title: Generation of muscle aquaporin 4 overexpressing transgenic mouse: its characterization at RNA and protein levels including freeze-fracture study.
abstract: 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.
authors.affiliation: Department of Neurology, Showa University Fujigaoka Hospital, 1-30 Fujigaoka, Aoba-ku, Yokohama 227-8501, Japan. wakayama@showa-university-fujigaoka.gr.jp
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Wakayama
authors.forename: Joji
authors.initials: J
authors.lastname: Takahashi
authors.forename: Seiji
authors.initials: S
authors.lastname: Shibuya
authors.forename: Masahiko
authors.initials: M
authors.lastname: Inoue
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kojima
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Oniki
authors.forename: Satoru
authors.initials: S
authors.lastname: Arata
authors.forename: Hajime
authors.initials: H
authors.lastname: Hara
authors.forename: Takahiro
authors.initials: T
authors.lastname: Jimi
authors.forename: Seiji
authors.initials: S
authors.lastname: Shioda
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Sunada
authors.forename: Hiroko
authors.initials: H
authors.lastname: Ohi
authors.forename: Teruo
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D051401:Aquaporin 4 / Q000096:biosynthesis* / Q000235:genetics*; D005614:Freeze Fracturing; D015870:Gene Expression; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D008822:Mice, Transgenic; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry*; D011506:Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D051401:Aquaporin 4; D011506:Proteins; D012333:RNA, Messenger