Takao Hijikata

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMDJ) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle

BackgroundSkeletal muscles are composed of heterogeneous collections of muscle fiber types, the arrangement of which contributes to a variety of functional capabilities in many muscle types. Furthermore, skeletal muscles can adapt individual myofibers under various circumstances, such as disease and exercise, by changing fiber types. This study was performed to examine the influence of dystrophin deficiency on fiber type composition of skeletal muscles in canine X-linked muscular dystrophy in Japan (CXMDJ), a large animal model for Duchenne muscular dystrophy.MethodsWe used tibialis cranialis (TC) muscles and diaphragms of normal dogs and those with CXMDJ at various ages from 1 month to 3 years old. For classification of fiber types, muscle sections were immunostained with antibodies against fast, slow, or developmental myosin heavy chain (MHC), and the number and size of these fibers were analyzed. In addition, MHC isoforms were detected by gel electrophoresis.ResultsIn comparison with TC muscles of CXMDJ, the number of fibers expressing slow MHC increased markedly and the number of fibers expressing fast MHC decreased with growth in the affected diaphragm. In populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of CXMDJ muscles, slow MHC fibers were predominant in number and showed selective enlargement. Especially, in CXMDJ diaphragms, the proportions of slow MHC fibers were significantly larger in populations of myofibers with non-expression of developmental MHC. Analyses of MHC isoforms also indicated a marked increase of type I and decrease of type IIA isoforms in the affected diaphragm at ages over 6 months. In addition, expression of developmental (embryonic and/or neonatal) MHC decreased in the CXMDJ diaphragm in adults, in contrast to continuous high-level expression in affected TC muscle.ConclusionThe CXMDJ diaphragm showed marked changes in fiber type composition unlike TC muscles, suggesting that the affected diaphragm may be effectively adapted toward dystrophic stress by switching to predominantly slow fibers. Furthermore, the MHC expression profile in the CXMDJ diaphragm was markedly different from that in mdx mice, indicating that the dystrophic dog is a more appropriate model than a murine one, to investigate the mechanisms of respiratory failure in DMD.

Plectin 1 links intermediate filaments to costameric sarcolemma through β-synemin, α-dystrobrevin and actin

In skeletal muscles, the sarcolemma is possibly stabilized and protected against contraction-imposed stress by intermediate filaments (IFs) tethered to costameric sarcolemma. Although there is emerging evidence that plectin links IFs to costameres through dystrophin-glycoprotein complexes (DGC), the molecular organization from plectin to costameres still remains unclear. Here, we show that plectin 1, a plectin isoform expressed in skeletal muscle, can interact with β-synemin, actin and a DGC component, α-dystrobrevin, in vitro. Ultrastructurally, β-synemin molecules appear to be incorporated into costameric dense plaques, where they seem to serve as actin-associated proteins rather than IF proteins. In fact, they can bind actin and α-dystrobrevin in vitro. Moreover, in vivo immunoprecipitation analyses demonstrated that β-synemin- and plectin-immune complexes from lysates of muscle light microsomes contained α-dystrobrevin, dystrophin, nonmuscle actin, metavinculin, plectin and β-synemin. These findings suggest a model in which plectin 1 interacts with DGC and integrin complexes directly, or indirectly through nonmuscle actin and β-synemin within costameres. The DGC and integrin complexes would cooperate to stabilize and fortify the sarcolemma by linking the basement membrane to IFs through plectin 1, β-synemin and actin. Besides, the two complexes, together with plectin and IFs, might have their own functions as platforms for distinct signal transduction.

Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.

Zebrafish protocadherin 10 is involved in paraxial mesoderm development and somitogenesis

Here, we present the first report of the molecular cloning of zebrafish protocadherin 10 (Pcdh10, OL‐protocadherin) and describe its functional analyses in the development of segmental plate. Epitope‐tagged Pcdh10 expressed in embryos was localized on cell peripheries of adjacent cells. In situ hybridization showed that pcdh10 was expressed in the paraxial mesoderm (PAM) and developing somites, and in the pineal body, the diencephalon, and the vicinity of otocysts. Expression in PAM increased in the last few presumptive somites, reached the maximum level in the latest segmenting somites, and decreased thereafter during somite maturation. These expression patterns suggested that Pcdh10 is involved in development of PAM and somites. This was confirmed by morpholino knockdown and dominant‐negative inhibition of Pcdh10 in embryos, which disturbed movements of PAM cells and somite segmentation. Comparative studies showed that pcdh10 expression lasted up to approximately three times longer in maturing somites than that of paraxial protocadherin (pcdh8). They also indicated that the adaxial cells expressed pcdh8 but not pcdh10. We propose that Pcdh10 is involved in the morphogenic movements of PAM cells and somite segmentation and that differential adhesion of Pcdh8 and Pcdh10 plays a role in the morphogenic machinery of somites and adaxial cells. Developmental Dynamics 235:506–514, 2006.

Functional morphology of serially linked skeletal muscle fibers.

In the skeletal muscle fiber organization of many vertebrate muscles, serial arrangements or linkages of muscle fibers along the muscle or fascicle are commonly found. These serially linked muscle fibers employ distinct junctional morphologies from muscle to muscle. Notable are the end-to-end linkages of muscle fibers through tendinous intersections (TIs), where many fibers end onto a continuous connective tissue plate with folded terminations similar to myotendinous junctions. Besides this end-to-end linkage, overlapping linkages or arrangements occur among nonspanning fibers terminating intrafascicularly. These nonspanning fibers bear tapering terminations with direct cell-cell (myomuscular) junctions or without any specialized junctions. Despite their overlapping linkages or tapering profiles, nonspanning fibers maintain a uniform sarcomere length along the linked fibers, suggesting that the overlapping-linked nonspanning fibers are equivalent to the end-to-end linked fibers in their mechanical capacity. However, the junctional compliance could differ in their extracellular elastic components and their organization at junctional sites, e.g., direct mechanical (myomuscular) junctions vs. indirect linkages through connective tissue. Increasing evidence suggests that the elastic components, including muscle fibers as well as connective tissues, are more critical than previously thought for the mode and/or the efficiency of tension transmission among serially arranged fibers and thus for the mechanical properties of the muscle.

Distal regulatory element of the STAT1 gene potentially mediates positive feedback control of STAT1 expression

We previously identified a distal regulatory element located approximately 5.5‐kb upstream of the signal transducer and activator of transcription 1 (STAT1) gene, thereafter designating it as 5.5‐kb upstream regulatory region (5.5URR). In this study, we investigated the functional roles of 5.5URR in the transcriptional regulation of STAT1 gene. A chromosome conformation capture assay indicated physical interaction of 5.5URR with the STAT1 core promoter. In luciferase reporter assays, 5.5URR‐combined STAT1 core promoter exhibited significant increase in reporter activity enhanced by forced STAT1 expression or interferon (IFN) treatment, but STAT1 core promoter alone did not. The 5.5URR contained IFN‐stimulated response element and GAS sites, which bound STAT1 complexes in electrophoretic mobility shift assays. Consistently, chromatin immunoprecipitation (ChIP) assays of HEK293 cells with Halo‐tagged STAT1 expression indicated the association of Halo‐tagged STAT1 with 5.5URR. ChIP assays with IFN treatment demonstrated that IFNs promoted the recruitment of Halo‐tagged STAT1 to 5.5URR. Forced STAT1 expression or IFN treatment increased the expression of endogenous STAT1 and other IFN signaling pathway components, such as STAT2, IRF9 and IRF1, besides IFN‐responsive genes. Collectively, the results suggest that 5.5URR may provide a regulatory platform for positive feedback control of STAT1 expression possibly to amplify or sustain the intracellular IFN signals.

A conserved regulatory element located far downstream of the gls locus modulates gls expression through chromatin loop formation during myogenesis

Chromatin loops formed between distant regulatory elements and promoters modulate gene expression. We identified a novel distant regulatory element located approximately 120 kb downstream of the gls promoter, and examined its regulatory relevance to gls gene expression in C2C12 cells by a chromosome conformation capture assay. The distant element physically interacted with the gls promoter in myoblasts but not in myotubes. Semiquantitative analysis by real‐time PCR showed more abundant gls transcripts in myoblasts than in myotubes. These findings suggest that this distant element differentially regulates gls gene expression through dynamic formation and abrogation of a chromatin loop during myogenesis.

The direct visualization of structural array from laminin to dystrophin in sarcolemmal vesicles prepared from rat skeletal muscles

It has been biochemically shown that dystrophin and α‐ and β‐dystroglycan form an oligomeric complex which links laminin, a component of the basement membrane, to components of the subsarcolemmal cytoskeleton in skeletal muscle fibers. In the present study the dystrophin‐glycoprotein complex and its structural relationships to laminin and subsarcolemmal cytoskeleton were ultrastructurally examined in crude surface membranes prepared from rat skeletal muscles. Sarcolemmal vesicles within crude surface membranes were identified and characterized by fine protrusions on their outer surface and electron‐dense materials or patches associated with the inner surface. These two components were seen to be in register with each other across the sarcolemma. The fine protrusions were immunolabeled by anti‐α‐dystroglycan and reassociated with exogenous laminin. Immunolabeling in combination with laminin reassociation demonstrated that the electron‐dense materials contained dystrophin at laminin‐binding domains of the membrane. In addition, they were often associated with very fine filaments. These results provide morphological evidence for the biochemically proposed model of molecular array of dystrophin complex from the basement membrane to the subsarcolemmal cytoskeleton.

Staging of disuse atrophy of skeletal muscles on immunofluorescence microscopy.

The Japanese population is rapidly aging, thereby causing excess demand for facilities for elderly invalids. It is imperative that social measures and scientific studies be carried out to enable better care of bedridden elderly people. The purpose of the present study was to review the histological changes that occur in disuse atrophy of skeletal muscles, the primary pathophysiology of bedridden invalids, with the object of developing a staging standard to be used by researchers and clinicians. Rat hindlimb suspension was used as an experimental model. Atrophy of the soleus muscle was evaluated qualitatively and quantitatively on immunofluorescence microscopy. The myofibrils decreased significantly in the first 2-3 weeks of disuse atrophy. The earliest morphological change was fan-shaped multistep forking of sarcomeres, which appeared by the first week. This type of muscular lesion, designated here as ‘sarcomeric disarray’, was first described in the present study. Central-core lesions appeared mainly in slow muscle fibers by the second week. These lesions disappeared by the fourth or fifth week. Nerves remained intact and no inflammation or regeneration occurred up to the fifth week. Methods and criteria were compiled for staging of disuse atrophy based on the present results and a diagnosis kit designed for studies on disuse atrophy of skeletal muscles.

JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes. AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis.