Naruki Sato

Identification of Cardiac-Specific Myosin Light Chain Kinase

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca2+/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.

TBP-interacting protein 120B (TIP120B)/cullin-associated and neddylation-dissociated 2 (CAND2) inhibits SCF-dependent ubiquitination of myogenin and accelerates myogenic differentiation.

Despite fast protein degradation in muscles, protein concentrations remain constant during differentiation and maintenance of muscle tissues. Myogenin, a basic helix-loop-helix-type myogenic transcription factor, plays a critical role through transcriptional activation in myogenesis as well as muscle maintenance. TBP-interacting protein 120/cullin-associated neddylation-dissociated (TIP120/CAND) is known to bind to cullin and negatively regulate SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase, although its physiological role has not been elucidated. We have identified a muscle-specific isoform of TIP120, named TIP120B/CAND2. In this study, we found that TIP120B is not only induced in association with myogenic differentiation but also actively accelerates the myogenic differentiation of C2C12 cells. Although myogenin is a short lived protein and is degraded by a ubiquitin-proteasome system, TIP120B suppressed its ubiquitination and subsequent degradation of myogenin. TIP120B bound to cullin family proteins, especially Cullin 1 (CUL1), and was associated with SCF complex in cells. It was demonstrated that myogenin was also associated with SCF and that CUL1 small interference RNA treatment inhibited ubiquitination of myogenin and stabilized it. TIP120B was found to break down the SCF-myogenin complex. Consequently suppression of SCF-dependent ubiquitination of myogenin by TIP120B, which leads to stabilization of myogenin, can account for the TIP120B-directed accelerated differentiation of C2C12 cells. TIP120B is proposed to be a novel regulator for myogenesis.

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 α-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.

Effects of BTS (N-benzyl-p-toluene sulphonamide), an Inhibitor for Myosin-Actin Interaction, on Myofibrillogenesis in Skeletal Muscle Cells in Culture

Abstract 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 myosinactin 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 μM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of α-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.

Differential expression of C-protein isoforms in developing and degenerating mouse striated muscles.

With the aim of clarifying the roles of C‐protein isoforms in developing mammalian skeletal muscle, we cloned the complementary DNA (cDNAs) encoding mouse fast (F) and slow (S) skeletal muscle C‐proteins and determined their entire sequences. Northern blotting with these cDNAs together with mouse cardiac (C) C‐protein cDNA was performed. It revealed that in adult mice, C, F, and S isoforms are expressed in a tissue‐specific fashion, although the messages for both F and S isoforms are transcribed in extensor digitorum longus muscle, which has been categorized as a fast muscle. In addition, although C isoform is expressed first and transiently during development of chicken skeletal muscles, C isoform is not expressed in mouse skeletal muscles at all through the developmental stages; S isoform is first expressed, followed by the appearance of F isoform. Finally, in dystrophic mouse skeletal muscles, the expression of S isoform is increased as it is in dystrophic chicken muscle. These observations suggest that mutations in C isoform (MyBP‐C) do not lead to any disturbance in skeletal muscle, although they may lead to familial hypertrophic cardiomyopathy. We also suggest that the expression of S isoform may be stimulated in degenerating human dystrophic muscles.

Expression and Localization of Type II Diacylglycerol Kinase Isozymes δ and η in the Developing Mouse Brain

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.

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.

Comparative studies on troponin, a Ca2+-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.

Sea Lily Muscle Lacks a Troponin-Regulatory System, While It Contains Paramyosin

Troponin, a Ca2+-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 Ca2+-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 Mg2+-ATPase activity of rabbit skeletal muscle myosin irrespective of the presence or absence of Ca2+. The results indicate that Ca2+-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.

Functional Characteristics of Amphioxus Troponin in Regulation of Muscle Contraction

Troponin regulates contraction of vertebrate striated muscle in a Ca2+-dependent manner. More specifically, it acts as an inhibitor of actin-myosin interaction in the absence of Ca2+ 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 Ca2+, not as an inhibitor in the absence of Ca2+ 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 Ca2+-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.