Hiroko Kojima

Immunocytochemical studies of aquaporin 4 in the skeletal muscle of mdx mouse

Immunostainability of anti aquaporin 4 antiserum was investigated in the muscles of dystrophin deficient mdx mice. Western blot analysis showed that the rabbit antiserum against aquaporin 4 reacted with a 28 kDa protein in extracts of normal mouse quadriceps femoris muscles but did not react with the protein in extracts of quadriceps femoris muscles of mdx mice. Immunoperoxidase staining of the muscles from normal and mdx mice revealed the positive immunoreaction at the myofiber surface of normal mice and the negative, or the faint and discontinuous immunostaining at the surface of mdx myofibers. Immunogold electron microscopy disclosed the localization of aquaporin 4 molecules at the myofiber plasma membranes of normal mice and the localization was consistent with that of orthogonal array particles in the protoplasmic face of normal muscle plasma membrane seen in freeze fracture replicas. This study demonstrated that the density of aquaporin 4 molecules was decreased in the muscle plasma membranes of mdx mice, resulting in the faulty function of mdx myofibers.

Ultrastructural localization of α1-syntrophin and neuronal nitric oxide synthase in normal skeletal myofiber, and their relation to each other and to dystrophin

Abstract We investigated the ultrastructural localization of α1-syntrophin and neuronal nitric oxide synthase (nNOS) in normal human skeletal myofibers and analyzed their relation to each other and to dystrophin using single and double immunogold-labeling electron microscopy. Single immunolabeling showed antibodies to α1-syntrophin and nNOS on the inner surface of the muscle plasma membrane, the sarcoplasmic side of plasma membrane invaginations, and the sarcoplasm near mitochondria of subsarcolemmal areas. The epitopes of α1-syntrophin and nNOS tended to be present in clusters. Double immunolabeling revealed that epitope combinations of α1-syntrophin-dystrophin, α1-syntrophin-nNOS, and nNOS-dystrophin occurred more frequently in doublet form than did other epitope combinations, such as α1-syntrophin-β-spectrin and nNOS-β-spectrin. These increased frequencies were noted both at the muscle plasma membrane undercoat and near mitochondria of subsarcolemmal areas. A significantly higher percentage of doublets comprised antibodies against α1-syntrophin and dystrophin (28.5 ± 1.5%, group mean ± SE) than those against α1-syntrophin and β-spectrin (9.2 ± 0.8%, P < 0.01). Furthermore, nNOS formed doublets significantly more frequently with dystrophin (25.2 ± 3.3%) and α1-syntrophin (26.0 ± 4.1%) than with β-spectrin (13.9 ± 2.3%; P < 0.05). These data support the association of dystrophin, α1-syntrophin, and nNOS at the inner surface of the muscle plasma membrane and near mitochondria of subsarcolemmal areas of normal human skeletal myofibers.

Expression of aquaporin 3 and its localization in normal skeletal myofibres

The question whether aquaporin 3 (AQP3) is expressed in normal human skeletal muscle at mRNA and protein levels has been examined, since AQP3 has been reported to be coexpressed with AQP4 in various kinds of tissues other than skeletal muscle. The gel electrophoresis of the reverse transcription polymerase chain reaction (RT-PCR) product of total RNA samples extracted from normal human muscle specimens by using the oligonucleotide primers for AQP3 contained a band of 629 base pairs which corresponded to the base pair length between two primers of AQP3. The nucleotide sequence of this RT-PCR product coincided with that of AQP3. At the protein level, immunoblot, immunohistochemical and immunoelectron microscopical studies were done by using rabbit antibody against the synthetic peptide of the cytoplasmic domain of the human AQP3 molecule. Immunoblot analysis showed that rabbit antibody against the human AQP3 reacted with a protein of approximately 30 kDa molecular weight in extracts of normal human skeletal muscles. The immunoreaction for the anti-AQP3 antibody with normal human muscle was noted at the myofibre surface. Immunogold labelling electron microscopy revealed that the gold particles indicating the presence of AQP3 molecules were located mainly at the inside surface of muscle plasma membrane.

Merosin (laminin-2) localization in basal lamina of normal skeletal muscle fibers and changes in plasma membrane of merosin-deficient skeletal muscle fibers

Primary deficiency of merosin causes a severe congenital muscular dystrophy (CMD) and a mouse dystrophy (dy/dy mouse). Also, its secondary deficiency is seen in some CMD with abnormal glycosylation of Α-dystroglycan, an extracellular membrane protein, which is the receptor of merosin and binds to dystrophin underlying the sarcolenma via Β-dystroglycan, a transmembrane protein. In immunogold and freeze-etch electron microscopic studies, merosin in basal lamina of normal skeletal muscles has a zonation in the distribution and is localized at the lamina lucida of muscle basal lamina, and dystrophin molecules are often closed to merosin molecules at the inside and outside surface of muscle plasma membrane. Moreover, merosin molecules exist as the short fine cross-bridge fibrils connecting the basal lamina to the neighboring outer leaflet of the muscle plasma membrane. In freeze-fracture studies, the changes in muscle plasma membranes of dy/dy mice reveal a markedly decreased density of orthogonal arrays (OAs) but normal density of intramembranous particles (IMPs), whereas depletions of IMPs with decreased OAs have been found in Fukyama-type congenital muscular dystrophy, Duchenne muscular dystrophy, and mdx mice. Thus, further studies including the functional role of OAs would be required to understand the pathomechanism of merosin-deficient CMD.

Electron microscopic observations of triple immunogold labelling for dystrophin, β-dystroglycan and adhalin in human skeletal myofibers

Abstract Dystrophin is the Duchenne muscular dystrophy gene product and is a membrane cytoskeletal protein present in the network of the plasma membrane undercoat. Adhalin (50 kDa dystrophin-associated glycoprotein) and β-dystroglycan (43 kDa dystrophin-associated glycoprotein) are the transmembrane components of the normal muscle plasma membrane, and β-dystroglycan has been demonstrated to bind dystrophin at the inside surface of normal muscle plasma membrane. This investigation was undertaken to test whether the epitopes of dystrophin, β-dystroglycan and adhalin are closely associated with each other by using triple immunogold labelling electron microscopy on normal human skeletal myofibers. Although closely associated signals of triplet immunogold particles were observed, there were less numerous than expected. However, closely associated signals of two epitopes of dystrophin and β-dystroglycan, dystrophin and adhalin, or adhalin and β-dystroglycan were frequently observed. These ultrastructural findings are consistent with biochemical evidence implying that dystrophin, β-dystroglycan and adhalin are closely associated with each other at the normal muscle plasma membrane.

Ultrastructural localization of α-, β- and γ-sarcoglycan and their mutual relation, and their relation to dystrophin, β-dystroglycan and β-spectrin in normal skeletal myofiber

Abstract Ultrastructural localization of α-, β- and γ-sarcoglycan and their mutual relation, and their relation to dystrophin, β-dystroglycan and β-spectrin were investigated in normal skeletal myofibers. Single-immunogold labeling electron microscopy showed that the signals of rabbit and sheep polyclonal antibodies against the synthetic peptide of the cytoplasmic domain of α-, β or γ-sarcoglycan were present along the inside surface of muscle plasma membrane and at the sarcoplasmic side of plasma membrane invaginations and vesicular structures in subsarcolemmal areas. These localizations were similar to that of dystrophin, β-dystroglycan and β-spectrin. Double-immunogold labeling disclosed the close association of α-, β- and γ-sarcoglycan each other and α-, β-, γ-sarcoglycan with dystrophin or β-dystroglycan, and this was confirmed by statistical analysis. Monoclonal antibody against the extracellular domain of α-sarcoglycan was used with above-mentioned polyclonal anti-β- and -γ-sarcoglycan antibodies for triple-immunogold labeling, in which signals of α-sarcoglycan localized at the outer surface of muscle plasmalemma and those of β- and γ-sarcoglycans were present at the inside surface of plasma membrane. The triple immunolabeling showed an occasional closely associated presence of the three signals for α-, β- and γ-sarcoglycans, and a more frequent association for two signals out of α-, β- and γ-sarcoglycans. This study demonstrated that α-, β- and γ-sarcoglycan are closely located to one another and to dystrophin and β-dystroglycan at the muscle plasma membrane.

Aquaporin 1: Examination of Its Expression and Localization in Normal Human Skeletal Muscle Tissue

To examine aquaporin 1 (AQP1) expression in skeletal muscle tissue precisely, we performed reverse transcription-polymerase chain reaction (RT-PCR) at RNA level and immunoblot analysis, immunohistochemistry and immunoelectron microscopy at protein level. The RT-PCR study of total RNA from normal human skeletal muscle showed a strong single band of AQP1. At the protein level we used two commercially available antibodies, both of which recognize the cytoplasmic domain of the AQP1 molecule. One antibody gave positive results. Immunoblot of muscle extract showed a 30-kDa band protein, the molecular weight of which corresponded to that of AQP1. Immunohistochemically, AQP1 was immunostained at the myofiber surface both in type 1 and type 2 myofibers with almost the same intensity, and its staining pattern was rather diffuse and irregular compared with that of the anti-dystrophin antibody. The endomysial endothelial cells were also immunolabeled. Immunoelectron microscopy revealed that the immunogold particles indicating the presence of the AQP1 molecule were present along the inside surface of the muscle plasma membrane. However, another antibody showed negative results except for the endomysial endothelial cells which were positively stained. We drew the conclusion that AQP1 is expressed at the endomysial capillary endothelial cell and further AQP1 may be expressed at the human skeletal myofiber plasma membrane.

Altered aquaporin 4 expression in muscles of Fukuyama-type congenital muscular dystrophy

This study was undertaken to investigate the expression of aquaporin 4 (AQP4) in the muscle plasma membrane of children with Fukuyama-type congenital muscular dystrophy (FCMD) at protein and mRNA levels. The biopsied six muscles with FCMD, six histochemically normal muscles and eight disease control muscles were analyzed by means of immunoblots, immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR). Immunoblots showed that the band of FCMD muscle extracts stained with anti-AQP4 antibody was faint in comparison with that of normal muscle extracts. The immunohistochemistry revealed that most of the FCMD myofibers showed negative immunostaining with anti-AQP4 antibody, although the partially positive immunostaining of sporadic FCMD myofibers was noted. The immunoreactivity was positive with anti-dystrophin and anti-β-spectrin antibodies in almost all FCMD myofibers. The quantitative RT-PCR demonstrated that the AQP4 mRNA level of the FCMD muscles was markedly reduced. On the basis of these findings, we conclude that the expression of AQP4 in FCMD myofibers is reduced and the reduced content of AQP4 mRNA in FCMD muscles may be related to the decreased expression of AQP4 at the muscle plasma membrane of FCMD myofibers.

A comparative freeze-fracture study of plasma membrane of dystrophic skeletal muscles in dy/dy mice with merosin (laminin 2) deficiency and mdx mice with dystrophin deficiency

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities of skeletal muscle plasma membranes of merosin deficient dy/dy mice and their control mice at 7, 14 and 28 days after birth were analysed by freeze‐fracture electron microscopy. Similar studies were performed on dystrophin‐deficient mdx mice with mild muscle weakness at 28 days after birth for the comparison with those of dy/dy mice with severe muscle weakness at the same age. In the pre‐clinical stage of dy/dy mice at 14 days after birth, the membranes showed a significantly decreased density of OAs (P<0.01 by Wilcoxon rank‐sum test) as compared with control mice, while those in the clinical stage of dy/dy mice at 28 days after birth showed normal IMP density but a marked depletion of OA density (P<0.01). Moreover, at 28 days after birth, the reduction of OAs in the plasma membranes of dy/dy mice was more marked than that of mdx mice (P<0.05 by Wilcoxon rank‐sum test). These results provided us with the information that the OA density was affected more severely with merosin deficiency than with dystrophin deficiency, and again supported our previously proposed concept that the clinical severity in muscular dystrophies correlated with the OA density.

Aquaporin 4 Expression in the mdx Mouse Diaphragm

Expression of aquaporin (AQP) 4 in the surface membranes of skeletal myofibers is well established; however, its functional significance is still unknown. The alterations of AQP4 expressions in dystrophic muscles at RNA and protein levels have been reported in various dystrophic muscles such as dystrophinopathy, dysferlinopathy, and sarcoglycanopathy. We are interested in the relationship between the severity of dystrophic muscle degeneration and the expression of AQP4. Here we compared the AQP4 expression of the limb muscles with that of diaphragms in both mdx and control mice. The dystrophic muscle degeneration, such as rounding profile of cross sectional myofiber shape, dense eosin staining, central nuclei, and endomysial fibrosis in mdx mice, were more marked in diaphragms than in limb muscles. The decrease of AQP4 expression at protein level was more marked in diaphragms than in the limb muscles of mdx mice. However, the expression of AQP4 mRNA in the diaphragms of mdx mice was not reduced in comparison with limb muscles of mdx mice. The present study revealed that AQP4 expression at protein level was correlated with the severity of dystrophic changes in muscle tissues of mdx mice.