From the a Departments of Molecular Pharmacology, h Pathology, and i Cell Biology and the b Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461 and the f Servizio Malattie Neuro-Muscolari, Università di Genova, Istituto Gaslini, Largo Gaslini 5, 16147 Genova, Italy
Received for publication, January 29, 2001
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ABSTRACT |
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Caveolin-3, a muscle-specific caveolin-related
protein, is the principal structural protein of caveolae membrane
domains in striated muscle cells. Recently, we identified a novel
autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C) in
humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an ~95% reduction in caveolin-3 protein expression, i.e. a
caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 Caveolae are 50-100-nm vesicular invaginations of the plasma
membrane that participate in vesicular trafficking events and signal
transduction processes (1-5). Caveolin, a 21-24-kDa integral membrane
protein, is a principal component of caveolae membranes in
vivo (6-10). Caveolin is only the first member of a new gene family; as a consequence, caveolin has been re-termed caveolin-1 (11).
The mammalian caveolin gene family now consists of caveolin-1, -2, and
-3 (3, 11-13). Caveolin-1 and -2 are co-expressed and form a
hetero-oligomeric complex (14) in many cell types, with particularly
high levels in adipocytes, whereas expression of caveolin-3 is
muscle-specific and found in both cardiac and skeletal muscle, as well
as smooth muscle cells (15). Expression of caveolin-3 is induced during
the differentiation of skeletal myoblasts, and caveolin-3 is localized
to the muscle cell plasma membrane (sarcolemma), where it forms a
complex with dystrophin and its associated glycoproteins (15). It has
been proposed that caveolin family members function as scaffolding
proteins (16) to organize and concentrate specific lipids (cholesterol and glycosphingolipids; Refs. 17-19) and lipid modified signaling molecules (Src-like kinases, Ha-Ras, endothelial nitric-oxide synthase,
and G-proteins; Refs. 17 and 20-24) within caveolae membranes.
Caveolin-3 is most closely related to caveolin-1 based on protein
sequence homology; caveolin-1 and caveolin-3 are ~65% identical and
~85% similar (13). However, caveolin-3 mRNA is expressed predominantly in muscle tissue types (skeletal muscle, diaphragm, and
heart) (13). Identification of a muscle-specific member of the caveolin
gene family has implications for understanding the role of caveolins in
different muscle cell types, as previous morphological studies have
demonstrated that caveolae are abundant in these cells. This indicates
that muscle cell caveolae may play an important role in muscle membrane biology.
Tight regulation of caveolin-3 expression appears essential for
maintaining normal muscle homeostasis, as we have demonstrated that
transgenic overexpression of wild-type
(WT)1 caveolin-3 in mouse
skeletal muscle fibers induces a Duchenne-like muscular dystrophy
phenotype (25). Analysis of skeletal muscle tissue from transgenic mice
overexpressing caveolin-3 revealed: (i) a dramatic increase in
sarcolemmal caveolae; (ii) hypertrophic, necrotic, and regenerating
skeletal muscle fibers with central nuclei; and (iii) down-regulation
of dystrophin and One possibility is that overexpression of wild-type caveolin-3 disrupts
the normal processing or stoichiometry of the dystrophin complex,
leading to its degradation. In support of this hypothesis, we have
recently demonstrated that a novel WW-like domain within caveolin-3
directly recognizes the extreme C terminus of In collaboration with Minetti and colleagues (27), we have identified
an autosomal dominant form of limb-girdle muscular dystrophy (LGMD-1C)
in two Italian families that is due to a deficiency in caveolin-3
expression. Analysis of their genomic DNA reveals two distinct
mutations in caveolin-3: (i) a 9-base pair microdeletion that removes
the sequence TFT from the caveolin scaffolding domain, and (ii) a
missense mutation that changes a proline to a leucine (Pro Using heterologous expression in cultured cells, we have recently
demonstrated that LGMD-1C mutants of caveolin-3 behave in a
dominant-negative fashion, causing the intracellular retention and
degradation of wild-type caveolin-3 via the proteasome system (28).
Interestingly, treatment with proteasomal inhibitors blocks the
dominant negative effect of LGMD-1C mutants and rescues wild-type caveolin-3 (29).
Here, using a gene targeting approach, we generated mice lacking
caveolin-3 protein expression (CAV3 Materials--
Antibodies and their sources were as follows:
anti-caveolin-3 IgG (mAb 26 (15); gift of Dr. Roberto Campos-Gonzalez,
Transduction Laboratories, Inc.); anti-caveolin-1 IgG (mAb 2297 (30);
gift of Dr. Roberto Campos-Gonzalez); anti-caveolin-2 IgG (mAb 65 (14); gift of Dr. Roberto Campos-Gonzalez); anti- Assembly of the Caveolin-3 Construct for Targeted Gene
Disruption--
Briefly, an ~200-kb BAC clone containing murine
caveolin-3 was previously isolated using the murine caveolin-3 cDNA
as a probe for colony hybridizations (31). An 8-kb BamHI
subclone containing the second exon of caveolin-3 was isolated and
cloned into the BamHI site of Bluescript (pBS SK+). This was
used for the 3' end of the construct. From this clone, a 4.0-kb
XbaI/BglII fragment was isolated and cloned into
pCB7. This fragment was isolated by double digestion with
XbaI and SacI and cloned into the PGT-N29 vector
(New England Biolabs, Inc.) that contains the neomycin resistance gene.
From the original BAC clone, a 10-kb KpnI subclone was
isolated and cloned into Bluescript (pBS SK+). This clone contains exon
2 of caveolin-3 and was used for the 5' end of the construct. An
internal 4.4-kb BamHI fragment was subcloned into Bluescript, which then was liberated with EcoRI (yielding a
4.2-kb fragment) and subcloned into pGT-N29 that already possessed the 3' end of the construct. The final knockout construct was linearized with XhoI.
Derivation of ES Cell Clones Harboring a Disrupted Caveolin-3
Gene--
After electroporation of the WW6 ES cell line (gift of Dr.
Pamela Stanley) with the linearized DNA containing the caveolin-3 K/O
construct, G418-resistant clones were selected for analysis. Homologous
recombination was detected by Southern blot analysis. Genomic DNA from
approximately 1000 G418-resistant ES cell clones was prepared. For each
clone, ~10 µg of genomic DNA was digested with EcoRI and
separated on an 0.8% agarose gel. The DNA was transferred to
nitrocellulose and probed with a 1.5-kb BglII fragment that is 3' to the knockout construct. Homologous recombination was detected
by the presence of a ~8.5-kb band (K/O band) in addition to a
~13-kb band (WT band). This is due to the introduction of an
additional EcoRI site into the caveolin-3 locus via
homologous recombination (see Fig. 1).
Generation of Caveolin-3 Knock-out Mice--
We injected
positive ES cell clones into blastocysts in order to obtain chimeric
mice with germline transmission of the caveolin-3 gene disruption. We
next back-crossed the resulting heterozygote mice at least three times
with C57BL/6 mice (Jackson Laboratories). Homozygous null mice were
identified by Southern blot analysis by the presence of the ~8.5-kb
band (K/O band) and the absence of the ~13-kb band (WT band). In
addition, homozygous mice were identified by PCR analysis using primers
specific for the neomycin resistance gene and exon-2 of caveolin-3.
Amplification of only the neomycin resistance gene, but not
caveolin-3/exon-2, indicated that the mice were homozygous null. CAV3
+/+ (WT) and CAV3 Immunoblot Analysis--
Mouse tissues were harvested, minced
with a scissors, homogenized in a Polytron tissue grinder for 30 s
at a medium range speed, and solubilized in a buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 60 mM octyl glucoside for 45 min at 4 °C. In addition, samples were centrifuged at 13,000 × g for 10 min at 4 °C to remove
insoluble proteins. Soluble proteins were resolved by SDS-PAGE (12.5%
or 8% acrylamide) and transferred to BA83 nitrocellulose membranes
(0.2 µm, Schleicher & Schuell). Blots were incubated for 2 h in
TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0, 2% Tween 20) containing 2% powdered skim milk and 1% bovine serum
albumin. After three washes with TBST, membranes were incubated for
2 h with the primary antibody (~1,000-fold diluted in TBST) and
for 1 h with horseradish peroxidase-conjugated goat
anti-rabbit/mouse IgG (~5,000-fold diluted). Bound antibodies were
detected using an ECL detection kit (Amersham Pharmacia Biotech).
Histological and Histochemical Analyses--
Muscle tissue
sections were subjected to hematoxylin/eosin staining, essentially as
described (32).
Immunostaining of Murine Skeletal Muscle Tissue
Sections--
Samples were isolated from the extensor digitorum
longus, the soleus, and the gastrocnemius muscle, rapidly frozen in
liquid nitrogen-cooled isopentane, sectioned, and stored in liquid
nitrogen. Unfixed serial sections (4 µm thick) of frozen
muscle were incubated with a given primary antibody for 1 h at
room temperature (~1000-fold diluted in PBS with 0.1% Triton X-100,
0.2% bovine serum albumin). After three washes with PBS (10 min each),
sections were incubated with the secondary antibody for 1 h at
room temperature: lissamine rhodamine B sulfonyl chloride-conjugated
goat anti-rabbit antibody (5 µg/ml)/fluorescein
isothiocyanate-conjugated goat anti-mouse antibody (5 µg/ml).
Finally, the sections were washed three times with PBS (10 min each
wash), and slides were mounted with Slow-Fade anti-fade reagent
(Molecular Probes, Inc., Eugene, OR) and observed under a Bio-Rad MR
600 confocal microscope. Three-dimensional reconstructions were
performed using National Institutes of Health Image software.
Transmission Electron Microscopy--
Mouse skeletal muscle
tissue samples were fixed with glutaraldehyde, post-fixed with
OsO4, and stained with uranyl acetate and lead citrate.
Samples were examined under a JEOL 1200EX transmission electron
microscope and photographed (magnification, ×25,000) (33-35).
Caveolae were identified by their characteristic flask shape, size
(50-100 nm), and location at or near the plasma membrane (36).
T-tubule System Staining--
T-tubule system staining was
performed as described previously (37). Briefly, mouse skeletal muscle
tissue samples were fixed in 2% paraformaldehyde, 2.5%
glutaraldehyde, 0.1 M cacodylate, and 50 mM
CaCl2, pH 7.4. Samples were post-fixed with 2%
OsO4, 0.8% K3Fe(CN)6, followed by
incubation with saturated uranyl acetate. Samples were dehydrated in a
graded series of ethanol and embedded in LX112 (Ladd Research
Industries). Sections were cut on Reichert UCT ultramicrotome and
examined under a JEOL 1200EX transmission electron microscope and
photographed (magnification, ×12,000).
Preparation of Caveolae-enriched Membrane Fractions--
Mouse
skeletal muscle tissue was harvested, minced with a scissors, and
homogenized in 2 ml of Mes-buffered saline containing 1% (v/v) Triton
X-100. Homogenization was carried out with a Polytron tissue grinder
for 30 s at a medium range speed at 4 °C. Samples were
centrifuged at low speed for 5 min at 4 °C, and the supernatant was
adjusted to 40% sucrose by the addition of 2 ml of 80% sucrose prepared in Mes-buffered saline and placed at the bottom of an ultracentrifuge tube. A 5-30% linear sucrose gradient was formed above the homogenate and centrifuged at 39,000 rpm for 16-20 h in a
SW41 rotor (Beckman Instruments). A light scattering band confined to
the 15-20% sucrose region was observed that contained endogenous
caveolin-3, but excluded most of other cellular proteins. From the top
of each gradient, 1-ml gradient fractions were collected to yield a
total of 12 fractions. An equal amount of protein from each gradient
fraction was separated by SDS-PAGE and subjected to immunoblot analysis.
Generating Caveolin-3 Null Mice via Homologous
Recombination--
A BAC clone containing murine caveolin-3 was
previously isolated using the murine caveolin-3 cDNA as a probe for
colony hybridizations, and its intron/exon organization was determined;
the caveolin-3 gene consists of only two coding exons (31). The
knockout construct for murine caveolin-3 was designed to replace exon-2
of caveolin-3 with the neomycin resistance gene cassette, but in the
opposite transcriptional orientation (Fig.
1A). Importantly, exon-2 of caveolin-3 encodes the bulk of the caveolin-3 protein and all of its
functional domains (13, 31).
After electroporation of the WW6 ES cell line (38) with the linearized
DNA containing the caveolin-3 K/O construct, G418-resistant clones were
selected for analysis. Homologous recombination was detected by
Southern blot analysis (Fig. 1B). Next, positive clones were
injected into blastocysts in order to obtain chimeric mice with
germline transmission of this caveolin-3 gene disruption. We next
crossed the resulting heterozygote mice (CAV3 +/
Homozygous null mice were identified by the presence of the ~8.5-kb
band (K/O band) and the absence of the ~13-kb band (WT band) on
Southern blots (Fig. 1C). In addition, homozygous null mice
were identified by PCR analysis using primers specific for the neomycin
resistance gene and exon-2 of caveolin-3. Amplification of only the
neomycin resistance gene, but not caveolin-3/exon2, indicated that the
mice were homozygous null (Fig. 1D).
CAV3 (
As the first exon of caveolin-3 was not removed by our recombination
strategy, we also analyzed the possible expression of Cav-3/exon-1 in
these mice. Cav-3/exon-1 (114 base pairs) is predicted to encode a
truncated protein that contains residues 1-38 of caveolin-3, which do
not contain any of the known functional domains of the caveolins. All
of the functional domains of caveolin-3 are contained within exon-2
(residues 39-151), which has been replaced by our cloning strategy.
Importantly, the monospecific mAb probe we used to detect caveolin-3
protein expression is directed against the unique N terminus of
caveolin-3 (residues 3-24) (15). Therefore, this antibody would
recognize a truncated Cav-3/exon-1 protein, if it was expressed.
However, smaller truncated forms of the caveolin-3 protein were not observed.
Next, skeletal muscle tissue sections from WT and
caveolin-3 null mice were immunostained with anti-caveolin-3 IgG. Fig.
2B shows that caveolin-3 is correctly expressed at the
sarcolemma in WT mice, but is clearly absent in caveolin-3 null mice.
Recombinant expression of caveolin-3 in cultured cells is sufficient to
drive caveolae formation (39). As caveolin-3 is the only member of the
caveolin gene family expressed in striated muscle cells, caveolin-3 is
thought to be responsible for caveolae formation in skeletal muscle
cells in vivo. In fact, transgenic overexpression of
caveolin-3 in skeletal muscle fibers dramatically increases the number
of plasmalemmal caveolae (25). Thus, we would predict that loss of
caveolin-3 expression in caveolin-3 null mice would ablate sarcolemmal
caveolae formation.
As predicted, electron microscopic analysis of skeletal muscle fibers
revealed an absence of sarcolemmal caveolae in caveolin-3 null mice
(Fig. 2C, upper panels). Importantly,
caveolae were still present in endothelial cells from caveolin-3 null
mice (Fig. 2C, lower panels). This is
consistent with our previous observations demonstrating that
endothelial cells express only caveolin-1 and -2, but not caveolin-3
(14).
Histological Analysis of Muscle Tissues from Caveolin-3 Null Mice
Reveals Mild Myopathic Changes--
Interestingly, caveolin-3 null
mice did not show an overt "clinical" phenotype. In order to
identify a phenotype associated with loss of caveolin-3 expression in
caveolin-3 null mice, tissue sections from these mice were
hematoxylin/eosin-stained and examined by light microscopy.
Interestingly, no pathological changes were observed, with the
exception of skeletal muscle tissue (Fig.
3; data not shown). Importantly, several
pathologists carefully assessed the other tissues (including the heart)
where caveolin-3 is endogenously expressed and did not observe any
noticeable pathologic changes.
Hematoxylin/eosin staining of skeletal muscle tissue sections from
caveolin-3 null mice revealed only mild myopathic changes (Fig. 3).
These changes included variability in the size of the muscle fibers and
the presence of necrotic fibers. In addition to muscle biopsies, serum
creatine kinase levels are used clinically to diagnose muscular
dystrophy. As creatine kinase is a cytosolic muscle enzyme, elevated
serum creatine kinase levels indicate lysis or necrosis of muscle
fibers, with a subsequent release of the enzyme into the blood.
Interestingly, caveolin-3 null mice showed sporadic elevations (an
~3-4-fold increase) in serum creatine kinase activity (data not
shown), consistent with the skeletal muscle fiber degeneration observed
in tissue sections (Fig. 3).
Importantly, the mild muscle damage we observed in the caveolin-3 null
mice is very similar to the findings we described earlier for human
LGMD-1C, where caveolin-3 expression is reduced by ~90-95% (27).
Taken together, these results indicate that a deficiency in caveolin-3
expression (CAV3 The Dystrophin-Glycoprotein Complex Is Excluded from
Detergent-resistant Membrane Microdomains in Caveolin-3 Null
Mice--
As caveolin-3 is found associated with dystrophin and
dystrophin-associated glycoproteins at the level of the sarcolemma (15, 40), one possibility is that loss of caveolin-3 affects the level of
expression and/or the localization of dystrophin and dystrophin-associated glycoproteins. To test this hypothesis, we next
examined the level of expression and localization of these proteins in
caveolin-3 null mice.
Western blot analysis of lysates prepared from skeletal muscle tissue
revealed that caveolin-3 null mice expressed normal levels of
dystrophin,
We and others have shown previously that dystrophin and its associated
glycoproteins co-purify with caveolin-3 and are normally targeted to
cholesterol-sphingolipid raft domains/caveolae (15, 26, 40, 41). Next,
we evaluated if dystrophin and dystrophin-associated glycoproteins were
enriched in cholesterol-sphingolipid-rich raft domains in WT and
caveolin-3 null mice. When caveolin-3 is expressed, it is targeted to
these lipid rafts and induces the formation of morphological caveolae
(39), as caveolins are cholesterol-binding proteins (5, 42). However,
cholesterol-sphingolipid rafts exist in the absence of caveolin protein
expression (30, 33, 43). Interactions between cholesterol and
shingolipids make these plasma membrane microdomains resistant to
non-ionic detergents at low temperatures, thereby facilitating their
rapid purification (5, 44, 45).
Cholesterol-sphingolipid rafts/caveolae were purified using an
established equilibrium sucrose density gradient system that separates
these detergent resistant membranes from the bulk of cellular membranes
and cytosolic proteins (see "Experimental Procedures") (20, 30, 33,
34, 46-52). In this fractionation scheme, immunoblotting with
anti-caveolin-3 IgG can be used to track the position of
caveolae-derived membranes within these bottom-loaded sucrose gradients
(13, 15). These caveolae-enriched membranes (fractions 4-6) exclude
>99.95% of total cellular proteins and also markers for non-caveolar
plasma membrane, Golgi, lysosomes, mitochondria, and endoplasmic
reticulum (retained in fractions 8-12) (33, 34, 48).
Fig. 4C shows that dystrophin,
Thus, we conclude that caveolin-3 protein expression is required for
the correct targeting of the dystrophin-glycoprotein complex to
cholesterol-sphingolipid raft domains/caveolae in normal muscle fibers.
These results are consistent with our recent observation that
caveolin-3 interacts directly with the dystrophin-glycoprotein complex
by recognizing a PPXY motif in the C-terminal tail of Caveolin-3 Deficiency Is Associated with Abnormalities of the
T-tubule System--
Caveolin-3 has been shown to be transiently
associated with the T-tubule system during skeletal muscle development
(53). However, it remains unknown whether caveolin-3 expression is
required for or greatly facilitates the proper development of the
T-tubule system. To test this hypothesis, we analyzed the protein
expression and localization of two well known T-tubule markers,
dihydropyridine receptor-1
However, the localization of dihydropyridine receptor-1
In striking contrast, dihydropyridine receptor-1
In order to further explore the organization of the T-tubule system in
detail, we next employed a well established method to specifically
stain the T-tubule system so that it can be visualized by electron
microscopy, i.e. the potassium ferrocyanate method (see
"Experimental Procedures") (37). Fig.
7A shows that T-tubules have
an orderly transverse orientation in WT mice (left
panel), as is expected. However, electron micrographs of the
longitudinal sections from caveolin-3 null mice indicate that the
T-tubules are dilated/swollen and run in irregular directions (Fig.
7A, right panel). Transmission
electron micrographs at a higher magnification better illustrate the
abnormal organization of the T-tubule system in caveolin-3 null mice
(Fig. 7B). Interestingly, T-tubules in a predominantly
longitudinal orientation are typical of immature muscle in normal
mice.
LGMD-1C is an autosomal dominant form of limb-girdle muscular
dystrophy that is genetically caused by mutations within the coding
regions of the caveolin-3 gene. In collaboration with Minetti and
colleagues (27), we recently identified two different Italian families
with this autosomal dominant form of limb-girdle muscular dystrophy
that is due to a deficiency in caveolin-3 expression. In these
patients, by quantitative immunofluorescence and Western blot analysis,
the levels of the caveolin-3 protein were reduced by ~90-95%.
Additionally, muscle biopsies from these patients showed muscle damage
of mild-to-moderate severity (27).
Here, we created a caveolin-3-deficient (CAV3 In patients with LGMD-1C, the level of expression and sarcolemma
localization of dystrophin and dystrophin-associated glycoproteins is
not affected by loss of caveolin-3 expression (27). Similarly, analysis
of skeletal muscle fibers from caveolin-3 null mice showed that the
expression levels and macroscopic localization of dystrophin, However, the precise distribution of dystrophin and its associated
glycoproteins within the sarcolemma in absence of caveolin-3 protein
expression has not been addressed. Here, we demonstrate that
dystrophin, In fully differentiated skeletal muscle fibers, caveolin-3 is
associated with sarcolemmal caveolae (53). However, early morphological
studies suggested that T-tubules form from the repeated budding of
caveolae (37, 54). In addition, caveolin-3 is transiently associated
with T-tubules during the differentiation of primary cultured cells and
the development of mouse skeletal muscle fibers (53). These results
suggest that a functional relationship may exist between caveolin-3
expression, caveolae formation, and T-tubule biogenesis. It remains
unknown whether caveolin-3 expression is required for proper T-tubule biogenesis.
Thus, we next assessed the status of the T-tubule system in caveolin-3
null mice. Here, we show that two T-tubule marker proteins (dihydropyridine receptor-1 In accordance with this interpretation, electron micrographs of
longitudinal sections from caveolin-3 null mice indicate that the
T-tubules are dilated/swollen and run in irregular directions (Fig. 7,
A and B). Interestingly, the T-tubule network has
an exclusively longitudinal orientation at early stages of muscle differentiation and only becomes transversely oriented in fully differentiated skeletal muscle fibers (37). As the T-tubule system in
caveolin-3 null mice showed a clear tendency to run longitudinally,
these results suggest that caveolin-3 expression and caveolae formation
are required to generate a highly organized/fully mature T-tubule
system in vivo. Thus, a disorganized immature T-tubule
system may also contribute to the pathogenesis of LGMD-1C in humans.
/
)
mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice
reveals: (i) mild myopathic changes; (ii) an exclusion of the
dystrophin-glycoprotein complex from lipid raft domains; and (iii)
abnormalities in the organization of the T-tubule system, with dilated
and longitudinally oriented T-tubules. These results have clear
mechanistic implications for understanding the pathogenesis of LGMD-1C
at a molecular level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystroglycan protein expression.
-dystroglycan that
contains a PPXY motif (26). As the WW domain of dystrophin recognizes the same site within
-dystroglycan, caveolin-3 can effectively block the interaction of dystrophin with
-dystroglycan in vitro (26), suggesting competitive regulation of the
recruitment of dystrophin to the sarcolemma in vivo.
Leu) in
the transmembrane domain (27). Both mutations lead to a loss of
~90-95% of caveolin-3 protein expression.
/
). Analysis of skeletal muscle
fibers from these caveolin-3 null mice reveals mild myopathic changes,
with a loss of sarcolemmal caveolae, that is consistent with what is
observed in patients with LGMD-1C. In addition, skeletal muscle fibers
from these caveolin-3 null mice are characterized by alterations in
targeting of the dystrophin-glycoprotein complex to lipid raft
microdomains and abnormalities in the organization of the T-tubule
system. These data suggest that mislocalization of the dystrophin
complex and abnormal T-tubule development may underlie the pathogenesis
of LGMD-1C in humans.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dystroglycan IgG (mAb, NCL-b-DG; Novocastra, Newcastle, United Kingdom);
anti-
-sarcoglycan (mAb, NCL-a-SARC; Novocastra); anti-dystrophin IgG
(mAb, NCL-DYS3; Novocastra); anti-DHPR-1
IgG (for
immunofluorescence, polyclonal antibody DHPR-1
N19 (Santa Cruz
Biotechnology); for Western blotting, mAb DHPR-1
D-218 (Sigma));
anti-ryanodine-R IgG (for immunofluorescence, polyclonal antibody RyR
N19 (Santa Cruz Biotechnology); for Western blotting, mAb RyR R-129
(Sigma)). All other biochemicals used were of the highest purity
available and were obtained from regular commercial sources.
/
(caveolin-3 null) mice comprising the
F4 generation were subjected to detailed analysis. For all
experiments described herein, each experiment was repeated at least
twice, and four mice were analyzed in a given experimental group.
Animals were analyzed at 8-12 weeks of age; however, virtually
identical results were also obtained with 6-month-old mice.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Generating caveolin-3 null (CAV3
/
) mice via homologous
recombination. A, generation of the caveolin-3
construct for targeted gene disruption. The knockout construct for
murine caveolin-3 was designed to replace exon-2 of caveolin-3 with the
neomycin resistance gene cassette, but in the opposite transcriptional
orientation. A BAC clone containing murine caveolin-3 was previously
isolated using the murine caveolin-3 cDNA as a probe for colony
hybridizations and its intron/exon organization was determined (31). An
8-kb BamHI subclone containing the second exon of caveolin-3
was isolated and cloned into the BamHI site of Bluescript
(pBS SK+). It was used for the 3' end of the construct. From this
clone, a 4.0-kb XbaI/BglII fragment was isolated
and cloned into pCB7. This fragment was then isolated by double
digestion with XbaI and SacI and cloned into the
PGT-N29 vector (New England Biolabs, Inc.) that contains the neomycin
resistance gene. From the original BAC clone, a 10-kb KpnI
subclone was isolated and cloned into Bluescript (pBS SK+). This clone
contained exon 2 of caveolin-3 and was used for the 5' end of the
construct. An internal 4.4-kb BamHI fragment was subcloned
into Bluescript, which was then liberated with EcoRI
(yielding a 4.2-kb fragment) and subcloned into pGT-N29 that already
possessed the 3' end of the construct. The final knockout construct was
linearized with XhoI. B, derivation of ES cell
clones harboring a disrupted caveolin-3 gene. After electroporation of
the WW6 ES cell line (38) with the linearized DNA containing the
caveolin-3 K/O construct, G418-resistant clones were selected for
analysis. Homologous recombination was detected by Southern blot
analysis. Genomic DNA from approximately 1000 G418-resistant ES cell
clones was prepared. For each clone, ~10 µg of genomic DNA was
digested with EcoRI and separated on a 0.8% agarose gel.
The DNA was transferred to nitrocellulose and probed with a 1.5-kb
BglII fragment that is 3' to the knockout construct (Fig.
1A). Homologous recombination was detected in several
independent clones by the presence of a ~8.5-kb band (K/O band) in
addition to a ~13-kb band (WT band). This is due to the introduction
of an additional EcoRI site into the caveolin-3 locus via
homologous recombination (see panel A).
C, identification of homozygous null mice harboring a
disrupted caveolin-3 gene by Southern blot analysis. Positive clones
were injected into blastocysts in order to obtain chimeric mice with
germline transmission of this caveolin-3 gene disruption. We next
crossed the resulting heterozygote mice to obtain homozygote mice that
harbored the intended gene disruption. Approximately 10 µg of genomic
DNA was extracted from mouse tail biopsies, digested with
EcoRI, and separated on a 0.8% agarose gel. The DNA was
transferred to nitrocellulose and probed with a 1.5-kb BglII
fragment that is 3' to the knockout construct. Homozygous null mice
were identified by the presence of the ~8.5-kb band (K/O band) and
the absence of the ~13-kb band (WT band). D,
identification of homozygous null mice harboring a disrupted caveolin-3
gene by PCR analysis. In addition, homozygous null mice were identified
by PCR analysis using primers specific for the neomycin resistance gene
and exon-2 of caveolin-3. Amplification of only the neomycin resistance
gene, but not caveolin-3/exon2, indicated that the mice were
homozygous null. bp, base pairs.
) to obtain
homozygote mice (CAV3
/
) that harbored the intended gene disruption.
/
) Mice Do Not Express the Caveolin-3 Protein Product and
Lack Sarcolemmal Caveolae Membrane Domains--
To demonstrate the
loss of caveolin-3 protein expression, tissues were harvested from WT
and caveolin-3 null mice and examined by Western blot analysis using a
caveolin-3 specific mAb probe. In all cases, CAV3 +/
mice were
crossed and the resulting CAV3 +/+ and CAV3
/
littermates were
compared. Fig. 2A shows that caveolin-3 is only expressed in skeletal muscle and heart tissues from
WT mice, but is clearly absent in caveolin-3 null mice. In contrast,
the expression of caveolin-1 and -2, the other members of the caveolin
gene family, was not affected in caveolin-3 null mice.
View larger version (42K):
[in a new window]
Fig. 2.
Caveolin-3 null mice do not express the
caveolin-3 protein and lack sarcolemmal caveolae. A,
Western blot analysis. Protein lysates were prepared from skeletal
muscle, heart, and lung tissues of WT mice and caveolin-3
KO mice. After SDS-PAGE and transfer to nitrocellulose,
immunoblotting was performed with monospecific antibodies probes that
recognize only caveolin-1, caveolin-2, or caveolin-3. Note that
caveolin-3 is only expressed in skeletal muscle and in the heart of WT
mice. Interestingly, caveolin-1 and -2 are equally expressed in WT mice
and caveolin-3 null mice. The presence of caveolin-1 and -2 in skeletal
muscle and cardiac tissues reflects their expression in fibroblasts,
endothelial cells, and smooth muscle cells, but caveolin-1 and -2 are
not expressed in the striated muscle cells themselves (14, 15). Each
lane contains an equal amount of total protein. B,
immunocytochemistry. Skeletal muscle tissue sections were prepared from
WT mice (upper panel) and caveolin-3 KO mice
(lower panel). After immunostaining with
antibodies directed against caveolin-3, samples were observed under a
confocal microscope. Note that caveolin-3 is expressed only in WT mice.
C, transmission electron microscopy analysis. Transmission
electron micrographs of skeletal muscle cells (upper
panels) and neighboring endothelial cells (lower
panels) from WT mice (left panels) and
caveolin-3 KO mice (right panels) are shown. Note
that a lack of caveolin-3 expression in caveolin-3 KO mice results in
an absence of caveolae at the sarcolemma (muscle cell plasma membrane).
As expected, caveolae are still present in endothelial cells from WT
mice and caveolin-3 KO mice.
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Fig. 3.
Histological analysis of skeletal muscle
fibers from caveolin-3 null mice. Muscle tissue sections
from WT mice (upper panel) and caveolin-3 KO mice
(lower panel) were stained with hematoxylin and
eosin (H&E). Note that muscle tissue sections from
caveolin-3 KO mice exhibit variability in muscle fiber size and reveal
the presence of some necrotic fibers (see arrows).
/
) in mice is sufficient to induce a mild
myopathic phenotype that is consistent with LGMD-1C in humans.
-sarcoglycan, and
-dystroglycan (Fig. 4A). Virtually identical
results were obtained by immunocytochemistry, indicating that
dystrophin,
-sarcoglycan, and
-dystroglycan protein expression is
not affected in caveolin-3 null mice (Fig. 4B).
Interestingly, these proteins were still localized at the sarcolemma in
skeletal muscle fibers lacking caveolin-3 expression (Fig.
4B). These results are consistent with our previous data showing that the expression level and the macroscopic
localization of dystrophin and its associated glycoproteins are not
affected by the loss of caveolin-3 expression in human LGMD-1C
(27).
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Fig. 4.
Caveolin-3 protein expression is required for
the correct targeting of the dystrophin-glycoprotein complex to
cholesterol-sphingolipid raft domains/caveolae in normal muscle
fibers. A, Western blot analysis. Protein lysates were
prepared from skeletal muscle of WT mice and caveolin-3 null mice.
After SDS-PAGE and transfer to nitrocellulose, immunoblotting was
performed with monospecific antibody probes that recognize dystrophin
(upper panel), -sarcoglycan (middle
panel), and
-dystroglycan (lower
panel). Note that dystrophin,
-sarcoglycan, and
-dystroglycan are expressed at normal levels in the skeletal muscle
of caveolin-3 KO mice. Each lane contains an equal amount of total
protein. B, immunocytochemistry. Skeletal muscle tissue
sections were prepared from WT mice (left panels)
and caveolin-3 KO mice (right panels). After
immunostaining with antibodies directed against dystrophin,
-sarcoglycan, and
-dystroglycan, samples were observed under a
confocal microscope. Note that dystrophin,
-sarcoglycan, and
-dystroglycan are expressed at normal levels at the plasma membrane
in WT mice and caveolin-3 KO mice. C, targeting to
cholesterol-sphingolipid-rich raft domains/caveolae. Skeletal muscle
tissue samples from WT mice and caveolin-3 KO mice were subjected to
subcellular fractionation. To separate membranes enriched in lipid
rafts/caveolae from the bulk of cellular membranes and cytosolic
proteins, an established equilibrium sucrose density gradient system
was utilized (see "Experimental Procedures"). In this fractionation
scheme, immunoblotting with anti-caveolin IgG can be used to track the
position of lipid rafts/caveolae (fractions 4-6) within these
bottom-loaded sucrose gradients. Note that dystrophin,
-sarcoglycan,
and
-dystroglycan are excluded from lipid raft domains in caveolin-3
KO mice.
-sarcoglycan, and
-dystroglycan are all excluded from these cholesterol-sphingolipid
raft domains in caveolin-3 null mice. These results indicate that, although the expression levels of dystrophin and dystrophin-associated glycoproteins do not change in caveolin-3 null mice, their microscopic localization within cholesterol-sphingolipid rafts/caveolae domains is
compromised. As the dystrophin-glycoprotein complex is important for
normal skeletal muscle functioning, our results suggest that mislocalization of dystrophin,
-sarcoglycan, and
-dystroglycan in
caveolin-3 null mice may contribute to the myopathic changes we observe
in skeletal muscle tissue sections.
-dystroglycan (26).
and ryanodine receptor in WT and
caveolin-3 null mice. Skeletal muscle tissue lysates from WT and
caveolin-3 null mice were subjected to Western blot analysis using
monoclonal antibody probes specific for dihydropyridine receptor-1
and ryanodine receptor. Fig.
5A shows that the protein
expression of these T-tubule markers is not altered in caveolin-3 null
mice.
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Fig. 5.
Dihydropyridine receptor-1
and ryanodine receptor are expressed at normal levels, but are
mislocalized in skeletal muscle fibers from caveolin-3 null mice.
A, Western blot analysis. Protein lysates were prepared from
skeletal muscle tissue of WT mice and caveolin-3 null mice. After
SDS-PAGE and transfer to nitrocellulose, immunoblotting was performed
with monospecific antibodies probes that recognize DHPR-1
(upper panel) and ryanodine receptor
(Ryanodine-R) (Lower panel). Note that DHPR-1
and
ryanodine receptor are expressed at normal levels in caveolin-3 null
mice. Each lane contains an equal amount of total protein.
B-E, immunocytochemistry. Skeletal muscle tissue sections
were prepared from WT mice and caveolin-3 null mice. After
immunostaining with antibodies directed against DHPR-1
(B
and C), and ryanodine receptor (Ryanodine-R)
(D and E) samples were observed under a confocal
microscope. Note that DHPR-1
and ryanodine R show the characteristic
double row staining pattern in skeletal muscle fibers from WT mice.
However, DHPR-1
and ryanodine R show a more diffuse staining pattern
in skeletal muscle fibers from caveolin-3 null mice. The
boxed areas in panels B and
D are shown at a higher magnification level in
panels C and E, respectively.
and
ryanodine receptor was severely affected in the skeletal muscle fibers
of caveolin-3 null mice. In WT mice, double rows of discrete punctate
immunostaining, representing pairs of triads on the opposite sides of
the Z-lines, were observed using monoclonal antibody probes specific
for dihydropyridine receptor-1
(Fig. 5, B and C, left panels) and ryanodine receptor
(Fig. 5, D and E, left panels).
(Fig. 5,
B and C, right panels) and
ryanodine receptor (Fig. 5, D and E, right panels) showed a diffuse pattern of
expression in skeletal muscle sections from caveolin-3 null mice. A
three-dimensional reconstruction of these sections is shown in Fig.
6. These results suggest that the
organization of the T-tubule system is clearly abnormal or immature in
caveolin-3 null mice.
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Fig. 6.
Three-dimensional reconstruction of
dihydropyridine receptor-1
immunostaining. A, skeletal muscle tissue
sections were prepared from WT mice and caveolin-3 KO mice. After
immunostaining with antibodies directed against DHPR-1
, Z-series
were acquired using a Bio-Rad MR 600 confocal microscope. Images shown
represent the three-dimensional reconstructions of 4-µm-thick
sections as viewed in the x-y plane. B,
boxed regions in panel A are also
shown as viewed from the x-z plane. Note that DHPR-1
is
localized diffusely along the length of the muscle fiber in caveolin-3
KO mice, while it is concentrated in discrete double rows in WT
mice.
View larger version (72K):
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Fig. 7.
Caveolin-3 protein expression is required for
the development of a mature highly organized T-tubule system.
Skeletal muscle tissue samples from WT mice (A,
left panel) and caveolin-3 KO mice (A,
right panel) were subjected to T-tubule system
staining using potassium ferrocyanate (see "Experimental
Procedures"). As a consequence, the T-tubules appear as
electron-dense structures (stained black). Note that the
T-tubule system is dilated and longitudinally oriented in caveolin-3 KO
mice. Transmission electron micrographs of skeletal muscle sections
from caveolin-3 KO mice are also shown in panel B
at a higher magnification.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) mouse model, using
standard homologous recombination techniques, to mimic the situation in
patients with LGMD-1C. Importantly, loss of caveolin-3 protein
expression resulted in loss of caveolae at the sarcolemma (Fig.
2C). This result clearly indicates that caveolin-3 is
required for caveolae formation in skeletal muscle cells in
vivo. Analysis of skeletal muscle tissue from caveolin-3 null mice
revealed mild myopathic changes, with variability in the size of the
muscle fibers and the presence of necrotic fibers (Fig. 3). Taken
together, these results indicate that loss of caveolin-3 and
sarcolemmal caveolae is sufficient to induce a mild myopathy that is
similar in its severity to that seen in patients with LGMD-1C.
-sarcoglycan, and
-dystroglycan was not affected (Fig. 4,
A and B).
-sarcoglycan, and
-dystroglycan are all excluded from
cholesterol-sphingolipid rafts, in the absence of caveolin-3 expression
(Fig. 4C). Thus, one function of caveolin-3 is to recruit the dystrophin-glycoprotein complex to cholesterol-sphingolipid rafts/caveolae in normal muscle fibers. As dystrophin and
dystrophin-associated glycoproteins are important for normal skeletal
muscle functioning, these findings suggest a possible mechanism for
understanding the pathogenesis of LGMD-1C in humans.
and ryanodine receptor) are mislocalized in skeletal muscle fibers from caveolin-3 null mice. The localization of these marker proteins also provides an indication that the T-tubule
system is disorganized or immature in caveolin-3 null mice (Figs. 5
(B-E) and 6).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Frank Macaluso for expertise in T-tubule staining and Jorge Bermudez for help in collecting frozen sections of skeletal muscle tissue.
![]() |
Addendum |
---|
While this work was being completed, another paper appeared describing the generation of caveolin-3 null mice (55). In accordance with our results, these authors demonstrate that loss of caveolin-3 expression prevents caveolae formation and induces a mild myopathy (55), as is known to occur in patients with LGMD-1C (27). However, these authors did not provide any additional mechanistic insights into how caveolin-3 deficiency causes muscular dystrophy. In contrast, we show here that caveolin-3 expression is required for (i) the proper targeting of the dystrophin-glycoprotein complex to lipid rafts/caveolae and (ii) the development of a highly organized T-tubule system. As the dystrophin-glycoprotein complex and the T-tubule system represent important elements in normal muscle functioning, we speculate that alterations in their organization may contribute to the mild myopathic changes that we observed in caveolin-3 null mice and in patients with LGMD-1C.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health, the Muscular Dystrophy Association, the American Heart Association, and the Susan B. Komen Breast Cancer Foundation (to M. P. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
c Recipient of Fellowship 470/bi from Telethon-Italia. Current address: The University of Pittsburgh School of Medicine, Dept. of Pharmacology, Biomedical Science Tower East, Rm. 1302, Pittsburgh, PA 15261-0001.
d These authors contributed equally to this work.
e Current address: The University of Pittsburgh School of Medicine, Dept. of Pharmacology, Biomedical Science Tower East, Rm. 1356, Pittsburgh, PA 15261-0001.
g Supported by grants from Telethon-Italia and the Italian Ministry of Health (G. Gaslini Institute, Ricerca Finalizzata).
j Supported by National Institutes of Health Grant R01-CA-76329.
k Recipient of a Hirsch/Weil-Caulier Career Scientist Award. To whom correspondence should be addressed: Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8828; Fax: 718-430-8830; E-mail: lisanti@aecom.yu.edu.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M100828200
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ABBREVIATIONS |
---|
The abbreviations used are: WT, wild-type; LGMD, limb-girdle muscular dystrophy; K/O band, knockout band; KO mice, knockout mice; mAb, monoclonal antibody; kb, kilobase pair(s); TBST, Tris-buffered saline with Tween 20; Mes, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; RyR, ryanodine receptor; DHPR, dihydropyridine receptor.
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