© 2000 American Heart Association, Inc.
Cellular Biology |
Dystrophin Associates With Caveolae of Rat Cardiac Myocytes
Relationship to Dystroglycan
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 Children’s 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—The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3–enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan. Further purification of cav-3–containing membranes by immunoprecipitation using anti–cav-3–coated 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 anti–cav-3 antibodies. In contrast, immunoprecipitation of membranes with anti-dystrophin–coated beads yielded both cav-3 and dystroglycan. Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti–cav-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-3–containing 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. Immuno–electron 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 membrane–associated vesicles called caveolae are non–clathrin-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 Ca2+ 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 Immuno–electron 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-3–enriched 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
anti–cav-3–coupled 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-3–containing membranes (above) were incubated at 4°C with
polyclonal anti–cav-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 fragment–specific
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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Coprecipitation of Dystrophin With Immunopurified Cardiac Caveolae
We previously reported that Triton X-100–insoluble 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-3–containing membranes by anti–cav-3–coated 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
anti–cav-3–coated 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 anti–cav-1
antibodies from extracts of sonicated A431 cells. Other vesicles were
electron-opaque, resembling some of the anti–cav-1–immunoprecipitated
membranes from rat lung seen by Stan et
al.17
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When detergent-resistant membranes prepared from whole rat
ventricle were subjected to immunoprecipitation using
anti–cav-3–coated 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 anti–cav-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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In correlative experiments, we examined whether
anti-dystrophin–coated 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 anti–cav-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.
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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 high–ionic-strength
buffers similar in manner to our approach to whole ventricle.
Immunoisolation of cav-3–containing 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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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-3–containing 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-3–containing 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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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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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 anti–cav-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 anti–cav-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 signal–regulated 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.
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Acknowledgments |
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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.
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