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.


OBJECTIVE
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.


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.


CONCLUSIONS
The reduction in AQP4 in DMD muscles results from decreased levels of AQP4 mRNA in DMD myofibers.
Wakayama
Yoshihiro
Y
Department of Neurology, Showa University Fujigaoka Hospital, 1-30 Fujigaoka, Aoba-ku, Yokohama 227-8501, Japan. wakayama@showa-university-fujigaoka.gr.jp
Jimi
Takahiro
T
Inoue
Masahiko
M
Kojima
Hiroko
H
Murahashi
Makoto
M
Kumagai
Toshiyuki
T
Yamashita
Sumimasa
S
Hara
Hajime
H
Shibuya
Seiji
S
D051401:Aquaporin 4; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000378:metabolism; D016678:Genome; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry / Q000379:methods; D007223:Infant; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism*; D009468:Neuromuscular Diseases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling
C496030:AQP4 protein, human; D051401:Aquaporin 4; D020346:Aquaporins; D012333:RNA, Messenger; D004247:DNA