Periodic Stretching of Cultured Myotubes Enhances Myofibril Assembly.

The effects of mechanical stress on cultured muscle cells were examined with particular interest in myofibril assembly by using a cell-stretching system. We observed that formation and maintenance of cross-striated myofibrils in chick muscle cell cultures was suppressed in the media containing higher concentration of KCl, tetrodotoxin, or ML-9 (an inhibitor of myosin light chain kinase), but periodic stretching of myotubes for several days enabled formation of striated myofibrils just as in standard muscle cultures. However, ryanodine (a blocker of the Ca2 + channel in sarcoplasmic reticulum) and BDM (an inhibitor of myosin-actin interaction) suppressed the stretch-induced myofibrillogenesis. We further found that stretching of myotubes causes quick and transient elevation of the intracellular Ca2 + concentration and this elevation is disturbed by inhibition of Ca2 + channels of sarcoplasmic reticulum and suppression of Ca2 + influx from culture medium. These observations indicate that periodic stretching induces elevation of intracellular Ca2 + concentration and that this elevation may be due to release of Ca2 + from sarcoplasmic reticulum and Ca2 + influx from outside of the cells. The increased Ca2 + may activate actin-myosin interaction by interacting with troponin that is located along actin filaments and/or inducing phosphorylation of myosin light chains and thereby promote myofibril assembly.

Chimeric Mice with Deletion of Cfl2 that Encodes Muscle-Type Cofilin (MCF or Cofilin-2) Results in Defects of Striated Muscles, Both Skeletal and Cardiac Muscles.

Cofilin, a member of the ADF/cofilin family, is an actin-binding protein which is widely distributed among eukaryotic organisms and involved in actin filament dynamics in a variety of cell types. In mammalian striated muscles, muscle-type cofilin (MCF or cofilin-2) is predominantly expressed. Previous investigations have shown that MCF plays an essential role in the regulation of assembly of contractile apparatus in skeletal muscle, but its role in cardiac muscle has remained unclear. In the present study, in order to further clarify the role of MCF in organization of myofibrillar structure in vivo, we generated chimeric mice with a combination of MCF-deficient cells that were generated by Cfl2-knockout (Cfl2-/-) and wild type cells containing MCF, and examined the effect of MCF deficiency on striated muscles, especially on the fine structures of contractile apparatus in cardiac muscle. We found that mice chimeric for MCF deficient cells exhibited structural defects in their skeletal muscles as previously reported. Histological analysis showed that MCF deficiency leads to degradation of myofibers and promotion of muscle regeneration. Electron microscopic observation of cardiac muscle of the chimeric mice showed coexistence of the cells with normal sarcomeres and those with disorganized myofibrils in a chimeric pattern. In these cofilin-deficient cells, myofilaments were scattered in the cytoplasm and myofibrillar structures were severely disrupted. These results provide strong evidence for that MCF plays a critical role in the formation and the maintenance of myofibril structure not only in skeletal muscle but also in cardiac muscle.

Characterization of paramyosin and thin filaments in the smooth muscle of acorn worm, a member of hemichordates.

Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca2+-dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca2+-independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles.

UNC-87 isoforms, Caenorhabditis elegans calponin-related proteins, interact with both actin and myosin and regulate actomyosin contractility.

Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ~190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.

Sea lily muscle lacks a troponin-regulatory system, while it contains paramyosin.

Troponin, a Ca(2+)-dependent regulator of striated muscle contraction, has been characterized in vertebrates, protochordates (amphioxus and ascidian), and many invertebrate animals that are categorized in protostomes, but it has not been detected in echinoderms, such as sea urchin and sea cucumber, members of subphylum Eleutherozoa. In this study, we examined the muscle of a species of isocrinid sea lilies, a member of subphylum Pelmatozoa, that constitute the most basal group of extant echinoderms to clarify whether troponin is lacking from the early evolution of echinoderms. Native thin filaments were released from the muscle homogenates in a relaxing buffer containing ATP and EGTA, a Ca(2+)-chelator, and were collected by ultra-centrifugation. Actin and tropomyosin, but not a troponin-like protein, were detected in the filament preparation. The filaments increased Mg(2+)-ATPase activity of rabbit skeletal muscle myosin irrespective of the presence or absence of Ca(2+). The results indicate that Ca(2+)-sensitive factor, troponin, is lacking in the thin filaments of sea lily muscle as in those of the other echinoderms, sea urchin and sea cucumber. On the other hand, a paramyosin-like protein that is absent from chordates was detected in sea lily muscle as in the muscles of the other echinoderms and invertebrate animals of protostomes.

Cofilin is required for organization of sarcomeric actin filaments in chicken skeletal muscle cells.

Cofilin is an actin regulatory protein that plays a critical role in actin filament dynamics in a variety of cells. We have previously demonstrated that excess cofilin in skeletal muscle cells leads to disruption of actin filaments, followed by actin-cofilin rod formation in the cytoplasm. In this study, to further clarify the role of cofilin in actin assembly during myofibrillogenesis, cofilin expression was suppressed in cultured chicken skeletal muscle cells. First, we confirmed that turnover of cofilin in myotubes was much higher than that of actin, and that the cofilin level could be decreased drastically within 2 days when cofilin de novo synthesis was suppressed. Next, cofilin expression in individual myotubes was suppressed by introducing antisense morpholino oligonucleotides into the cells by microinjection. Cofilin depletion at the early phase of myofibrillogenesis caused abnormal actin aggregates in myotubes and impaired actin organization into cross-striated myofibril structures. However, when cofilin expression was suppressed in developed myotubes, actin localization in striated myofibrils was scarcely affected. These results indicate that cofilin plays a critical role in the regulation of actin assembly at the early process of myofibrillogenesis.

Comparative studies on troponin, a Ca²âº-dependent regulator of muscle contraction, in striated and smooth muscles of protochordates.

Troponin is well known as a Ca(2+)-dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin-myosin interaction at low Ca(2+) concentrations, and Ca(2+) at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca(2+)-dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca(2+). Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals.

Detection of a troponin I-like protein in non-striated muscle of the tardigrades (water bears).

Tardigrades, also known as water bears, have somatic muscle fibers that are responsible for movement of their body and legs. These muscle fibers contain thin and thick filaments in a non-striated pattern. However, the regulatory mechanism of muscle contraction in tardigrades is unknown. In the absence of extensive molecular and genomic information, we detected a protein of 31 kDa in whole lysates of tardigrades that cross-reacted with the antibody raised against nematode troponin I (TnI). TnI is a component of the troponin complex that regulates actin-myosin interaction in a Ca(2+)-dependent and actin-linked manner. This TnI-like protein was co-extracted with actin in a buffer containing ATP and EGTA, which is known to induce relaxation of a troponin-regulated contractile system. The TnI-like protein was specifically expressed in the somatic muscle fibers in adult animals and partially co-localized with actin filaments in a non-striated manner. Interestingly, the pharyngeal muscle did not express this protein. These observations suggest that the non-striated somatic muscle of tardigrades has an actin-linked and troponin-regulated system for muscle contraction.

Troponin in both smooth and striated muscles of Ascidian Ciona intestinalis functions as a Ca2+-dependent accelerator of actin−myosin interaction.

Troponin, a Ca2+-dependent regulator of muscle contraction, acts as an inhibitor of the actin−myosin interaction in the absence of Ca2+ during contraction in vertebrate striated muscle. However, variation has been observed in the mode of troponin-dependent regulation among the animals belonging to Protochordata, the taxon most closely related to Vertebrata. Although troponin in striated muscle of a cephalochordate amphioxus functions as an inhibitor in the absence of Ca2+ as in vertebrates [Dennisson, J. G., et al. (2010) Zool. Sci. 27, 461−469], troponin in the smooth muscle of a urochordate ascidian (Halocynthia roretzi) regulates actin−myosin interaction as an activator in the presence of Ca2+ and not an inhibitor in the absence of Ca2+ as in vertebrates [Endo, T., and Obinata, T. (1981) J. Biochem. 89, 1599−1608]. In this study, to further clarify the functional diversity of troponin, we examined the role of troponin in Ca2+-dependent regulation of the actin−myosin interaction in striated and smooth muscles in another member of Ascidiacea (Ciona inetestinalis) using three recombinant troponin components, TnT, TnI, and TnC, produced using an Escherichia coli expression system. On the basis of actomyosin ATPase assays, we show here that troponins in both smooth and striated muscles of ascidian function as a Ca2+-dependent activator of the actin−myosin interaction and TnT is the component responsible for this activation. These results indicate that troponin of ascidian has evolved in a manner different from that of amphioxus and vertebrates in terms of function.

Functional characteristics of amphioxus troponin in regulation of muscle contraction.

Troponin regulates contraction of vertebrate striated muscle in a Ca(2+)-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca(2+) during contraction. In vertebrates, this regulatory mechanism is unlike that in some less highly derived taxa. Troponin in the smooth muscle of the protochordate ascidian species Halocynthia roretzi regulates actinmyosin contraction as an activator in the presence of Ca(2+), not as an inhibitor in the absence of Ca(2+) as is the case in vertebrates. In this study, contractile regulation of striated muscle from another protochordate, the amphioxus Branchiostoma belcheri, was analyzed using recombinant troponin components TnT, TnI, and TnC that were produced in an Escherichia coli expression system to further elucidate their roles in Ca(2+)-dependent regulation of the actin-myosin interaction. Combination of these troponin components in an actin-myosin ATPase activity assay showed that troponin in amphioxus striated muscle functions in a similar manner to troponin in vertebrate striated muscle, and differently from ascidian smooth muscle troponin. Thus, troponin function appears to have evolved differently in different protochordate muscles.

Troponin I controls ovulatory contraction of non-striated actomyosin networks in the C. elegans somatic gonad.

The myoepithelial sheath of the Caenorhabditis elegans somatic gonad has non-striated actomyosin networks that provide contractile forces during ovulation, a process in which a mature oocyte is expelled from the ovary. Troponin T and troponin C are known regulators of contraction of the myoepithelial sheath. These are two of the three components of the troponin complex that is generally considered as a striated-muscle-specific regulator of actomyosin contraction. Here, we report identification of troponin I as the third component of the troponin complex that regulates ovulatory contraction of the myoepithelial sheath. C. elegans has four genes encoding troponin-I isoforms. We found that tni-1 and unc-27 (also known as tni-2) encode two major troponin-I isoforms in the myoepithelial sheath. Combination of RNA interference and mutation of tni-1 and unc-27 resulted in loss of the troponin-I protein in the gonad and caused sterility due to defective contraction of the myoepithelial sheath. Troponin-I-depleted gonads were hypercontracted, which is consistent with the function of troponin I as an inhibitor of actomyosin contraction. Troponin I was associated with non-striated actin networks in a tropomyosin-dependent manner. Our results demonstrate that troponin I regulates contraction of non-striated actomyosin networks and is an essential cytoskeletal component of the C. elegans reproductive system.

Differential expression of two cardiac myosin-binding protein-C isoforms in developing chicken cardiac and skeletal muscle cells.

Myosin-binding protein-C (MyBP-C), also known as C-protein, is a major myosin-binding protein characteristic of striated muscle, and plays a critical role in myofibril organization, especially in registration of thick filaments in the sarcomeres during myofibrillogenesis. We previously demonstrated that cardiac-type MyBP-C is involved early in the process of myofibrillogenesis in both cardiac and skeletal muscle during chicken muscle development. Two variants (type I and type II) have been detected in chicken cardiac MyBP-C; they differ only in the presence or absence of a sequence of 15 amino acid residues (termed P-seq) that includes a phosphorylation site for cyclic AMP-dependent kinase in the cardiac MyBP-C motif ( Yasuda et al, 1995 ). Therefore, types I and II are regarded as phosphorylatable and non-phosphorylatable isoforms, respectively. In this study, an antibody specific for P-seq was prepared. With this and other monoclonal antibodies to cardiac MyBP-C (C-315), expression and localization of the two MyBP-C isoforms in developing chicken cardiac and skeletal muscle were examined by immunocytochemistry and immunoblotting. The results showed that type I is predominantly expressed in the heart and is localized in myofibrils of both atrial and ventricular muscles through development. In contrast, type II is mainly expressed in embryonic skeletal muscle, although type I is faintly expressed in cultured skeletal muscle. These observations were confirmed by RT-PCR.

Activity of cofilin can be regulated by a mechanism other than phosphorylation/dephosphorylation in muscle cells in culture.

Cofilin plays a critical role in actin filament dynamics in a variety of eukaryotic cells. Its activity is regulated by phosphorylation/dephosphorylation of a Ser3 residue on the N-terminal side and/or its binding to a phosphoinositide, PIP(2). To clarify how cofilin activity is regulated in muscle cells, we generated analogues of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser3) of cofilin to Ala and Asp, respectively. These mutated proteins, as well as the cofilin having Ser3 residue (S3-cofilin), were produced in an E. coli expression system and conjugated with fluorescent dyes. In an in vitro functional assay, A3-cofilin retained the ability to bind to F-actin. Upon injection into cultured muscle cells, A3-cofilin and S3-cofilin promptly disrupted actin filaments in the cytoplasm, and many cytoplasmic rods containing both the exogenous cofilin and actin were generated, while D3-cofilin was simply diffused in the cytoplasm without affecting actin filaments. Several hours after the injection, however, the activity of A3-cofilin and S3-cofilin was suppressed: the actin-A3-cofilin (or S3-cofilin) rods disappeared, the cofilin diffused in the cytoplasm like D3-cofilin, and actin filaments reformed. Both GFP-fused A3-cofilin and S3-cofilin that were produced by cDNA transfection were also suppressed in the cytoplasm of muscle cells in culture. Thus, some mechanism(s) other than phosphorylation can suppress A3-cofilin activity. We observed that PIP(2) can bind to A3-cofilin just as to S3-cofilin and inhibits the interaction of A3-cofilin with actin. Our results suggest that the activity of A3-cofilin and also S3-cofilin can be regulated by PIP(2) in the cytoplasm of muscle cells.

Effects of BTS (N-benzyl-p-toluene sulphonamide), an inhibitor for myosin-actin interaction, on myofibrillogenesis in skeletal muscle cells in culture.

Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosin-actin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10-50 microM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of alpha-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2-3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.

Two mouse cofilin isoforms, muscle-type (MCF) and non-muscle type (NMCF), interact with F-actin with different efficiencies.

Two cofilin isoforms, a muscle-type (MCF) and a non-muscle-type (NMCF), are co-expressed in developing mammalian skeletal and cardiac muscles. To clarify how they are involved in the actin filament dynamics during myofibrillogenesis, we examined their localization in muscle tissues and cultured muscle cells using immunocytochemical methods, and their interaction with F-actin in vitro. NMCF was mostly detected in a diffuse pattern in the cytoplasm but MCF was partly localized to the striated structures in myofibrils. The location of chicken cofilin, a homologue of MCF, in the I-bands of myofibrils was determined by an immunocytochemical method. It is suggested that MCF could be associated with actin filaments in muscle cells more efficiently than NMCF. Using purified recombinant MCF and NMCF, their interaction with F-actin was examined in vitro by a cosedimentation assay method. We observed that MCF was precipitated with F-actin more effectively than NMCF. When MCF and NMCF were simultaneously incubated with F-actin, MCF was preferentially associated with F-actin. MCF and NMCF inhibited the interaction of F-actin with tropomyosin, but the former suppressed the actin-tropomyosin interaction more strongly than the latter. These results suggest that MCF interacts with F-actin with higher affinity than NMCF, and although both of them are involved in the regulation of actin assembly in developing myotubes, the two proteins may play somewhat different roles.

Myocyte differentiation generates nuclear invaginations traversed by myofibrils associating with sarcomeric protein mRNAs.

Certain types of cell both in vivo and in vitro contain invaginated or convoluted nuclei. However, the mechanisms and functional significance of the deformation of the nuclear shape remain enigmatic. Recent studies have suggested that three types of cytoskeleton, microfilaments, microtubules and intermediate filaments, are involved in the formation of nuclear invaginations, depending upon cell type or conditions. Here, we show that undifferentiated mouse C2C12 skeletal muscle myoblasts had smoothsurfaced spherical or ellipsoidal nuclei, whereas prominent nuclear grooves and invaginations were formed in multinucleated myotubes during terminal differentiation. Conversion of mouse fibroblasts to myocytes by the transfection of MyoD also resulted in the formation of nuclear invaginations after differentiation. C2C12 cells prevented from differentiation did not have nuclear invaginations, but biochemically differentiated cells without cell fusion exhibited nuclear invaginations. Thus, biochemical differentiation is sufficient for the nuclear deformation. Although vimentin markedly decreased both in the biochemically and in the terminally differentiated cells, exogenous expression of vimentin in myotubes did not rescue nuclei from the deformation. On the other hand, non-striated premyofibrils consisting of sarcomeric actinmyosin filament bundles and cross-striated myofibrils traversed the grooves and invaginations. Time-lapse microscopy showed that the preformed myofibrillar structures cut horizontally into the nuclei. Prevention of myofibril formation retarded the generation of nuclear invaginations. These results indicate that the myofibrillar structures are, at least in part, responsible for the formation of nuclear grooves and invaginations in these myocytes. mRNA of sarcomeric proteins including myosin heavy chain and alpha-actin were frequently associated with the myofibrillar structures running along the nuclear grooves and invaginations. Consequently, the grooves and invaginations might function in efficient sarcomeric protein mRNA transport from the nucleus along the traversing myofibrillar structures for active myofibril formation.

A novel variant of cardiac myosin-binding protein-C that is unable to assemble into sarcomeres is expressed in the aged mouse atrium.

Cardiac myosin-binding protein-C (MyBP-C), also known as C-protein, is one of the major myosin-binding proteins localizing at A-bands. MyBP-C has three isoforms encoded by three distinct genes: fast-skeletal, slow-skeletal, and cardiac type. Herein, we are reporting a novel alternative spliced form of cardiac MyBP-C, MyBP-C(+), which includes an extra 30 nucleotides, encoding 10 amino acids in the carboxyl-terminal connectin/titin binding region. This alternative spliced form of MyBP-C(+) has a markedly decreased binding affinity to myosin filaments and connectin/titin in vitro and does not localize to A-bands in cardiac myocytes. When MyBP-C(+) was expressed in chicken cardiac myocytes, sarcomere structure was markedly disorganized, suggesting it has possible dominant negative effects on sarcomere organization. Expression of MyBP-C(+) is hardly detected in ventricles through cardiac development, but its expression gradually increases in atria and becomes the dominant form after 6 mo of age. The present study demonstrates an age-induced new isoform of cardiac MyBP-C harboring possible dominant negative effects on sarcomere assembly.

Uptake of albumin is coupled with stretch-induced hypertrophy of skeletal muscle cells in culture.

Hypertrophy is induced in skeletal muscle when mechanical overload, for example repetitive stretching, is presented. This is a well-known phenomenon and the molecular mechanism involved has been investigated from various aspects. In this study, with a system that enables periodic stretching of cultured skeletal muscle cells, myotubes, along the long cellular axis uni-directionally at a constant frequency, we examined the effects of stretching on skeletal muscle using mouse C2 myotubes in culture as a model. Significant hypertrophy was observed in the myotubes after several days of periodic stretching and this was accompanied by the accumulation of a protein of about 67kDa. This protein was identified with albumin, which was present in the culture medium, based on its antigenicity, size and pI. When bovine serum albumin tagged with biotin was added to the culture medium, it became detectable in the cytoplasm of the stretched myotubes. mRNA encoding albumin was not detectable in the myotubes by northern blotting irrespective of their stretching or non-stretching, indicating that transcription of the albumin gene was not induced in the stretched muscle cells. From these results, we conclude that the accumulation of albumin in stretched myotubes was due to uptake of the protein from the culture medium not to de novo synthesis of the protein in myotubes. We suggest that albumin uptake may be involved in skeletal muscular hypertrophy.