Aquaporin 9 expression and its localization in normal skeletal myofiber.

The examination was performed whether aquaporin (AQP) 9 is expressed in normal skeletal muscle at mRNA and protein levels. Gel electrophoresis of the reverse transcription-polymerase chain reaction (RT-PCR) product of total RNA samples of human normal muscles by oligonucleotide primers for human AQP9 showed a band of 221 basepairs, which corresponded to the basepair length between two primers of AQP9. The nucleotide sequence of RT-PCR product coincided with that of human AQP9. Immunoblot analysis revealed that the rabbit and sheep antibodies against the synthetic peptide of the C-terminal cytoplasmic domain of human AQP9 molecule reacted with a protein of approximately 30 kDa molecular weight in extracts of human normal skeletal muscles. Immunohistochemistry with our anti-AQP9 antibodies showed an immunoreaction at the myofiber surface of both type 1 and type 2 fibers with almost equal staining intensity in human skeletal muscles. The implication of AQP9 expression in skeletal myofibers was discussed.

Skeletal muscle syntrophin interactors revealed by yeast two-hybrid assay.

Syntrophins are the cytoplasmic peripheral proteins of dystrophin glycoprotein complex, of which five (alpha l, beta 1, beta 2, gamma 1 and gamma 2) isoforms have been identified so far. Respective syntrophin isoforms are encoded by different genes but have similar domain structures. At the sarcolemma of skeletal muscle, the most abundant alpha l-syntrophin was shown to interact at its PDZ domain with many membrane proteins. Among them, the AQP4 interaction with alpha 1-syntrophin PDZ domain was demonstrated by a Tg mouse study, prompting us to investigate the interaction between mouse alpha l-syntrophin (BC018546: nt.267-492, PDZ domain) pEXP-AD502 as prey vector and mouse AQP4 (NM009700: nt.805-969) pDBLeu as bait vector by the yeast two-hybrid assay, resulting in a negative study. We further studied the binding partner of another sarcolemma located beta 1-syntrophin, and performed a yeast two-hybrid experiment. With human beta 1-syntrophin as bait and human skeletal muscle cDNA library as prey, we obtained one positive clone which turned out to be alpha-dystrobrevin. Although the interaction of human beta 1-syntrophin with alpha-dystrobrevin has already been shown by immunoprecipitation assay, we have here confirmed this interaction by a yeast two-hybrid experiment.

Reduced expression of sarcospan in muscles of Fukuyama congenital muscular dystrophy.

Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.

Aquaporin 4 mRNA levels in neuromuscular tissues of wild-type and dystrophin-deficient mice.

Aquaporin (AQP) 4 is a water-specific channel protein and is abundant in central nervous tissues and skeletal muscles. Recently, the AQP4 molecule has been increasingly highlighted in its pathophysiological role of several neurological diseases, such as stroke, muscular dystrophy and neuromyelitis optica. We therefore measured the levels of AQP4 mRNA and glyceraldehyde-3 phosphate dehydrogenase mRNA (an internal control) in muscle and brain tissues of wild-type mice (C57BL10/ScSn) and age-matched dystrophin-deficient mdx mice (C57BL10/ScSn mdx) by real-time quantitative RT-PCR. The relative AQP4 mRNA level was highest in the spinal cord among the neuromuscular tissues examined in wild-type mice. Among the muscle tissues of wild-type mice, the relative AQP4 mRNA level was higher in extensor digitorum longus (EDL) muscles, and its descending order was EDL, quadriceps femoris, soleus and heart muscles. It is noteworthy that there was no difference in the relative AQP4 mRNA levels in the brain tissues between wild-type mice and age-matched mdx mice. In contrast, the AQP4 mRNA level in the quadriceps femoris muscle was significantly lower in mdx mice than in wild-type mice. The fact that the spinal cord contains the highest AQP4 mRNA may be related to the pathogenesis of neuromyelitis optica, in which AQP4 protein is the target antigen. In addition, the low expression level of AQP4 mRNA in the mdx mouse muscle suggests a functional link between AQP4 and dystrophin in the muscle tissue. We suggest that a similar pathomechanism may underlie the phenotypic consequences of the mdx mouse and Duchenne muscular dystrophy.

Generation of muscle aquaporin 4 overexpressing transgenic mouse: its characterization at RNA and protein levels including freeze-fracture study.

We generated the muscle aquaporin 4 (AQP4) overexpressing transgenic mice in order to investigate the skeletal muscle pathology at RNA and protein levels. At RNA level, the AQP4 mRNA expression of soleus, EDL and cardiac muscles in Tg mice was statistically significantly higher than that in wild mice by the real-time reverse transcription polymerase chain reaction method. At protein level examinations, we used the immunoblot, immunohistochemistry and freeze-fracture electron microscopy. The immunoblot showed the single band of 31kDa with anti-AQP4 antibody in the extracts of soleus and EDL muscles of wild mice but not in extract of wild cardiac muscle; while the reaction band was noted in cardiac muscle of Tg mice and the reaction band was stronger in the extracts of soleus and EDL muscles of Tg mice. The immunohistochemistry showed that the expression of AQP4 at the myofiber surface of soleus and EDL muscles of Tg mice was more marked than that of wild mice and, interestingly, the AQP4 expression of these muscles of Tg mice appeared to be more remarkable in type 1 slow twitch myofibers as judged by the positive slow myosin immunostaining of adjacent serial sections. The immunofluorescence staining with anti-AQP4 antibody of cardiac muscles of wild mice revealed the scarcely immunopositive myofibers; whereas the immunostaining cardiac muscles of Tg mice contained the numerous AQP4 immunopositive myofibers. The freeze-fracture electron microscopy demonstrated that the orthogonal array densities in soleus and EDL muscle plasma membranes of Tg mice were significantly higher than those of wild mice and that the orthogonal array like particle density of cardiac muscle plasma membranes of Tg mice appeared to be more numerous than that of cardiac myofibers of wild mice. Finally the clinical phenotype of Tg mice appeared to be similar to that of wild mice. Further physiological examination with devices may suggest some about the physiological difference.

The relationship between intramembranous particles and aquaporin molecules in the plasma membranes of normal rat skeletal muscles: a fracture-label study.

The plasma membrane of skeletal muscles contains water channels such as aquaporin 4 (AQP4), aquaporin 3 (AQP3) and aquaporin 7 (AQP7). In dehydrated mice, we have recently reported the altered distribution of the aggregations of intramembranous particles (IMPs), such as orthogonal array (a crystal-like structure) and IMP cluster (a rosette-like structure) on the freeze-fractured skeletal muscle plasma membranes. In this fracture-label study, we first tested whether the orthogonal arrays (OAs) were composed of AQP4 in skeletal muscles and further analyzed the relationship between IMPs including IMP clusters and AQP3 molecules. As a result, many of the gold particles indicating AQP4 was associated with OAs (79%) by our fracture-label technique. On the other hand, approximately 50% of gold particles indicating AQP3 were associated with IMP clusters. Thus we confirmed that the OAs are composed of AQP4 in skeletal muscles, and further demonstrated that some of the IMP clusters are composed of AQP3 and may participate in maintaining osmotic homeostasis in skeletal myofibers. The fracture-label method is useful in investigating the molecular identification of membrane proteins such as AQP3 and AQP4.

Expression of myoferlin in skeletal muscles of patients with dysferlinopathy.

Myoferlin is a novel protein of unknown function with high homology to dysferlin, the gene mutations of which cause limb girdle muscular dystrophy type 2B and Miyoshi myopathy. The myoferlin gene seems to be a candidate for the modifier, and because of the high homology to dysferlin myoferlin may work as a compensator for the absence of dysferlin in dysferlinopathy. This hypothesis is based on the observation that utrophin, which has 80% homology with dystrophin, is overexpressing in the dystrophin deficient myofibers. To test this hypothesis, we investigated the myoferlin expression by immunoblot and immunohistochemical analysis in muscles of five patients with dysferlinopathy. For this aim, we generated a myoferlin specific antibody that does not cross react with dysferlin, and performed the immunoblot, immunohistochemical and immunoelectron microscopic studies. Immunohistochemical analysis showed that the antibodies against myoferlin and dysferlin clearly stained the normal human myofiber surface membranes. The electron microscopy of single immunogold labeled samples for myoferlin showed the presence of the molecular signal along the normal muscle cell membrane. Immunoblot analysis showed that the intensity of 230-kDa myoferlin band of dysferlinopathy muscle extracts was similar to that of normal muscle extracts. The immunostaining of dysferlinopathy muscles with anti-myoferlin antibody revealed a weak immunoreactivity along the muscle cell surface. Thus, the compensatory overexpression of myoferlin was not detected in muscles with dysferlinopathy.

Sarcospan: ultrastructural localization and its relation to the sarcoglycan subcomplex.

Sarcospan is a 25 kDa transmembrane component of dystrophin-associated glycoprotein. We generated a rabbit polyclonal antibody against synthetic peptide of the N-terminal domain of human sarcospan. Using this antibody we investigated the localization of sarcospan and its spacial relation to the components of sarcoglycan subcomplex in normal human skeletal myofibers by immunofluorescent microscopy and immunogold electron microscopy. In immunofluorescence the reaction was observed continuously at the myofiber surface. Ultrastructurally the gold signals of rabbit anti sarcospan antibody were present along the muscle plasma membrane, mainly at its inside surface. The triple immunogold labeled muscle samples showed that the signals of rabbit or sheep polyclonal anti alpha-, beta-, gamma- and delta-sarcoglycan antibodies and/or mouse monoclonal anti beta-, gamma- and delta-sarcoglycan antibodies were located along the muscle plasma membrane, and the cluster formation of different two or three sarcoglycan molecules was observed. The triple immunogold labeling also revealed that the signal of sarcospan molecules are present frequently in doublets and/or triplets with the components of sarcoglycan subcomplex, resulting in the cluster formation of signals of sarcoglycan and sarcospan molecules. The result of this study showed that sarcospan was expressed at the myofiber surface and that sarcospan was present in close association with alpha-, beta-, gamma- and delta-sarcoglycans and formed a functional unit with sarcoglycan subcomplex.

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.

Alterations of molecular architectures in skeletal muscle plasma membranes of dehydrated and water-loaded mice.

It is likely that orthogonal arrays (OAs) and caveolae seen on the replicas of freeze-fractured muscle plasma membranes are involved in maintaining osmotic homeostasis. Therefore, using the freeze-fracture technique, we examined the ultrastructural changes in OAs and caveolae of the skeletal muscle plasma membrane of dehydrated and water-loaded mice. In the muscle plasma membranes of 6 dehydrated and 6 water-loaded mice, caveolar distribution was not altered, and the densities of caveolae and OAs did not show statistically significant differences when compared with those in 6 control mice, although the skeletal muscles of water-loaded mice sometimes had muscle plasma membranes with extremely numerous OAs. In contrast, the muscle plasma membranes of dehydrated mice often revealed changes in the distribution of OAs, which existed in a group at the confined area of the muscle plasma membranes and were frequently accompanied by the aggregations of intramembranous particles (IMPs) around OAs. Thus, on the basis of the present study, we suggest that OAs in skeletal muscles as well as those in brain may play an important role in maintaining osmotic homeostasis of these organs under abnormal water balance.

Reduced expression of aquaporin 4 in human muscles with amyotrophic lateral sclerosis and other neurogenic atrophies.

Aquaporin 4 (AQP4) is a water channel protein that is widely distributed in human tissues. However, the precise functional role of AQP4 in skeletal muscle tissue has not yet been determined. Expression of AQP4 was reported to be reduced in muscle tissue from Duchenne muscular dystrophy patients. In the regenerating phase of skeletal muscle, AQP4 expression was reduced when nerve supply was not present. However, in diseased human muscles with neurogenic atrophy including amyotrophic lateral sclerosis, there has been no data on the changes in AQP4 expression. In the present study, we investigated the expression of AQP4 at mRNA and protein levels in human muscles with neurogenic atrophy. The mean level of AQP4 mRNA was significantly lower in muscles with neurogenic atrophy than that in muscles from normal controls. The myofiber surface immunostaining with anti-AQP4 antibody in muscles with neurogenic atrophy was reduced on the surface of scattered myofibers, small angulated myofibers, and myofibers in small- and large-group atrophy despite the presence of dystrophin. Based on the present findings, we conclude that the expression of AQP4 is affected by nerve supply and is down-regulated in human muscles with neurogenic atrophy.

Expression and localization of aquaporin 7 in normal skeletal myofiber.

We examined whether AQP7 molecules are expressed in the normal skeletal muscle at mRNA and protein levels. Gel electrophoresis of the reverse transcription-polymerase chain reaction (RT-PCR) product of total RNA samples of normal human or mouse muscles by using oligonucleotide primers for human or mouse AQP7 showed a band of 328 or 369 basepairs, which corresponded to the basepair length between two primers of AQP7. The nucleotide sequence of these RT-PCR products coincided with those of human and mouse AQP7. Immunoblot, immunohistochemical and immunoelectron-microscopic studies of the protein were done by using the rabbit antibody against the synthetic peptide of the N-terminal cytoplasmic domain of the human AQP7 molecule. Immunoblot analysis showed that the rabbit antibody against human AQP7 reacted with a protein of approximately 30 kDa molecular weight in extracts of normal human and mouse skeletal muscles, and normal mouse liver. Immunohistochemistry with our anti-AQP7 antibody showed an immunoreaction at the myofiber surface of type 1 and type 2 fibers in human muscles and of type 2 fibers in mouse muscles.

Reduced density of intramembranous particle clusters: freeze-fracture study of mdx mouse muscle plasma membrane.

Our previous freeze-fracture study demonstrated the decreased density of intramembranous particles (IMPs) on the protoplasmic (P) face of muscle plasma membranes in mdx mice. However, the molecular mechanism is unknown. In the present freeze-fracture study, we examined whether the reduced P-face IMP density in mdx mice would be caused by depletion of the rosette-like IMP clusters, which are IMP aggregations differing from crystal-like orthogonal arrays (OAs). By comparison with control mice, the P-face plasma membranes of extensor digitorum longus (EDL) and soleus (SOL) muscles of mdx mice demonstrated the following findings: (1) decreased IMP density with subunit particles of OAs and IMP clusters, (2) decreased IMP density without subunit particles of OAs, (3) normal IMP density without subunit particles of OAs and IMP clusters, (4) decreased OA density in EDL muscles and normal OA density in SOL muscles, and (5) decreased IMP cluster densities in both muscles. Thus, the reduced IMP density of P-face muscle plasma membranes in mdx mice may result from the decreased IMP clusters, suggesting the relationship between IMP clusters and the integral membrane proteins is influenced by dystrophin deficiency such as that of dystrophin-associated glycoproteins or other membrane proteins.

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 Alpha-dystroglycan, an extracellular membrane protein, which is the receptor of merosin and binds to dystrophin underlying the sarcolenma via Beta-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.

[ACE inhibitors and its usefulness in the prevention of aspiration pneumonia in chronic cerebrovascular disease patients with asymptomatic swallowing dysfunction].

The double contrast pharyngogram by use of computed radiography (DCP-CR) has been found to be useful in detection of asymptomatic swallowing dysfunction. Following the DCP-CR examination, we investigated the incidence of aspiration pneumonia in 143 patients with chronic cerebrovascular disease (CVD) for 3 years and the effects of ACE inhibitors on the prevention of pneumonia. Aspiration pneumonia occurred in 29 out of 143 patients, and more frequently in the elderly chronic CVD patients with multiple brain lesions. Aspiration pneumonia was confirmed in 26 out of 85 patients (30.6%) with abnormal barium adhesion to the pharyngeal wall on the double contrast pharyngogram image by DCP-CR; whereas pneumonia occurred in 3 out of 58 patients (5.2%) with normal findings of DCP-CR pharyngogram. Among chronic CVD patients with abnormal findings of DCP-CR pharyngogram, the incidence of aspiration pneumonia was significantly lower in the patients treated with ACE inhibitors than in those treated with other antihypertensive agents or without antihypertensive agents (chi 2 value = 7.163, p < 0.05). Accordingly, ACE inhibitors may prevent the aspiration pneumonia and reduce the incidence of aspiration pneumonia in the chronic CVD patients with abnormal DCP-CR pharyngogram images.

Ultrastructural localization of aquaporin 4 and alpha1-syntrophin in the vascular feet of brain astrocytes.

Aquaporin 4 (AQP4) is a recently discovered membrane bound water-selective channel and has been described at the light microscopic level to be predominantly expressed in the astrocytes of the brain, especially at the perivascular astrocyte endfoot processes. Alpha1-syntrophin, a member of dystrophin-associated protein, has also been reported at the light microscopic to be expressed level in the same site of astrocytes as AQP4 and interacts with other molecules through its PDZ domain. AQP4 expression has been reported to be absent at the sarcolemma and the perivascular astrocyte endfoot processes of alpha1-syntrophin knockout mice. Based on these observations, the molecular association between AQP4 and alpha1-syntrophin could be speculated. To test this hypothesis, we investigated the ultrasturctural localization of AQP4 and alpha1-syntrophin in the brain astrocytes by using double immunogold labeled electron microscopy. The results showed that AQP4 and alpha1-syntrophin colocalized frequently at the astrocyte membrane, especially at the perivascular astrocyte endfoot processes and suggested the presence of linkage between AQP4 and alpha1-syntrophin at the astrocyte plasma membrane.

Changes in the distribution and density of caveolin 3 molecules at the plasma membrane of mdx mouse skeletal muscles: a fracture-label electron microscopic study.

To analyze the molecular mechanism of the increased caveolin 3 activities in dystrophin-deficient muscles, we investigated three-dimensionally the changes in caveolin 3 molecular distribution and density at the sarcolemma of mdx mice by the fracture-label electron microscopic technique. At the sarcolemma of skeletal muscles from mdx mice, the densities of gold particles associated with caveolae, non-associated with caveolae and arranged circularly without caveolae were higher than those in control mice (P<0.01, P<0.01 and P<0.05 by two-tailed t-test), although in mdx mice, the overall arrangement of gold particles appeared to be irregular. These findings may reflect the active process of caveolar formation and the results of the disrupted protein-protein interaction in dystrophin-deficient muscle plasma membrane.

Reduced aquaporin 4 expression in the muscle plasma membrane of patients with Duchenne muscular dystrophy.

BACKGROUND: In Duchenne muscular dystrophy (DMD), previous freeze-fracture electron microscopic studies demonstrated that muscle plasma membrane contained markedly decreased numbers of orthogonal arrays. Recent investigations showed that orthogonal arrays were composed of aquaporin 4 (AQP4) molecules, a member of the water channel protein family. OBJECTIVES: To study whether the immunostainability of anti-AQP4 antibody is reduced in muscles of patients with DMD and whether, if it is reduced, the problem is at the genomic DNA, messenger RNA (mRNA), or posttranscriptional level. PATIENTS AND METHODS: We analyzed the muscle and blood samples from 6 boys with DMD, 6 normal control subjects, and 12 patients with neuromuscular diseases at the protein, genomic DNA, and mRNA levels. At the protein level, immunohistochemical staining and immunoblot analysis were performed. At the genomic DNA and mRNA levels, the polymerase chain reaction and reverse transcription polymerase chain reaction, respectively, were used to screen for mutations in the AQP4 gene. RESULTS: At the protein level, immunohistochemical staining of our originally generated rabbit anti-AQP4 antibody in DMD muscles was markedly reduced. Most of the DMD myofibers showed negative staining with sporadic partially positive fibers at their myofiber surface, whereas the control muscles displayed continuous myofiber surface staining. Immunoblot analysis showed that the content of AQP4 in DMD muscles was remarkably decreased. Amplification of leukocyte genomic DNA by polymerase chain reaction showed that the patients with DMD had genomic DNA of the AQP4 molecule. Quantitative reverse transcription polymerase chain reaction demonstrated that DMD skeletal muscles contained markedly decreased AQP4 mRNA compared with controls. CONCLUSION: The reduction in AQP4 in DMD muscles results from decreased levels of AQP4 mRNA in DMD myofibers.