Circulation Research. 2000;87:480-488
(Circulation Research. 2000;87:480.)
© 2000 American Heart Association, Inc.
Dystrophin Associates With Caveolae of Rat Cardiac Myocytes
Relationship to Dystroglycan
Donald D. Doyle,
Gwen Goings,
Judy Upshaw-Earley,
S. Kelly Ambler,
Alison Mondul,
H. Clive Palfrey,
Ernest Page
From the Departments of Medicine (E.P.) and Neurobiology, Pharmacology
and Physiology (D.D.D., G.G., J.U.-E., S.K.A., A.M., H.C.P., E.P.), University
of Chicago, Chicago, Ill. G.G.s present address is Childrens
Memorial Institute for Education and Research, Chicago, Ill. S.K.A.s
present address is Cardiology Section, Denver Health Medical Center, Denver,
Colo.
Correspondence to Ernest Page, Department of Medicine MC5085, 5841 S Maryland Ave, Chicago, IL 60637. E-mail page{at}hearts.bsd.uchicago.edu
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Abstract
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AbstractThe possibility of an
interaction between the
cytoskeletal protein dystrophin and cell
surface caveolae in
the mammalian myocardium was
investigated by several techniques.
Caveolin (cav)-3enriched,
detergent-insoluble membranes
isolated from purified
ventricular sarcolemma by density-gradient
fractionation
were found to contain dystrophin and dystroglycan.
Further purification
of cav-3containing membranes by
immunoprecipitation using
anticav-3coated magnetic
beads yielded dystrophin but not always
dystroglycan. Electron
microscopic analysis of precipitated
material revealed caveola-sized
vesicular profiles that could be
double-labeled with anti-dystrophin
and anticav-3 antibodies. In
contrast, immunoprecipitation
of membranes with anti-dystrophincoated
beads yielded
both cav-3 and dystroglycan. Electron microscopic
analysis of
this material showed heterogeneous
membrane profiles, some of
which could be decorated with anticav-3
antibodies. To
confirm that dystrophin and cav-3 were closely
associated in
cardiac myocytes, we verified that dystrophin was also
present
in immunoprecipitated cav-3containing membranes from
detergent
extracts, as well as in sonicated extracts of purified
ventricular
myocytes. Confocal
immunofluorescence microscopy of
ventricular
and atrial cardiac myocytes showed that the
cellular distributions
of cav-3 and dystrophin partially overlapped.
Immunoelectron
micrographs of thin sections of rat atrial myocytes
revealed
a fraction of dystrophin molecules that are in apparently
close
apposition to caveolae. These results suggest that a
subpopulation
of dystrophin molecules interacts with cardiac myocyte
caveolae
in vivo and that some of the dystrophin is engaged in linking
cav-3
with the dystroglycan complex.
Key Words: heart myocyte caveolae dystrophin
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Introduction
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The flask-shaped plasma membraneassociated vesicles
called
caveolae are nonclathrin-coated organelles first described
by
Palade in 1953.
1 Recently, caveolae have been proposed to
be
multifunctional organelles that, in diverse cell types, participate
in
one or more of the following: as sites for concentration and
subsequent
internalization of small molecules and as binding sites for
Ca
2+ ions,
2 as the loci of multiple
signal-transducing molecules,
3 and as sites of
cytoskeletal attachment to the plasma membrane.
4
Presumptive caveolar properties or "markers" include the caveolar
coat
protein caveolin (cav), an 18- to 24-kDa protein (depending
on the
isoform),
5 and a high concentration of
cholesterol and
sphingolipids.
6 Four isoforms
of caveolin encoded by 3 distinct
genes have so far been identified.
Cav-1

,
7 also known as VIP21
or VCAV,
8 is
found in a variety of tissues, as is cav-1ß,
a truncated version also
derived from the same gene.
9 cav-2,
the isoform most
abundant in fat cells, coexpresses with cav-1
in many nonmuscle
tissues,
10 whereas cav-3,
11 or
MCAV,
12 is found predominantly if not exclusively in
muscle cells. Thus,
in the heart, cav-3 is the isoform expressed in
cardiac myocytes
and vascular smooth muscle cells, whereas the cav-1
isoform
is predominantly expressed in nonmuscle
cells.
13
Several laboratories have isolated cav-enriched supramolecular
complexes from diverse cell types, using either detergent insolubility
or sonication of whole-cell or crude membrane fractions followed by
sucrose or OptiPrep (Gibco-BRL) density flotation as a final
purification step.14 However, a number of reports have
suggested that density gradient flotation of such preparations is by
itself insufficient to obtain caveolae free of contamination with
noncaveolar membranes.15 16 17 For example, we previously
showed18 that sucrose density flotation of
detergent-insoluble membrane preparations derived from highly purified
sheep ventricular sarcolemma yields a diverse population of
proteins including cav-1 and cav-3 and the glycophosphoinositol
(GPI)-linked protein T-cadherin. To determine whether T-cadherin
was a bona fide caveolar protein, we purified the cav-enriched
complexes of cardiac membranes further by immunoprecipitation using
magnetic beads coated with an antibody to cav-3.19
Immunoblot analysis of the material thus bound
showed that, whereas a substantial fraction of cav-3 was
immunoprecipitated, other detergent-insoluble membrane fragments
containing T-cadherin were not precipitated, which indicated that
T-cadherin is not directly associated with cardiac caveolae.
Dystrophin, a large (430-kDa), rod-shaped protein, is a component of
the subsarcolemmal cytoskeleton in both striated and smooth muscle
cells and is abundant in cardiac myocytes.20 Although it
is established that dystrophin is implicated in muscular dystrophy, its
role in normal cells is incompletely understood.21
Dystrophin is attached at a cysteine-rich region of its C terminus to
the trans-sarcolemmal ß-subunit of dystroglycan, a member of the
protein complex dystrophin-associated glycoproteins, which
link dystrophin to the extracellular matrix.22 Dystrophin
is also associated with other elements of the cytoskeleton through
binding to filamentous actin.23 Dystrophin and
dystroglycan have been previously shown to comigrate with cav-3 in
sucrose density gradients of crude detergent extracts from skeletal
muscle cells, and cav-3 was shown to coimmunoprecipitate with
dystrophin.11 However, cav-3 was subsequently shown not to
be a component of the dystroglycan complex.24
Immunoelectron microscopy (EM) of smooth muscle cells has shown
dystrophin to be present in the caveola-rich region of the
sarcolemma, but no direct association of dystrophin with caveolae was
demonstrated.25
To determine whether dystrophin is associated with cav-3 in cardiac
myocytes and whether dystroglycan also participates, we studied the
relationship between these proteins in mammalian heart muscle cells
using immunoprecipitation, immuno-EM, and
immunofluorescence microscopy. Our results suggest
that a component of dystrophin associates with caveolae in cardiac
myocytes and that at least a portion of this dystrophin is
simultaneously linked to dystroglycan.
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Materials and Methods
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Materials
An antibody to the N-terminal residues 2 to 18 of the rat cav-3
sequence
(QCB) was made commercially. Polyclonal and monoclonal
antibodies
to cav-1 and cav-3 were obtained from Transduction
Laboratories.
Polyclonal antibodies to rat T-cadherin were provided by
Dr
Barbara Ranscht (Burnham Institute, La Jolla, Calif). A polyclonal
antibody
to dystrophin was the gift of Dr Timothy Byers (Indiana
University
School of Medicine, Indianapolis, Ind); 2 monoclonal
antibodies
to dystrophin (Dys1 and Dys2) and a monoclonal antibody to
ß-dystroglycan
were purchased from Vector Laboratories. Anti-rabbit
and anti-mouse
IgG Fc-specific gold-labeled antibodies were purchased
from
E-Y Laboratories. Tosylated magnetic polystyrene beads were
obtained
from Dynal Corp.
Preparation of cav-Enriched Membrane Fragments From Sheep and Rat
Whole Ventricles
To produce membranes enriched in plasmalemma from
sheep and rat whole ventricles, we used our previously reported
approach, which is based on the method of Jones.26
Triton-insoluble membrane fragments were prepared and isolated from
these preparations as previously described.19
Preparation of cav-Enriched Membrane Fragments From Rat Heart
Isolated Ventricular Myocytes
Rat ventricular cells were dispersed by
collagenase treatment of whole heart via perfusion on the
Langendorff cannula using MEM (Gibco-BRL). Viable myocytes were further
purified by Percoll density gradient
centrifugation.27 By visual count, >95%
of the cells were found to be myocytes. Cav-enriched membranes were
prepared by detergent dissolution or by sonication.
Detergent Extraction
Myocytes were incubated with 0.5% Triton X-100 detergent on
ice.
Extraction by Sonication
Cav-3enriched membranes were also prepared from acutely
isolated myocytes by a detergent-free procedure based on methods
developed by Lasley et al.28 One portion of the samples
prepared as described above was reserved as starting material, and an
aliquot was subjected to immunoprecipitation as described below.
Immunopurification of Membranes Containing cav-3
Membrane fragments, prepared from whole ventricle or from
isolated myocytes as described above, were incubated with
anticav-3coupled magnetic beads to separate bound from
nonbound (NB) material. Pelletable nonbound material (NBp) was
collected by centrifugation at 100 000g for
1 hour. NBp and some bound material were dissolved in sample buffer for
subsequent SDS-PAGE. Proteins in an equivalent volume of starting
incubation material and, in some cases, nonbound nonpelletable material
were precipitated with trichloroacetic acid before dissolution in
sample buffer. Some bound material was incubated with primary
antibodies and colloidal gold-labeled secondary antibodies and then
fixed and prepared for viewing by EM.
Immuno-EM
Rat atria were thin sectioned and processed for immuno-EM as
described by Chang et al.29 Magnetic beads with captured
cav-3containing membranes (above) were incubated at 4°C with
polyclonal anticav-3 and monoclonal anti-dystrophin (Dys1 and Dys2)
antibodies or, as a control, with equivalent concentrations of rabbit
and mouse IgGs, decorated with gold-labeled Fc fragmentspecific
anti-rabbit and anti-mouse secondary antibodies, fixed with 3%
glutaraldehyde, postfixed with 0.8% osmium tetroxide,
treated with 1% tannic acid, dehydrated, and embedded in Epon for
subsequent thin sectioning.
Confocal Immunofluorescence Microscopy
Rat atria frozen in liquid nitrogen were sectioned and prepared
for confocal microscopy as previously described.30
Gel Electrophoresis and Antibody Staining
Proteins were separated on 8% or 10% SDS-PAGE gels. The
proteins were transferred to nitrocellulose filters for antibody
staining as previously described.19
An expanded Materials and Methods section can be found in an online
data supplement available at http://www.circresaha.org.
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Results
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Coprecipitation of Dystrophin With Immunopurified Cardiac
Caveolae
We previously reported that Triton X-100insoluble floatable
membrane
fragments (the TIFF or T-CAV fractions) obtained from
plasmalemmal
preparations of sheep heart ventricle isolated
by sucrose density
gradient centrifugation are enriched
in cav-1 and cav-3, as
well as in a variety of other proteins including
T-cadherin,
the major GPI-linked protein in sarcolemma from sheep
heart.
19 However, immunoprecipitation of cav-3containing
membranes
by anticav-3coated magnetic beads did not coprecipitate
T-cadherin
or cav-1, each of which remained in other
detergent-insoluble
membrane fragments. These results suggested that
the TIFF fraction
is heterogeneous.
Analysis of the same TIFF fraction revealed the presence
of substantial dystrophin and of a small fraction of the dystroglycan
originally present in the plasmalemmal preparation, as
well as cav-1 and cav-3 (Figure 1A
). As
visualized by EM of thin-sectioned pellets, the TIFF fraction from
heart contains a heterogeneous mixture of membranes that
includes electron-lucent and electron-opaque vesicles of about the same
profile size as that of caveolae in situ (50 to 100 nm). Vesicles of
larger mean profile size were also seen (Figure 1B
). When these
detergent-insoluble membranes were subjected to immunoprecipitation by
anticav-3coated magnetic beads, vesicles having the approximate
profile size of caveolae were captured (Figure 1C
). Many of
these captured vesicles were electron-lucent, thus resembling vesicles
immunoprecipitated by Waugh et al31 using anticav-1
antibodies from extracts of sonicated A431 cells. Other vesicles were
electron-opaque, resembling some of the anticav-1immunoprecipitated
membranes from rat lung seen by Stan et
al.17

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Figure 1. On discontinuous sucrose density gradients,
dystrophin coisolates with cav-1 and cav-3 in Triton-insoluble
floatable membrane fragments from sheep heart. Sarcolemma-enriched
membranes prepared from sheep heart were solubilized by treatment with
cold Triton X-100 (0.5% final). They were then made to be 40% with
respect to sucrose in 2 mL, layered under a 10% to 30% sucrose
gradient, and centrifuged at 140 000g
overnight, after which they were fractionated into 12 1-mL fractions.
Fractions 1 to 10 contained floatable complexes. Fractions 10 to 12
contained soluble protein. A, Immunoblots of proteins from
each fraction of a gradient (equal volumes loaded) shown with a
previously reported profile19 of a similarly produced
gradient. Floatable membrane fragments are enriched in cav-1 and cav-3,
as well as in dystrophin, an abundant cytoskeletal protein. Also
present is a minority portion of the ß-dystroglycan resident in
the starting sarcolemma-enriched fraction. B, Electron micrograph of
membranes in the cav-enriched floatable fractions. Note the presence of
electron-lucent and electron-dense vesicular profiles about the size
and shape as caveolae, as well as profiles larger than caveolae and/or
of inappropriate shape to be caveolae. C, Vesicular fragments the
appropriate size and shape of caveolae immunoprecipitated by
anticav-3coated magnetic beads. Some profiles have sharply defined
circumferences yet are interiorly electron-lucent (long arrow), whereas
some are more electron-opaque throughout (short arrow). Bar=200
nm.
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When detergent-resistant membranes prepared from whole rat
ventricle were subjected to immunoprecipitation using
anticav-3coated magnetic beads, a major portion of the cav-3 and a
significant portion of the dystrophin were captured (Figure 2A
). In agreement with our
previous results, detergent-insoluble membranes containing cav-1 or
T-cadherin were not retained by the beads. In 3 of 6 experiments no
detectable ß-dystroglycan was immunoprecipitated (Figure 2A
).
In 3 of 6 experiments a small portion of the ß-dystroglycan was
immunoprecipitated (data not shown). The bead-captured vesicles could
be codecorated with anticav-3 and anti-dystrophin antibodies (Figures 2B
through 2E
). In view of the background associated with
double-label EM studies of this type, quantification of electron
micrographs was attempted by carefully counting the numbers of vesicles
that immunostained for either epitope and for both epitopes
together. These studies confirmed that caveola-sized vesicles were
significantly immunolabeled with antibodies to both cav-3 and
dystrophin (Table
). These results suggest that we
are isolating a fraction that is enriched in bona fide cardiac caveolae
and that some of the cellular dystrophin is bound to these organelles.
The interaction between dystrophin and caveolae is sufficiently strong
to survive washes of both low (10 mmol/L HEPES-EDTA) and high (0.6
mol/L KCl) ionic strength.

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Figure 2. Dystrophin coimmunoprecipitates with rat heart
caveolae in the presence of anticav-3coated magnetic beads. A, An
aliquot of the starting material (S) consists of Triton-solubilized
sarcolemma-enriched membranes prepared from whole rat ventricle.
Starting-material membranes were incubated with anticav-3coated
magnetic beads. Bound membranes (B) were separated from nonbound
material by a magnet. Nonbound pelletable material, NBp, was collected
by centrifugation at 100 000g for 1
hour. Proteins from equal aliquots of each fraction were separated by
SDS-PAGE and transferred to nitrocellulose membranes. Membrane filters
were immunoblotted with primary antibodies against cav-3
and cav-1 (Transduction Laboratories), T-cadherin, dystroglycan,
dystrophin, and horseradish peroxidaseconjugated secondary
antibodies. Detection was by enhanced chemiluminescence amplification.
Cav-3 present in the starting Triton-treated sarcolemma (S) was
found to bind to the beads (B), as well as to a significant subset of
the dystrophin in the starting material. Not bound (NBp) to the beads
but present in pelletable (100 000g) material were
cav-1, T-cadherin, and ß-dystroglycan. B, Electron micrograph of a
thin section of membranes adhered to immunobeads, such as in panel A
(ie, to the bound fraction), immunolabeled by rabbit anticav-3
polyclonal antibody and mouse anti-dystrophin monoclonal antibodies
(Dys1 and Dys2). A few membrane fragments (<5% by rough estimate),
apparently not of the appropriate shape or size of caveolae, were
observed to be associated with caveola-like profiles. C, Enlargement
(2x) of indicated region of panel B. Initial primary antibody labeling
was followed by 5 nm goldlabeled Fc chain-specific anti-rabbit
secondary antibody (long arrows) and 10 nm goldlabeled Fc
chain-specific anti-mouse secondary antibody (short arrows). D,
Bead-captured membranes incubated with equivalent concentrations of
rabbit and mouse IgG as a control. E, Enlargement of indicated region
of D. Gold particles of both species are specifically (see
Table ) associated with well-defined vesicular membranes the size
of caveolae. Bars=200 nm.
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Table 1. Quantification of Immunogold Labeling of Membrane Vesicles
Immunoprecipitated by Anticav-3 or Anti-Dystrophin (Dys2) Antibodies
on Protein A/Gcoupled Magnetic Beads
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In correlative experiments, we examined whether
anti-dystrophincoated beads could precipitate membranes that were
immunoreactive toward cav-3. This turned out to be the case, as shown
in Figure 3A
, and such beads captured
dystroglycan but virtually no cav-1 or T-cadherin. Examination of
precipitated membranes by EM revealed a heterogeneous
mixture of fragments, some of which could be decorated by anticav-3
antibodies (Figures 3B
through 3D
; Table
). These results
again suggest that a fraction of dystrophin is associated with a
membranous organelle that contains cav-3.
Dystrophin Is Present in cav-Enriched Membranes Prepared by
Either Detergent Extraction or Sonication of Isolated Cardiac
Ventricular Myocytes
Sarcolemma-enriched membrane preparations isolated from
mammalian whole ventricle contain membranes derived from all cell types
present in the heart, including vascular smooth muscle cells,
which, like cardiac myocytes, also contain caveola-associated cav-3. To
substantiate further that dystrophin associates with cav-3 in cardiac
myocytes, we first enzymatically dispersed the hearts into their
component cells and then isolated the myocytes by Percoll density
gradient centrifugation. The resultant, highly (>95%)
cardiomyocyte-enriched preparations were examined by
confocal immunofluorescence microscopy, which
confirmed that both cav-3 and dystrophin were present in close
association with the cardiac myocyte sarcolemma (data not shown).
Membrane fragments were prepared from these cells by
homogenization in low and highionic-strength
buffers similar in manner to our approach to whole ventricle.
Immunoisolation of cav-3containing membranes from detergent extracts
of these membranes resulted in coprecipitation of dystrophin (Figure 4A
). As with the caveolar material
prepared from whole ventricle, in some experiments such as those shown
in Figure 2
, dystroglycan was not associated with the
dystrophin-cav-3 complexes captured by the beads. Dystrophin and
dystroglycan not associated with caveolae were in soluble form, not
pelletable by centrifugation at
100 000g.

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Figure 4. Dystrophin coimmunoprecipitates with cav-3 in
Triton X-100treated as well as sonicated lysates of isolated rat
ventricular myocytes immunoprecipitated by
anticav-3coated magnetic beads. A, From a Triton extract most of
the cav-3, as well as dystrophin, in the starting incubation mixture
(S) was recovered in the fraction bound to the beads (B). Dystroglycan
is not found in the fraction bound to the beads or in pelletable
(100 000g) material not bound to the beads (NBp).
Instead, it is present in the nonpelletable (ie, soluble) nonbound
fraction (NBs). B, From a detergent-free sonicated extract most of the
cav-3, as well as dystrophin, in the starting incubation mixture was
also recovered in the fraction bound to the beads. However, some
dystroglycan coimmunopurified with the cav-3dystrophin complex. Some
dystrophin and dystroglycan was found in the pelletable nonbound
fraction, NBp, in contrast to the detergent-treated material in panel
A. C, Electron micrographs of sonicated membranes captured by the beads
and immunolabeled with anti-dystrophin monoclonal antibodies (Dys1 and
Dys2; left) or mouse IgG as control (right). Primary antibodies were
decorated with Fc chainspecific anti-mouse secondary antibodies
conjugated with 15 nm colloidal gold (arrows). Micrographs show
vesicular profiles similar to those immunoprecipitated from
detergent-treated material. Some vesicles are in close apposition to
one another, which may be the result of undissolved plasma membrane
associated with the dystroglycan complex linked to caveolae attached to
the beads. Bar=200 nm.
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Because we were concerned about possible molecular rearrangement
induced by detergent extraction,16 we applied an alternate
approach to caveolar isolation. Acutely isolated cells suspended in an
isotonic sucrose buffer were homogenized with a
polytetrafluoroethylene (Teflon) pestle,
and a plasmalemma-enriched fraction was isolated by Percoll
density gradient centrifugation.28 The
resulting membranes were sonicated, and a cav-enriched fraction was
isolated by OptiPrep gradient centrifugation.
Immunoisolation of cav-3containing membranes from these sonicated
extracts resulted in coprecipitaion of dystrophin (Figure 4B
). Under these milder preparative conditions, dystroglycan was
seen to coprecipitate with cav-3 in all experiments. Examination of
precipitated sonicated membranes by EM revealed vesicles similar in
size and appearance to those prepared with detergent (Figure 4C
). These results support the notion that dystrophin associates
with cav-3containing membranes in cardiac myocytes. These results
also emphasize that the relative association of cav-3, dystrophin, and
dystroglycan in cellular fragments is dependent on conditions of
preparation.
Association of Dystrophin With Caveolae in Rat Atrial
Tissue
The above data suggest that a fraction of dystrophin is somehow
linked to caveolae from rat ventricle. Because the status of dystrophin
in rat atrial tissue has not been probed in depth, we investigated the
disposition of this protein in frozen sections of atria from mature
rats that lack or have only vestigial T-tubules.32 We
confirmed by confocal immunofluorescence that cav-3
labeling in rat atria is limited to the surface of the cells (Figures 5A
and 5C
), whereas dystrophin appears
both at the cell surface and in the cell interior (Figure 5B
).
Dystroglycan labeling was also limited to the cell surface (Figure 5D
).

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Figure 5. Cav-3, dystrophin, and dystroglycan in rat atrial
myocytes. In thin sections of frozen rat atrial myocytes, which have a
much less developed T-tubular system than ventricular
myocytes, anticav-3 labeling is seen at the cell surface but not
intracellularly (A and C). Dystrophin labeling, however, is seen
intracellularly as well as at the surface (B). Dystroglycan colocalizes
with cav-3 at the cell surface (D).
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To assess the relationship between dystrophin, dystroglycan, and
morphologically identified caveolae, we immunostained thin
sections of rat atria with anti-dystrophin and anti-dystroglycan
antibodies, followed by exposure to gold-labeled secondary antibodies
and examination by EM (Figure 6A
).
Some dystrophin labeling was found to be in close proximity to
optically well-resolved myocyte caveolae, which suggests that this
dystrophin may reside in or in close apposition to caveolae of atrial
myocytes. That the distribution of dystroglycan labeling was distinct
from that of dystrophin was suggested by the finding that dystroglycan
was located closer to the cell surface sarcolemma, as might be expected
for a transmembrane, laminin-binding protein (Figure 6B
).

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Figure 6. Electron microscopic localization of
dystrophin and dystroglycan in rat atrial myocytes. Rat atria, fixed
overnight in 3% paraformaldehyde, were sectioned with
a Vibratome (Technical Products, Intl), labeled with anti-dystrophin
polyclonal antibody (A) or anti-ß-dystroglycan monoclonal antibody
(B), exposed to gold-labeled secondary antibody, fixed with osmium
tetroxide, embedded in Epon, and thin sectioned. Overnight
paraformaldehyde fixation preserved structural
integrity well enough that caveolae (short arrows) could be seen in
cross section of cardiomyocytes, while the antigenicity of
the probed-for proteins was sufficiently retained to allow for
labeling. A, Some dystrophin (arrowheads) is localized well within a
caveolar diameter (50 to 100 nmol/L), above and at the circumference of
the caveolae. Asterisk denotes a region where the sarcolemmal profile
is outside the plane of the section. B, In contrast, ß-dystroglycan,
although likewise in the vicinity of caveolae (arrowheads), is more
closely localized to the plasmalemma (long arrow).
Bars=100 nm.
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Discussion
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For about 10 years it has been widely assumed that numerous
proteins
are present within or closely associated with caveolae;
this
assumption is based on studies of crude Triton-insoluble as
well
as sonication-impervious floatable fragments (TIFFs and
SIFFs), some of
which were isolated from whole cells. It is
now clear that such
fractions are heterogeneous and that the
presence of a
protein in a TIFF or SIFF light membrane fraction
is insufficient to
conclude that it is within or bound to caveolae.
One approach to this
problem is to purify caveolae further by
immunoprecipitation. Others
have shown that this procedure can
separate caveolar from noncaveolar
membranes in lung endothelial
cells and A431
cells.
17 31 Indeed, in previous work,
18 we
showed
that the major GPI-linked protein in the mammalian
myocardium,
T-cadherin, is present in the TIFF fraction
but can be readily
separated from caveolae by immunoprecipitation with
anticav-3
beads. In this report we apply a similar methodology to
show
that dystrophin in mammalian heart muscle is reproducibly
associated
with cardiac myocyte caveolae. This conclusion is further
supported
by electron microscopic observations both on immunopurified
membranes
and in in situ heart muscle preparations.
The principal association of dystrophin with the plasma membrane
of muscle cells is thought to be via its link to the dystroglycan
complex, a link mediated by the C-terminal cysteine-rich
domain.20 Indeed, when immunoprecipitation was performed
with anti-dystrophin antibodies, ß-dystroglycan was found in the
complex along with cav-3 (Figure 3A
). In addition, apparently
noncaveolar membrane fragments were observed to be attached to caveolar
profiles in micrographs of precipitated caveolae (Figure 2B
).
Such results suggest that dystrophin can bind
simultaneously to the dystroglycan complex and to caveolae.
This conclusion is qualified, however, by the
heterogeneity of the membrane fragments precipitated by
anti-dystrophin antibodies (Figure 3D
) and by the failure to
find significant dystroglycan in anticav-3 immunoprecipitates in some
experiments (Figure 2A
). Nevertheless, although we are not
excluding other interpretations, we think it likely that the vigorous
disruption of rat ventricle necessary to isolate and immunopurify
cardiac myocyte caveolae is capable of producing several populations of
dystrophin-containing complexes, including dystrophin bound to caveolae
but not bound to dystroglycan; dystrophin bound to dystroglycan; and,
in some cases, all 3 components together in a complex. Previous studies
on skeletal muscle suggested that dystrophin and dystroglycan appear
together with cav-3 in the crude TIFF fraction.11 Although
cav-3 was also shown to coimmunoprecipitate with dystrophin, it was
subsequently shown that dystroglycan-containing complexes, which
include dystrophin, can be isolated independently of
cav-3.24 Our data remain consistent with the dual
hypotheses that, in heart muscle, ß-dystroglycan does not reside in
caveolae, whereas some dystrophin links caveolae to ß-dystroglycan
and thereby to other dystrophin-binding proteins present in the
cardiac dystroglycan complex. Our data do not exclude the possibility
that in situ, either permanently or intermittently, some dystrophin may
be bound to caveolae and not to dystroglycan, or to dystroglycan and
not to caveolae. Cav-3 and dystrophin may associate by direct
binding33 or indirectly through mutual binding to a third
entity.34 The region of the dystrophin molecule involved
in such an interaction remains to be determined; the protein also
associates with actin, probably through sequences located in the
spectrin-like repeat domain. The roles of other domains, such as the
N-terminal, are less well understood. Interestingly, filamin binding to
cav-1 has recently been implicated in interaction between caveolae and
actin, which occurs in 3T3 fibroblasts and T4.5
trophoblasts.35 Filamin 2, a muscle-specific isoform, has
been shown to interact with the dystrophin-glycoprotein
complex of skeletal muscle cells.36
Several proteins have been shown to be associated with cardiac
myocyte caveolae. Endothelial NO synthase has been
shown to be targeted to caveolae of adult mammalian
ventricular myocytes.37 Endothelin has been
shown to induce the translocation of protein kinase C isoforms to
caveolae of neonatal ventricular myocytes, where members of
the extracellular signalregulated receptor kinases (ERKs) were shown
to reside.38 Muscarinic M2 receptors display
agonist-induced translocation to caveolae of adult rat
ventricular myocytes.39 Neuregulin receptor
erbB4 has been localized to caveolae of neonatal
ventricular myocytes.40
Natriuretic peptide receptor type B41 and
monocarboxylate transporter (MCT1)42 have been
immunohistochemically localized to myocyte caveolae in situ. Most
recently, activated adenosine A1
receptors have been shown to translocate out of adult rat
ventricular myocyte caveolae on agonist
binding.28 Here we report the first association of a
cytoskeletal protein with caveolae of heart myocytes.
Although the functional significance of the dystrophin-caveolar
association in heart muscle remains to be clarified, it may be relevant
that a correlation between mutations in cav-3 and clinically observed
muscular dystrophies has recently been reported.34 43 It
has been proposed that recruitment of proteins to caveolae, possibly by
the direct binding of some of these proteins to cav,33 may
play a role in caveolar function.14 Whether the
association of dystrophin with caveolae in cardiac myocytes is static
and serves a structural role or is dynamic and serves a functional role
or roles is an important question for further study.
 |
Acknowledgments
|
|---|
This study was supported by NIH Grant HL54302. The
Gastroenterology
Section of the Department of Medicine and the
Department of
Molecular Genetics and Cell Biology (University of
Chicago,
Chicago, Ill) kindly provided confocal microscopes and
valuable
assistance.
Received July 17, 2000;
revision received August 9, 2000;
accepted August 9, 2000.
 |
References
|
|---|
-
Palade GE. Fine structure of blood capillaries.
J Appl Physiol. 1953;24:1424a.
-
Anderson RG. Caveolae: where incoming and
outgoing messengers meet. Proc Natl Acad Sci U S A. 1993;90:1090910913.[Abstract/Free Full Text]
-
Sargiacomo M, Sudol M, Tang Z, Lisanti MP.
Signal transducing molecules and glycosyl-phosphatidylinositol-linked
proteins form a caveolin-enriched insoluble complex in MDCK cells.
J Cell Biol. 1993;122:789807.[Abstract/Free Full Text]
-
Lisanti MP, Scherer PE, Vidugiriene J, Tang Z,
Hermanowski-Vosatka A, Tu Y-H, Cook RF, Sargiacomo M. Characterization
of caveolin-rich membrane domains isolated from an
endothelial-rich source: implications for human
disease. J Cell Biol. 1994;126:111126.[Abstract/Free Full Text]
-
Rothberg KG, Heuser JE, Donzell WC, Ying Y-S,
Glenney JR, Anderson RGW. Caveolin, a protein component of caveolae
membrane coats. Cell. 1992;68:673682.[Medline]
[Order article via Infotrieve]
-
Brown DA, Rose JK. Sorting of GPI-anchored
proteins to glycolipid-enriched membrane subdomains during transport to
the apical cell surface. Cell. 1992;68:533544.[Medline]
[Order article via Infotrieve]
-
Glenney JR Jr, Soppet D. Sequence and expression
of caveolin, a protein component of caveolae plasma membrane domains
phosphorylated on tyrosine in Rous sarcoma
virus-transformed fibroblasts. Proc Natl Acad Sci U S A. 1992;89:1051710521.[Abstract/Free Full Text]
-
Monier S, Parton RG, Vogel F, Behlke J, Henske
A, Kurzchalia TV. VIP21-caveolin, a membrane protein constituent of the
caveolar coat, oligomerizes in vivo and in vitro. Mol Biol
Cell. 1995;6:911927.[Abstract]
-
Scherer PE, Tang Z, Chun M, Sargiacomo M, Lodish
HF, Lisanti MP. Caveolin isoforms differ in their N-terminal protein
sequence and subcellular distribution. J Biol Chem. 1995;270:1639516401.[Abstract/Free Full Text]
-
Scheiffele P, Verkade P, Fra AM, Virta H, Simons
K, Ikonen E. Caveolin-1 and -2 in the exocytic pathway of MDCK cells.
J Cell Biol. 1998;140:795806.[Abstract/Free Full Text]
-
Song K, Li S, Okamoto T, Quilliam LA, Sargiacomo
M, Lisanti MP. Co-purification and direct interaction of Ras with
caveolin, an integral membrane protein of caveolae microdomains.
J Biol Chem. 1996;271:96909697.[Abstract/Free Full Text]
-
Way M, Parton RG. M-caveolin, a muscle-specific
caveolin-related protein. FEBS Lett. 1995;376:108112.[Medline]
[Order article via Infotrieve]
-
Parton RG, Way M, Zorzi N, Stang E. Caveolin-3
associates with developing T-tubules during muscle differentiation.
J Cell Biol. 1997;136:137154.[Abstract/Free Full Text]
-
Schlegel A, Volonte D, Engelman JA, Galbiati F,
Mehta P, Zhang X-L, Scherer PE, Lisanti MP. Crowded little caves:
structure and function of caveolae. Cell. 1998;10:457463.
-
Mayor S, Rothberg KG, Maxfield FR. Sequestration
of GPI-anchored proteins in caveolae triggered by cross-linking.
Science. 1994;264:19481951.[Abstract/Free Full Text]
-
Mayor S, Maxfield FR. Insolubility and
redistribution of GPI-anchored proteins at the cell surface after
detergent treatment. Mol Biol Cell. 1995;6:929944.[Abstract]
-
Stan R-V, Roberts WG, Predescu D, Ihida K, Saucan
L, Ghitescu L, Palade GE. Immunoisolation and partial characterization
of endothelial plasmalemmal vesicles
(caveolae). Mol Biol Cell. 1997;8:595605.[Abstract]
-
Doyle DD, Ranscht B, Page E, Palfrey HC.
T-cadherin is a major phosphorylated and
ADP-ribosylated component of cardiac caveolae. Mol Biol
Cell. 1995;6:403a. Abstract.
-
Doyle DD, Goings GE, Upshaw-Earley J, Page E,
Ranscht B, Palfrey HC. T-cadherin is a major
glycophosphoinositol-anchored protein associated with
noncaveolar detergent-insoluble domains of the cardiac sarcolemma.
J Biol Chem. 1998;273:69376943.[Abstract/Free Full Text]
-
Small JV, Furst DO, Thornell L-E. The
cytoskeletal lattice of muscle cells. Eur J Biochem. 1992;208:559572.[Medline]
[Order article via Infotrieve]
-
Straub V, Campbell KP. Muscular dystrophies and
the dystrophin-glycoprotein complex. Curr Opin
Neurol. 1997;10:168175.[Medline]
[Order article via Infotrieve]
-
Klietsch R, Ervasti JM, Arnold W, Campbell KP,
Jorgensen AO. Dystrophin-glycoprotein complex and lamini
colocalize to the sarcolemma and transverse tubules of cardiac muscle.
Circ Res. 1993;72:349360.[Abstract/Free Full Text]
-
Rybakova IN, Ervasti JM.
Dystrophin-glycoprotein complex is monomeric and stabilizes
actin filaments in vitro through a lateral association. J
Biol Chem. 1997;272:2877128778.[Abstract/Free Full Text]
-
Crosbie RH, Yamada H, Venzke DP, Lisanti MP,
Campbell KP. Caveolin-3 is not an integral component of the dystrophin
glycoprotein complex. FEBS Lett. 1998;427:279282.[Medline]
[Order article via Infotrieve]
-
North AJ, Galazkiewicz B, Byers TJ, Glenney JR,
Small JV. Complementary distributions of vinculin and dystrophin define
two distinct sarcolemma domains in smooth muscle. J Cell
Biol. 1993;120:11591167.[Abstract/Free Full Text]
-
Jones LR. Rapid preparation of canine cardiac
sarcolemmal vesicles by sucrose flotation. Methods Enzymol. 1998;157:8591.
-
Maisch B. Enrichment of vital adult cardiac
muscle cells by continuous silica sol gradient
centrifugation. Basic Res Cardiol. 1981;76:622629.[Medline]
[Order article via Infotrieve]
-
Lasley RD, Narayan P, Uittenbogaard A, Smart EJ.
Activated cardiac adenosine A1 receptors translocate
out of caveolae. J Biol Chem. 2000;275:44174421.[Abstract/Free Full Text]
-
Chang W-J, Ying Y-S, Rothberg KG, Hooper NM,
Turner AJ, Gambliel HA, Degunzberg J, Mumby SM, Gilman AG, Anderson
RGW. Purification and characterization of smooth muscle cell caveolae.
J Cell Biol. 1994;126:127138.[Abstract/Free Full Text]
-
Page E, Winterfield J, Goings G, Bastawrous A,
Upshaw-Earley J, Doyle D. Water channel proteins in rat cardiac myocyte
caveolae: osmolarity-dependent reversible internalization.
Am J Physiol. 1998;274:H1988H2000.[Abstract/Free Full Text]
-
Waugh MG, Lawson D, Tan SK, Hsuan JJ.
Phosphatidylinositol 4-phosphate synthesis in immunoisolated
caveolae-like vesicles and low buoyant density non-caveolar membranes.
J Biol Chem. 1998;273:1711517121.[Abstract/Free Full Text]
-
Page E, Upshaw-Earley J, Goings GE, Hanck DA.
Fluid-phase endocytosis by in situ cardiac myocytes of rat atria.
Am J Physiol. 1993;265:C986C996.[Abstract/Free Full Text]
-
Couet J, Li S, Okamoto T, Ikezu T, Lisanti MP.
Identification of peptide and protein ligands for the
caveolin-scaffolding domain: implications for the interaction of
caveolin with caveolae-associated proteins. J Biol
Chem. 1997;272:65256533.[Abstract/Free Full Text]
-
McNally EM, de Sa Moreira E, Duggan DJ, Bonnemann
CG, Lisanti MP, Lidov HGW, Vainzof M, Passos-Bueno MR, Hoffman EP, Zatz
M, Kunkel LM. Caveolin-3 in muscular dystrophy. Hum Mol
Genet. 1998;7:871877.[Abstract/Free Full Text]
-
Stahlhut M, Deurs BV. Identification of filamin
as a novel ligand for caveolin-1: evidence for the organization of
caveolin-1 associated membrane domains by the actin cytoskeleton.
Mol Biol Cell. 2000;11:325337.[Abstract/Free Full Text]
-
Thompson TG, Chan Y-M, Hack AA, Brosius M,
Rajala M, Lidov HGW, McNally EM, Watkins S, Kunkel LM. Filamin 2(FLN2):
a muscle-specific sarcoglycan interacting protein. J Cell
Biol. 2000;148:115126.[Abstract/Free Full Text]
-
Feron O, Belhassen L, Kobzik L, Smith TW, Kelly
RA, Michel T. Endothelial nitric oxide synthase
targeting to caveolae: specific interactions with caveolin isoforms in
cardiac myocytes and endothelial cells. J
Biol Chem. 1996;271:2281022814.[Abstract/Free Full Text]
-
Rybin VO, Xu X, Steinberg SF. Activated
protein kinase C isoforms target to cardiac myocyte caveolae.
Circ Res. 1999;84:980988.[Abstract/Free Full Text]
-
Kijima Y, Saito A, Jetton TL, Magnuson MA,
Fleischer S. Different intracellular localization of inositol
1,4,5-triphosphate and ryanodine receptors in
cardiomyocytes. J Biol Chem. 1993;268:34993506.[Abstract/Free Full Text]
-
Zhao Y-y, Feron O, Dessy C, Han H, Marchionni MA,
Kelly RA. Neuregulin signaling in the heart: dynamic targeting of erbB4
to caveolar microdomains in cardiac myocyte caveolae. Circ
Res. 1999;84:13801387.[Abstract/Free Full Text]
-
Doyle DD, Ambler SK, Upshaw-Earley J, Bastawrous
A, Goings GE, Page E. Type B atrial natriuretic peptide
receptor in cardiac myocyte caveolae. Circ Res. 1997;81:8691.[Abstract/Free Full Text]
-
Johannsson E, Nagelhus EA, McCullagh KJ,
Sejersted OM, Blackstad TW, Bonen A, Ottersen OP. Cellular and
subcellular expression of the monocarboxylate transporter MCT1 in rat
heart: a high-resolution immunogold analysis. Circ
Res. 1997;80:400407.
-
Minetti C, Sotgia F, Bruno C, Scartezzini P,
Broda P, Bado M, Masetti E, Mazzocco M, Egeo A, Donati MA, Volonte D,
Galbiati F, Cordone G, Bricarelli FD, Lisanti MP, Zara F. Mutations in
the caveolin-3 gene cause autosomal dominant limb-girdle muscular
dystrophy. Nat Genet. 1998;18:365368.[Medline]
[Order article via Infotrieve]
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