Aquaporin-4 deficiency in skeletal muscle and brain of dystrophic mdx mice

Abstract

We report a detailed study of AQP4 expression in the neuromuscular system of mdx mice. Immunocytochemical analysis performed by double immunostaining revealed that mdx mice manifest a progressive reduction in AQP4 at the sarcolemmal level of skeletal muscle fast fibers and that type IIB fibers are the first to manifest this reduction in AQP4 expression. No labeling was observed in the cytoplasm of muscle fibers, indicating that the reduction in sarcolemma staining is not associated with an intracellular compartmentalization of mistargeted protein. By Western blot and RT-PCR analysis, we found that whereas the total content of AQP4 protein decreased (by 90% in adult mdx mice), mRNA levels for AQP4 remained unchanged. A similar age-related reduction in AQP4 expression was found in brain astrocytic end-feet surrounding capillaries of mdx mice. Morphometric analysis performed after immunogold electron microscopy indicated a reduction of approximately 85% in gold particles (32+/-2/microm vs. 4.7+/-0.61/microm). Western blot experiments conducted using membrane fractions from brain cortex revealed a strong reduction (of 70%) in AQP4 protein in adult mdx mice, and RT-PCR experiments demonstrated that the reduction was not at transcription level. More interesting was the finding that AQP4 reduction was associated with swelling of astrocytic perivascular processes whose ultrastructural modifications are commonly indicated as an important and early event in the development of brain edema. No apparent reduction in AQP4 was found in mdx stomach and kidney. Our data provide evidence that dystrophin deficiency in mdx mice leads to disturbances in AQP4 assembly in the plasma membrane of fast skeletal muscle fibers and brain astrocytic end-feet, suggesting that changes in the osmotic equilibrium of the neuromuscular apparatus may be involved in the pathology of muscular dystrophy.