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Aquaporins in skeletal muscle: reassessment of the functional role of aquaporin-4¹

ANTONIO FRIGERI^*,2, GRAZIA PAOLA NICCHIA^*, ROSALBA BALENA^*, BEATRICE NICO and MARIA SVELTO^*

^* Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, Bari, Italy; and
Department of Human Anatomy, University of Bari, Bari, Italy

²Correspondence: 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

Aquaporin-4 (AQP4) is the major water channel of the neuromuscular system but its physiological function in perivascular astrocytes and skeletal muscle sarcolemma is unclear. The purpose of the present study was to assess in skeletal muscle 1) the expression of all cloned water channels; 2) the functional role of AQP4 using sarcolemma vesicles purified by means of several fractionation methods; 3) the functional effect of AQP4 reduction in mdx mice, the animal model of Duchenne muscular dystrophy (DMD).

PRINCIPAL FINDINGS

1. Two aquaporins (AQPs) are expressed in mouse skeletal muscle
To determine the AQP expression pattern in mouse skeletal muscle, we initially performed immunolocalization and Western blot experiments using affinity purified antibodies for each of the known AQPs. By immunofluorescence, specific staining was found only for AQP1 and AQP4. In agreement with previous studies, AQP1 was detected on endothelial cells of capillaries arranged between muscle fibers whereas AQP4 antibodies were found to label the entire plasma membrane of fast fibers. No specific staining was found using AQP0, AQP2, AQP3, AQP5, AQP6, AQP7, AQP8, and AQP9 antibodies. Immunoblot analysis confirmed the presence of a 30 kDa band corresponding to AQP4 protein and a 28 kDa band corresponding to AQP1 protein.

2. AQP4 expression in skeletal muscle sarcolemma is associated with high water permeability
To analyze the physiological role of AQP4 in skeletal muscle sarcolemma, we purified a plasma membrane fraction using two protocols. We compared the protocol used to isolate the dystrophin glycoprotein (DGP) complex with the one used by other groups to assess AQP4 function in skeletal muscle using KO mice. The first protocol yields a light microsome (LM) fraction, the second four sucrose membrane vesicle fractions (F1-F4). Figure 1 A illustrates immunoblot results of experiments carried out on samples of the four sucrose gradient fractions compared with those obtained using the sarcolemma (LM) fraction. A weak AQP4 signal was detected in all sucrose gradient fractions (F1-F4), with no significant differences between them. Low levels of ß-dystroglycan, used as sarcolemma markers, were detected in all the sucrose fractions. In contrast, ß-dystroglycan and the other markers were highly detected in the LM fraction obtained using the classic protocol to purify sarcolemma fractions (Fig. 1A ). Densitometric analysis performed using ß-dystroglycan levels indicates that the F1 fraction contains <10% of the sarcolemma membrane proteins of that found in the LM fraction.

View larger version (61K): [in this window] [in a new window]   Figure 1. Immunoblot and functional analysis of rabbit skeletal muscle sarcolemma vesicles. A) Top: Coomassie blue-stained PVDF membrane containing the four sucrose gradient fractions (F1-F4) obtained with the protocol of Yang et al. and the LM fraction obtained with the protocol of Ohlendieck et al. Blots were labeled with AQP4 (middle) and ß-dystroglycan (ß-DG, bottom) antibodies. B) Time course of scattered light intensity in response to a 50 mOsm sucrose gradient at 12°C. The osmotic gradient causes water efflux, vesicle shrinkage, and an increase in scattered light intensity. Top panel: representative curves are shown for F1 and LM vesicles (in 1 set of experiment typical of 3). Bottom panel, data are mean values +/– SE from 3 independent vesicle preparations. *P < 0.001 compared with F1-F4 vesicles.

To analyze the osmotic water permeability of skeletal muscle sarcolemma, we performed stopped-flow light scattering measurements on vesicle preparations obtained using two different procedures. Vesicles were exposed to an inwardly directed osmotic gradient created by sucrose and the time course of the scattered light intensity was measured. Kinetic of osmotic vesicle swelling was much faster for the LM fraction than that of the F1-F4 fractions of the sucrose gradient (Fig. 1B ). The osmotic permeability coefficient P_f calculated from fitting of the data to exponential function and from the vesicle diameter (130+/– 12 nm for LM vesicles and 100 +/– 15 nm for F1 vesicles) was 156 µm/s for the LM preparation and 43 µm/s for the F1 fraction. The high P_f in LM fraction is typical of membranes containing water channels whereas the P_f value in the F1 vesicle preparation indicates the absence of a water channel pathway, in agreement with the different levels of sarcolemma vesicles and AQP4 present in the two fractions.

3. Mdx mice sarcolemma vesicles show reduced osmotic water permeability
We recently reported that AQP4 expression decreased in skeletal muscle of mdx mice and DMD patients. Myotubes from mdx mice have been shown to be more sensitive to osmotic stress than that of normal myotubes. To determine whether AQP4 reduction in mdx mice results in decreased sarcolemma water transport, LM vesicles were prepared using the procedure described above. As illustrated in Fig. 2 A, the expression level of AQP4 was found to be drastically reduced in mdx mice. Quantitative analysis by densitometry revealed that the total content of AQP4 protein decreased by 90%. The muscular dystrophin isoform (DP427) was absent from mdx muscle LM, in agreement with previous studies. Caveolin-3 expression levels were strongly increased in mdx mouse muscle, in agreement with previous reports. Electron microscopy images of the LM fraction indicate a homogeneous population of vesicles (Fig. 2B ) in wild-type and mdx samples, with no contamination by mitochondria and rough endoplasmic reticulum.

View larger version (68K): [in this window] [in a new window]   Figure 2. Analysis of mdx sarcolemma vesicles. A) Immunoblot analysis of LM fraction from control (WT) and mdx skeletal muscles using AQP4, dystrophin, ß-dystroglycan, and caveolin-3 antibodies. B) Electron microscopy micrographs of LM vesicles from mdx and wild-type (WT) mice. Note the homogeneous size of vesicles and the absence of contamination by mitochondria and ER. Original magnification: x20,000. C) Time course of scattered light intensity in response to a 100 mOsm sucrose gradient at 12°C. Representative curves for LM vesicles of mdx and WT. D) Pf data are mean values +/– SE from 3 independent vesicle preparations. *P < 0.001.

Water transport measurements performed by stopped-flow light scattering from plasmalemma vesicles of mdx mice subjected to an inward osmotic sucrose gradient displayed a slower shrinkage rate than that of wild-type LM vesicles (Fig. 2C ). Computation of the P_f value from the fitted data indicates that the water permeability of mdx vesicles is reduced by 70% compared with wild-type mice, consistent with the reduction in AQP4 expression.

CONCLUSIONS

It is well established that fast fluid shift occurs during skeletal muscle stimulation. These studies demonstrated that volume changes of muscle cells occurring during mechanical activity are consequences of water influx due to osmolite production. Thus, rapid water transport seems to have a physiological role in contraction-induced muscle swelling.

Using affinity purified antibodies, we found that only two AQPs are expressed in skeletal muscle, AQP1 and AQP4. AQP1 was found in endothelial cells of continuous capillaries and AQP4 was found on the plasma membrane of muscle fiber. For rapid water transport between blood and muscle fibers, water channels need to be expressed on the side where water will encounter two series limiting barriers: the endothelial cell membrane and the sarcolemma. Thus, AQP1 and AQP4 represent the endothelial membrane and the sarcolemma water channel pathways, respectively. These two water channels would function in cooperation to determine high water transport between the blood and myofibrils during muscle activity.

The physiological role of AQP4 in skeletal muscle has been questioned by the results obtained using AQP4 null mice. No differences in water transport and muscle function have been found between wild-type and AQP4 knockout mice, indicating that AQP4 contribution to sarcolemma water transport is negligible.

We found that the procedure used by Yang and collaborators to purify sarcolemma vesicles was inadequate for skeletal muscle plasma membrane fractionation (see Fig. 1 ). Their protocol is a modification of a previous procedure reported by Grimditch and colleagues to isolate sarcolemma vesicles for glucose transport studies. However, these modifications transformed the original protocol into a completely different procedure that resembles the one used to purify plasma membranes from CHO cells. The procedure missed important steps, such as KCl extraction, essential for sarcolemma purification with minimal contamination of contractile proteins.

We studied AQP4 mediated water transport in skeletal muscle sarcolemma by means of the method commonly used to purify and characterize proteins of the plasma membrane associated with dystrophin. This procedure allowed us to purify an enriched sarcolemma fraction (LM) as confirmed by the expression of high levels of membrane proteins, including AQP4. In contrast, the plasma membrane content in the vesicle preparation obtained with the Yang method represents only a minor fraction. Water transport studies demonstrated high water permeability of LM vesicles, consistent with AQP-mediated pathway for water movement. In fact, the P_f value was >twofold higher than that found from small intestine (60 µm/s) membrane vesicles, hepatocyte basolateral (67 µm/s at 12°C) and canalicular (57 µm/s at 12°C) plasma membrane vesicles. These data indicate that AQP4 in skeletal muscle sarcolemma is functionally expressed as a water channel protein and provides a molecular explanation for numerous studies that show the occurrence of fast water transport in muscle during mechanical activity.

The close correspondence between AQP4 expression and water transport is indicated by studies in the mdx mouse. Sarcolemma vesicles prepared from mdx mice had reduced AQP4 levels and lower P_f compared with wild-type mice. Although we cannot deny with certitude that other affected sarcolemma proteins may contribute to determine the low P_f observed in mdx sarcolemma vesicles, the drastic reduction of AQP4 protein determined by immunoblot and the known highest water transport capacity of AQP4 strongly suggest that AQP4 disappearance from the plasma membrane is the cause of the reduced water permeability. These results provide a rationale for the findings that dystrophin deficient muscle fibers and myotubes exhibit osmotic fragility and that AQP4 plays a role in the pathogenesis of muscular dystrophy (Fig. 3 ).

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic diagram of the AQP4 possible role in normal and dystrophic skeletal muscle.

In conclusion, the results support the hypothesis that AQP4 plays an important role in the high water permeability of the plasmalemma of fast twitch fibers. Sarcolemmal AQP4 together with the vascular AQP1 may be responsible for the fast water transfer from the blood to the muscle during intense activity. These data imply an important role for aquaporins in skeletal muscle physiology as well as an involvement of AQP4 in molecular alterations occurring in the muscle of DMD patients.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0987fje

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