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Muscle loading modulates aquaporin-4 expression in skeletal muscle¹

ANTONIO FRIGERI^*,2,3, GRAZIA PAOLA NICCHIA^*,2, JEAN-FRANÇOIS DESAPHY, SABATA PIERNO, ANNAMARIA DE LUCA, DIANA CONTE CAMERINO and MARIA SVELTO^*

^* Department of General and Environmental Physiology and
Unit of Pharmacology, Department of Pharmacobiology, University of Bari, I-70126 Bari, Italy

SPECIFIC AIMS

The purpose of this investigation was to determine the effect of hindlimb suspension (HS) on the expression of aquaporin-4 in rat soleus. We used this model of muscle disuse to determine 1) whether AQP4 might be regulated in skeletal muscle depending on the changes in muscle use and 2) whether its expression parallels the transition of muscle fibers, particularly when a slow fiber turns fast.

PRINCIPAL FINDINGS

1. Hindlimb suspension determines slow to fast conversion of soleus muscle fibers
Unloading of rats for up to 21 days led to a progressive weight loss in the soleus muscle accompanied by changes to the pattern of MHC isoforms. The major effect of HS consisted of an increase (twofold) in fast MHC after 8 days of HS, concomitant with a similar decrease in the relative amount of slow MHC isoforms. This level did not significantly increase further after 14 and 21 days of tail suspension. This result confirmed the slow-to-fast twitch fiber conversion and shows that 8 days of tail suspension are sufficient to obtain the maximal increase in fast MHC isoform.

2. Immunofluorescence analysis reveals a consistent increase in AQP4/type IIA-expressing fibers after HS
To analyze AQP4 expression in rat soleus after tail suspension, we performed immunofluorescence experiments using AQP4-purified antibodies together with specific antibodies against types IIA, IIB, and type I MHC isoforms. After 8 days of HS, the number of fast-type IIA fibers increased more than twofold and remained at the same level after 14 and 21 days of suspension. In parallel, the number of type I fibers decreased after suspension. No type IIB fibers were detected in either control or hindlimb suspended soleus muscle. By double immunofluorescence experiments (Fig. 1A ), we found that the increase in AQP4 expression was strictly associated with the increase in type IIA fibers. After 8 days, the number of AQP4-expressing fibers increased by twofold. Quantitative/qualitative analysis demonstrated that all the new AQP4-expressing fibers also expressed type IIA MHC isoforms and that the number of AQP4 positive as well as type IIA fibers did not change after 2 and 3 wk of suspension.

View larger version (95K): [in this window] [in a new window]   Figure 1. Analysis of the AQP4 expression in skeletal muscle after HS. A) Double immunofluorescence analysis of AQP4 and type IIA MHC isoform after HS. Experiments were performed on soleus from control and 1, 2, and 3 wk hindlimb suspended rats (Ctrl, 1 w, 2 w, and 3 w HS, respectively). Top: cross sections stained with antibodies against type IIA MHC isoform. Bottom: the same sections stained with AQP4 antibodies. B) AQP4 immunoprecipitation from control and 3 wk hindlimb suspended rat soleus. After immunoprecipitation, the sample was analyzed by Western blot. Densitometric analysis of AQP4 bands from control (Crl) and 3 wk hindlimb suspended (3 w HS). Negative control (N C). C) AQP4 mRNA analysis by semiquantitative RT-PCR experiments. Soleus muscles of control (Crl) and 1, 2, and 3 wk hindlimb suspended rats (1 w, 2 w, and 3 w HS) were analyzed. The 18S rRNA (488 bp) used as internal standard was coamplified with the specific fragment of AQP4 mRNA (310 bp). Note the strong increase of AQP4 mRNA in hindlimb suspended rats compared with control. St: 1 kb DNA ladder.

3. The increase in AQP4-expressing fibers is due to an increase in AQP4 protein content
Immunoprecipitation experiments were subsequently performed to determine whether the increased AQP4 immunofluorescence signal reflects an increase in AQP4 protein content or secondary conformational modifications of AQP4 protein (Fig. 1B ). Soleus from 3 wk hindlimb suspended rats and matched controls were used and the content of AQP4 protein was determined. Densitometric analysis of the immunoprecipitated protein revealed that compared with control soleus, HS suspension determined a twofold increase in AQP4 protein, indicating that HS determines an increase in AQP4 protein expression.

4. AQP4 expression is regulated pretranslationally
To test whether AQP4 expression is regulated pretranslationally, changes at the AQP4 mRNA levels were measured by semiquantitative RT-PCR experiments. Experiments were carried out at 8, 14, and 21 days of HS to assess the relative expression of AQP4 by comparison with control (Fig. 1C ). Results showed that AQP4 mRNA up-regulation peaked at 8 days of HS and remained stable at 14 and 21 days. This experiment demonstrates that AQP4 mRNA was strongly induced in the HS rat.

5. AQP4 sarcolemma expression requires the concomitant expression of type IIA MHC isoform
To further examine the temporal correlation between AQP4 expression and the appearance of type IIA MHC protein, we analyzed soleus from 4-day-old HS rats. Double immunofluorescence experiments were performed using serial sections. Many fibers were found to coexpress slow MHC and fast-type IIA MHC proteins at different expression levels (Fig. 2 ). The analysis of these transitional fibers revealed that AQP4 sarcolemma expression appeared in those fibers that expressed even low levels of type IIA MHC, indicating that AQP4 expression is temporally strictly related with the expression of type IIA MHC.

View larger version (57K): [in this window] [in a new window]   Figure 2. Expression of AQP4 in transitional fibers. Double immunofluorescence experiments performed on two consecutive cross sections of soleus from rat suspended for 4 days. AQP4 is in red, type IIA MHC isoform in green, and type I MHC isoform in blue. Note that some AQP4-positive fibers coexpress both type IIA and I MHC isoforms at different levels of expression.

CONCLUSIONS

This study demonstrates that AQP4 gene expression in skeletal muscle is acutely up-regulated during adaptive changes induced by muscle unloading. Previous studies reported that only very few genes other than myosin isoforms were found to be up-regulated. Our results show that the absence of a mechanical stimulus positively modulates the expression of AQP4 protein in the sarcolemma of soleus muscle fibers, indicating that AQP4 is involved in the adaptation mechanism of muscle unloading.

Another interesting finding of the present study is that long-term AQP4-regulated expression is associated with slow- to fast-twitch conversion of soleus muscle fibers (Fig. 3 ). As previously reported and confirmed by this study, HS unloading results in muscle atrophy and reduces muscle regeneration, suggesting that the overexpression of AQP4 in soleus was not a phenomenon involving new generated fibers but occurred in preexisting fibers that maintained their integrity and transformed during the suspension period. This indicates that the ability to modulate the expression of AQP4 in nonregenerating muscle fibers is strictly linked to a transformation from type I to type II fibers. Up-regulation of AQP4 mRNA transcript and protein indicates that it is a component involved in fast-type transformation of soleus muscle and correlates with the glycolytic energetic metabolism (Fig. 3) . Recent studies have demonstrated that the glycolytic pathway is selectively activated in unweighted soleus muscle, determining the specific induction of some genes as well others that are indirectly involved. This phenomenon determines changes in the substrate profile toward a greater utilization of glycogen, which causes accumulation of lactate and increases in fatigability. This confirms our idea that the expression of AQP4 is associated with the glycolytic metabolism of the fiber and that its functional role may be linked to muscle fatigue.

View larger version (58K): [in this window] [in a new window]   Figure 3. Schematic diagram of the processes occurring during hindlimb suspension that lead to AQP4 over-expression.

Unloading by HS suspension causes MHC adaptations in the rat soleus similar to the effect of microgravity occurring during space flight and over prolonged periods of time without weight bearing, such as those associated with bed rest. Since these modifications also occur during normal muscle activity, skeletal muscle fibers are capable of adjusting their phenotypic properties to an altered functional demand. This muscle plasticity relates to the expression of many muscle proteins; the fact that AQP4 expression can be modulated indicates that AQP4 is a component of the ensemble of muscle protein involved in muscle plasticity.

Skeletal muscle is a tissue of central importance in the osmotic equilibrium of the human body, and rapid fluid shifts occur in skeletal muscle after intense muscle use. Thus, the role of AQP4 in the sarcolemma may be to move water into stimulated muscle cells at high rates.

In many pathological situations such as hemorrhage, hypertonic fluid resuscitation, or hyperglycemia, acute increases in plasma osmolality occur. In these situations, the maintenance of plasma volume is crucial and water is absorbed from skeletal muscle. Hence, the presence of AQP4 would be important to determine rapid water flux into the hypertonic blood and thus to quickly reestablish the plasma volume.

In contrast to this view is a recent study reporting that transgenic AQP4-deficient mice did not manifest significant abnormalities in skeletal muscle water transport and function. The authors concluded that the expression of AQP4 in skeletal muscle may represent a residual sarcolemma protein that is no longer important for muscle function. Our data strongly clash with this hypothesis, since it is highly unlikely that an ancient protein no longer used for muscle function can be regulated and involved in the biochemical modifications occurring during muscle fiber transition that reflect the dynamic remodeling of a muscle.

In conclusion, our results show that AQP4 expression in skeletal muscle can be subjected to regulation depending on the functional demand. Moreover, its expression is temporally associated with the transition from the slow phenotype to the fast and thus to the glycolytic metabolism of the fiber.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0525fje ; to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0525fje

² These authors contributed equally to this study.

³ Correspondence: 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

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