| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2002 as doi:10.1096/fj.01-0797fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Dipartimento di Fisiologia Generale ed Ambientale and Centro di Eccellenza in Genomica Comparata, Università degli Studi di Bari, I-70126 Bari, Italy; and
Servizio Malattie Neuro-Muscolari, Università di Genova, Istituto G. Gaslini, Genova, Italy
3Correspondence: Dipartimento di Fisiologia Generale ed Ambientale, Università degli Studi di Bari, via Amendola 165/A, I-70126 Bari, Italy. E-mail: a.frigeri{at}biologia.uniba.it
SPECIFIC AIMS
Duchenne muscular dystrophy (DMD) and the milder form Becker muscular dystrophy (BMD) are X-linked genetic neuromuscular disorders that affect 
1 in 3500 boys. The purpose of this investigation was to 1) determine whether the expression of aquaporin-4 in DMD/BMD muscle sarcolemma is affected as we previously reported for mdx mice, the animal model of DMD; 2) determine the extent of the alteration, if any, in relationship to the type of dystrophin gene mutation and age of the patients; 3) compare the alteration of AQP4 with the expression of 
-syntrophin; 4) analyze the presence of AQP4 in different forms of muscular dystrophy such as the limb girdle muscular dystrophy (LGMD-1C) and the facioscapulohumeral muscular dystrophy (FSH-MD) to determine whether AQP4 reduction is a common feature of dystrophic muscles.
PRINCIPAL FINDINGS
1. AQP4 immunoreactivity is severely altered in DMD muscles
Immunofluorescence experiments using AQP4 affinity-purified antibodies were performed on the DMD muscle biopsies and biopsies from age-matched controls. AQP4 immunoreactivity was localized to the sarcolemma of normal quadriceps muscle fibers similar to dystrophin and other members of the DAPs complex. In all DMD patients analyzed, AQP4 immunoreactivity was either absent or severely reduced at the sarcolemma compared with normal control. No increase in intracellular AQP4 immunofluorescence staining was revealed.
The expression of caveolin-3 was analyzed in parallel in DMD samples to determine the level of sarcolemma integrity. Immunofluorescence experiments revealed an increase in the expression of caveolin-3, suggesting that sarcolemma deterioration is not a determining cause of AQP4 deficiency in these patients. We found no correlation between the age of patients or different mutations of the dystrophin gene and the level of AQP4 expression in muscle fibers.
2. Reduction of AQP4 expression is not due to a reduction of fast fibers
We reported that muscle unloading/reloading influences slow-to-fast and fast-to-slow conversion of muscle fibers in parallel with increase/decrease in sarcolemma AQP4 expression. To analyze whether AQP4 protein reduction in DMD patients is due to a fast-to-slow conversion of muscle fibers, we performed double immunofluorescence experiments using AQP4 affinity-purified antibodies together with specific antibodies against fast-type IIA MHC isoform. Experiments revealed that many type IIA fast fibers were found in DMD muscles and that the corresponding AQP4 staining was lost or strongly reduced. These data indicate that the reduced AQP4 expression is not a consequence of the reduction in the number of fast fibers.
3. Altered AQP4 expression in BMD muscles does not always correlate with -syntrophin expression
As DMD, BMD is also characterized by defects of the dystrophin gene; however, mutations do not determine the absence of dystrophin but result in its lower abundance. Six different BMD muscle biopsies were analyzed by immunofluorescence to determine whether AQP4 expression is altered as in DMD. Experiments revealed (Fig. 1
) that three of six muscles displayed strong AQP4 reduction whereas the other three muscle biopsies displayed near-normal AQP4 immunostaining. Although to a different extent, all six muscles revealed reduction in dystrophin immunofluorescence signal.
|
Recent studies suggest an -syntrophin-dependent localization of AQP4 in skeletal muscle. To analyze whether AQP4 expression in DMD/BMD muscles is strictly associated with -syntrophin, double immunofluorescence experiments were performed. Although some muscle biopsies from BMD (B1, B3) and DMD patients (D3) displayed a matching reduction in AQP4 and -syntrophin, others (B2, B4, B6) revealed largely normal AQP4 staining and reduced -syntrophin. In one case (B5), AQP4 reduction is more dramatic than that of -syntrophin. Thus, whereas -syntrophin appears constantly reduced in BMD and DMD muscles, AQP4 is normally expressed in half of the BMD patients. These results suggest that in BMD, AQP4 reduction is not the consequence of -syntrophin altered expression. Moreover, considering the patients’ age we can exclude a correlation between the AQP4 alteration and the progression of the pathology.
4. AQP4 expression is not altered in LGMD-1C and FSH-MD
To determine whether the alteration in AQP4 expression is a common characteristic of dystrophic muscle, we analyzed the expression of AQP4 in two other forms of muscular dystrophy not related to dystrophin gene mutations: LGMD-1C and the FSH-MD (Fig. 2
). Caused by the absence of caveolin-3, LGMD-1C is also characterized by normal expression of dystrophin and reduced expression of -dystroglycan. FSH-MD is an autosomal dominant neuromuscular disorder associated with large deletions of variable size on chromosome 4q35. These deletions, however, do not appear to disrupt a transcribed gene but are thought to interfere with the expression of a gene or genes located proximal to the deletions.
|
Immunofluorescenceanalysis revealed that AQP4 is normally expressed at the sarcolemma of LGMD-1C and FSH-MD biopsies. LGMD-1C results indicate that AQP4 expression is not reduced by the absence of caveolin-3, suggesting (Fig. 2)
these two proteins probably do not directly interact in the sarcolemma.
5. Total AQP4 protein content is reduced only in DMD/BMD muscles
To confirm that the reduction in AQP4 sarcolemma immunoreactivity was due to a reduction in total AQP4 content and not to an improper assembly of AQP4 protein in the membrane, protein levels of muscle biopsies were analyzed by Western blot. AQP4 was detected by a method commonly used to analyze the level of DAPs. As shown in Fig. 2
, no AQP4 signal was detected in samples of DMD (D3) and BMD (B1, B3, B5) patients. In contrast, patients B2, B4, and B6 as well as LGMD-1C and FSH-MD showed no major alteration in AQP4 protein. Western blot experiments indicate that decreased immunofluorescence AQP4 signal observed is due to a reduced total AQP4 protein content.
CONCLUSIONS
Here we present the first description of altered AQP4 expression in skeletal muscle of human patients affected by DMD and BMD. These results, together with our recent report on mdx mice, confirm an involvement of AQP4 in the molecular alterations occurring in the skeletal muscle of DMD/BMD patients. The immunofluorescence analysis of AQP4 expression in the six DMD patients used for the present study did not show any correlation between the type of dystrophin gene mutation and the severity of AQP4 reduction, indicating that the alteration in AQP4 expression is a common trait in DMD. Moreover, immunofluorescence experiments using human biopsies of patients affected by LGMD-1C and FSH-MD showed normal sarcolemma expression of AQP4, indicating that AQP4 deficiency is not a common feature of all the muscular dystrophies.
As extensively reported in the literature, the absence of dystrophin in dystrophic muscle results in a consistent reduction of the proteins of the DAPs complex. Although the precise function of many of these proteins is unknown, the complex is clearly important for normal muscle physiology, since the absence of some of these proteins causes many forms of muscular dystrophy. The parallel reduction of AQP4 and DAPs suggests that AQP4 could directly or indirectly interact with the DAPs complex. An interesting information comes from -syntrophin null mice. These mice show the absence of AQP4 in muscle fiber sarcolemma and brain astrocytes as well as of nNOS from the sarcolemma. Recently, two papers reported that, in mice, the sarcolemma expression/stability of AQP4 in vivo depends on the presence of the -syntrophin PDZ domain. Chemical cross-linking followed by coimmunoprecitation experiments showed AQP4 in association with dystrophin, ß-dystroglycan, and syntrophin. To analyze the relationship between AQP4 and syntrophin, we took advantage of the BMD biopsies in which the reduction in syntrophin expression is not always accompanied by a parallel AQP4 reduction, suggesting that AQP4 sarcolemma expression does not necessarily requires the presence of syntrophin. Furthermore, Western blot results indicate that in those BMD muscles where -syntrophin is reduced, neither membrane stability nor the degradation rate of AQP4 were grossly influenced. Together with the literature reports, our data allow us to speculate that AQP4 and -syntrophin interaction would occur via an unknown adaptor protein. This may explain the need for a cross-linker to preserve the interaction between AQP4, -syntrophin, and the dystrophin complex. Alternatively, AQP4 and -syntrophin interaction in the human sarcolemma membrane occurs directly but it is not sufficient for stable retention of AQP4, suggesting the presence of other AQP4-anchoring proteins specific to human skeletal muscle. Consequently, we can hypothesize that a different sarcolemma proteins assembly between mouse and human could be at the basis of the different severity of the pathology.
The absence of -syntrophin, nNOS, and AQP4 at the sarcolemma did not determine gross histological changes and showed no significant differences in muscle contractile properties in -syntrophin mutant mice. Thus, it is not surprising that AQP4 knockout mice manifested neither gross skeletal muscle abnormalities nor apparent functional alterations. Although mdx mice and DMD are genetically homologous and characterized by a complete absence of dystrophin, the limb muscles of adult mdx mice suffer neither the detectable weakness nor the progressive degeneration seen in DMD. Thus, species-specific differences involving the capacity of sustained muscle fiber regeneration, as occur in mdx mice, could explain the differences.
In conclusion, our work demonstrates for the first time that DMD patients manifest a drastic reduction in AQP4 expression in skeletal muscle, suggesting its interaction with the DAPs complex. It is not clear how this interaction occurs, whether the loss of AQP4 in dystrophic muscle is a consequence of muscle pathology, or whether and to what extent the loss of AQP4 contributes to the progress of DMD pathogenesis. However, analysis of LGMD-1C and FSH-MD shows that AQP4 reduction is not a general phenomenon related to the dystrophic process but it is a peculiarity of those muscular dystrophies caused by the absence/reduction of dystrophin.
Understanding the pathophysiological role of the various proteins involved in the disease will provide better insight into the mechanism leading to muscle degradation in dystrophy and facilitate pharmacological intervention.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0797fje; to cite this article, use FASEB J. (May 8, 2002) 
2 A.F. and G.P.N. contributed equally to this study.
This article has been cited by other articles:
|
|
 |
|
  M. E. Adams, Y. Tesch, J. M. Percival, D. E. Albrecht, J. I. Conhaim, K. Anderson, and S. C. Froehner Differential targeting of nNOS and AQP4 to dystrophin-deficient sarcolemma by membrane-directed {alpha}-dystrobrevin J. Cell Sci., January 1, 2008; 121(1): 48 - 54. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Vajda, M. Pedersen, E.-M. Fuchtbauer, K. Wertz, H. Stodkilde-Jorgensen, E. Sulyok, T. Doczi, J. D. Neely, P. Agre, J. Frokiar, et al. Delayed onset of brain edema and mislocalization of aquaporin-4 in dystrophin-null transgenic mice PNAS, October 1, 2002; 99(20): 13131 - 13136. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
