[The Interaction of miRNA-5p and miRNA-3p with the mRNAs of Orthologous Genes].

miRNAs regulate the expression of many genes and are involved in the development of diseases. We studied miRNAs that interact partly or fully complementarily with the 5'UTR, CDS and 3'UTR of mRNAs of target genes. The MirTarget program used in this study allows for the discovery of miRNA binding sites (BS) in the entire nucleotide sequence of the mRNA and for determining the characteristics of the interactions of miRNAs with mRNAs. We identified five pairs of fully complementary BS for miR-127-5p and miR-127-3p, miR-136-5p and miR-136-3p, miR-431-5p and miR-431-3p, miR-432-5p and miR-432-3p, and miR-433-5p and miR-433-3p in the CDS of the human and animal mRNA of RTL1 gene. The fully complementary BS for miR-6720-5p, miR-6720-3p were identified in the CDS of the FOXF2 gene; BS for miR-3187-5p, miR-3187-3p were found in the CDS of the PLPPR3 gene; BS for miR-4665-5p, miR-4665-3p were found in the 5'UTR of the KIAA2026 gene; BS for miR-135a-5p, miR-135a-3p were found in the 3'UTR of the GLYCTK gene; BS for miR-7106-5p, miR-7106-3p were found in the 3'UTR of the CCDC42B gene. The miRNA-5p and miRNA-3p associated with the RTL1 gene have BS in the mRNAs of 32 target human genes. The miRNA-5p and miRNA-3p associated with the FOXF2, PLPPR3, KIAA2026, GLYCTK and CCDC42B genes have BS in the mRNAs of 27 target genes, involved in development of several diseases. Nucleotide sequences of miRNA-5p and miRNA-3p and BS are conserved over tens of millions of years of divergence of the studied animal species. Binding characteristics of miR-3120-3p and miR-3120-5p, miR-196b-3p and miR-196b-5p, miR-125a-3p and miR-125a-3p, let-7e-3p and let-7e-5p, miR-99b-3p in fully complementary BS of non-coding DMN3OS, HOXA10-AS, SPACA6P-AS genes have been established.

Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by a ventricular hypertrophy predominantly affecting the interventricular septum and associated with a large extent of myocardial and myofibrillar disarray. It is the most common cause of sudden death in the young. In the four disease loci found, three genes have been identified which code for beta-myosin heavy chain, cardiac troponin T and alpha-tropomyosin. Recently the human cardiac myosin binding protein-C (MyBP-C) gene was mapped to chromosome 11p11.2 (ref. 8), making this gene a good candidate for the fourth locus, CMH4 (ref. 5). Indeed, MyBP-C is a substantial component of the myofibrils that interacts with several proteins of the thick filament of the sarcomere. In two unrelated French families linked to CMH4, we found a mutation in a splice acceptor site of the MyBP-C gene, which causes the skipping of the associated exon and could produce truncated cardiac MyBP-Cs. Mutations in the cardiac MyBP-C gene likely cause chromosome 11-linked hypertrophic cardiomyopathy, further supporting the hypothesis that hypertrophic cardiomyopathy results from mutations in genes encoding contractile proteins.

Muscle assembly: a titanic achievement?

The formation of perfectly aligned myofibrils in striated muscle represents a dramatic example of supramolecular assembly in eukaryotic cells. Recently, considerable progress has been made in deciphering the roles that titin, the third most abundant protein in muscle, has in this process. An increasing number of sarcomeric proteins (ligands) are being identified that bind to specific titin domains. Titin may serve as a molecular blueprint for sarcomere assembly and turnover by specifying the precise position of its ligands within each half-sarcomere in addition to functioning as a molecular spring that maintains the structural integrity of the contracting myofibrils.

Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments.

Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (approximately 2.0 micron) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from approximately 2.0 to 2.7 micron, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond approximately 2.7 micron, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.

Distinct families of Z-line targeting modules in the COOH-terminal region of nebulin.

To learn how nebulin functions in the assembly and maintenance of I-Z-I bands, MYC- and GFP- tagged nebulin fragments were expressed in primary cultured skeletal myotubes. Their sites of incorporation were visualized by double staining with anti-MYC, antibodies to myofibrillar proteins, and FITC- or rhodamine phalloidin. Contrary to expectations based on in vitro binding studies, none of the nebulin fragments expressed in maturing myotubes were incorporated selectively into I-band approximately 1.0-micrometer F-alpha-actin-containing thin filaments. Four of the MYC/COOH-terminal nebulin fragments were incorporated exclusively into periodic approximately 0.1-micrometer Z-bands. Whereas both anti-MYC and Rho-phalloidin stained intra-Z-band F-alpha-actin oligomers, only the latter stained the pointed ends of the polarized approximately 1.0-micrometer thin filaments. Z-band incorporation was independent of the nebulin COOH-terminal Ser or SH3 domains. In vitro cosedimentation studies also demonstrated that nebulin SH3 fragments did not bind to F-alpha-actin or alpha-actinin. The remaining six fragments were not incorporated into Z-bands, but were incorporated (a) diffusely throughout the sarcoplasm and into (b) fibrils/patches of varying lengths and widths nested among normal striated myofibrils. Over time, presumably in response to the mediation of muscle-specific homeostatic controls, many of the ectopic MYC-positive structures were resorbed. None of the tagged nebulin fragments behaved as dominant negatives; they neither blocked the assembly nor induced the disassembly of mature striated myofibrils. Moreover, they were not cytotoxic in myotubes, as they were in the fibroblasts and presumptive myoblasts in the same cultures.

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

In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.

The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

Titins: giant proteins in charge of muscle ultrastructure and elasticity.

In addition to thick and thin filaments, vertebrate striated muscle contains a third filament system formed by the giant protein titin. Single titin molecules extend from Z discs to M lines and are longer than 1 micrometer. The titin filament contributes to muscle assembly and resting tension, but more details are not known because of the large size of the protein. The complete complementary DNA sequence of human cardiac titin was determined. The 82-kilobase complementary DNA predicts a 3-megadalton protein composed of 244 copies of immunoglobulin and fibronectin type III (FN3) domains. The architecture of sequences in the A band region of titin suggests why thick filament structure is conserved among vertebrates. In the I band region, comparison of titin sequences from muscles of different passive tension identifies two elements that correlate with tissue stiffness. This suggests that titin may act as two springs in series. The differential expression of the springs provides a molecular explanation for the diversity of sarcomere length and resting tension in vertebrate striated muscles.

The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts.

Fibroblasts are a highly heterogeneous population of cells, being found in a large number of different tissues. These cells produce the extracellular matrix, which is essential to preserve structural integrity of connective tissues. Fibroblasts are frequently engaged in migration and remodeling, exerting traction forces in the extracellular matrix, which is crucial for matrix deposition and wound healing. In addition, previous studies performed on primary myoblasts suggest that the E3 ligase MuRF2 might function as a cytoskeleton adaptor. Here, we hypothesized that MuRF2 also plays a functional role in skeletal muscle fibroblasts. We found that skeletal muscle fibroblasts express MuRF2 and its siRNA knock-down promoted decreased fibroblast migration, cell border accumulation of polymerized actin, and down-regulation of the phospho-Akt expression. Our results indicated that MuRF2 was necessary to maintain the actin cytoskeleton functionality in skeletal muscle fibroblasts via Akt activity and exerted an important role in extracellular matrix remodeling in the skeletal muscle tissue.

Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy. Characterization Of cardiac transcript and protein.

Familial hypertrophic cardiomyopathy is a disease generally believed to be caused by mutations in sarcomeric proteins. In a family with hypertrophic cardiomyopathy linked to polymorphic markers on chromosome 11, we found a new mutation of a splice donor site of the cardiac myosin-binding protein-C gene. This mutation causes the skipping of the associated exon in mRNA from lymphocytes and myocardium. Skipping of the exon with a consecutive reading frame shift leads to premature termination of translation and is thus expected to produce a truncated cardiac myosin-binding protein-C with loss of the myosin- and titin-binding COOH terminus. However, Western blot analysis of endomyocardial biopsies from histologically affected left ventricular myocardium failed to show the expected truncated protein. These data show for the first time that a splice donor site mutation in the myosin-binding protein-C gene is transcribed to cardiac mRNA. Truncated cardiac myosin-binding protein-C does not act as a "poison polypeptide," since it seems not to be incorporated into the sarcomere in significant amounts. The absence of mutant protein and of significantly reduced amounts of wild-type protein in the presence of the mutated mRNA argues against the "poison protein" and the "null allele" hypotheses and suggests yet unknown mechanisms relevant to the genesis of chromosome-11- associated familial hypertrophic cardiomyopathy.

Protein kinase A phosphorylates titin's cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes.

beta-Adrenergic stimulation of cardiac muscle activates protein kinase A (PKA), which is known to phosphorylate proteins on the thin and thick filaments of the sarcomere. Cardiac muscle sarcomeres contain a third filament system composed of titin, and here we demonstrate that titin is also phosphorylated by the beta-adrenergic pathway. Titin phosphorylation was observed after beta-receptor stimulation of intact cardiac myocytes and incubation of skinned cardiac myocytes with PKA. Mechanical experiments with isolated myocytes revealed that PKA significantly reduces passive tension. In vitro phosphorylation of recombinant titin fragments and immunoelectron microscopy suggest that PKA targets a subdomain of the elastic segment of titin, referred to as the N2B spring element. The N2B spring element is expressed only in cardiac titins, in which it plays an important role in determining the level of passive tension. Because titin-based passive tension is a determinant of diastolic function, these results suggest that titin phosphorylation may modulate cardiac function in vivo.

The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system.

Titin is a giant vertebrate striated muscle protein with critical importance for myofibril elasticity and structural integrity. We show here that the complete sequence of the human titin gene contains 363 exons, which together code for 38 138 residues (4200 kDa). In its central I-band region, 47 novel PEVK exons were found, which contribute to titin's extensible spring properties. Additionally, 3 unique I-band titin exons were identified (named novex-1 to -3). Novex-3 functions as an alternative titin C-terminus. The novex-3 titin isoform is approximately 700 kDa in size and spans from Z1-Z2 (titin's N-terminus) to novex-3 (C-terminal exon). Novex-3 titin specifically interacts with obscurin, a 721-kDa myofibrillar protein composed of 57 Ig/FN3 domains, followed by one IQ, SH3, DH, and a PH domain at its C-terminus. The obscurin domains Ig48/Ig49 bind to novex-3 titin and target to the Z-line region when expressed as a GFP fusion protein in live cardiac myocytes. Immunoelectron microscopy detected the C-terminal Ig48/Ig49 obscurin epitope near the Z-line edge. The distance from the Z-line varied with sarcomere length, suggesting that the novex-3 titin/obscurin complex forms an elastic Z-disc to I-band linking system. This system could link together calcium-dependent, SH3-, and GTPase-regulated signaling pathways in close proximity to the Z-disc, a structure increasingly implicated in the restructuring of sarcomeres during cardiomyopathies.

Series of exon-skipping events in the elastic spring region of titin as the structural basis for myofibrillar elastic diversity.

Titins are megadalton-sized filamentous polypeptides of vertebrate striated muscle. The I-band region of titin underlies the myofibrillar passive tension response to stretch. Here, we show how titins with highly diverse I-band structures and elastic properties are expressed from a single gene. The differentially expressed tandem-Ig, PEVK, and N2B spring elements of titin are coded by 158 exons, which are contained within a 106-kb genomic segment and are all subject to tissue-specific skipping events. In ventricular heart muscle, exons 101 kb apart are joined, leading to the exclusion of 155 exons and the expression of a 2.97-MDa cardiac titin N2B isoform. The atria of mammalian hearts also express larger titins by the exclusion of 90 to 100 exons (cardiac N2BA titin with 3.3 MDa). In the soleus and psoas skeletal muscles, different exon-skipping pathways produce titin transcripts that code for 3.7- and 3.35-MDa titin isoforms, respectively. Mechanical and structural studies indicate that the exon-skipping pathways modulate the fractional extensions of the tandem Ig and PEVK segments, thereby influencing myofibrillar elasticity. Within the mammalian heart, expression of different levels of N2B and N2BA titins likely contributes to the elastic diversity of atrial and ventricular myofibrils.

Titin antibodies in "seronegative" myasthenia gravis--A new role for an old antigen.

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction of skeletal muscles. Triple-seronegative MG (tSN-MG, without detectable AChR, MuSK and LRP4 antibodies), which accounts for ~10% of MG patients, presents a serious gap in MG diagnosis and complicates differential diagnosis of similar disorders. Several AChR antibody positive patients (AChR-MG) also have antibodies against titin, usually detected by ELISA. We have developed a very sensitive radioimmunoprecipitation assay (RIPA) for titin antibodies, by which many previously negative samples were found positive, including several from tSN-MG patients. The validity of the RIPA results was confirmed by western blots. Using this RIPA we screened 667 MG sera from 13 countries; as expected, AChR-MG patients had the highest frequency of titin antibodies (40.9%), while MuSK-MG and LRP4-MG patients were positive in 14.6% and 16.4% respectively. Most importantly, 13.4% (50/372) of the tSN-MG patients were also titin antibody positive. None of the 121 healthy controls or the 90 myopathy patients, and only 3.6% (7/193) of other neurological disease patients were positive. We thus propose that the present titin antibody RIPA is a useful tool for serological MG diagnosis of tSN patients.

The complete primary structure of human nebulin and its correlation to muscle structure.

Nebulin is a giant filamentous protein specific for vertebrate skeletal muscles. The correlation of its size to thin filament lengths in vertebrates suggests that nebulin may function as a molecular ruler to determine thin filament length. We have isolated a full-length cDNA of 20.8 kb encoding human nebulin and determined its sequence. The cDNA's predicted peptide has a molecular weight of 773 kDa, and 97% of its mass consists of 185 copies of -35-residue module. Within the molecule, different sub-families of modules can be distinguished, and their arrangement is correlated to the structure of the thin filament. The central 154 copies are grouped into 22 seven-module super repeats corresponding to 38.5 nm thin filament repeats. In the thin filament ruler region, multiple isoforms are generated by alternative exon usage which is likely to explain the developmental and tissue-specific size variations of nebulins previously found in vertebrate skeletal muscles. We propose that different types of nebulin molecular rulers are expressed in the different types of skeletal muscles by differential splicing. Outside the super repeat region, the presence of distinct module arrangements implies functional diversity of the nebulin module family. A novel "simple repeat" family together with an SH3 domain at the C-terminus appear to anchor the nebulin filament system in the Z-disc. Nebulin's SH3 domain is highly related in sequence to the SH3 domains in yeast actin binding protein ABP-1 and to the src substrate p80/85 in chicken, both proteins which are involved in regulating actin assembly of the cytoskeleton in non-muscle cells. Study of nebulins terminal sequences is likely to reveal how integration of the nebulin filament into the sarcomere is regulated.

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.

Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs.

Nebulin is an 800 kDa large actin-binding protein specific to skeletal muscle and thought to act as a molecular template that regulates the length of thin filaments. Recently, a 100 kDa nebulin-like protein has been described in the avian cardiac muscle and referred to as nebulette. We have determined the full-length (8 kb) cDNA sequence of the human nebulette. Its open reading frame (3044 bp) encodes a 109 kDa protein that shares extensive similarity with the C-terminal region of human nebulin. The C-terminal regions of nebulin and nebulette are identical in domain organization and share a family of highly related C-terminal repeats, a serine-rich domain with potential phosphorylation sites, and an SH3 domain. Immunoelectron-microscopy suggests that the C-terminal 30 kDa of nebulin and nebulette filaments integrate into the Z-disc lattice, whereas their N termini appear to project into the I-band. Gene mapping studies assign the human nebulette gene to chromosome 10p12, whereas the nebulin gene has been previously assigned to 2q21. Evolutionary constraints appear to have maintained identical modular arrangements in these two independent genes. Comparison of nebulin and nebulette cDNAs demonstrates that a subgroup of repeats within the C-terminal regions is regulated tissue-specifically and stage-dependently during development of both molecules. This leads to a substantial diversity of nebulin and nebulette isoforms. Their further study is likely to provide insights into how they contribute to the molecular diversity of Z-discs from different muscle tissues and fiber types.

Genomic organization of M line titin and its tissue-specific expression in two distinct isoforms.

Titin is a 3000 kDa large protein of vertebrate striated muscle which extends from Z discs to M lines. Within the segment of titin that locates in the I band, tissue-specific isoforms are expressed by differential splicing in correlation to the sarcomeric ultrastructure. We have now searched the M-line region of titin for differential expression. The 20 kb section from the 3' end of the gene has been sequenced and contains 23 exons. Exon/intron organization is correlated to the modular organization of the titin protein. The six exons at the 3' end of the gene encode the M-line section of titin and are referred to as Mex1 to Mex6. Analysis of the RNAs expressed in different rabbit striated muscles reveals that the exon Mex5 is either included or excluded in the titin mRNA during splicing. The levels of inclusion of Mex5 vary between different types of striated muscles. Heart expresses (Mex5+)-titin, skeletal muscles co-express tissue-specifically distinct ratios of (Mex5+) and (Mex5-)-titins. In situ hybridization of whole-mount mouse embryos with Mex5 antisense RNA provide no evidence for the exclusion of Mex5 during embryonic development. We speculate that the establishment of differential splicing pathways of M-line titin late during development may correlate with and explain the postnatal development of different M-line fine structures in the different muscles. Comparison of titin gene sequences from different vertebrates reveals that the intron sequences located upstream of Mex3 and Mex5, referred to as Min-2 and Min-4, respectively, have remained strongly conserved during evolution. While the conservation of Min-4 may be explained by its participation in the regulation of the differential skipping of Mex5, the functional significance of the conservation of the Min-2 intron located upstream of Mex3 is yet unknown.

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.