The reduction in AQP4 in DMD muscles results from decreased levels of AQP4 mRNA in DMD myofibers.

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.

We investigated the ultrastructural localization of alpha 1-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 alpha 1-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 alpha 1-syntrophin and nNOS tended to be present in clusters. Double immunolabeling revealed that epitope combinations of alpha 1-syntrophin-dystrophin, alpha 1-syntrophin-nNOS, and nNOS-dystrophin occurred more frequently in doublet form than did other epitope combinations, such as alpha 1-syntrophin-beta- spectrin and nNOS-beta-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 alpha 1-syntrophin and dystrophin (28.5 +/- 1.5%, group mean +/- SE) than those against alpha 1-syntrophin and beta-spectrin (9.2 +/- 0.8%, P < 0.01). Furthermore, nNOS formed doublets significantly more frequently with dystrophin (25.2 +/- 3.3%) and alpha 1-syntrophin (26.0 +/- 4.1%) than with beta-spectrin (13.9 +/- 2.3%; P < 0.05). These data support the association of dystrophin, alpha 1-syntrophin, and nNOS at the inner surface of the muscle plasma membrane and near mitochondria of subsarcolemmal areas of normal human skeletal myofibers.

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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-beta-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.