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Reduced Aquaporin 4 Expression in the Muscle Plasma Membrane of Patients With Duchenne Muscular Dystrophy
Yoshihiro Wakayama, MD, PhD;
Takahiro Jimi, MD;
Masahiko Inoue, MD;
Hiroko Kojima, BS;
Makoto Murahashi, MD;
Toshiyuki Kumagai, MD;
Sumimasa Yamashita, MD;
Hajime Hara, MD;
Seiji Shibuya, MD
Arch Neurol. 2002;59:431-437.
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.
From the Department of Neurology, Showa University Fujigaoka Hospital,
Yokohama (Drs Wakayama, Jimi, Inoue, Murahashi, Hara, and Shibuya and Ms Kojima);
Department of Pediatric Neurology, Aichi Prefectural Colony, Kasugai (Dr Kumagai);
and Department of Neurology, Kanagawa Children's Medical Center, Yokohama
(Dr Yamashita), Japan.
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