared from chicken muscle, was found to contain the reducible cross-links derived from lysine and hydroxylysine aldehydes. The aldimine form of lysinonorleucine is the most abundant reducible cross-link in this elastic protein. A smaller proportion of the reduced cross-link, histidino-hydroxymerodesmosine, is also detected. Since collagen contamination in the connectin preparation was if any negligible, it is concluded that connectin and connective tissue proteins, collagen and elastin, share common features of coss-linking.     

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.     

Binding of actin filaments to connectin

The binding of actin filaments to connectin, a muscle elastic protein, was investigated by means of turbidity and sedimentation measurements and electron microscopy. In the presence of less than 0.12 M KCl at pH 7.0, actin filaments bound to connectin. Long actin filaments formed bundles. Short actin filaments also aggregated into irregular bundles or a meshwork, and were frequently attached perpendicularly to long bundles. The binding of F-actin to connectin was saturated at an equal weight ratio (molar ratio, 50 : 1), as determined by a cosedimentation assay. Larger amounts of sonicated short actin filaments appeared to bind to connectin than intact F-actin. Myosin S1-decorated actin filaments did not bind to connectin. The addition of S1 to connectin-induced actin bundles resulted in partial disaggregation. Thus, connectin does not appear to interfere with actin-myosin interactions, since myosin S1 binds to actin more strongly than connectin.     

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I- band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.     

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.     

Post-mortem changes in skeletal muscle connectin.

Changes in connectin and elasticity of skeletal muscle were determined during post-mortem ageing. The amount of connectin decreased with increasing time of post-mortem storage whereas the rate of the decrease depended on the source of muscles. The loss in elasticity of muscle coincided well with the decrease in connectin contents. Electron microscopically, a network structure between the Z discs vanished when the amount of connectin fell to zero. We have concluded that the continuous net structure of connectin is responsible for about 30% of the total elasticity of living skeletal muscle and its degradtaion is responsible for post-mortem tenderization of meat.     

Molecular size and shape of beta-connectin, an elastic protein of striated muscle

Connectin is an elastic protein of vertebrate striated muscle, and consists of doublet components, alpha and beta (also called titins 1 and 2). In the present study, beta-connectin isolated in the native state was investigated in order to characterize its molecular size and shape. The molecular weight was approximately 2.1 X 10(6) (SDS gel electrophoresis) or 2.7 X 10(6) (sedimentation equilibrium). The sedimentation coefficient (SO20, w) was 17S in 0.1 M phosphate buffer, pH 7.0. The intrinsic viscosity measured in an Ostwald-type viscometer was 1.8 dl/g. However, the viscosity was greatly dependent on the velocity gradient, and at a very low velocity gradient of 0.0007 s-1, a solution of connectin (0.3 mg/ml) showed a viscosity value of 17,000 cp. Flow birefringence measurements suggested a length distribution ranging from 300 to 450 nm. Electron microscopic observations revealed that connectin is a long flexible filament and the peaks of frequency of length distribution were at 150, 300, 450, and 600 nm. It was tentatively assumed that the connectin molecule is 300-400 nm long and 34-38 nm wide. It is likely that beta-connectin is derived from alpha-connectin, which has an apparent molecular weight of 2.8 X 10(6).     

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.     

Oxidative stress and the mechanical properties of naturally occurring chimeric collagen-containing fibers.

The byssal threads of marine mussels are a fiber-reinforced composite material. Fibers are continuous, separated by matrix, and consist of chimeric collagens that encompass within the same primary protein structure domains corresponding to collagen, polyhistidine, and either elastin or dragline spider silk. The elastic modulus (stiffness) of the proximal portion of byssal threads was measured by cyclic stress-strain analysis at 50% extension. Before measurement, the threads were conditioned by various treatments, particularly agitation in aerated or nitrogen-sparged seawater. Stiffness can be permanently increased by more than two times, e.g., from 25 MPa to a maximum of 65 MPa, by simple agitation in aerated seawater. Much but not all of this stiffening can be prevented by agitation under nitrogen. Reversible strain stiffening would seem to be a useful adaptation to lower residual stresses arising from the deformation of two joined materials, i.e., distal and proximal portions with rather different elastic moduli. The permanent strain stiffening that characterizes proximal byssal threads subjected to oxidative stress is probably due to protein cross-linking. In the short term, this results in a stronger thread but at the expense of dynamic interactions between the molecules in the structure.     

Genomic- and protein-based approaches for connectin (titin) identification in the ascidian Ciona intestinalis

Determining the complete primary structure of large proteins is difficult because of the large sequence size and low sequence homology among animals, as is the case with connectin (titin)-like proteins in invertebrate muscles. Conventionally, large proteins have been investigated using immuno-screenings and plaque hybridization screenings that require significant time and labor. Recently, however, the genomic sequences of various invertebrates have been determined, leading to changes in the strategies used to elucidate the complete primary structures of large proteins. In this paper, we describe our methods for determining the sequences of large proteins by elucidating the primary structure of connectin from the ascidian Ciona intestinalis as an example. We searched for genes that encode connectin-like proteins in the C. intestinalis genome using the BLAST search program. Subsequently, we identified some domains present in connectin and connectin-like proteins, such as immunoglobulin (Ig), fibronectin type 3 (Fn) and kinase domains in C. intestinalis using the SMART program and manual estimation. The existence of these domains and the unique sequences between each domain were confirmed using RT-PCR. We also examined the localization of mRNA using whole-mount in situ hybridization (WISH) and protein expression using SDS-PAGE. These analyses indicate that the domain structure and molecular weight of ascidian connectin are similar to those of vertebrate connectin and that ascidian connectin is also expressed in heart muscle, similarly to vertebrate connectin. The methods described in this study can be used to determine the primary structures of large proteins, such as novel connectin-like proteins in invertebrates.     

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.     

Connectin filaments link thick filaments and Z lines in frog skeletal muscle as revealed by immunoelectron microscopy

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397). In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region approximately 0.15 micron from the center of the A band.     

Four-stranded coiled-coil elastic protein in the byssus of the giant clam, Tridacna maxima

An elastic protein with a secondary structure distinct from all well-known load-bearing proteins is found in the byssus of the giant clam, Tridacna maxima . The byssus consists of a bundle of hundreds of individual threads, each measuring about about 100 μm in diameter, which exhibit a tendon-like mechanical response. The amino acid composition of Tridacna byssus, however, is unlike tendon collagen, lacking high glycine, proline, and hydroxyproline. Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) measurements suggest that the constituent nanofibrils of the byssal threads are distinct from known secondary structure motifs previously reported for elastic proteins including the collagen triple-helix, the β-sheet nanocrystalline domains of silks, or the double-stranded coiled-coil regions of intermediate filaments. Instead, X-ray diffraction data indicate a structural organization in which four coiled-coil α-helices form a stable rope-like structure, which then further pack in a pseudohexagonal lattice to form nanofibrils. Amino acid composition analysis shows unusually high concentrations of acidic as well as basic residues, suggesting that the four-helix structure is stabilized by strong ionic interactions between oppositely charged residues in neighboring strands. The composition also suggests additional stabilization by disulfide cross-linking. On a larger scale, scanning and conventional transmission electron microscope (STEM and TEM) observations indicate that the nanofibrils exhibit an alternating periodicity of about 500 nm along the axial direction. A molecular model that combines the mechanical properties with the structural characteristics of the Tridacna byssal threads is proposed.     

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.     

Changes in contractile properties with selective digestion of connectin (titin) in skinned fibers of frog skeletal muscle.

Changes in contractile properties of mechanically skinned fibers were examined when connectin in the fibers was selectively digested by a low concentration (0.25 microgram/ml) of trypsin. Resting tension and isometric active tension were reduced as the digestion of the connectin progressed; the rate of reduction of active tension was larger than that of resting tension. Maximum shortening speed and calcium ion sensitivity of active tension were not changed by the digestion. Electron micrographs showed that A-bands in the fibers treated with trypsin are dislocated from I-bands. These results suggest that the digestion of connectin does not directly influence the reaction of actin-myosin-regulatory proteins, and thus the resultant reduction in the active tension is mainly due to disordering of the regular structure in a sarcomere.